# Patent

## Title
Conjugated toxin peptide therapeutic agents

## Abstract

Disclosed is a composition of matter comprising an OSK1 peptide analog, and in some embodiments, a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprises the composition and a pharmaceutically acceptable carrier. Also disclosed are DNAs encoding the inventive composition of matter, an expression vector comprising the DNA, and host cells comprising the expression vector. Methods of treating an autoimmune disorder and of preventing or mitigating a relapse of a symptom of multiple sclerosis are also disclosed.

## Background
This application claims priority from U.S. Provisional Application No. 60/854,674, filed Oct. 25, 2006, and U.S. Application No. 60/995,370, filed Sep. 25, 2007, both of which are hereby incorporated by reference.
This application is related to U.S. Non-provisional application Ser. No. 11/978,104, filed Oct. 25, 2007, U.S. Non-provisional application Ser. No. 11/978,105, filed Oct. 25, 2007, U.S. Non-provisional application Ser. No. 11/978,110 filed Oct. 25, 2007, U.S. Non-provisional application Ser. No. 11/978,111 filed Oct. 25, 2007, and U.S. Non-provisional application Ser. No. 11/978,119, filed Oct. 25, 2007, all which applications are hereby incorporated by reference. This application is also related to U.S. Non-provisional application Ser. No. 11/584,177, filed Oct. 19, 2006, which claims priority from U.S. Provisional Application No. 60/729,083, filed Oct. 21, 2005, both of which applications are hereby incorporated by reference.
The instant application contains an ASCII “txt” compliant sequence listing submitted via EFS-WEB on May 18, 2010, which serves as both the computer readable form (CRF) and the paper copy required by 37 C.F.R. Section 1.821(c) and 1.821(e), and is hereby incorporated by reference in its entirety. The name of the “txt” file created on Sep. 10, 2009, is: A-1186-US-NP-RevSeqList091109.txt, and is 2,607 kB in size.
Throughout this application various publications are referenced within parentheses or brackets. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is related to the biochemical arts, in particular to therapeutic peptides and conjugates.
2. Discussion of the Related Art
Ion channels are a diverse group of molecules that permit the exchange of small inorganic ions across membranes. All cells require ion channels for function, but this is especially so for excitable cells such as those present in the nervous system and the heart. The electrical signals orchestrated by ion channels control the thinking brain, the beating heart and the contracting muscle. Ion channels play a role in regulating cell volume, and they control a wide variety of signaling processes.
The ion channel family includes Na+, K+, and Ca2+ cation and Cl− anion channels. Collectively, ion channels are distinguished as either ligand-gated or voltage-gated. Ligand-gated channels include both extracellular and intracellular ligand-gated channels. The extracellular ligand-gated channels include the nicotinic acetylcholine receptor (nAChR), the serotonin (5-hdroxytryptamine, 5-HT) receptors, the glycine and γ-butyric acid receptors (GABA) and the glutamate-activated channels including kanate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate receptors (NMDA) receptors. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34). Intracellular ligand gated channels include those activated by cyclic nucleotides (e.g. cAMP, cGMP), Ca2+ and G-proteins. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34). The voltage-gated ion channels are categorized by their selectivity for inorganic ion species, including sodium, potassium, calcium and chloride ion channels. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34).
A unified nomenclature for classification of voltage-gated channels was recently presented. (Catterall et al. (2000), Pharmacol. Rev. 55: 573-4; Gutman et al. (2000), Pharmacol. Rev. 55, 583-6; Catterall et al. (2000) Pharmacol. Rev. 55: 579-81; Catterall et al. (2000), Pharmacol. Rev. 55: 575-8; Hofmann et al. (2000), Pharmacol. Rev. 55: 587-9; Clapham et al. (2000), Pharmacol Rev. 55: 591-6; Chandy (1991), Nature 352: 26; Goldin et al. (2000), Neuron 28: 365-8; Ertel et al. (2000), Neuron 25: 533-5).
The K+ channels constitute the largest and best characterized family of ion channels described to date. Potassium channels are subdivided into three general groups: the 6 transmembrane (6TM) K+ channels, the 2TM-2TM/leak K+ channels and the 2TM/K+ inward rectifying channels. (Tang et al. (2004), Ann. Rev. Physiol. 66, 131-159). These three groups are further subdivided into families based on sequence similarity. The voltage-gated K+ channels, including (Kv1-6, Kv8-9), EAG, KQT, and Slo (BKCa), are family members of the 6TM group. The 2TM-2TM group comprises TWIK, TREK, TASK, TRAAK, and THIK, whereas the 2TM/Kir group consists of Kir1-7. Two additional classes of ion channels include the inward rectifier potassium (IRK) and ATP-gated purinergic (P2X) channels. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34).
Toxin peptides produced by a variety of organisms have evolved to target ion channels. Snakes, scorpions, spiders, bees, snails and sea anemone are a few examples of organisms that produce venom that can serve as a rich source of small bioactive toxin peptides or “toxins” that potently and selectively target ion channels and receptors. In most cases, these toxin peptides have evolved as potent antagonists or inhibitors of ion channels, by binding to the channel pore and physically blocking the ion conduction pathway. In some other cases, as with some of the tarantula toxin peptides, the peptide is found to antagonize channel function by binding to a region outside the pore (e.g., the voltage sensor domain).
The toxin peptides are usually between about 20 and about 80 amino acids in length, contain 2-5 disulfide linkages and form a very compact structure (see, e.g., FIG. 10). Toxin peptides (e.g., from the venom of scorpions, sea anemones and cone snails) have been isolated and characterized for their impact on ion channels. Such peptides appear to have evolved from a relatively small number of structural frameworks that are particularly well suited to addressing the critical issues of potency and stability. The majority of scorpion and Conus toxin peptides, for example, contain 10-40 amino acids and up to five disulfide bonds, forming extremely compact and constrained structure (microproteins) often resistant to proteolysis. The conotoxin and scorpion toxin peptides can be divided into a number of superfamilies based on their disulfide connections and peptide folds. The solution structure of many of these has been determined by NMR spectroscopy, illustrating their compact structure and verifying conservation of their family fold. (E.g., Tudor et al., Ionisation behaviour and solution properties of the potassium-channel blocker ShK toxin, Eur. J. Biochem. 251(1-2):133-41 (1998); Pennington et al., Role of disulfide bonds in the structure and potassium channel blocking activity of ShK toxin, Biochem. 38(44): 14549-58 (1999); Jaravine et al., Three-dimensional structure of toxin OSK1 from Orthochirus scrobiculosus scorpion venom, Biochem. 36(6):1223-32 (1997); del Rio-Portillo et al.; NMR solution structure of Cn12, a novel peptide from the Mexican scorpion Centruroides noxius with a typical beta-toxin sequence but with alpha-like physiological activity, Eur. J. Biochem. 271(12): 2504-16 (2004); Prochnicka-Chalufour et al., Solution structure of discrepin, a new K+-channel blocking peptide from the alpha-KTx15 subfamily, Biochem. 45(6):1795-1804 (2006)).
Conserved disulfide structures can also reflect the individual pharmacological activity of the toxin family. (Nicke et al. (2004), Eur. J. Biochem. 271: 2305-19, Table 1; Adams (1999), Drug Develop. Res. 46: 219-34). For example, α-conotoxins have well-defined four cysteine/two disulfide loop structures (Loughnan, 2004) and inhibit nicotinic acetylcholine receptors. In contrast, ω-conotoxins have six cysteine/three disulfide loop consensus structures (Nielsen, 2000) and block calcium channels. Structural subsets of toxins have evolved to inhibit either voltage-gated or calcium-activated potassium channels. FIG. 9 shows that a limited number of conserved disulfide architectures shared by a variety of venomous animals from bee to snail and scorpion to snake target ion channels. FIG. 7A-7B shows alignment of alpha-scorpion toxin family and illustrates that a conserved structural framework is used to derive toxins targeting a vast array of potassium channels.
Due to their potent and selective blockade of specific ion channels, toxin peptides have been used for many years as tools to investigate the pharmacology of ion channels. Other than excitable cells and tissues such as those present in heart, muscle and brain, ion channels are also important to non-excitable cells such as immune cells. Accordingly, the potential therapeutic utility of toxin peptides has been considered for treating various immune disorders, in particular by inhibition of potassium channels such as Kv1.3 and IKCa1 since these channels indirectly control calcium signaling pathway in lymphocytes. [e.g., Kem et al., ShK toxin compositions and methods of use, U.S. Pat. No. 6,077,680; Lebrun et al., Neuropeptides originating in scorpion, U.S. Pat. No. 6,689,749; Beeton et al., Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases, Molec. Pharmacol. 67(4):1369-81 (2005); Mouhat et al., K+ channel types targeted by synthetic OSK1, a toxin from Orthochirus scrobiculosus scorpion venom, Biochem. J. 385:95-104 (2005); Mouhat et al., Pharmacological profiling of Orthochirus scrobiculosus toxin 1 analogs with a trimmed N-terminal domain, Molec. Pharmacol. 69:354-62 (2006); Mouhat et al., OsK1 derivatives, WO 2006/002850 A2; B. S. Jensen et al. The Ca2+-activated K+ Channel of Intermediate Conductance: A Molecular Target for Novel Treatments?, Current Drug Targets 2:401-422 (2001); Rauer et al., Structure-guided Transformation of Charybdotoxin Yields an Analog That Selectively Targets Ca2+-activated over Voltage-gated K+ Channels, J. Biol. Chem. 275: 1201-1208 (2000); Castle et al., Maurotoxin: A Potent Inhibitor of Intermediate Conductance Ca2+-Activated Potassium Channels, Molecular Pharmacol. 63: 409-418 (2003); Chandy et al., K+ channels as targets for specific Immunomodulation, Trends in Pharmacol. Sciences 25: 280-289 (2004); Lewis & Garcia, Therapeutic Potential of Venom Peptides, Nat. Rev. Drug Discov. 2: 790-802 (2003)].
Small molecules inhibitors of Kv1.3 and IKCa1 potassium channels and the major calcium entry channel in T cells, CRAC, have also been developed to treat immune disorders [A. Schmitz et al. (2005) Molecul. Pharmacol. 68, 1254; K. G. Chandy et al. (2004) TIPS 25, 280; H. Wulff et al. (2001) J. Biol. Chem. 276, 32040; C. Zitt et al. (2004) J. Biol. Chem. 279, 12427], but obtaining small molecules with selectivity toward some of these targets has been difficult.
Calcium mobilization in lymphocytes is known to be a critical pathway in activation of inflammatory responses [M. W. Winslow et al. (2003) Current Opinion Immunol. 15, 299]. Compared to other cells, T cells show a unique sensitivity to increased levels of intracellular calcium and ion channels both directly and indirectly control this process. Inositol triphosphate (IP3) is the natural second messenger which activates the calcium signaling pathway. IP3 is produced following ligand-induced activation of the T cell receptor (TCR) and upon binding to its intracellular receptor (a channel) causes unloading of intracellular calcium stores. The endoplasmic reticulum provides one key calcium store. Thapsigargin, an inhibitor of the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA), also causes unloading of intracellular stores and activation of the calcium signaling pathway in lymphocytes. Therefore, thapsigargin can be used as a specific stimulus of the calcium signaling pathway in T cells. The unloading of intracellular calcium stores in T cells is known to cause activation of a calcium channel on the cell surface which allows for influx of calcium from outside the cell. This store operated calcium channel (SOCC) on T cells is referred to as “CRAC” (calcium release activated channel) and sustained influx of calcium through this channel is known to be critical for full T cell activation [S. Feske et al. (2005) J. Exp. Med. 202, 651 and N. Venkatesh et al. (2004) PNAS 101, 8969]. For many years it has been appreciated that in order to maintain continued calcium influx into T cells, the cell membrane must remain in a hyperpolarized condition through efflux of potassium ions. In T cells, potassium efflux is accomplished by the voltage-gated potassium channel Kv1.3 and the calcium-activated potassium channel IKCa1 [K. G. Chandy et al. (2004) TIPS 25, 280]. These potassium channels therefore indirectly control the calcium signaling pathway, by allowing for the necessary potassium efflux that allows for a sustained influx of calcium through CRAC.
Sustained increases in intracellular calcium activate a variety of pathways in T cells, including those leading to activation of NFAT, NF-kB and AP-1 [Quintana-A (2005) Pflugers Arch.—Eur. J. Physiol. 450, 1]. These events lead to various T cell responses including alteration of cell size and membrane organization, activation of cell surface effector molecules, cytokine production and proliferation. Several calcium sensing molecules transmit the calcium signal and orchestrate the cellular response. Calmodulin is one molecule that binds calcium, but many others have been identified (M. J. Berridge et al. (2003) Nat. Rev. Mol. Cell. Biol. 4, 517). The calcium-calmodulin dependent phosphatase calcineurin is activated upon sustained increases in intracellular calcium and dephosphorylates cytosolic NFAT. Dephosphorylated NFAT quickly translocates to the nucleus and is widely accepted as a critical transcription factor for T cell activation (F. Macian (2005) Nat. Rev. Immunol. 5, 472 and N. Venkatesh et al. (2004) PNAS 101, 8969). Inhibitors of calcineurin, such as cyclosporin A (Neoral, Sandimmune) and FK506 (Tacrolimus) are a main stay for treatment of severe immune disorders such as those resulting in rejection following solid organ transplant (I. M. Gonzalez-Pinto et al. (2005) Transplant. Proc. 37, 1713 and D. R. J. Kuypers (2005) Transplant International 18, 140). Neoral has been approved for the treatment of transplant rejection, severe rheumatoid arthritis (D. E. Yocum et al. (2000) Rheumatol. 39, 156) and severe psoriasis (J. Koo (1998) British J. Dermatol. 139, 88). Preclinical and clinical data has also been provided suggesting calcineurin inhibitors may have utility in treatment of inflammatory bowel disease (IBD; Baumgart D C (2006) Am. J. Gastroenterol. March 30; Epub ahead of print), multiple sclerosis (Ann. Neurol. (1990) 27, 591) and asthma (S. Rohatagi et al. (2000) J. Clin. Pharmacol. 40, 1211). Lupus represents another disorder that may benefit from agents blocking activation of helper T cells. Despite the importance of calcineurin in regulating NFAT in T cells, calcineurin is also expressed in other tissues (e.g. kidney) and cyclosporine A & FK506 have a narrow safety margin due to mechanism based toxicity. Renal toxicity and hypertension are common side effects that have limited the promise of cyclosporine & FK506. Due to concerns regarding toxicity, calcineurin inhibitors are used mostly to treat only severe immune disease (Bissonnette-R et al. (2006) J. Am. Acad. Dermatol. 54, 472). Kv1.3 inhibitors offer a safer alternative to calcineurin inhibitors for the treatment of immune disorders. This is because Kv1.3 also operates to control the calcium signaling pathway in T cells, but does so through a distinct mechanism to that of calcineurin inhibitors, and evidence on Kv1.3 expression and function show that Kv1.3 has a more restricted role in T cell biology relative to calcineurin, which functions also in a variety of non-lymphoid cells and tissues.
Calcium mobilization in immune cells also activates production of the cytokines interleukin 2 (IL-2) and interferon gamma (IFNg) which are critical mediators of inflammation. IL-2 induces a variety of biological responses ranging from expansion and differentiation of CD4+ and CD8+ T cells, to enhancement of proliferation and antibody secretion by B cells, to activation of NK cells [S. L. Gaffen & K. D. Liu (2004) Cytokine 28, 109]. Secretion of IL-2 occurs quickly following T cell activation and T cells represent the predominant source of this cytokine. Shortly following activation, the high affinity IL-2 receptor (IL2-R) is upregulated on T cells endowing them with an ability to proliferate in response to IL-2. T cells, NK cells, B cells and professional antigen presenting cells (APCs) can all secrete IFNg upon activation. T cells represent the principle source of IFNg production in mediating adaptive immune responses, whereas natural killer (NK) cells & APCs are likely an important source during host defense against infection [K. Schroder et al. (2004) J. Leukoc. Biol. 75, 163]. IFNg, originally called macrophage-activating factor, upregulates antigen processing and presentation by monocytes, macrophages and dendritic cells. IFNg mediates a diverse array of biological activities in many cell types [U. Boehm et al. (1997) Annu. Rev. Immunol. 15, 749] including growth & differentiation, enhancement of NK cell activity and regulation of B cell immunoglobulin production and class switching.
CD40L is another cytokine expressed on activated T cells following calcium mobilization and upon binding to its receptor on B cells provides critical help allowing for B cell germinal center formation, B cell differentiation and antibody isotype switching. CD40L-mediated activation of CD40 on B cells can induce profound differentiation and clonal expansion of immunoglobulin (Ig) producing B cells [S. Quezada et al. (2004) Annu. Rev. Immunol. 22, 307]. The CD40 receptor can also be found on dendritic cells and CD40L signaling can mediate dendritic cell activation and differentiation as well. The antigen presenting capacity of B cells and dendritic cells is promoted by CD40L binding, further illustrating the broad role of this cytokine in adaptive immunity. Given the essential role of CD40 signaling to B cell biology, neutralizing antibodies to CD40L have been examined in preclinical and clinical studies for utility in treatment of systemic lupus erythematosis (SLE),—a disorder characterized by deposition of antibody complexes in tissues, inflammation and organ damage [J. Yazdany and J Davis (2004) Lupus 13, 377].
Production of toxin peptides is a complex process in venomous organisms, and is an even more complex process synthetically. Due to their conserved disulfide structures and need for efficient oxidative refolding, toxin peptides present challenges to synthesis. Although toxin peptides have been used for years as highly selective pharmacological inhibitors of ion channels, the high cost of synthesis and refolding of the toxin peptides and their short half-life in vivo have impeded the pursuit of these peptides as a therapeutic modality. Far more effort has been expended to identify small molecule inhibitors as therapeutic antagonists of ion channels, than has been given the toxin peptides themselves. One exception is the recent approval of the small ω-conotoxin MVIIA peptide (ZiconotideTM) for treatment of intractable pain. The synthetic and refolding production process for ZiconotideTM, however, is costly and only results in a small peptide product with a very short half-life in vivo (about 4 hours).
A cost-effective process for producing therapeutics, such as but not limited to, inhibitors of ion channels, is a desideratum provided by the present invention, which involves toxin peptides fused, or otherwise covalently conjugated to a vehicle.
SUMMARY OF THE INVENTION
The present invention relates to a composition of matter of the formula:

(X1)a-(F1)d-(X2)b-(F2)e-(X3)c  (I)

and multimers thereof, wherein:

F1 and F2 are half-life extending moieties, and d and e are each independently 0 or 1, provided that at least one of d and e is 1;
X1, X2, and X3 are each independently -(L)f-P-(L)g-, and f and g are each independently 0 or 1;
P is a toxin peptide of no more than about 80 amino acid residues in length, comprising at least two intrapeptide disulfide bonds, and at least one P is an OSK1 peptide analog;
L is an optional linker (present when f=1 and/or g=1); and
a, b, and c are each independently 0 or 1, provided that at least one of a, b and c is 1.
The present invention thus concerns molecules having variations on Formula I, such as the formulae:

P-(L)g-F1 (i.e., b, c, and e equal to 0);  (II)

F1-(L)f-P (i.e., a, c, and e equal to 0);  (III)

P-(L)g-F1-(L)f-P or (X1)a-F1-(X2)b (i.e., c and e equal to 0);  (IV)

F1-(L)f-P-(L)g-F2 (i.e., a and c equal to 0);  (V)

F1-(L)f-P-(L)g-F2-(L)f-P (i.e., a equal to 0);  (VI)

F1-F2-(L)f-P (i.e., a and b equal to 0);  (VII)

P-(L)g-F1-F2 (i.e., b and c equal to 0);  (VIII)

P-(L)g-F1-F2-(L)f-P (i.e., b equal to 0);  (IX)

and any multimers of any of these, when stated conventionally with the N-terminus of the peptide(s) on the left. All of such molecules of Formulae II-IX are within the meaning of Structural Formula I. Within the meaning of Formula I, the toxin peptide (P), if more than one is present, can be independently the same or different from the OSK1 peptide analog, or any other toxin peptide(s) also present in the inventive composition, and the linker moiety ((L)f and/or (L)g), if present, can be independently the same or different from any other linker, or linkers, that may be present in the inventive composition. Conjugation of the toxin peptide(s) to the half-life extending moiety, or moieties, can be via the N-terminal and/or C-terminal of the toxin peptide, or can be intercalary as to its primary amino acid sequence, F1 being linked closer to the toxin peptide's N-terminus than is linked F2. Examples of useful half-life extending moieties (F1 or F2) include immunoglobulin Fc domain (e.g., a human immunoglobulin Fc domain, including Fc of IgG1, IgG2, IgG3 or IgG4) or a portion thereof, human serum albumin (HSA), or poly(ethylene glycol) (PEG). These and other half-life extending moieties described herein are useful, either individually or in combination. A monovalent dimeric Fc-toxin peptide fusion (as represented schematically in FIG. 2B), for example, an Fc-OSK1 peptide analog fusion or Fc-ShK peptide analog fusion, is an example of the inventive composition of matter encompassed by Formula VII above.

The present invention also relates to a composition of matter, which includes, conjugated or unconjugated, a toxin peptide analog of ShK, OSK1, ChTx, or Maurotoxin modified from the native sequences at one or more amino acid residues, having greater Kv1.3 or IKCa1 antagonist activity, and/or target selectivity, compared to a ShK, OSK1, or Maurotoxin (MTX) peptides having a native sequence. The toxin peptide analogs comprise an amino acid sequence selected from any of the following:
SEQ ID NOS: 88, 89, 92, 148 through 200, 548 through 561, 884 through 949, or 1295 through 1300 as set forth in Table 2; or
SEQ ID NOS: 980 through 1274, 1303, or 1308 as set forth in Table 7; or any of SEQ ID NOS: 1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through 5015 as set forth in Table 7A, Table 7B, Table 7C, Table 7D, Table 7E, Table 7F, Table 7G, Table 7H, Table 7I or Table 7J.
SEQ ID NOS: 330 through 337, 341, 1301, 1302, 1304 through 1307, 1309, 1311, 1312, and 1315 through 1336 as set forth in Table 13; or
SEQ ID NOS: 36, 59, 344-346, or 1369 through 1390 as set forth in Table 14.
The present invention also relates to other toxin peptide analogs that comprise an amino acid sequence selected from, or comprise the amino acid primary sequence of, any of the following:
SEQ ID NOS: 201 through 225 as set forth in Table 3; or
SEQ ID NOS: 242 through 248 or 250 through 260 as set forth in Table 4; or
SEQ ID NOS: 261 through 275 as set forth in Table 5; or
SEQ ID NOS: 276 through 293 as set forth in Table 6; or
SEQ ID NOS: 299 through 315 as set forth in Table 8; or
SEQ ID NOS: 316 through 318 as set forth in Table 9; or
SEQ ID NO: 319 as set forth in Table 10; or
SEQ ID NO: 327 or 328 as set forth in Table 11; or
SEQ ID NOS: 330 through 337, 341, 1301, 1302, 1304 through 1307, 1309, 1311, 1312, or 1315 through 1336 as set forth in Table 13;
SEQ ID NOS: 1369 through 1390 as set forth in Table 14; or
SEQ ID NOS: 348 through 353 as set forth in Table 16; or
SEQ ID NOS: 357 through 362, 364 through 368, 370, 372 through 385, or 387 through 398 as set forth in Table 19; or
SEQ ID NOS: 399 through 408 as set forth in Table 20; or
SEQ ID NOS: 410 through 421 as set forth in Table 22; or
SEQ ID NOS: 422, 424, 426, or 428 as set forth in Table 23; or
SEQ ID NOS: 430 through 437 as set forth in Table 24; or
SEQ ID NOS: 438 through 445 as set forth in Table 25; or
SEQ ID NOS: 447, 449, 451, 453, 455, or 457 as set forth in Table 26; or
SEQ ID NOS: 470 through 482 or 484 through 493 as set forth in Table 28; or
SEQ ID NOS: 495 through 506 as set forth in Table 29; or
SEQ ID NOS: 507 through 518 as set forth in Table 30.
The present invention is also directed to a pharmaceutical composition that includes the inventive composition of matter and a pharmaceutically acceptable carrier.
The compositions of this invention can be prepared by conventional synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins well known in the art. Compositions of this invention that have non-peptide portions can be synthesized by conventional organic chemistry reactions, in addition to conventional peptide chemistry reactions when applicable. Thus the present invention also relates to DNAs encoding the inventive compositions and expression vectors and host cells for recombinant expression.
The primary use contemplated is as therapeutic and/or prophylactic agents. The inventive compositions incorporating the toxin peptide can have activity and/or ion channel target selectivity comparable to—or even greater than—the unconjugated peptide.
Accordingly, the present invention includes a method of treating an autoimmune disorder, which involves administering to a patient who has been diagnosed with an autoimmune disorder, such as multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease (IBD, including Crohn's Disease and ulcerative colitis), contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, or lupus, a therapeutically effective amount of the inventive composition of matter (preferably comprising a Kv1.3 antagonist peptide or IKCa1 antagonist peptide), whereby at least one symptom of the disorder is alleviated in the patient. In addition, the present invention also relates to the use of one or more of the inventive compositions of matter in the manufacture of a medicament for the treatment or prevention of an autoimmune disorder, such as, but not limited to, any of the above-listed autoimmune disorders, e.g. multiple sclerosis, type 1 diabetes or IBD.
The present invention is further directed to a method of preventing or mitigating a relapse of a symptom of multiple sclerosis, which method involves administering to a patient, who has previously experienced at least one symptom of multiple sclerosis, a prophylactically effective amount of the inventive composition of matter (preferably comprising a Kv1.3 antagonist peptide or IKCa1 antagonist peptide), such that the at least one symptom of multiple sclerosis is prevented from recurring or is mitigated.
Although mostly contemplated as therapeutic agents, compositions of this invention can also be useful in screening for therapeutic or diagnostic agents. For example, one can use an Fc-peptide in an assay employing anti-Fc coated plates. The half-life extending moiety, such as Fc, can make insoluble peptides soluble and thus useful in a number of assays.
Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention.
U.S. Nonprovisional patent application Ser. No. 11/406,454, filed Apr. 17, 2006, is hereby incorporated by reference in its entirety.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows schematic structures of some exemplary Fc dimers that can be derived from an IgG1 antibody. “Fc” in the figure represents any of the Fc variants within the meaning of “Fc domain” herein. “X1” and “X2” represent peptides or linker-peptide combinations as defined hereinafter. The specific dimers are as follows:
FIG. 1A and FIG. 1D: Single disulfide-bonded dimers;
FIG. 1B and FIG. 1E: Doubly disulfide-bonded dimers;
FIG. 1C and FIG. 1F: Noncovalent dimers.
FIG. 2A-C show schematic structures of some embodiments of the composition of the invention that shows a single unit of the pharmacologically active toxin peptide. FIG. 2A shows a single chain molecule and can also represent the DNA construct for the molecule. FIG. 2B shows a dimer in which the linker-peptide portion is present on only one chain of the dimer (i.e., a “monovalent” dimer). FIG. 2C shows a dimer having the peptide portion on both chains. The dimer of FIG. 2C will form spontaneously in certain host cells upon expression of a DNA construct encoding the single chain shown in FIG. 2A. In other host cells, the cells could be placed in conditions favoring formation of dimers or the dimers can be formed in vitro.
FIG. 3A-3B shows exemplary nucleic acid and amino acid sequences (SEQ ID NOS: 1 and 2, respectively) of human IgG1 Fc that is optimized for mammalian expression and can be used in this invention.
FIG. 4A-4B shows exemplary nucleic acid and amino acid sequences (SEQ ID NOS: 3 and 4, respectively) of human IgG1 Fc that is optimized for bacterial expression and can be used in this invention.
FIG. 5A shows the amino acid sequence of the mature ShK peptide (SEQ ID NO: 5), which can be encoded for by a nucleic acid sequence containing codons optimized for expression in mammalian cell, bacteria or yeast.
FIG. 5B shows the three disulfide bonds (—S—S—) formed by the six cysteines within the ShK peptide (SEQ ID NO: 10).
FIG. 6 shows an alignment of the voltage-gated potassium channel inhibitor Stichodactyla helianthus (ShK) with other closely related members of the sea anemone toxin family. The sequence of the 35 amino acid mature ShK toxin (Accession #P29187) isolated from the venom of Stichodactyla helianthus is shown aligned to other closely related members of the sea anemone family. The consensus sequence and predicted disulfide linkages are shown, with highly conserved residues being shaded. The HmK peptide toxin sequence shown (Swiss-Protein Accession #097436) is of the immature precursor from the Magnificent sea anemone (Radianthus magnifica; Heteractis maqnifica) and the putative signal peptide is underlined. The mature HmK peptide toxin would be predicted to be 35 amino acids in length and span residues 40 through 74. AeK is the mature peptide toxin, isolated from the venom of the sea anemone Actinia equine (Accession #P81897). The sequence of the mature peptide toxin AsKS (Accession #Q9TWG1) and BgK (Accession #P29186) isolated from the venom of the sea anemone Anemonia sulcata and Bunodosoma granulifera, respectively, are also shown. FIG. 6A shows the amino acid alignment (SEQ ID NO: 10) of ShK to other members of the sea anemone family of toxins, HmK (SEQ ID NO: 6 (Mature Peptide), (SEQ ID NO: 542, Signal and Mature Peptide portions)), AeK (SEQ ID NO: 7), AsKs (SEQ ID NO: 8), and BgK (SEQ ID NO: 9). The predicted disulfide linkages are shown and conserved residues are highlighted. (HmK, SEQ ID NO: 543; ShK, SEQ ID NO: 10; AeK, SEQ ID NO: 544; AsKS, SEQ ID NO: 545). FIG. 6B shows a disulfide linkage map for this family having 3 disulfide linkages (C1-C6, C2-C4, C3-C5).
FIG. 7A-7B shows an amino acid alignment of the alpha-scorpion toxin family of potassium channel inhibitors. (BmKK1, SEQ ID NO: 11; BmKK4, SEQ ID NO: 12; PBTx1, SEQ ID NO: 14; Tc32, SEQ ID NO: 13; BmKK6, SEQ ID NO: 15; P01, SEQ ID NO: 16; Pi2, SEQ ID NO: 17; Pi3, SEQ ID NO: 18; Pi4, SEQ ID NO: 19; MTX, SEQ ID NO: 20; Pi1, SEQ ID NO: 21; HsTx1, SEQ ID NO: 61; AgTx2, SEQ ID NO: 23; KTX1, SEQ ID NO: 24; OSK1, SEQ ID NO: 25; BmKTX, SEQ ID NO: 22; HgTX1, SEQ ID NO: 27; MgTx, SEQ ID NO: 28; C11Tx1, SEQ ID NO: 29; NTX, SEQ ID NO: 30; Tc30, SEQ ID NO: 31; TsTX-Ka, SEQ ID NO: 32; PBTx3, SEQ ID NO: 33; Lqh 15-1, SEQ ID NO: 34; MartenTx, SEQ ID NO: 37; ChTx, SEQ ID NO:36; ChTx-Lq2, SEQ ID NO: 42; IbTx, SEQ ID NO: 38; SloTx, SEQ ID NO: 39; BmTx1; SEQ ID NO: 43; BuTx, SEQ ID NO: 41; AmmTx3, SEQ ID NO: 44; AaTX1, SEQ ID NO: 45; BmTX3, SEQ ID NO: 46; Tc1, SEQ ID NO: 48; OSK2, SEQ ID NO: 49; TsK, SEQ ID NO: 54; CoTx1, SEQ ID NO:55; CoTx2, SEQ ID NO: 871; BmPo5, SEQ ID NO: 60; ScyTx, SEQ ID NO: 51; P05, SEQ ID NO: 52; Tamapin, SEQ ID NO: 53; and TmTx, SEQ ID NO: 691. Highly conserved residues are shaded and a consensus sequence is listed. Subfamilies of the α-KTx are listed and are from Rodriguez de la Vega, R. C. et al. (2003) TIPS 24: 222-227. A list of some ion channels reported to antagonized is listed (IK=IKCa, BK=BKCa, SK=SKCa, Kv=voltage-gated K+ channels). Although most family members in this alignment represent the mature peptide product, several represent immature or modified forms of the peptide and these include: BmKK1, BmKK4, BmKK6, BmKTX, MartenTx, ChTx, ChTx-Lq2, BmTx1, AaTx1, BmTX3, TsK, CoTx1, BmP05.
FIG. 8 shows an alignment of toxin peptides converted to peptibodies in this invention (Apamin, SEQ ID NO: 68; HaTx1, SEQ ID NO: 494; ProTx1, SEQ ID NO: 56; PaTx2, SEQ ID NO: 57; ShK[2-35], SEQ ID NO: 92; ShK[1-35], SEQ ID NO: 5; HmK, SEQ ID NO: 6; ChTx (K32E), SEQ ID NO: 59; ChTx, SEQ ID NO: 36; IbTx, SEQ ID NO: 38; OSK1 (E16K, K20D), SEQ ID NO: 296; OSK1, SEQ ID NO: 25; AgTx2, SEQ ID NO: 23; KTX1, SEQ ID NO: 24; MgTx, SEQ ID NO: 28; NTX, SEQ ID NO: 30; MTX, SEQ ID NO: 20; Pi2, SEQ ID NO: 17; HsTx1, SEQ ID NO: 61; Anuroctoxin [AnTx], SEQ ID NO: 62; BeKm1, SEQ ID NO: 63; ScyTx, SEQ ID NO: 51; ωGVIA, SEQ ID NO: 64; ωMVIIa, SEQ ID NO: 65; Ptu1, SEQ ID NO: 66; and CTX, SEQ ID NO: 67). The original sources of the toxins is indicated, as well as, the number of cysteines in each. Key ion channels targeted are listed. The alignment shows clustering of toxin peptides based on their source and ion channel target impact.
FIG. 9 shows disulfide arrangements within the toxin family. The number of disulfides and the disulfide bonding order for each subfamily is indicated. A partial list of toxins that fall within each disulfide linkage category is presented.
FIG. 10 illustrates that solution structures of toxins reveal a compact structure. Solution structures of native toxins from sea anemone (ShK), scorpion (MgTx, MTX, HsTx1), marine cone snail (wGVIA) and tarantula (HaTx1) indicate the 28 to 39 amino acid peptides all form a compact structure. The toxins shown have 3 or 4 disulfide linkages and fall within 4 of the 6 subfamilies shown in FIG. 9. The solution structures of native toxins from sea anemone (ShK), scorpion (MgTx, MTX, HsTx1), marine cone snail (wGVIA) and tarantula (HaTx1) were derived from Protein Data Bank (PDB) accession numbers 1ROO (mmdbld:5247), 1MTX (mmdbld:4064), 1TXM (mmdbld:6201), 1QUZ (mmdbld:36904), 1OMZ (mmdbld:1816) and 1D1H (mmdbld:14344) using the MMDB Entrez 3D-structure database [J. Chen et al. (2003) Nucleic Acids Res. 31, 474] and viewer.
FIG. 11A-C shows the nucleic acid (SEQ ID NO: 69 and SEQ ID NO: 1358) and encoded amino acid (SEQ ID NO:70, SEQ ID NO:1359 and SEQ ID NO: 1360) sequences of residues 5131-6660 of pAMG21ampR-Fc-pep. The sequences of the Fc domain (SEQ ID NOS: 71 and 72) exclude the five C-terminal glycine residues. This vector enables production of peptibodies in which the peptide-linker portion is at the C-terminus of the Fc domain.
FIG. 11D shows a circle diagram of a peptibody bacterial expression vector pAMG21ampR-Fc-pep having a chloramphenicol acetyltransferase gene (cat; “CmR” site) that is replaced with the peptide-linker sequence.
FIG. 12A-C shows the nucleic acid (SEQ ID NO: 73 and SEQ ID NO: 1361) and encoded amino acid (SEQ ID NO:74, SEQ ID NO: 1362 and SEQ ID NO: 1363) sequences of residues 5131-6319 of pAMG21ampR-Pep-Fc. The sequences of the Fc domain (SEQ ID NOS: 75 and 76) exclude the five N-terminal glycine residues. This vector enables production of peptibodies in which the peptide-linker portion is at the N-terminus of the Fc domain.
FIG. 12D shows a circle diagram of a peptibody bacterial expression vector having a zeocin resistance (ble; “ZeoR”) site that is replaced with the peptide-linker sequence.
FIG. 12E-G shows the nucleic acid (SEQ ID NO:1339) and encoded amino acid sequences of pAMG21ampR-Pep-Fc (SEQ ID NO:1340, SEQ ID NO:1341, and SEQ ID NO:1342). The sequences of the Fc domain (SEQ ID NOS: 75 and 76) exclude the five N-terminal glycine residues. This vector enables production of peptibodies in which the peptide-linker portion is at the N-terminus of the Fc domain.
FIG. 13A is a circle diagram of mammalian expression vector pCDNA3.1(+) CMVi.
FIG. 13B is a circle diagram of mammalian expression vector pCDNA3.1 (+) CMVi-Fc-2×G4S-Activin RIIb that contains a Fc region from human IgG1, a 10 amino acid linker and the activin RIIb gene.
FIG. 13C is a circle diagram of the CHO expression vector pDSRa22 containing the Fc-L10-ShK[2-35] coding sequence.
FIG. 14A-14B shows the nucleotide and encoded amino acid sequences (SEQ. ID. NOS: 77 and 78, respectively) of the molecule identified as “Fc-L10-ShK[1-35]” in Example 1 hereinafter. The L10 linker amino acid sequence (SEQ ID NO: 79) is underlined.
FIG. 15A-15B shows the nucleotide and encoded amino acid sequences (SEQ. ID. NOS: 80 and 81, respectively) of the molecule identified as “Fc-L10-ShK[2-35]” in Example 2 hereinafter. The same L10 linker amino acid sequence (SEQ ID NO: 79) as used in Fc-L10-ShK[1-35] (FIG. 14A-14B) is underlined.
FIG. 16A-16B shows the nucleotide and encoded amino acid sequences (SEQ. ID. NOS: 82 and 83, respectively) of the molecule identified as “Fc-L25-ShK[2-35]” in Example 2 hereinafter. The L25 linker amino acid sequence (SEQ ID NO: 84) is underlined.
FIG. 17 shows a scheme for N-terminal PEGylation of ShK peptide (SEQ ID NO: 5 and SEQ ID NO:10) by reductive amination, which is also described in Example 32 hereinafter.
FIG. 18 shows a scheme for N-terminal PEGylation of ShK peptide (SEQ ID NO: 5 and SEQ ID NO:10) via amide formation, which is also described in Example 34 hereinafter.
FIG. 19 shows a scheme for N-terminal PEGylation of ShK peptide (SEQ ID NO: 5 and SEQ ID NO:10) by chemoselective oxime formation, which is also described in Example 33 hereinafter.
FIG. 20A shows a reversed-phase HPLC analysis at 214 nm and FIG. 20B shows electrospray mass analysis of folded ShK[2-35], also described as folded-“Des-Arg1-ShK” (Peptide 2).
FIG. 21 shows reversed phase HPLC analysis at 214 nm of N-terminally PEGylated ShK[2-35], also referred to as N-Terminally PEGylated-“Des-Arg1-ShK”.
FIG. 22A shows a reversed-phase HPLC analysis at 214 nm of folded ShK[1-35], also referred to as “ShK”.
FIG. 22B shows electrospray mass analysis of folded ShK[1-35], also referred to as “ShK”.
FIG. 23 illustrates a scheme for N-terminal PEGylation of ShK[2-35] (SEQ ID NO: 92 and SEQ ID NO: 58, also referred to as “Des-Arg1-ShK” or “ShK d1”) by reductive amination, which is also described in Example 31 hereinafter.
FIG. 24A shows a western blot of conditioned medium from HEK 293 cells transiently transfected with Fc-L10-ShK[1-35]. Lane 1: molecular weight markers; Lane 2: 15 μl Fc-L10-ShK; Lane 3: 10 μl Fc-L10-ShK; Lane 4: 5 μl Fc-L10-ShK; Lane 5; molecular weight markers; Lane 6: blank; Lane 7: 15 μl No DNA control; Lane 8: 10 μl No DNA control; Lane 9: 5 μl No DNA control; Lane 10; molecular weight markers.
FIG. 24B shows a western blot of with 15 μl of conditioned medium from clones of Chinese Hamster Ovary (CHO) cells stably transfected with Fc-L-ShK[1-35]. Lanes 1-15 were loaded as follows: blank, BB6, molecular weight markers, BB5, BB4, BB3, BB2, BB1, blank, BD6, BD5, molecular weight markers, BD4, BD3, BD2.
FIG. 25A shows a western blot of a non-reducing SDS-PAGE gel containing conditioned medium from 293T cells transiently transfected with Fc-L-SmIIIA.
FIG. 25B shows a western blot of a reducing SDS-PAGE gel containing conditioned medium from 293T cells transiently transfected with Fc-L-SmIIIA.
FIG. 26A shows a Spectral scan of 10 μl purified bivalent dimeric Fc-L10-ShK[1-35] product from stably transfected CHO cells diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1-cm path length quartz cuvette.
FIG. 26B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final bivalent dimeric Fc-L10-ShK[1-35] product. Lanes 1-12 were loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.
FIG. 26C shows size exclusion chromatography on 20 μg of the final bivalent dimeric Fc-L10-ShK[1-35] product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, and pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 26D shows a MALDI mass spectral analysis of the final sample of bivalent dimeric Fc-L10-ShK[1-35] analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.
FIG. 26E shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final monovalent dimeric Fc-L10-ShK[1-35] product. Lanes 1-12 were loaded as follows: Novex Mark12 wide range protein standards (10 μL), 0.5 μg product non-reduced (1.3 μL), blank, 2.0 μg product non-reduced (5 μL), blank, 10 μg product non-reduced (25 μL), Novex Mark12 wide range protein standards (10 μL), 0.5 μg product reduced (1.3 μL), blank, 2.0 μg product reduced (5 μL), blank, and 10 μg product reduced (25 μL).
FIG. 26F shows size exclusion chromatography on 20 μg of the final monovalent dimeric Fc-L10-ShK[1-35] product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, and pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 27A shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final purified bivalent dimeric Fc-L10-ShK[2-35] product from stably transfected CHO cells. Lane 1-12 were loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.
FIG. 27B shows size exclusion chromatography on 50 μg of the purified bivalent dimeric Fc-L10-ShK[2-35] injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, and pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 27C shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of bivalent dimeric Fc-L5-ShK[2-35] purified from stably transfected CHO cells. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.
FIG. 27D shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of bivalent dimeric Fc-L25-ShK[2-35] purified from stably transfected CHO cells. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.
FIG. 27E shows a spectral scan of 10 μl of the bivalent dimeric Fc-L10-ShK[2-35] product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 27F shows a MALDI mass spectral analysis of the final sample of bivalent dimeric Fc-L110-ShK[2-35] analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from about 200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.
FIG. 27G shows a spectral scan of 10 μl of the bivalent dimeric Fc-L5-ShK[2-35] product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 27H shows the size exclusion chromatography on 50 mg of the final bivalent dimeric Fc-L5-ShK[2-35] product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 27I shows a MALDI mass spectral analysis of the final sample of bivalent dimeric Fc-L5-ShK[2-35] analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.
FIG. 27J shows a Spectral scan of 20 μl of the product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 27K shows the size exclusion chromatography on 50 μg of the final bivalent dimeric Fc-L25-ShK[2-35] product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 27L shows a MALDI mass spectral analysis of the final sample of bivalent dimeric Fc-L25-ShK[2-35] analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from about 200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.
FIG. 28A shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of Fc-L10-KTX1 purified and refolded from bacterial cells. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.
FIG. 28B shows size exclusion chromatography on 45 μg of purified Fc-L10-KTX1 injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 28C shows a Spectral scan of 20 μl of the Fc-L10-KTX1 product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 28D shows a MALDI mass spectral analysis of the final sample of Fc-L10-KTX1 analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.
FIG. 29A shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of Fc-L-AgTx2 purified and refolded from bacterial cells. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.
FIG. 29B shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of Fc-L10-HaTx1 purified and refolded from bacterial cells. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced, spectral scan of the purified material.
FIG. 29C shows a Spectral scan of 20 μl of the Fc-L10-AgTx2 product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 29D shows the Size exclusion chromatography on 20 μg of the final Fc-L10-AgTx2 product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 29E shows a MALDI mass spectral analysis of the final sample of Fc-L10-AgTx2 analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from about 200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.
FIG. 29F shows a Spectral scan of 20 μl of the Fc-L10-HaTx1 product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 29G shows the size exclusion chromatography on 20 μg of the final Fc-L10-HaTx1 product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 29H shows a MALDI mass spectral analysis of the final sample of Fc-L10-HaTx1 analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.
FIG. 30A shows Fc-L10-ShK[1-35] purified from CHO cells produces a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing the human Kv1.3 channel.
FIG. 30B shows the time course of potassium current block by Fc-L10-ShK[1-35] at various concentrations. The IC50 was estimated to be 15±2 pM (n=4 cells).
FIG. 30C shows synthetic ShK[1-35] (also referred to as “ShK” alone) produces a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel.
FIG. 30D shows the time course of ShK[1-35] block at various concentrations. The IC50 for ShK was estimated to be 12±1 pM (n=4 cells).
FIG. 31A shows synthetic peptide analog ShK[2-35] producing a concentration dependent block of the outward potassium current as recorded from HEK293 cells stably expressing human Kv1.3 channel with an IC50 of 49±5 pM (n=3 cells).
FIG. 31B shows the CHO-derived Fc-L10-ShK[2-35] peptibody producing a concentration dependent block of the outward potassium current as recorded from HEK293 cell stably expressing human Kv1.3 channel with an IC50 of 115±18 pM (n=3 cells).
FIG. 31C shows the Fc-L5-ShK[2-35] peptibody produces a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel with an IC50 of 100 pM (n=3 cells).
FIG. 32A shows Fc-L-KTX1 peptibody purified from bacterial cells producing a concentration dependent block of the outward potassium current as recorded from HEK293 cell stably expressing human Kv1.3 channel.
FIG. 32B shows the time course of potassium current block by Fc-L10-KTX1 at various concentrations.
FIG. 33 shows by immunohistochemistry that CHO-derived Fc-L10-ShK[1-35] peptibody stains HEK 293 cells stably transfected with human Kv1.3 (FIG. 33A), whereas untransfected HEK 293 cells are not stained with the peptibody (FIG. 33B).
FIG. 34 shows results of an enzyme-immunoassay using fixed HEK 293 cells stably transfected with human Kv1.3. FIG. 34A shows the CHO-derived Fc-L10-ShK[1-35] (referred to here simply as “Fc-L10-ShK”) peptibody shows a dose-dependent increase in response, whereas the CHO-Fc control (“Fc control”) does not. FIG. 34B shows the Fc-L10-ShK[1-35] peptibody (referred to here as “Fc-ShK”) does not elicit a response from untransfected HEK 293 cells using similar conditions and also shows other negative controls.
FIG. 35 shows the CHO-derived Fc-L10-ShK[1-35] peptibody shows a dose-dependent inhibition of IL-2 (FIG. 35A) and IFNγ (FIG. 35B) production from PMA and αCD3 antibody stimulated human PBMCs. The peptibody shows a novel pharmacology exhibiting a complete inhibition of the response, whereas the synthetic ShK[1-35] peptide alone shows only a partial inhibition.
FIG. 36 shows the mammalian-derived Fc-L10-ShK[1-35] peptibody inhibits T cell proliferation (3H-thymidine incorporation) in human PBMCs from two normal donors stimulated with antibodies to CD3 and CD28. FIG. 36A shows the response of donor 1 and FIG. 36B the response of donor 2. Pre-incubation with the anti-CD32 (FcgRII) blocking antibody did not alter the sensitivity toward the peptibody.
FIG. 37 shows the purified CHO-derived Fc-L10-ShK[1-35] peptibody causes a dose-dependent inhibition of IL-2 (FIG. 37A) and IFNγ (FIG. 37B) production from αCD3 and αCD28 antibody stimulated human PBMCs.
FIG. 38A shows the PEGylated ShK[2-35] synthetic peptide produces a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel and the time course of potassium current block at various concentrations is shown in FIG. 38B.
FIG. 39A shows a spectral scan of 50 μl of the Fc-L10-ShK(1-35) product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 39B shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-ShK(1-35) product. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.
FIG. 39C shows the Size exclusion chromatography on 50 μg of the final Fc-L10-ShK(1-35) product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 40A shows a Spectral scan of 20 μl of the Fc-L10-ShK(2-35) product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 40B shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-ShK(2-35) product. Lanes 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.
FIG. 40C shows the size exclusion chromatography on 50 μg of the final Fc-L10-ShK(2-35) product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 40D shows a MALDI mass spectral analysis of the final sample of Fc-L10-ShK(2-35) analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.
FIG. 41A shows spectral scan of 50 μl of the Fc-L10-OSK1 product diluted in 700 μl Formulation Buffer using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 41B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-OSK1 product. Lanes 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.
FIG. 41C shows size exclusion chromatography on 123 μg of the final Fc-L10-OSK1 product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 41D shows liquid chromatography—mass spectral analysis of approximately 4 μg of the final Fc-L110-OSK1 sample using a Vydac C4 column with part of the effluent directed into a LCQ ion trap mass spectrometer. The mass spectrum was deconvoluted using the Bioworks software provided by the mass spectrometer manufacturer.
FIG. 42A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1040 and SEQ ID NO: 1041, respectively) of Fc-L10-OSK1.
FIG. 43A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1042 and SEQ ID NO: 1043, respectively) of Fc-L10-OSK1[K7S].
FIG. 44A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1044 and SEQ ID NO: 1045, respectively) of Fc-L10-OSK1[E16K,K20D].
FIG. 45A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1046 and SEQ ID NO: 1047, respectively) of Fc-L10-OSK1[K7S,E16K,K20D].
FIG. 46 shows a Western blot (from tris-glycine 4-20% SDS-PAGE) with anti-human Fc antibodies. Lanes 1-6 were loaded as follows: 15 μl of Fc-L10-OSK1[K7S,E16K,K20D]; 15 μl of Fc-L10-OSK1[E16K,K20D]; 15 μl of Fc-L10-OSK1[K7S]; 15 μl of Fc-L10-OSK1; 15 μl of “No DNA” control; molecular weight markers.
FIG. 47 shows a Western blot (from tris-glycine 4-20% SDS-PAGE) with anti-human Fc antibodies. Lanes 1-5 were loaded as follows: 21 of Fc-L10-OSK1; 5 μl of Fc-L10-OSK1; 10 μl of Fc-L10-OSK1; 20 ng Human IgG standard; molecular weight markers.
FIG. 48 shows a Western blot (from tris-glycine 4-20% SDS-PAGE) with anti-human Fc antibodies. Lanes 1-13 were loaded as follows: 20 ng Human IgG standard; D1; C3; C2; B6; B5; B2; B1; A6; A5; A4; A3; A2 (5 μl of clone-conditioned medium loaded in lanes 2-13).
FIG. 49A shows a spectral scan of 50 μl of the Fc-L10-OSK1 product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 49B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-OSK1 product. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.
FIG. 49C shows Size exclusion chromatography on 149 μg of the final Fc-L10-OSK1 product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 49D shows MALDI mass spectral analysis of the final sample of Fc-L10-OsK1 analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.
FIG. 50A shows a spectral scan of 50 μl of the Fc-L10-OsK1(K7S) product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 50B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-OsK1(K7S) product. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.
FIG. 50C shows size exclusion chromatography on 50 μg of the final Fc-L10-OsK1(K7S) product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 50D shows MALDI mass spectral analysis of a sample of the final product Fc-L10-OsK1 (K7S) analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.
FIG. 51A shows a spectral scan of 50 μl of the Fc-L10-OsK1(E16K, K20D) product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 51B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-OsK1(E16K, K20D) product. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.
FIG. 51C shows size exclusion chromatography on 50 μg of the final Fc-L10-OsK1(E16K, K20D) product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 51D shows MALDI mass spectral analysis of a sample of the final product Fc-L10-OsK1 (E16K, K20D) analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.
FIG. 52A shows a spectral scan of 50 μl of the Fc-L110-OsK1 (K7S, E16K, K20D) product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 52B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-OsK1(K7S, E16K, K20D) product. Lanes 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.
FIG. 52C shows size exclusion chromatography on 50 μg of the final Fc-L10-OsK1(K7S, E16K, K20D) product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 52D shows MALDI mass spectral analysis of a sample of the final product Fc-L10-OsK1(K7S, E16K, K20D) analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.
FIG. 53 shows inhibition of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel by synthetic Osk1, a 38-residue toxin peptide of the Asian scorpion Orthochirus scrobiculosus venom. FIG. 53A shows a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel by the synthetic Osk1 toxin peptide. FIG. 53B shows the time course of the synthetic Osk1 toxin peptide block at various concentrations. The IC50 for the synthetic Osk1 toxin peptide was estimated to be 39±12 pM (n=4 cells).
FIG. 54 shows that modification of the synthetic OSK1 toxin peptide by fusion to the Fc-fragment of an antibody (OSK1-peptibody) retained the inhibitory activity against the human Kv1.3 channel. FIG. 54A shows a concentration dependent block of the outward potassium current recorded from HEK293 cells stably expressing human Kv1.3 channel by OSK1 linked to a human IgG1 Fc-fragment with a linker chain length of 10 amino acid residues (Fc-L10-OSK1). The fusion construct was stably expressed in Chinese Hamster Ovarian (CHO) cells. FIG. 54B shows the time course of the Fc-L10-OSK1 block at various concentrations. The IC50 for Fc-L10-OSK1 was estimated to be 198±35 pM (n=6 cells), approximately 5-fold less potent than the synthetic OSK1 toxin peptide.
FIG. 55 shows that a single amino-acid residue substitution of the OSK1-peptibody retained the inhibitory activity against the human Kv1.3 channel. FIG. 55A shows a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel by OSK1-peptibody with a single amino acid substitution (lysine to serine at the 7th position from N-terminal, [K7S]) and linked to a human IgG1 Fc-fragment with a linker chain length of 10 amino acid residues (Fc-L10-OSK1[K7S]). The fusion construct was stably expressed in Chinese Hamster Ovarian (CHO) cells. FIG. 55B shows the time course of potassium current block by Fc-L10-OSK1[K7S] at various concentrations. The IC50 was estimated to be 372±71 pM (n=4 cells), approximately 10-fold less potent than the synthetic OSK1 toxin peptide.
FIG. 56 shows that a two amino-acid residue substitution of the OSK1-peptibody retained the inhibitory activity against the human Kv1.3 channel. FIG. 56A shows a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel by OSK1-peptibody with two amino acid substitutions (glutamic acid to lysine and lysine to aspartic acid at the 16th and 20th position from N-terminal respectively, [E16KK20D]) and linked to a human IgG1 Fc-fragment with a linker chain length of 10 amino acid residues (Fc-L10-OSK1[E16KK20D]). The fusion construct was stably expressed in Chinese Hamster Ovarian (CHO) cells. FIG. 56B shows the time course of potassium current block by Fc-L10-OSK1[E16KK20D] at various concentrations. The IC50 was estimated to be 248±63 pM (n=3 cells), approximately 6-fold less potent than the synthetic OSK1 toxin peptide.
FIG. 57 shows that a triple amino-acid residue substitution of the OSK1-peptibody retained the inhibitory activity against the human Kv1.3 channel, but the potency of inhibition was significantly reduced when compared to the synthetic OSK1 toxin peptide. FIG. 57A shows a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel by OSK1-peptibody with triple amino acid substitutions (lysine to serine, glutamic acid to lysine and lysine to aspartic acid at the 7th, 16th and 20th position from N-terminal respectively, [K7SE16KK20D]) and linked to a human IgG1 Fc-fragment with a linker chain length of 10 amino acid residues (Fc-L10-OSK1[K7SE16KK20D]). The fusion construct was stably expressed in Chinese Hamster Ovarian (CHO) cells. FIG. 57B shows the time course of potassium current block by Fc-L10-OSK1[K7SE16KK20D] at various concentrations. The IC50 was estimated to be 812±84 pM (n=3 cells), approximately 21-fold less potent than the synthetic OSK1 toxin peptide.
FIG. 58 shows Standard curves for ShK (FIG. 58A) and 20K PEG-ShK[1-35] (FIG. 58B) containing linear regression equations for each Standard at a given percentage of serum.
FIG. 59 shows the pharmacokinetic profile in rats of 20K PEG ShK[1-35] molecule after IV injection.
FIG. 60 shows Kv1.3 inhibitory activity in serum samples (5%) of rats receiving a single equal molar IV injection of Kv1.3 inhibitors ShK versus 20K PEG-ShK[1-35].
FIG. 61 illustrates an Adoptive Transfer EAE model experimental design (n=5 rats per treatment group). Dosing values in microgram per kilogram (mg/kg) are based on peptide content.
FIG. 62 shows that treatment with PEG-ShK ameliorated disease in rats in the adoptive transfer EAE model. Clinical scoring: 0=No signs, 0.5=distal limp tail, 1.0=limp tail, 2.0=mild paraparesis, ataxia, 3.0=moderate paraparesis, 3.5=one hind leg paralysis, 4.0=complete hind leg paralysis, 5.0=complete hind leg paralysis and incontinence, 5.5=tetraplegia, 6.0=moribund state or death. Rats reaching a score of 5.5 to 6 died or were euthanized. Mean±sem values are shown. (n=5 rats per treatment group.)
FIG. 63 shows that treatment with PEG-ShK prevented loss of body weight in the adoptive transfer EAE model. Rats were weighed on days −1, 4, 6, and 8 (for surviving rats). Mean±sem values are shown.
FIG. 64 shows that thapsigargin-induced IL-2 production in human whole blood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] and Fc-L10-ShK[2-35]. The calcineurin inhibitor cyclosporine A also blocked the response. The BKCa channel inhibitor iberiotoxin (IbTx) showed no significant activity. The response of whole blood from two separate donors is shown in FIG. 64A and FIG. 64B.
FIG. 65 shows that thapsigargin-induced IFN-g production in human whole blood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] and Fc-L10-ShK[2-35]. The calcineurin inhibitor cyclosporine A also blocked the response. The BKCa channel inhibitor iberiotoxin (IbTx) showed no significant activity. The response of whole blood from two separate donors is shown in FIG. 65A and FIG. 65B.
FIG. 66 shows that thapsigargin-induced upregulation of CD40L on T cells in human whole blood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] and Fc-L10-ShK[1-35] (Fc-ShK). The calcineurin inhibitor cyclosporine A (CsA) also blocked the response. FIG. 66A shows results of an experiment looking at the response of total CD4+ T cells. FIG. 66B shows results of an experiment that looked at total CD4+ T cells, as well as CD4+CD45+ and CD4+CD45-T cells. In FIG. 66B, the BKCa channel inhibitor iberiotoxin (IbTx) and the Kv1.1 channel inhibitor dendrotoxin-K (DTX-K) showed no significant activity.
FIG. 67 shows that thapsigargin-induced upregulation of the IL-2R on T cells in human whole blood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] and Fc-L10-ShK[1-35] (Fc-ShK). The calcineurin inhibitor cyclosporine A (CsA) also blocked the response. FIG. 67A shows results of an experiment looking at the response of total CD4+ T cells. FIG. 67B shows results of an experiment that looked at total CD4+ T cells, as well as CD4+CD45+ and CD4+CD45-T cells. In FIG. 67B, the BKCa channel inhibitor iberiotoxin (IbTx) and the Kv1.1 channel inhibitor dendrotoxin-K (DTX-K) showed no significant activity.
FIG. 68 shows cation exchange chromatograms of PEG-peptide purification on SP Sepharose HP columns for PEG-Shk purification (FIG. 68A) and PEG-OSK-1 purification (FIG. 68B).
FIG. 69 shows RP-HPLC chromatograms on final PEG-peptide pools to demonstrate purity of PEG-Shk purity >99% (FIG. 69A) and PEG-Osk1 purity >97% (FIG. 69B).
FIG. 70 shows the amino acid sequence (SEQ ID NO: 976) of an exemplary FcLoop-L2-OsK1-L2 having three linked domains: Fc N-terminal domain (amino acid residues 1-139); OsK1 (underlined amino acid residues 142-179); and Fc C-terminal domain (amino acid residues 182-270).
FIG. 71 shows the amino acid sequence (SEQ ID NO: 977) of an exemplary FcLoop-L2-ShK-L2 having three linked domains: Fc N-terminal domain (amino acid residues 1-139); ShK (underlined amino acid residues 142-176); and Fc C-terminal domain (amino acid residues 179-267).
FIG. 72 shows the amino acid sequence (SEQ ID NO: 978) of an exemplary FcLoop-L2-ShK-L4 having three linked domains: Fc N-terminal domain (amino acid residues 1-139); ShK (underlined amino acid residues 142-176); and Fc C-terminal domain (amino acid residues 181-269).
FIG. 73 shows the amino acid sequence (SEQ ID NO: 979) of an exemplary FcLoop-L4-OsK1-L2 having three linked domains: Fc N-terminal domain (amino acid residues 1-139); OsK1(underlined amino acid residues 144-181); and Fc C-terminal domain (amino acid residues 184-272).
FIG. 74 shows that the 20K PEGylated ShK[1-35] provided potent blockade of human Kv1.3 as determined by whole cell patch clamp electrophysiology on HEK293/Kv1.3 cells. The data represents blockade of peak current.
FIG. 75 shows schematic structures of some other exemplary embodiments of the composition of matter of the invention. “X2” and “X3” represent toxin peptides or linker-toxin peptide combinations (i.e., -(L)f-P-(L)g-) as defined herein. As described herein but not shown in FIG. 75, an additional X1 domain and one or more additional PEG moieties are also encompassed in other embodiments. The specific embodiments shown here are as follows:
FIG. 75C, FIG. 75D, FIG. 75G and FIG. 75H: show a single chain molecule and can also represent the DNA construct for the molecule.
FIG. 75A, FIG. 75B, FIG. 75E and FIG. 75F: show doubly disulfide-bonded Fc dimers (in position F2); FIG. 75A and FIG. 75B show a dimer having the toxin peptide portion on both chains in position X3; FIG. 75E and FIG. 75F show a dimer having the toxin peptide portion on both chains In position X2.
FIG. 76A shows a spectral scan of 50 μl of the ShK[2-35]-Fc product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 76B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final ShK[2-35]-Fc product. Lanes 1-12 were loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.
FIG. 76C shows size exclusion chromatography on 70 μg of the final ShK[2-35]-Fc product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 76D shows LC-MS analysis of the final ShK[2-35]-Fc sample using an Agilent 1100 HPCL running reverse phase chromatography, with the column effluent directly coupled to an electrospray source of a Thermo Finnigan LCQ ion trap mass spectrometer. Relevant spectra were summed and deconvoluted to mass data with the Bioworks software package.
FIG. 77A shows a spectral scan of 20 μl of the met-ShK[1-35]-Fc product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 77B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final met-ShK[1-35]-Fc product. Lanes 1-12 were loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.
FIG. 77C shows size exclusion chromatography on 93 μg of the final met-ShK[1-35]-Fc product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 77D shows MALDI mass spectral analysis of the final met-ShK[1-35]-Fc sample analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.
FIG. 78 shows a spectral scan of 10 μl of the CH2-OSK1 fusion protein product diluted in 150 μl water (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.
FIG. 79 shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final CH2-OSK1 fusion protein product. Lane 1-7 were loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, and Novex Mark12 wide range protein standards.
FIG. 80 shows size exclusion chromatography on 50 μg of the final CH2-OSK1 fusion protein product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.
FIG. 81 shows liquid chromatography—mass spectral analysis of the CH2-OSK1 fusion protein sample using a Vydac C4 column with part of the effluent directed into a LCQ ion trap mass spectrometer. The mass spectrum was deconvoluted using the Bioworks software provided by the mass spectrometer manufacturer.
FIG. 82 shows cation exchange chromatogram of PEG-CH2-OSK1 reaction mixture. Vertical lines delineate fractions pooled to obtain mono-PEGylated CH2-OSK1.
FIG. 83 shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final PEGylated CH2-OSK1 pool. Lane 1-2 were loaded as follows: Novex Mark12 wide range protein standards, 2.0 μg product non-reduced.
FIG. 84 shows whole cell patch clamp (WCPC) and PatchXpress (PX) electrophysiology comparing the activity of OSK1[Ala-12] (SEQ ID No:1410) on human Kv1.3 and human Kv1.1 heterologously overexpressed on CHO and HEK293 cells, respectively. The table summarizes the calculated IC50 values and the plots show the individual traces of the impact of various concentrations of analog on the relative Kv1.3 or Kv1.1 current (percent of control, POC).
FIG. 85 shows whole cell patch clamp electrophysiology comparing the activity of OSK1[Ala-29] (SEQ ID No:1424) on human Kv1.3 and human Kv1.1 heterologously overexpressed on CHO and HEK293 cells, respectively. Concentration response curves of OSK1[Ala-29] on CHO/Kv1.3 (circle, square and diamond, IC50=0.033 nM, n=3) and on HEK/Kv1.1 (filled triangle, IC50=2.7 nM, n=1).
FIG. 86 shows a dose-response curve for OSK1[Ala-29] (SEQ ID No:1424) against human Kv1.3 (CHO) (panel A) and human Kv1.1 (HEK293) (panel B) as determined by high-throughput 384-well planar patch clamp electrophysiology using the IonWorks Quattro system.
FIG. 87A-B show Western blots of Tris-glycine 4-20% SDS-PAGE (FIG. 87A with longer exposure time and FIG. 87B with shorter exposure time) of a monovalent dimeric Fc-L-ShK(2-35) molecule product expressed by and released into the conditioned media from mammalian cells transiently transfected with pTT5-Fc-Fc-L10-Shk(2-35), which was sampled after the indicated number of days. Lanes 3-8 were loaded with 20 μL of conditioned medium per lane. The immunoblot was probed with anti-human IgG-Fc-HRP (Pierce). The lanes were loaded as follows: 1) MW Markers; 2) purified Fc-L10-ShK(2-35), 100 ng; 3) 293-6E-HD (5-day); 4) 293-6E-HD (6-day); 5) 293-6E-PEI (5-day); 6) 293-6E-PEI (6-day); 7) CHO-S (5-day); 8) CHO-S (6-day). Four bands were expected in the reduced gel: Linker-Fc-Shk(2-35) (one cut at 3′ furin site; predicted MW: 33.4 kDa); Fc-ShK(2-35) (both furin sites cut; predicted MW: 30.4 kDa); Fc-linker (one cut at 5′ furin site; predicted MW: 29.1 kDa); Fc (both furin sites cut; predicted MW: 25.8 kDa). Further mass spec or amino acid sequence analysis of the individual bands is needed to identify these bands and their relative ratios.
FIG. 88 shows a western blot of serum samples from a pharmacokinetic study on monovalent dimeric Fc-ShK(1-35) in SD rats. Various times (0.083-168 hours) after a single 1 mg/kg intravenous injection of monovalent dimeric Fc-L10-ShK(1-35) (see, Example 2), blood was drawn, and serum was collected. A Costar EIA/RIA 96 well plate was coated with 2 μg/ml polyclonal goat anti-human Fc antibody overnight at 4° C. Capture antibody was removed and the plate was washed with PBST and then blocked with Blotto. After the plate was washed, serum samples diluted in PBST/0.1% BSA were added. Binding was allowed to occur at room temperature for several hours, and then the plate was again washed. Samples were eluted from the plate with reducing Laemmle buffer, heated, then run on SDS-Page gels. Run in an adjacent lane (“5 ng Control”) of the gel as a standard was 5 ng of the purified monovalent dimeric Fc-L10-ShK(1-35) fusion protein used in the pharmacokinetic study. Proteins were transferred to PVDF membranes by western blot. Membranes were blocked with Blotto followed by incubation with goat anti-Human Fc-HRP conjugated antibody. After the membranes were washed, signal was detected via chemiluminescence using a CCD camera.
FIG. 89 shows the NMR solution structure of OSK1 and sites identified by analoging to be important for Kv1.3 activity and selectivity. Space filling structures are shown in FIGS. 89A, 89B and 89D. The color rendering in FIG. 89A depicts amino acid charge. In FIG. 89B, several key OSK1 amino acid residues found to be important for Kv1.3 activity (Tables 37-40) are lightly shaded and labeled Phe25, Gly26, Lys27, Met29 and Asn30. In FIG. 89D residues Ser11, Met29 and His34 are labeled. Some analogues of these residues were found to result in improved Kv1.3 selectivity over Kv1.1 (Tables 41). FIG. 89C shows the three beta strands and single alpha helix of OSK1. The amino acid sequence of native OSK1 (SEQ ID No: 25) is shown in FIG. 89E, with residues forming the molecules beta strands (β1, β2, β3) and alpha helix (al) underlined. The OSK1 structures shown were derived from PDB:1SCO, and were rendered using Cn3D vers4.1.
FIG. 90A-D illustrates that toxin peptide inhibitors of Kv1.3 provide potent blockade of the whole blood inflammatory response. The activity of the calcineurin inhibitor cyclosporin A (FIG. 90A) and Kv1.3 peptide inhibitors ShK-Ala22 (FIG. 90B; SEQ ID No: 123), OSK1-Ala29 (FIG. 90C; SEQ ID No: 1424) and OSK1-Ala12 (FIG. 90D; SEQ ID No: 1410) were compared in the whole blood assay of inflammation (Example 46) using the same donor blood sample. The potency (IC50) of each molecule is shown, where for each panel the left curve is the impact on IL-2 production and the right curve is the impact on IFNγ production.
FIG. 91A-B shows an immunoblot analysis of expression of monovalent dimeric IgG1-Fc-L-ShK(2-35) from non-reduced SDS-PAGE. FIG. 91A shows detection of human Fc expression with goat anti-human IgG (H+L)-HRP. FIG. 91B shows detection of ShK(2-35) expression with a goat anti-mouse IgG (H+L)-HRP that cross reacts with human IgG. Lane 1: purified Fc-L10-Shk(2-35); Lane 2: conditioned medium from 293EBNA cells transiently transfected with pTT5-huIgG1+pTT5-hKappa+pCMVi-Fc-L10-ShK(2-35); Lane 3: conditioned media from 293EBNA cells transiently transfected with pTT5-huIgG2+pTT5-hKappa+pCMVi-Fc-L10-Shk(2-35); Lane 4: conditioned media from 293EBNA cells transiently transfected with pTT14 vector alone. The two arrows point to the full length huIgG (mol. wt. ̃150 kDa) and monovalent dimeric huIgG-FcShK(2-35) (mol. wt. ̃100 kDa); the abundant 60-kDa band is the bivalent dimeric Fc-ShK(2-35).
FIG. 92A-C shows schematic representations of an embodiment of a monovalent “hemibody”-toxin peptide fusion protein construct; the single toxin peptide is represented by an oval. FIG. 92A, which can also represent the DNA construct for the fusion protein, represents an immunoglobulin light chain (LC, open rectangle), an immunoglobulin heavy chain (HC, longer cross-hatched rectangle), and an immunoglobulin Fc domain (Fc, shorter cross-hatched rectangle), each separated by an intervening peptidyl linker sequence (thick lines) comprising at least one protease cleavage site (arrows), e.g., a furin cleavage site. FIG. 92 illustrates the association of the recombinantly expressed LC, HC, and Fc-toxin peptide components connected by the peptidyl linker sequences (thick lines) and, in FIG. 92C, the final monovalent chimeric immunoglobulin (LC+HC)-Fc (i.e., “hemibody”)-toxin peptide fusion protein after cleavage (intracellularly or extracellularly) at the protease cleavage sites, to release the linkers, and formation of disulfide bridges between the light and heavy chains and between the heavy chain and the Fc components (shown as thin horizontal lines between the LC, HC, and Fc components in FIG. 92C).



DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
Definition of Terms
The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances. As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.
“Polypeptide” and “protein” are used interchangeably herein and include a molecular chain of two or more amino acids linked through peptide bonds. The terms do not refer to a specific length of the product. Thus, “peptides,” and “oligopeptides,” are included within the definition of polypeptide. The terms include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. In addition, protein fragments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide. The terms also include molecules in which one or more amino acid analogs or non-canonical or unnatural amino acids are included as can be synthesized, or expressed recombinantly using known protein engineering techniques. In addition, inventive fusion proteins can be derivatized as described herein by well-known organic chemistry techniques.
The term “fusion protein” indicates that the protein includes polypeptide components derived from more than one parental protein or polypeptide. Typically, a fusion protein is expressed from a fusion gene in which a nucleotide sequence encoding a polypeptide sequence from one protein is appended in frame with, and optionally separated by a linker from, a nucleotide sequence encoding a polypeptide sequence from a different protein. The fusion gene can then be expressed by a recombinant host cell as a single protein.
A “domain” of a protein is any portion of the entire protein, up to and including the complete protein, but typically comprising less than the complete protein. A domain can, but need not, fold independently of the rest of the protein chain and/or be correlated with a particular biological, biochemical, or structural function or location (e.g., a ligand binding domain, or a cytosolic, transmembrane or extracellular domain).
A “secreted” protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a secretory signal peptide sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a “mature” protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
The term “signal peptide” refers to a relatively short (3-60 amino acid residues long) peptide chain that directs the post-translational transport of a protein, e.g., its export to the extracellular space. Thus, secretory signal peptides are encompassed by “signal peptide”. Signal peptides may also be called targeting signals, signal sequences, transit peptides, or localization signals.
The term “recombinant” indicates that the material (e.g., a nucleic acid or a polypeptide) has been artificially or synthetically (i.e., non-naturally) altered by human intervention. The alteration can be performed on the material within, or removed from, its natural environment or state. For example, a “recombinant nucleic acid” is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other well known molecular biological procedures. A “recombinant DNA molecule,” is comprised of segments of DNA joined together by means of such molecular biological techniques. The term “recombinant protein” or “recombinant polypeptide” as used herein refers to a protein molecule which is expressed using a recombinant DNA molecule. A “recombinant host cell” is a cell that contains and/or expresses a recombinant nucleic acid.
A “polynucleotide sequence” or “nucleotide sequence” or “nucleic acid sequence,” as used interchangeably herein, is a polymer of nucleotides, including an oligonucleotide, a DNA, and RNA, a nucleic acid, or a character string representing a nucleotide polymer, depending on context. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence can be determined. Included are DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of ribonucleotides along the mRNA chain, and also determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the RNA sequence and for the amino acid sequence.
“Expression of a gene” or “expression of a nucleic acid” means transcription of DNA into RNA (optionally including modification of the RNA, e.g., splicing), translation of RNA into a polypeptide (possibly including subsequent post-translational modification of the polypeptide), or both transcription and translation, as indicated by the context.
The term “gene” is used broadly to refer to any nucleic acid associated with a biological function. Genes typically include coding sequences and/or the regulatory sequences required for expression of such coding sequences. The term “gene” applies to a specific genomic or recombinant sequence, as well as to a cDNA or mRNA encoded by that sequence. A “fusion gene” contains a coding region that encodes a fusion protein. Genes also include non-expressed nucleic acid segments that, for example, form recognition sequences for other proteins. Non-expressed regulatory sequences including transcriptional control elements to which regulatory proteins, such as transcription factors, bind, resulting in transcription of adjacent or nearby sequences.
As used herein the term “coding region” when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of an mRNA molecule. The coding region is bounded, in eukaryotes, on the 5′ side by the nucleotide triplet “ATG” which encodes the initiator methionine and on the 3′ side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA).
Transcriptional control signals in eukaryotes comprise “promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis, et al., Science 236:1237 (1987)). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review see Voss, et al., Trends Biochem. Sci., 11:287 (1986) and Maniatis, et al., Science 236:1237 (1987)).
The term “expression vector” as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host cell. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence can also, optionally, be encoded by the expression vector, operably linked to the coding sequence for the inventive recombinant fusion protein, so that the expressed fusion protein can be secreted by the recombinant host cell, for more facile isolation of the fusion protein from the cell, if desired. Such techniques are well known in the art. (E.g., Goodey, Andrew R.; et al., Peptide and DNA sequences, U.S. Pat. No. 5,302,697; Weiner et al., Compositions and methods for protein secretion, U.S. Pat. Nos. 6,022,952 and 6,335,178; Uemura et al., Protein expression vector and utilization thereof, U.S. Pat. No. 7,029,909; Ruben et al., 27 human secreted proteins, US 2003/0104400 A1).
The terms “in operable combination”, “in operable order” and “operably linked” as used herein refer to the linkage of nucleic acid sequences in such a manner or orientation that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced and/or transported.
Recombinant DNA- and/or RNA-mediated protein expression techniques, or any other methods of preparing peptides or, are applicable to the making of the inventive recombinant fusion proteins. For example, the peptides can be made in transformed host cells. Briefly, a recombinant DNA molecule, or construct, coding for the peptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences encoding the peptides can be excised from DNA using suitable restriction enzymes. Any of a large number of available and well-known host cells may be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial host cells in culture include bacteria (such as Escherichia coli sp.), yeast (such as Saccharomyces sp.) and other fungal cells, insect cells, plant cells, mammalian (including human) cells, e.g., CHO cells and HEK293 cells. Modifications can be made at the DNA level, as well. The peptide-encoding DNA sequence may be changed to codons more compatible with the chosen host cell. For E. coli, optimized codons are known in the art. Codons can be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. Next, the transformed host is cultured and purified. Host cells may be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art.
The term “half-life extending moiety” (i.e., F1 or F2 in Formula I) refers to a pharmaceutically acceptable moiety, domain, or “vehicle” covalently linked (“conjugated”) to the toxin peptide directly or via a linker, that prevents or mitigates in vivo proteolytic degradation or other activity-diminishing chemical modification of the toxin peptide, increases half-life or other pharmacokinetic properties such as but not limited to increasing the rate of absorption, reduces toxicity, improves solubility, increases biological activity and/or target selectivity of the toxin peptide with respect to a target ion channel of interest, increases manufacturability, and/or reduces immunogenicity of the toxin peptide, compared to an unconjugated form of the toxin peptide.
By “PEGylated peptide” is meant a peptide or protein having a polyethylene glycol (PEG) moiety covalently bound to an amino acid residue of the peptide itself or to a peptidyl or non-peptidyl linker (including but not limited to aromatic or aryl linkers) that is covalently bound to a residue of the peptide.
By “polyethylene glycol” or “PEG” is meant a polyalkylene glycol compound or a derivative thereof, with or without coupling agents or derivatization with coupling or activating moieties (e.g., with aldehyde, hydroxysuccinimidyl, hydrazide, thiol, triflate, tresylate, azirdine, oxirane, orthopyridyl disulphide, vinylsulfone, iodoacetamide or a maleimide moiety). In accordance with the present invention, useful PEG includes substantially linear, straight chain PEG, branched PEG, or dendritic PEG. (See, e.g., Merrill, U.S. Pat. No. 5,171,264; Harris et al., Multiarmed, monofunctional, polymer for coupling to molecules and surfaces, U.S. Pat. No. 5,932,462; Shen, N-maleimidyl polymer derivatives, U.S. Pat. No. 6,602,498).
The term “peptibody” refers to molecules of Formula I in which F1 and/or F2 is an immunoglobulin Fc domain or a portion thereof, such as a CH2 domain of an Fc, or in which the toxin peptide is inserted into a human IgG1 Fc domain loop, such that F1 and F2 are each a portion of an Fc domain with a toxin peptide inserted between them (See, e.g., FIGS. 70-73 and Example 49 herein). Peptibodies of the present invention can also be PEGylated as described further herein, at either an Fc domain or portion thereof, or at the toxin peptide(s) portion of the inventive composition, or both.
The term “native Fc” refers to molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of whole antibody, whether in monomeric or multimeric form. The original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins, although IgG1 or IgG2 are preferred. Native Fc's are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The term “native Fc” as used herein is generic to the monomeric, dimeric, and multimeric forms.
The term “Fc variant” refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn. Several published patent documents describe exemplary Fc variants, as well as interaction with the salvage receptor. See International Applications WO 97/34 631 (published 25 Sep. 1997; WO 96/32 478, corresponding to U.S. Pat. No. 6,096,891, issued Aug. 1, 2000, hereby incorporated by reference in its entirety; and WO 04/110 472. Thus, the term “Fc variant” includes a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that can be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, the term “Fc variant” includes a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in further detail hereinafter.
The term “Fc domain” encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc's, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.
The term “multimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two or more polypeptide chains associated covalently, noncovalently, or by both covalent and non-covalent interactions. IgG molecules typically form dimers; IgM, pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, or tetramers. One skilled in the art can form multimers by exploiting the sequence and resulting activity of the native Ig source of the Fc or by derivatizing (as defined below) such a native Fc.
The term “dimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two polypeptide chains associated covalently or non-covalently. Thus, exemplary dimers within the scope of this invention are as shown in FIG. 2. A “monovalent dimeric” Fc-toxin peptide fusion, or “monovalent dimer”, is a Fc-toxin peptide fusion that includes a toxin peptide conjugated with only one of the dimerized Fc domains (e.g., as represented schematically in FIG. 2B). A “bivalent dimeric” Fc-toxin peptide fusion, or “bivalent dimer”, is a Fc-toxin peptide fusion having both of the dimerized Fc domains each conjugated separately with a toxin peptide (e.g., as represented schematically in FIG. 2C).
The terms “derivatizing” and “derivative” or “derivatized” comprise processes and resulting compounds respectively in which (1) the compound has a cyclic portion; for example, cross-linking between cysteinyl residues within the compound; (2) the compound is cross-linked or has a cross-linking site; for example, the compound has a cysteinyl residue and thus forms cross-linked dimers in culture or in vivo; (3) one or more peptidyl linkage is replaced by a non-peptidyl linkage; (4) the N-terminus is replaced by —NRR1, NRC(O)R1, —NRC(O)OR1, —NRS(O)2R1, —NHC(O)NHR, a succinimide group, or substituted or unsubstituted benzyloxycarbonyl-NH—, wherein R and R1 and the ring substituents are as defined hereinafter; (5) the C-terminus is replaced by —C(O)R2 or —NR3R4 wherein R2, R3 and R4 are as defined hereinafter; and (6) compounds in which individual amino acid moieties are modified through treatment with agents capable of reacting with selected side chains or terminal residues. Derivatives are further described hereinafter.
The term “peptide” refers to molecules of 2 to about 80 amino acid residues, with molecules of about 10 to about 60 amino acid residues preferred and those of about 30 to about 50 amino acid residues most preferred. Exemplary peptides can be randomly generated by any known method, carried in a peptide library (e.g., a phage display library), or derived by digestion of proteins. In any peptide portion of the inventive compositions, for example a toxin peptide or a peptide linker moiety described herein, additional amino acids can be included on either or both of the N- or C-termini of the given sequence. Of course, these additional amino acid residues should not significantly interfere with the functional activity of the composition.
“Toxin peptides” include peptides having the same amino acid sequence of a naturally occurring pharmacologically active peptide that can be isolated from a venom, and also include modified peptide analogs (spelling used interchangeably with “analogues”) of such naturally occurring molecules.
The term “peptide analog” refers to a peptide having a sequence that differs from a peptide sequence existing in nature by at least one amino acid residue substitution, internal addition, or internal deletion of at least one amino acid, and/or amino- or carboxy-terminal end truncations, or additions). An “internal deletion” refers to absence of an amino acid from a sequence existing in nature at a position other than the N- or C-terminus. Likewise, an “internal addition” refers to presence of an amino acid in a sequence existing in nature at a position other than the N- or C-terminus. “Toxin peptide analogs”, such as, but not limited to, an OSK1 peptide analog, ShK peptide analog, or ChTx peptide analog, contain modifications of a native toxin peptide sequence of interest (e.g., amino acid residue substitutions, internal additions or insertions, internal deletions, and/or amino- or carboxy-terminal end truncations, or additions as previously described above) relative to a native toxin peptide sequence of interest, which is in the case of OSK1: GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK (SEQ ID NO:25).
Examples of toxin peptides useful in practicing the present invention are listed in Tables 1-32. The toxin peptide (“P”, or equivalently shown as “P1” in FIG. 2) comprises at least two intrapeptide disulfide bonds, as shown, for example, in FIG. 9. Accordingly, this invention concerns molecules comprising:

    
    
        a) C1-C3 and C2-C4 disulfide bonding in which C1, C2, C3, and C4 represent the order in which cysteine residues appear in the primary sequence of the toxin peptide stated conventionally with the N-terminus of the peptide on the left, with the first and third cysteines in the amino acid sequence forming a disulfide bond, and the second and fourth cysteines forming a disulfide bond. Examples of toxin peptides with such a C1-C3, C2-C4 disulfide bonding pattern include, but are not limited to, apamin peptides, α-conopeptides, PnIA peptides, PnIB peptides, and MII peptides, and analogs of any of the foregoing.
        b) C1-C6, C2-C4 and C3-C5 disulfide bonding in which, as described above, C1, C2, C3, C4, C5 and C6 represent the order of cysteine residues appearing in the primary sequence of the toxin peptide stated conventionally with the N-terminus of the peptide(s) on the left, with the first and sixth cysteines in the amino acid sequence forming a disulfide bond, the second and fourth cysteines forming a disulfide bond, and the third and fifth cysteines forming a disulfide bond. Examples of toxin peptides with such a C1-C6, C2-C4, C3-C5 disulfide bonding pattern include, but are not limited to, ShK, BgK, HmK, AeKS, AsK, and DTX1, and analogs of any of the foregoing.
        c) C1-C4, C2-C5 and C3-C6 disulfide bonding in which, as described above, C1, C2, C3, C4, C5 and C6 represent the order of cysteine residues appearing in the primary sequence of the toxin peptide stated conventionally with the N-terminus of the peptide(s) on the left, with the first and fourth cysteines in the amino acid sequence forming a disulfide bond, the second and fifth cysteines forming a disulfide bond, and the third and sixth cysteines forming a disulfide bond. Examples of toxin peptides with such a C1-C4, C2-C5, C3-C6 disulfide bonding pattern include, but are not limited to, ChTx, MgTx, OSK1, KTX1, AgTx2, Pi2, Pi3, NTX, HgTx1, BeKM1, BmKTX, P01, BmKK6, Tc32, Tc1, BmTx1, BmTX3, IbTx, P05, ScyTx, TsK, HaTx1, ProTX1, PaTX2, Ptu1, ωGVIA, ωMVIIA, and SmIIIa, and analogs of any of the foregoing.
        d) C1-C5, C2-C6, C3-C7, and C4-C8 disulfide bonding in which C1, C2, C3, C4, C5, C6, C7 and C8 represent the order of cysteine residues appearing in the primary sequence of the toxin peptide stated conventionally with the N-terminus of the peptide(s) on the left, with the first and fifth cysteines in the amino acid sequence forming a disulfide bond, the second and sixth cysteines forming a disulfide bond, the third and seventh cysteines forming a disulfide bond, and the fourth and eighth cysteines forming a disulfide bond. Examples of toxin peptides with such a C1-C5, C2-C6, C3-C7, C4-C8 disulfide bonding pattern include, but are not limited to, Anuoroctoxin (AnTx), Pi1, HsTx1, MTX (P12A, P20A), and Pi4 peptides, and analogs of any of the foregoing.
        e) C1-C4, C2-C6, C3-C7, and C5-C8 disulfide bonding in which C7, C2, C3, C4, C5, C6, C7 and C8 represent the order of cysteine residues appearing in the primary sequence of the toxin peptide stated conventionally with the N-terminus of the peptide(s) on the left, with the first and fourth cysteines in the amino acid sequence forming a disulfide bond, the second and sixth cysteines forming a disulfide bond, the third and seventh cysteines forming a disulfide bond, and the fifth and eighth cysteines forming a disulfide bond. Examples of toxin peptides with such a C1-C4, C2-C6, C3-C7, C5-C8 disulfide bonding pattern include, but are not limited to, Chlorotoxin, Bm-12b, and, and analogs of either.
        f) C1-C5, C2-C6, C3-C4, and C7-C8 disulfide bonding in which C1, C2, C3, C4, C5, C6, C7 and C8 represent the order of cysteine residues appearing in the primary sequence of the toxin peptide stated conventionally with the N-terminus of the peptide(s) on the left, with the first and fifth cysteines in the amino acid sequence forming a disulfide bond, the second and sixth cysteines forming a disulfide bond, the third and fourth cysteines forming a disulfide bond, and the seventh and eighth cysteines forming a disulfide bond. Examples of toxin peptides with such a C1-C5, C2-C6, C3-C4, C7-C8 disulfide bonding pattern include, but are not limited to, Maurotoxin peptides and analogs thereof.
    
    


The term “randomized” as used to refer to peptide sequences refers to fully random sequences (e.g., selected by phage display methods) and sequences in which one or more residues of a naturally occurring molecule is replaced by an amino acid residue not appearing in that position in the naturally occurring molecule. Exemplary methods for identifying peptide sequences include phage display, E. coli display, ribosome display, yeast-based screening, RNA-peptide screening, chemical screening, rational design, protein structural analysis, and the like.
The term “pharmacologically active” means that a substance so described is determined to have activity that affects a medical parameter (e.g., blood pressure, blood cell count, cholesterol level) or disease state (e.g., cancer, autoimmune disorders). Thus, pharmacologically active peptides comprise agonistic or mimetic and antagonistic peptides as defined below.
The terms “-mimetic peptide” and “-agonist peptide” refer to a peptide having biological activity comparable to a naturally occurring toxin peptide molecule, e.g., naturally occurring ShK toxin peptide. These terms further include peptides that indirectly mimic the activity of a naturally occurring toxin peptide molecule, such as by potentiating the effects of the naturally occurring molecule.
The term “antagonist peptide” or “inhibitor peptide” refers to a peptide that blocks or in some way interferes with the biological activity of a receptor of interest, or has biological activity comparable to a known antagonist or inhibitor of a receptor of interest (such as, but not limited to, an ion channel).
The term “acidic residue” refers to amino acid residues in D- or L-form having sidechains comprising acidic groups. Exemplary acidic residues include D and E.
The term “amide residue” refers to amino acids in D- or L-form having sidechains comprising amide derivatives of acidic groups. Exemplary residues include N and Q.
The term “aromatic residue” refers to amino acid residues in D- or L-form having sidechains comprising aromatic groups. Exemplary aromatic residues include F, Y, and W.
The term “basic residue” refers to amino acid residues in D- or L-form having sidechains comprising basic groups. Exemplary basic residues include H, K, R, N-methyl-arginine, ω-aminoarginine, ω-methyl-arginine, 1-methyl-histidine, 3-methyl-histidine, and homoarginine (hR) residues.
The term “hydrophilic residue” refers to amino acid residues in D- or L-form having sidechains comprising polar groups. Exemplary hydrophilic residues include C, S, T, N, Q, D, E, K, and citrulline (Cit) residues.
The term “nonfunctional residue” refers to amino acid residues in D- or L-form having sidechains that lack acidic, basic, or aromatic groups. Exemplary nonfunctional amino acid residues include M, G, A, V, I, L and norleucine (Nle).
The term “neutral polar residue” refers to amino acid residues in D- or L-form having sidechains that lack basic, acidic, or polar groups. Exemplary neutral polar amino acid residues include A, V, L, I, P, W, M, and F.
The term “polar hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains comprising polar groups. Exemplary polar hydrophobic amino acid residues include T, G, S, Y, C, Q, and N.
The term “hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains that lack basic or acidic groups. Exemplary hydrophobic amino acid residues include A, V, L, I, P, W, M, F, T, G, S, Y, C, Q, and N.
In some useful embodiments of the compositions of the invention, the amino acid sequence of the toxin peptide is modified in one or more ways relative to a native toxin peptide sequence of interest, such as, but not limited to, a native ShK or OSK1 sequence, their peptide analogs, or any other toxin peptides having amino acid sequences as set for in any of Tables 1-32. The one or more useful modifications can include amino acid additions or insertions, amino acid deletions, peptide truncations, amino acid substitutions, and/or chemical derivatization of amino acid residues, accomplished by known chemical techniques. Such modifications can be, for example, for the purpose of enhanced potency, selectivity, and/or proteolytic stability, or the like. Those skilled in the art are aware of techniques for designing peptide analogs with such enhanced properties, such as alanine scanning, rational design based on alignment mediated mutagenesis using known toxin peptide sequences and/or molecular modeling. For example, ShK analogs can be designed to remove protease cleavage sites (e.g., trypsin cleavage sites at K or R residues and/or chymotrypsin cleavage sites at F, Y, or W residues) in a ShK peptide- or ShK analog-containing composition of the invention, based partially on alignment mediated mutagenesis using HmK (see, e.g., FIG. 6) and molecular modeling. (See, e.g., Kalman et al., ShK-Dap22, a potent Kv1.3-specific immunosuppressive polypeptide, J. Biol. Chem. 273(49):32697-707 (1998); Kem et al., U.S. Pat. No. 6,077,680; Mouhat et al., OsK1 derivatives, WO 2006/002850 A2)).
The term “protease” is synonymous with “peptidase”. Proteases comprise two groups of enzymes: the endopeptidases which cleave peptide bonds at points within the protein, and the exopeptidases, which remove one or more amino acids from either N- or C-terminus respectively. The term “proteinase” is also used as a synonym for endopeptidase. The four mechanistic classes of proteinases are: serine proteinases, cysteine proteinases, aspartic proteinases, and metallo-proteinases. In addition to these four mechanistic classes, there is a section of the enzyme nomenclature which is allocated for proteases of unidentified catalytic mechanism. This indicates that the catalytic mechanism has not been identified.
Cleavage subsite nomenclature is commonly adopted from a scheme created by Schechter and Berger (Schechter I. & Berger A., On the size of the active site in proteases. I. Papain, Biochemical and Biophysical Research Communication, 27:157 (1967); Schechter I. & Berger A., On the active site of proteases. 3. Mapping the active site of papain; specific inhibitor peptides of papain, Biochemical and Biophysical Research Communication, 32:898 (1968)). According to this model, amino acid residues in a substrate undergoing cleavage are designated P1, P2, P3, P4 etc. in the N-terminal direction from the cleaved bond. Likewise, the residues in the C-terminal direction are designated P1′, P2′, P3′, P4′. etc.
The skilled artisan is aware of a variety of tools for identifying protease binding or protease cleavage sites of interest. For example, the PeptideCutter software tool is available through the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB; expasy.org/tools/peptidecutter). PeptideCutter searches a protein sequence from the SWISS-PROT and/or TrEMBL databases or a user-entered protein sequence for protease cleavage sites. Single proteases and chemicals, a selection or the whole list of proteases and chemicals can be used. Different forms of output of the results are available: tables of cleavage sites either grouped alphabetically according to enzyme names or sequentially according to the amino acid number. A third option for output is a map of cleavage sites. The sequence and the cleavage sites mapped onto it are grouped in blocks, the size of which can be chosen by the user. Other tools are also known for determining protease cleavage sites. (E.g., Turk, B. et al., Determination of protease cleavage site motifs using mixture-based oriented peptide libraries, Nature Biotechnology, 19:661-667 (2001); Barrett A. et al., Handbook of proteolytic enzymes, Academic Press (1998)).
The serine proteinases include the chymotrypsin family, which includes mammalian protease enzymes such as chymotrypsin, trypsin or elastase or kallikrein. The serine proteinases exhibit different substrate specificities, which are related to amino acid substitutions in the various enzyme subsites interacting with the substrate residues. Some enzymes have an extended interaction site with the substrate whereas others have a specificity restricted to the P1 substrate residue.
Trypsin preferentially cleaves at R or K in position P1. A statistical study carried out by Keil (1992) described the negative influences of residues surrounding the Arg- and Lys-bonds (i.e. the positions P2 and P1′, respectively) during trypsin cleavage. (Keil, B., Specificity of proteolysis, Springer-Verlag Berlin-Heidelberg-New York, 335 (1992)). A proline residue in position P1′ normally exerts a strong negative influence on trypsin cleavage. Similarly, the positioning of R and K in P1′ results in an inhibition, as well as negatively charged residues in positions P2 and P1′.
Chymotrypsin preferentially cleaves at a W, Y or F in position P1 (high specificity) and to a lesser extent at L, M or H residue in position P1. (Keil, 1992). Exceptions to these rules are the following: When W is found in position P1, the cleavage is blocked when M or P are found in position P1′ at the same time. Furthermore, a proline residue in position P1′ nearly fully blocks the cleavage independent of the amino acids found in position P1. When an M residue is found in position P1, the cleavage is blocked by the presence of a Y residue in position P1′. Finally, when H is located in position P1, the presence of a D, M or W residue also blocks the cleavage.
Membrane metallo-endopeptidase (NEP; neutral endopeptidase, kidney-brush-border neutral proteinase, enkephalinase, EC 3.4.24.11) cleaves peptides at the amino side of hydrophobic amino acid residues. (Connelly, J C et al., Neutral Endopeptidase 24.11 in Human Neutrophils: Cleavage of Chemotactic Peptide, PNAS, 82(24):8737-8741 (1985)).
Thrombin preferentially cleaves at an R residue in position P1. (Keil, 1992). The natural substrate of thrombin is fibrinogen. Optimum cleavage sites are when an R residue is in position P1 and Gly is in position P2 and position P1′. Likewise, when hydrophobic amino acid residues are found in position P4 and position P3, a proline residue in position P2, an R residue in position P1, and non-acidic amino acid residues in position P1′ and position P2′. A very important residue for its natural substrate fibrinogen is a D residue in P10.
Caspases are a family of cysteine proteases bearing an active site with a conserved amino acid sequence and which cleave peptides specifically following D residues. (Eamshaw W C et al., Mammalian caspases: Structure, activation, substrates, and functions during apoptosis, Annual Review of Biochemistry, 68:383-424 (1999)).
The Arg-C proteinase preferentially cleaves at an R residue in position P1. The cleavage behavior seems to be only moderately affected by residues in position P1′. (Keil, 1992). The Asp-N endopeptidase cleaves specifically bonds with a D residue in position P1′. (Keil, 1992).
Furin is a ubiquitous subtilisin-like proprotein convertase. It is the major processing enzyme of the secretory pathway and intracellularly is localized in the trans-golgi network (van den Ouweland, A. M. W. et al. (1990) Nucl. Acids Res., 18, 664; Steiner, D. F. (1998) Curr. Opin. Chem. Biol., 2, 31-39). The minimal furin cleavage site is Arg-X-X-Arg′. However, the enzyme prefers the site Arg-X-(Lys/Arg)-Arg′. An additional arginine at the P6 position appears to enhance cleavage (Krysan, D. J. et al. (1999) J. Biol. Chem., 274, 23229-23234).
The foregoing is merely exemplary and by no means an exhaustive treatment of knowledge available to the skilled artisan concerning protease binding and/or cleavage sites that the skilled artisan may be interested in eliminating in practicing the invention.
Additional useful embodiments of the toxin peptide, e.g., the OSK1 peptide analog, can result from conservative modifications of the amino acid sequences of the peptides disclosed herein. Conservative modifications will produce peptides having functional, physical, and chemical characteristics similar to those of the parent peptide from which such modifications are made. Such conservatively modified forms of the peptides disclosed herein are also contemplated as being an embodiment of the present invention.
In contrast, substantial modifications in the functional and/or chemical characteristics of the toxin peptides may be accomplished by selecting substitutions in the amino acid sequence that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the region of the substitution, for example, as an α-helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the molecule.
For example, a “conservative amino acid substitution” may involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for “alanine scanning mutagenesis” (see, for example, MacLennan et al., Acta Physiol. Scand. Suppl., 643:55-67 (1998); Sasaki et al., 1998, Adv. Biophys. 35:1-24 (1998), which discuss alanine scanning mutagenesis).
Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the peptide sequence, or to increase or decrease the affinity of the peptide or vehicle-conjugated peptide molecules described herein.
Naturally occurring residues may be divided into classes based on common side chain properties:
1) hydrophobic: norleucine (Nor), Met, Ala, Val, Leu, Ile;
2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
3) acidic: Asp, Glu;
4) basic: His, Lys, Arg;
5) residues that influence chain orientation: Gly, Pro; and
6) aromatic: Trp, Tyr, Phe.
Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.
Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into regions of the human antibody that are homologous with non-human antibodies, or into the non-homologous regions of the molecule.
In making such changes, according to certain embodiments, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (see, for example, Kyte et al., 1982, J. MOL Biol. 157:105-131). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices are within ±2 is included. In certain embodiments, those that are within ±1 are included, and in certain embodiments, those within ±0.5 are included.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as disclosed herein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.
The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5) and tryptophan (−3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 is included, in certain embodiments, those that are within ±1 are included, and in certain embodiments, those within ±0.5 are included. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as “epitopic core regions.”
Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine norleucine, alanine, or methionine for another, the substitution of one polar (hydrophilic) amino acid residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic amino acid residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. The phrase “conservative amino acid substitution” also includes the use of a chemically derivatized residue in place of a non-derivatized residue, provided that such polypeptide displays the requisite biological activity. Other exemplary amino acid substitutions that can be useful in accordance with the present invention are set forth in Table 1A.







TABLE 1A







Some Useful Amino Acid Substitutions










Original Residues
Exemplary Substitutions






Ala
Val, Leu, Ile



Arg
Lys, Gln, Asn



Asn
Gln



Asp
Glu



Cys
Ser, Ala



Gln
Asn



Glu
Asp



Gly
Pro, Ala



His
Asn, Gln, Lys, Arg



Ile
Leu, Val, Met, Ala, Phe, Norleucine



Leu
Norleucine, Ile, Val, Met, Ala, Phe



Lys
Arg, 1,4-Diaminobutyric Acid, Gln, Asn



Met
Leu, Phe, Ile



Phe
Leu, Val, Ile, Ala, Tyr



Pro
Ala



Ser
Thr, Ala, Cys



Thr
Ser



Trp
Tyr, Phe



Tyr
Trp, Phe, Thr, Ser



Val
Ile, Met, Leu, Phe, Ala, Norleucine









In other examples, a toxin peptide amino acid sequence, e.g., an OSK1 peptide analog sequence, modified from a naturally occurring toxin peptide amino acid sequence includes at least one amino acid residue inserted or substituted therein, relative to the amino acid sequence of the native toxin peptide sequence of interest, in which the inserted or substituted amino acid residue has a side chain comprising a nucleophilic or electrophilic reactive functional group by which the peptide is conjugated to a linker or half-life extending moiety. In accordance with the invention, useful examples of such a nucleophilic or electrophilic reactive functional group include, but are not limited to, a thiol, a primary amine, a seleno, a hydrazide, an aldehyde, a carboxylic acid, a ketone, an aminooxy, a masked (protected) aldehyde, or a masked (protected) keto functional group. Examples of amino acid residues having a side chain comprising a nucleophilic reactive functional group include, but are not limited to, a lysine residue, an α,β-diaminopropionic acid residue, an α,γ-diaminobutyric acid residue, an ornithine residue, a cysteine, a homocysteine, a glutamic acid residue, an aspartic acid residue, or a selenocysteine residue. In some embodiments, the toxin peptide amino acid sequence (or “primary sequence”) is modified at one, two, three, four, five or more amino acid residue positions, by having a residue substituted therein different from the native primary sequence (e.g., OSK1 SEQ ID NO:25) or omitted (e.g., an OSK1 peptide analog optionally lacking a residue at positions 36, 37, 36-38, 37-38, or 38).
In further describing toxin peptides herein, a one-letter abbreviation system is frequently applied to designate the identities of the twenty “canonical” amino acid residues generally incorporated into naturally occurring peptides and proteins (Table 1B). Such one-letter abbreviations are entirely interchangeable in meaning with three-letter abbreviations, or non-abbreviated amino acid names. Within the one-letter abbreviation system used herein, an uppercase letter indicates a L-amino acid, and a lower case letter indicates a D-amino acid, unless otherwise noted herein. For example, the abbreviation “R” designates L-arginine and the abbreviation “r” designates D-arginine.







TABLE 1B





One-letter abbreviations for the canonical amino acids


Three-letter abbreviations are in parentheses


















Alanine (Ala)
A



Glutamine (Gln)
Q



Leucine (Leu)
L



Serine (Ser)
S



Arginine (Arg)
R



Glutamic Acid (Glu)
E



Lysine (Lys)
K



Threonine (Thr)
T



Asparagine (Asn)
N



Glycine (Gly)
G



Methionine (Met)
M



Tryptophan (Trp)
W



Aspartic Acid (Asp)
D



Histidine (His)
H



Phenylalanine (Phe)
F



Tyrosine (Tyr)
Y



Cysteine (Cys)
C



Isoleucine (Ile)
I



Proline (Pro)
P



Valine (Val)
V









An amino acid substitution in an amino acid sequence is typically designated herein with a one-letter abbreviation for the amino acid residue in a particular position, followed by the numerical amino acid position relative to the native toxin peptide sequence of interest, which is then followed by the one-letter symbol for the amino acid residue substituted in. For example, “T30D” symbolizes a substitution of a threonine residue by an aspartate residue at amino acid position 30, relative to a hypothetical native toxin peptide sequence. By way of further example, “R18hR” or “R18Cit” indicates a substitution of an arginine residue by a homoarginine or a citrulline residue, respectively, at amino acid position 18, relative to the hypothetical native toxin peptide. An amino acid position within the amino acid sequence of any particular toxin peptide (or peptide analog) described herein may differ from its position relative to the native sequence, i.e., as determined in an alignment of the N-terminal or C-terminal end of the peptide's amino acid sequence with the N-terminal or C-terminal end, as appropriate, of the native toxin peptide sequence. For example, amino acid position 1 of the sequence SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK(2-35); SEQ ID NO:92), a N-terminal truncation of the native ShK sequence, thus aligned with the C-terminal of native ShK(1-35) (SEQ ID NO:5), corresponds to amino acid position 2 relative to the native sequence, and amino acid position 34 of SEQ ID NO:92 corresponds to amino acid position 35 relative to the native sequence (SEQ ID NO:5).
In certain embodiments of the present invention, amino acid substitutions encompass, non-canonical amino acid residues, which include naturally rare (in peptides or proteins) amino acid residues or unnatural amino acid residues. Non-canonical amino acid residues can be incorporated into the peptide by chemical peptide synthesis rather than by synthesis in biological systems, such as recombinantly expressing cells, or alternatively the skilled artisan can employ known techniques of protein engineering that use recombinantly expressing cells. (See, e.g., Link et al., Non-canonical amino acids in protein engineering, Current Opinion in Biotechnology, 14(6):603-609 (2003)). The term “non-canonical amino acid residue” refers to amino acid residues in D- or L-form that are not among the 20 canonical amino acids generally incorporated into naturally occurring proteins, for example, β-amino acids, homoamino acids, cyclic amino acids and amino acids with derivatized side chains. Examples include (in the L-form or D-form; abbreviated as in parentheses): citrulline (Cit), homocitrulline (hCit), Nα-methylcitrulline (NMeCit), Nα-methylhomocitrulline (Nα-MeHoCit), ornithine (Orn), Nα-Methylornithine (Nα-MeOrn or NMeOrn), sarcosine (Sar), homolysine (hLys or hK), homoarginine (hArg or hR), homoglutamine (hQ), Nα-methylarginine (NMeR), Nα-methylleucine (Nα-MeL or NMeL), N-methylhomolysine (NMeHoK), Nα-methylglutamine (NMeQ), norleucine (Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic), Octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal), 3-(2-naphthyl)alanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline (Tic), 2-indanylglycine (Igl), para-iodophenylalanine (pI-Phe), para-aminophenylalanine (4AmP or 4-Amino-Phe), 4-guanidino phenylalanine (Guf), glycyllysine (abbreviated herein “K(N∈-glycyl)” or “K(glycyl)” or “K(gly)”), nitrophenylalanine (nitrophe), aminophenylalanine (aminophe or Amino-Phe), benzylphenylalanine (benzylphe), γ-carboxyglutamic acid (γ-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine (Cpa), α-aminoadipic acid (Aad), Nα-methyl valine (NMeVal), N-α-methyl leucine (NMeLeu), Nα-methylnorleucine (NMeNle), cyclopentylglycine (Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), α,β-diaminopropionoic acid (Dpr), α,γ-diaminobutyric acid (Dab), diaminopropionic acid (Dap), cyclohexylalanine (Cha), 4-methyl-phenylalanine (MePhe), β,β-diphenyl-alanine (BiPhA), aminobutyric acid (Abu), 4-phenyl-phenylalanine (or biphenylalanine; 4Bip), α-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionic acid, piperidinic acid, aminocaproic acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine, N-ethylaspargine, hydroxylysine, allo-hydroxylysine, isodesmosine, allo-isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline, 4-hydroxyproline (Hyp), γ-carboxyglutamate, ∈-N,N,N-trimethyllysine, ∈-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-Amino-O-Phthalic Acid (4APA), and other similar amino acids, and derivatized forms of any of these as described herein. Table 1B contains some exemplary non-canonical amino acid residues that are useful in accordance with the present invention and associated abbreviations as typically used herein, although the skilled practitioner will understand that different abbreviations and nomenclatures may be applicable to the same substance and my appear interchangeably herein.
Table 1B. Useful non-canonical amino acids for amino acid addition, insertion, or substitution into toxin peptide sequences, including OSK1 peptide analog sequences, in accordance with the present invention. In the event an abbreviation listed in Table 1B differs from another abbreviation for the same substance disclosed elsewhere herein, both abbreviations are understood to be applicable.











Abbreviation
Amino Acid







Sar
Sarcosine


Nle
norleucine


Ile
isoleucine


1-Nal
3-(1-naphthyl)alanine


2-Nal
3-(2-naphthyl)alanine


Bip
4,4′-biphenyl alanine


Dip
3,3-diphenylalanine


Nvl
norvaline


NMe-Val
Nα-methyl valine


NMe-Leu
Nα-methyl leucine


NMe-Nle
Nα-methyl norleucine


Cpg
cyclopentyl glycine


Chg
cyclohexyl glycine


Hyp
hydroxy proline


Oic
Octahydroindole-2-Carboxylic Acid


Igl
Indanyl glycine


Aib
aminoisobutyric acid


Aic
2-aminoindane-2-carboxylic acid


Pip
pipecolic acid


BhTic
β-homo Tic


BhPro
β-homo proline


Sar
Sarcosine


Cpg
cyclopentyl glycine


Tiq
1,2,3,4-L-Tetrahydroisoquinoline-1-Carboxylic acid


Nip
Nipecotic Acid


Thz
Thiazolidine-4-carboxylic acid


Thi
3-thienyl alanine


4GuaPr
4-guanidino proline


4Pip
4-Amino-1-piperidine-4-carboxylic acid


Idc
indoline-2-carboxylic acid


Hydroxyl-Tic
1,2,3,4-Tetrahydroisoquinoline-7-hydroxy-3-



carboxylic acid


Bip
4,4′-biphenyl alanine


Ome-Tyr
O-methyl tyrosine


I-Tyr
Iodotyrosine


Tic
1,2,3,4-L-Tetrahydroisoquinoline-3-Carboxylic



acid


Igl
Indanyl glycine


BhTic
β-homo Tic


BhPhe
β-homo phenylalanine


AMeF
α-methyl Phenyalanine


BPhe
β-phenylalanine


Phg
phenylglycine


Anc
3-amino-2-naphthoic acid


Atc
2-aminotetraline-2-carboxylic acid


NMe-Phe
Nα-methyl phenylalanine


NMe-Lys
Nα-methyl lysine


Tpi
1,2,3,4-Tetrahydronorharman-3-Carboxylic acid


Cpg
cyclopentyl glycine


Dip
3,3-diphenylalanine


4Pal
4-pyridinylalanine


3Pal
3-pyridinylalanine


2Pal
2-pyridinylalanine


4Pip
4-Amino-1-piperidine-4-carboxylic acid


4AmP
4-amino-phenylalanine


Idc
indoline-2-carboxylic acid


Chg
cyclohexyl glycine


hPhe
homophenylalanine


BhTrp
β-homotryptophan


pI-Phe
4-iodophenylalanine


Aic
2-aminoindane-2-carboxylic acid


NMe-Lys
Nα-methyl lysine


Orn
ornithine


Dpr
2,3-Diaminopropionic acid


Dbu
2,4-Diaminobutyric acid


homoLys
homolysine


N-eMe-K
Nε-methyl-lysine


N-eEt-K
Nε-ethyl-lysine


N-eIPr-K
Nε-isopropyl-lysine


bhomoK
β-homolysine


rLys
Lys ψ (CH2NH)-reduced amide bond


rOrn
Orn ψ (CH2NH)-reduced amide bond


Acm
acetamidomethyl


Ahx
6-aminohexanoic acid


ε Ahx
6-aminohexanoic acid


K(NPeg11)
Nε-(O-(aminoethyl)-O′-(2-propanoyl)-



undecaethyleneglycol)-Lysine


K(NPeg27)
Nε-(O-(aminoethyl)-O′-(2-propanoyl)-



(ethyleneglycol)27-Lysine


Cit
Citrulline


hArg
homoarginine


hCit
homocitrulline


NMe-Arg
Nα-methyl arginine (NMeR)


Guf
4-guanidinyl phenylalanine


bhArg
β-homoarginine


3G-Dpr
2-amino-3-guanidinopropanoic acid


4AmP
4-amino-phenylalanine


4AmPhe
4-amidino-phenylalanine


4AmPig
2-amino-2-(1-carbamimidoylpiperidin-4-



yl)acetic acid


4GuaPr
4-guanidino proline


N-Arg
Nα-[(CH2)3NHCH(NH)NH2] substituted glycine


rArg
Arg ψ(CH2NH) -reduced amide bond


4PipA
4-Piperidinyl alanine


NMe-Arg
Nα-methyl arginine (or NMeR)


NMe-Thr
Nα-methyl threonine(or NMeThr)









Nomenclature and Symbolism for Amino Acids and Peptides by the UPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) have been published in the following documents: Biochem. J., 1984, 219, 345-373; Eur. J. Biochem., 1984, 138, 9-37; 1985, 152, 1; 1993, 213, 2; Internat. J. Pept. Prot. Res., 1984, 24, following p 84; J. Biol. Chem., 1985, 260, 14-42; Pure Appl. Chem., 1984, 56, 595-624; Amino Acids and Peptides, 1985, 16, 387-410; Biochemical Nomenclature and Related Documents, 2nd edition, Portland Press, 1992, pages 39-69].
As stated herein, in accordance with the present invention, peptide portions of the inventive compositions, such as the toxin peptide or a peptide linker, can also be chemically derivatized at one or more amino acid residues. Peptides that contain derivatized amino acid residues can be synthesized by known organic chemistry techniques. “Chemical derivative” or “chemically derivatized” in the context of a peptide refers to a subject peptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty canonical amino acids, whether in L- or D-form. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine maybe substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
Useful derivatizations include, in some embodiments, those in which the amino terminal of the toxin peptide, such as but not limited to the OSK1 peptide analog, is chemically blocked so that conjugation with the vehicle will be prevented from taking place at an N-terminal free amino group. There may also be other beneficial effects of such a modification, for example a reduction in the toxin peptide's susceptibility to enzymatic proteolysis. The N-terminus of the toxin peptide, e.g., the OSK1 peptide analog, can be acylated or modified to a substituted amine, or derivatized with another functional group, such as an aromatic or aryl moiety (e.g., an indole acid, benzyl (Bzl or Bn), dibenzyl (DiBzl or Bn2), benzoyl, or benzyloxycarbonyl (Cbz or Z)), N,N-dimethylglycine or creatine. For example, in some embodiments, an acyl moiety, such as, but not limited to, a formyl, acetyl (Ac), propanoyl, butanyl, heptanyl, hexanoyl, octanoyl, or nonanoyl, can be covalently linked to the N-terminal end of the peptide, e.g., the OSK1 peptide analog, which can prevent undesired side reactions during conjugation of the vehicle to the peptide. Alternatively, a fatty acid (e.g. butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic or the like) or polyethylene glycol moiety can be covalently linked to the N-terminal end of the peptide, e.g., the OSK1 peptide analog. Other exemplary N-terminal derivative groups include —NRR1 (other than —NH2), —NRC(O)R1, —NRC(O)OR1, —NRS(O)2R1, —NHC(O)NHR1, succinimide, or benzyloxycarbonyl-NH-(Cbz-NH—), wherein R and R1 are each independently hydrogen or lower alkyl and wherein the phenyl ring may be substituted with 1 to 3 substituents selected from C1-C4 alkyl, C1-C4 alkoxy, chloro, and bromo.
In some embodiments of the present invention, basic residues (e.g., lysine) of the toxin peptide of interest can be replaced with other residues (nonfunctional residues preferred). Such molecules will be less basic than the molecules from which they are derived and otherwise retain the activity of the molecules from which they are derived, which can result in advantages in stability and immunogenicity; the present invention should not, however, be limited by this theory.
Additionally, physiologically acceptable salts of the inventive compositions are also encompassed, including when the inventive compositions are referred to herein as “molecules” or “compounds.”. By “physiologically acceptable salts” is meant any salts that are known or later discovered to be pharmaceutically acceptable. Some non-limiting examples of pharmaceutically acceptable salts are: acetate; trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide; sulfate; citrate; maleate; tartrate; glycolate; gluconate; succinate; mesylate; besylate; salts of gallic acid esters (gallic acid is also known as 3, 4, 5 trihydroxybenzoic acid) such as PentaGalloylGlucose (PGG) and epigallocatechin gallate (EGCG), salts of cholesteryl sulfate, pamoate, tannate and oxalate salts.
Structure of Compounds:
In general. Recombinant proteins have been developed as therapeutic agents through, among other means, covalent attachment to half-life extending moieties. Such moieties include the “Fc” domain of an antibody, as is used in Enbrel® (etanercept), as well as biologically suitable polymers (e.g., polyethylene glycol, or “PEG”), as is used in Neulasta® (pegfilgrastim). Feige et al. described the use of such half-life extenders with peptides in U.S. Pat. No. 6,660,843, issued Dec. 9, 2003 (hereby incorporated by reference in its entirety).
The present inventors have determined that molecules of this invention-peptides of about 80 amino acids or less with at least two intrapeptide disulfide bonds-possess therapeutic advantages when covalently attached to half-life extending moieties. Molecules of the present invention can further comprise an additional pharmacologically active, covalently bound peptide, which can be bound to the half-life extending moiety (F1 and/or F2) or to the peptide portion (P). Embodiments of the inventive compositions containing more than one half-life extending moiety (F1 and F2) include those in which F1 and F2 are the same or different half-life extending moieties. Examples (with or without a linker between each domain) include structures as illustrated in FIG. 75 as well as the following embodiments (and others described herein and in the working Examples):
20KPEG—toxin peptide—Fc domain, consistent with the formula [(F1)1-(X2)1-(F2)1];
20KPEG—toxin peptide—Fc CH2 domain, consistent with the formula [(F1)1-(X2)1-(F2)1];
20KPEG—toxin peptide—HSA, consistent with the formula [(F1)1(X2)1-(F2)1];
20KPEG—Fc domain—toxin peptide, consistent with the formula [(F1)1-(F2)1-(X3)1];
20KPEG—Fc CH2 domain—toxin peptide, consistent with the formula [(F1)1-(F2)1-(X3)1]; and
20KPEG—HSA—toxin peptide, consistent with the formula [(F1)1-(F2)1-(X3)1].
Toxin peptides. Any number of toxin peptides (i.e., “P”, or equivalently shown as “P1” in FIG. 2) can be used in conjunction with the present invention. Of particular interest are the toxin peptides ShK, HmK, MgTx, AgTx2, Agatoxins, and HsTx1, as well as modified analogs of these, in particular OsK1 (also referred to as “OSK1”) peptide analogs of the present invention, and other peptides that mimic the activity of such toxin peptides. As stated herein above, if more than one toxin peptide “P” is present in the inventive composition, “P” can be independently the same or different from any other toxin peptide(s) also present in the inventive composition. For example, in a composition having the formula P-(L)g-F1-(L)f-P, both of the toxin peptides, “P”, can be the same peptide analog of ShK, different peptide analogs of ShK, or one can be a peptide analog of ShK and the other a peptide analog of OSK1. In a preferred embodiment, at least one P is a an OSK1 peptide analog as further described herein.
In some embodiments of the invention, other peptides of interest are especially useful in molecules having additional features over the molecules of structural Formula I. In such molecules, the molecule of Formula I further comprises an additional pharmacologically active, covalently bound peptide, which is an agonistic peptide, an antagonistic peptide, or a targeting peptide; this peptide can be conjugated to F1 or F2 or P. Such agonistic peptides have activity agonistic to the toxin peptide but are not required to exert such activity by the same mechanism as the toxin peptide. Peptide antagonists are also useful in embodiments of the invention, with a preference for those with activity that can be complementary to the activity of the toxin peptide. Targeting peptides are also of interest, such as peptides that direct the molecule to particular cell types, organs, and the like. These classes of peptides can be discovered by methods described in the references cited in this specification and other references. Phage display, in particular, is useful in generating toxin peptides for use in the present invention. Affinity selection from libraries of random peptides can be used to identify peptide ligands for any site of any gene product. Dedman et al. (1993), J. Biol. Chem. 268: 23025-30. Phage display is particularly well suited for identifying peptides that bind to such proteins of interest as cell surface receptors or any proteins having linear epitopes. Wilson et al. (1998), Can. J. Microbiol. 44: 313-29; Kay et al. (1998), Drug Disc. Today 3: 370-8. Such proteins are extensively reviewed in Herz et al. (1997), J. Receptor and Signal Transduction Res. 17(5): 671-776, which is hereby incorporated by reference in its entirety. Such proteins of interest are preferred for use in this invention.
Particularly preferred peptides appear in the following tables. These peptides can be prepared by methods disclosed in the art or as described hereinafter. Single letter amino acid abbreviations are used. Unless otherwise specified, each X is independently a nonfunctional residue.







TABLE 1







Kv1.3 inhibitor peptide sequences











SEQ



Short-hand
ID


Sequence/structure
designation
NO:












LVKCRGTSDCGRPCQQQTGCPNSKCINRMCKCYGC
Pi1
21


 


TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGR
Pi2
17


 


TISCTNEKQCYPHCKKETGYPNAKCMNRKCKCFGR
Pi3
18


 


IEAIRCGGSRDCYRPCQKRTGCPNAKCINKTCKCY
Pi4
19


GCS




 


ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNRC
HsTx1
61


 


GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHC
AgTx2
23


TPK




 


GVPINVKCTGSPQCLKPCKDAGMRFGKCINGKCHC
AgTx1
85


TPK




 


GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC
OSK1
25


TPK




 


ZKECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK
Anuroctoxin
62


 


TIINVKCTSPKQCSKPCKELYGSSAGAKCMNGKCK
NTX
30


CYNN




 


TVIDVKCTSPKQCLPPCKAQFGIRAGAKCMNGKCK
HgTx1
27


CYPH




 


QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRC
ChTx
36


YS




 


VFINAKCRGSPECLPKCKEAIGKAAGKCMNGKCKC
Titystoxin-
86


YP
Ka



 


VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCE
BgK
9


LC




 


VGINVKCKHSGQCLKPCKDAGMRFGKCINGKCDCT
BmKTX
26


PKG




 


QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRC
BmTx1
40


YS




 


VFINVKCRGSKECLPACKAAVGKAAGKCMNGKCKC
Tc30
87


YP




 


TGPQTTCQAAMCEAGCKGLGKSMESCQGDTCKCKA
Tc32
13
















TABLE 2







ShK peptide and ShK peptide analog sequences










Short-hand
SEQ


Sequence/structure
designation
ID NO:












RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK
5


 


RSCIDTIPKSRCTAFQSKHSMKYRLSFCRKTSGTC
ShK-S17/S32
88


 


RSSIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTS
ShK-S3/S35
89


 


SSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-S1
90


 


(N-acetylarg)
ShK-N-acetylarg1
91


SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC




 


 SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-d1
92


 


  CIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-d2
93


 


ASCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A1
94


 


RSCADTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A4
95


 


RSCADTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC
ShK-A4/A15
96


 


RSCADTIPKSRCTAAQCKHSMKYRASFCRKTCGTC
ShK-A4/A15/A25
97


 


RSCIDAIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A6
98


 


RSCIDTAPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A7
99


 


RSCIDTIAKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A8
100


 


RSCIDTIPASRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A9
101


 


RSCIDTIPESRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-E9
102


 


RSCIDTIPQSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-Q9
103


 


RSCIDTIPKARCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A10
104


 


RSCIDTIPKSACTAFQCKHSMKYRLSFCRKTCGTC
ShK-A11
105


 


RSCIDTIPKSECTAFQCKHSMKYRLSFCRKTCGTC
ShK-E11
106


 


RSCIDTIPKSQCTAFQCKHSMKYRLSFCRKTCGTC
ShK-Q11
107


 


RSCIDTIPKSRCAAFQCKHSMKYRLSFCRKTCGTC
ShK-A13
108


 


RSCIDTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC
ShK-A15
109


 


RSCIDTIPKSRCTAWQCKHSMKYRLSFCRKTCGTC
ShK-W15
110


 


RSCIDTIPKSRCTAXs15QCKHSMKYRLSFCRKTCGTC
ShK-X15
111


 


RSCIDTIPKSRCTAAQCKHSMKYRASFCRKTCGTC
ShK-A15/A25
112


 


RSCIDTIPKSRCTAFACKHSMKYRLSFCRKTCGTC
ShK-A16
113


 


RSCIDTIPKSRCTAFECKHSMKYRLSFCRKTCGTC
ShK-E16
114


 


RSCIDTIPKSRCTAFQCAHSMKYRLSFCRKTCGTC
ShK-A18
115


 


RSCIDTIPKSRCTAFQCEHSMKYRLSFCRKTCGTC
ShK-E18
116


 


RSCIDTIPKSRCTAFQCKASMKYRLSFCRKTCGTC
ShK-A19
117


 


RSCIDTIPKSRCTAFQCKKSMKYRLSFCRKTCGTC
ShK-K19
118


 


RSCIDTIPKSRCTAFQCKHAMKYRLSFCRKTCGTC
ShK-A20
119


 


RSCIDTIPKSRCTAFQCKHSAKYRLSFCRKTCGTC
ShK-A21
120


 


RSCIDTIPKSRCTAFQCKHSXs21KYRLSFCRKTCGTC
ShK-X21
121


 


RSCIDTIPKSRCTAFQCKHS(norleu)KYRLSFCRKTCGTC
ShK-Nle21
122


 


RSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-A22
123


 


RSCIDTIPKSRCTAFQCKHSMEYRLSFCRKTCGTC
ShK-E22
124


 


RSCIDTIPKSRCTAFQCKHSMRYRLSFCRKTCGTC
ShK-R22
125


 


RSCIDTIPKSRCTAFQCKHSMXs22YRLSFCRKTCGTC
ShK-X22
126


 


RSCIDTIPKSRCTAFQCKHSM(norleu)YRLSFCRKTCGTC
ShK-Nle22
127


 


RSCIDTIPKSRCTAFQCKHSM(orn)YRLSFCRKTCGTC
ShK-Orn22
128


 


RSCIDTIPKSRCTAFQCKHSM(homocit)YRLSFCRKTCGTC
ShK-Homocit22
129


 


RSCIDTIPKSRCTAFQCKHSM(diaminopropionic)YRLS
ShK-Diamino-
130


FCRKTCGTC
propionic22



 


RSCIDTIPKSRCTAFQCKHSMKARLSFCRKTCGTC
ShK-A23
131


 


RSCIDTIPKSRCTAFQCKHSMKSRLSFCRKTCGTC
ShK-S23
132


 


RSCIDTIPKSRCTAFQCKHSMKFRLSFCRKTCGTC
ShK-F23
133


 


RSCIDTIPKSRCTAFQCKHSMKXs23RLSFCRKTCGTC
ShK-X23
134


 


RSCIDTIPKSRCTAFQCKHSMK(nitrophe)RLSFCRKTCGTC
ShK-Nitrophe23
135


 


RSCIDTIPKSRCTAFQCKHSMK(aminophe)RLSFCRKTCGTC
ShK-Aminophe23
136


 


RSCIDTIPKSRCTAFQCKHSMK(benzylphe)RLSFCRKTCG
ShK-Benzylphe23
137


TC




 


RSCIDTIPKSRCTAFQCKHSMKYALSFCRKTCGTC
ShK-A24
138


 


RSCIDTIPKSRCTAFQCKHSMKYELSFCRKTCGTC
ShK-E24
139


 


RSCIDTIPKSRCTAFQCKHSMKYRASFCRKTCGTC
ShK-A25
140


 


RSCIDTIPKSRCTAFQCKHSMKYRLAFCRKTCGTC
ShK-A26
141


 


RSCIDTIPKSRCTAFQCKHSMKYRLSACRKTCGTC
ShK-A27
142


 


RSCIDTIPKSRCTAFQCKHSMKYRLSXs27CRKTCGTC
ShK-X27
143


 


RSCIDTIPKSRCTAFQCKHSMKYRLSFCAKTCGTC
ShK-A29
144


 


RSCIDTIPKSRCTAFQCKHSMKYRLSFCRATCGTC
ShK-A30
145


 


RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKACGTC
ShK-A31
146


 


RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGAC
ShK-A34
147


 


 SCADTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A4d1
148


 


 SCADTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC
ShK-A4/A15d1
149


 


 SCADTIPKSRCTAAQCKHSMKYRASFCRKTCGTC
ShK-A4/A15/A25
150



d1



 


 SCIDAIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A6 d1
151


 


 SCIDTAPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A7 d1
152


 


 SCIDTIAKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A8 d1
153


 


 SCIDTIPASRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A9 d1
154


 


 SCIDTIPESRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-E9 d1
155


 


 SCIDTIPQSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-Q9 d1
156


 


 SCIDTIPKARCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A10 d1
157


 


 SCIDTIPKSACTAFQCKHSMKYRLSFCRKTCGTC
ShK-A11 d1
158


 


 SCIDTIPKSECTAFQCKHSMKYRLSFCRKTCGTC
ShK-E11 d1
159


 


 SCIDTIPKSQCTAFQCKHSMKYRLSFCRKTCGTC
ShK-Q11 d1
160


 


 SCIDTIPKSRCAAFQCKHSMKYRLSFCRKTCGTC
ShK-A13 d1
161


 


 SCIDTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC
ShK-A15 d1
162


 


 SCIDTIPKSRCTAWQCKHSMKYRLSFCRKTCGTC
ShK-W15 d1
163


 


 SCIDTIPKSRCTAXs15QCKHSMKYRLSFCRKTCGTC
ShK-X15 d1
164


 


 SCIDTIPKSRCTAAQCKHSMKYRASFCRKTCGTC
ShK-A15/A25 d1
165


 


 SCIDTIPKSRCTAFACKHSMKYRLSFCRKTCGTC
ShK-A16 d1
166


 


 SCIDTIPKSRCTAFECKHSMKYRLSFCRKTCGTC
ShK-E16 d1
167


 


 SCIDTIPKSRCTAFQCAHSMKYRLSFCRKTCGTC
ShK-A18 d1
168


 


 SCIDTIPKSRCTAFQCEHSMKYRLSFCRKTCGTC
ShK-E18 d1
169


 


 SCIDTIPKSRCTAFQCKASMKYRLSFCRKTCGTC
ShK-A19 d1
170


 


 SCIDTIPKSRCTAFQCKKSMKYRLSFCRKTCGTC
ShK-K19 d1
171


 


 SCIDTIPKSRCTAFQCKHAMKYRLSFCRKTCGTC
ShK-A20 d1
172


 


 SCIDTIPKSRCTAFQCKHSAKYRLSFCRKTCGTC
ShK-A21 d1
173


 


 SCIDTIPKSRCTAFQCKHSXs21KYRLSFCRKTCGTC
ShK-X21 d1
174


 


SCIDTIPKSRCTAFQCKHS(norleu)KYRLSFCRKTCGTC
ShK-Nle21 d1
175


 


 SCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-A22 d1
176


 


 SCIDTIPKSRCTAFQCKHSMEYRLSFCRKTCGTC
ShK-E22 d1
177


 


 SCIDTIPKSRCTAFQCKHSMRYRLSFCRKTCGTC
ShK-R22 d1
178


 


 SCIDTIPKSRCTAFQCKHSMXs22YRLSFCRKTCGTC
ShK-X22 d1
179


 


SCIDTIPKSRCTAFQCKHSM(norleu)YRLSFCRKTCGTC
ShK-Nle22 d1
180


 


SCIDTIPKSRCTAFQCKHSM(orn)YRLSFCRKTCGTC
ShK-Orn22 d1
181


 


SCIDTIPKSRCTAFQCKHSM(homocit)YRLSFCRKTCGTC
ShK-Homocit22
182



d1



 


SCIDTIPKSRCTAFQCKHSM(diaminopropionic)YRLSF
ShK-Diamino-
183


CRKTCGTC
propionic22 d1



 


 SCIDTIPKSRCTAFQCKHSMKARLSFCRKTCGTC
ShK-A23 d1
184


 


 SCIDTIPKSRCTAFQCKHSMKSRLSFCRKTCGTC
ShK-S23 d1
185


 


 SCIDTIPKSRCTAFQCKHSMKFRLSFCRKTCGTC
ShK-F23 d1
186


 


 SCIDTIPKSRCTAFQCKHSMKXs23RLSFCRKTCGTC
ShK-X23 d1
187


 


SCIDTIPKSRCTAFQCKHSMK(nitrophe)RLSFCRKTCGTC
ShK-Nitrophe23
188



d1



 


SCIDTIPKSRCTAFQCKHSMK(aminophe)RLSFCRKTCGTC
ShK-Aminophe23
189



d1



 


SCIDTIPKSRCTAFQCKHSMK(benzylphe)RLSFCRKTCGTC
ShK-Benzylphe23
190



d1



 


 SCIDTIPKSRCTAFQCKHSMKYALSFCRKTCGTC
ShK-A24 d1
191


 


 SCIDTIPKSRCTAFQCKHSMKYELSFCRKTCGTC
ShK-E24 d1
192


 


 SCIDTIPKSRCTAFQCKHSMKYRASFCRKTCGTC
ShK-A25 d1
193


 


 SCIDTIPKSRCTAFQCKHSMKYRLAFCRKTCGTC
ShK-A26 d1
194


 


 SCIDTIPKSRCTAFQCKHSMKYRLSACRKTCGTC
ShK-A27 d1
195


 


 SCIDTIPKSRCTAFQCKHSMKYRLSXs27CRKTCGTC
ShK-X27 d1
196


 


 SCIDTIPKSRCTAFQCKHSMKYRLSFCAKTCGTC
ShK-A29 d1
197


 


 SCIDTIPKSRCTAFQCKHSMKYRLSFCRATCGTC
ShK-A30 d1
198


 


 SCIDTIPKSRCTAFQCKHSMKYRLSFCRKACGTC
ShK-A31 d1
199


 


 SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGAC
ShK-A34 d1
200


 


YSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-Y1
548


 


KSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-K1
549


 


HSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-H1
550


 


QSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-Q1
551


 


PPRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
PP-ShK
552


 


MRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
M-ShK
553


 


GRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
G-ShK
554


 


YSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-Y1/A22
555


 


KSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-K1/A22
556


 


HSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-H1/A22
557


 


QSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-Q1/A22
558


 


PPRSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
PP-ShK-A22
559


 


MRSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
M-ShK-A22
560


 


GRSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
G-ShK-A22
561


 


RSCIDTIPASRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-A9/A22
884


 


SCIDTIPASRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-A9/A22 d1
885


 


RSCIDTIPVSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-V9
886


 


RSCIDTIPVSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-V9/A22
887


 


SCIDTIPVSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-V9 d1
888


 


SCIDTIPVSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-V9/A22 d1
889


 


RSCIDTIPESRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-E9/A22
890


 


SCIDTIPESRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-E9/A22 d1
891


 


RSCIDTIPKSACTAFQCKHSMAYRLSFCRKTCGTC
ShK-A11/A22
892


 


SCIDTIPKSACTAFQCKHSMAYRLSFCRKTCGTC
ShK-A11/22 d1
893


 


RSCIDTIPKSECTAFQCKHSMAYRLSFCRKTCGTC
ShK-E11/A22
894


 


SCIDTIPKSECTAFQCKHSMAYRLSFCRKTCGTC
ShK-E11/A22 d1
895


 


RSCIDTIPKSRCTDFQCKHSMKYRLSFCRKTCGTC
ShK-D14
896


 


RSCIDTIPKSRCTDFQCKHSMAYRLSFCRKTCGTC
ShK-D14/A22
897


 


SCIDTIPKSRCTDFQCKHSMKYRLSFCRKTCGTC
ShK-D14 d1
898


 


SCIDTIPKSRCTDFQCKHSMAYRLSFCRKTCGTC
ShK-D14/A22 d1
899


 


RSCIDTIPKSRCTAAQCKHSMAYRLSFCRKTCGTC
ShK-A15A/22
900


 


SCIDTIPKSRCTAAQCKHSMAYRLSFCRKTCGTC
ShK-A15/A22 d1
901


 


RSCIDTIPKSRCTAIQCKHSMKYRLSFCRKTCGTC
ShK-I15
902


 


RSCIDTIPKSRCTAIQCKHSMAYRLSFCRKTCGTC
ShK-I15/A22
903


 


SCIDTIPKSRCTAIQCKHSMKYRLSFCRKTCGTC
ShK-I15 d1
904


 


SCIDTIPKSRCTAIQCKHSMAYRLSFCRKTCGTC
ShK-I15/A22 d1
905


 


RSCIDTIPKSRCTAVQCKHSMKYRLSFCRKTCGTC
ShK-V15
906


 


RSCIDTIPKSRCTAVQCKHSMAYRLSFCRKTCGTC
ShK-V15/A22
907


 


SCIDTIPKSRCTAVQCKHSMKYRLSFCRKTCGTC
ShK-V15 d1
908


 


SCIDTIPKSRCTAVQCKHSMAYRLSFCRKTCGTC
ShK-V15/A22 d1
909


 


RSCIDTIPKSRCTAFRCKHSMKYRLSFCRKTCGTC
ShK-R16
910


 


RSCIDTIPKSRCTAFRCKHSMAYRLSFCRKTCGTC
ShK-R16/A22
911


 


SCIDTIPKSRCTAFRCKHSMKYRLSFCRKTCGTC
ShK-R16 d1
912


 


SCIDTIPKSRCTAFRCKHSMAYRLSFCRKTCGTC
ShK-R16/A22 d1
913


 


RSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC
ShK-K16
914


 


RSCIDTIPKSRCTAFKCKHSMAYRLSFCRKTCGTC
ShK-K16/A22
915


 


SCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC
ShK-K16 d1
916


 


SCIDTIPKSRCTAFKCKHSMAYRLSFCRKTCGTC
ShK-K16/A22 d1
917


 


RSCIDTIPASECTAFQCKHSMKYRLSFCRKTCGTC
ShK-A9/E11
918


 


RSCIDTIPASECTAFQCKHSMAYRLSFCRKTCGTC
ShK-A9/E11/A22
919


 


SCIDTIPASECTAFQCKHSMKYRLSFCRKTCGTC
ShK-A9/E11 d1
920


 


SCIDTIPASECTAFQCKHSMAYRLSFCRKTCGTC
ShK-A9/E11/A22
921



d1



 


RSCIDTIPVSECTAFQCKHSMKYRLSFCRKTCGTC
ShK-V9/E11
922


 


RSCIDTIPVSECTAFQCKHSMAYRLSFCRKTCGTC
ShK-V9/E11/A22
923


 


SCIDTIPVSECTAFQCKHSMKYRLSFCRKTCGTC
ShK-V9/E11 d1
924


 


SCIDTIPVSECTAFQCKHSMAYRLSFCRKTCGTC
ShK-V9/E11/A22
925



d1



 


RSCIDTIPVSACTAFQCKHSMKYRLSFCRKTCGTC
ShK-V9/A11
926


 


RSCIDTIPVSACTAFQCKHSMAYRLSFCRKTCGTC
ShK-V9/A11/A22
927


 


SCIDTIPVSACTAFQCKHSMKYRLSFCRKTCGTC
ShK-V9/A11 d1
928


 


SCIDTIPVSACTAFQCKHSMAYRLSFCRKTCGTC
ShK-V9/A11/A22
929



d1



 


RSCIDTIPASACTAFQCKHSMKYRLSFCRKTCGTC
ShK-A9/A11
930


 


RSCIDTIPASACTAFQCKHSMAYRLSFCRKTCGTC
ShK-A9/A11/A22
931


 


SCIDTIPASACTAFQCKHSMKYRLSFCRKTCGTC
ShK-A9/A11 d1
932


 


SCIDTIPASACTAFQCKHSMAYRLSFCRKTCGTC
ShK-A9/A11/A22
933



d1



 


RSCIDTIPKSECTDIRCKHSMKYRLSFCRKTCGTC
ShK-
934



E11/D14/I15/R16



 


RSCIDTIPKSECTDIRCKHSMAYRLSFCRKTCGTC
ShK-
935



E11/D14/I15/R16/




A22



 


SCIDTIPKSECTDIRCKHSMKYRLSFCRKTCGTC
ShK-
936



E11/D14/I15/R16




d1



 


SCIDTIPKSECTDIRCKHSMAYRLSFCRKTCGTC
ShK-
937



E11/D14/I15//R16




A22 d1



 


RSCIDTIPVSECTDIRCKHSMKYRLSFCRKTCGTC
ShK-
938



V9/E11/D14/I15/




R16



 


RSCIDTIPVSECTDIRCKHSMAYRLSFCRKTCGTC
ShK-
939



V9/E11/D14/I15/




R16/A22



 


SCIDTIPVSECTDIRCKHSMKYRLSFCRKTCGTC
ShK-
940



V9/E11/D14/I15/




R16 d1



 


SCIDTIPVSECTDIRCKHSMAYRLSFCRKTCGTC
ShK-
941



V9/E11/D14/I15/




R16/A22 d1



 


RSCIDTIPVSECTDIQCKHSMKYRLSFCRKTCGTC
ShK-
942



V9/E11/D14/I15



 


RSCIDTIPVSECTDIQCKHSMAYRLSFCRKTCGTC
ShK-
943



V9/E11/D14/I15/A22



 


SCIDTIPVSECTDIQCKHSMKYRLSFCRKTCGTC
ShK-
944



V9/E11/D14/I15




d1



 


SCIDTIPVSECTDIQCKHSMAYRLSFCRKTCGTC
ShK-
945



V9/E11/D14/I15/A




22 d1



 


RTCKDLIPVSECTDIRCKHSMKYRLSFCRKTCGTC
ShK-
946



T2/K4/L6/V9/E11/




D14/I15/R16



 


RTCKDLIPVSECTDIRCKHSMAYRLSFCRKTCGTC
ShK-
947



T2/K4/L6/V9/E11/




D14/I15/R16/A22



 


TCKDLIPVSECTDIRCKHSMKYRLSFCRKTCGTC
ShK-
948



T2/K4/L6/V9/E11/




D14/I15/R16 d1



 


TCKDLIPVSECTDIRCKHSMAYRLSFCRKTCGTC
ShK-
949



T2/K4/L6/V9/E11/




D14/I15/R16/A22




d1



 


(L-Phosphotyrosine)-
ShK(L5)
950


AEEARSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC




 


QSCADTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC
ShK Q1/A4/A15
1295


 


QSCADTIPKSRCTAAQCKHSMAYRLSFCRKTCGTC
ShK
1296



Q1/A4/A15/A22



 


QSCADTIPKSRCTAAQCKHSM(Dap)YRLSFCRKTCGTC
ShK
1297



Q1/A4/A15/Dap22



 


QSCADTIPKSRCTAAQCKHSMKYRASFCRKTCGTC
ShK
1298



Q1/A4/A15/A25



 


QSCADTIPKSRCTAAQCKHSMAYRASFCRKTCGTC
ShK
1299



Q1/A4/A15/A22/A25



 


QSCADTIPKSRCTAAQCKHSM(Dap)YRASFCRKTCGTC
ShK
1300



Q1/A4/A15/Dap22/




A25









Many peptides as described in Table 2 can be prepared as described in U.S. Pat. No. 6,077,680 issued Jun. 20, 2000 to Kem et al., which is hereby incorporated by reference in its entirety. Other peptides of Table 2 can be prepared by techniques known in the art. For example, ShK(L5) (SEQ ID NO: 950) can be prepared as described in Beeton et al., Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases, Molec. Pharmacol. 67(4): 1369-81 (2005), which is hereby incorporated by reference in its entirety. In Table 2 and throughout the specification, Xs15, Xs21, Xs22, Xs23 and Xs27 each independently refer to nonfunctional amino acid residues.







TABLE 3







HmK, BgK, AeK and AsKS peptide and peptide


analog sequences










Short-




hand
SEQ



desig-
ID


Sequence/structure
nation
NO:












RTCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC
HmK
6


 


ATCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC
HmK-A1
201


 


STCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC
HmK-S1
202


 


 TCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC
HmK-d1
203


 


 SCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC
HmK-
204



d1/S2



 


 TCIDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC
HmK-
205



d1/I4



 


 TCKDTIPVSECTDIRCRTSMKYRLNLCRKTCGSC
HmK-
206



d1/T6



 


 TCKDLIPKSECTDIRCRTSMKYRLNLCRKTCGSC
HmK-
207



d1/K9



 


 TCKDLIPVSRCTDIRCRTSMKYRLNLCRKTCGSC
HmK-
208



d1/R11



 


 TCKDLIPVSECTAIRCRTSMKYRLNLCRKTCGSC
HmK-
209



d1/A14



 


 TCKDLIPVSECTDFRCRTSMKYRLNLCRKTCGSC
HmK-
210



d1/F15



 


 TCKDLIPVSECTDIQCRTSMKYRLNLCRKTCGSC
HmK-
211



d1/Q16



 


 TCKDLIPVSECTDIRCKTSMKYRLNLCRKTCGSC
HmK-
212



d1/K18



 


 TCKDLIPVSECTDIRCRHSMKYRLNLCRKTCGSC
HmK-
213



d1/H19



 


 TCKDLIPVSECTDIRCRTSMKYRLSLCRKTCGSC
HmK-
214



d1/S26



 


 TCKDLIPVSECTDIRCRTSMKYRLNFCRKTCGSC
HmK-
215



d1/F27



 


 TCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGTC
HmK-
216



d1/T34



 


 TCKDLIPVSRCTDIRCRTSMKYRLNFCRKTCGSC
HmK-
217



d1/R11/




F27



 


ATCKDLIPVSRCTDIRCRTSMKYRLNFCRKTCGSC
HmK-
218



A1/R11/




F27



 


 TCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGSC
HmK-
219



d1/Z1



 


 TCIDTIPKSRCTAFQCRTSMKYRLNFCRKTCGSC
HmK-
220



d1/Z2



 


 TCADLIPASRCTAIACRTSMKYRLNFCRKTCGSC
HmK-
221



d1/Z3



 


 TCADLIPASRCTAIACKHSMKYRLNFCRKTCGSC
HmK-
222



d1/Z4



 


 TCADLIPASRCTAIACAHSMKYRLNFCRKTCGSC
HmK-
223



d1/Z5



 


RTCKDLIPVSECTDIRCRTSMXh22YRLNLCRKTCGSC
HmK-X22
224


 


ATCKDLXh6PVSRCTDIRCRTSMKXh22RLNXh26CR
HmK-X6,
225


KTCGSC
22, 26



 


VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCELC
BgK
9


 


ACRDWFKETACRHAKSLGNCRTSQKYRANCAKTCELC
BgK-A1
226


 


VCADWFKETACRHAKSLGNCRTSQKYRANCAKTCELC
BgK-A3
227


 


VCRDAFKETACRHAKSLGNCRTSQKYRANCAKTCELC
BgK-A5
228


 


VCRDWFKATACRHAKSLGNCRTSQKYRANCAKTCELC
BgK-A8
229


 


VCRDWFKEAACRHAKSLGNCRTSQKYRANCAKTCELC
BgK-A9
230


 


VCRDWFKETACAHAKSLGNCRTSQKYRANCAKTCELC
BgK-A12
231


 


VCRDWFKETACRHAASLGNCRTSQKYRANCAKTCELC
BgK-A15
232


 


VCRDWFKETACRHAKALGNCRTSQKYRANCAKTCELC
BgK-A16
233


 


VCRDWFKETACRHAKSAGNCRTSQKYRANCAKTCELC
BgK-A17
234


 


VCRDWFKETACRHAKSLGNCATSQKYRANCAKTCELC
BgK-A21
235


 


VCRDWFKETACRHAKSLGNCRASQKYRANCAKTCELC
BgK-A22
236


 


VCRDWFKETACRHAKSLGNCRTSQKYAANCAKTCELC
BgK-A27
237


 


VCRDWFKETACRHAKSLGNCRTSQKYRANCAATCELC
BgK-A32
238


 


VCRDWFKETACRHAKSLGNCRTSQKYRANCAKACELC
BgK-A33
239


 


VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCALC
BgK-A35
240


 


VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCEAC
BgK-A37
241


 


GCKDNFSANTCKHVKANNNCGSQKYATNCAKTCGKC
AeK
7


 


ACKDNFAAATCKHVKENKNCGSQKYATNCAKTCGKC
AsKS
8









In Table 3 and throughout the specification, Xh6, Xh22, Xh26 are each independently nonfunctional residues.







TABLE 4







MgTx peptide and MgTx peptide analog sequences










Short-




hand
SEQ



desig-
ID


Sequence/structure
nation
NO:












TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx
28


 


TIINVACTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-
242



A6



 


TIINVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-
243



S6



 


TIINVKCTSPAQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-
244



A11



 


TIINVKCTSPKQCLPPCAAQFGQSAGAKCMNGKCKCYPH
MgTx-
245



A18



 


TIINVKCTSPKQCLPPCKAQFGQSAGAACMNGKCKCYPH
MgTx-
246



A28



 


TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGACKCYPH
MgTx-
247



A33



 


TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCACYPH
MgTx-
248



A35



 


TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPN
MgTx-
249



H39N



 


TIINVACTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPN
MgTx-
250



A6/




H39N



 


TIINVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYS
MgTx-
251



S6/




38/d39



 


TIITISCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-
252



T4/I5/




S6



 


   TISCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-
253



d3/T4/




I5/S6



 


   TISCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCFGR
MgTx-
254



Pi2



 


   NVACTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-
255



d3/A6



 


QFTNVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYS
MgTx-
256



ChTx



 


QFTDVDCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYQ
MgTx-
257



IbTx



 


 IINVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-
258



Z1



 


 IITISCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-
259



Z2



 


GVIINVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCY
MgTx-
260


PH
Z3









Many peptides as described in Table 4 can be prepared as described in WO 95/03065, published Feb. 2, 1995, for which the applicant is Merck & Co., Inc. That application corresponds to U.S. Ser. No. 07/096,942, filed 22 Jul. 1993, which is hereby incorporated by reference in its entirety.







TABLE 5







AgTx2 peptide and AgTx2 peptide analog


sequences










Short-




hand
SEQ



desig-
ID


Sequence/structure
nation
NO:












GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK
AgTx2
23


 


GVPIAVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK
AgTx2-
261



A5



 


GVPINVSCTGSPQCIAPCKDAGMRFGKCMNRKCHCTPK
AgTx2-
262



A16



 


GVPINVSCTGSPQCIKPCADAGMRFGKCMNRKCHCTPK
AgTx2-
263



A19



 


GVPINVSCTGSPQCIKPCKDAGMAFGKCMNRKCHCTPK
AgTx2-
264



A24



 


GVPINVSCTGSPQCIKPCKDAGMRFGACMNRKCHCTPK
AgTx2-
265



A27



 


GVPINVSCTGSPQCIKPCKDAGMRFGKCMNAKCHCTPK
AgTx2-
266



A31



 


GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRACHCTPK
AgTx2-
267



A32



 


GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPA
AgTx2-
268



A38



 


GVPIAVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPA
AgTx2-
269



A5/A38



 


GVPINVSCTGSPQCIKPCKDAGMRFGKCMNGKCHCTPK
AgTx2-
270



G31



 


GVPIIVSCKGSRQCIKPCKDAGMRFGKCMNGKCHCTPK
AgTx2-
271



OSK_z1



 


GVPIIVSCKISRQCIKPCKDAGMRFGKCMNGKCHCTPK
AgTx2-
272



OSK_z2



 


GVPIIVKCKGSRQCIKPCKDAGMRFGKCMNGKCHCTPK
AgTx2-
273



OSK_z3



 


GVPIIVKCKISRQCIKPCKDAGMRFGKCMNGKCHCTPK
AgTx2-
274



OSK_z4



 


GVPIIVKCKISRQCIKPCKDAGMRFGKCMNGKCHCTPK
AgTx2-
275



OSK_z5
















TABLE 6







Heteromitrus spinnifer (HsTx1) peptide and HsTx1


peptide analog sequences











SEQ



Short-hand
ID


Sequence/structure
designation
NO:












ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNRC
HsTx1
61


 


ASCXTPKDCADPCRKETGCPYGKCMNRKCKCNRC
HsTx1-X4
276


 


ASCATPKDCADPCRKETGCPYGKCMNRKCKCNRC
HsTx1-A4
277


 


ASCRTPXDCADPCRKETGCPYGKCMNRKCKCNRC
HsTx1-X7
278


 


ASCRTPADCADPCRKETGCPYGKCMNRKCKCNRC
HsTx1-A7
279


 


ASCRTPKDCADPCXKETGCPYGKCMNRKCKCNRC
HsTx1-X14
280


 


ASCRTPKDCADPCAKETGCPYGKCMNRKCKCNRC
HsTx1-A14
281


 


ASCRTPKDCADPCRXETGCPYGKCMNRKCKCNRC
HsTx1-X15
282


 


ASCRTPKDCADPCRAETGCPYGKCMNRKCKCNRC
HsTx1-A15
283


 


ASCRTPKDCADPCRKETGCPYGXCMNRKCKCNRC
HsTx1-X23
284


 


ASCRTPKDCADPCRKETGCPYGACMNRKCKCNRC
HsTx1-A23
285


 


ASCRTPKDCADPCRKETGCPYGKCMNXKCKCNRC
HsTx1-X27
286


 


ASCRTPKDCADPCRKETGCPYGKCMNAKCKCNRC
HsTx1-A27
287


 


ASCRTPKDCADPCRKETGCPYGKCMNRXCKCNRC
HsTx1-X28
288


 


ASCRTPKDCADPCRKETGCPYGKCMNRACKCNRC
HsTx1-A28
289


 


ASCRTPKDCADPCRKETGCPYGKCMNRKCXCNRC
HsTx1-X30
290


 


ASCRTPKDCADPCRKETGCPYGKCMNRKCACNRC
HsTx1-A30
291


 


ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNXC
HsTx1-X33
292


 


ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNAC
HsTx1-A33
293









Peptides as described in Table 5 can be prepared as described in U.S. Pat. No. 6,689,749, issued Feb. 10, 2004 to Lebrun et al., which is hereby incorporated by reference in its entirety.







TABLE 7







Orthochirus scrobiculosus (OSK1) peptide and OSK1 peptide analog sequences











SEQ



Short-hand
ID


Sequence/structure
designation
NO:












GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK
OSK1
25


 


GVIINVSCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK
OSK1-S7
1303


 


GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK
OSK1-K16
294


 


GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK
OSK1-D20
295


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
OSK1-K16, D20
296


 


GVIINVSCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
OSK1-S7, K16, D20
1308


 


GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK
OSK1-P12, K16, D20
297


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK
OSK1-K16, D20, Y36
298


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-OSK1-P12,
562



K16, D20



 


GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-P12, K16,
563



D20-NH2



 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-OSK1-P12,
564



K16, D20-NH2



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK-NH2
OSK1-K16, D20,
565



Y36-NH2



 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK
Ac-OSK1-K16,
566



D20, Y36



 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK-NH2
Ac-OSK1-K16, D20,
567



Y36-NH2



 


GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK-NH2
OSK1-K16-NH2
568


 


Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK
Ac-OSK1-K16
569


 


Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK-NH2
Ac-OSK1-K16-NH2
570


 


Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK
Ac-OSK1-D20
571


 


GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-D20-NH2
572


 


Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-OSK1-D20-NH2
573


 


GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH2
OSK1-NH2
574


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK
Ac-OSK1
575


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH2
Ac-OSK1-NH2
576


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-K16, D20-NH2
577


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-OSK1-K16,
578



D20



 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-OSK1-K16, D20-
579



NH2



 


VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK
Δ1-OSK1
580


 


Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK
Ac-Δ1-OSK1
581


 


VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH2
Δ1-OSK1-NH2
582


 


Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH2
Ac-Δ1-OSK1-NH2
583


 


GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK
OSK1-A34
584


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK
Ac-OSK1-A34
585


 


GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH2
OSK1-A34-NH2
586


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH2
Ac-OSK1-A34-NH2
587


 


VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Δ1-OSK1-K16, D20
588


 


Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-Δ1-OSK1-K16,
589



D20



 


VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Δ1-OSK1-K16, D20-
590



NH2



 


Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-Δ1-OSK1-K16,
591



D20-NH2



 


NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
(Δ1-4)-OSK1-K16,
592



D20



 


Ac-NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-(Δ1-4)-OSK1-
593



K16, D20



 


NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
(Δ1-4)-OSK1-K16,
594



D20-NH2



 


Ac-NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-(Δ1-4)-OSK1-
595



K16, D20-NH2



 


KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
(Δ1-6)-OSK1-K16,
596



D20



 


Ac-KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-(Δ1-6)-OSK1-
597



K16, D20



 


KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
(Δ1-6)-OSK1-K16,
598



D20-NH2



 


Ac-KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-(Δ1-6)-OSK1-
599



K16, D20-NH2



 


CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
(Δ1-7)-OSK1-K16,
600



D20



 


Ac-CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-(Δ1-7)-OSK1-
601



K16, D20



 


CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
(Δ1-7)-OSK1-K16,
602



D20-NH2



 


Ac-CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-(Δ1-7)-OSK1-
603



K16, D20-NH2



 


GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK
OSK1-K16, D20,
604



N25



 


GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK-NH2
OSK1-K16, D20,
605



N25-NH2



 


Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK
Ac-OSK1-K16,
606



D20, N25



 


Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK-NH2
Ac-OSK1-K16, D20,
607



N25-NH2



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
OSK1-K16, D20,
608



R31



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-NH2
OSK1-K16, D20,
609



R31-NH2



 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
Ac-OSK1-K16,
610



D20, R31



 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-NH2
Ac-OSK1-K16, D20,
611



R31-NH2



 


GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK
OSK1-K12, K16,
612



R19, D20



 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK
Ac-OSK1-K12, K16,
613



R19, D20



 


GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-NH2
OSK1-K12, K16,
614



R19, D20-NH2



 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-NH2
Ac-OSK1-K12, K16,
615



R19, D20-NH2



 


TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Δ1-OSK1-T2, K16,
616



D20



 


Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-Δ1-OSK1-T2,
617



K16, D20



 


TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Δ1-OSK1-T2, K16,
618



D20-NH2



 


Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-Δ1-OSK1-T2,
619



K16, D20-NH2



 


GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK
OSK1-K3
620


 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK
Ac-OSK1-K3
621


 


GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH2
OSK1-K3-NH2
622


 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH2
Ac-OSK1-K3-NH2
623


 


GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK
OSK1-K3, A34
624


 


GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
OSK1-K3, K16, D20
625


 


GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK
OSK1-K3, K16, D20,
626



A34



 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK
Ac-OSK1-K3, A34
627


 


GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH2
OSK1-K3, A34-NH2
628


 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH2
Ac-OSK1-K3, A34-
629



NH2



 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK
Ac-OSK1-K3, K16,
630



D20, A34



 


GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK-NH2
OSK1-K3, K16, D20,
631



A34-NH2



 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK-NH2
Ac-OSK1-K3, K16,
632



D20, A34-NH2



 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-OSK1-K3, K16,
633



D20



 


GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-K3, K16, D20-
634



NH2



 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-OSK1-K3, K16,
635



D20-NH2



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCT
Δ36-38-OSK1-K16,
636



D20



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCTPK
OSK1-O16, D20
980


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK
OSK1-hLys
981



16, D20



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK
OSK1-hArg
982



16, D20



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK
OSK1-Cit 16, D20
983


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK
OSK1-hCit
984



16, D20



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK
OSK1-Dpr 16, D20
985


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK
OSK1-Dab 16, D20
986


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYPK
OSK1-O16, D20, Y36
987


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK
OSK1-hLys
988



16, D20, Y36



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK
OSK1-hArg
989



16, D20, Y36



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK
OSK1-Cit
990



16, D20, Y36



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK
OSK1-hCit
991



16, D20, Y36



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK
OSK1-Dpr
992



16, D20, Y36



 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK
OSK1-Dab
993



16, D20, Y36



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYPK
OSK1-
994



K16, D20, A34, Y36



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCGCYPK
OSK1-
995



K16, D20, G34, Y36



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACFPK
OSK1-
996



K16, D20, A34, F36



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACWPK
OSK1-
997



K16, D20, A34, W36



 


GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYPK
OSK1-
998



K16, E20, A34, Y36



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACTPK
OSK1-O16, D20, A34
999


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACTPK
OSK1-hLys
1000



16, D20, A34



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACTPK
OSK1-hArg
1001



16, D20, A34



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACTPK
OSK1-Cit
1002



16, D20, A34



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK
OSK1-hCit
1003



16, D20, A34



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK
OSK1-Dpr
1004



16, D20, A34



 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACTPK
OSK1-Dab
1005



16, D20, A34



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1006



O16, D20,



 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1007



hLys 16, D20



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1008



hArg 16, D20



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-Cit
1009



16, D20



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1010



hCit 16, D20



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-Dpr
1011



16, D20



 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1012



Dab16, D20



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCAC
Δ36-38, OSK1-
1013



O16, D20, A34



 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCAC
Δ36-38, OSK1-
1014



hLys 16, D20, A34



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCAC
Δ36-38, OSK1-
1015



hArg 16, D20, A34



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCAC
Δ36-38, OSK1-Cit
1016



16, D20, A34



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1017



hCit 16, D20, A34



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCAC
Δ36-38, OSK1-Dpr
1018



16, D20, A34



 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCAC
Δ36-38, OSK1-Dab
1019



16, D20, A34



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCGCYGG
OSK1-
1020



K16, D20, G34, Y36, G37,




G38



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYGG
OSK1-
1021



O16, D20, Y36, G37, G38



 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG
OSK1-hLys
1022



16, D20, Y36, G37, G38



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG
OSK1-hArg
1023



16, D20, Y36, G37, G38



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG
OSK1-Cit
1024



16, D20, Y36, G37, G38



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG
OSK1-hCit
1025



16, D20, Y36, G37, G38



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG
OSK1-Dpr
1026



16, D20, Y36, G37, G38



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYGG
OSK1-
1027



K16, D20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACYGG
OSK1-
1028



O16, D20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACYGG
OSK1-hLys
1029



16, D20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACYGG
OSK1-hArg
1030



16, D20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACYGG
OSK1-Cit
1031



16, D20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG
OSK1-hCit
1032



16, D20, A34, Y3, G37,




G38



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG
OSK1-Dpr
1033



16, D20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACYGG
OSK1-Dab
1034



16, D20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYG
Δ38, OSK1-
1035



K16, D20, A34, Y36, G37



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCGGG
OSK1-
1036



O16, D20, G36-38



 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG
OSK1-hLys
1037



16, D20, G36-38



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG
OSK1-hArg
1038



16, D20, G36-38



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG
OSK1-Cit
1039



16, D20, G36-38



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG
OSK1-hCit
1040



16, D20, G36-38



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG
OSK1-Dpr
1041



16, D20, G36-38



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACFGG
OSK1-
1042



K16, D20, A34, F36, G37,




G38



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACGGG
OSK1-
1043



O16, D20, A34, G36-




38



 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACGGG
OSK1-hLys
1044



16, D20, A34, G36-38



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACGGG
OSK1-hArg
1045



16, D20, A34, G36-38



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACGGG
OSK1-Cit
1046



16, D20, A34, G36-38



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACGGG
OSK1-hCit
1047



16, D20, A34, G36-38



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG
OSK1-Dpr
1048



16, D20, A34, G36-38



 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACGGG
OSK1-Dab
1049



16, D20, A34, G36-38



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACGG
Δ38, OSK1-
1050



K16, D20, A34, G36-




37



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYG
Δ38, OSK1-
1051



K16, D20, A35, Y36, G37



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACGG
Δ38, OSK1-
1052



O16, D20, A35, Y36,




G37



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK
OSK1-hLys
1053



16, E20



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK
OSK1-hArg
1054



16, E20



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK
OSK1-Cit 16, E20
1055


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK
OSK1-hCit
1056



16, E20



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK
OSK1-Dpr 16, E20
1057


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK
OSK1-Dab 16, E20
1058


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYPK
OSK1-O16, E20, Y36
1059


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK
OSK1-hLys
1060



16, E20, Y36



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK
OSK1-hArg
1061



16, E20, Y36



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK
OSK1-Cit
1062



16, E20, Y36



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK
OSK1-hCit
1063



16, E20, Y36



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK
OSK1-Dpr
1064



16, E20, Y36



 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK
OSK1-Dab
1065



16, E20, Y36



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACTPK
OSK1-O16, E20, A34
1066


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACTPK
OSK1-hLys
1067



16, E20, A34



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACTPK
OSK1-hArg
1068



16, E20, A34



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACTPK
OSK1-Cit
1069



16, E20, A34



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK
OSK1-hCit
1070



16, E20, A34



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK
OSK1-Dpr
1071



16, E20, A34



 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACTPK
OSK1-Dab
1072



16, E20, A34



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1073



O16, E20,



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1074



hLys 16, E20



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1075



hArg 16, E20



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHC
Δ36-38, OSK1-Cit
1076



16, E20



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1077



hCit16, E20



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHC
Δ36-38, OSK1-Dpr
1078



16, E20



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCAC
Δ36-38, OSK1-
1079



O16, E20, A34



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCAC
Δ36-38, OSK1-
1080



hLys 16, E20, A34



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCAC
Δ36-38, OSK1-
1081



hArg 16, E20, A34



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCAC
Δ36-38, OSK1-Cit
1082



16, E20, A34



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1083



hCit 16, E20, A34



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCAC
Δ36-38, OSK1-Dpr
1084



16, E20, A34



 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCAC
Δ36-38, OSK1-Dab
1085



16, E20, A34



 


GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCHCYGG
OSK1-
1086



K16, E20, Y36, G37, G38



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYGG
OSK1-
1087



O16, E20, Y36, G37, G38



 


GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCHCYG
Δ38 OSK1-
1088



K16, E20, Y36, G37



 


GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYG
Δ38 OSK1-
1089



K16, E20, A34,




Y36, G37



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG
OSK1-hLys
1090



16, E20, Y36, G37, G38



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG
OSK1-hArg
1091



16, E20, Y36, G37, G38



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG
OSK1-Cit
1092



16, E20, Y36, G37, G38



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG
Δ37-38, OSK1-
1093



hCit




16, E20, Y36, G37, G38



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG
OSK1-Dpr
1094



16, E20, Y36, G37, G38



 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG
OSK1-Dab
1095



16, E20, Y36, G37, G38



 


GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYG
Δ38, OSK1-
1096



K16, E20, A34, Y36, G37



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACYGG
OSK1-
1097



O16, E20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACYGG
OSK1-hLys
1098



16, E20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACYGG
OSK1-hArg
1099



16, E20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACYGG
OSK1-Cit
1100



16, E20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG
OSK1-hCit
1101



16, E20, A34, Y3, G37,




G38



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG
OSK1-Dpr
1102



16, E20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACYGG
OSK1-Dab
1103



16, E20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACFGG
OSK1-
1104



K16, D20, A34, F36, G37,




G38



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCGGG
OSK1-
1105



O16, E20, G36-38



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG
OSK1-hLys
1106



16, E20, G36-38



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG
OSK1-hArg
1107



16, E20, G36-38



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG
OSK1-Cit
1108



16, E20, G36-38



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG
OSK1-hCit
1109



16, E20, G36-38



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG
OSK1-Dpr
1110



16, E20, G36-38



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACGGG
OSK1-
1111



O16, E20, A34, G36-




38



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACGGG
OSK1-hLys
1112



16, E20, A34, G36-38



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACGGG
OSK1-hArg
1113



16, E20, A34, G36-38



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACGGG
OSK1-Cit
1114



16, E20, A34, G36-38



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCACTP
OSK1-hCit
1115



16, E20, A34, G36-38



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACTP
OSK1-Dpr
1116



16, E20, A34, G36-38



 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACTP
OSK1-Dab
1117



16, E20, A34, G36-38



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-O16, D20-
1118



amide



 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK-
OSK1-hLys
1119


NH2
16, D20-amide



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK-
OSK1-hArg
1120


NH2
16, D20-amide



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-Cit
1121



16, D20-amide



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK-
OSK1-hCit
1122


NH2
16, D20-amide



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-Dpr
1123



16, D20-amide



 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-Dab 16, D20
1124


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYPK-NH2
OSK1-
1125



O16, D20, Y36-




amide



 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK-
OSK1-hLys
1126


NH2
16, D20, Y36-




amide



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK-
OSK1-hArg
1127


NH2
16, D20, Y36-




amide



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK-NH2
OSK1-Cit
1128



16, D20, Y36-




amide



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK-
OSK1-hCit
1129


NH2
16, D20, Y36-




amide



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK-NH2
OSK1-Dpr
1130



16, D20, Y36-




amide



 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK-NH2
OSK1-Dab
1131



16, D20, Y36-




amide



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACTPK-NH2
OSK1-
1132



O16, D20, A34-




amide



 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACTPK-
OSK1-hLys
1133


NH2
16, D20, A34-




amide



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACTPK-
OSK1-hArg
1134


NH2
16, D20, A34-




amide



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACTPK-NH2
OSK1-Cit
1135



16, D20, A34-




amide



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACTPK-
OSK1-hCit
1136


NH2
16, D20, A34-




amide



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK-NH2
OSK1-Dpr
1137



16, D20, A34-




amide



 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACTPK-NH2
OSK1-Dab
1138



16, D20, A34-




amide



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1139



O16, D20, -




amide



 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1140



hLys 16, D20-




amide



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1141



hArg 16, D20-




amide



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-Cit
1142



16, D20-amide



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1143



hCit16, D20-




amide



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-Dpr
1144



16, D20-amide



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-
1145



O16, D20, A34-




amide



 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-
1146



hLys 16, D20, A34-




amide



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-
1147



hArg 16, D20, A34-




amide



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-Cit
1148



16, D20, A34-




amide



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1149



hCit16, D20, A34



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-Dpr
1150



16, D20, A34-




amide



 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-Dab
1151



16, D20, A34-




amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYGG-NH2
OSK1-
1152



O16, D20, Y36, G37, G38-




amide



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYGG-NH2
OSK1-
1153



O16, D20, Y36, G37, G38



 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG-
OSK1-hLys
1154


NH2
16, D20, Y36, G37, G38-




amide



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG-
OSK1-hArg
1155


NH2
16, D20, Y36, G37, G38-




amide



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG-NH2
OSK1-Cit
1156



16, D20, Y36, G37, G38-




amide



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG-
OSK1-
1157


NH2
hCit16, D20, Y36, G37,




G38-amide



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG-NH2
OSK1-Dpr
1158



16, D20, Y36, G37, G38-




amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCFGG-NH2
OSK1-
1159



K16, D20, F36, G37, G38-




amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYG-NH2
Δ38-OSK1-
1160



K16, D20, Y36, G37-




amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYG-NH2
Δ38-OSK1-
1161



K16, D20, A34,




Y36, G37-amide



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACYGG-NH2
OSK1-
1162



O16, D20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACYGG-
OSK1-hLys
1163


NH2
16, D20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACYGG-
OSK1-hArg
1164


NH2
16, D20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACYGG-NH2
OSK1-Cit
1165



16, D20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACYGG-
OSK1-
1166


NH2
hCit16, D20, A34, Y3,




G37, G38-amide



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG-NH2
OSK1-Dpr
1167



16, D20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACYGG-NH2
OSK1-Dab
1168



16, D20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYGG-NH2
OSK1-
1169



K16, D20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCGGG-NH2
OSK1-
1170



O16, D20, G36-38-




amide



 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG-
OSK1-hLys
1171


NH2
16, D20, G36-38-




amide



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG-
OSK1-hArg
1172


NH2
16, D20, G36-38-




amide



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG-NH2
OSK1-Cit
1173



16, D20, G36-38-




amide



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG-
OSK1-
1174


NH2
hCit16, D20, G36-




38-amide



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG-NH2
OSK1-Dpr
1175



16, D20, G36-38-




amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACGGG-NH2
OSK1-
1176



K16, D20, A34, G36-




38-amide



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACFGG-NH2
OSK1-
1177



O16, D20, A34, F36,




G37-38-amide



 


GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACGGG-NH2
OSK1-
1178



O16, D20, A34, G36-




38-amide



 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACGGG-
OSK1-hLys
1179


NH2
16, D20, A34, G36-




38-amide



 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACGGG-
OSK1-hArg
1180


NH2
16, D20, A34, G36-




38-amide



 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACGGG-NH2
OSK1-Cit
1181



16, D20, A34, G36-




38-amide



 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACGGG-
OSK1-
1182


NH2
hCit16, D20, A34, G36-




38-amide



 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG-NH2
OSK1-Dpr
1183



16, D20, A34, G36-




38-amide



 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACGGG-NH2
OSK1-Dab
1184



16, D20, A34, G36-




38-amide



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCTPK-NH2
OSK1-O16, E20-
1185



amide



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK-
OSK1-hLys
1186


NH2
16, E20-amide



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK-
OSK1-hArg
1187


NH2
16, E20-amide



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK-NH2
OSK1-Cit
1188



16, E20-amide



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK-
OSK1-
1189


NH2
hCit16, E20-




amide



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK-NH2
OSK1-Dpr
1190



16, E20-amide



 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK-NH2
OSK1-Dab
1191



16, E20-amide



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYPK-NH2
OSK1-
1192



O16, E20, Y36-




amide



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK-
OSK1-hLys
1193


NH2
16, E20, Y36-




amide



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK-
OSK1-hArg
1194


NH2
16, E20, Y36-




amide



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK-NH2
OSK1-Cit
1195



16, E20, Y36-




amide



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK-
OSK1-
1196


NH2
hCit16, E20, Y36-




amide



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK-NH2
OSK1-Dpr
1197



16, E20, Y36-




amide



 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK-NH2
OSK1-Dab
1198



16, E20, Y36-




amide



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACTPK-NH2
OSK1-
1199



O16, E20, A34-




amide



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACTPK-
OSK1-hLys
1200


NH2
16, E20, A34-




amide



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACTPK-
OSK1-hArg
1201


NH2
16, E20, A34-




amide



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACTPK-NH2
OSK1-Cit
1202



16, E20, A34-




amide



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCACTPK-
OSK1-
1203


NH2
hCit16, E20, A34-




amide



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK-NH2
OSK1-Dpr
1204



16, E20, A34-




amide



 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACTPK-NH2
OSK1-Dab
1205



16, E20, A34-




amide



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1206



O16, E20, -




amide



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1207



hLys 16, E20-




amide



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1208



hArg 16, E20-




amide



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-Cit
1209



16, E20-amide



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1210



hCit16, E20-




amide



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-Dpr
1211



16, E20-amide



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-
1212



O16, E20, A34-




amide



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-
1213



hLys16, E20, A34-




amide



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-
1214



hArg16, E20, A34-




amide



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-Cit
1215



16, E20, A34-




amide



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1216



hCit16, E20, A34-




amide



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-Dpr
1217



16, E20, A34-




amide



 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-Dab
1218



16, E20, A34-




amide



 


GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCHCWGG-NH2
OSK1-
1219



O16, E20, W36, G37,




G38-amide



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYGG-NH2
OSK1-
1220



O16, E20, Y36, G37, G38-




amide



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG-
OSK1-hLys
1221


NH2
16, E20, Y36, G37, G38-




amide



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG-
OSK1-hArg
1222


NH2
16, E20, Y36, G37, G38-




amide



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG-NH2
OSK1-Cit
1223



16, E20, Y36, G37, G38-




amide



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG-
OSK1-hCit
1224


NH2
16, E20, Y36, G37, G38-




amide



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG-NH2
OSK1-Dpr
1225



16, E20, Y36, G37, G38-




amide



 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG-NH2
OSK1-Dpr
1226



16, E20, Y36, G37, G38-




amide



 


GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYGG-NH2
OSK1-
1227



K16, E20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACYGG-NH2
OSK1-
1228



O16, E20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACYGG-
OSK1-hLys
1229


NH2
16, E20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACYGG-
OSK1-hArg
1230


NH2
16, E20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACYGG-NH2
OSK1-Cit
1231



16, E20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG-
OSK1-hCit
1232


NH2
16, E20, A34, Y3, G37,




G38-amide



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG-NH2
OSK1-Dpr
1233



16, E20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACYGG-NH2
OSK1-Dab
1234



16, E20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCGGG-NH2
OSK1-
1235



O16, E20, G36-38-




amide



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG-
OSK1-hLys
1236


NH2
16, E20, G36-38-




amide



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG-
OSK1-hArg
1237


NH2
16, E20, G36-38-




amide



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG-NH2
OSK1-Cit
1238



16, E20, G36-38-




amide



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG-
OSK1-hCit
1239


NH2
16, E20, G36-38-




amide



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG-NH2
OSK1-Dpr
1240



16, E20, G36-38-




amide



 


GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACGGG-NH2
OSK1-
1241



O16, E20, A34, G36-




38-amide



 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACGGG-
OSK1-hLys
1242


NH2
16, E20, A34, G36-




38-amide



 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACGGG-
OSK1-hArg
1243


NH2
16, E20, A34, G36-




38-amide



 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACGGG-NH2
OSK1-Cit
1244



16, E20, A34, G36-




38-amide



 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCACTP-NH2
Δ38 OSK1-hCit
1245



16, E20, A34-




amide



 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACGGG-NH2
OSK1-Dpr
1246



16, E20, A34, G36-




38-amide



 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACGGG-NH2
OSK1-Dab
1247



16, E20, A34, G36-




38-amide



 


GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMNGKCACYGG-NH2
OSK1-K
1248



16, CPA20, A34, Y36,




G37, G38-amide



 


GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMNGKCACGGG-NH2
OSK1-K
1249



16, CPA20, A34, G36-




38-amide



 


GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMNGKCACY-NH2
Δ37-38OSK1-K
1250



16, CPA20, A34, Y36-




amide



 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYGG-NH2
Acetyl-OSK1-K
1251



16, D20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMNGKCACYGG-NH2
OSK1-K 16,
1252



Aad20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMNGKCHCYGG-NH2
OSK1-K 16,
1253



Aad20, Y36, G37, G38-




amide



 


GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMNGKCACYGG
OSK1-K 16,
1254



Aad20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACYGG-NH2
OSK1-H
1255



16, D20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACYGG
OSK1-H
1256



16, D20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACY-NH2
Δ37-38-OSK1-H
1257



16, D20, A34, Y36-




amide



 


GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCHCYGG-NH2
OSK1-H
1258



16, D20, A34, Y36, G37,




G38-amide



 


GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCHCYGG
OSK1-H
1259



16, D20, A34, Y36, G37,




G38



 


GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCHCYPK
OSK1-H
1260



16, D20, A34, Y36,



 


GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCAC
Δ36-38 OSK1-H
1261



16, D20, A34, Y36,



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[1Nal]GG-
OSK1-K
1262


NH2
16, D20, A34, 1Nal36,




G37, G38-amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[1Nal]PK-
OSK1-K
1263


NH2
16, D20, A34, 1Nal36-




amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[2Nal]GG-
OSK1-K
1264


NH2
16, D20, A34, 2Nal36,




G37, G38-amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[Cha]GG-NH2
OSK1-K
1265



16, D20, A34, Cha36,




G37, G38-amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[MePhe]GG-
OSK1-K
1266


NH2
16, D20, A34,




MePhe36, G37, G38-




amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[BiPhA]GG-
OSK1-K
1267


NH2
16, D20, A34,




BiPhA36, G37, G38-




amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKC[Aib]CYGG-NH2
OSK1-K 16, D20,
1268



Aib34, Y36, G37, G38-




amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKC[Abu]CYGG-NH2
OSK1-K 16, D20,
1269



Abu34, Y36, G37, G38-




amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[1Nal]
Δ37-38 OSK1-H
1270



16, D20, A34, 1Nal36, -




amide



 


GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCAC[1Nal]GG-
OSK1-H
1271


NH2
16, D20, A34,




1Nal36, G37, G38-




amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[4Bip]-NH2
Δ37-38 OSK1-H
1272



16, D20, A34, 4Bip




36, -amide



 


GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCAC[4Bip]GG-
OSK1-H
1273


NH2
16, D20, A34, 4Bip




36, G37, G38-




amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCGGG
OSK1-K16, E20, G36-
1274



38
















TABLE 7A







Additional useful OSK1 peptide analog sequences








SEQ ID



NO
Sequence





1391
GVIINVKCKISAQCLKPCRDAGMRFGKCMNGKCACTPK


 


1392
GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK


 


1393
GVIINVKCKISPQCLKPCKDAGIRFGKCINGKCACTPK


 


1394
GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACTPK


 


1395
GGGGSGVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1396
GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHC


 


1397
GVIINVKCKISPQCLOPCKEAGMRFGKCMNGKCHCTY[Nle]


 


1398
GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTY[Nle]


 


1399
GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCGGG


 


1400
AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1401
GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1402
GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1403
GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1404
GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1405
GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1406
GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1407
GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1408
GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1409
GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1410
GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1411
GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCHCTPK


 


1412
GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCHCTPK


 


1413
GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCHCTPK


 


1414
GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCHCTPK


 


1415
GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCHCTPK


 


1416
GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCHCTPK


 


1417
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1418
GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCHCTPK


 


1419
GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCHCTPK


 


1420
GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCHCTPK


 


1421
GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCHCTPK


 


1422
GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCHCTPK


 


1423
GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK


 


1424
GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK


 


1425
GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK


 


1426
GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCHCTPK


 


1427
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACHCTPK


 


1428
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK


 


1429
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCAPK


 


1430
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTAK


 


1431
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPA


 


1432
RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1433
GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1434
GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1435
GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1436
GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1437
GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1438
GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1439
GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1440
GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1441
GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1442
GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCHCTPK


 


1443
GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCHCTPK


 


1444
GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCHCTPK


 


1445
GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCHCTPK


 


1446
GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCHCTPK


 


1447
GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCHCTPK


 


1448
GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCHCTPK


 


1449
GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCHCTPK


 


1450
GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCHCTPK


 


1451
GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCHCTPK


 


1452
GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCHCTPK


 


1453
GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCHCTPK


 


1454
GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCHCTPK


 


1455
GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCHCTPK


 


1456
GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCHCTPK


 


1457
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTPK


 


1458
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCRCTPK


 


1459
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCRPK


 


1460
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTRK


 


1461
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPR


 


1462
EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1463
GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1464
GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1465
GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1466
GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1467
GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1468
GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1469
GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1470
GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1471
GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1472
GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1473
GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCHCTPK


 


1474
GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCHCTPK


 


1475
GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCHCTPK


 


1476
GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCHCTPK


 


1477
GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCHCTPK


 


1478
GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCHCTPK


 


1479
GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCHCTPK


 


1480
GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCHCTPK


 


1481
GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCHCTPK


 


1482
GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCHCTPK


 


1483
GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCHCTPK


 


1484
GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCHCTPK


 


1485
GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCHCTPK


 


1486
GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCHCTPK


 


1487
GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCHCTPK


 


1488
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECHCTPK


 


1489
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCECTPK


 


1490
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCEPK


 


1491
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTEK


 


1492
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPE


 


1493
[1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1494
G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1495
GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1496
GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1497
GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1498
GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1499
GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1500
GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1501
GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1502
GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1503
GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGKCHCTPK


 


1504
GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGKCHCTPK


 


1505
GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGKCHCTPK


 


1506
GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGKCHCTPK


 


1507
GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGKCHCTPK


 


1508
GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGKCHCTPK


 


1509
GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGKCHCTPK


 


1510
GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGKCHCTPK


 


1511
GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGKCHCTPK


 


1512
GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGKCHCTPK


 


1513
GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGKCHCTPK


 


1514
GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGKCHCTPK


 


1515
GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGKCHCTPK


 


1516
GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGKCHCTPK


 


1517
GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGKCHCTPK


 


1518
GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GKCHCTPK


 


1519
GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]KCHCTPK


 


1520
GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal]CHCTPK


 


1521
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC[1-Nal]CTPK


 


1522
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC[1-Nal]PK


 


1523
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT[1-Nal]K


 


1524
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP[1-Nal]


 


1525
Ac-AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1526
Ac-GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1527
Ac-GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1528
Ac-GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1529
Ac-GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1530
Ac-GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1531
Ac-GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1532
Ac-GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1533
Ac-GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1534
Ac-GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1535
Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1536
Ac-GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCHCTPK


 


1537
Ac-GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCHCTPK


 


1538
Ac-GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCHCTPK


 


1539
Ac-GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCHCTPK


 


1540
Ac-GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCHCTPK


 


1541
Ac-GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCHCTPK


 


1542
Ac-GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCHCTPK


 


1543
Ac-GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCHCTPK


 


1544
Ac-GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCHCTPK


 


1545
Ac-GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCHCTPK


 


1546
Ac-GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCHCTPK


 


1547
Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK


 


1548
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK


 


1549
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK


 


1550
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCHCTPK


 


1551
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACHCTPK


 


1552
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK


 


1553
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCAPK


 


1554
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTAK


 


1555
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPA


 


1556
Ac-RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1557
Ac-GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1558
Ac-GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1559
Ac-GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1560
Ac-GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1561
Ac-GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1562
Ac-GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1563
Ac-GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1564
Ac-GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1565
Ac-GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1566
Ac-GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCHCTPK


 


1567
Ac-GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCHCTPK


 


1568
Ac-GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCHCTPK


 


1569
Ac-GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCHCTPK


 


1570
Ac-GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCHCTPK


 


1571
Ac-GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCHCTPK


 


1572
Ac-GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCHCTPK


 


1573
Ac-GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCHCTPK


 


1574
Ac-GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCHCTPK


 


1575
Ac-GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCHCTPK


 


1576
Ac-GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCHCTPK


 


1577
Ac-GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCHCTPK


 


1578
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCHCTPK


 


1579
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCHCTPK


 


1580
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCHCTPK


 


1581
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTPK


 


1582
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCRCTPK


 


1583
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCRPK


 


1584
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTRK


 


1585
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPR


 


1586
Ac-EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1587
Ac-GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1588
Ac-GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1589
Ac-GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1590
Ac-GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1591
Ac-GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1592
Ac-GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1593
Ac-GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1594
Ac-GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1595
Ac-GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1596
Ac-GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCHCTPK


 


1597
Ac-GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCHCTPK


 


1598
Ac-GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCHCTPK


 


1599
Ac-GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCHCTPK


 


1600
Ac-GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCHCTPK


 


1601
Ac-GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCHCTPK


 


1602
Ac-GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCHCTPK


 


1603
Ac-GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCHCTPK


 


1604
Ac-GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCHCTPK


 


1605
Ac-GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCHCTPK


 


1606
Ac-GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCHCTPK


 


1607
Ac-GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCHCTPK


 


1608
Ac-GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCHCTPK


 


1609
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCHCTPK


 


1610
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCHCTPK


 


1611
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCHCTPK


 


1612
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECHCTPK


 


1613
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCECTPK


 


1614
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCEPK


 


1615
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTEK


 


1616
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPE


 


1617
Ac-[1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGK



CHCTPK


 


1618
Ac-G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGK



CHCTPK


 


1619
Ac-GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGK



CHCTPK


 


1620
Ac-GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGK



CHCTPK


 


1621
Ac-GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGK



CHCTPK


 


1622
Ac-GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGK



CHCTPK


 


1623
Ac-GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGK



CHCTPK


 


1624
Ac-GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGK



CHCTPK


 


1625
Ac-GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGK



CHCTPK


 


1626
Ac-GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGK



CHCTPK


 


1627
Ac-GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGK



CHCTPK


 


1628
Ac-GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGK



CHCTPK


 


1629
Ac-GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGK



CHCTPK


 


1630
Ac-GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGK



CHCTPK


 


1631
Ac-GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGK



CHCTPK


 


1632
Ac-GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGK



CHCTPK


 


1633
Ac-GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGK



CHCTPK


 


1634
Ac-GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGK



CHCTPK


 


1635
Ac-GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGK



CHCTPK


 


1636
Ac-GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGK



CHCTPK


 


1637
Ac-GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGK



CHCTPK


 


1638
Ac-GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGK



CHCTPK


 


1639
Ac-GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGK



CHCTPK


 


1640
Ac-GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGK



CHCTPK


 


1641
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGK



CHCTPK


 


1642
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GK



CHCTPK


 


1643
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]K



CHCTPK


 


1644
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal]



CHCTPK


 


1645
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC



[1-Nal]CTPK


 


1646
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC



[1-Nal]PK


 


1647
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT



[1-Nal]K


 


1648
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP



[1-Nal]


 


1649
AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1650
GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1651
GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1652
GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1653
GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1654
GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1655
GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1656
GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1657
GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1658
GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1659
GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1660
GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1661
GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1662
GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCHCTPK-amide


 


1663
GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCHCTPK-amide


 


1664
GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCHCTPK-amide


 


1665
GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCHCTPK-amide


 


1666
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1667
GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCHCTPK-amide


 


1668
GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCHCTPK-amide


 


1669
GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCHCTPK-amide


 


1670
GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCHCTPK-amide


 


1671
GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCHCTPK-amide


 


1672
GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide


 


1673
GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide


 


1674
GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide


 


1675
GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCHCTPK-amide


 


1676
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACHCTPK-amide


 


1677
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-amide


 


1678
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCAPK-amide


 


1679
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTAK-amide


 


1680
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPA-amide


 


1681
RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1682
GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1683
GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1684
GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1685
GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1686
GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1687
GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1688
GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1689
GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1690
GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1691
GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1692
GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCHCTPK-amide


 


1693
GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCHCTPK-amide


 


1694
GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCHCTPK-amide


 


1695
GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCHCTPK-amide


 


1696
GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCHCTPK-amide


 


1697
GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCHCTPK-amide


 


1698
GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCHCTPK-amide


 


1699
GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCHCTPK-amide


 


1700
GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCHCTPK-amide


 


1701
GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCHCTPK-amide


 


1702
GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCHCTPK-amide


 


1703
GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCHCTPK-amide


 


1704
GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCHCTPK-amide


 


1705
GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCHCTPK-amide


 


1706
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTPK-amide


 


1707
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCRCTPK-amide


 


1708
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCRPK-amide


 


1709
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTRK-amide


 


1710
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPR-amide


 


1711
EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1712
GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1713
GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1714
GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1715
GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1716
GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1717
GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1718
GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1719
GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1720
GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1721
GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1722
GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1723
GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCHCTPK-amide


 


1724
GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCHCTPK-amide


 


1725
GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCHCTPK-amide


 


1726
GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCHCTPK-amide


 


1727
GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCHCTPK-amide


 


1728
GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCHCTPK-amide


 


1729
GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCHCTPK-amide


 


1730
GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCHCTPK-amide


 


1731
GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCHCTPK-amide


 


1732
GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCHCTPK-amide


 


1733
GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCHCTPK-amide


 


1734
GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCHCTPK-amide


 


1735
GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCHCTPK-amide


 


1736
GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCHCTPK-amide


 


1737
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECHCTPK-amide


 


1738
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCECTPK-amide


 


1739
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCEPK-amide


 


1740
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTEK-amide


 


1741
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPE-amide


 


1742
[1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1743
G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1744
GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1745
GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1746
GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1747
GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1748
GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1749
GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1750
GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1751
GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1752
GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1753
GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1754
GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1755
GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGKCH



CTPK-amide


 


1756
GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGKCH



CTPK-amide


 


1757
GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGKCH



CTPK-amide


 


1758
GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGKCH



CTPK-amide


 


1759
GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGKCH



CTPK-amide


 


1760
GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGKCH



CTPK-amide


 


1761
GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGKCH



CTPK-amide


 


1762
GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGKCH



CTPK-amide


 


1763
GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGKCH



CTPK-amide


 


1764
GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGKCH



CTPK-amide


 


1765
GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGKCH



CTPK-amide


 


1766
GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGKCH



CTPK-amide


 


1767
GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GKCH



CTPK-amide


 


1768
GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]KCH



CTPK-amide


 


1769
GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal]CH



CTPK-amide


 


1770
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC[1-Nal]



CTPK-amide


 


1771
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC



[1-Nal]PK-amide


 


1772
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT



[1-Nal]K-amide


 


1773
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC



TP[1-Nal]-amide


 


1774
Ac-AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1775
Ac-GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1776
Ac-GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1777
Ac-GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1778
Ac-GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1779
Ac-GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1780
Ac-GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1781
Ac-GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1782
Ac-GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1783
Ac-GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1784
Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1785
Ac-GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1786
Ac-GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1787
Ac-GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1788
Ac-GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCH



CTPK-amide


 


1789
Ac-GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCH



CTPK-amide


 


1790
Ac-GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCH



CTPK-amide


 


1791
Ac-GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCH



CTPK-amide


 


1792
Ac-GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCH



CTPK-amide


 


1793
Ac-GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCH



CTPK-amide


 


1794
Ac-GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCH



CTPK-amide


 


1795
Ac-GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCH



CTPK-amide


 


1796
Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCH



CTPK-amide


 


1797
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCH



CTPK-amide


 


1798
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCH



CTPK-amide


 


1799
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCH



CTPK-amide


 


1800
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACH



CTPK-amide


 


1801
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCA



CTPK-amide


 


1802
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CAPK-amide


 


1803
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTAK-amide


 


1804
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPA-amide


 


1805
Ac-RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1806
Ac-GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1807
Ac-GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1808
Ac-GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1809
Ac-GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1810
Ac-GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1811
Ac-GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1812
Ac-GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1813
Ac-GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1814
Ac-GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1815
Ac-GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1816
Ac-GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1817
Ac-GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1818
Ac-GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCH



CTPK-amide


 


1819
Ac-GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCH



CTPK-amide


 


1820
Ac-GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCH



CTPK-amide


 


1821
Ac-GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCH



CTPK-amide


 


1822
Ac-GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCH



CTPK-amide


 


1823
Ac-GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCH



CTPK-amide


 


1824
Ac-GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCH



CTPK-amide


 


1825
Ac-GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCH



CTPK-amide


 


1826
Ac-GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCH



CTPK-amide


 


1827
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCH



CTPK-amide


 


1828
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCH



CTPK-amide


 


1829
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCH



CTPK-amide


 


1830
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCH



CTPK-amide


 


1831
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCR



CTPK-amide


 


1832
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CRPK-amide


 


1833
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTRK-amide


 


1834
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPR-amide


 


1835
Ac-EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1836
Ac-GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1837
Ac-GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1838
Ac-GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1839
Ac-GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1840
Ac-GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1841
Ac-GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1842
Ac-GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1843
Ac-GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1844
Ac-GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1845
Ac-GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1846
Ac-GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1847
Ac-GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1848
Ac-GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCH



CTPK-amide


 


1849
Ac-GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCH



CTPK-amide


 


1850
Ac-GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCH



CTPK-amide


 


1851
Ac-GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCH



CTPK-amide


 


1852
Ac-GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCH



CTPK-amide


 


1853
Ac-GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCH



CTPK-amide


 


1854
Ac-GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCH



CTPK-amide


 


1855
Ac-GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCH



CTPK-amide


 


1856
Ac-GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCH



CTPK-amide


 


1857
Ac-GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCH



CTPK-amide


 


1858
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCH



CTPK-amide


 


1859
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCH



CTPK-amide


 


1860
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCH



CTPK-amide


 


1861
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECH



CTPK-amide


 


1862
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCE



CTPK-amide


 


1863
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CEPK-amide


 


1864
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTEK-amide


 


1865
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPE-amide


 


1866
Ac-[1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGK



CHCTPK-amide


 


1867
Ac-G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGK



CHCTPK-amide


 


1868
Ac-GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1869
Ac-GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1870
Ac-GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1871
Ac-GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1872
Ac-GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1873
Ac-GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1874
Ac-GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1875
Ac-GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1876
Ac-GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1877
Ac-GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1878
Ac-GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGKCH



CTPK-amide


 


1879
Ac-GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGKCH



CTPK-amide


 


1880
Ac-GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGKCH



CTPK-amide


 


1881
Ac-GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGKCH



CTPK-amide


 


1882
Ac-GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGKCH



CTPK-amide


 


1883
Ac-GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGKCH



CTPK-amide


 


1884
Ac-GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGKCH



CTPK-amide


 


1885
Ac-GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGKCH



CTPK-amide


 


1886
Ac-GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGKCH



CTPK-amide


 


1887
Ac-GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGKCH



CTPK-amide


 


1888
Ac-GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGKCH



CTPK-amide


 


1889
Ac-GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGKCH



CTPK-amide


 


1890
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGKCH



CTPK-amide


 


1891
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GKCH



CTPK-amide


 


1892
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]KCH



CTPK-amide


 


1893
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal]CH



CTPK-amide


 


1894
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC[1-Nal]



CTPK-amide


 


1895
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC



[1-Nal]PK-amide


 


1896
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT



[1-Nal]K-amide


 


1897
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP



[1-Nal]-amide
















TABLE 7B







Additional useful OSK1 peptide analog


sequences: Ala-12 Substituted Series









SEQ ID


Sequence/structure
NO:





GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK
1898


 


GVIINVSCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK
1899


 


GVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK
1900


 


GVIINVKCKISAQCLEPCKDAGMRFGKCMNGKCHCTPK
1901


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1902


 


GVIINVSCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1903


 


GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK
1904


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK
1905


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK
1906


 


GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK-
1907


amide



 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK-
1908


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK-amide
1909


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK
1910


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK-
1911


amide



 


GVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK-amide
1912


 


Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK
1913


 


Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK-
1914


amide



 


Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCMNGKCHCTPK
1915


 


GVIINVKCKISAQCLEPCKDAGMRFGKCMNGKCHCTPK-amide
1916


 


Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCMNGKCHCTPK-
1917


amide



 


GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide
1918


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK
1919


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-
1920


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1921


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1922


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-
1923


amide



 


VIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK
1924


 


Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK
1925


 


VIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide
1926


 


Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-
1927


amide



 


GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK
1928


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK
1929


 


GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK-amide
1930


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK-
1931


amide



 


VIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1932


 


Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1933


 


VIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1934


 


Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-
1935


amide



 


NVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1936


 


Ac-NVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1937


 


NVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1938


 


Ac-NVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1939


 


KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1940


 


Ac-KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1941


 


KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1942


 


Ac-KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1943


 


CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1944


 


Ac-CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1945


 


CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1946


 


Ac-CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1947


 


GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK
1948


 


GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK-amide
1949


 


Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK
1950


 


Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK-
1951


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
1952


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
1953


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
1954


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-
1955


amide



 


GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK
1956


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK
1957


 


GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-amide
1958


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-
1959


amide



 


TIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1960


 


Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1961


 


TIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1962


 


Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-
1963


amide



 


GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK
1964


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK
1965


 


GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide
1966


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-
1967


amide



 


GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK
1968


 


GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1969


 


GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK
1970


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK
1971


 


GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK-amide
1972


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK-
1973


amide



 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK
1974


 


GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK-amide
1975


 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK-
1976


amide



 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1977


 


GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1978


 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-
1979


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCT
1980


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCTPK
1981


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK
1982


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK
1983


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK
1984


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK
1985


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK
1986


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK
1987


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYPK
1988


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK
1989


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK
1990


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK
1991


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK
1992


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK
1993


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK
1994


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYPK
1995


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCGCYPK
1996


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACFPK
1997


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACWPK
1998


 


GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYPK
1999


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACTPK
2000


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACTPK
2001


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACTPK
2002


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACTPK
2003


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK
2004


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK
2005


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACTPK
2006


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHC
2007


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHC
2008


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHC
2009


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHC
2010


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC
2011


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHC
2012


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHC
2013


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCAC
2014


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCAC
2015


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCAC
2016


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCAC
2017


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC
2018


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCAC
2019


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCAC
2020


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCGCYGG
2021


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYGG
2022


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG
2023


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG
2024


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG
2025


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG
2026


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG
2027


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYGG
2028


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACYGG
2029


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACYGG
2030


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACYGG
2031


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACYGG
2032


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG
2033


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG
2034


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACYGG
2035


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYG
2036


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCGGG
2037


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG
2038


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG
2039


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG
2040


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG
2041


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG
2042


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACFGG
2043


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACGGG
2044


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACGGG
2045


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACGGG
2046


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACGGG
2047


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACGGG
2048


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG
2049


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACGGG
2050


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACGG
2051


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYG
2052


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACGG
2053


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK
2054


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK
2055


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK
2056


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK
2057


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK
2058


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK
2059


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYPK
2060


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK
2061


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK
2062


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK
2063


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK
2064


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK
2065


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK
2066


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACTPK
2067


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACTPK
2068


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACTPK
2069


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACTPK
2070


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK
2071


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK
2072


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACTPK
2073


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHC
2074


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHC
2075


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHC
2076


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHC
2077


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC
2078


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHC
2079


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCAC
2080


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCAC
2081


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCAC
2082


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCAC
2083


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC
2084


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCAC
2085


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCAC
2086


 


GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCHCYGG
2087


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYGG
2088


 


GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCHCYG
2089


 


GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYG
2090


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG
2091


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG
2092


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG
2093


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG
2094


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG
2095


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG
2096


 


GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYG
2097


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACYGG
2098


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACYGG
2099


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACYGG
2100


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACYGG
2101


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG
2102


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG
2103


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACYGG
2104


 


GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACFGG
2105


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCGGG
2106


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG
2107


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG
2108


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG
2109


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG
2110


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG
2111


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACGGG
2112


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACGGG
2113


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACGGG
2114


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACGGG
2115


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCACTP
2116


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACTP
2117


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACTP
2118


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCTPK-amide
2119


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK-
2120


amide



 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK-
2121


amide



 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK-
2122


amide



 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK-
2123


amide



 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK-
2124


amide



 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK-
2125


amide



 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYPK-amide
2126


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK-
2127


amide



 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK-
2128


amide



 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK-
2129


amide



 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK-
2130


amide



 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK-
2131


amide



 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK-
2132


amide



 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACTPK-amide
2133


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACTPK-
2134


amide



 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACTPK-
2135


amide



 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACTPK-
2136


amide



 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACTPK-
2137


amide



 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK-
2138


amide



 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACTPK-
2139


amide



 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHC-amide
2140


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHC-
2141


amide



 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHC-
2142


amide



 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHC-
2143


amide



 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC-
2144


amide



 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHC-
2145


amide



 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCAC-amide
2146


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCAC-
2147


amide



 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCAC-
2148


amide



 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCAC-
2149


amide



 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC-
2150


amide



 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCAC-
2151


amide



 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCAC-
2152


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYGG-
2153


amide



 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYGG-
2154


amide



 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG-
2155


amide



 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG-
2156


amide



 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG-
2157


amide



 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG-
2158


amide



 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG-
2159


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCFGG-amide
2160


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYG-amide
2161


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYG-amide
2162


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACYGG-amide
2163


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACYGG-
2164


amide



 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACYGG-
2165


amide



 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACYGG-
2166


amide



 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACYGG-
2167


amide



 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG-
2168


amide



 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACYGG-
2169


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYGG-amide
2170


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCGGG-amide
2171


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG-
2172


amide



 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG-
2173


amide



 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG-
2174


amide



 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG-
2175


amide



 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG-
2176


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACGGG-amide
2177


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACFGG-amide
2178


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACGGG-amide
2179


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACGGG-
2180


amide



 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACGGG-
2181


amide



 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACGGG-
2182


amide



 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACGGG-
2183


amide



 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG-
2184


amide



 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACGGG-
2185


amide



 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCTPK-amide
2186


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK-
2187


amide



 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK-
2188


amide



 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK-
2189


amide



 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK-
2190


amide



 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK-
2191


amide



 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK-
2192


amide



 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYPK-amide
2193


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK-
2194


amide



 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK-
2195


amide



 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK-
2196


amide



 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK-
2197


amide



 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK-
2198


amide



 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK-
2199


amide



 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACTPK-amide
2200


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACTPK-
2201


amide



 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACTPK-
2202


amide



 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACTPK-
2203


amide



 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCACTPK-
2204


amide



 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK-
2205


amide



 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACTPK-
2206


amide



 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHC-amide
2207


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHC-
2208


amide



 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHC-
2209


amide



 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHC-
2210


amide



 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC-
2211


amide



 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHC-
2212


amide



 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCAC-amide
2213


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCAC-
2214


amide



 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCAC-
2215


amide



 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCAC-
2216


amide



 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC-
2217


amide



 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCAC-
2218


amide



 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCAC-
2219


amide



 


GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCHCWGG-
2220


amide



 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYGG-
2221


amide



 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG-
2222


amide



 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG-
2223


amide



 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG-
2224


amide



 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG-
2225


amide



 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG-
2226


amide



 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG-
2227


amide



 


GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYGG-amide
2228


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACYGG-amide
2229


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACYGG-
2230


amide



 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACYGG-
2231


amide



 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACYGG-
2232


amide



 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG-
2233


amide



 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG-
2234


amide



 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACYGG-
2235


amide



 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCGGG-amide
2236


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG-
2237


amide



 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG-
2238


amide



 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG-
2239


amide



 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG-
2240


amide



 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG-
2241


amide



 


GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACGGG-amide
2242


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACGGG-
2243


amide



 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACGGG-
2244


amide



 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACGGG-
2245


amide



 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCACTP-
2246


amide



 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACGGG-
2247


amide



 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACGGG-
2248


amide



 


GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMNGKCACYGG-
2249


amide



 


GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMNGKCACGGG-
2250


amide



 


GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMNGKCACY-
2251


amide



 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYGG-
2252


amide



 


GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMNGKCACYGG-
2253


amide



 


GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMNGKCHCYGG-
2254


amide



 


GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMNGKCACYGG
2255


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCACYGG-amide
2256


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCACYGG
2257


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCACY-amide
2258


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCHCYGG-amide
2259


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCHCYGG
2260


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCHCYPK
2261


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCAC
2262


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[1Nal]GG-
2263


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[1Nal]PK-
2264


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[2Nal]GG-
2265


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[Cha]GG-
2266


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[MePhe]
2267


GG-amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[BiPhA]
2268


GG-amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKC[Aib]CYGG-
2269


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKC[Abu]CYGG-
2270


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[1Nal]
2271


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCAC[1Nal]GG-
2272


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[4Bip]-
2273


amide



 


GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCAC[4Bip]GG-
2274


amide



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCGGG
2275
















TABLE 7C







Additional useful OSK1 peptide analog


sequences: Ala-12 & Ala27 Substituted Series









SEQ



ID


Sequence/structure
NO:





GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK
2276


 


GVIINVSCKISAQCLEPCKKAGMRFGACMNGKCHCTPK
2277


 


GVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK
2278


 


GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK
2279


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2280


 


GVIINVSCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2281


 


GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK
2282


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK
2283


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK
2284


 


GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2285


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK-
2286


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK-amide
2287


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK
2288


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK-
2289


amide


 


GVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK-amide
2290


 


Ac-GVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK
2291


 


Ac-GVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK-
2292


amide


 


Ac-GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK
2293


 


GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK-amide
2294


 


Ac-GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK-
2295


amide


 


GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-amide
2296


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK
2297


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-
2298


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2299


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2300


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-
2301


amide


 


VIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK
2302


 


Ac-VIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK
2303


 


VIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-amide
2304


 


Ac-VIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-
2305


amide


 


GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK
2306


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK
2307


 


GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK-amide
2308


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK-
2309


amide


 


VIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2310


 


Ac-VIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2311


 


VIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2312


 


Ac-VIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-
2313


amide


 


NVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2314


 


Ac-NVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2315


 


NVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2316


 


Ac-NVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2317


 


KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2318


 


Ac-KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2319


 


KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2320


 


Ac-KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2321


 


CKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2322


 


Ac-CKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2323


 


CKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2324


 


Ac-CKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2325


 


GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK
2326


 


GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK-amide
2327


 


Ac-GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK
2328


 


Ac-GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK-
2329


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
2330


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
2331


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
2332


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-
2333


amide


 


GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK
2334


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK
2335


 


GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK-amide
2336


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK-
2337


amide


 


TIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2338


 


Ac-TIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2339


 


TIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2340


 


Ac-TIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-
2341


amide


 


GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK
2342


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK
2343


 


GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-amide
2344


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-
2345


amide


 


GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK
2346


 


GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2347


 


GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK
2348


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK
2349


 


GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK-amide
2350


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK-
2351


amide


 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK
2352


 


GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK-amide
2353


 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK-
2354


amide


 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2355


 


GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2356


 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-
2357


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCT
2358


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCTPK
2359


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCTPK
2360


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCTPK
2361


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCTPK
2362


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCTPK
2363


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK
2364


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCTPK
2365


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYPK
2366


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYPK
2367


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYPK
2368


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYPK
2369


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYPK
2370


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK
2371


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCYPK
2372


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYPK
2373


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCGCYPK
2374


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACFPK
2375


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACWPK
2376


 


GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYPK
2377


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACTPK
2378


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACTPK
2379


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACTPK
2380


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACTPK
2381


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCTPK
2382


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACTPK
2383


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACTPK
2384


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHC
2385


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHC
2386


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHC
2387


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHC
2388


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC
2389


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHC
2390


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHC
2391


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCAC
2392


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCAC
2393


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCAC
2394


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCAC
2395


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC
2396


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCAC
2397


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCAC
2398


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCGCYGG
2399


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYGG
2400


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYGG
2401


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYGG
2402


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYGG
2403


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYGG
2404


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG
2405


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYGG
2406


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACYGG
2407


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACYGG
2408


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACYGG
2409


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACYGG
2410


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYGG
2411


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACYGG
2412


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACYGG
2413


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYG
2415


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCGGG
2416


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCGGG
2417


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCGGG
2418


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCGGG
2419


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCGGG
2420


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG
2421


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACFGG
2422


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACGGG
2423


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACGGG
2424


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACGGG
2425


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACGGG
2426


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACGGG
2427


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACGGG
2428


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACGGG
2429


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACGG
2430


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYG
2431


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACGG
2432


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCTPK
2433


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCTPK
2434


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCTPK
2435


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCTPK
2436


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK
2437


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCTPK
2438


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYPK
2439


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYPK
2440


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYPK
2441


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYPK
2442


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYPK
2443


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK
2444


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYPK
2445


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACTPK
2446


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACTPK
2447


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACTPK
2448


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACTPK
2449


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCTPK
2450


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACTPK
2451


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACTPK
2452


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHC
2453


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHC
2454


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHC
2455


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHC
2456


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC
2457


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHC
2458


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCAC
2459


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCAC
2460


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCAC
2461


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCAC
2462


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC
2463


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCAC
2464


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCAC
2465


 


GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCHCYGG
2466


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYGG
2467


 


GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCHCYG
2468


 


GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYG
2469


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYGG
2470


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYGG
2471


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYGG
2472


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG
2473


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG
2474


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYGG
2475


 


GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYG
2476


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACYGG
2477


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACYGG
2478


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACYGG
2479


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACYGG
2480


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG
2481


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACYGG
2482


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACYGG
2483


 


GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACFGG
2484


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCGGG
2485


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCGGG
2486


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCGGG
2487


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCGGG
2488


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCGGG
2489


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG
2490


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACGGG
2491


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACGGG
2492


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACGGG
2493


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACGGG
2494


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCACTP
2495


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACTP
2496


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACTP
2497


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCTPK-amide
2498


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCTPK-
2499


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCTPK-
2500


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCTPK-
2501


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCTPK-
2502


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK-
2503


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCTPK-
2504


amide


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYPK-amide
2505


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYPK-
2506


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYPK-
2507


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYPK-
2508


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYPK-
2509


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK-
2510


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCYPK-
2511


amide


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACTPK-amide
2512


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACTPK-
2513


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACTPK-
2514


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACTPK-
2515


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACTPK-
2516


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACTPK-
2517


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACTPK-
2518


amide


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHC-amide
2519


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHC-
2520


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHC-
2521


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHC-
2522


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC-
2523


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHC-amide
2524


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCAC-amide
2525


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCAC-
2526


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCAC-
2527


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCAC-amide
2528


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC-
2529


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCAC-
2530


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCAC-amide
2531


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYGG-amide
2532


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYGG-amide
2533


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYGG-
2534


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYGG-
2535


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYGG-
2536


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYGG-
2537


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG-
2538


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCFGG-amide
2539


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYG-amide
2540


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYG-amide
2541


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACYGG-amide
2542


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACYGG-
2543


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACYGG-
2544


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACYGG-
2545


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACYGG-
2546


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACYGG-
2547


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACYGG-
2548


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYGG-amide
2549


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCGGG-amide
2550


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCGGG-
2551


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCGGG-
2552


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCGGG-
2553


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCGGG-
2554


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG-
2555


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACGGG-amide
2556


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACFGG-amide
2557


 


GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACGGG-amide
2558


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACGGG-
2559


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACGGG-
2560


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACGGG-
2561


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACGGG-
2562


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACGGG-
2563


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACGGG-
2564


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCTPK-amide
2565


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCTPK-
2566


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCTPK-
2567


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCTPK-
2568


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCTPK-
2569


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK-
2570


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCTPK-
2571


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYPK-amide
2572


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYPK-
2573


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYPK-
2574


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYPK-
2575


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYPK-
2576


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK-
2577


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYPK-
2578


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACTPK-amide
2579


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACTPK-
2580


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACTPK-
2581


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACTPK-
2582


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCACTPK-
2583


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACTPK-
2584


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACTPK-
2585


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHC-amide
2586


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHC-
2587


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHC-
2588


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHC-amide
2589


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC-
2590


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHC-
2591


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCAC-amide
2592


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCAC-
2593


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCAC-
2594


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCAC-amide
2595


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC-
2596


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCAC-
2597


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCAC-amide
2598


 


GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCHCWGG-amide
2599


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYGG-amide
2600


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYGG-
2601


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYGG-
2602


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYGG-
2603


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG-
2604


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG-
2605


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYGG-
2606


amide


 


GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYGG-amide
2607


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACYGG-amide
2608


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACYGG-
2609


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACYGG-
2610


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACYGG-
2611


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG-
2612


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACYGG-
2613


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACYGG-
2614


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCGGG-amide
2615


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCGGG-
2616


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCGGG-
2617


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCGGG-
2618


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCGGG-
2619


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG-
2620


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACGGG-amide
2621


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACGGG-
2622


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACGGG-
2623


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACGGG-
2624


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCACTP-
2625


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACGGG-
2626


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACGGG-
2627


amide


 


GVIINVKCKISAQCLKPCK[Cpa]AGMRFGACMNGKCACYGG-
2628


amide


 


GVIINVKCKISAQCLKPCK[Cpa]AGMRFGACMNGKCACGGG-
2629


amide


 


GVIINVKCKISAQCLKPCK[Cpa]AGMRFGACMNGKCACY-
2630


amide


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYGG-
2631


amide


 


GVIINVKCKISAQCLKPCK[Aad]AGMRFGACMNGKCACYGG-
2632


amide


 


GVIINVKCKISAQCLKPCK[Aad]AGMRFGACMNGKCHCYGG-
2633


amide


 


GVIINVKCKISAQCLKPCK[Aad]AGMRFGACMNGKCACYGG
2634


 


GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCACYGG-amide
2635


 


GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCACYGG
2636


 


GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCACY-amide
2637


 


GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCHCYGG-amide
2638


 


GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCHCYGG
2639


 


GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCHCYPK
2640


 


GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCAC
2641


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[1Nal]GG-
2642


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[1Nal]PK-
2643


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[2Nal]GG-
2644


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[Cha]GG-
2645


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[MePhe]GG-
2646


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[BiPhA]GG-
2647


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKC[Aib]CYGG-
2648


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKC[Abu]CYGG-
2649


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[1Nal]
2650


 


GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCAC[1Nal]GG-
2651


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[4Bip]-
2652


amide


 


GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCAC[4Bip]GG-
2653


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCGGG
2654
















TABLE 7D







Additional useful OSK1 peptide analog


sequences: Ala-12 & Ala29 Substituted Series









SEQ



ID


Sequence/structure
NO:





GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK
2655


 


GVIINVSCKISAQCLEPCKKAGMRFGKCANGKCHCTPK
2656


 


GVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK
2657


 


GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK
2658


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2659


 


GVIINVSCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2660


 


GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK
2661


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK
2662


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK
2663


 


GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2664


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK-
2665


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK-amide
2666


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK
2667


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK-
2668


amide


 


GVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK-amide
2669


 


Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK
2670


 


Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK-
2671


amide


 


Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK
2672


 


GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK-amide
2673


 


Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK-
2674


amide


 


GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-amide
2675


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK
2676


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-
2677


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2678


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2679


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-
2680


amide


 


VIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK
2681


 


Ac-VIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK
2682


 


VIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-amide
2683


 


Ac-VIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-
2684


amide


 


GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK
2685


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK
2686


 


GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK-amide
2687


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK-
2688


amide


 


VIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2689


 


Ac-VIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2690


 


VIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2691


 


Ac-VIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-
2692


amide


 


NVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2693


 


Ac-NVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2694


 


NVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2695


 


Ac-NVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2696


 


KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2697


 


Ac-KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2698


 


KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2699


 


Ac-KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2700


 


CKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2701


 


Ac-CKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2702


 


CKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2703


 


Ac-CKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2704


 


GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK
2705


 


GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK-amide
2706


 


Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK
2707


 


Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK-
2708


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
2709


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
2710


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
2711


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-
2712


amide


 


GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK
2713


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK
2714


 


GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK-amide
2715


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK-
2716


amide


 


TIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2717


 


Ac-TIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2718


 


TIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2719


 


Ac-TIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-
2720


amide


 


GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK
2721


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK
2722


 


GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-amide
2723


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-
2724


amide


 


GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK
2725


 


GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2726


 


GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK
2727


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK
2728


 


GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK-amide
2729


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK-
2730


amide


 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK
2731


 


GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK-amide
2732


 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK-
2733


amide


 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2734


 


GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2735


 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-
2736


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCT
2737


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCTPK
2738


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCTPK
2739


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCTPK
2740


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCTPK
2741


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCTPK
2742


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK
2743


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCTPK
2744


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYPK
2745


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYPK
2746


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYPK
2747


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYPK
2748


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYPK
2749


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK
2750


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCYPK
2751


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYPK
2752


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCGCYPK
2753


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACFPK
2754


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACWPK
2755


 


GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYPK
2756


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACTPK
2757


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACTPK
2758


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACTPK
2759


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACTPK
2760


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCTPK
2761


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACTPK
2762


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACTPK
2763


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHC
2764


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHC
2765


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHC
2766


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHC
2767


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC
2768


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHC
2769


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHC
2770


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCAC
2771


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCAC
2772


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCAC
2773


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCAC
2774


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC
2775


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCAC
2776


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCAC
2777


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCGCYGG
2778


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYGG
2779


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYGG
2780


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYGG
2781


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYGG
2782


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYGG
2783


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG
2784


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYGG
2785


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACYGG
2786


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACYGG
2787


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACYGG
2788


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACYGG
2789


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYGG
2790


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACYGG
2791


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACYGG
2792


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYG
2793


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCGGG
2794


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCGGG
2795


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCGGG
2796


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCGGG
2797


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCGGG
2798


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG
2799


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACFGG
2800


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACGGG
2801


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACGGG
2802


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACGGG
2803


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACGGG
2804


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACGGG
2805


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACGGG
2806


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACGGG
2807


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACGG
2808


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYG
2809


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACGG
2810


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCTPK
2811


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCTPK
2812


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCTPK
2813


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCTPK
2814


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK
2815


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCTPK
2816


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYPK
2817


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYPK
2818


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYPK
2819


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYPK
2820


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYPK
2821


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK
2822


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYPK
2823


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACTPK
2824


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACTPK
2825


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACTPK
2826


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACTPK
2827


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCTPK
2828


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACTPK
2829


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACTPK
2830


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHC
2831


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHC
2832


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHC
2833


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHC
2834


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC
2835


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHC
2836


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCAC
2837


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCAC
2838


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCAC
2839


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCAC
2840


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC
2841


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCAC
2842


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCAC
2843


 


GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCHCYGG
2844


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYGG
2845


 


GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCHCYG
2846


 


GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYG
2847


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYGG
2848


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYGG
2849


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYGG
2850


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG
2851


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG
2852


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYGG
2853


 


GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYG
2854


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACYGG
2855


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACYGG
2856


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACYGG
2857


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACYGG
2858


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG
2859


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACYGG
2860


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACYGG
2861


 


GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACFGG
2862


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCGGG
2863


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCGGG
2864


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCGGG
2865


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCGGG
2866


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCGGG
2867


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG
2868


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACGGG
2869


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACGGG
2870


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACGGG
2871


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACGGG
2872


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCACTP
2873


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACTP
2874


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACTP
2875


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCTPK-amide
2876


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCTPK-
2877


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCTPK-
2878


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCTPK-
2879


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCTPK-
2880


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK-
2881


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCTPK-
2882


amide


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYPK-amide
2883


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYPK-
2884


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYPK-
2885


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYPK-
2886


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYPK-
2887


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK-
2888


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCYPK-
2889


amide


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACTPK-amide
2890


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACTPK-
2891


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACTPK-
2892


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACTPK-
2893


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACTPK-
2894


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACTPK-
2895


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACTPK-
2896


amide


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHC-amide
2897


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHC-
2898


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHC-
2899


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHC-amide
2900


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC-
2901


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHC-amide
2902


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCAC-amide
2903


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCAC-
2904


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCAC-
2905


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCAC-amide
2906


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC-
2907


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCAC-
2908


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCAC-amide
2909


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYGG-amide
2910


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYGG-amide
2911


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYGG-
2912


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYGG-
2913


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYGG-
2914


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYGG-
2915


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG-
2916


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCFGG-amide
2917


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYG-amide
2918


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYG-amide
2919


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACYGG-amide
2920


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACYGG-
2921


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACYGG-
2922


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACYGG-
2923


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACYGG-
2924


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACYGG-
2925


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACYGG-
2926


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYGG-amide
2927


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCGGG-amide
2928


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCGGG-
2929


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCGGG-
2930


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCGGG-
2931


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCGGG-
2932


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG-
2933


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACGGG-amide
2934


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACFGG-amide
2935


 


GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACGGG-amide
2936


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACGGG-
2937


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACGGG-
2938


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACGGG-
2939


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACGGG-
2940


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACGGG-
2941


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACGGG-
2942


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCTPK-amide
2943


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCTPK-
2944


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCTPK-
2945


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCTPK-
2946


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCTPK-
2947


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK-
2948


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCTPK-
2949


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYPK-amide
2950


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYPK-
2951


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYPK-
2952


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYPK-
2953


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYPK-
2954


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK-
2955


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYPK-
2956


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACTPK-amide
2957


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACTPK-
2958


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACTPK-
2959


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACTPK-
2960


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCACTPK-
2961


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACTPK-
2962


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACTPK-
2963


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHC-amide
2964


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHC-
2965


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHC-
2966


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHC-amide
2967


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC-
2968


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHC-amide
2969


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCAC-amide
2970


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCAC-
2971


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCAC-
2972


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCAC-amide
2973


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC-
2974


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCAC-amide
2975


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCAC-amide
2976


 


GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCHCWGG-amide
2977


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYGG-amide
2978


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYGG-
2979


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYGG-
2980


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYGG-
2981


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG-
2982


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG-
2983


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYGG-
2984


amide


 


GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYGG-amide
2985


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACYGG-amide
2986


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACYGG-
2987


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACYGG-
2988


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACYGG-
2989


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG-
2990


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACYGG-
2991


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACYGG-
2992


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCGGG-amide
2993


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCGGG-
2994


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCGGG-
2995


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCGGG-
2996


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCGGG-
2997


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG-
2998


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACGGG-amide
2999


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACGGG-
3000


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACGGG-
3001


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACGGG-
3002


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCACTP-
3003


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACGGG-
3004


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACGGG-
3005


amide


 


GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCANGKCACYGG-
3006


amide


 


GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCANGKCACGGG-
3007


amide


 


GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCANGKCACY-
3008


amide


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYGG-
3009


amide


 


GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCANGKCACYGG-
3010


amide


 


GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCANGKCHCYGG-
3011


amide


 


GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCANGKCACYGG
3012


 


GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCACYGG-amide
3013


 


GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCACYGG
3014


 


GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCACY-amide
3015


 


GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCHCYGG-amide
3016


 


GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCHCYGG
3017


 


GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCHCYPK
3018


 


GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCAC
3019


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[1Nal]GG-
3020


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[1Nal]PK-
3021


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[2Nal]GG-
3022


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[Cha]GG-
3023


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[MePhe]GG-
3024


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[BiPhA]GG-
3025


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKC[Aib]CYGG-
3026


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKC[Abu]CYGG-
3027


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[1Nal]
3028


 


GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCAC[1Nal]GG-
3029


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[4Bip]-
3030


amide


 


GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCAC[4Bip]GG-
3031


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCGGG
3032
















TABLE 7E







Additional useful OSK1 peptide analogs:


Ala-12 & Ala30 Substituted Series









SEQ



ID


Sequence/structure
NO:





GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK
3033


 


GVIINVSCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK
3034


 


GVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK
3035


 


GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK
3036


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3037


 


GVIINVSCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3038


 


GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK
3039


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK
3040


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK
3041


 


GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3042


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK-
3043


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK-amide
3044


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK
3045


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK-
3046


amide


 


GVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK-amide
3047


 


Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK
3048


 


Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK-
3049


amide


 


Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK
3050


 


GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK-amide
3051


 


Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK-
3052


amide


 


GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-amide
3053


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK
3054


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-
3055


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3056


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3057


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-
3058


amide


 


VIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK
3059


 


Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK
3060


 


VIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-amide
3061


 


Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-
3062


amide


 


GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK
3063


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK
3064


 


GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK-amide
3065


 


Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK-
3066


amide


 


VIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3067


 


Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3068


 


VIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3069


 


Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-
3070


amide


 


NVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3071


 


Ac-NVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3072


 


NVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3073


 


Ac-NVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3074


 


KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3075


 


Ac-KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3076


 


KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3077


 


Ac-KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3078


 


CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3079


 


Ac-CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3080


 


CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3081


 


Ac-CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3082


 


GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK
3083


 


GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK-amide
3084


 


Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK
3085


 


Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK-
3086


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
3087


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
3088


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
3089


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-
3090


amide


 


GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK
3091


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK
3092


 


GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK-amide
3093


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK-
3094


amide


 


TIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3095


 


Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3096


 


TIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3097


 


Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-
3098


amide


 


GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK
3099


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK
3100


 


GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-amide
3101


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-
3102


amide


 


GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK
3103


 


GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3104


 


GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK
3105


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK
3106


 


GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK-amide
3107


 


Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK-
3108


amide


 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK
3109


 


GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK-amide
3110


 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK-
3111


amide


 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3112


 


GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3113


 


Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-
3114


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCT
3115


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCTPK
3116


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK
3117


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCTPK
3118


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK
3119


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK
3120


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK
3121


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK
3122


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYPK
3123


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK
3124


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYPK
3125


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK
3126


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK
3127


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK
3128


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK
3129


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYPK
3130


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCGCYPK
3131


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACFPK
3132


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACWPK
4920


 


GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYPK
4921


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACTPK
4922


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACTPK
4923


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACTPK
4924


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACTPK
4925


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK
4926


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK
4927


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACTPK
4928


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHC
4929


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHC
3133


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHC
3134


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHC
3135


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC
3136


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHC
3137


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHC
3138


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCAC
3139


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCAC
3140


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCAC
3141


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCAC
3142


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC
3143


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCAC
3144


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCAC
3145


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCGCYGG
3146


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYGG
3147


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG
3148


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG
3149


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG
3150


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG
3151


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG
3152


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYGG
3153


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACYGG
3154


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACYGG
3155


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACYGG
3156


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACYGG
3157


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG
3158


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG
3159


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACYGG
3160


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYG
3161


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCGGG
3162


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG
3163


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG
3164


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG
3165


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG
3166


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG
3167


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACFGG
3168


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACGGG
3169


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACGGG
3170


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACGGG
3171


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACGGG
3172


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACGGG
3173


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG
3174


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACGGG
3175


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACGG
3176


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYG
3177


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACGG
3178


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK
3179


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK
3180


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK
3181


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK
3182


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK
3183


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK
3184


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYPK
3185


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK
3186


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCYPK
3187


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK
3188


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK
3189


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK
3190


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCYPK
3191


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACTPK
3192


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACTPK
3193


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACTPK
3194


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACTPK
3195


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK
3196


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK
3197


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACTPK
3198


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHC
3199


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHC
3200


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHC
3201


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHC
3202


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC
3203


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHC
3204


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCAC
3205


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCAC
3206


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCAC
3207


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCAC
3208


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC
3209


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCAC
3210


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCAC
3211


 


GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCHCYGG
3212


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYGG
3213


 


GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCHCYG
3214


 


GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYG
3215


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG
3216


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG
3217


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG
3218


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG
3219


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG
3220


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG
3221


 


GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYG
3222


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACYGG
3223


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACYGG
3224


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACYGG
3225


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACYGG
3226


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG
3227


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG
3228


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACYGG
3229


 


GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACFGG
3230


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCGGG
3231


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG
3232


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCGGG
3233


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG
3234


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG
3235


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG
3236


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACGGG
3237


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACGGG
3238


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACGGG
3239


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACGGG
3240


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCACTP
3241


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACTP
3242


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACTP
3243


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCTPK-amide
3244


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK-
3245


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCTPK-
3246


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK-
3247


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK-
3248


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK-
3249


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK-
3250


amide


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYPK-amide
3251


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK-
3252


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYPK-
3253


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK-
3254


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK-
3255


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK-
3256


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK-
3257


amide


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACTPK-amide
3258


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACTPK-
3259


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACTPK-
3260


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACTPK-
3261


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACTPK-
3262


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK-
3263


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACTPK-
3264


amide


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHC-amide
3265


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHC-
3266


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHC-
3267


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHC-amide
3268


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC-
3269


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHC-amide
3270


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCAC-amide
3271


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCAC-
3272


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCAC-
3273


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCAC-amide
3274


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC-
3275


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCAC-
3276


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCAC-amide
3277


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYGG-amide
3278


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYGG-amide
3279


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG-
3280


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG-
3281


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG-
3282


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG-
3283


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG-
3284


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCFGG-amide
3285


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYG-amide
3286


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYG-amide
3287


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACYGG-amide
3288


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACYGG-
3289


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACYGG-
3290


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACYGG-
3291


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACYGG-
3292


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG-
3293


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACYGG-
3294


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYGG-amide
3295


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCGGG-amide
3296


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG-
3297


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG-
3298


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG-
3299


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG-
3300


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG-
3301


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACGGG-amide
3302


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACFGG-amide
3303


 


GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACGGG-amide
3304


 


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACGGG-
3305


amide


 


GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACGGG-
3306


amide


 


GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACGGG-
3307


amide


 


GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACGGG-
3308


amide


 


GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG-
3309


amide


 


GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACGGG-
3310


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCTPK-amide
3311


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK-
3312


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK-
3313


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK-
3314


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK-
3315


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK-
3316


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK-
3317


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYPK-amide
3318


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK-
3319


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCYPK-
3320


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK-
3321


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK-
3322


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK-
3323


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCYPK-
3324


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACTPK-amide
3325


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACTPK-
3326


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACTPK-
3327


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACTPK-
3328


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCACTPK-
3329


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK-
3330


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACTPK-
3331


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHC-amide
3332


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHC-
3333


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHC-
3334


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHC-amide
3335


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC-
3336


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHC-amide
3337


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCAC-amide
3338


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCAC-
3339


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCAC-
3340


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCAC-amide
3341


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC-
3342


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCAC-amide
3343


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCAC-amide
3344


 


GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCHCWGG-amide
3345


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYGG-amide
3346


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG-
3347


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG-
3348


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG-
3349


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG-
3350


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG-
3351


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG-
3352


amide


 


GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYGG-amide
3353


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACYGG-amide
3354


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACYGG-
3355


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACYGG-
3356


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACYGG-
3357


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG-
3358


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG-
3359


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACYGG-
3360


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCGGG-amide
3361


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG-
3362


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCGGG-
3363


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG-
3364


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG-
3365


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG-
3366


amide


 


GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACGGG-amide
3367


 


GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACGGG-
3368


amide


 


GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACGGG-
3369


amide


 


GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACGGG-
3370


amide


 


GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCACTP-
3371


amide


 


GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACGGG-
3372


amide


 


GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACGGG-
3373


amide


 


GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMAGKCACYGG-
3374


amide


 


GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMAGKCACGGG-
3375


amide


 


GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMAGKCACY-
3376


amide


 


Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYGG-
3377


amide


 


GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMAGKCACYGG-
3378


amide


 


GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMAGKCHCYGG-
3379


amide


 


GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMAGKCACYGG
3380


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCACYGG-amide
3381


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCACYGG
3382


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCACY-amide
3383


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCHCYGG-amide
3384


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCHCYGG
3385


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCHCYPK
3386


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCAC
3387


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[1Nal]GG-
3388


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[1Nal]PK-
3389


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[2Nal]GG-
3390


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[Cha]GG-
3391


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[MePhe]GG-
3392


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[BiPhA]GG-
3393


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKC[Aib]CYGG-
3394


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKC[Abu]CYGG-
3395


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[1Nal]
3396


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCAC[1Nal]GG-
3397


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[4Bip]-
3398


amide


 


GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCAC[4Bip]GG-
3399


amide


 


GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCGGG
3400
















TABLE 7F







Addit6ional useful OSK1 peptide analogs:


Ala27Substituted Series









SEQ



ID


Sequence/structure
NO:





GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK
3401


 


GVIINVSCKISRQCLEPCKKAGMRFGACMNGKCHCTPK
3402


 


GVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK
3403


 


GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK
3404


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3405


 


GVIINVSCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3406


 


GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK
3407


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK
3408


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK
3409


 


GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3410


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK-
3411


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK-amide
3412


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK
3413


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK-
3414


amide


 


GVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK-amide
3415


 


Ac-GVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK
3416


 


Ac-GVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK-
3417


amide


 


Ac-GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK
3418


 


GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK-amide
3419


 


Ac-GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK-
3420


amide


 


GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide
3421


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK
3422


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-
3423


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3424


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3425


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-
3426


amide


 


VIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK
3427


 


Ac-VIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK
3428


 


VIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide
3429


 


Ac-VIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-
3430


amide


 


GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK
3431


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK
3432


 


GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK-amide
3433


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK-
3434


amide


 


VIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3435


 


Ac-VIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3436


 


VIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3437


 


Ac-VIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-
3438


amide


 


NVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3439


 


Ac-NVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3440


 


NVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3441


 


Ac-NVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3442


 


KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3443


 


Ac-KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3444


 


KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3445


 


Ac-KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3446


 


CKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3447


 


Ac-CKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3448


 


CKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3449


 


Ac-CKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3450


 


GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK
3451


 


GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK-amide
3452


 


Ac-GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK
3453


 


Ac-GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK-
3454


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
3455


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
3456


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
3457


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-
3458


amide


 


GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK
3459


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK
3460


 


GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK-amide
3461


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK-
3462


amide


 


TIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3463


 


Ac-TIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3464


 


TIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3465


 


Ac-TIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-
3466


amide


 


GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK
3467


 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK
3468


 


GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide
3469


 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-
3470


amide


 


GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK
3471


 


GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3472


 


GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK
3473


 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK
3474


 


GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK-amide
3475


 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK-
3476


amide


 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK
3477


 


GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK-amide
3478


 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK-
3479


amide


 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3480


 


GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3481


 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-
3482


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCT
3483


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCTPK
3484


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCTPK
3485


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCTPK
3486


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCTPK
3487


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCTPK
3488


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK
3489


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCTPK
3490


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYPK
3491


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYPK
3492


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYPK
3493


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYPK
3494


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYPK
3495


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK
3496


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCYPK
3497


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYPK
3498


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCGCYPK
3499


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACFPK
3500


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACWPK
3501


 


GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYPK
3502


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACTPK
3503


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACTPK
3504


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACTPK
3505


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACTPK
3506


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCTPK
3507


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACTPK
3508


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACTPK
3509


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHC
3510


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHC
3511


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHC
3512


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHC
3513


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC
3514


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHC
3515


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHC
3516


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCAC
3517


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCAC
3518


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCAC
3519


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCAC
3520


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC
3521


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCAC
3522


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCAC
3523


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCGCYGG
3524


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYGG
3525


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYGG
3526


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYGG
3527


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYGG
3528


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYGG
3529


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG
3530


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYGG
3531


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACYGG
3532


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACYGG
3533


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACYGG
3534


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACYGG
3535


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYGG
3536


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACYGG
3537


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACYGG
3538


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYG
3539


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCGGG
3540


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCGGG
3541


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCGGG
3542


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCGGG
3543


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCGGG
3544


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG
3545


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACFGG
3546


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACGGG
3547


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACGGG
3548


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACGGG
3549


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACGGG
3550


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACGGG
3551


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACGGG
3552


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACGGG
3553


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACGG
3554


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYG
3555


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACGG
3556


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCTPK
3557


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCTPK
3558


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCTPK
3559


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCTPK
3560


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK
3561


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCTPK
3562


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYPK
3563


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYPK
3564


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYPK
3565


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYPK
3566


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYPK
3567


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK
3568


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYPK
3569


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACTPK
3570


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACTPK
3571


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACTPK
3572


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACTPK
3573


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCTPK
3574


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACTPK
3575


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACTPK
3576


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHC
3577


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHC
3578


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHC
3579


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHC
3580


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC
3581


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHC
3582


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCAC
3583


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCAC
3584


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCAC
3585


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCAC
3586


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC
3587


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCAC
3588


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCAC
3589


 


GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCHCYGG
3590


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYGG
3591


 


GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCHCYG
3592


 


GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYG
3593


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYGG
3594


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYGG
3595


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYGG
3596


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG
3597


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG
3598


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYGG
3599


 


GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYG
3600


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACYGG
3601


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACYGG
3602


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACYGG
3603


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACYGG
3604


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG
3605


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACYGG
3606


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACYGG
3607


 


GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACFGG
3608


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCGGG
3609


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCGGG
3610


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCGGG
3611


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCGGG
3612


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCGGG
3613


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG
3614


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACGGG
3615


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACGGG
3616


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACGGG
3617


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACGGG
3618


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCACTP
3619


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACTP
3620


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACTP
3621


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCTPK-amide
3622


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCTPK-
3623


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCTPK-
3624


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCTPK-
3625


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCTPK-
3626


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK-
3627


amide


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCTPK-
3628


amide


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYPK-amide
3629


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYPK-
3630


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYPK-
3631


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYPK-
3632


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYPK-
3633


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK-
3634


amide


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCYPK-
3635


amide


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACTPK-amide
3636


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACTPK-
3637


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACTPK-
3638


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACTPK-
3639


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACTPK-
3640


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACTPK-
3641


amide


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACTPK-
3642


amide


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHC-amide
3643


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHC-
3644


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHC-
3645


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHC-amide
3646


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC-
3647


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHC-amide
3648


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCAC-amide
3649


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCAC-
3650


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCAC-
3651


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCAC-amide
3652


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC-
3653


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCAC-amide
3654


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCAC-amide
3655


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYGG-amide
3656


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYGG-amide
3657


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYGG-
3658


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYGG-
3659


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYGG-
3660


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYGG-
3661


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG-
3662


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCFGG-amide
3663


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYG-amide
3664


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYG-amide
3665


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACYGG-amide
3666


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACYGG-
3667


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACYGG-
3668


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACYGG-
3669


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACYGG-
3670


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACYGG-
3671


amide


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACYGG-
3672


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYGG-amide
3673


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCGGG-amide
3674


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCGGG-
3675


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCGGG-
3676


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCGGG-
3677


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCGGG-
3678


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG-
3679


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACGGG-amide
3680


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACFGG-amide
3681


 


GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACGGG-amide
3682


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACGGG-
3683


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACGGG-
3684


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACGGG-
3685


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACGGG-
3686


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACGGG-
3687


amide


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACGGG-
3688


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCTPK-amide
3689


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCTPK-
3690


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCTPK-
3691


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCTPK-
3692


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCTPK-
3693


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK-
3694


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCTPK-
3695


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYPK-amide
3696


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYPK-
3697


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYPK-
3698


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYPK-
3699


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYPK-
3700


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK-
3701


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYPK-
3702


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACTPK-amide
3703


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACTPK-
3704


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACTPK-
3705


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACTPK-
3706


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCACTPK-
3707


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACTPK-
3708


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACTPK-
3709


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHC-amide
3710


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHC-
3711


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHC-
3712


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHC-amide
3713


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC-
3714


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHC-amide
3715


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCAC-amide
3716


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCAC-
3717


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCAC-
3718


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCAC-amide
3719


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC-
3720


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCAC-amide
3721


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCAC-amide
3722


 


GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCHCWGG-amide
3723


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYGG-amide
3724


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYGG-
3725


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYGG-
3726


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYGG-
3727


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG-
3728


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG-
3729


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYGG-
3730


amide


 


GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYGG-amide
3731


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACYGG-amide
3732


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACYGG-
3733


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACYGG-
3734


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACYGG-
3735


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG-
3736


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACYGG-
3737


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACYGG-
3738


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCGGG-amide
3739


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCGGG-
3740


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCGGG-
3741


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCGGG-
3742


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCGGG-
3743


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG-
3744


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACGGG-amide
3745


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACGGG-
3746


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACGGG-
3747


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACGGG-
3748


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCACTP-
3749


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACGGG-
3750


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACGGG-
3751


amide


 


GVIINVKCKISRQCLKPCK[Cpa]AGMRFGACMNGKCACYGG-
3752


amide


 


GVIINVKCKISRQCLKPCK[Cpa]AGMRFGACMNGKCACGGG-
3753


amide


 


GVIINVKCKISRQCLKPCK[Cpa]AGMRFGACMNGKCACY-
3754


amide


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYGG-
3755


amide


 


GVIINVKCKISRQCLKPCK[Aad]AGMRFGACMNGKCACYGG-
3756


amide


 


GVIINVKCKISRQCLKPCK[Aad]AGMRFGACMNGKCHCYGG-
3757


amide


 


GVIINVKCKISRQCLKPCK[Aad]AGMRFGACMNGKCACYGG
3758


 


GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCACYGG-amide
3759


 


GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCACYGG
3760


 


GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCACY-amide
3761


 


GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCHCYGG-amide
3762


 


GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCHCYGG
3763


 


GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCHCYPK
3764


 


GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCAC
3765


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[1Nal]GG-
3766


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[1Nal]PK-
3767


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[2Nal]GG-
3768


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[Cha]GG-
3769


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[MePhe]GG-
3770


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[BiPhA]GG-
3771


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKC[Aib]CYGG-
3772


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKC[Abu]CYGG-
3773


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[1Nal]
3774


 


GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCAC[1Nal]GG-
3775


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[4Bip]-
3776


amide


 


GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCAC[4Bip]GG-
3777


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCGGG
3778
















TABLE 7G







Additional useful OSK1 peptide analogs:


Ala 29 Substituted Series









SEQ



ID


Sequence/structure
NO:





GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK
3779


 


GVIINVSCKISRQCLEPCKKAGMRFGKCANGKCHCTPK
3780


 


GVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK
3781


 


GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK
3782


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3783


 


GVIINVSCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3784


 


GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK
3785


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK
3786


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK
3787


 


GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3788


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK-
3789


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK-amide
3790


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK
3791


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK-
3792


amide


 


GVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK-amide
3793


 


Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK
3794


 


Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK-
3795


amide


 


Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK
3796


 


GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK-amide
3797


 


Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK-
3798


amide


 


GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide
3799


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK
3800


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-
3801


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3802


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3803


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-
3804


amide


 


VIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK
3805


 


Ac-VIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK
3806


 


VIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide
3807


 


Ac-VIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-
3808


amide


 


GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK
3809


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK
3810


 


GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK-amide
3811


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK-
3812


amide


 


VIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3813


 


Ac-VIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3814


 


VIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3815


 


Ac-VIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-
3816


amide


 


NVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3817


 


Ac-NVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3818


 


NVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3819


 


Ac-NVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3820


 


KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3821


 


Ac-KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3822


 


KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3823


 


Ac-KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3824


 


CKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3825


 


Ac-CKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3826


 


CKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3827


 


Ac-CKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3828


 


GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK
3829


 


GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK-amide
3830


 


Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK
3831


 


Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK-
3832


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
3833


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
3834


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
3835


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-
3836


amide


 


GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK
3837


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK
3838


 


GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK-amide
3839


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK-
3840


amide


 


TIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3841


 


Ac-TIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3842


 


TIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3843


 


Ac-TIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-
3844


amide


 


GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK
3845


 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK
3846


 


GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide
3847


 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-
3848


amide


 


GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK
3849


 


GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3850


 


GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK
3851


 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK
3852


 


GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK-amide
3853


 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK-
3854


amide


 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK
3855


 


GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK-amide
3856


 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK-
3857


amide


 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3858


 


GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3859


 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-
3860


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCT
3861


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCTPK
3862


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCTPK
3863


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCTPK
3864


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCTPK
3865


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCTPK
3866


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK
3867


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCTPK
3868


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYPK
3869


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYPK
3870


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYPK
3871


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYPK
3872


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYPK
3873


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK
3874


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCYPK
3875


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYPK
3876


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCGCYPK
3877


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACFPK
3878


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACWPK
3879


 


GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYPK
3880


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACTPK
3881


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACTPK
3882


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACTPK
3883


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACTPK
3884


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCTPK
3885


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACTPK
3886


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACTPK
3887


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHC
3888


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHC
3889


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHC
3890


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHC
3891


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC
3892


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHC
3893


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHC
3894


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCAC
3895


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCAC
3896


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCAC
3897


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCAC
3898


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC
3899


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCAC
3900


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCAC
3901


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCGCYGG
3902


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYGG
3903


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYGG
3904


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYGG
3905


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYGG
3906


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYGG
3907


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG
3908


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYGG
3909


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACYGG
3910


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACYGG
3911


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACYGG
3912


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACYGG
3913


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYGG
3914


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACYGG
3915


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACYGG
3916


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYG
3917


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCGGG
3918


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCGGG
3919


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCGGG
3920


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCGGG
3921


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCGGG
3922


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG
3923


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACFGG
3924


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACGGG
3925


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACGGG
3926


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACGGG
3927


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACGGG
3928


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACGGG
3929


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACGGG
3930


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACGGG
3931


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACGG
3932


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYG
3933


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACGG
3934


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCTPK
3935


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCTPK
3936


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCTPK
3937


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCTPK
3938


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK
3939


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCTPK
3940


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYPK
3941


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYPK
3942


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYPK
3943


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYPK
3944


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYPK
3945


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK
3946


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYPK
3947


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACTPK
3948


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACTPK
3949


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACTPK
3950


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACTPK
3951


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCTPK
3952


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACTPK
3953


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACTPK
3954


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHC
3955


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHC
3956


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHC
3957


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHC
3958


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC
3959


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHC
3960


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCAC
3961


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCAC
3962


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCAC
3963


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCAC
3964


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC
3965


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCAC
3966


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCAC
3967


 


GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCHCYGG
3968


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYGG
3969


 


GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCHCYG
3970


 


GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYG
3971


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYGG
3972


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYGG
3973


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYGG
3974


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG
3975


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG
3976


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYGG
3977


 


GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYG
3978


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACYGG
3979


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACYGG
3980


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACYGG
3981


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACYGG
3982


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG
3983


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACYGG
3984


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACYGG
3985


 


GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACFGG
3986


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCGGG
3987


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCGGG
3988


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCGGG
3989


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCGGG
3990


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCGGG
3991


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG
3992


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACGGG
3993


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACGGG
3994


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACGGG
3995


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACGGG
3996


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCACTP
3997


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACTP
3998


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACTP
3999


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCTPK-amide
4000


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCTPK-
4001


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCTPK-
4002


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCTPK-
4003


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCTPK-
4004


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK-
4005


amide


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCTPK-
4006


amide


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYPK-amide
4007


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYPK-
4008


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYPK-
4009


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYPK-
4010


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYPK-
4011


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK-
4012


amide


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCYPK-
4013


amide


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACTPK-amide
4014


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACTPK-
4015


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACTPK-
4016


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACTPK-
4017


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACTPK-
4018


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACTPK-
4019


amide


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACTPK-
4020


amide


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHC-amide
4021


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHC-
4022


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHC-
4023


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHC-amide
4024


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC-
4025


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHC-amide
4026


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCAC-amide
4027


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCAC-
4028


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCAC-
4029


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCAC-amide
4030


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC-
4031


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCAC-amide
4032


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCAC-amide
4033


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYGG-amide
4034


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYGG-amide
4035


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYGG-
4036


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYGG-
4037


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYGG-
4038


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYGG-
4039


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG-
4040


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCFGG-amide
4041


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYG-amide
4042


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYG-amide
4043


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACYGG-amide
4044


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACYGG-
4045


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACYGG-
4046


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACYGG-
4047


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACYGG-
4048


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACYGG-
4049


amide


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACYGG-
4050


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYGG-amide
4051


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCGGG-amide
4052


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCGGG-
4053


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCGGG-
4054


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCGGG-
4055


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCGGG-
4056


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG-
4057


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACGGG-amide
4058


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACFGG-amide
4059


 


GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACGGG-amide
4060


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACGGG-
4061


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACGGG-
4062


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACGGG-
4063


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACGGG-
4064


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACGGG-
4065


amide


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACGGG-
4066


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCTPK-amide
4067


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCTPK-
4068


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCTPK-
4069


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCTPK-
4070


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCTPK-
4071


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK-
4072


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCTPK-
4073


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYPK-amide
4074


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYPK-
4075


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYPK-
4076


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYPK-
4077


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYPK-
4078


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK-
4079


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYPK-
4080


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACTPK-amide
4081


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACTPK-
4082


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACTPK-
4083


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACTPK-
4084


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCACTPK-
4085


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACTPK-
4086


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACTPK-
4087


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHC-amide
4088


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHC-
4089


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHC-
4090


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHC-amide
4091


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC-
4092


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHC-amide
4093


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCAC-amide
4094


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCAC-
4095


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCAC-
4096


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCAC-amide
4097


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC-
4098


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCAC-amide
4099


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCAC-amide
4100


 


GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCHCWGG-amide
4101


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYGG-amide
4102


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYGG-
4103


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYGG-
4104


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYGG-
4105


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG-
4106


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG-
4107


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYGG-
4108


amide


 


GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYGG-amide
4109


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACYGG-amide
4110


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACYGG-
4111


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACYGG-
4112


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACYGG-
4113


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG-
4114


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACYGG-
4115


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACYGG-
4116


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCGGG-amide
4117


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCGGG-
4118


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCGGG-
4119


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCGGG-
4120


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCGGG-
4121


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG-
4122


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACGGG-amide
4123


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACGGG-
4124


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACGGG-
4125


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACGGG-
4126


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCACTP-
4127


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACGGG-
4128


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACGGG-
4129


amide


 


GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCANGKCACYGG-
4130


amide


 


GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCANGKCACGGG-
4131


amide


 


GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCANGKCACY-
4132


amide


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYGG-
4133


amide


 


GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCANGKCACYGG-
4134


amide


 


GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCANGKCHCYGG-
4135


amide


 


GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCANGKCACYGG
4136


 


GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCACYGG-amide
4137


 


GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCACYGG
4138


 


GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCACY-amide
4139


 


GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCHCYGG-amide
4140


 


GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCHCYGG
4141


 


GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCHCYPK
4142


 


GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCAC
4143


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[1Nal]GG-
4144


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[1Nal]PK-
4145


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[2Nal]GG-
4146


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[Cha]GG-
4147


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[MePhe]GG-
4148


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[BiPhA]GG-
4149


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKC[Aib]CYGG-
4150


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKC[Abu]CYGG-
4151


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[1Nal]
4152


 


GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCAC[1Nal]GG-
4153


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[4Bip]-
4154


amide


 


GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCAC[4Bip]GG-
4155


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCGGG
4156
















TABLE 7H







Additional useful OSK1 peptide analogs:


Ala 30 Substituted Series









SEQ



ID


Sequence/structure
NO:





GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK
4157


 


GVIINVSCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK
4158


 


GVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK
4159


 


GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK
4160


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4161


 


GVIINVSCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4162


 


GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK
4163


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK
4164


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK
4165


 


GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4166


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK-
4167


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK-amide
4168


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK
4169


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK-
4170


amide


 


GVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK-amide
4171


 


Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK
4172


 


Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK-
4173


amide


 


Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK
4174


 


GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK-amide
4175


 


Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK-
4176


amide


 


GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide
4177


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK
4178


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-
4179


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4180


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4181


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-
4182


amide


 


VIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK
4183


 


Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK
4184


 


VIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide
4185


 


Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-
4186


amide


 


GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK
4187


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK
4188


 


GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK-amide
4189


 


Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK-
4190


amide


 


VIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4191


 


Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4192


 


VIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4193


 


Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-
4194


amide


 


NVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4195


 


Ac-NVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4196


 


NVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4197


 


Ac-NVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4198


 


KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4199


 


Ac-KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4200


 


KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4201


 


Ac-KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4202


 


CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4203


 


Ac-CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4204


 


CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4205


 


Ac-CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4206


 


GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK
4207


 


GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK-amide
4208


 


Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK
4209


 


Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK-
4210


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
4211


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
4212


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
4213


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-
4214


amide


 


GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK
4215


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK
4216


 


GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK-amide
4217


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK-
4218


amide


 


TIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4219


 


Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4220


 


TIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4221


 


Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-
4222


amide


 


GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK
4223


 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK
4224


 


GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide
4225


 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-
4226


amide


 


GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK
4227


 


GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4228


 


GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK
4229


 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK
4230


 


GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK-amide
4231


 


Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK-
4232


amide


 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK
4233


 


GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK-amide
4234


 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK-
4235


amide


 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4236


 


GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4237


 


Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-
4238


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCT
4239


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCTPK
4240


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK
4241


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCTPK
4242


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK
4243


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK
4244


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK
4245


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK
4246


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYPK
4247


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK
4248


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCYPK
4249


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK
4250


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK
4251


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK
4252


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK
4253


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYPK
4254


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCGCYPK
4255


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACFPK
4256


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACWPK
4257


 


GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYPK
4258


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACTPK
4259


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACTPK
4260


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACTPK
4261


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACTPK
4262


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK
4263


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK
4264


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACTPK
4265


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHC
4266


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHC
4267


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHC
4268


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHC
4269


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC
4270


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHC
4271


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHC
4272


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCAC
4273


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCAC
4274


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCAC
4275


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCAC
4276


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC
4277


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCAC
4278


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCAC
4279


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCGCYGG
4280


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYGG
4281


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG
4282


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG
4283


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG
4284


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG
4285


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG
4286


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYGG
4287


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACYGG
4288


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACYGG
4289


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACYGG
4290


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACYGG
4291


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG
4292


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG
4293


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACYGG
4294


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYG
4295


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCGGG
4296


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG
4297


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG
4298


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG
4299


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG
4300


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG
4301


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACFGG
4302


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACGGG
4303


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACGGG
4304


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACGGG
4305


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACGGG
4306


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACGGG
4307


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG
4308


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACGGG
4309


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACGG
4310


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYG
4311


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACGG
4312


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK
4313


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK
4314


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK
4315


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK
4316


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK
4317


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK
4318


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYPK
4319


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK
4320


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYPK
4321


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK
4322


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK
4323


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK
4324


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYPK
4325


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACTPK
4326


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACTPK
4327


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACTPK
4328


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACTPK
4329


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK
4330


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK
4331


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACTPK
4332


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHC
4333


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHC
4334


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHC
4335


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHC
4336


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC
4337


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHC
4338


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCAC
4339


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCAC
4340


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCAC
4341


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCAC
4342


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC
4343


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCAC
4344


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCAC
4345


 


GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCHCYGG
4346


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYGG
4347


 


GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCHCYG
4348


 


GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYG
4349


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG
4350


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG
4351


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG
4352


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG
4353


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG
4354


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG
4355


 


GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYG
4356


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACYGG
4357


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACYGG
4358


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACYGG
4359


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACYGG
4360


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG
4361


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG
4362


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACYGG
4363


 


GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACFGG
4364


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCGGG
4365


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG
4366


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCGGG
4367


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG
4368


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG
4369


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG
4370


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACGGG
4371


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACGGG
4372


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACGGG
4373


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACGGG
4374


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCACTP
4375


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACTP
4376


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACTP
4377


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCTPK-amide
4378


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK-
4379


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCTPK-
4380


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK-
4381


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK-
4382


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK-
4383


amide


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK-
4384


amide


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYPK-amide
4385


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK-
4386


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCYPK-
4387


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK-
4388


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK-
4389


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK-
4390


amide


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK-
4391


amide


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACTPK-amide
4392


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACTPK-
4393


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACTPK-
4394


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACTPK-
4395


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACTPK-
4396


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK-
4397


amide


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACTPK-
4398


amide


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHC-amide
4399


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHC-
4400


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHC-
4401


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHC-amide
4402


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC-
4403


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHC-amide
4404


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCAC-amide
4405


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCAC-
4406


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCAC-
4407


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCAC-amide
4408


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC-
4409


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCAC-amide
4410


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCAC-amide
4411


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYGG-amide
4412


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYGG-amide
4413


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG-
4414


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG-
4415


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG-
4416


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG-
4417


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG-
4418


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCFGG-amide
4419


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYG-amide
4420


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYG-amide
4421


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACYGG-amide
4422


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACYGG-
4423


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACYGG-
4424


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACYGG-
4425


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACYGG-
4426


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG-
4427


amide


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACYGG-
4428


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYGG-amide
4429


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCGGG-amide
4430


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG-
4431


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG-
4432


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG-
4433


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG-
4434


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG-
4435


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACGGG-amide
4436


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACFGG-amide
4437


 


GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACGGG-amide
4438


 


GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACGGG-
4439


amide


 


GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACGGG-
4440


amide


 


GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACGGG-
4441


amide


 


GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACGGG-
4442


amide


 


GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG-
4443


amide


 


GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACGGG-
4444


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCTPK-amide
4445


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK-
4446


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK-
4447


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK-
4448


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK-
4449


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK-
4450


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK-
4451


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYPK-amide
4452


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK-
4453


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYPK-
4454


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK-
4455


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK-
4456


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK-
4457


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYPK-
4458


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACTPK-amide
4459


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACTPK-
4460


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACTPK-
4461


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACTPK-
4462


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCACTPK-
4463


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK-
4464


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACTPK-
4465


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHC-amide
4466


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHC-
4467


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHC-
4468


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHC-amide
4469


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC-
4470


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHC-amide
4471


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCAC-amide
4472


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCAC-
4473


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCAC-
4474


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCAC-amide
4475


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC-
4476


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCAC-amide
4477


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCAC-amide
4478


 


GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCHCWGG-amide
4479


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYGG-amide
4480


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG-
4481


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG-
4482


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG-
4483


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG-
4484


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG-
4485


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG-
4486


amide


 


GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYGG-amide
4487


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACYGG-amide
4488


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACYGG-
4489


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACYGG-
4490


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACYGG-
4491


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG-
4492


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG-
4493


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACYGG-
4494


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCGGG-amide
4495


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG-
4496


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCGGG-
4497


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG-
4498


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG-
4499


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG-
4500


amide


 


GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACGGG-amide
4501


 


GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACGGG-
4502


amide


 


GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACGGG-
4503


amide


 


GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACGGG-
4504


amide


 


GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCACTP-
4505


amide


 


GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACGGG-
4506


amide


 


GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACGGG-
4507


amide


 


GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMAGKCACYGG-
4508


amide


 


GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMAGKCACGGG-
4509


amide


 


GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMAGKCACY-
4510


amide


 


Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYGG-
4511


amide


 


GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMAGKCACYGG-
4512


amide


 


GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMAGKCHCYGG-
4513


amide


 


GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMAGKCACYGG
4514


 


GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCACYGG-amide
4515


 


GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCACYGG
4516


 


GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCACY-amide
4517


 


GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCHCYGG-amide
4518


 


GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCHCYGG
4519


 


GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCHCYPK
4520


 


GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCAC
4521


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[1Nal]GG-
4522


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[1Nal]PK-
4523


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[2Nal]GG-
4524


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[Cha]GG-
4525


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[MePhe]GG-
4526


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[BiPhA]GG-
4527


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKC[Aib]CYGG-
4528


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKC[Abu]CYGG-
4529


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[1Nal]
4530


 


GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCAC[1Nal]GG-
4531


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[4Bip]-
4532


amide


 


GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCAC[4Bip]GG-
4533


amide


 


GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCGGG
4534
















TABLE 7I







Additional useful OSK1 peptide analogs: Combined


Ala-11, 12, 27, 29, 30 Substituted Series









SEQ



ID


Sequence/structure
NO:





GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK
4535


 


GVIINVSCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK
4536


 


GVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK
4537


 


GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK
4538


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4539


 


GVIINVSCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4540


 


GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK
4541


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK
4542


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK
4543


 


GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4544


 


Ac-GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK-
4545


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK-amide
4546


 


Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK
4547


 


Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK-
4548


amide



 


GVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK-amide
4549


 


Ac-GVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK
4550


 


Ac-GVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK-
4551


amide



 


Ac-GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK
4552


 


GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK-amide
4553


 


Ac-GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK-
4554


amide



 


GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-amide
4555


 


Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK
4556


 


Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-
4557


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4558


 


Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4559


 


Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-
4560


amide



 


VIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK
4561


 


Ac-VIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK
4562


 


VIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-amide
4563


 


Ac-VIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-
4564


amide



 


GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK
4565


 


Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK
4566


 


GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK-amide
4567


 


Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK-
4568


amide



 


VIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4569


 


Ac-VIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4570


 


VIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4571


 


Ac-VIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-
4572


amide



 


NVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4573


 


Ac-NVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4574


 


NVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4575


 


Ac-NVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4576


 


KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4577


 


Ac-KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4578


 


KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4579


 


Ac-KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4580


 


CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4581


 


Ac-CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4582


 


CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4583


 


Ac-CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4584


 


GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK
4585


 


GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK-amide
4586


 


Ac-GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK
4587


 


Ac-GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK-
4588


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK
4589


 


GVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
4590


 


Ac-GVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK
4591


 


Ac-GVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK-
4592


amide



 


GVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK
4593


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK
4594


 


GVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK-amide
4595


 


Ac-GVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK-
4596


amide



 


TIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4597


 


Ac-TIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4598


 


TIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4599


 


Ac-TIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-
4600


amide



 


GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK
4601


 


Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK
4602


 


GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-amide
4603


 


Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-
4604


amide



 


GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK
4605


 


GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4606


 


GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK
4607


 


Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK
4608


 


GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK-amide
4609


 


Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK-
4610


amide



 


Ac-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK
4611


 


GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK-amide
4612


 


Ac-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK-
4613


amide



 


Ac-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4614


 


GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4615


 


Ac-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-
4616


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCT
4617


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCTPK
4618


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCTPK
4619


 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCTPK
4620


 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCTPK
4621


 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCTPK
4622


 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCTPK
4623


 


GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCTPK
4624


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYPK
4625


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYPK
4626


 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYPK
4627


 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYPK
4628


 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYPK
4629


 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYPK
4630


 


GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCYPK
4631


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYPK
4632


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCGCYPK
4633


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACFPK
4634


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACWPK
4635


 


GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYPK
4636


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACTPK
4637


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACTPK
4638


 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACTPK
4639


 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACTPK
4640


 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCTPK
4641


 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACTPK
4642


 


GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACTPK
4643


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHC
4644


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHC
4645


 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHC
4646


 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHC
4647


 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC
4648


 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHC
4649


 


GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHC
4650


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCAC
4651


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCAC
4652


 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCAC
4653


 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCAC
4654


 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC
4655


 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCAC
4656


 


GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCAC
4657


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCGCYGG
4658


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYGG
4659


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYGG
4660


 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYGG
4661


 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYGG
4662


 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYGG
4663


 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYGG
4664


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYGG
4665


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACYGG
4666


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACYGG
4667


 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACYGG
4668


 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACYGG
4669


 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYGG
4670


 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACYGG
4671


 


GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACYGG
4672


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYG
4673


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCGGG
4674


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCGGG
4675


 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCGGG
4676


 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCGGG
4677


 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCGGG
4678


 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCGGG
4679


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACFGG
4680


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACGGG
4681


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACGGG
4682


 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACGGG
4683


 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACGGG
4684


 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACGGG
4685


 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACGGG
4686


 


GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACGGG
4687


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACGG
4688


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYG
4689


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACGG
4690


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCTPK
4691


 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCTPK
4692


 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCTPK
4693


 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCTPK
4694


 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCTPK
4695


 


GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCTPK
4696


 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYPK
4697


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYPK
4698


 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYPK
4699


 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYPK
4700


 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYPK
4701


 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYPK
4702


 


GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYPK
4703


 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACTPK
4704


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACTPK
4705


 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACTPK
4706


 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACTPK
4707


 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCTPK
4708


 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACTPK
4709


 


GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACTPK
4710


 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHC
4711


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHC
4712


 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHC
4713


 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHC
4714


 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC
4715


 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHC
4716


 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCAC
4717


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCAC
4718


 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCAC
4719


 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCAC
4720


 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC
4721


 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCAC
4722


 


GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCAC
4723


 


GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCHCYGG
4724


 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYGG
4725


 


GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCHCYG
4726


 


GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYG
4727


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYGG
4728


 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYGG
4729


 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYGG
4730


 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG
4731


 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYGG
4732


 


GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYGG
4733


 


GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYG
4734


 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACYGG
4735


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACYGG
4736


 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACYGG
4737


 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACYGG
4738


 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG
4739


 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACYGG
4740


 


GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACYGG
4741


 


GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACFGG
4742


 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCGGG
4743


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCGGG
4744


 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCGGG
4745


 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCGGG
4746


 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCGGG
4747


 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCGGG
4748


 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACGGG
4749


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACGGG
4750


 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACGGG
4751


 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACGGG
4752


 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCACTP
4753


 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACTP
4754


 


GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACTP
4755


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCTPK-amide
4756


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCTPK-
4757


amide



 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCTPK-
4758


amide



 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCTPK-
4759


amide



 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCTPK-
4760


amide



 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCTPK-
4761


amide



 


GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCTPK-
4762


amide



 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYPK-amide
4763


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYPK-
4764


amide



 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYPK-
4765


amide



 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYPK-
4766


amide



 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYPK-
4767


amide



 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYPK-
4768


amide



 


GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCYPK-
4769


amide



 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACTPK-amide
4770


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACTPK-
4771


amide



 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACTPK-
4772


amide



 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACTPK-
4773


amide



 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACTPK-
4774


amide



 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACTPK-
4775


amide



 


GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACTPK-
4776


amide



 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHC-amide
4777


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHC-
4778


amide



 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHC-
4779


amide



 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHC-amide
4780


 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC-
4781


amide



 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHC-
4782


amide



 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCAC-amide
4783


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCAC-
4784


amide



 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCAC-
4785


amide



 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCAC-amide
4786


 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC-
4787


amide



 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCAC-amide
4788


 


GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCAC-amide
4789


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYGG-amide
4790


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYGG-amide
4791


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYGG-
4792


amide



 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYGG-
4793


amide



 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYGG-
4794


amide



 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYGG-
4795


amide



 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYGG-
4796


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCFGG-amide
4797


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYG-amide
4798


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYG-amide
4799


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACYGG-amide
4800


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACYGG-
4801


amide



 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACYGG-
4802


amide



 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACYGG-
4803


amide



 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACYGG-
4804


amide



 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACYGG-
4805


amide



 


GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACYGG-
4806


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYGG-amide
4807


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCGGG-amide
4808


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCGGG-
4809


amide



 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCGGG-
4810


amide



 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCGGG-
4811


amide



 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCGGG-
4812


amide



 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCGGG-
4813


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACGGG-amide
4814


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACFGG-amide
4815


 


GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACGGG-amide
4816


 


GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACGGG-
4817


amide



 


GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACGGG-
4818


amide



 


GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACGGG-
4819


amide



 


GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACGGG-
4820


amide



 


GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACGGG-
4821


amide



 


GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACGGG-
4822


amide



 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCTPK-amide
4823


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCTPK-
4824


amide



 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCTPK-
4825


amide



 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCTPK-
4826


amide



 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCTPK-
4827


amide



 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCTPK-
4828


amide



 


GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCTPK-
4829


amide



 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYPK-amide
4830


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYPK-
4831


amide



 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYPK-
4832


amide



 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYPK-
4833


amide



 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYPK-
4834


amide



 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYPK-
4835


amide



 


GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYPK-
4836


amide



 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACTPK-amide
4837


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACTPK-
4838


amide



 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACTPK-
4839


amide



 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACTPK-
4840


amide



 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCACTPK-
4841


amide



 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACTPK-
4842


amide



 


GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACTPK-
4843


amide



 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHC-amide
4844


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHC-
4845


amide



 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHC-
4846


amide



 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHC-amide
4847


 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC-
4848


amide



 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHC-amide
4849


 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCAC-amide
4850


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCAC-
4851


amide



 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCAC-
4852


amide



 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCAC-amide
4853


 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC-
4854


amide



 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCAC-amide
4855


 


GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCAC-amide
4856


 


GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCHCWGG-amide
4857


 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYGG-amide
4858


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYGG-
4859


amide



 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYGG-
4860


amide



 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYGG-
4861


amide



 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG-
4862


amide



 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYGG-
4863


amide



 


GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYGG-
4864


amide



 


GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYGG-amide
4865


 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACYGG-amide
4866


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACYGG-
4867


amide



 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACYGG-
4868


amide



 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACYGG-
4869


amide



 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG-
4870


amide



 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACYGG-
4871


amide



 


GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACYGG-
4872


amide



 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCGGG-amide
4873


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCGGG-
4874


amide



 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCGGG-
4875


amide



 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCGGG-
4876


amide



 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCGGG-
4877


amide



 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCGGG-
4878


amide



 


GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACGGG-amide
4879


 


GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACGGG-
4880


amide



 


GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACGGG-
4881


amide



 


GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACGGG-
4882


amide



 


GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCACTP-
4883


amide



 


GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACGGG-
4884


amide



 


GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACGGG-
4885


amide



 


GVIINVKCKIAAQCLKPCK[Cpa]AGMRFGACAAGKCACYGG-
4886


amide



 


GVIINVKCKIAAQCLKPCK[Cpa]AGMRFGACAAGKCACGGG-
4887


amide



 


GVIINVKCKIAAQCLKPCK[Cpa]AGMRFGACAAGKCACY-
4888


amide



 


Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYGG-
4889


amide



 


GVIINVKCKIAAQCLKPCK[Aad]AGMRFGACAAGKCACYGG-
4890


amide



 


GVIINVKCKIAAQCLKPCK[Aad]AGMRFGACAAGKCHCYGG-
4891


amide



 


GVIINVKCKIAAQCLKPCK[Aad]AGMRFGACAAGKCACYGG
4892


 


GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCACYGG-amide
4893


 


GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCACYGG
4894


 


GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCACY-amide
4895


 


GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCHCYGG-amide
4896


 


GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCHCYGG
4897


 


GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCHCYPK
4898


 


GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCAC
4899


 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[1Nal]GG-
4900


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[1Nal]PK-
4901


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[2Nal]GG-
4902


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[Cha]GG-
4903


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[MePhe]GG-
4904


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[BiPhA]GG-
4905


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKC[Aib]CYGG-
4906


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKC[Abu]CYGG-
4907


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[1Nal]
4908


 


GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCAC[1Nal]GG-
4909


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[4Bip]-
4910


amide



 


GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCAC[4Bip]GG-
4911


amide



 


GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCGGG
4912


 


GIINVKCKISAQCLKPCRDAGMRFGKCMNGKCACTPK
4916
















TABLE 7J







Additional useful OSK1 peptide analogs











SEQ



Short-hand
ID


Sequence/Structure
designation
NO:





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCGC
[Gly34]OSK1
4930


TPK




 


GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCSC
[Ser34]OSK1
4931


TPK




 


GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCTC
[Thr34]OSK1
4932


TPK




 


GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCNC
[Asn34]OSK1
4933


TPK




 


GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCVC
[Val34]OSK1
4934


TPK




 


GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCLC
[Leu34]OSK1
4935


TPK




 


GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCIC
[Ile34]OSK1
4936


TPK




 


GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCPC
[Pro34]OSK1
4937


TPK




 


GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCMC
[Met34]OSK1
4938


TPK




 


GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCQC
[Gln34]OSK1
4939


TPK




 


GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCKC
[Lys34]OSK1
4940


TPK




 


GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCDC
[Asp34]OSK1
4941


TPK




 


WVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC
[Trp1]OSK1
4942


WPK




 


GVWINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC
[Trp3]OSK1
4943


TPK




 


GVIIWVKCKISRQCLEPCKKAGMRFGKCMNGKCHC
[Trp5]OSK1
4944


TPK




 


[1Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMN
[1Nal1]OSK1
4945


GKCHCWPK




 


GV[1Nal]INVKCKISRQCLEPCKKAGMRFGKCMN
[1Nal3]OSK1
4946


GKCHCTPK




 


GVII[1Nal]VKCKISRQCLEPCKKAGMRFGKCMN
[1Nal5]OSK1
4947


GKCHCTPK




 


GVIKNVKCKISRQCLEPCKKAGMRFGKCMNGKCHC
[Lys4]OSK1
4948


TPK




 


GVIKNVKCKISRQCLEPCKKAGMRFGKCMNGKCAC
[Lys4, Ala34]OSK1
4949


TPK




 


[1Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMN
[1Nal1;
4950


GKCACWPK
Ala34]OSK1



 


GV[1Nal]INVKCKISRQCLEPCKKAGMRFGKCMN
[1Nal3;
4951


GKCACTPK
Ala34]OSK1



 


GVII[1Nal]VKCKISRQCLEPCKKAGMRFGKCMN
[1Nal5;
4952


GKCACTPK
Ala34]OSK1



 


WVIINVKCKISRQCLEPCKKAGMRFCKCMNGKCAC
[Trp1;
4953


WPK
Ala34]OSK1



 


GVWINVKCKISRQCLEPCKKAGMRFGKCMNGKCAC
[Trp3;
4954


TPK
Ala34]OSK1



 


GVIIWVKCKISRQCLEPCKKAGMRFGKCMNGKCAC
[Trp5;
4955


TPK
Ala34]OSK1



 


WVWIWVKCKISRQCLEPCKKAGMRFGKCMNGKCAC
[Trp1, 3, 5;
4956


TPK
Ala34]OSK1



 


[1Nal]V[1Nal]I[1Nal]VKCKISRQCLEPCKK
[1Nal1, 3, 5;
4957


AGMRFGKCMNGKCACTPK
Ala34]OSK1



 


CKISRQCLEPCKKAGMRFGKCMNGKCACTPK
Δ1-7,
4958



[Ala34]OSK1



 


KCKISRQCLEPCKKAGMRFGKCMNGKCACTPK
Δ1-6,
4959



[Ala34]OSK1



 


GVIINVKCKI[1Nal]RQCLEPCKKAGMRFGKCAN
[1Nal11; Ala29,
4960


GKCACWPK
34]




Osk-1



 


GVIINVKCKIRRQCLEPCKKAGMRFGKCANGKCAC
[Arg11; Ala29,
4961


WPK
34]




Osk-1



 


GVIINVKCKISRQCEEPCKKAGMRFGKCANGKCAC
[Glu15; Ala29,
4962


WPK
34]




Osk-1



 


GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCAC
[Arg11;
4963


WPK
Ala34]Osk-1



 


GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCAC
[Glu15;
4964


WPK
Ala34]Osk-1



 


CKIRRQCEEPCKKAGMRFGKCANGKCACTPK
Δ1-7, [Arg11;
4965



Glu15; Ala29, 34]




OSK1



 


GVIINVKCKIRRQCEEPCKKAGMRFGKCANGKCAC
[Arg11; Glu15;
4966


TPK
Ala29, 34]OSK1



 


CVIINVKCKIRRQCEEPCKKAGMRFGKCANGKCAC
[Cys1, 37; Arg11;
4967


TCK
Glu15;




Ala29, 34]OSK1



 


GVIINVKCKIRAQCEEPCKKAGMRFGKCANGKCAC
[Arg11; Ala12,
4968


TPK
29, 34; Glu15]




OSK1



 


GVIINVKCKIRAQCEEPCKKAGMRFGKCANGKCAC
[Arg11; Glu15;
4969


TPK-NH2
Ala12, 29, 34]OSK1-




amide



 


Ac-
Ac-[Arg11;
4970


GVIINVKCKIRAQCEEPCKKAGMRFGKCANGKCAC
Glu15; Ala12,



TPK-NH2
29, 34]OSK1-




amide



 


GVIINVKCKI[1Nal]AQCEEPCKKAGMRFGKCAN
[1Nal11];
4971


GKCACTPK
Glu15;




Ala12, 29, 34]OSK1



 


GVIINVKCKISRQCLEPCKKAGMRFGKCANGKC[1Nal]
[Ala29; 1Nal34]
4972


CWPK
Osk-1



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHC
[Ala12; Lys16;
4973


TPK
Asp20]Osk-1



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC
[Ala12, 34; Lys16;
4974


TPK
Asp20]Osk-1



 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC
[Ala12, 29, 34; Lys16;
4975


TPK
Asp20]Osk-1



 


GVIINVKCKIRAQCLKPCKDAGMRFGKCANGKCAC
[Arg11; Ala12,
4976


TPK
29, 34; Lys16; Asp20]




Osk-1



 


GVIINVKCKISAQCEKPCKDAGMRFGKCANGKCAC
[Ala12, 29, 34;
4977


TPK
Glu15, Lys16;




Asp20]Osk-1



 


GVIINVKCKI[1Nal]AQCLKPCKDAGMRFGKCAN
[1Nal11; Ala12,
4978


GKCACTPK
29, 34; Lys16;




Asp20]Osk-1



 


GVIINVKCKIRAQCEKPCKDAGMRFGKCANGKCAC
[Arg11; Ala12,
4979


TPK
29, 34; Glu15; Lys16;




Asp20]Osk-1



 


GVIINVKCKIRAQCEKPCKDAGMRFGKCMNGKCAC
[Arg11; Ala12,
4980


TPK
34; Glu15; Lys16;




Asp20]Osk-1



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKC[1Nal]
[A12, K16,
4981


CTPK
D20, Nal34]-




OSK1



 


GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC
[A12, K16,
4982


TPK
D20, A34]-




OSK1



 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKC[1Nal]
[A12, K16,
4983


CTPK
D20, A29,




Nal34]-OSK1



 


GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC
[A12, K16,
4984


TPK
D20, A29,




A34]-OSK1



 


{Acetyl}GVIINVKCKISRQCLEPCK(Glycyl)
Ac-
4985


KAGMRFGKCMNGKCACTPK
[K(Gly)19, Ala34]-




Osk1



 


{Acetyl}GVIK(Glycyl)NVKCKISRQCLEPCK
Ac-
4986


KAGMRFGKCMNGKCACTPK
[K(Gly)4, Ala34]-




Osk1



 


{Acetyl}GVIINVKCKISRQCLEPCKKAGMRFGK
Ac-
4987


CMNGKCACTPK(Glycyl)
[Ala34, K(Gly)




38]-Osk1



 


GVIINVKCKISRQCLEPCKKAGMRFGKCANGKC[1Nal]
[A29, Nal34]-
4988


CTPK
OSK1



 


CKISRQCLKPCKDAGMRFGKCMNGKCHC{Amide}
OSK1[des1-7,
4989



E16K,




K20D, des36-38]-




amide



 


GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC
OSK1-
4990


TPK
K16, D20, A34



 


GVIINVKCKI[1Nal]AQCLEPCKKAGMRFGKCAN
Osk-
4991


GKC[1Nal]CTPK
1[1Nal11, A12,




A29, 1-Na134]



 


GVIINVKCKI[1Nal]AQCLEPCKKAGMRFGKCAN
[1Nal11, A12, A29,
4992


GKC[1Nal]CTEK
1Nal34, E37]




Osk-1



 


GVIINVKCKI[1Nal]AQCEEPCKKAGMRFGKCAN
Osk-1[1-
4993


GKC[1Nal]CEEK
Nal11, A12, E15,




A29, 1Nal34,




E36, E37]



 


GVIINVKCKI[1Nal]AQCLEPCKKAGFRFGKCAN
[1Nal11, A12, F23,
4994


GKC[1Nal]CTPK
A29, 1Nal34]




Osk-1



 


GVIINVKCKI[1Nal]AQCLEPCKKAG[Nle]RFG
[1Nal11, A12,
4995


KCANGKC[1Nal]CTEK
Nle23, A29, 1Nal34,




E37]Osk-1



 


GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCAC
[Pro12, Lys16,
4996


TY[Nle]
Asp20,




Ala34, Tyr37, Nle38]




Osk-1-




amide



 


GVIINVKCKISPQCLOPCKEAGMRFGKCMNGKCAC
[P12, Orn16, E20,
4997


TY[Nle]
A34, Y37, Nle38]




Osk-1-




amide



 


NVKCKISRQCLEPCKKAGMRFGKCANGKC[1Nal]
des1-4, [A29,
4998


CTPK
Nal34]-OSK1



 


NVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK
des1-4, [A29,
4999



A34]-OSK1



 


GVIINVKCKIRRQCLEPCKKAGMRFGKCANGKCAC
[R11, A29,
5000


TPK
A34]-OSK1



 


GVIINVKCKIRAQCLEPCKKAGMRFGKCANGKCAC
[R11, A12,
5001


TPK
A29, A34]-




OSK1



 


CKISRQCLEPCKKAGMRFGKCMNGKCACTPK
[Ala34]OSK1
5002



(8-35)



 


CKISRQCLEPCKKAGMRFGKCMNGKCAC
[Ala34]OSK1
5003



(8-35)



 


CKIRRQCLEPCKKAGMRFGKCANGKCAC
[Arg11; Ala29,
5004



34]




Osk-1(8-35)



 


CKISAQCLEPCKKAGMRFGKCANGKCAC
[Ala12; Ala29,
5005



34]




Osk-1(8-35)



 


CKISAQCLEPCKKAGMRFGKCMNGKCAC
[Ala12; Ala34]
5006



Osk-1(8-35)



 


GVI[Dpr(AOA)]NVKCKISRQCLEPCKKAGMRFGKCM
[Dpr(AOA)4]
5009


NGKCHCTPK
Osk1



 


GVI[Dpr(AOA-PEG)]NVKCKISRQCLEPCKKAGMRF
[Dpr(AOA)-PEG)4]
5010


GKCMNGKCHCTPK
Osk1



 


GCIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTCK
[C2, C37]-
5012



OSK1



 


SCIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTCK
[S1, C2, C37]-
5013



OSK1



 


SCIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTCK
[S1, C2, A34,
5014



C37]-OSK1



 


SCVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTCK
Ser-[C1, C37]-
5015



OSK1









Any OSK1 peptide analog that comprises an amino acid sequence selected from SEQ ID NOS: 1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through 5015 as set forth in Tables 7, 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H or 7I, is useful in accordance with the present invention. Any of these can also be derivatized at either its N-terminal or C-terminal, e.g., with a fatty acid having from 4 to 10 carbon atoms and from 0 to 2 carbon-carbon double bonds, or a derivative thereof such as an ω-amino-fatty acid. (E.g., Mouhat et al., WO 2006/002850 A2, which is incorporated by reference in its entirety). Examples of such fatty acids include valeric acid or (for the C-terminal) co-amino-valeric acid.
Among useful OSK1 peptide analog sequences of the present invention are analog sequences that introduce amino acid residues that can form an intramolecular covalent bridge (e.g., a disulfide bridge) or non-covalent interactions (e.g. hydrophobic, ionic, stacking) between the first and last beta strand, which may enhance the stability of the structure of the unconjugated or conjugated (e.g., PEGylated) OSK1 peptide analog molecule. Examples of such sequences include SEQ ID NOS: 4985-4987 and 5012-5015.
In some embodiments of the composition of matter, the C-terminal carboxylic acid moiety of the OSK1 peptide analog is replaced with a moiety selected from:
(A) —COOR, where R is independently (C1-C8)alkyl, haloalkyl, aryl or heteroaryl;
(B) —C(═O)NRR, where R is independently hydrogen, (C1-C8)alkyl, haloalkyl, aryl or heteroaryl; and
(C) —CH2OR where R is hydrogen, (C1-C8) alkyl, aryl or heteroaryl.
“Aryl” is phenyl or phenyl vicinally-fused with a saturated, partially-saturated, or unsaturated 3-, 4-, or 5 membered carbon bridge, the phenyl or bridge being substituted by 0, 1, 2 or 3 substituents selected from C18 alkyl, C14 haloalkyl or halo.
“Heteroaryl” is an unsaturated 5, 6 or 7 membered monocyclic or partially-saturated or unsaturated 6-, 7-, 8-, 9-, 10- or 11 membered bicyclic ring, wherein at least one ring is unsaturated, the monocyclic and the bicyclic rings containing 1, 2, 3 or 4 atoms selected from N, O and S, wherein the ring is substituted by 0, 1, 2 or 3 substituents selected from C18 alkyl, C14 haloalkyl and halo.
In other embodiments of the composition of matter comprising a half-life extending moiety, the OSK1 peptide analog comprises an amino acid sequence of the formula:







SEQ ID NO: 5011







G1V2I3I4N5V6K7C8K9I10Xaa11Xaa12Q13C14Xaa15Xaa16P17


 


C18Xaa19Xaa20A21G22M23R24F25G26Xaa27C28Xaa29Xaa30


 


G31Xaa32C33Xaa34C35Xaa36Xaa37Xaa38






wherein:
amino acid residues 1 through 7 are optional (Thus, the OSK1 peptide analog optionally includes residues 1-7 as indicated above in SEQ ID NO:5011, or a N-terminal truncation leaving present residues 2-7, 3-7, 4-7, 5-7, 6-7, or 7, or alternatively, a N-terminal truncation wherein all of residues 1-7 are entirely absent.);

    
    
        Xaa11 is a neutral, basic, or acidic amino acid residue (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit, Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and 4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
        Xaa12 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
        Xaa15 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
        Xaa16 is a neutral or basic amino acid residue (e.g., Lys, His, Arg, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Cit, Nα-Methyl-Cit, Homocitrulline, His, Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);
        Xaa19 is a neutral or basic amino acid residue (e.g., Lys, His, Arg, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Cit, Nα-Methyl-Cit, Homocitrulline, His, Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);
        Xaa20 is a neutral or basic amino acid residue (e.g., Lys, His, Arg, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Cit, Nα-Methyl-Cit, Homocitruiline, His, Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);
        Xaa27 is a neutral, basic, or acidic amino acid residue (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit, Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and 4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
        Xaa29 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
        Xaa30 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
        Xaa32 is a neutral, basic, or acidic amino acid residue (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit, Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and 4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
        Xaa34 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
        Xaa36 is optional, and if present, is a neutral amino acid residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic, D-Tic, D-Pro, Thz, Nα-Methyl-Cit, Homocitrulline, Aib, Sar, Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip;
        Xaa37 is optional, and if present, is a neutral amino acid residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic, D-Tic, D-Pro, Thz, Nα-Methyl-Cit, Homocitrulline, Aib, Sar, Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip); and
        Xaa38 is optional, and if present, is a basic amino acid residue (e.g., Lys, His, Orn, D-Orn, Arg, Nα Methyl-Arg; homoarginine, Cit, Nα-Methyl-Cit, Homocitrulline, Guf, and 4-Amino-Phe).
    
    


In some other embodiments of the composition of matter comprising a half-life extending moiety, the OSK1 peptide analog comprises an amino acid sequence of the formula:







SEQ ID NO: 4913







G1V2I3I4N5V6K7C8K9I10Xaa11Xaa12Q13C14L15Xaa16P17


 


C18K19Xaa20A21G22M23R24F25G26Xaa27C28Xaa29Xaa30G31


 


K32C33Xaa34C35Xaa36Xaa37Xaa38






wherein:

    
    
        amino acid residues 1 to 7 are optional (Thus, the OSK1 peptide analog optionally includes residues 1-7 as indicated above in SEQ ID NO:4913, or a N-terminal truncation leaving present residues 2-7, 3-7, 4-7, 5-7, 6-7, or 7, or alternatively, a N-terminal truncation wherein all of residues 1-7 are entirely absent.);
        Xaa11 is a neutral, basic or acidic amino acid residue (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit, Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and 4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
        Xaa12 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
        Xaa16 is a neutral or basic amino acid residue (e.g., Lys, His, Arg, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Cit, Nα-Methyl-Cit, Homocitrulline, His, Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);
        Xaa20 is a neutral or basic amino acid residue (e.g., Lys, His, Arg, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Cit, Nα-Methyl-Cit, Homocitrulline, His, Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);
        Xaa27 is a neutral, basic, or acidic amino acid residue (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit, Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and 4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
        Xaa29 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
        Xaa30 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
        Xaa35 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
        Xaa36 is optional, and if present, is a neutral amino acid residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic, D-Tic, D-Pro, Thz, Nα-Methyl-Cit, Homocitrulline, Aib, Sar, Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip;
        Xaa37 is optional, and if present, is a neutral amino acid residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic, D-Tic, D-Pro, Thz, Nα-Methyl-Cit, Homocitrulline, Aib, Sar, Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip); and
        Xaa38 is optional, and if present, is a basic amino acid residue (e.g., Lys, His, Orn, D-Orn, Arg, Nα Methyl-Arg; homoarginine, Cit, Nα-Methyl-Cit, Homocitrulline, Guf, and 4-Amino-Phe).
    
    









TABLE 8







Pi2 peptide and PiP2 s peptide analog equences










Short-




hand




desig-
SEQ ID


Sequence/structure
nation
NO:












TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGR
Pi2
17


 


TISCTNPXQCYPHCKKETGYPNAKCMNRKCKCFGR
Pi2-X8
299


 


TISCTNPAQCYPHCKKETGYPNAKCMNRKCKCFGR
Pi2-A8
300


 


TISCTNPKQCYPHCXKETGYPNAKCMNRKCKCFGR
Pi2-X15
301


 


TISCTNPKQCYPHCAKETGYPNAKCMNRKCKCFGR
Pi2-A15
302


 


TISCTNPKQCYPHCKXETGYPNAKCMNRKCKCFGR
Pi2-X16
303


 


TISCTNPKQCYPHCKAETGYPNAKCMNRKCKCFGR
Pi2-A16
304


 


TISCTNPKQCYPHCKKETGYPNAXCMNRKCKCFGR
Pi2-X24
305


 


TISCTNPKQCYPHCKKETGYPNAACMNRKCKCFGR
Pi2-A24
306


 


TISCTNPKQCYPHCKKETGYPNAKCMNXKCKCFGR
Pi2-X28
307


 


TISCTNPKQCYPHCKKETGYPNAKCMNAKCKCFGR
Pi2-A28
308


 


TISCTNPKQCYPHCKKETGYPNAKCMNRXCKCFGR
Pi2-X29
309


 


TISCTNPKQCYPHCKKETGYPNAKCMNRACKCFGR
Pi2-A29
310


 


TISCTNPKQCYPHCKKETGYPNAKCMNRKCXCFGR
Pi2-X31
311


 


TISCTNPKQCYPHCKKETGYPNAKCMNRKCACFGR
Pi2-A31
312


 


TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGX
Pi2-X35
313


 


TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGA
Pi2-A35
314


 


TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFG
Pi2-d35
315
















TABLE 9







Anuroctoxin (AnTx) peptide and peptide


analog sequences











SEQ



Short-hand
ID


Sequence/structure
designation
NO:












ZKECTGPQHCTNFCRKNKCTHGKCMNRKC
Anuroctoxin
62


KCFNCK
(AnTx)



 


KECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK
AnTx-d1
316


 


XECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK
AnTx-d1, X2
317


 


AECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK
AnTx-d1, A2
318
















TABLE 10







Noxiustoxin (NTX) peptide and NTX peptide


analog sequences










Short-hand
SEQ ID


Sequence/structure
designation
NO:












TIINVKCTSPKQCSKPCKELYGSSAGAKCMNG
NTX
30


KCKCYNN




 


TIINVACTSPKQCSKPCKELYGSSAGAKCMNG
NTX-A6
319


KCKCYNN




 


TIINVKCTSPKQCSKPCKELYGSSRGAKCMNG
NTX-R25
320


KCKCYNN




 


TIINVKCTSSKQCSKPCKELYGSSAGAKCMNG
NTX-S10
321


KCKCYNN




 


TIINVKCTSPKQCWKPCKELYGSSAGAKCMNG
NTX-W14
322


KCKCYNN




 


TIINVKCTSPKQCSKPCKELYGSSGAKCMNG
NTX-A25d
323


KCKCYNN




 


TIINVKCTSPKQCSKPCKELFGVDRGKCMNG
NTX-IbTx1
324


KCKCYNN




 


TIINVKCTSPKQCWKPCKELFGVDRGKCMNG
NTX-IBTX2
325


KCKCYN
















TABLE 11







Kaliotoxin1 (KTX1) peptide and KTX1 peptide


analog sequences










Short-hand
SEQ ID


Sequence/structure
designation
NO:












GVEINVKCSGSPQCLKPCKDAGMRFGKCMNRK
KTX1
24


CHCTPK




 


 VRIPVSCKHSGQCLKPCKDAGMRFGKCMNGK
KTX2
326


CDCTPK




 


GVEINVSCSGSPQCLKPCKDAGMRFGKCMNRK
KTX1-S7
327


CHCTPK




 


GVEINVACSGSPQCLKPCKDAGMRFGKCMNRK
KTX1-A7
328


CHCTPK
















TABLE 12







IKCa1 inhibitor peptide sequences











SEQ



Short-hand
ID


Sequence/structure
designation
NO:












VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX
20


 


  QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKK
ChTx
36


CRCYS




 


  QFTQESCTASNQCWSICKRLHNTNRGKCMNKK
ChTx-Lq2
329


CRCYS
















TABLE 13







Maurotoxin (MTx) peptide amd MTx peptide


analog sequences










Short-




hand
SEQ



desig-
ID


Sequence/structure
nation
NO:












VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX
20


 


VSCAGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-A4
330


 


VSCTGAKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-A6
331


 


VSCTGSADCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-A7
332


 


VSCTGSKDCAAPCRKQTGCPNAKCINKSCKCYGC
MTX-A10
333


 


VSCTGSKDCYAPCQKQTGCPNAKCINKSCKCYGC
MTX-Q14
334


 


VSCTGSKDCYAPCRQQTGCPNAKCINKSCKCYGC
MTX-Q15
335


 


VSCTGSKDCYAPCQQQTGCPNAKCINKSCKCYGC
MTX-Q14,
336



15



 


VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYAC
MTX-A33
337


 


VSCTGSKDCYAPCRKQTGCPYGKCMNRKCKCNRC
MTX-HsTx1
338


 


VSCTGSKDCYAACRKQTGCANAKCINKSCKCYGC
MTX-A12,
339



20



 


  VSCTGSKDCYAPCRKQTGXM19PNAKCIN
MTX-X19,
340


KSCKCYGXM34
34



 


VSCTGSKDCYAPCRKQTGSPNAKCINKSCKCYGS
MTX-S19,
341



34



 


VSCTGSADCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-A7
342


 


VVIGQRCTGSKDCYAPCRKQTGCPNAKCINKSCK
TsK-MTX
343


CYGC




 


VSCRGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-R4
1301


 


VSCGGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-G4
1302


 


VSCTTSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-T5
1304


 


VSCTASKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-A5
1305


 


VSCTGTKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-T6
1306


 


VSCTGPKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-P6
1307


 


VSCTGSKDCGAPCRKQTGCPNAKCINKSCKCYGC
MTX-G10
1309


 


VSCTGSKDCYRPCRKQTGCPNAKCINKSCKCYGC
MTX-R11
1311


 


VSCTGSKDCYDPCRKQTGCPNAKCINKSCKCYGC
MTX-D11
1312


 


VSCTGSKDCYAPCRKRTGCPNAKCINKSCKCYGC
MTX-R16
1315


 


VSCTGSKDCYAPCRKETGCPNAKCINKSCKCYGC
MTX-E16
1316


 


VSCTGSKDCYAPCRKQTGCPYAKCINKSCKCYGC
MTX-Y21
1317


 


VSCTGSKDCYAPCRKQTGCPNSKCINKSCKCYGC
MTX-S22
1318


 


VSCTGSKDCYAPCRKQTGCPNGKCINKSCKCYGC
MTX-G22
1319


 


VSCTGSKDCYAPCRKQTGCPNAKCINRSCKCYGC
MTX-R27
1320


 


VSCTGSKDCYAPCRKQTGCPNAKCINKTCKCYGC
MTX-T28
1321


 


VSCTGSKDCYAPCRKQTGCPNAKCINKMCKCYGC
MTX-M28
1322


 


VSCTGSKDCYAPCRKQTGCPNAKCINKKCKCYGC
MTX-K28
1323


 


VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCNGC
MTX-N32
1324


 


VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYRC
MTX-R33
1325


 


VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-S35
1326


S




 


 SCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-d1
1327


 


 SCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-S35
1328


S
d1



 


VSCTGSKDCYAPCAKQTGCPNAKCINKSCKCYGC
MTX-A14
1329


 


VSCTGSKDCYAPCRAQTGCPNAKCINKSCKCYGC
MTX-A15
1330


 


VSCTGSKDCYAPCRKQTGCPNAACINKSCKCYGC
MTX-A23
1331


 


VSCTGSKDCYAPCRKQTGCPNAKCINASCKCYGC
MTX-A27
1332


 


VSCTGSKDCYAPCRKQTGCPNAKCINKSCACYGC
MTX-A30
1333


 


VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCAGC
MTX-A32
1334


 


ASCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-A1
1335


 


MSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-M1
1336









In Table 13 and throughout this specification, Xm19 and Xm34 are each independently nonfunctional residues.







TABLE 14







Charybdotoxin(ChTx) peptide and ChTx peptide


analog sequences










Short-hand



Sequence/structure
designation
SEQ ID NO:












QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx
36


 


QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKECRCYS
ChTx-E32
59


 


QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKDCRCYS
ChTx-D32
344


 


      CTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-d1-d6
345


 


QFTNVSCTTSKECWSVCQRLFGVDRGKCMGKKCRCYQ
ChTx-IbTx
346


 


QFTNVSCTTSKECWSVCQRLHNTSRGKCMNGKCRCYS
ChTx-G31
1369


 


QFTNVSCTTSKECLSVCQRLHNTSRGKCMNKKCRCYS
ChTx-L14
1370


 


QFTNVSCTTSKECASVCQRLHNTSRGKCMNKKCRCYS
ChTx-A14
1371


 


QFTNVSCTTSKECWAVCQRLHNTSRGKCMNKKCRCYS
ChTx-A15
1372


 


QFTNVSCTTSKECWPVCQRLHNTSRGKCMNKKCRCYS
ChTx-P15
1373


 


QFTNVSCTTSKECWSACQRLHNTSRGKCMNKKCRCYS
ChTx-A16
1374


 


QFTNVSCTTSKECWSPCQRLHNTSRGKCMNKKCRCYS
ChTx-P16
1375


 


QFTNVSCTTSKECWSVCQRLHNTSAGKCMNKKCRCYS
ChTx-A25
1376


 


QFTNVACTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-A6
1377


 


QFTNVKCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-K6
1378


 


QFTNVSCTTAKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-A10
1379


 


QFTNVSCTTPKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-P10
1380


 


QFTNVSCTTSKACWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-A12
1381


 


QFTNVSCTTSKQCWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-Q12
1382


 


AFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-A1
1383


 


TFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-T1
1384


 


QATNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-A2
1385


 


QITNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-I2
1386


 


QFANVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-A3
1387


 


QFINVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-I3
1388


 


TIINVKCTSPKQCLPPCKAQFGTSRGKCMNKKCRCYSP
ChTx-MgTx
1389


 


TIINVSCTSPKQCLPPCKAQFGTSRGKCMNKKCRCYSP
ChTx-MgTx-b
1390
















TABLE 15







SKCa inhibitor peptide sequences











SEQ



Short-hand
ID


Sequence/structure
designation
NO:





           CNCKAPETALCARRCQQHG
Apamin
68


 


AFCNLRMCQLSCRSLGLLGKCIGDKCECVKH
ScyTx
51


 


   AVCNLKRCQLSCRSLGLLGKCIGDKCECVKHG
BmP05
50


 


    TVCNLRRCQLSCRSLGLLGKCIGVKCECVKH
P05
52


 


    AFCNLRRCELSCRSLGLLGKCIGEECKCVPY
Tamapin
53


 


VSCEDCPEHCSTQKAQAKCDNDKCVCEPI
P01
16


 


VVIGQRCYRSPDCYSACKKLVGKATGKCTNGRCDC
TsK
47
















TABLE 16







Apamin peptide and peptide analog


inhibitor sequences










Short-hand
SEQ


Sequence/structure
designation
ID NO:












CNCKAPETALCARRCQQHG
Apamin (Ap)
68


 


CNCXAPETALCARRCQQHG
Ap-X4
348


 


CNCAAPETALCARRCQQHG
Ap-A4
349


 


CNCKAPETALCAXRCQQHG
Ap-X13
350


 


CNCKAPETALCAARCQQHG
Ap-A13
351


 


CNCKAPETALCARXCQQHG
Ap-X14
352


 


CNCKAPETALCARACQQHG
Ap-A14
353
















TABLE 17







Scyllatoxin (ScyTx), BmP05, P05, Tamapin, P01


peptide and peptide analog inhibitor sequences










Short-hand
SEQ ID


Sequence/structure
designation
NO:












AFCNLRMCQLSCRSLGLLGKCIGDKCECVKH
ScyTx
51


 


AFCNLRRCQLSCRSLGLLGKCIGDKCECVKH
ScyTx-R7
354


 


AFCNLRMCQLSCRSLGLLGKCMGKKCRCVKH
ScyTx-IbTx
355


 


AFSNLRMCQLSCRSLGLLGKSIGDKCECVKH
ScyTx-C/S
356


 


AFCNLRRCELSCRSLGLLGKCIGEECKCVPY
Tamapin
53
















TABLE 18







BKCa inhibitor peptide sequences










Short-hand



Sequence/structure
designation
SEQ ID NO:












QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCRCYQ
IbTx
38


 


  TFIDVDCTVSKECWAPCKAAFGVDRGKCMGKKCKCYV
Slotoxin
39



(SloTx)



 


QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS
BmTx1
40


 


WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCRCYTN
BuTx
41


 


  FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY
MartenTx
35


 


  ITINVKCTSPQQCLRPCKDRFGQHAGGKCINGKCKCYP
CIITx1
29
















TABLE 19







IbTx, Slotoxin, BmTx1, & BuTX (Slotoxin family) peptide


and peptide analog inhibitor sequences










Short-hand



Sequence/structure
designation
SEQ ID NO:












QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCRCYQ
IbTx
38


 


QFTDVDCSVSXECWSVCKDLFGVDRGKCMGKKCRCYQ
IbTx-X11
357


 


QFTDVDCSVSAECWSVCKDLFGVDRGKCMGKKCRCYQ
IbTx-A11
358


 


QFTDVDCSVSKECWSVCXDLFGVDRGKCMGKKCRCYQ
IbTx-X18
359


 


QFTDVDCSVSKECWSVCADLFGVDRGKCMCKKCRCYQ
IbTx-A18
360


 


QFTDVDCSVSKECWSVCKDLFGVDXGKCMGKKCRCYQ
IbTx-X25
361


 


QFTDVDCSVSKECWSVCKDLFGVDAGKCMGKKCRCYQ
IbTx-A25
362


 


QFTDVDCSVSKECWSVCKDLFGVDRGXCMGKKCRCYQ
IbTx-X27
363


 


QFTDVDCSVSKECWSVCKDLFGVDRGACMGKKCRCYQ
IbTx-A27
364


 


QFTDVDCSVSKECWSVCKDLFGVDRGKCMGXKCRCYQ
IbTx-X31
365


 


QFTDVDCSVSKECWSVCKDLFGVDRGKCMGAKCRCYQ
IbTx-A31
366


 


QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKXCRCYQ
IbTx-X32
367


 


QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKACRCYQ
IbTx-A32
368


 


QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCXCYQ
IbTx-X34
369


 


QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCACYQ
IbTx-A34
370


 


QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS
BmTx1
371


 


QFTDVXCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS
BmTx1-X6
372


 


QFTDVACTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS
BmTx1-A6
373


 


QFTDVKCTGSXQCWPVCKQMFGKPNGKCMNGKCRCYS
BmTx1-X11
374


 


QFTDVKCTGSAQCWPVCKQMFGKPNGKCMNGKCRCYS
BmTx1-A11
375


 


QFTDVKCTGSKQCWPVCXQMFGKPNGKCMNGKCRCYS
BmTx1-X18
376


 


QFTDVKCTGSKQCWPVCAQMFGKPNGKCMNGKCRCYS
BmTx1-A18
377


 


QFTDVKCTGSKQCWPVCKQMFGXPNGKCMNGKCRCYS
BmTx1-X23
378


 


QFTDVKCTGSKQCWPVCKQMFGAPNGKCMNGKCRCYS
BmTx1-A23
379


 


QFTDVKCTGSKQCWPVCKQMFGKPNGXCMNGKCRCYS
BmTx1-X27
380


 


QFTDVKCTGSKQCWPVCKQMFGKPNGACMNGKCRCYS
BmTx1-A27
381


 


QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGXCRCYS
BmTx1-X32
382


 


QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGARCYS
BmTx1-A32
383


 


QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCXYS
BmTx1-X34
384


 


QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCAYS
BmTx1-A34
385


 


WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCRCYTN
BuTx
386


 


WCSTCLDLACGASXCYDPCFKAFGRAHGKCMNNKCRCYTN
BuTx-X14
387


 


WCSTCLDLACGASACYDPCFKAFGRAHGKCMNNKCRCYTN
BuTx-A14
388


 


WCSTCLDLACGASRECYDPCFXFGRAHGKCMNNKCRCYTN
BuTx-X22
389


 


WCSTCLDLACGASRECYDPCFAGRAHGKCMNNKCRCYTN
BuTx-A22
390


 


WCSTCLDLACGASRECYDPCFKAFGXHGKCMNNKCRCYTN
BuTx-X26
391


 


WCSTCLDLACGASRECYDPCFKAFGAHGKCMNNKCRCYTN
BuTx-A26
392


 


WCSTCLDLACGASRECYDPCFKAFGRAHGXMNNKCRCYTN
BuTx-X30
393


 


WCSTCLDLACGASRECYDPCFKAFGRAHGANNNKCRCYTN
BuTx-A30
394


 


WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNXRCYTN
BuTx-X35
395


 


WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNARCYTN
BuTx-A35
396


 


WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCXYTN
BuTx-X37
397


 


WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCAYTN
BuTx-A37
398
















TABLE 20







Martentoxin peptide and peptide analog


inhibitor sequences










Short-hand



Sequence/structure
designation
SEQ ID NO:












FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY
MartenTx
35


 


FGLIDVXCFASSECWTACKKVTGSGQGKCQNNQCRCY
MartenTx-X7
399


 


FGLIDVACFASSECWTACKKVTGSGQGKCQNNQCRCY
MartenTx-A7
400


 


FGLIDVKCFASSECWTACXKVTGSGQGKCQNNQCRCY
MartenTx-X19
401


 


FGLIDVKCFASSECWTACAKVTGSGQGKCQNNQCRCY
MartenTx-A19
402


 


FGLIDVKCFASSECWTACKXVTGSGQGKCQNNQCRCY
MartenTx-X20
403


 


FGLIDVKCFASSECWTACKAVTGSGQGKCQNNQCRCY
MartenTx-A20
404


 


FGLIDVKCFASSECWTACKKVTGSGQGXCQNNQCRCY
MartenTx-X28
405


 


FGLIDVKCFASSECWTACKKVTGSGQGACQNNQCRCY
MartenTx-A28
406


 


FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCXCY
MartenTx-X35
407


 


FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCACY
MartenTx-A35
408
















TABLE 21







N type Ca2+ channel inhibitor peptide sequences










Short-




hand des-
SEQ ID


Sequence/structure
ignation
NO:












CKGKGAKCSRLMYDCCTGSCRSGKC
MVIIA
65


 


CKSPGSSCSPTSYNCCRSCNPYTKRCY
GVIA
64


 


CKSKGAKCSKLMYDCCTGSCSGTVGRC
CVIA
409


 


CKLKGQSCRKTSYDCCSGSCGRSGKC
SVIB
347


 


AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANKCL
Ptu1
66


 


CKGKGASCRKTMYDCCRGSCRSGRC
CVIB
1364


 


CKGKGQSCSKLMYDCCTGSCSRRGKC
CVIC
1365


 


CKSKGAKCSKLMYDCCSGSCSGTVGRC
CVID
1366


 


CLSXGSSCSXTSYNCCRSCNXYSRKCY
TVIA
1367
















TABLE 22







ωMVIIA peptide and peptide analog


inhibitor sequences










Short-hand



Sequence/structure
designation
SEQ ID NO:












CKGKGAKCSRLMYDCCTGSCRSGKC
MVIIA
65


 


CXGKGAKCSRLMYDCCTGSCRSGKC
MVIIA-X2
410


 


CAGKGAKCSRLMYDCCTGSCRSGKC
MVIIA-A2
411


 


CKGXGAKCSRLMYDCCTGSCRSGKC
MVIIA-X4
412


 


CKGAGAKCSRLMYDCCTGSCRSGKC
MVIIA-A4
413


 


CKGKGAXCSRLMYDCCTGSCRSGKC
MVIIA-X7
414


 


CKGKGAACSRLMYDCCTGSCRSGKC
MVIIA-A7
415


 


CKGKGAKCSXLMYDCCTGSCRSGKC
MVIIA-X10
416


 


CKGKGAKCSALMYDCCTGSCRSGKC
MVIIA-A10
417


 


CKGKGAKCSRLMYDCCTGSCXSGKC
MVIIA-X21
418


 


CKGKGAKCSRLMYDCCTGSCASGKC
MVIIA-A21
419


 


CKGKGAKCSRLMYDCCTGSCRSGXC
MVIIA-X24
420


 


CKGKGAKCSRLMYDCCTGSCRSGAC
MVIIA-A24
421
















TABLE 23







□GVIA peptide and peptide analog


inhibitor sequences










Short-hand



Sequence/structure
designation
SEQ ID NO:












CKSPGSSCSPTSYNCCRSCNPYTKRCY
GVIA
64


 


CXSPGSSCSPTSYNCCRSCNPYTKRCY
GVIA-X2
422


 


CASPGSSCSPTSYNCCRSCNPYTKRCY
GVIA-A2
423


 


CKSPGSSCSPTSYNCCXSCNPYTKRCY
GVIA-X17
424


 


CKSPGSSCSPTSYNCCASCNPYTKRCY
GVIA-A17
425


 


CKSPGSSCSPTSYNCCRSCNPYTXRCY
GVIA-X24
426


 


CKSPGSSCSPTSYNCCRSCNPYTARCY
GVIA-A24
427


 


CKSPGSSCSPTSYNCCRSCNPYTKXCY
GVIA-X25
428


 


CKSPGSSCSPTSYNCCRSCNPYTKACY
GVIA-A25
429
















TABLE 24







Ptu1 peptide and peptide analog


inhibitor sequences










Short-




hand des-
SEQ ID


Sequence/structure
ignation
NO:












AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANKCL
Ptu1
66


 


AEXDCIAPGAPCFGTDKPCCNPRAWCSSYANKCL
Ptu1-X3
430


 


AEADCIAPGAPCFGTDKPCCNPRAWCSSYANKCL
Ptu1-A3
431


 


AEKDCIAPGAPCFGTDXPCCNPRAWCSSYANKCL
Ptu1-X17
432


 


AEKDCIAPGAPCFGTDAPCCNPRAWCSSYANKCL
Ptu1-A17
433


 


AEKDCIAPGAPCFGTDKPCCNPXAWCSSYANKCL
Ptu1-X23
434


 


AEKDCIAPGAPCFGTDKPCCNPAAWCSSYANKCL
Ptu1-A23
435


 


AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANXCL
Ptu1-X32
436


 


AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANACL
Ptu1-A32
437
















TABLE 25







Thrixopelma pruriens (ProTx1) and ProTx1 peptide analogs


and other T type Ca2+ channel inhibitor peptide sequences










Short-hand



Sequence/structure
designation
SEQ ID NO:












ECRYWLGGCSAGQTCCKHLVCSRRHGWCVWDGTFS
ProTx1
56


 


ECXYWLGGCSAGQTCCKHLVCSRRHGWCVWDGTFS
ProTx1-X3
438


 


ECAYWLGGCSAGQTCCKHLVCSRRHGWCVWDGTFS
ProTx1-A3
439


 


ECRYWLGGCSAGQTCCXHLVCSRRHGWCVWDGTFS
ProTx1-X17
440


 


ECRYWLGGCSAGQTCCAHLVCSRRHGWCVWDGTFS
ProTx1-A17
441


 


ECRYWLGGCSAGQTCCKHLVCSXRHGWCVWDGTFS
ProTx1-X23
442


 


ECRYWLGGCSAGQTCCKHLVCSARHGWCVWDGTFS
ProTx1-A23
443


 


ECRYWLGGCSAGQTCCKHLVCSRXHGWCVWDGTFS
ProTx1-X24
444


 


ECRYWLGGCSAGQTCCKHLVCSRAHGWCVWDGTFS
ProTx1-A24
445


 


KIDGYPVDYW NCKRICWYNN KYCNDLCKGL
Kurtoxin
1276


KADSGYCWGW TLSCYCQGLP DNARIKRSGR CRA
















TABLE 26







BeKM1 M current inhibitor peptide and BeKM1


peptide analog sequences










Short-hand



Sequence/structure
designation
SEQ ID NO:












RPTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF
BeKM1
63


 


 PTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF
BeKM1-d1
446


 


XPTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF
BeKM1-X1
447


 


APTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF
BeKM1-A1
448


 


RPTDIXCSESYQCFPVCKSRFGKTNGRCVNGFCDCF
BeKM1-X6
449


 


RPTDIACSESYQCFPVCKSRFGKTNGRCVNGFCDCF
BeKM1-A6
450


 


RPTDIKCSESYQCFPVCXSRFGKTNGRCVNGFCDCF
BeKM1-X18
451


 


RPTDIKCSESYQCFPVCASRFGKTNGRCVNGFCDCF
BeKM1-A18
452


 


RPTDIKCSESYQCFPVCKSXFGKTNGRCVNGFCDCF
BeKM1-X20
453


 


RPTDIKCSESYQCFPVCKSAFGKTNGRCVNGFCDCF
BeKM1-A20
454


 


RPTDIKCSESYQCFPVCKSRFGXTNGRCVNGFCDCF
BeKM1-X23
455


 


RPTDIKCSESYQCFPVCKSRFGATNGRCVNGFCDCF
BeKM1-A23
456


 


RPTDIKCSESYQCFPVCKSRFGKTNGXCVNGFCDCF
BeKM1-X27
457


 


RPTDIKCSESYQCFPVCKSRFGKTNGACVNGFCDCF
BeKM1-A27
458
















TABLE 27







Na+ channel inhibitor peptide sequences










Short-hand
SEQ ID


Sequence/structure
designation
NO:





QRCCNGRRGCSSRWCRDHSRCC
SmIIIa
459


 


RDCCTOOKKCKDRQCKOQRCCA
μ-GIIIA
460


 


RDCCTOORKCKDRRCKOMRCCA
μ-GIIIB
461


 


ZRLCCGFOKSCRSRQCKOHRCC
μ-PIIIA
462


 


ZRCCNGRRGCSSRWCRDHSRCC
μ-SmIIIA
463


 


ACRKKWEYCIVPIIGFIYCCPGLICGPFVCV
μO-MrVIA
464


 


ACSKKWEYCIVPIIGFIYCCPGLICGPFVCV
μO-MrVIB
465


 


EACYAOGTFCGIKOGLCCSEFCLPGVCFG
δ-PVIA
466


 


DGCSSGGTFCGIHOGLCCSEFCFLWCITFID
δ-SVIE
467


 


WCKQSGEMCNLLDQNCCDGYCIVLVCT
δ-TxVIA
468


 


VKPCRKEGQLCDPIFQNCCRGWNCVLFCV
δ-GmVIA
469
















TABLE 28







Cl− channel inhibitor peptide sequences










Short-hand



Sequence/structure
designation
SEQ ID NO:












  MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR
CTX
67


 


MCMPCFTTDHQMAXKCDDCCGGKGRGKCYGPQCLCR
CTX-X14
470


 


MCMPCFTTDHQMAAKCDDCCGGKGRGKCYGPQCLCR
CTX-A14
471


 


MCMPCFTTDHQMARXCDDCCGGKGRGKCYGPQCLCR
CTX-X15
472


 


MCMPCFTTDHQMARACDDCCGGKGRGKCYGPQCLCR
CTX-A15
473


 


MCMPCFTTDHQMARKCDDCCGGXGRGKCYGPQCLCR
CTX-X23
474


 


MCMPCFTTDHQMARKCDDCCGGAGRGKCYGPQCLCR
CTX-A23
475


 


MCMPCFTTDHQMARKCDDCCGGKGXGKCYGPQCLCR
CTX-X25
476


 


MCMPCFTTDHQMARKCDDCCGGKGAGKCYGPQCLCR
CTX-A25
477


 


MCMPCFTTDHQMARKCDDCCGGKGRGXCYGPQCLCR
CTX-X27
478


 


MCMPCFTTDHQMARKCDDCCGGKGRGACYGPQCLCR
CTX-A27
479


 


  MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCX
CTX-X36
480


 


MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCA
CTX-A36
481


 


   MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLC
CTX-d36
482


 


QTDGCGPCFTTDANMARKCRECCGGNGKCFGPQCLCNRE
Bm-12b
483


 


QTDGCGPCFTTDANMAXKCRECCGGNGKCFGPQCLCNRE
Bm-12b-X17
484


 


QTDGCGPCFTTDANMAAKCRECCGGNGKCFGPQCLCNRE
Bm-12b-A17
485


 


QTDGCGPCFTTDANMARXCRECCGGNGKCFGPQCLCNRE
Bm-12b-X18
486


 


QTDGCGPCFTTDANMARACRECCGGNGKCFGPQCLCNRE
Bm-12b-A18
487


 


QTDGCGPCFTTDANMARKCXECCGGNGKCFGPQCLCNRE
Bm-12b-X20
488


 


 QTDGCGPCFTTDANMARKCAECCGGNGKCFGPQCLCNRE
Bm-12b-A20
489


 


QTDGCGPCFTTDANMARKCRECCGGNGXCFGPQCLCNRE
Bm-12b-X28
490


 


QTDGCGPCFTTDANMARKCRECCGGNGACFGPQCLCNRE
Bm-12b-A28
491


 


QTDGCGPCFTTDANMARKCRECCGGNGKCFGPQCLCNXE
Bm-12b-X38
492


 


QTDGCGPCFTTDANMARKCRECCGGNGKCFGPQCLCNAE
Bm-12b-A38
493
















TABLE 29







Kv2.1 inhibitor peptide sequences










Short-hand



Sequence/structure
designation
SEQ ID NO:





ECRYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFS
HaTx1
494


 


ECXYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFS
HaTx1-X3
495


 


ECAYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFS
HaTx1-A3
496


 


ECRYLFGGCXTTSDCCKHLGCKFRDKYCAWDFTFS
HaTx1-X10
497


 


ECRYLFGGCATTSDCCKHLGCKFRDKYCAWDFTFS
HaTx1-A10
498


 


ECRYLFGGCKTTSDCCXHLGCKFRDKYCAWDFTFS
HaTx1-X17
499


 


ECRYLFGGCKTTSDCCAHLGCKFRDKYCAWDFTFS
HaTx1-A17
500


 


ECRYLFGGCKTTSDCCKHLGCXFRDKYCAWDFTFS
HaTx1-X22
501


 


ECRYLFGGCKTTSDCCKHLGCAFRDKYCAWDFTFS
HaTx1-A22
502


 


ECRYLFGGCKTTSDCCKHLGCKFXDKYCAWDFTFS
HaTx1-X24
503


 


ECRYLFGGCKTTSDCCKHLGCKFADKYCAWDFTFS
HaTx1-A24
504


 


ECRYLFGGCKTTSDCCKHLGCKFRDXYCAWDFTFS
HaTx1-X26
505


 


ECRYLFGGCKTTSDCCKHLGCKFRDAYCAWDFTFS
HaTx1-A26
506
















TABLE 30







Kv4.3 & Kv4.2 inhibitor peptide sequences










Short-hand
SEQ ID


Sequence/structure
designation
NO:












YCQKWMWTCDEERKCCEGLVCRLWCKRIINM
PaTx2
57


 


YCQXWMWTCDEERKCCEGLVCRLWCKRIINM
PaTx2-X4
507


 


YCQAWMWTCDEERKCCEGLVCRLWCKRIINM
PaTx2-A4
508


 


YCQKWMWTCDEEXKCCEGLVCRLWCKRIINM
PaTx2-X13
509


 


YCQKWMWTCDEEAKCCEGLVCRLWCKRIINM
PaTx2-A13
510


 


YCQKWMWTCDEERXCCEGLVCRLWCKRIINM
PaTx2-X14
511


 


YCQKWMWTCDEERACCEGLVCRLWCKRIINM
PaTx2-A14
512


 


YCQKWMWTCDEERKCCEGLVCXLWCKRIINM
PaTx2-X22
513


 


YCQKWMWTCDEERKCCEGLVCALWCKRIINM
PaTx2-A22
514


 


YCQKWMWTCDEERKCCEGLVCRLWCXRIINM
PaTx2-X26
515


 


YCQKWMWTCDEERKCCEGLVCRLWCARIINM
PaTx2-A26
516


 


YCQKWMWTCDEERKCCEGLVCRLWCKXIINM
PaTx2-X27
517


 


YCQKWMWTCDEERKCCEGLVCRLWCKAIINM
PaTx2-A27
518
















TABLE 31







nACHR channel inhibitor peptide sequences











SEQ



Short-hand
ID


Sequence/structure
designation
NO:





GCCSLPPCAANNPDYC
PnIA
519


 


GCCSLPPCALNNPDYC
PnIA-L10
520


 


GCCSLPPCAASNPDYC
PnIA-S11
521


 


GCCSLPPCALSNPDYC
PnIB
522


 


GCCSLPPCAASNPDYC
PnIB-A10
523


 


GCCSLPPCALNNPDYC
PnIB-N11
524


 


GCCSNPVCHLEHSNLC
MII
525


 


GRCCHPACGKNYSC
α-MI
526


 


RD(hydroxypro)CCYHPTCNMSNPQIC
α-EI
527


 


GCCSYPPCFATNPDC
α-AuIB
528


 


RDPCCSNPVCTVHNPQIC
α-PIA
529


 


GCCSDPRCAWRC
α-ImI
530


 


ACCSDRRCRWRC
α-ImII
531


 


ECCNPACGRHYSC
α-GI
532


 


GCCGSY(hydroxypro)NAACH(hydroxypro)
αA-PIVA
533


CSCKDR(hydroxypro)SYCGQ




 


GCCPY(hydroxypro)
αA-EIVA
534


NAACH(hydroxypro)CGCKVCR




(hydroxypro)(hydroxypro)




YCDR(hydroxypro)SGG




 


H(hydroxypro)(hydroxypro)
ψ-PIIIE
535


CCLYGKCRRY(hydroxypro)




GCSSASCCQR




 


GCCSDPRCNMNNPDYC
EpI
536


 


GCCSHPACAGNNQHIC
GIC
537


 


IRD(γ-carboxyglu) CCSNPACRVNN
GID
538


(hydroxypro)HVC




 


GGCCSHPACAANNQDYC
AnIB
539


 


GCCSYPPCFATNSDYC
AuIA
540


 


GCCSYPPCFATNSGYC
AuIC
541
















TABLE 32







Agelenopsis aperta (Agatoxin) toxin peptides and peptide


analogs and other Ca2+


channel inhibiter peptides










Short-hand



Sequence/structure
designation
SEQ ID NO:












KKKCIAKDYG RCKWGGTPCC RGRGCICSIM
ω-Aga-IVA
959


GTNCECKPRL IMEGLGLA




 


EDNCIAEDYG KCTWGGTKCC RGRPCRCSMI
ω-Aga-IVB
960


GTNCECTPRL IMEGLSFA




 


SCIDIGGDCD GEKDDCQCCR RNGYCSCYSL
ω-Aga-IIIA
961


FGYLKSGCKC VVGTSAEFQG ICRRKARQCY




NSDPDKCESH NKPKRR




 


SCIDIGGDCD GEKDDCQCCR RNGYCSCYSL
ω-Aga-IIIA-
962


FGYLKSGCKC VVGTSAEFQG ICRRKARTCY
T58



NSDPDKCESH NKPKRR




 


SCIDFGGDCD GEKDDCQCCR SNGYCSCYSL
ω-Aga-IIIB
963


FGYLKSGCKC EVGTSAEFRR ICRRKAKQCY




NSDPDKCVSV YKPKRR




 


SCIDFGGDCD GEKDDCQCCR SNGYCSCYNL
ω-Aga-IIIB-
964


FGYLKSGCKC EVGTSAEFRR
N29



ICRRKAKQCYNSDPDKCVSV YKPKRR




 


SCIDFGGDCD GEKDDCQCCR SNGYCSCYNL
ω-Aga-IIIB-
965


FGYLRSGCKC EVGTSAEFRR ICRRKAKQCY
N29/R35



NSDPDKCVSV YKPKRR




 


NCIDFGGDCD GEKDDCQCCX RNGYCSCYNL
ω-Aga-IIIC
966


FGYLKRGCKX EVG




 


SCIKIGEDCD GDKDDCQCCR TNGYCSXYXL FGYLKSG
ω-Aga-IIID
967


 


GCIEIGGDCD GYQEKSYCQC CRNNGFCS
ω-Aga-IIA
968


 


AKAL PPGSVCDGNE SDCKCYGKWH KCRCPWKWHF
ω-Aga-IA
969


TGEGPCTCEK GMKHTCITKL HCPNKAEWGL DW
(major chain)



 


ECVPENGHCR DWYDECCEGF YCSCRQPPKC ICRNNNX
μ-Aga
970


 


DCVGESQQCA DWAGPHCCDG YYCTCRYFPK CICVNNN
μ-Aga-6
971


 


ACVGENKQCA DWAGPHCCDG YYCTCRYFPK CICRNNN
μ-Aga-5
972


 


ACVGENQQCA DWAGPHCCDG YYCTCRYFPK CICRNNN
μ-Aga-4
973


 


ADCVGDGQRC ADWAGPYCCS GYYCSCRSMP
μ-Aga-3
1275


YCRCRSDS




 


ECATKNKRCA DWAGPWCCDG LYCSCRSYPG CMCRPSS
μ-Aga-2
974


 


ECVPENGHCR DWYDECCEGF YCSCRQPPKC ICRNNN
μ-Aga-1
975


 


AELTSCFPVGHECDGDASNCNCCGDDVYCGCGWGRWNCKC
Tx-1
1277


KVADQSYAYGICKDKVNCPNRHLWPAKVCKKPCRREC




 


GCANAYKSCNGPHTCCWGYNGYKKACICSGXNWK
Tx3-3
1278


 


SCINVGDFCDGKKDCCQCDRDNAFCSCSVIFGYKTNCRCE
Tx3-4
1279


 


SCINVGDFCDGKKDDCQCCRDNAFCSCSVIFGYKTNCRCE
ω-PtXIIA
1280


VGTTATSYGICMAKHKCGRQTTCTKPCLSKRCKKNH




 


AECLMIGDTSCVPRLGRRCCYGAWCYCDQQLSCRRVGRKR
Dw13.3
1281


ECGWVEVNCKCGWSWSQRIDDWRADYSCKCPEDQ




 


GGCLPHNRFCNALSGPRCCSGLKCKELSIWDSRCL
Agelenin
1282


 


DCVRFWGKCSQTSDCCPHLACKSKWPRNICVWDGSV
ω-GTx-SIA
1283


 


GCLEVDYFCG IPFANNGLCC SGNCVFVCTP Q
ω-conotoxin
1284



PnVIA



 


DDDCEPPGNF CGMIKIGPPC CSGWCFFACA
ω-conotoxin
1285



PnVIB



 


VCCGYKLCHP C
Lambda-
1286



conotoxin




CMrVIA



 


MRCLPVLIIL LLLTASAPGV VVLPKTEDDV
Lambda-
1287


PMSSVYGNGK SILRGILRNG VCCGYKLCHP C
conotoxin




CMrVIB



 


KIDGYPVDYW NCKRICWYNN KYCNDLCKGL
Kurtoxin
1276


KADSGYCWGW TLSCYCQGLP DNARIKRSGR CRA




 


CKGKGAPCRKTMYDCCSGSCGRRGKC
MVIIC
1368









In accordance with this invention are molecules in which at least one of the toxin peptide (P) portions of the molecule comprises a Kv1.3 antagonist peptide. Amino acid sequences selected from ShK, HmK, MgTx, AgTx1, AgTx2, Heterometrus spinnifer (HsTx1), OSK1, Anuroctoxin (AnTx), Noxiustoxin (NTX), KTX1, Hongotoxin, ChTx, Titystoxin, BgK, BmKTX, BmTx, AeK, AsKS Tc30, Tc32, Pi1, Pi2, and/or Pi3 toxin peptides and peptide analogs of any of these are preferred. Examples of useful Kv1.3 antagonist peptide sequences include those having any amino acid sequence set forth in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, and/or Table 11 herein above;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is an IKCa1 antagonist peptide. Useful IKCa1 antagonist peptides include Maurotoxin (MTx), ChTx, peptides and peptide analogs of either of these, examples of which include those having any amino acid sequence set forth in Table 12, Table 13, and/or Table 14;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a SKCa inhibitor peptide. Useful SKCa inhibitor peptides include, Apamin, ScyTx, BmP05, P01, P05, Tamapin, TsK, and peptide analogs of any of these, examples of which include those having any amino acid sequence set forth in Table 15;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is an apamin peptide, and peptide analogs of apamin, examples of which include those having any amino acid sequence set forth in Table 16;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Scyllotoxin family peptide, and peptide analogs of any of these, examples of which include those having any amino acid sequence set forth in Table 17;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a BKCa inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 18;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Slotoxin family peptide, and peptide analogs of any of these, examples of which include those having any amino acid sequence set forth in Table 19;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Martentoxin peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 20;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a N-type Ca2+ channel inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 21;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a ωMVIIA peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 22;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a ωGVIA peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 23;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Ptu1 peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 24;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a ProTx1 peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 25;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a BeKM1 peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 26;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Na+ channel inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 27;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Cl− channel inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 28;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Kv2.1 inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 29;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Kv4.2/Kv4.3 inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 30;
Other embodiments of the inventive composition include at least one toxin peptide (P) that is a nACHR inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 31; and
Other embodiments of the inventive composition include at least one toxin peptide (P) that is an Agatoxin peptide, a peptide analog thereof or other calcium channel inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 32.
Half-life extending moieties. This invention involves the presence of at least one half-life extending moiety (F1 and/or F2 in Formula I) attached to a peptide through the N-terminus, C-terminus or a sidechain of one of the intracalary amino acid residues. Multiple half-life extending moieties can also be used; e.g., Fc's at each terminus or an Fc at a terminus and a PEG group at the other terminus or at a sidechain. In other embodiments the Fc domain can be PEGylated (e.g., in accordance with the formulae F1-F2-(L)f-P; P-(L)g-F1-F2; or P-(L)9-F1-F2-(L)f-P).
The half-life extending moiety can be selected such that the inventive composition achieves a sufficient hydrodynamic size to prevent clearance by renal filtration in vivo. For example, a half-life extending moiety can be selected that is a polymeric macromolecule, which is substantially straight chain, branched-chain, or dendritic in form. Alternatively, a half-life extending moiety can be selected such that, in vivo, the inventive composition of matter will bind to a serum protein to form a complex, such that the complex thus formed avoids substantial renal clearance. The half-life extending moiety can be, for example, a lipid; a cholesterol group (such as a steroid); a carbohydrate or oligosaccharide; or any natural or synthetic protein, polypeptide or peptide that binds to a salvage receptor.
Exemplary half-life extending moieties that can be used, in accordance with the present invention, include an immunoglobulin Fc domain, or a portion thereof, or a biologically suitable polymer or copolymer, for example, a polyalkylene glycol compound, such as a polyethylene glycol or a polypropylene glycol. Other appropriate polyalkylene glycol compounds include, but are not limited to, charged or neutral polymers of the following types: dextran, polylysine, colominic acids or other carbohydrate based polymers, polymers of amino acids, and biotin derivatives. In some monomeric fusion protein embodiments an immunoglobulin (including light and heavy chains) or a portion thereof, can be used as a half-life-extending moiety, preferably an immunoglobulin of human origin, and including any of the immunoglobulins, such as, but not limited to, IgG1, IgG2, IgG3 or IgG4.
Other examples of the half-life extending moiety, in accordance with the invention, include a copolymer of ethylene glycol, a copolymer of propylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid (e.g., polylysine), a dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinyl alcohol, a linear or branched glycosylated chain, a polyacetal, a long chain fatty acid, a long chain hydrophobic aliphatic group, an immunoglobulin light chain and heavy chain, an immunoglobulin Fc domain or a portion thereof (see, e.g., Feige et al., Modified peptides as therapeutic agents, U.S. Pat. No. 6,660,843), a CH2 domain of Fc, an albumin (e.g., human serum albumin (HSA)); see, e.g., Rosen et al., Albumin fusion proteins, U.S. Pat. No. 6,926,898 and US 2005/0054051; Bridon et al., Protection of endogenous therapeutic peptides from peptidase activity through conjugation to blood components, U.S. Pat. No. 6,887,470), a transthyretin (TTR; see, e.g., Walker et al., Use of transthyretin peptide/protein fusions to increase the serum half-life of pharmacologically active peptides/proteins, US 2003/0195154 A1; 2003/0191056 A1), or a thyroxine-binding globulin (TBG). Thus, exemplary embodiments of the inventive compositions also include HSA fusion constructs such as but not limited to: HSA fusions with ShK, OSK1, or modified analogs of those toxin peptides. Examples include HSA-L10-ShK(2-35); HSA-L10-OsK1(1-38); HSA-L10-ShK(2-35); and HSA-L10-OsK1(1-38).
Other embodiments of the half-life extending moiety, in accordance with the invention, include peptide ligands or small (organic) molecule ligands that have binding affinity for a long half-life serum protein under physiological conditions of temperature, pH, and ionic strength. Examples include an albumin-binding peptide or small molecule ligand, a transthyretin-binding peptide or small molecule ligand, a thyroxine-binding globulin-binding peptide or small molecule ligand, an antibody-binding peptide or small molecule ligand, or another peptide or small molecule that has an affinity for a long half-life serum protein. (See, e.g., Blaney et al., Method and compositions for increasing the serum half-life of pharmacologically active agents by binding to transthyretin-selective ligands, U.S. Pat. No. 5,714,142; Sato et al., Serum albumin binding moieties, US 2003/0069395 A1; Jones et al., Pharmaceutical active conjugates, U.S. Pat. No. 6,342,225). A “long half-life serum protein” is one of the hundreds of different proteins dissolved in mammalian blood plasma, including so-called “carrier proteins” (such as albumin, transferrin and haptoglobin), fibrinogen and other blood coagulation factors, complement components, immunoglobulins, enzyme inhibitors, precursors of substances such as angiotensin and bradykinin and many other types of proteins. The invention encompasses the use of any single species of pharmaceutically acceptable half-life extending moiety, such as, but not limited to, those described herein, or the use of a combination of two or more different half-life extending moieties, such as PEG and immunoglobulin Fc domain or a CH2 domain of Fc, albumin (e.g., HSA), an albumin-binding protein, transthyretin or TBG, or a combination such as immunoglobulin (light chain+heavy chain) and Fc domain (the combination so-called “hemibody”).
In some embodiments of the invention an Fc domain or portion thereof, such as a CH2 domain of Fc, is used as a half-life extending moiety. The Fc domain can be fused to the N-terminal (e.g., in accordance with the formula F1-(L)f-P) or C-terminal (e.g., in accordance with the formula P-(L)g-F1) of the toxin peptides or at both the N and C termini (e.g., in accordance with the formulae F1-(L)f-P-(L)g-F2 or P-(L)g-F1-(L)f-P). A peptide linker sequence can be optionally included between the Fc domain and the toxin peptide, as described herein. Examples of the formula F1-(L)f-P include: Fc-L10-ShK(K22A)[2-35]; Fc-L10-ShK(R1K/K22A)[1-35]; Fc-L10-ShK(R1H/K22A)[1-35]; Fc-L110-ShK(R1Q/K22A)[1-35]; Fc-L110-ShK(R1Y/K22A)[1-35]; Fc-L10-PP-ShK(K22A) [1-35]; and any other working examples described herein. Examples of the formula P-(L)g-F1 include: ShK(1-35)-L10-Fc; OsK1(1-38)-L10-Fc; Met-ShK(1-35)-L10-Fc; ShK(2-35)-L10-Fc; Gly-ShK(1-35)-L10-Fc; Osk1(1-38)-L10-Fc; and any other working examples described herein.
Fc variants are suitable half-life extending moieties within the scope of this invention. A native Fc can be extensively modified to form an Fc variant in accordance with this invention, provided binding to the salvage receptor is maintained; see, for example WO 97/34631, WO 96/32478, and WO 04/110 472. In such Fc variants, one can remove one or more sites of a native Fc that provide structural features or functional activity not required by the fusion molecules of this invention. One can remove these sites by, for example, substituting or deleting residues, inserting residues into the site, or truncating portions containing the site. The inserted or substituted residues can also be altered amino acids, such as peptidomimetics or D-amino acids. Fc variants can be desirable for a number of reasons, several of which are described below. Exemplary Fc variants include molecules and sequences in which:

    
    
        1. Sites involved in disulfide bond formation are removed. Such removal can avoid reaction with other cysteine-containing proteins present in the host cell used to produce the molecules of the invention. For this purpose, the cysteine-containing segment at the N-terminus can be truncated or cysteine residues can be deleted or substituted with other amino acids (e.g., alanyl, seryl). In particular, one can truncate the N-terminal 20-amino acid segment of SEQ ID NO: 2 or delete or substitute the cysteine residues at positions 7 and 10 of SEQ ID NO: 2. Even when cysteine residues are removed, the single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently.
        2. A native Fc is modified to make it more compatible with a selected host cell. For example, one can remove the PA sequence near the N-terminus of a typical native Fc, which can be recognized by a digestive enzyme in E. coli such as proline iminopeptidase. One can also add an N-terminal methionine residue, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli. The Fc domain of SEQ ID NO: 2 (FIG. 4A-4B) is one such Fc variant.
        3. A portion of the N-terminus of a native Fc is removed to prevent N-terminal heterogeneity when expressed in a selected host cell. For this purpose, one can delete any of the first 20 amino acid residues at the N-terminus, particularly those at positions 1, 2, 3, 4 and 5.
        4. One or more glycosylation sites are removed. Residues that are typically glycosylated (e.g., asparagine) can confer cytolytic response. Such residues can be deleted or substituted with unglycosylated residues (e.g., alanine).
        5. Sites involved in interaction with complement, such as the C1q binding site, are removed. For example, one can delete or substitute the EKK sequence of human IgG1. Complement recruitment may not be advantageous for the molecules of this invention and so can be avoided with such an Fc variant.
        6. Sites are removed that affect binding to Fc receptors other than a salvage receptor. A native Fc can have sites for interaction with certain white blood cells that are not required for the fusion molecules of the present invention and so can be removed.
        7. The ADCC site is removed. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgG1. These sites, as well, are not required for the fusion molecules of the present invention and so can be removed.
        8. When the native Fc is derived from a non-human antibody, the native Fc can be humanized. Typically, to humanize a native Fc, one will substitute selected residues in the non-human native Fc with residues that are normally found in human native Fc. Techniques for antibody humanization are well known in the art.
    
    


Preferred Fc variants include the following. In SEQ ID NO: 2, the leucine at position 15 can be substituted with glutamate; the glutamate at position 99, with alanine; and the lysines at positions 101 and 103, with alanines. In addition, phenyalanine residues can replace one or more tyrosine residues.
An alternative half-life extending moiety would be a protein, polypeptide, peptide, antibody, antibody fragment, or small molecule (e.g., a peptidomimetic compound) capable of binding to a salvage receptor. For example, one could use as a half-life extending moiety a polypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also be selected by phage display for binding to the FcRn salvage receptor. Such salvage receptor-binding compounds are also included within the meaning of “half-life extending moiety” and are within the scope of this invention. Such half-life extending moieties should be selected for increased half-life (e.g., by avoiding sequences recognized by proteases) and decreased immunogenicity (e.g., by favoring non-immunogenic sequences, as discovered in antibody humanization).
As noted above, polymer half-life extending moieties can also be used for F1 and F2. Various means for attaching chemical moieties useful as half-life extending moieties are currently available, see, e.g., Patent Cooperation Treaty (“PCT”) International Publication No. WO 96/11953, entitled “N-Terminally Chemically Modified Protein Compositions and Methods,” herein incorporated by reference in its entirety. This PCT publication discloses, among other things, the selective attachment of water-soluble polymers to the N-terminus of proteins.
In some embodiments of the inventive compositions, the polymer half-life extending moiety is polyethylene glycol (PEG), as F1 and/or F2, but it should be understood that the inventive composition of matter, beyond positions F1 and/or F2, can also include one or more PEGs conjugated at other sites in the molecule, such as at one or more sites on the toxin peptide. Accordingly, some embodiments of the inventive composition of matter further include one or more PEG moieties conjugated to a non-PEG half-life extending moiety, which is F1 and/or F2, or to the toxin peptide(s) (P), or to any combination of any of these. For example, an Fc domain or portion thereof (as F1 and/or F2) in the inventive composition can be made mono-PEGylated, di-PEGylated, or otherwise multi-PEGylated, by the process of reductive alkylation.
Covalent conjugation of proteins and peptides with poly(ethylene glycol) (PEG) has been widely recognized as an approach to significantly extend the in vivo circulating half-lives of therapeutic proteins. PEGylation achieves this effect predominately by retarding renal clearance, since the PEG moiety adds considerable hydrodynamic radius to the protein. (Zalipsky, S., et al., Use of functionalized poly(ethylene glycol)s for modification of polypeptides., in poly(ethylene glycol) chemistry: Biotechnical and biomedical applications., J. M. Harris, Ed., Plenum Press: New York., 347-370 (1992)). Additional benefits often conferred by PEGylation of proteins and peptides include increased solubility, resistance to proteolytic degradation, and reduced immunogenicity of the therapeutic polypeptide. The merits of protein PEGylation are evidenced by the commercialization of several PEGylated proteins including PEG-Adenosine deaminase (AdagenTM/Enzon Corp.), PEG-L-asparaginase (OncasparTM/Enzon Corp.), PEG-Interferon α-2b (PEG-IntronTM/Schering/Enzon), PEG-Interferon α-2a (PEGASYSTM/Roche) and PEG-G-CSF (NeulastaTM/Amgen) as well as many others in clinical trials.
Briefly, the PEG groups are generally attached to the peptide portion of the composition of the invention via acylation or reductive alkylation (or reductive amination) through a reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the inventive compound (e.g., an aldehyde, amino, or ester group).
A useful strategy for the PEGylation of synthetic peptides consists of combining, through forming a conjugate linkage in solution, a peptide and a PEG moiety, each bearing a special functionality that is mutually reactive toward the other. The peptides can be easily prepared with conventional solid phase synthesis (see, for example, FIGS. 5 and 6 and the accompanying text herein). The peptides are “preactivated” with an appropriate functional group at a specific site. The precursors are purified and fully characterized prior to reacting with the PEG moiety. Ligation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC. The PEGylated peptides can be easily purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry.
PEG is a well-known, water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). In the present application, the term “PEG” is used broadly to encompass any polyethylene glycol molecule, in mono-, bi-, or poly-functional form, without regard to size or to modification at an end of the PEG, and can be represented by the formula:

X—O(CH2CH2O)n-1CH2CH2OH,  (X)

where n is 20 to 2300 and X is H or a terminal modification, e.g., a C1-4 alkyl.
In some useful embodiments, a PEG used in the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CH3 (“methoxy PEG”). It is noted that the other end of the PEG, which is shown in formula (II) terminating in OH, covalently attaches to an activating moiety via an ether oxygen bond, an amine linkage, or amide linkage. When used in a chemical structure, the term “PEG” includes the formula (II) above without the hydrogen of the hydroxyl group shown, leaving the oxygen available to react with a free carbon atom of a linker to form an ether bond. More specifically, in order to conjugate PEG to a peptide, the peptide must be reacted with PEG in an “activated” form. Activated PEG can be represented by the formula:

(PEG)-(A)  (XI)

where PEG (defined supra) covalently attaches to a carbon atom of the activation moiety (A) to form an ether bond, an amine linkage, or amide linkage, and (A) contains a reactive group which can react with an amino, azido, alkyne, imino, maleimido, N-succinimidyl, carboxyl, aminooxy, seleno, or thiol group on an amino acid residue of a peptide or a linker moiety covalently attached to the peptide, e.g., the OSK1 peptide analog. Residues baring chemoselective reactive groups can be introduced into the toxin peptide, e.g., an OSK1 peptide analog during assembly of the peptide sequence solid-phase synthesis as protected derivatives. Alternatively, chemoselective reactive groups can be introduced in the toxin peptide after assembly of the peptide sequence by solid-phase synthesis via the use of orthogonal protecting groups at specific sites. Examples of amino acid residues useful for chemoselective reactions include, but are not limited to, (amino-oxyacetyl)-L-diaminopropionic acid, p-azido-phenylalanine, azidohomolalanine, para-propargyloxy-phenylalanine, selenocysteine, para-acetylphenylalanine, (Nε-levulinyl)-Lysine, (Nε-pyruvyl)-Lysine, selenocysteine, and orthogonally protected cysteine and homocysteine.

Accordingly, in some embodiments of the composition of matter, the toxin peptide, e.g., the OSK1 peptide analog, is conjugated to a polyethylene glycol (PEG) at:
(a) 1, 2, 3 or 4 amino functionalized sites in the toxin peptide;
(b) 1, 2, 3 or 4 thiol functionalized sites in the toxin peptide; (c) 1 or 2 ketone functionalized sites in the toxin peptide; (d) 1 or 2 azido functionalized sites of the toxin peptide; (e) 1 or 2 carboxyl functionalized sites in the toxin peptide; (f) 1 or 2 aminooxy functionalized sites in the toxin peptide; or (g) 1 or 2 seleno functionalized sites in the toxin peptide.
In other embodiments of the composition of matter, the toxin peptide, e.g., the OSK1 peptide analog, is conjugated to a polyethylene glycol (PEG) at:
(a) 1, 2, 3 or 4 amino functionalized sites of the PEG;
(b) 1, 2, 3 or 4 thiol functionalized sites of the PEG;
(c) 1, 2, 3 or 4 maleimido functionalized sites of the PEG;
(d) 1, 2, 3 or 4 N-succinimidyl functionalized sites of the PEG;
(e) 1, 2, 3 or 4 carboxyl functionalized sites of the PEG; or
(f) 1, 2, 3 or 4 p-nitrophenyloxycarbonyl functionalized sites of the PEG.
Techniques for the preparation of activated PEG and its conjugation to biologically active peptides are well known in the art. (E.g., see U.S. Pat. Nos. 5,643,575, 5,919,455, 5,932,462, and 5,990,237; Thompson et al., PEGylation of polypeptides, EP 0575545 B1; Petit, Site specific protein modification, U.S. Pat. Nos. 6,451,986, and 6,548,644; S. Herman et al., Poly(ethylene glycol) with reactive endgroups: I. Modification of proteins, J. Bioactive Compatible Polymers, 10:145-187 (1995); Y. Lu et al., Pegylated peptides III: Solid-phase synthesis with PEGylating reagents of varying molecular weight: synthesis of multiply PEGylated peptides, Reactive Polymers, 22:221-229 (1994); A. M. Felix et al., PEGylated Peptides IV: Enhanced biological activity of site-directed PEGylated GRF analogs, Int. J. Peptide Protein Res., 46:253-264 (1995); A. M. Felix, Site-specific poly(ethylene glycol)ylation of peptides, ACS Symposium Series 680(poly(ethylene glycol)): 218-238 (1997); Y. Ikeda et al., Polyethylene glycol derivatives, their modified peptides, methods for producing them and use of the modified peptides, EP 0473084 B1; G. E. Means et al., Selected techniques for the modification of protein side chains, in: Chemical modification of proteins, Holden Day, Inc., 219 (1971)).
Activated PEG, such as PEG-aldehydes or PEG-aldehyde hydrates, can be chemically synthesized by known means or obtained from commercial sources, e.g., Shearwater Polymers, (Huntsville, Ala.) or Enzon, Inc. (Piscataway, N.J.).
An example of a useful activated PEG for purposes of the present invention is a PEG-aldehyde compound (e.g., a methoxy PEG-aldehyde), such as PEG-propionaldehyde, which is commercially available from Shearwater Polymers (Huntsville, Ala.). PEG-propionaldehyde is represented by the formula PEG-CH2CH2CHO. (See, e.g., U.S. Pat. No. 5,252,714). Other examples of useful activated PEG are PEG acetaldehyde hydrate and PEG bis aldehyde hydrate, which latter yields a bifunctionally activated structure. (See., e.g., Bentley et al., Poly(ethylene glycol) aldehyde hydrates and related polymers and applications in modifying amines, U.S. Pat. No. 5,990,237).
Another useful activated PEG for generating the PEG-conjugated peptides of the present invention is a PEG-maleimide compound, such as, but not limited to, a methoxy PEG-maleimide, such as maleimido monomethoxy PEG, are particularly useful for generating the PEG-conjugated peptides of the invention. (E.g., Shen, N-maleimidyl polymer derivatives, U.S. Pat. No. 6,602,498; C. Delgado et al., The uses and properties of PEG-linked proteins., Crit. Rev. Therap. Drug Carrier Systems, 9:249-304 (1992); S. Zalipsky et al., Use of functionalized poly(ethylene glycol)s for modification of polypeptides, in: Poly(ethylene glycol) chemistry: Biotechnical and biomedical applications (J. M. Harris, Editor, Plenum Press: New York, 347-370 (1992); S. Herman et al., Poly(ethylene glycol) with reactive endgroups: I. Modification of proteins, J. Bioactive Compatible Polymers, 10:145-187 (1995); P. J. Shadle et al., Conjugation of polymer to colony stimulating factor-1, U.S. Pat. No. 4,847,325; G. Shaw et al., Cysteine added variants IL-3 and chemical modifications thereof, U.S. Pat. No. 5,166,322 and EP 0469074 B1; G. Shaw et al., Cysteine added variants of EPO and chemical modifications thereof, EP 0668353 A1; G. Shaw et al., Cysteine added variants G-CSF and chemical modifications thereof, EP 0668354 A1; N. V. Katre et al., Interleukin-2 muteins and polymer conjugation thereof, U.S. Pat. No. 5,206,344; R. J. Goodson and N. V. Katre, Site-directed pegylation of recombinant interleukin-2 at its glycosylation site, Biotechnology, 8:343-346 (1990)).
A poly(ethylene glycol) vinyl sulfone is another useful activated PEG for generating the PEG-conjugated peptides of the present invention by conjugation at thiolated amino acid residues, e.g., at C residues. (E.g., M. Morpurgo et al., Preparation and characterization of poly(ethylene glycol) vinyl sulfone, Bioconj. Chem., 7:363-368 (1996); see also Harris, Functionalization of polyethylene glycol for formation of active sulfone-terminated PEG derivatives for binding to proteins and biologically compatible materials, U.S. Pat. Nos. 5,446,090; 5,739,208; 5,900,461; 6,610,281 and 6,894,025; and Harris, Water soluble active sulfones of poly(ethylene glycol), WO 95/13312 A1).
Another activated form of PEG that is useful in accordance with the present invention, is a PEG-N-hydroxysuccinimide ester compound, for example, methoxy PEG-N-hydroxysuccinimidyl (NHS) ester.
Heterobifunctionally activated forms of PEG are also useful. (See, e.g., Thompson et al., PEGylation reagents and biologically active compounds formed therewith, U.S. Pat. No. 6,552,170).
Typically, a toxin peptide or, a fusion protein comprising the toxin peptide, is reacted by known chemical techniques with an activated PEG compound, such as but not limited to, a thiol-activated PEG compound, a diol-activated PEG compound, a PEG-hydrazide compound, a PEG-oxyamine compound, or a PEG-bromoacetyl compound. (See, e.g., S. Herman, Poly(ethylene glycol) with Reactive Endgroups: I. Modification of Proteins, J. Bioactive and Compatible Polymers, 10:145-187 (1995); S. Zalipsky, Chemistry of Polyethylene Glycol Conjugates with Biologically Active Molecules, Advanced Drug Delivery Reviews, 16:157-182 (1995); R. Greenwald et al., Poly(ethylene glycol) conjugated drugs and prodrugs: a comprehensive review, Critical Reviews in Therapeutic Drug Carrier Systems, 17:101-161 (2000)).
Methods for N-terminal PEGylation are exemplified herein in Examples 31-34, 45 and 47-48, but these are in no way limiting of the PEGylation methods that can be employed by one skilled in the art.
Any molecular mass for a PEG can be used as practically desired, e.g., from about 1,000 or 2,000 Daltons (Da) to about 100,000 Da (n is 20 to 2300). Preferably, the combined or total molecular mass of PEG used in a PEG-conjugated peptide of the present invention is from about 3,000 Da or 5,000 Da, to about 50,000 Da or 60,000 Da (total n is from 70 to 1,400), more preferably from about 10,000 Da to about 40,000 Da (total n is about 230 to about 910). The most preferred combined mass for PEG is from about 20,000 Da to about 30,000 Da (total n is about 450 to about 680). The number of repeating units “n” in the PEG is approximated for the molecular mass described in Daltons. It is preferred that the combined molecular mass of PEG on an activated linker is suitable for pharmaceutical use. Thus, the combined molecular mass of the PEG molecule should not exceed about 100,000 Da.
Polysaccharide polymers are another type of water-soluble polymer that can be used for protein modification. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by α1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kDa to about 70 kDa. Dextran is a suitable water-soluble polymer for use in the present invention as a half-life extending moiety by itself or in combination with another half-life extending moiety (e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456, which is hereby incorporated by reference in its entirety. Dextran of about 1 kDa to about 20 kDa is preferred when dextran is used as a half-life extending moiety in accordance with the present invention.
Linkers. Any “linker” group or moiety (i.e., “-(L)f-” or “-(L)g-” in Formulae I-IX) is optional. When present, its chemical structure is not critical, since it serves primarily as a spacer. As stated herein above, the linker moiety (-(L)f- and/or -(L)g-), if present, can be independently the same or different from any other linker, or linkers, that may be present in the inventive composition. For example, an “(L)f” can represent the same moiety as, or a different moiety from, any other “(L)f” or any “(L)g” in accordance with the invention. The linker is preferably made up of amino acids linked together by peptide bonds. Some of these amino acids can be glycosylated, as is well understood by those in the art. For example, a useful linker sequence constituting a sialylation site is X1X2NX4X5G (SEQ ID NO: 637), wherein X1, X2, X4 and X5 are each independently any amino acid residue.
As stated above, in some embodiments, a peptidyl linker is present (i.e., made up of amino acids linked together by peptide bonds) that is made in length, preferably, of from 1 up to about 40 amino acid residues, more preferably, of from 1 up to about 20 amino acid residues, and most preferably of from 1 to about 10 amino acid residues. Preferably, but not necessarily, the amino acid residues in the linker are from among the twenty canonical amino acids, more preferably, cysteine, glycine, alanine, proline, asparagine, glutamine, and/or serine. Even more preferably, a peptidyl linker is made up of a majority of amino acids that are sterically unhindered, such as glycine, serine, and alanine linked by a peptide bond. It is also desirable that, if present, a peptidyl linker be selected that avoids rapid proteolytic turnover in circulation in vivo. Thus, preferred linkers include polyglycines (particularly (Gly)4 (SEQ ID NO: 4918), (Gly)5) (SEQ ID NO: 4919), poly(Gly-Ala), and polyalanines. Other preferred linkers are those identified herein as “L5” (GGGGS; SEQ ID NO: 638), “L10” (GGGGSGGGGS; SEQ ID NO:79), “L25” GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID NO:84) and any linkers used in the working examples hereinafter. The linkers described herein, however, are exemplary; linkers within the scope of this invention can be much longer and can include other residues.
In some embodiments of the compositions of this invention, which comprise a peptide linker moiety (L), acidic residues, for example, glutamate or aspartate residues, are placed in the amino acid sequence of the linker moiety (L). Examples include the following peptide linker sequences:










GGEGGG;
(SEQ ID NO: 639)


 



GGEEEGGG;
(SEQ ID NO: 640)


 



GEEEG;
(SEQ ID NO: 641)


 



GEEE;
(SEQ ID NO: 642)


 



GGDGGG;
(SEQ ID NO: 643)


 



GGDDDGG;
(SEQ ID NO: 644)


 



GDDDG;
(SEQ ID NO: 645)


 



GDDD;
(SEQ ID NO: 646)


 



GGGGSDDSDEGSDGEDGGGGS;
(SEQ ID NO: 647)


 



WEWEW;
(SEQ ID NO: 648)


 



FEFEF;
(SEQ ID NO: 649)


 



EEEWWW;
(SEQ ID NO: 650)


 



EEEFFF;
(SEQ ID NO: 651)


 



WWEEEWW;
(SEQ ID NO: 652)



or



 



FFEEEFF.
(SEQ ID NO: 653)






In other embodiments, the linker constitutes a phosphorylation site, e.g., X1X2YX3X4G (SEQ ID NO: 654), wherein X1, X2, X3 and X4 are each independently any amino acid residue; X1X2SX3X4G (SEQ ID NO: 655), wherein X1, X2, X3 and X4 are each independently any amino acid residue; or X1X2TX3X4G (SEQ ID NO: 656), wherein X1, X2, X3 and X4 are each independently any amino acid residue.
Non-peptide linkers are also possible. For example, alkyl linkers such as —NH—(CH2)s—C(O)—, wherein s=2-20 could be used. These alkyl linkers can further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C1-C6) lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. An exemplary non-peptide linker is a PEG linker,




wherein n is such that the linker has a molecular weight of 100 to 5000 kDa, preferably 100 to 500 kDa. The peptide linkers can be altered to form derivatives in the same manner as described above.

Useful linker embodiments also include aminoethyloxyethyloxy-acetyl linkers as disclosed by Chandy et al. (Chandy et al., WO 2006/042151 A2, incorporated herein by reference in its entirety).
Derivatives. The inventors also contemplate derivatizing the peptide and/or half-life extending moiety portion of the compounds. Such derivatives can improve the solubility, absorption, biological half-life, and the like of the compounds. The moieties can alternatively eliminate or attenuate any undesirable side-effect of the compounds and the like. Exemplary derivatives include compounds in which:

    1. The compound or some portion thereof is cyclic. For example, the peptide portion can be modified to contain two or more Cys residues (e.g., in the linker), which could cyclize by disulfide bond formation.
    2. The compound is cross-linked or is rendered capable of cross-linking between molecules. For example, the peptide portion can be modified to contain one Cys residue and thereby be able to form an intermolecular disulfide bond with a like molecule. The compound can also be cross-linked through its C-terminus, as in the molecule shown below.






    3. Non-peptidyl linkages (bonds) replace one or more peptidyl [—C(O)NR—] linkages. Exemplary non-peptidyl linkages are —CH2-carbamate [—CH2—OC(O)NR—], phosphonate, —CH2-sulfonamide [—CH2—S(O)2NR—], urea [—NHC(O)NH—], —CH2-secondary amine, and alkylated peptide [—C(O)NR6— wherein R6 is lower alkyl].
    4. The N-terminus is chemically derivatized. Typically, the N-terminus can be acylated or modified to a substituted amine. Exemplary N-terminal derivative groups include —NRR1 (other than —NH2), —NRC(O)R1, —NRC(O)OR1, —NRS(O)2R1, —NHC(O)NHR1, succinimide, or benzyloxycarbonyl-NH— (CBZ-NH—), wherein R and R1 are each independently hydrogen or lower alkyl and wherein the phenyl ring can be substituted with 1 to 3 substituents selected from the group consisting of C1-C4 alkyl, C1-C4 alkoxy, chloro, and bromo.
    5. The free C-terminus is derivatized. Typically, the C-terminus is esterified or amidated. For example, one can use methods described in the art to add (NH—CH2—CH2—NH2)2 to compounds of this invention having any of SEQ ID NOS: 504 to 508 at the C-terminus. Likewise, one can use methods described in the art to add —NH2 to compounds of this invention having any of SEQ ID NOS: 924 to 955, 963 to 972, 1005 to 1013, or 1018 to 1023 at the C-terminus. Exemplary C-terminal derivative groups include, for example, —C(O)R2 wherein R2 is lower alkoxy or —NR3R4 wherein R3 and R4 are independently hydrogen or C1-C8 alkyl (preferably C1-C4 alkyl).
    6. A disulfide bond is replaced with another, preferably more stable, cross-linking moiety (e.g., an alkylene). See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am. Pep. Symp., 357-9.
    7. One or more individual amino acid residues are modified. Various derivatizing agents are known to react specifically with selected sidechains or terminal residues, as described in detail below.


Lysinyl residues and amino terminal residues can be reacted with succinic or other carboxylic acid anhydrides, which reverse the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
Arginyl residues can be modified by reaction with any one or combination of several conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents can react with the groups of lysine as well as the arginine epsilon-amino group.
Specific modification of tyrosyl residues has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
Carboxyl sidechain groups (aspartyl or glutamyl) can be selectively modified by reaction with carbodiimides (R′—N═C═N—R′) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
Glutaminyl and asparaginyl residues can be deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
Cysteinyl residues can be replaced by amino acid residues or other moieties either to eliminate disulfide bonding or, conversely, to stabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9.
Derivatization with bifunctional agents is useful for cross-linking the peptides or their functional derivatives to a water-insoluble support matrix or to other macromolecular half-life extending moieties. Commonly used cross-linking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
Carbohydrate (oligosaccharide) groups can conveniently be attached to sites that are known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids other than proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, can confer acidic properties to the glycosylated compound. Such site(s) can be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). However, such sites can further be glycosylated by synthetic or semi-synthetic procedures known in the art.
Other possible modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, oxidation of the sulfur atom in Cys, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains. Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman and Co., San Francisco), pp. 79-86 (1983).
Compounds of the present invention can be changed at the DNA level, as well. The DNA sequence of any portion of the compound can be changed to codons more compatible with the chosen host cell. For E. coli, which is the preferred host cell, optimized codons are known in the art. Codons can be substituted to eliminate restriction sites or to include silent restriction sites, which can aid in processing of the DNA in the selected host cell. The half-life extending moiety, linker and peptide DNA sequences can be modified to include any of the foregoing sequence changes.
A process for preparing conjugation derivatives is also contemplated. Tumor cells, for example, exhibit epitopes not found on their normal counterparts. Such epitopes include, for example, different post-translational modifications resulting from their rapid proliferation. Thus, one aspect of this invention is a process comprising:

    
    
        a) selecting at least one randomized peptide that specifically binds to a target epitope; and
        b) preparing a pharmacologic agent comprising (i) at least one half-life extending moiety (Fc domain preferred), (ii) at least one amino acid sequence of the selected peptide or peptides, and (iii) an effector molecule.

The target epitope is preferably a tumor-specific epitope or an epitope specific to a pathogenic organism. The effector molecule can be any of the above-noted conjugation partners and is preferably a radioisotope.

    
    


Methods of Making
The present invention also relates to nucleic acids, expression vectors and host cells useful in producing the polypeptides of the present invention. Host cells can be eukaryotic cells, with mammalian cells preferred and CHO cells most preferred. Host cells can also be prokaryotic cells, with E. coli cells most preferred.
The compounds of this invention largely can be made in transformed host cells using recombinant DNA techniques. To do so, a recombinant DNA molecule coding for the peptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences coding for the peptides could be excised from DNA using suitable restriction enzymes. Alternatively, the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidate method. Also, a combination of these techniques could be used.
The invention also includes a vector capable of expressing the peptides in an appropriate host. The vector comprises the DNA molecule that codes for the peptides operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation.
The resulting vector having the DNA molecule thereon is used to transform an appropriate host. This transformation can be performed using methods well known in the art.
Any of a large number of available and well-known host cells can be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts can be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (such as E. coli sp.), yeast (such as Saccharomyces sp.) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.
Next, the transformed host is cultured and purified. Host cells can be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art. Finally, the peptides are purified from culture by methods well known in the art.
In some embodiments of the inventive DNA, the DNA encodes a recombinant fusion protein composition of the invention, preferably, but not necessarily, monovalent with respect to the toxin peptide, for expression in a mammalian cell, such as, but not limited to, CHO or HEK293. The encoded fusion protein includes (a)-(c) immediately below, in the N-terminal to C-terminal direction:
(a) an immunoglobulin, which includes the constant and variable regions of the immunoglobulin light and heavy chains, or a portion of an immunoglobulin (e.g., an Fc domain, or the variable regions of the light and heavy chains); if both immunoglobulin light chain and heavy chain components are to be included in the construct, then a peptidyl linker, as further described in (b) immediately below, is also included to separate the immunoglobulin components (See, e.g., FIG. 92A-C); useful coding sequences for immunoglobulin light and heavy chains are well known in the art;
(b) a peptidyl linker, which is at least 4 (or 5) amino acid residues long and comprises at least one protease cleavage site (e.g., a furin cleavage site, which is particularly useful for intracellular cleavage of the expressed fusion protein); typically, the peptidyl linker sequence can be up to about 35 to 45 amino acid residues long (e.g., a 7x L5 linker modified to include the desired protease cleavage site(s)), but linkers up to about 100 to about 300 amino acid residues long are also useful; and
(c) an immunoglobulin Fc domain or a portion thereof. The Fc domain of (c) can be from the same type of immunoglobulin in (a), or different. In such embodiments, the DNA encodes a toxin peptide covalently linked to the N-terminal or C-terminal end of (a) or (c) above, either directly or indirectly via a peptidyl linker (a linker minus a protease cleavage site). Any toxin peptide or peptide analog thereof as described herein can be encoded by the DNA (e.g., but not limited to, ShK, HmK, MgTx, AgTx1, AgTx2, HsTx1, OSK1, Anuroctoxin, Noxiustoxin, Hongotoxin, HsTx1, ChTx, MTx, Titystoxin, BgK, BmKTX, BmTx, Tc30, Tc32, Pi1, Pi2, Pi3 toxin peptide, or a peptide analog of any of these). For example, an OSK1 peptide analog comprising an amino acid sequence selected from SEQ ID NOS: 25, 294 through 298, 562 through 636, 980 through 1274, 1303, 1308, 1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through 5015, as set forth in Tables 7 and Tables 7A-J, can be employed. Alternatively, an ShK peptide analog comprising an amino acid sequence selected from SEQ ID NOS: 5, 88 through 200, 548 through 561, 884 through 950, and 1295 through 1300 as set forth in Table 2, can be employed. Any other toxin peptide sequence described herein that can alternatively be expressed recombinantly using recombinant and protein engineering techniques known in the art can also be used. The immunoglobulin of (a) and (c) above can be in each instance independently selected from any desired type, such as but not limited to, IgG1, IgG2, IgG3, and IgG4. The variable regions can be non-functional in vivo (e.g., CDRs specifically binding KLH), or alternatively, if targeting enhancement function is also desired, the variable regions can be chosen to specifically bind (non-competitively) the ion channel target of the toxin peptide (e.g., Kv1.3) or specifically bind another antigen typically found associated with, or in the vicinity of, the target ion channel. In addition, the inventive DNA optionally further encodes, 5′ to the coding region of (a) above, a signal peptide sequence (e.g., a secretory signal peptide) operably linked to the expressed fusion protein. An example of the inventive DNA encoding a recombinant fusion protein for expression in a mammalian cell, described immediately above, is a DNA that encodes a fusion protein comprising, in the N-terminal to C-terminal direction:
(a) an immunoglobulin light chain;
(b) a first peptidyl linker at least 4 amino acid residues long comprising at least one protease cleavage site, as described above;
(c) an immunoglobulin heavy chain;
(d) a second peptidyl linker at least 4 amino acid residues long comprising at least one protease cleavage site, as described above; and
(e) an immunoglobulin Fc domain or a portion thereof. Here, the Fc domain of (e) can be from the same type of immunoglobulin as the heavy chain in (c), or different. The DNA encodes a toxin peptide covalently linked to the N-terminal or C-terminal end of (a), (c), or (e) of the expressed fusion protein, either directly or indirectly via a peptidyl linker (a linker minus a protease cleavage site). FIG. 92A-C illustrates schematically an embodiment, in which the toxin peptide (e.g., an OSK1, ShK, or a peptide analog of either of these) is covalently linked to the C-terminal end of the Fc domain of (e). In FIG. 92A-C, a linker is shown covalently linking the toxin peptide to the rest of the molecule, but as previously described, this linker is optional.
In some embodiments particularly suited for the recombinant expression of monovalent dimeric Fc-toxin peptide fusions or “peptibodies” (see, FIG. 2B and Example 56) by mammalian cells, such as, but not limited to, CHO or HEK293, the inventive DNA encodes a recombinant expressed fusion protein that comprises, in the N-terminal to C-terminal direction:

    
    
        (a) a first immunoglobulin Fc domain or portion thereof;
        (b) a peptidyl linker at least 4 (or 5) amino acid residues long comprising at least one protease cleavage site (e.g., a furin cleavage site, which is particularly useful for intracellular cleavage of the expressed fusion protein); typically, the peptidyl linker sequence can be up to about 35 to 45 amino acid residues long (e.g., a 7x L5 linker modified to include the desired protease cleavage site(s)), but linkers up to about 100 to about 300 amino acid residues long are also useful; and
        (c) a second immunoglobulin Fc domain or portion thereof (which may be the same or different from the first Fc domain, but should be expressed in the same orientation as the first Fc domain).

For such embodiments, the DNA encodes a toxin peptide covalently linked to the N-terminal or C-terminal end of (a) or (c) of the expressed fusion protein, either directly or indirectly via a peptidyl linker (a linker minus a protease cleavage site); Example 56 describes an embodiment in which the toxin peptide is conjugated to the C-terminal end of the second immunoglobulin Fc domain (c). Any toxin peptide or peptide analog thereof as described herein can be encoded by the DNA (e.g., but not limited to, ShK, HmK, MgTx, AgTx1, AgTx2, HsTx1, OSK1, Anuroctoxin, Noxiustoxin, Hongotoxin, HsTx1, ChTx, MTx, Titystoxin, BgK, BmKTX, BmTx, Tc30, Tc32, Pi1, Pi2, Pi3 toxin peptide, or a peptide analog of any of these). For example, an OSK1 peptide analog comprising an amino acid sequence selected from SEQ ID NOS: 25, 294 through 298, 562 through 636, 980 through 1274, 1303, 1308, 1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through 5015, as set forth in Tables 7 and Tables 7A-J, can be employed. Alternatively, an ShK peptide analog comprising an amino acid sequence selected from SEQ ID NOS: 5, 88 through 200, 548 through 561, 884 through 950, and 1295 through 1300 as set forth in Table 2, can be employed. Any other toxin peptide sequence described herein that can alternatively be expressed using recombinant and protein engineering techniques known in the art can also be used. In addition, the inventive DNA optionally further encodes, 5′ to the coding region of (a) above, a signal peptide sequence (e.g., a secretory signal peptide) operably linked to the expressed fusion protein.

    
    


DNA constructs similar to those described above are also useful for recombinant expression by mammalian cells of other dimeric Fc fusion proteins (“peptibodies”) or chimeric immunoglobulin (light chain+heavy chain)-Fc heterotrimers (“hemibodies”), conjugated to pharmacologically active peptides (e.g., agonist or antagonist peptides) other than toxin peptides.
Peptide compositions of the present invention can also be made by synthetic methods. Solid phase synthesis is the preferred technique of making individual peptides since it is the most cost-effective method of making small peptides. For example, well known solid phase synthesis techniques include the use of protecting groups, linkers, and solid phase supports, as well as specific protection and deprotection reaction conditions, linker cleavage conditions, use of scavengers, and other aspects of solid phase peptide synthesis. Suitable techniques are well known in the art. (E.g., Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527; “Protecting Groups in Organic Synthesis,” 3rd Edition, T. W. Greene and P. G. M. Wuts, Eds., John Wiley & Sons, Inc., 1999; NovaBiochem Catalog, 2000; “Synthetic Peptides, A User's Guide,” G. A. Grant, Ed., W.H. Freeman & Company, New York, N.Y., 1992; “Advanced Chemtech Handbook of Combinatorial & Solid Phase Organic Chemistry,” W. D. Bennet, J. W. Christensen, L. K. Hamaker, M. L. Peterson, M. R. Rhodes, and H. H. Saneii, Eds., Advanced Chemtech, 1998; “Principles of Peptide Synthesis, 2nd ed.,” M. Bodanszky, Ed., Springer-Verlag, 1993; “The Practice of Peptide Synthesis, 2nd ed.,” M. Bodanszky and A. Bodanszky, Eds., Springer-Verlag, 1994; “Protecting Groups,” P. J. Kocienski, Ed., Georg Thieme Verlag, Stuttgart, Germany, 1994; “Fmoc Solid Phase Peptide Synthesis, A Practical Approach,” W. C. Chan and P. D. White, Eds., Oxford Press, 2000, G. B. Fields et al., Synthetic Peptides: A User's Guide, 1990, 77-183).
Whether the compositions of the present invention are prepared by synthetic or recombinant techniques, suitable protein purification techniques can also be involved, when applicable. In some embodiments of the compositions of the invention, the toxin peptide portion and/or the half-life extending portion, or any other portion, can be prepared to include a suitable isotopic label (e.g., 125I, 14C, 13C, 35S, 3H, 2H, 13N, 15N, 18O, 17O, etc.), for ease of quantification or detection.
Compounds that contain derivatized peptides or which contain non-peptide groups can be synthesized by well-known organic chemistry techniques.
Uses of the Compounds
In general. The compounds of this invention have pharmacologic activity resulting from their ability to bind to proteins of interest as agonists, mimetics or antagonists of the native ligands of such proteins of interest. Heritable diseases that have a known linkage to ion channels (“channelopathies”) cover various fields of medicine, some of which include neurology, nephrology, myology and cardiology. A list of inherited disorders attributed to ion channels includes:

    
    
        cystic fibrosis (Cl− channel; CFTR),
        Dent's disease (proteinuria and hypercalciuria; Cl− channel; CLCN5),
        osteopetrosis (Cl− channel; CLCN7),
        familial hyperinsulinemia (SUR1; KCNJ11; K channel),
        diabetes (KATP/SUR channel),
        Andersen syndrome (KCNJ2, Kir2.1 K channel),
        Bartter syndrome (KCNJ1; Kir1.1/ROMK; K channel),
        hereditary hearing loss (KCNQ4; K channel),
        hereditary hypertension (Liddle's syndrome; SCNN1; epithelial Na channel),
        dilated cardiomyopathy (SUR2, K channel),
        long-QT syndrome or cardiac arrhythmias (cardiac potassium and sodium channels),
        Thymothy syndrome (CACNA1C, Cav1.2),
        myasthenic syndromes (CHRNA, CHRNB, CNRNE; nAChR), and a variety of other myopathies,
        hyperkalemic periodic paralysis (Na and K channels),
        epilepsy (Na+ and K+ channels),
        hemiplegic migraine (CACNA1A, Cav2.1 Ca2+ channel and ATP1A2),
        central core disease (RYR1, RYR1; Ca2+ channel), and
        paramyotonia and myotonia (Na+, Cl− channels)

See L. J. Ptacek and Y—H Fu (2004), Arch. Neurol. 61: 166-8; B. A. Niemeyer et al. (2001), EMBO reports 21: 568-73; F. Lehmann-Horn and K. Jurkat-Rott (1999), Physiol. Rev. 79: 1317-72. Although the foregoing list concerned disorders of inherited origin, molecules targeting the channels cited in these disorders can also be useful in treating related disorders of other, or indeterminate, origin.

    
    


In addition to the aforementioned disorders, evidence has also been provided supporting ion channels as targets for treatment of:

    
    
        sickle cell anemia (IKCa1)—in sickle cell anemia, water loss from erythrocytes leads to hemoglobin polymerization and subsequent hemolysis and vascular obstruction. The water loss is consequent to potassium efflux through the so-called Gardos channel i.e., IKCa1. Therefore, block of IKCa1 is a potential therapeutic treatment for sickle cell anemia.
        glaucoma (BKCa),—in glaucoma the intraocular pressure is too high leading to optic nerve damage, abnormal eye function and possibly blindness. Block of BKCa potassium channels can reduce intraocular fluid secretion and increase smooth muscle contraction, possibly leading to lower intraocular pressure and neuroprotection in the eye.
        multiple sclerosis (Kv, KCa),
        psoriasis (Kv, KCa),
        arthritis (Kv, KCa),
        asthma (KCa, Kv),
        allergy (KCa, Kv),
        COPD (KCa, Kv, Ca),
        allergic rhinitis (KCa, Kv),
        pulmonary fibrosis,
        lupus (IKCa1, Kv),
        transplantation, GvHD (KCa, Kv),
        inflammatory bone resorption (KCa, Kv),
        periodontal disease (KCa, Kv),
        diabetes, type I (Kv),—type I diabetes is an autoimmune disease that is characterized by abnormal glucose, protein and lipid metabolism and is associated with insulin deficiency or resistance. In this disease, Kv1.3-expressing T-lymphocytes attack and destroy pancreatic islets leading to loss of beta-cells. Block of Kv1.3 decreases inflammatory cytokines. In addition block of Kv1.3 facilitates the translocation of GLUT4 to the plasma membrane, thereby increasing insulin sensitivity.
        obesity (Kv),—Kv1.3 appears to play a critical role in controlling energy homeostasis and in protecting against diet-induced obesity. Consequently, Kv1.3 blockers could increase metabolic rate, leading to greater energy utilization and decreased body weight.
        restenosis (KCa, Ca2+),—proliferation and migration of vascular smooth muscle cells can lead to neointimal thickening and vascular restenosis. Excessive neointimal vascular smooth muscle cell proliferation is associated with elevated expression of IKCa1. Therefore, block of IKCa1 could represent a therapeutic strategy to prevent restenosis after angioplasty.
        ischaemia (KCa, Ca2+),—in neuronal or cardiac ischemia, depolarization of cell membranes leads to opening of voltage-gated sodium and calcium channels. In turn this can lead to calcium overload, which is cytotoxic. Block of voltage-gated sodium and/or calcium channels can reduce calcium overload and provide cytoprotective effects. In addition, due to their critical role in controlling and stabilizing cell membrane potential, modulators of voltage- and calcium-activated potassium channels can also act to reduce calcium overload and protect cells.
        renal incontinence (KCa), renal incontinence is associated with overactive bladder smooth muscle cells. Calcium-activated potassium channels are expressed in bladder smooth muscle cells, where they control the membrane potential and indirectly control the force and frequency of cell contraction. Openers of calcium-activated potassium channels therefore provide a mechanism to dampen electrical and contractile activity in bladder, leading to reduced urge to urinate.
        osteoporosis (Kv),
        pain, including migraine (Nav, TRP [transient receptor potential channels], P2X, Ca2+), N-type voltage-gated calcium channels are key regulators of nociceptive neurotransmission in the spinal cord. Ziconotide, a peptide blocker of N-type calcium channels reduces nociceptive neurotransmission and is approved worldwide for the symptomatic alleviation of severe chronic pain in humans. Novel blockers of nociceptor-specific N-type calcium channels would be improved analgesics with reduced side-effect profiles.
        hypertension (Ca2+),—L-type and T-type voltage-gated calcium channels are expressed in vascular smooth muscle cells where they control excitation-contraction coupling and cellular proliferation. In particular, T-type calcium channel activity has been linked to neointima formation during hypertension. Blockers of L-type and T-type calcium channels are useful for the clinical treatment of hypertension because they reduce calcium influx and inhibit smooth muscle cell contraction.
        wound healing, cell migration serves a key role in wound healing. Intracellular calcium gradients have been implicated as important regulators of cellular migration machinery in keratinocytes and fibroblasts. In addition, ion flux across cell membranes is associated with cell volume changes. By controlling cell volume, ion channels contribute to the intracellular environment that is required for operation of the cellular migration machinery. In particular, IKCa1 appears to be required universally for cell migration. In addition, Kv1.3, Kv3.1, NMDA receptors and N-type calcium channels are associated with the migration of lymphocytes and neurons.
        stroke,
        Alzheimer's,
        Parkenson's Disease (nACHR, Nav)
        Bipolar Disorder (Nav, Cav)
        cancer, many potassium channel genes are amplified and protein subunits are upregulated in many cancerous condition. Consistent with a pathophysiological role for potassium channel upregulation, potassium channel blockers have been shown to suppress proliferation of uterine cancer cells and hepatocarcinoma cells, presumably through inhibition of calcium influx and effects on calcium-dependent gene expression.
        a variety of neurological, cardiovascular, metabolic and autoimmune diseases.
    
    


Both agonists and antagonists of ion channels can achieve therapeutic benefit. Therapeutic benefits can result, for example, from antagonizing Kv1.3, IKCa1, SKCa, BKCa, N-type or T-type Ca2+ channels and the like. Small molecule and peptide antagonists of these channels have been shown to possess utility in vitro and in vivo. Limitations in production efficiency and pharmacokinetics, however, have largely prevented clinical investigation of inhibitor peptides of ion channels.
Compositions of this invention incorporating peptide antagonists of the voltage-gated potassium channel Kv1.3, in particular OSK1 peptide analogs, whether or not conjugated to a half-life extending moiety, are useful as immunosuppressive agents with therapeutic value for autoimmune diseases. For example, such molecules are useful in treating multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, and rheumatoid arthritis. (See, e.g., H. Wulff et al. (2003) J. Clin. Invest. 111, 1703-1713 and H. Rus et al. (2005) PNAS 102, 11094-11099; Beeton et al., Targeting effector memory T cells with a selective inhibitor peptide of Kv1.3 channels for therapy of autoimmune diseases, Molec. Pharmacol. 67(4):1369-81 (2005); 1 Beeton et al. (2006), Kv1.3: therapeutic target for cell-mediated autoimmune disease, electronic preprint at //webfiles.uci.edu/xythoswfs/webui/2670029.1). Inhibitors of the voltage-gated potassium channel Kv1.3 have been examined in a variety of preclinical animal models of inflammation. Small molecule and peptide inhibitors of Kv1.3 have been shown to block delayed type hypersensitivity responses to ovalbumin [C. Beeton et al. (2005) Mol. Pharmacol. 67, 1369] and tetanus toxoid [G. C. Koo et al. (1999) Clin. Immunol. 197, 99]. In addition to suppressing inflammation in the skin, inhibitors also reduced antibody production [G. C. Koo et al. (1997) J. Immunol. 158, 5120]. Kv1.3 antagonists have shown efficacy in a rat adoptive-transfer experimental autoimmune encephalomyelitis (AT-EAE) model of multiple sclerosis (MS). The Kv1.3 channel is overexpressed on myelin-specific T cells from MS patients, lending further support to the utility Kv1.3 inhibitors may provide in treating MS. Inflammatory bone resorption was also suppressed by Kv1.3 inhibitors in a preclinical adoptive-transfer model of periodontal disease [P. Valverde et al. (2004) J. Bone Mineral Res. 19, 155]. In this study, inhibitors additionally blocked antibody production to a bacterial outer membrane protein,—one component of the bacteria used to induce gingival inflammation. Recently in preclinical rat models, efficacy of Kv1.3 inhibitors was shown in treating pristane-induced arthritis and diabetes [C. Beeton et al. (2006) preprint available at //webfiles.uci.edu/xythoswfs/webui/_xy-2670029—1.]. The Kv1.3 channel is expressed on all subsets of T cells and B cells, but effector memory T cells and class-switched memory B cells are particularly dependent on Kv1.3 [H. Wulff et al. (2004) J. Immunol. 173, 776]. Gad5/insulin-specific T cells from patients with new onset type 1 diabetes, myelin-specific T cells from MS patients and T cells from the synovium of rheumatoid arthritis patients all overexpress Kv1.3 [C. Beeton et al. (2006) preprint at //webfiles.uci.edu/xythoswfs/webui/_xy-2670029—1.]. Because mice deficient in Kv1.3 gained less weight when placed on a high fat diet [J. Xu et al. (2003) Human Mol. Genet. 12, 551] and showed altered glucose utilization [J. Xu et al. (2004) Proc. Natl. Acad. Sci. 101, 3112], Kv1.3 is also being investigated for the treatment of obesity and diabetes. Breast cancer specimens [M. Abdul et al. (2003) Anticancer Res. 23, 3347] and prostate cancer cell lines [S. P. Fraser et al. (2003) Pflugers Arch. 446, 559] have also been shown to express Kv1.3, and Kv1.3 blockade may be of utility for treatment of cancer. Disorders that can be treated in accordance with the inventive method of treating an autoimmune disorder, involving Kv1.3 inhibitor toxin peptide(s), include multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, and systemic lupus erythematosus (SLE) and other forms of lupus.
Some of the cells that express the calcium-activated potassium of intermediate conductance IKCa1 include T cells, B cells, mast cells and red blood cells (RBCs). T cells and RBCS from mice deficient in IKCa1 show defects in volume regulation [T. Begenisich et al. (2004) J. Biol. Chem. 279, 47681]. Preclinical and clinical studies have demonstrated IKCa1 inhibitors utility in treating sickle cell anemia [J. W. Stocker et al. (2003) Blood 101, 2412]. Blockers of the IKCa1 channel have also been shown to block EAE, indicating they may possess utility in treatment of MS [E. P. Reich et al. (2005) Eur. J. Immunol. 35, 1027]. IgE-mediated histamine production from mast cells is also blocked by IKCa1 inhibitors [S. Mark Duffy et al. (2004) J. Allergy Clin. Immunol. 114, 66], therefore they may also be of benefit in treating asthma. The IKCa1 channel is overexpressed on activated T and B lymphocytes [H. Wulff et al. (2004) J. Immunol. 173, 776] and thus may show utility in treatment of a wide variety of immune disorders. Outside of the immune system, IKCa1 inhibitors have also shown efficacy in a rat model of vascular restinosis and thus might represent a new therapeutic strategy to prevent restenosis after angioplasty [R. Kohler et al. (2003) Circulation 108, 1119]. It is also thought that IKCa1 antagonists are of utility in treatment of tumor angiogenesis since inhibitors suppressed endothelial cell proliferation and angionenesis in vivo [I. Grgic et al. (2005) Arterioscler. Thromb. Vasc. Biol. 25, 704]. The IKCa1 channel is upregulated in pancreatic tumors and inhibitors blocked proliferation of pancreatic tumor cell lines [H. Jager et al. (2004) Mol Pharmacol. 65, 630]. IKCa1 antagonists may also represent an approach to attenuate acute brain damage caused by traumatic brain injury [F. Mauler (2004) Eur. J. Neurosci. 20, 1761]. Disorders that can be treated with IKCa1 inhibitors include multiple sclerosis, asthma, psoriasis, contact-mediated dermatitis, rheumatoid & psoriatic arthritis, inflammatory bowel disease, transplant rejection, graft-versus-host disease, Lupus, restinosis, pancreatic cancer, tumor angiogenesis and traumatic brain injury.
Accordingly, molecules of this invention incorporating peptide antagonists of the calcium-activated potassium channel of intermediate conductance, IKCa can be used to treat immune dysfunction, multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, and lupus.
Accordingly, the present invention includes a method of treating an autoimmune disorder, which involves administering to a patient who has been diagnosed with an autoimmune disorder, such as multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, or lupus, a therapeutically effective amount of the inventive composition of matter, whereby at least one symptom of the disorder is alleviated in the patient. “Alleviated” means to be lessened, lightened, diminished, softened, mitigated (i.e., made more mild or gentle), quieted, assuaged, abated, relieved, nullified, or allayed, regardless of whether the symptom of interest is entirely erased, eradicated, eliminated, or prevented in a particular patient.
The present invention is further directed to a method of preventing or mitigating a relapse of a symptom of multiple sclerosis, which method involves administering to a patient, who has previously experienced at least one symptom of multiple sclerosis, a prophylactically effective amount of the inventive composition of matter, such that the at least one symptom of multiple sclerosis is prevented from recurring or is mitigated.
The inventive compositions of matter preferred for use in practicing the inventive method of treating an autoimmune disorder, e.g., inflammatory bowel disease (IBD, including Crohn's Disease and ulcerative colitis), and the method of preventing or mitigating a relapse of a symptom of multiple sclerosis include as P (conjugated as in Formula I), a Kv1.3 or IKCa1 antagonist peptide, such as a ShK peptide, an OSK1 peptide or an OSK1 peptide analog, a ChTx peptide and/or a Maurotoxin (MTx) peptide, or peptide analogs of any of these.
For example, the conjugated ShK peptide or ShK peptide analog can comprise an amino acid sequence selected from the following:

    
    
        SEQ ID NOS: 5, 88 through 200, 548 through 561, 884 through 950, or 1295 through 1300 as set forth in Table 2.
    
    


The conjugated OSK1 peptide, or conjugated or unconjugated OSK1 peptide analog, can comprise an amino acid sequence selected from the following:

    
    
        SEQ ID NOS: 25, 294 through 298, 562 through 636, 980 through 1274, GVIINVSCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK (OSK1-S7) (SEQ ID NO: 1303), or GVIINVSCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK (OSK1-S7,K16,D20) (SEQ ID NO: 1308) as set forth in Table 7, or any of SEQ ID NOS: 1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through 5015 as set forth in Table 7A, Table 7B, Table 7C, Table 7D, Table 7E, Table 7F, Table 7G, Table 7H, Table 7I, or Table 7J.
    
    


Also by way of example, a the conjugated MTX peptide, MTX peptide analog, ChTx peptide or ChTx peptide analog can comprise an amino acid sequence selected from:

    
    
        SEQ ID NOS: 20, 330 through 343, 1301, 1302, 1304 through 1307, 1309, 1311, 1312, or 1315 through 1336 as set forth in Table 13; or SEQ ID NOS: 36, 59, 344 through 346, or 1369 through 1390 as set forth in Table 14.
    
    


Also useful in these methods conjugated, or unconjugated, are a Kv1.3 or IKCa1 inhibitor toxin peptide analog that comprises an amino acid sequence selected from:

    
    
        SEQ ID NOS: 88, 89, 92, 148 through 200, 548 through 561, 884 through 949, or 1295 through 1300 as set forth in Table 2; or
        SEQ ID NOS: 980 through 1274, GVIINVSCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK (OSK1-S7) (SEQ ID NO: 1303), or GVIINVSCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK (OSK1-S7,K16,D20) (SEQ ID NO: 1308) as set forth in Table 7; or
        SEQ ID NOS: 330 through 337, 341, 1301, 1302, 1304 through 1307, 1309, 1311, 1312, and 1315 through 1336 as set forth in Table 13.
    
    


In accordance with these inventive methods, a patient who has been diagnosed with an autoimmune disorder, such as, but not limited to multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoratic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, or lupus, or a patient who has previously experienced at least one symptom of multiple sclerosis, are well-recognizable and/or diagnosed by the skilled practitioner, such as a physician, familiar with autoimmune disorders and their symptoms.
For example, symptoms of multiple sclerosis can include the following:

    
    
        visual symptoms, such as, optic neuritis (blurred vision, eye pain, loss of color vision, blindness); diplopia (double vision); nystagmus (jerky eye movements); ocular dysmetria (constant under- or overshooting eye movements); internuclear opthalmoplegia (lack of coordination between the two eyes, nystagmus, diplopia); movement and sound phosphenes (flashing lights when moving eyes or in response to a sudden noise); afferent pupillary defect (abnormal pupil responses);
        motor symptoms, such as, paresis, monoparesis, paraparesis, hemiparesis, quadraparesis (muscle weakness—partial or mild paralysis); plegia, paraplegia, hemiplegia, tetraplegia, quadraplegia (paralysis—total or near total loss of muscle strength); spasticity (loss of muscle tone causing stiffness, pain and restricting free movement of affected limbs); dysarthria (slurred speech and related speech problems); muscle atrophy (wasting of muscles due to lack of use); spasms, cramps (involuntary contraction of muscles); hypotonia, clonus (problems with posture); myoclonus, myokymia (jerking and twitching muscles, tics); restless leg syndrome (involuntary leg movements, especially bothersome at night); footdrop (foot drags along floor during walking); dysfunctional reflexes (MSRs, Babinski's, Hoffman's, Chaddock's);
        sensory symptoms, such as, paraesthesia (partial numbness, tingling, buzzing and vibration sensations); anaesthesia (complete numbness/loss of sensation); neuralgia, neuropathic and neurogenic pain (pain without apparent cause, burning, itching and electrical shock sensations); L'Hermitte's (electric shocks and buzzing sensations when moving head); proprioceptive dysfunction (loss of awareness of location of body parts); trigeminal neuralgia (facial pain);
        coordination and balance symptoms, such as, ataxia (loss of coordination); intention tremor (shaking when performing fine movements); dysmetria (constant under- or overshooting limb movements); vestibular ataxia (abnormal balance function in the inner ear); vertigo (nausea/vomitting/sensitivity to travel sickness from vestibular ataxia); speech ataxia (problems coordinating speech, stuttering); dystonia (slow limb position feedback); dysdiadochokinesia (loss of ability to produce rapidly alternating movements, for example to move to a rhythm);
        bowel, bladder and sexual symptoms, such as, frequent micturation, bladder spasticity (urinary urgency and incontinence); flaccid bladder, detrusor-sphincter dyssynergia (urinary hesitancy and retention); erectile dysfunction (male and female impotence); anorgasmy (inability to achieve orgasm); retrograde ejaculation (ejaculating into the bladder); frigidity (inability to become sexually aroused); constipation (infrequent or irregular bowel movements); fecal urgency (bowel urgency); fecal incontinence (bowel incontinence);
        cognitive symptoms, such as, depression; cognitive dysfunction (short-term and long-term memory problems, forgetfulness, slow word recall); dementia; mood swings, emotional lability, euphoria; bipolar syndrome; anxiety; aphasia, dysphasia (impairments to speech comprehension and production); and
        other symptoms, such as, fatigue; Uhthoff's Symptom (increase in severity of symptoms with heat); gastroesophageal reflux (acid reflux); impaired sense of taste and smell; epileptic seizures; swallowing problems, respiratory problems; and sleeping disorders.
    
    


By way of further example, symptoms of inflammatory bowel disease can include the following symptoms of Crohn's Disease or ulcerative colitis:
A. Symptoms of Crohn's disease can include:

    
    
        Abdominal pain. The pain often is described as cramping and intermittent, and the abdomen may be sore when touched. Abdominal pain may turn to a dull, constant ache as the condition progresses.
        Diarrhea. Some patients may have diarrhea 10 to 20 times per day. They may wake up at night and need to go to the bathroom. Crohn's disease may cause blood in stools, but not always.
        Loss of appetite.
        Fever. In severe cases, fever or other symptoms that affect the entire body may develop. A high fever may indicate a complication involving infection, such as an abscess.
        Weight loss. Ongoing symptoms, such as diarrhea, can lead to weight loss. Too few red blood cells (anemia). Some patients with Crohn's disease develop anemia because of low iron levels caused by bloody stools or the intestinal inflammation itself.
    
    


B. The symptoms of ulcerative colitis may include:

    
    
        Diarrhea or rectal urgency. Some patients may have diarrhea 10 to 20 times per day. The urge to defecate may wake patients at night.
        Rectal bleeding. Ulcerative colitis usually causes bloody diarrhea and mucus. Patients also may have rectal pain and an urgent need to empty the bowels.
        Abdominal pain, often described as cramping. The patient's abdomen may be sore when touched.
        Constipation. This symptom may develop depending on what part of the colon is affected.
        Loss of appetite.
        Fever. In severe cases, fever or other symptoms that affect the entire body may develop.
        Weight loss. Ongoing (chronic) symptoms, such as diarrhea, can lead to weight loss.
        Too few red blood cells (anemia). Some patients develop anemia because of low iron levels caused by bloody stools or intestinal inflammation.
    
    


The symptoms of multiple sclerosis and inflammatory bowel disease (including Crohn's Disease and ulcerative colitis) enumerated above, are merely illustrative and are not intended to be an exhaustive description of all possible symptoms experienced by a single patient or by several sufferers in composite, and to which the present invention is directed. Those skilled in the art are aware of various clinical symptoms and constellations of symptoms of autoimmune disorders suffered by individual patients, and to those symptoms are also directed the present inventive methods of treating an autoimmune disorder or of preventing or mitigating a relapse of a symptom of multiple sclerosis.
The therapeutically effective amount, prophylactically effective amount, and dosage regimen involved in the inventive methods of treating an autoimmune disorder or of preventing or mitigating a relapse of a symptom of multiple sclerosis, will be determined by the attending physician, considering various factors which modify the action of therapeutic agents, such as the age, condition, body weight, sex and diet of the patient, the severity of the condition being treated, time of administration, and other clinical factors. Generally, the daily amount or regimen should be in the range of about 1 to about 10,000 micrograms (μg) of the vehicle-conjugated peptide per kilogram (kg) of body mass, preferably about 1 to about 5000 μg per kilogram of body mass, and most preferably about 1 to about 1000 μg per kilogram of body mass.
Molecules of this invention incorporating peptide antagonists of the voltage-gated potassium channel Kv2.1 can be used to treat type II diabetes.
Molecules of this invention incorporating peptide antagonists of the M current (e.g., BeKm-1) can be used to treat Alzheimer's disease and enhance cognition.
Molecules of this invention incorporating peptide antagonists of the voltage-gated potassium channel Kv4.3 can be used to treat Alzheimer's disease.
Molecules of this invention incorporating peptide antagonists of the calcium-activated potassium channel of small conductance, SKCa can be used to treat epilepsy, memory, learning, neuropsychiatric, neurological, neuromuscular, and immunological disorders, schizophrenia, bipolar disorder, sleep apnea, neurodegeneration, and smooth muscle disorders.
Molecules of this invention incorporating N-type calcium channel antagonist peptides are useful in alleviating pain. Peptides with such activity (e.g., ZiconotideTM, ω-conotoxin-MVIIA) have been clinically validated.
Molecules of this invention incorporating T-type calcium channel antagonist peptides are useful in alleviating pain. Several lines of evidence have converged to indicate that inhibition of Cav3.2 in dorsal root ganglia may bring relief from chronic pain. T-type calcium channels are found at extremely high levels in the cell bodies of a subset of neurons in the DRG; these are likely mechanoreceptors adapted to detect slowly-moving stimuli (Shin et al., Nature Neuroscience 6:724-730, 2003), and T-type channel activity is likely responsible for burst spiking (Nelson et al., J Neurosci 25:8766-8775, 2005). Inhibition of T-type channels by either mibefradil or ethosuximide reverses mechanical allodynia in animals induced by nerve injury (Dogrul et al., Pain 105:159-168, 2003) or by chemotherapy (Flatters and Bennett, Pain 109:150-161, 2004). Antisense to Cav3.2, but not Cav3.1 or Cav3.3, increases pain thresholds in animals and also reduces expression of Cav3.2 protein in the DRG (Bourinet et al., EMBO J 24:315-324, 2005). Similarly, locally injected reducing agents produce pain and increase Cav3.2 currents, oxidizing agents reduce pain and inhibit Cav3.2 currents, and peripherally administered neurosteroids are analgesic and inhibit T-type currents from DRG (Todorovic et al., Pain 109:328-339, 2004; Pathirathna et al., Pain 114:429-443, 2005). Accordingly, it is thought that inhibition of Cav3.2 in the cell bodies of DRG neurons can inhibit the repetitive spiking of these neurons associated with chronic pain states.
Molecules of this invention incorporating L-type calcium channel antagonist peptides are useful in treating hypertension. Small molecules with such activity (e.g., DHP) have been clinically validated.
Molecules of this invention incorporating peptide antagonists of the NaV1 (TTXs-type) channel can be used to alleviate pain. Local anesthetics and tricyclic antidepressants with such activity have been clinically validated. Such molecules of this invention can in particular be useful as muscle relaxants.
Molecules of this invention incorporating peptide antagonists of the NaV1 (TTXR-type) channel can be used to alleviate pain arising from nerve and or tissue injury.
Molecules of this invention incorporating peptide antagonists of glial & epithelial cell Ca2+-activated chloride channel can be used to treat cancer and diabetes.
Molecules of this invention incorporating peptide antagonists of NMDA receptors can be used to treat pain, epilepsy, brain and spinal cord injury.
Molecules of this invention incorporating peptide antagonists of nicotinic receptors can be used as muscle relaxants. Such molecules can be used to treat pain, gastric motility disorders, urinary incontinence, nicotine addiction, and mood disorders.
Molecules of this invention incorporating peptide antagonists of 5HT3 receptor can be used to treat Nausea, pain, and anxiety.
Molecules of this invention incorporating peptide antagonists of the norepinephrine transporter can be used to treat pain, anti-depressant, learning, memory, and urinary incontinence.
Molecules of this invention incorporating peptide antagonists of the Neurotensin receptor can be used to treat pain.
In addition to therapeutic uses, the compounds of the present invention can be useful in diagnosing diseases characterized by dysfunction of their associated protein of interest. In one embodiment, a method of detecting in a biological sample a protein of interest (e.g., a receptor) that is capable of being activated comprising the steps of: (a) contacting the sample with a compound of this invention; and (b) detecting activation of the protein of interest by the compound. The biological samples include tissue specimens, intact cells, or extracts thereof. The compounds of this invention can be used as part of a diagnostic kit to detect the presence of their associated proteins of interest in a biological sample. Such kits employ the compounds of the invention having an attached label to allow for detection. The compounds are useful for identifying normal or abnormal proteins of interest.
The therapeutic methods, compositions and compounds of the present invention can also be employed, alone or in combination with other molecules in the treatment of disease.
Pharmaceutical Compositions
In General. The present invention also provides pharmaceutical compositions comprising the inventive composition of matter and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can be configured for administration to a patient by a wide variety of delivery routes, e.g., an intravascular delivery route such as by injection or infusion, subcutaneous, intramuscular, intraperitoneal, epidural, or intrathecal delivery routes, or for oral, enteral, pulmonary (e.g., inhalant), intranasal, transmucosal (e.g., sublingual administration), transdermal or other delivery routes and/or forms of administration known in the art. The inventive pharmaceutical compositions may be prepared in liquid form, or may be in dried powder form, such as lyophilized form. For oral or enteral use, the pharmaceutical compositions can be configured, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, elixirs or enteral formulas.
In the practice of this invention the “pharmaceutically acceptable carrier” is any physiologically tolerated substance known to those of ordinary skill in the art useful in formulating pharmaceutical compositions, including, any pharmaceutically acceptable diluents, excipients, dispersants, binders, fillers, glidants, anti-frictional agents, compression aids, tablet-disintegrating agents (disintegrants), suspending agents, lubricants, flavorants, odorants, sweeteners, permeation or penetration enhancers, preservatives, surfactants, solubilizers, emulsifiers, thickeners, adjuvants, dyes, coatings, encapsulating material(s), and/or other additives singly or in combination. Such pharmaceutical compositions can include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween® 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol®, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, which are herein incorporated by reference in their entirety. The compositions can be prepared in liquid form, or can be in dried powder, such as lyophilized form. Implantable sustained release formulations are also useful, as are transdermal or transmucosal formulations. Additionally (or alternatively), the present invention provides compositions for use in any of the various slow or sustained release formulations or microparticle formulations known to the skilled artisan, for example, sustained release microparticle formulations, which can be administered via pulmonary, intranasal, or subcutaneous delivery routes.
One can dilute the inventive compositions or increase the volume of the pharmaceutical compositions of the invention with an inert material. Such diluents can include carbohydrates, especially, mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may also be used as fillers, including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.
A variety of conventional thickeners are useful in creams, ointments, suppository and gel configurations of the pharmaceutical composition, such as, but not limited to, alginate, xanthan gum, or petrolatum, may also be employed in such configurations of the pharmaceutical composition of the present invention. A permeation or penetration enhancer, such as polyethylene glycol monolaurate, dimethyl sulfoxide, N-vinyl-2-pyrrolidone, N-(2-hydroxyethyl)-pyrrolidone, or 3-hydroxy-N-methyl-2-pyrrolidone can also be employed. Useful techniques for producing hydrogel matrices are known. (E.g., Feijen, Biodegradable hydrogel matrices for the controlled release of pharmacologically active agents, U.S. Pat. No. 4,925,677; Shah et al., Biodegradable pH/thermosensitive hydrogels for sustained delivery of biologically active agents, WO 00/38651 A1). Such biodegradable gel matrices can be formed, for example, by crosslinking a proteinaceous component and a polysaccharide or mucopolysaccharide component, then loading with the inventive composition of matter to be delivered.
Liquid pharmaceutical compositions of the present invention that are sterile solutions or suspensions can be administered to a patient by injection, for example, intramuscularly, intrathecally, epidurally, intravascularly (e.g., intravenously or intraarterially), intraperitoneally or subcutaneously. (See, e.g., Goldenberg et al., Suspensions for the sustained release of proteins, U.S. Pat. No. 6,245,740 and WO 00/38652 A1). Sterile solutions can also be administered by intravenous infusion. The inventive composition can be included in a sterile solid pharmaceutical composition, such as a lyophilized powder, which can be dissolved or suspended at a convenient time before administration to a patient using sterile water, saline, buffered saline or other appropriate sterile injectable medium.
Implantable sustained release formulations are also useful embodiments of the inventive pharmaceutical compositions. For example, the pharmaceutically acceptable carrier, being a biodegradable matrix implanted within the body or under the skin of a human or non-human vertebrate, can be a hydrogel similar to those described above. Alternatively, it may be formed from a poly-alpha-amino acid component. (Sidman, Biodegradable, implantable drug delivery device, and process for preparing and using same, U.S. Pat. No. 4,351,337). Other techniques for making implants for delivery of drugs are also known and useful in accordance with the present invention.
In powder forms, the pharmaceutically acceptable carrier is a finely divided solid, which is in admixture with finely divided active ingredient(s), including the inventive composition. For example, in some embodiments, a powder form is useful when the pharmaceutical composition is configured as an inhalant. (See, e.g., Zeng et al., Method of preparing dry powder inhalation compositions, WO 2004/017918; Trunk et al., Salts of the CGRP antagonist BIBN4096 and inhalable powdered medicaments containing them, U.S. Pat. No. 6,900,317).
One can dilute or increase the volume of the compound of the invention with an inert material. These diluents could include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts can also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-FloTM, EmdexTM, STA-RxTM 1500, EmcompressTM and AvicellTM.
Disintegrants can be included in the formulation of the pharmaceutical composition into a solid dosage form. Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, ExplotabTM. Sodium starch glycolate, AmberliteTM, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite can all be used. Insoluble cationic exchange resin is another form of disintegrant. Powdered gums can be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.
Binders can be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.
An antifrictional agent can be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants can be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants can also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.
Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants can include starch, talc, pyrogenic silica and hydrated silicoaluminate.
To aid dissolution of the compound of this invention into the aqueous environment a surfactant might be added as a wetting agent. Surfactants can include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethonium chloride. The list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.
Oral dosage forms. Also useful are oral dosage forms of the inventive compositions. If necessary, the composition can be chemically modified so that oral delivery is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the compound and increase in circulation time in the body. Moieties useful as covalently attached half-life extending moieties in this invention can also be used for this purpose. Examples of such moieties include: PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, for example, Abuchowski and Davis (1981), Soluble Polymer-Enzyme Adducts, Enzymes as Drugs (Hocenberg and Roberts, eds.), Wiley-Interscience, New York, N.Y., pp 367-83; Newmark, et al. (1982), J. Appl. Biochem. 4:185-9. Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are PEG moieties.
For oral delivery dosage forms, it is also possible to use a salt of a modified aliphatic amino acid, such as sodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhance absorption of the therapeutic compounds of this invention. The clinical efficacy of a heparin formulation using SNAC has been demonstrated in a Phase II trial conducted by Emisphere Technologies. See U.S. Pat. No. 5,792,451, “Oral drug delivery composition and methods.”
In one embodiment, the pharmaceutically acceptable carrier can be a liquid and the pharmaceutical composition is prepared in the form of a solution, suspension, emulsion, syrup, elixir or pressurized composition. The active ingredient(s) (e.g., the inventive composition of matter) can be dissolved, diluted or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both, or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as detergents and/or solubilizers (e.g., Tween 80, Polysorbate 80), emulsifiers, buffers at appropriate pH (e.g., Tris-HCl, acetate, phosphate), adjuvants, anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol), sweeteners, flavoring agents, suspending agents, thickening agents, bulking substances (e.g., lactose, mannitol), colors, viscosity regulators, stabilizers, electrolytes, osmolutes or osmo-regulators. Additives can also be included in the formulation to enhance uptake of the inventive composition. Additives potentially having this property are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.
Useful are oral solid dosage forms, which are described generally in Remington's Pharmaceutical Sciences (1990), supra, in Chapter 89, which is hereby incorporated by reference in its entirety. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Also, liposomal or proteinoid encapsulation can be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation can be used and the liposomes can be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given in Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. T. Rhodes, in Chapter 10, which is hereby incorporated by reference in its entirety. In general, the formulation will include the inventive compound, and inert ingredients that allow for protection against the stomach environment, and release of the biologically active material in the intestine.
The composition of this invention can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.
Colorants and flavoring agents can all be included. For example, the protein (or derivative) can be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.
In tablet form, the active ingredient(s) are mixed with a pharmaceutically acceptable carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
The powders and tablets preferably contain up to 99% of the active ingredient(s). Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
Controlled release formulation can be desirable. The composition of this invention could be incorporated into an inert matrix that permits release by either diffusion or leaching mechanisms e.g., gums. Slowly degenerating matrices can also be incorporated into the formulation, e.g., alginates, polysaccharides. Another form of a controlled release of the compositions of this invention is by a method based on the OrosTM therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect.
Other coatings can be used for the formulation. These include a variety of sugars that could be applied in a coating pan. The therapeutic agent could also be given in a film-coated tablet and the materials used in this instance are divided into 2 groups. The first are the nonenteric materials and include methylcellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxymethyl cellulose, providone and the polyethylene glycols. The second group consists of the enteric materials that are commonly esters of phthalic acid.
A mix of materials might be used to provide the optimum film coating. Film coating can be carried out in a pan coater or in a fluidized bed or by compression coating.
Pulmonarv delivery forms. Pulmonary delivery of the inventive compositions is also useful. The protein (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. (Other reports of this include Adjei et al., Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990), Internatl. J. Pharmaceutics 63: 135-44 (leuprolide acetate); Braquet et al. (1989), J. Cardiovasc. Pharmacol. 13 (supp1.5): s.143-146 (endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12 (α1-antitrypsin); Smith et al. (1989), J. Clin. Invest. 84: 1145-6 (α1-proteinase); Oswein et al. (March 1990), “Aerosolization of Proteins,” Proc. Symp. Resp. Drug Delivery II, Keystone, Colo. (recombinant human growth hormone); Debs et al. (1988), J. Immunol. 140: 3482-8 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor).
Useful in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass. (See, e.g., Helgesson et al., Inhalation device, U.S. Pat. No. 6,892,728; McDerment et al., Dry powder inhaler, WO 02/11801 A1; Ohki et al., Inhalant medicator, U.S. Pat. No. 6,273,086).
All such devices require the use of formulations suitable for the dispensing of the inventive compound. Typically, each formulation is specific to the type of device employed and can involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy.
The inventive compound should most advantageously be prepared in particulate form with an average particle size of less than 10 μm (or microns), most preferably 0.5 to 5 μm, for most effective delivery to the distal lung.
Pharmaceutically acceptable carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations can include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants can be used. PEG can be used (even apart from its use in derivatizing the protein or analog). Dextrans, such as cyclodextran, can be used. Bile salts and other related enhancers can be used. Cellulose and cellulose derivatives can be used. Amino acids can be used, such as use in a buffer formulation.
Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.
Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise the inventive compound dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation can also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation can also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.
Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the inventive compound suspended in a propellant with the aid of a surfactant. The propellant can be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid can also be useful as a surfactant. (See, e.g., Bäckström et al., Aerosol drug formulations containing hydrofluoroalkanes and alkyl saccharides, U.S. Pat. No. 6,932,962).
Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and can also include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
Nasal delivery forms. In accordance with the present invention, intranasal delivery of the inventive composition of matter and/or pharmaceutical compositions is also useful, which allows passage thereof to the blood stream directly after administration to the inside of the nose, without the necessity for deposition of the product in the lung. Formulations suitable for intransal administration include those with dextran or cyclodextran, and intranasal delivery devices are known. (See, e.g, Freezer, Inhaler, U.S. Pat. No. 4,083,368).
Transdermal and transmucosal (e.g., buccal) delivery forms). In some embodiments, the inventive composition is configured as a part of a pharmaceutically acceptable transdermal or transmucosal patch or a troche. Transdermal patch drug delivery systems, for example, matrix type transdermal patches, are known and useful for practicing some embodiments of the present pharmaceutical compositions. (E.g., Chien et al., Transdermal estrogen/progestin dosage unit, system and process, U.S. Pat. Nos. 4,906,169 and 5,023,084; Cleary et al., Diffusion matrix for transdermal drug administration and transdermal drug delivery devices including same, U.S. Pat. No. 4,911,916; Teillaud et al., EVA-based transdermal matrix system for the administration of an estrogen and/or a progestogen, U.S. Pat. No. 5,605,702; Venkateshwaran et al., Transdermal drug delivery matrix for coadministering estradiol and another steroid, U.S. Pat. No. 5,783,208; Ebert et al., Methods for providing testosterone and optionally estrogen replacement therapy to women, U.S. Pat. No. 5,460,820). A variety of pharmaceutically acceptable systems for transmucosal delivery of therapeutic agents are also known in the art and are compatible with the practice of the present invention. (E.g., Heiber et al., Transmucosal delivery of macromolecular drugs, U.S. Pat. Nos. 5,346,701 and 5,516,523; Longenecker et al., Transmembrane formulations for drug administration, U.S. Pat. No. 4,994,439).
Buccal delivery of the inventive compositions is also useful. Buccal delivery formulations are known in the art for use with peptides. For example, known tablet or patch systems configured for drug delivery through the oral mucosa (e.g., sublingual mucosa), include some embodiments that comprise an inner layer containing the drug, a permeation enhancer, such as a bile salt or fusidate, and a hydrophilic polymer, such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, dextran, pectin, polyvinyl pyrrolidone, starch, gelatin, or any number of other polymers known to be useful for this purpose. This inner layer can have one surface adapted to contact and adhere to the moist mucosal tissue of the oral cavity and can have an opposing surface adhering to an overlying non-adhesive inert layer. Optionally, such a transmucosal delivery system can be in the form of a bilayer tablet, in which the inner layer also contains additional binding agents, flavoring agents, or fillers. Some useful systems employ a non-ionic detergent along with a permeation enhancer. Transmucosal delivery devices may be in free form, such as a cream, gel, or ointment, or may comprise a determinate form such as a tablet, patch or troche. For example, delivery of the inventive composition can be via a transmucosal delivery system comprising a laminated composite of, for example, an adhesive layer, a backing layer, a permeable membrane defining a reservoir containing the inventive composition, a peel seal disc underlying the membrane, one or more heat seals, and a removable release liner. (E.g., Ebert et al., Transdermal delivery system with adhesive overlay and peel seal disc, U.S. Pat. No. 5,662,925; Chang et al., Device for administering an active agent to the skin or mucosa, U.S. Pat. Nos. 4,849,224 and 4,983,395). These examples are merely illustrative of available transmucosal drug delivery technology and are not limiting of the present invention.
Dosages. The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, the daily regimen should be in the range of 0.1-1000 micrograms of the inventive compound per kilogram of body weight, preferably 0.1-150 micrograms per kilogram.
WORKING EXAMPLES
The compositions described above can be prepared as described below. These examples are not to be construed in any way as limiting the scope of the present invention.
Example 1
Fc-L10-ShK[1-35] Mammalian Expression
Fc-L10-ShK[1-35], also referred to as “Fc-2xL-ShK[1-35]”, an inhibitor of Kv1.3. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a linker sequence and a monomer of the Kv1.3 inhibitor peptide ShK[1-35] was constructed as described below. Methods for expressing and purifying the peptibody from mammalian cells (HEK 293 and Chinese Hamster Ovary cells) are disclosed herein.
The expression vector pcDNA3.1(+) CMVi (FIG. 13A) was constructed by replacing the CMV promoter between MluI and HindIII in pcDNA3.1(+) with the CMV promoter plus intron (Invitrogen). The expression vector pcDNA3.1 (+) CMVi-hFc-ActivinRIIB (FIG. 13B) was generated by cloning a HindIII-NotI digested PCR product containing a 5′ Kozak sequence, a signal peptide and the human Fc-linker-ActivinRIIB fusion protein with the large fragment of HindIII-NotI digested pcDNA3.1(+) CMVi. The nucleotide and amino acid sequence of the human IgG1 Fc region in pcDNA3.1(+) CMVi-hFc-ActivinRIIB is shown in FIG. 3A-3B. This vector also has a GGGGSGGGGS (“L10”; SEQ ID NO:79) linker split by a BamHI site thus enabling with the oligo below formation of the 10 amino acid linker between Fc and the ShK[1-35] peptide (see FIG. 14A-14B) for the final Fc-L10-ShK[1-35] nucleotide and amino acid sequence (FIG. 14A-14B and SEQ ID NO: 77 and SEQ ID NO:78).
The Fc-L10-ShK[1-35] expression vector was constructing using PCR stategies to generate the full length ShK gene linked to a four glycine and one serine amino acid linker (lower case letters here indicate linker sequence of L-form amino acid residues) with two stop codons and flanked by BamHI and NotI restriction sites as shown below.









BamHI




GGATCCGGAGGAGGAGGAAGCCGCAGCTGCATCGACACCATCCCCAAGAGCCGCTGCACCGCCTTCCAG
//SEQ ID NO: 657


      g  g  g  s  R  S  C  I  D  T  I  P  K  S  R  C  T  A  F  Q
//SEQ ID NO: 658


 


TGCAAGCACAGCATGAAGTACCGCCTGAGCTTCTGCCGCAAGACCTGCGGCACCTGCTAATGAGCGGCCGC



C  K  H  S  M  K  Y  R  L  S  F  C  R  K  T  C  G  T  C          NotI







Two oligos with the sequence as depicted below were used in a PCR reaction with HerculaseTM polymerase (Stratagene) at 94° C.-30 sec, 50° C.-30 sec, and 72° C.-1 min for 30 cycles.







//SEQ ID NO: 659







cat gga tcc gga gga gga gga agc cgc agc tgc atc


gac acc atc ccc aag agc cgc tgc acc gcc ttc cag


tgc aag cac


 







//SEQ ID NO: 660







cat gcg gcc gct cat tag cag gtg ccg cag gtc ttg


cgg cag aag ctc agg cgg tac ttc atg ctg tgc ttg


cac tgg aag g






The resulting PCR products were resolved as the 150 bp bands on a one percent agarose gel. The 150 bp PCR product was digested with BamHI and NotI (Roche) restriction enzymes and agarose gel purified by Gel Purification Kit (Qiagen). At the same time, the pcDNA3.1 (+) CMVi-hFc-ActivinRIIB vector (FIG. 13B) was digested with BamHI and NotI restriction enzymes and the large fragment was purified by Gel Purification Kit. The gel purified PCR fragment was ligated to the purified large fragment and transformed into XL-1 blue bacteria (Stratagene). DNAs from transformed bacterial colonies were isolated and digested with BamHI and NotI restriction enzyme digestion and resolved on a one percent agarose gel. DNAs resulting in an expected pattern were submitted for sequencing. Although, analysis of several sequences of clones yielded a 100% percent match with the above sequence, only one clone was selected for large scaled plasmid purification. The DNA from Fc-2xL-ShK in pcDNA3.1(+) CMVi clone was resequenced to confirm the Fc and linker regions and the sequence was 100% identical to the predicted coding sequence, which is shown in FIG. 14A-14B.
HEK-293 cells used in transient transfection expression of Fc-2xL-ShK[1-35] in pcDNA3.1(+) CMVi protein were cultured in growth medium containing DMEM High Glucose (Gibco), 10% fetal bovine serum (FBS from Gibco) and 1× non-essential amino acid (NEAA from Gibco). 5.6 ug of Fc-2xL-ShK[1-35] in pcDNA3.1(+) CMVi plasmid that had been phenol/chloroform extracted was transfected into HEK-293 cells using Fugene 6 (Roche). The cells recovered for 24 hours, and then placed in DMEM High Glucose and 1× NEAA medium for 48 hours. The conditioned medium was concentrated 50× by running 30 ml through Centriprep YM-10 filter (Amicon) and further concentrated by a Centricon YM-10 (Amicon) filter. Various amounts of concentrated medium were mixed with an in-house 4× Loading Buffer (without B-mercaptoethanol) and electrophoresed on a Novex 4-20% tris-glycine gel using a Novex Xcell II apparatus at 101V/46 mA for 2 hours in a 5× Tank buffer solution (0.123 Tris Base, 0.96M Glycine) along with 10 ul of BenchMark Pre-Stained Protein ladder (Invitrogen). The gel was then soaked in Electroblot buffer (35 mM Tris base, 20% methanol, 192 mM glycine) for 30 minutes. A PVDF membrane from Novex (Cat. No. LC2002, 0.2 um pores size) was soaked in methanol for 30 seconds to activate the PVDF, rinsed with deionized water, and soaked in Electroblot buffer. The pre-soaked gel was blotted to the PVDF membrane using the XCell II Blot module according to the manufacturer instructions (Novex) at 40 mA for 2 hours. Then, the blot was first soaked in a 5% milk (Carnation) in Tris buffered saline solution pH7.5 (TBS) for 1 hour at room temperature and incubated with 1:500 dilution in TBS with 0.1% Tween-20 (TBST Sigma) and 1% milk buffer of the HRP-conjugated murine anti-human Fc antibody (Zymed Laboratores Cat. no. 05-3320) for two hours shaking at room temperature. The blot was then washed three times in TBST for 15 minutes per wash at room temperature. The primary antibody was detected using Amersham Pharmacia Biotech's ECL western blotting detection reagents according to manufacturer's instructions. Upon ECL detection, the western blot analysis displayed the expected size of 66 kDa under non-reducing gel conditions (FIG. 24A).
AM1 CHOd- (Amgen Proprietary) cells used in the stable expression of Fc-L10-ShK[1-35] protein were cultured in AM1 CHOd- growth medium containing DMEM High Glucose, 10% fetal bovine serum, 1× hypoxantine/thymidine (HT from Gibco) and 1×NEAA. 6.5 ug of pcDNA3.1(+) CMVi-Fc-ShK plasmid was also transfected into AM1 CHOd- cells using Fugene 6. The following day, the transfected cells were plated into twenty 15 cm dishes and selected using DMEM high glucose, 10% FBS, 1xHT, 1xNEAA and Geneticin (800 μg/ml G418 from Gibco) for thirteen days. Forty-eight surviving colonies were picked into two 24-well plates. The plates were allowed to grow up for a week and then replicated for freezing. One set of each plate was transferred to AM1 CHOd- growth medium without 10% FBS for 48 hours and the conditioned media were harvested. Western Blot analysis similar to the transient Western blot analysis with detection by the same anti-human Fc antibody was used to screen 15 ul of conditioned medium for expressing stable CHO clones. Of the 48 stable clones, more than 50% gave ShK expression at the expected size of 66 kDa. The BB6, BD5 and BD6 clones were selected with BD5 and BD6 as a backup to the primary clone BB6 (FIG. 24B).
The BB6 clone was scaled up into ten roller bottles (Corning) using AM1 CHOd- growth medium and grown to confluency as judged under the microscope. Then, the medium was exchanged with a serum-free medium containing to 50% DMEM high glucose and 50% Ham's F12 (Gibco) with 1xHT and 1xNEAA and let incubate for one week. The conditioned medium was harvested at the one-week incubation time, filtered through 0.45 μm filter (Corning) and frozen. Fresh serum-free medium was added and incubated for an additional week. The conditioned serum-free medium was harvested like the first time and frozen.
Purification of monovalent and bivalent dimeric Fc-L10-ShK(1-35). Approximately 4 L of conditioned medium was thawed in a water bath at room temperature. The medium was concentrated to about 450 ml using a Satorius Sartocon Polysulfon 10 tangential flow ultra-filtration cassette (0.1 m2) at room temperature. The retentate was then filtered through a 0.22 μm cellulose acetate filter with a pre-filter. The retentate was then loaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7° C., and the column was washed with several column volumes of Dulbecco's phosphate buffered saline without divalent cations (PBS) and sample was eluted with a step to 100 mM glycine pH 3.0. The protein A elution pool (approximately 9 ml) was diluted to 50 ml with water and loaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mM NaH2PO4, pH 7.0) at 5 ml/min and 7° C. The column was then washed with several column volumes S-Buffer A, and then developed using a linear gradient from 25% to 75% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pooled material was then concentrated to about 3.4 ml using a Pall Life Sciences Macrosep 10K Omega centrifugal ultra-filtration device and then filtered though a Costar 0.22 μm cellulose acetate syringe filter.
A spectral scan was then conducted on 10 μl of the filtered material diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 26A). The concentration of the filtered material was determined to be 5.4 mg/ml using a calculated molecular mass of 32,420 g/mol and extinction coefficient of 47,900 M−1 cm−1.
The purity of the filtered bivalent dimeric Fc-L10-ShK(1-35) product was assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 26B). The monvalent dimeric Fc-L10-ShK(1-35) product was analyzed using reducing and non-reducing sample buffers by SDS-PAGE on a 1.0 mm TRIS-glycine 4-20% gel developed at 220 V and stained with Boston Biologicals QuickBlue (FIG. 26E). The endotoxin levels were then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 108-fold dilution of the sample in PBS yielding a result of <1 EU/mg protein. The macromolecular state of the products was then determined using size exclusion chromatography on 20 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 26C, bivalent dimeric Fc-L10-ShK(1-35); FIG. 26F, monovalent dimeric Fc-L10-SHK(1-35)). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 26D) and confirmed (within experimental error) the integrity of the purified peptibody. The product was then stored at −80° C.
Purified bivalent dimeric Fc-L10-ShK[1-35] potently blocked human Kv1.3 (FIG. 30A and FIG. 30B) as determined by electrophysiology (see Example 36). The purified bivalent dimeric Fc-L10-ShK[1-35] molecule also blocked T cell proliferation (FIG. 36A and FIG. 36B) and production of the cytokines IL-2 (FIG. 35A and FIG. 37A) and IFN-g (FIG. 35B and FIG. 37B).
Example 2
Fc-L-ShK[2-35] Mammalian Expression
A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a monomer of the Kv1.3 inhibitor peptide ShK[2-35] was constructed using standard PCR technology. The ShK[2-35] and the 5, 10, or 25 amino acid linker portion of the molecule were generated in a PCR reaction using the original Fc-2xL-ShK[1-35] in pcDNA3.1(+) CMVi as a template (Example 1, FIG. 14A-14B). All ShK constructs should have the following amino acid sequence of








SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
(SEQ ID NO: 92)






with the first amino acid of the wild-type sequence deleted.

The sequences of the primers used to generate Fc-L5-ShK[2-35], also referred to as “Fc-1xL-ShK[2-35]”, are shown below:







//SEQ ID NO: 661









cat gga tcc agc tgc atc gac acc atc;


 







//SEQ ID NO: 662









cat gcg gcc gct cat tag c;






The sequences of the primers used to generate Fc-L10-ShK[2-35], also referred to as “Fc-2xL-ShK[2-35]” are shown below:







//SEQ ID NO: 663









cat gga tcc gga gga gga gga agc agc tgc a;


 







//SEQ ID NO: 664









cat gcg gcc gct cat tag cag gtg c;






The sequences of the primers used to generate Fc-L25-ShK[2-35], also referred to as “Fc-5xL-ShK[2-35]”, are shown below:







//SEQ ID NO: 665







cat gga tcc ggg ggt ggg ggt tct ggg ggt ggg ggt


tct gga gga gga gga agc gga gga gga gga agc agc


tgc a;


 







//SEQ ID NO: 666







cat gcg gcc gct cat tag cag gtg c;






The PCR products were digested with BamHI and NotI (Roche) restriction enzymes and agarose gel purified by Gel Purification Kit. At the same time, the pcDNA3.1(+) CMVi-hFc-ActivinRIIB vector was digested with BamHI and NotI restriction enzymes and the large fragment was purified by Gel Purification Kit. Each purified PCR product was ligated to the large fragment and transformed into XL-1 blue bacteria. DNAs from transformed bacterial colonies were isolated and subjected to BamHI and NotI restriction enzyme digestions and resolved on a one percent agarose gel. DNAs resulting in an expected pattern were submitted for sequencing. Although, analysis of several sequences of clones yielded a 100% percent match with the above sequence, only one clone was selected for large scaled plasmid purification. The DNA from this clone was resequenced to confirm the Fc and linker regions and the sequence was 100% identical to the expected sequence.
Plasmids containing the Fc-1xL-Shk[2-35], Fc-2xL-Shk[2-35] and Fc-5xL-Shk[2-35] inserts in pcDNA3.1 (+) CMVi vector were digested with Xba1 and Xho1 (Roche) restriction enzymes and gel purified. The inserts were individually ligated into Not1 and SalI (Roche) digested pDSRα-22 (Amgen Proprietary) expression vector. Integrity of the resulting constructs were confirmed by DNA sequencing. The final plasmid DNA expression vector constructs were pDSRα-22-Fc-1xL-Shk[2-35], pDSRα-22-Fc-2xL-Shk[2-35] (FIG. 13C and FIG. 15A-15B) and pDSRα-22-Fc-5xL-Shk[2-35] (FIG. 16A-16B) and contained 5, 10 and 25 amino acid linkers, respectively.
Twenty-four hours prior to transfection, 1.2e7 AM-1/D CHOd- (Amgen Proprietary) cells were plated into a T-175 cm sterile tissue culture flask, to allow 70-80% confluency on the day of transfection. The cells had been maintained in the AM-1/D CHOd- culture medium containing DMEM High Glucose, 5% FBS, 1× Glutamine Pen/Strep (Gibco), 1×HT, 1×NEAA's and 1× sodium pyruvate (Gibco). The following day, eighteen micrograms of each of the linearized pDSRα22:Fc-1xL-ShK[2-35], pDSRα22:Fc-2xL-ShK[2-35] and pDSRα22:Fc-5xL-ShK[2-35] (RDS's #20050037685, 20050053709, 20050073295) plasmids were mixed with 72 μg of linearized Selexis MAR plasmid and pPAGO1 (RDS 20042009896) and diluted into 6 ml of OptiMEM in a 50 ml conical tube and incubate for five minutes. LF2000 (210 μl) was added to 6 ml of OptiMEM and incubated for five minutes. The diluted DNA and LF2000 were mixed together and incubated for 20 minutes at room temperature. In the meantime, the cells were washed one time with PBS and then 30 ml OptiMEM without antibiotics were added to the cells. The OptiMEM was aspirated off, and the cells were incubated with 12 ml of DNA/LF2000 mixture for 6 hours or overnight in the 37° C. incubator with shaking. Twenty-four hours post transfection, the cells were split 1:5 into AM-1/D CHOd- culture medium and at differing dilutions for colony selection. Seventy-two hours post transfection, the cell medium was replaced with DHFR selection medium containing 10% Dialyzed FBS (Gibco) in DMEM High Glucose, plus 1× Glutamine Pen/Strep, 1×NEAA's and 1× Na Pyr to allow expression and secretion of protein into the cell medium. The selection medium was changed two times a week until the colonies are big enough to pick. The pDSRa22 expression vector contains a DHFR expression cassette, which allows transfected cells to grow in the absence of hypoxanthine and thymidine. The five T-175 pools of the resulting colonies were scaled up into roller bottles and cultured under serum free conditions. The conditioned media were harvested and replaced at one-week intervals. The resulting 3 liters of conditioned medium was filtered through a 0.45 μm cellulose acetate filter (Corning, Acton, Mass.) and transferred to Protein Chemistry for purification. As a backup, twelve colonies were selected from the 10 cm plates after 10-14 days on DHFR selection medium and expression levels evaluated by western blot using HRP conjugated anti human IgGFc as a probe. The three best clones expressing the highest level of each of the different linker length Fc-L-ShK[2-35] fusion proteins were expanded and frozen for future use.
Purification of Fc-L10-ShK(2-35). Approximately 1 L of conditioned medium was thawed in a water bath at room temperature. The medium was loaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7° C., and the column was washed with several column volumes of Dulbecco's phosphate buffered saline without divalent cations (PBS) and sample was eluted with a step to 100 mM glycine pH 3.0. The protein A elution pool (approximately 8.5 ml) combined with 71 μl 3 M sodium acetate and then diluted to 50 ml with water. The diluted material was then loaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mM NaH2PO4, pH 7.0) at 5 ml/min 7° C. The column was then washed with several column volumes S-Buffer A, and then developed using a linear gradient from 0% to 75% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pooled material was then filtered through a 0.22 μm cellulose acetate filter and concentrated to about 3.9 ml using a Pall Life Sciences Macrosep 10K Omega centrifugal ultra-filtration device. The concentrated material was then filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang E membrane at 2 ml/min room temperature. A spectral scan was then conducted on 10 μl of the filtered material diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 27E). The concentration of the filtered material was determined to be 2.76 mg/ml using a calculated molecular mass of 30,008 g/mol and extinction coefficient of 36,900 M−1 cm−1. Since material was found in the permeate, repeated concentration step on the permeate using a new Macrosep cartridge. The new batch of concentrated material was then filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang E membrane at 2 ml/min room temperature. Both lots of concentrated material were combined into one pool.
A spectral scan was then conducted on 10 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer. The concentration of the filtered material was determined to be 3.33 mg/ml using a calculated molecular mass of 30,008 g/mol and extinction coefficient of 36,900 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 27A). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 67-fold dilution of the sample in PBS yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 27B). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 27F) and the experiment confirmed the integrity of the peptibody, within experimental error. The product was then stored at −80° C.
FIG. 31B shows that purified Fc-L10-ShK[2-35] potently blocks human Kv1.3 current (electrophysiology was done as described in Example 36). The purified Fc-L10-ShK[2-35] molecule also blocked IL-2 (FIG. 64A and FIG. 64B) and IFN-g (FIG. 65A and FIG. 65B) production in human whole blood, as well as, upregulation of CD40L (FIG. 66A and FIG. 66B) and IL-2R (FIG. 67A and FIG. 67B) on T cells.
Purification of Fc-L5-ShK(2-35). Approximately 1 L of conditioned medium was loaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7° C., and the column was washed with several column volumes of Dulbecco's phosphate buffered saline without divalent cations (PBS) and sample was eluted with a step to 100 mM glycine pH 3.0. The protein A elution pool (approximately 9 ml) combined with 450 μl 1 M tris HCl pH 8.5 followed by 230 μl 2 M acetic acid then diluted to 50 ml with water. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mM NaH2PO4, pH 7.0) at 5 ml/min 7° C. The column was then washed with several column volumes S-Buffer A, and then developed using a linear gradient from 0% to 75% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pooled material was then concentrated to about 5.5 ml using a Pall Life Sciences Macrosep 10K Omega centrifugal ultra-filtration device. The concentrated material was then filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang E membrane at 2 ml/min room temperature.
A spectral scan was then conducted on 10 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 27G). The concentration of the filtered material was determined to be 4.59 mg/ml using a calculated molecular mass of 29,750 g/mol and extinction coefficient of 36,900 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 27C). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 92-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 27H). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 27I) and confirmed the integrity of the peptibody, within experimental error. The product was then stored at −80° C.
FIG. 31C shows that purified Fc-L5-ShK[2-35] is highly active and blocks human Kv1.3 as determined by whole cell patch clamp electrophysiology (see Example 36).
Purification of Fc-L25-ShK(2-35). Approximately 1 L of conditioned medium was loaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7° C., and the column was washed with several column volumes of Dulbecco's phosphate buffered saline without divalent cations (PBS) and sample was eluted with a step to 100 mM glycine pH 3.0. The protein A elution pool (approximately 9.5 ml) combined with 119 μl 3 M sodium acetate and then diluted to 50 ml with water. The pH adjusted material was then loaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mM NaH2PO4, pH 7.0) at 5 ml/min 7° C. The column was then washed with several column volumes S-Buffer A, and then developed using a linear gradient from 0% to 75% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions containing the main peak from the chromatogram were pooled and filtered through a 0.22 μm cellulose acetate filter.
A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer FIG. 27J. The concentration of the filtered material was determined to be 1.40 mg/ml using a calculated molecular mass of 31,011 g/mol and extinction coefficient of 36,900 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 27D). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 28-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 27K). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 27L) and this confirmed the integrity of the peptibody, within experimental error. The product was then stored at −80° C.
Purified Fc-L25-ShK[2-35] inhibited human Kv1.3 with an IC50 of  ̃150 pM by whole cell patch clamp electrophysiology on HEK293/Kv1.3 cells (Example 36).
Example 3
Fc-L-ShK[1-35] Bacterial Expression
Description of bacterial peptibody expression vectors and procedures for cloning and expression of peptibodies. The cloning vector used for bacterial expression (Examples 3-30) is based on pAMG21 (originally described in U.S. Patent 2004/0044188). It has been modified in that the kanamycin resistance component has been replaced with ampicillin resistance by excising the DNA between the unique BstBI and NsiI sites of the vector and replacing with an appropriately digested PCR fragment bearing the beta-lactamase gene using PCR primers CCA ACA CAC TTC GAA AGA CGT TGA TCG GCA C (SEQ ID NO: 667) and CAC CCA ACA ATG CAT CCT TAA AAA AAT TAC GCC C (SEQ ID NO: 668) with pUC19 DNA as the template source of the beta-lactamase gene conferring resistance to ampicillin. The new version is called pAMG21ampR.
Description of cloning vector pAMG21ampR-Fc-Pep used in examples 3 to 30, excluding 15 and 16. FIG. 11A-C and FIG. 11D (schematic diagram) show the ds-DNA that has been added to the basic vector pAMG21ampR to permit the cloning of peptide fusions to the C-terminus of the Fc gene. The DNA has been introduced between the unique NdeI and BamHI sites in the pAMG21ampR vector. This entire region of DNA is shown in FIG. 11A-C. The coding region for Fc extends from nt 5134 to 5817 and the protein sequence appears below the DNA sequence. This is followed in frame by a glyX5 linker (nt's 5818-5832). A BsmBI site (GAGACG) spansnucleotides 5834-5839. DNA cleavage occurs between nucleotides 5828 and 5829 on the upper DNA strand and between nucleotides 5832 and 5833 on the lower DNA strand. Digestion creates 4 bp cohesive termini as shown here. The BsmBI site is underlined.











AGGTGG
TGGTTGAGACG
 SEQ ID NO: 683



 



TCCACCACCA
    ACTCTGC
SEQ ID NO: 684






A second BsmBI site occurs at nucleotides 6643 through 6648; viz., CGTCTC. DNA cleavage occurs between nucleotides 6650 and 6651 on the upper strand and between 6654 and 6655 on the lower strand.











CGTCTCT
TAAGGATCCG
 SEQ ID NO: 685



 



GCAGAGAATTC
    CTAGGC
SEQ ID NO: 686






Between the two BsmBI sites is a dispensable chloramphenicol resistance cassette constitutively expressing chloramphenicol acetyltransferase (cat gene). The cat protein sequence:










1
MEKKITGYTT VDISQWHRKE HFEAFQSVAQ CTYNQTVQLD ITAFLKTVKK
SEQ ID NO: 1337



 


51
NKHKFYPAFI HILARLMNAH PEFRMAMKDG ELVIWDSVHP CYTVFHEQTE




 


101
TFSSLWSEYH DDFRQFLHIY SQDVACYGEN LAYFPKGFIE NMFFVSANPW




 


151
VSFTSFDLNV ANMDNFFAPV FTMGKYYTQG DKVLMPLAIQ VHHAVCDGFH




 


201
VGRMLNELQQ YCDEWQGGA//








is shown in FIG. 11A-C and extends from nucleotides 5954 to 6610. The peptide encoding duplexes in each example (except Examples 15 and 16) bear cohesive ends complementary to those presented by the vector.

Description of the cloning vector pAMG21ampR-Pep-Fc used in examples 15 and 16. FIG. 12A-C, and the schematic diagram in FIG. 12D, shows the ds-DNA sequence that has been added to the basic vector pAMG21ampR to permit the cloning of peptide fusions to the N-terminus of the Fc gene. The DNA has been introduced between the unique NdeI and BamHI sites in the pAMG21ampR vector. The coding region for Fc extends from nt 5640 to 6309 and the protein sequence appears below the DNA sequence. This is preceded in frame by a glyX5 linker (nt's 5614-5628). A BsmBI site spans nucleotides 5138 to 5143; viz., GAGACG. The cutting occurs between nucleotides 5132 and 5133 on the upper DNA strand and between 5136 and 5137 on the lower DNA strand.
Digestion creates 4 bp cohesive termini as shown. The BsmBI site is underlined.











AATAACA
TATGCGAGACG
SEQ ID NO: 687


 



TTATTGTATAC
    GCTCTGC
SEQ ID NO: 688






A second BsmBI site occurs at nucleotides 5607 through 5612; viz., CGTCTC. Cutting occurs between nucleotides 5613 and 5614 on the upper strand and between 5617 and 5618 on the lower strand.












CGTCTCA
GGTGGTGGT
SEQ ID NO: 689


 



GCAGAGTCCAC
    CACCA







Between the BsmBI sites is a dispensable zeocin resistance cassette constitutively expressing the Shigella ble protein. The ble protein sequence:











1
MAKLTSAVPV LTARDVAGAV EFWTDRLGFS RDFVEDDFAG VVRDDVTLFI
//SEQ ID NO: 1338



 


51
SAVQDQVVPD NTLAWVWVRG LDELYAEWSE VVSTNFRDAS GPAMTEIGEQ




 


101
PWGREFALRD PAGNCVHFVA EEQD








is shown extending from nucleotides 5217 to 5588 in FIG. 12A-C. The peptide encoding duplexes in Examples 15 and 16 bear cohesive ends complementary to those presented by the vector.

Description of the cloning vector pAMG21ampR-Pep-Fc used in Examples 52 and 53. FIG. 12E-G shows the ds-DNA sequence that has been added to the basic vector pAMG21ampR to permit the cloning of peptide fusions to the N-terminus of the Fc gene in which the first two codons of the peptide are to be met-gly. The DNA has been introduced between the unique NdeI and BamHI sites in the pAMG21ampR vector. The coding region for Fc extends from nt 5632 to 6312 and the protein sequence appears below the DNA sequence. This is preceded in frame by a glyX5 linker (nt's 5617-5631). A BsmBI site spans nucleotides 5141 to 5146; viz., GAGACG. The cutting occurs between nucleotides 5135 and 5136 on the upper DNA strand and between 5139 and 5140 on the lower DNA strand.
Digestion creates 4 bp cohesive termini as shown. The BsmBI site is underlined.











AATAACATAT
GGGTCGAGACG
 SEQ ID NO: 1344





SEQ ID NO: 1343



 



TTATTGTATACCCA
    GCTCTGC
SEQ ID NO: 1345






A second BsmBI site occurs at nucleotides 5607 through 5612; viz., CGTCTC. Cutting occurs between nucleotides 5613 and 5614 on the upper strand and between 5617 and 5618 on the lower strand.











CGTCTCA     GGTGGTGGT
SEQ ID NO: 1346



 



GCAGAGTCCAC     CACCA







Between the BsmBI sites is a dispensable zeocin resistance cassette constitutively expressing the Shigella ble protein. The ble protein sequence, as described above, is shown extending from nucleotide positions 5220 to 5591. The peptide encoding duplexes in Examples 52 and 53 herein below bear cohesive ends complementary to those presented by the vector.

For Examples 3 to 30 for which all are for bacterial expression, cloned peptide sequences are all derived from the annealing of oligonucleotides to create a DNA duplex that is directly ligated into the appropriate vector. Two oligos suffice for Example 20, four are required for all other examples. When the duplex is to be inserted at the N-terminus of Fc (see, Examples 15, 16, 52, and 53 herein) the design is as follows with the ordinal numbers matching the listing of oligos in each example:




When the duplex is to be inserted at the C-terminus of Fc (Examples 3, 4, 5, 10, 11, 12, 13, and 30) the design is as follows:




All remaining examples have the duplex inserted at the C-terminus of Fc and utilize the following design.




No kinasing step is required for the phosphorylation of any of the oligos. A successful insertion of a duplex results in the replacement of the dispensable antibiotic resistance cassette (Zeocin resistance for pAMG21ampR-Pep-Fc and chloramphenicol resistance for pAMG21ampR-Fc-Pep). The resulting change in phenotype is useful for discriminating recombinant from nonrecombinant clones.
The following description gives the uniform method for carrying out the cloning of all 30 bacterially expressed recombinant proteins exemplified herein. Only the set of oligonucleotides and the vector are varied. These specifications are given below in each example.
An oligonucleotide duplex containing the coding region for a given peptide was formed by annealing the oligonucleotides listed in each example. Ten picomoles of each oligo was mixed in a final volume of 10 μl containing 1× ligation buffer along with 0.3 μg of appropriate vector that had been previously digested with restriction endonuclease BsmBI. The mix was heated to 80° C. and allowed to cool at 0.1 degree/sec to room temperature. To this was added 10 μl of 1× ligase buffer plus 400 units of T4 DNA ligase. The sample was incubated at 14 C for 20 min. Ligase was inactivated by heating at 65° C. for 10 minutes. Next, 10 units of restriction endonucleases BsmBI were added followed by incubation at 55 C for one hour to cleave any reformed parental vector molecules. Fifty ul of chemically competent E. coli cells were added and held at 2 C for 20 minutes followed by heat shock at 42 C for 5 second. The entire volume was spread onto Luria Agar plates supplemented with carbenicillin at 200 μg/ml and incubated overnight at 37 C. Colonies were tested for the loss of resistance to the replaceable antibiotic resistance marker. A standard PCR test can be used to confirm the expected size of the duplex insert. Plasmid preparations were obtained and the recombinant insert was verified by DNA sequencing. Half liter cultures of a sequence confirmed construct were grown in Terrific Broth, expression of the peptibody was induced by addition of N-(3-oxo-hexanoyl)-homoserine lactone at 50 ng/ml and after 4-6 hours of shaking at 37 C the cells were centrifuged and the cell paste stored at −20 C.
The following gives for each example the cloning vector and the set of oligonucleotides used for constructing each fusion protein. Also shown is a DNA/protein map.
Bacterial expression of Fc-L-ShK[1-35] inhibitor of Kv1.3. The methods to clone and express the peptibody in bacteria are described above. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ShK[1-35].
Oligos used to form the duplex:







//SEQ ID NO: 669







TGGTTCCGGTGGTGGTGGTTCCCGTTCCTGCATCGACACCAT;


 







//SEQ ID NO: 670







CCCGAAATCCCGTTGCACCGCTTTCCAGTGCAAACACTCCATGAAATACC


 


GTCTGTCCTTCTGCCGTAAAACCTGCGGTACCTGC;


 







//SEQ ID NO: 671







CTTAGCAGGTACCGCAGGTTTTACGGCAGAAGGACAGACGGT;


 







//SEQ ID NO: 672







ATTTCATGGAGTGTTTGCACTGGAAAGCGGTGCAACGGGATTTCGGGATG


 


GTGTCGATGCAGGAACGGGAACCACCACCACCGGA;






The oligo duplex is shown below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen. Purification of bacterially expressed Fc-L10-ShK(1-35) is further described in Example 38 herein below.
Example 4
Fc-L-ShK[2-35] Bacterial Expression
Bacterial expression of Fc-L-ShK[2-35]. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ShK[2-35].
Oligos used to form duplex are shown below:







//SEQ ID NO: 676







TGGTTCCGGTGGTGGTGGTTCCTGCATCGACACCATCCCGAAATCCCGTT


 


GCACCGCTTTCCAGTGCAAACACTCCATGAAAT;


 







//SEQ ID NO: 677







ACCGTCTGTCCTTCTGCCGTAAAACCTGCGGTACCTGC;


 







//SEQ ID NO: 678







CTTAGCAGGTACCGCAGGTTTTACGGCAGAAGGACAGACGGTATTTCATG


 


GAGTGTTTGCACTGGAAAGCGGTGCAACGGGA;


 







//SEQ ID NO: 679







TTTCGGGATGGTGTCGATGCAGGAACCACCACCACCGGA;






The oligo duplex formed is shown below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen. Purification of bacterially expressed Fc-L10-ShK(2-35) is further described in Example 39 herein below.
Example 5
Fc-L-HmK Bacterial Expression
Bacterial expression of Fc-L-HmK. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-HmK.
Oligos used to form duplex are shown below:







//SEQ ID NO: 690







TGGTTCCGGTGGTGGTGGTTCCCGTACCTGCAAAGACCTGAT;


 







SEQ ID NO: 692







CCCGGTTTCCGAATGCACCGACATCCGTTGCCGTACCTCCATGAAATACC


 


GTCTGAACCTGTGCCGTAAAACCTGCGGTTCCTGC;


 







//SEQ ID NO: 693







CTTAGCAGGAACCGCAGGTTTTACGGCACAGGTTCAGACGGT;


 







//SEQ ID NO: 694







ATTTCATGGAGGTACGGCAACGGATGTCGGTGCATTCGGAAACCGGGATC


 


AGGTCTTTGCAGGTACGGGAACCACCACCACCGGA;






The oligo duplex formed is shown below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 6
Fc-L-KTX1 Bacterial Expression
Bacterial expression of Fc-L-KTX1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-KTX1.
Oligos used to form duplex are shown below:







//SEQ ID NO: 698







TGGTTCCGGTGGTGGTGGTTCCGGTGTTGAAATCAACGTTAAATGCT;


 







//SEQ ID NO: 699







CCGGTTCCCCGCAGTGCCTGAAACCGTGCAAAGACGCTGGTATGCGTTTC


 


GGTAAATGCATGAACCGTAAATGCCACTGCACCCCGAAA;


 







//SEQ ID NO: 700







CTTATTTCGGGGTGCAGTGGCATTTACGGTTCATGCATTTACCGAAA;


 







//SEQ ID NO: 701







CGCATACCAGCGTCTTTGCACGGTTTCAGGCACTGCGGGGAACCGGAGCA


 


TTTAACGTTGATTTCAACACCGGAACCACCACCACCGGA;






The oligo duplex formed is shown below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Purification and refolding of Fc-L-KTX1 expressed in bacteria. Frozen, E. coli paste (28 g) was combined with 210 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml water using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet (4.8 g) was then dissolved in 48 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The dissolved pellet was then reduced by adding 30 μl 1 M dithiothreitol to 3 ml of the solution and incubating at 37° C. for 30 minutes. The reduced pellet solution was then centrifuged at 14,000 g for 5 min at room temperature, and then 2.5 ml of the supernatant was transferred to 250 ml of the refolding buffer (2 M urea, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl, 4 mM cysteine, pH 8.5) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 2 days at 4° C. The refolding solution was then filtered through a 0.22 μm cellulose acetate filter and stored at 4° C. for 3 days.
The stored refold was then diluted with 1 L of water and the pH was adjusted to 7.5 using 1 M H3PO4. The pH adjusted material was then loaded on to a 10 ml Amersham SP-HP HiTrap column at 10 ml/min in S-Buffer A (20 mM NaH2PO4, pH 7.3) at 7° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3) followed by a step to 100% S-Buffer B at 5 ml/min 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data (45 ml). The pool was then loaded on to a 1 ml Amersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was then washed with several column volumes of PBS and eluted with 100 mM glycine pH 3.0. To the elution peak (2.5 ml), 62.5 μl 2 M tris base was added, and then the pH adjusted material was filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang E membrane at 2 ml/min room temperature.
A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 28C). The concentration of the filtered material was determined to be 2.49 mg/ml using a calculated molecular mass of 30,504 g/mol and extinction coefficient of 35,410 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 28A). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 50-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 45 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 28B). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 28D) and these studies confirmed the integrity of the purified peptibody, within experimental error. The product was then stored at −80° C.
Purified Fc-L-KTX1 blocked the human Kv1.3 current in a dose-dependent fashion (FIG. 32A and FIG. 32B) by electrophysiology (method was as described in Example 36).
Example 7
Fc-L-HsTx1 Bacterial Expression
Bacterial expression of Fc-L-HsT1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-HsTx1.
Oligos used to form duplex are shown below:







//SEQ ID NO: 705







TGGTTCCGGTGGTGGTGGTTCCGCTTCCTGCCGTACCCCGAAAGAC;


 







//SEQ ID NO: 706







TGCGCTGACCCGTGCCGTAAAGAAACCGGTTGCCCGTACGGTAAATGCAT


 


GAACCGTAAATGCAAATGCAACCGTTGC;


 







//SEQ ID NO: 707







CTTAGCAACGGTTGCATTTGCATTTACGGTTCATGCATTTACCGTACG;


 







//SEQ ID NO: 708







GGCAACCGGTTTCTTTACGGCACGGGTCAGCGCAGTCTTTCGGGGTACGG


 


CAGGAAGCGGAACCACCACCACCGGA;






The duplex formed by the oligos above is shown below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 8
Fc-L-MgTx Bacterial Expression
Bacterial expression of Fc-L-MgTx. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-MgTx.
Oligos used to form duplex are shown below:







//SEQ ID NO: 712







TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGTTAAATGCACCTC;


 







//SEQ ID NO: 713







CCCGAAACAGTGCCTGCCGCCGTGCAAAGCTCAGTTCGGTCAGTCCGCTG


 


GTGCTAAATGCATGAACGGTAAATGCAAATGCTACCCGCAC;


 







//SEQ ID NO: 714







CTTAGTGCGGGTAGCATTTGCATTTACCGTTCATGCATTTAGCACCAG;


 







//SEQ ID NO: 715







CGGACTGACCGAACTGAGCTTTGCACGGCGGCAGGCACTGTTTCGGGGAG


 


GTGCATTTAACGTTGATGATGGTGGAACCACCACCACCGGA;






The oligos above were used to form the duplex shown below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 9
Fc-L-AgTx2 Bacterial Expression
Bacterial expression of Fc-L-AgTx2. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-AgTx2.
Oligos used to form duplex are shown below:







//SEQ ID NO: 719







TGGTTCCGGTGGTGGTGGTTCCGGTGTTCCGATCAACGTTTCCTGCACCG


 


GT;


 







//SEQ ID NO: 720







TCCCCGCAGTGCATCAAACCGTGCAAAGACGCTGGTATGCGTTTCGGTAA


 


ATGCATGAACCGTAAATGCCACTGCACCCCGAAA;


 







//SEQ ID NO: 721







CTTATTTCGGGGTGCAGTGGCATTTACGGTTCATGCATTTACCGAAACGC


 


ATA;


 







//SEQ ID NO: 722







CCAGCGTCTTTGCACGGTTTGATGCACTGCGGGGAACCGGTGCAGGAAAC


 


GTTGATCGGAACACCGGAACCACCACCACCGGA;






The oligos listed above were used to form the duplex shown below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Refolding and purification of Fc-L-AgTx2 expressed in bacteria. Frozen, E. coli paste (15 g) was combined with 120 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml water using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet (4.6 g) was then dissolved in 46 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The dissolved pellet was then reduced by adding 30 μl 1 M dithiothreitol to 3 ml of the solution and incubating at 37° C. for 30 minutes. The reduced pellet solution was then centrifuged at 14,000 g for 5 min at room temperature, and then 2.5 ml of the supernatant was transferred to 250 ml of the refolding buffer (2 M urea, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl, 4 mM cysteine, pH 9.5) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 2 days at 4° C. The refolding solution was then filtered through a 0.22 μm cellulose acetate filter and stored at −70° C.
The stored refold was defrosted and then diluted with 1 L of water and the pH was adjusted to 7.5 using 1 M H3PO4. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 10 ml Amersham SP-HP HiTrap column at 10 ml/min in S-Buffer A (20 mM NaH2PO4, pH 7.3) at 7° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3) followed by a step to 100% S-Buffer B at 5 ml/min 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data (15 ml). The pool was then loaded on to a 1 ml Amersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was then washed with several column volumes of 20 mM NaH2PO4 pH 6.5, 1 M NaCl and eluted with 100 mM glycine pH 3.0. To the elution peak (1.5 ml), 70 μl 1 M tris HCl pH 8.5 was added, and then the pH-adjusted material was filtered though a 0.22 μm cellulose acetate filter.
A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 29C). The concentration of the filtered material was determined to be 1.65 mg/ml using a calculated molecular mass of 30,446 g/mol and extinction coefficient of 35,410 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 29A). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 33-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <4 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 20 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 29D). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 29E) and these studies confirmed the integrity of the purified peptibody, within experimental error. The product was then stored at −80° C.
Example 10
Fc-L-OSK1 Bacterial Expression
Bacterial expression of Fc-L-OSK1. The methods used to clone and express the peptibody in bacteria were as described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-OSK1.
Oligos used to form duplex are shown below:







//SEQ ID NO: 726







TGGTTCCGGTGGTGGTGGTTCCGGTGTTATCATCAACGTTAAATGCAAAA


 


TCTCCCGTCAGTGCCTGGAACCGTGCAAAAAAG;


 







//SEQ ID NO: 727







CTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATGCCACTGCACCCCG


 


AAA;


 







//SEQ ID NO: 728







CTTATTTCGGGGTGCAGTGGCATTTACCGTTCATGCATTTACCGAAACGC


 


ATACCAGCTTTTTTGCACGGTTCCAGGCACTGA;


 







//SEQ ID NO: 729







CGGGAGATTTTGCATTTAACGTTGATGATAACACCGGAACCACCACCACC


 


GGA;






The oligos shown above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen for later use. Purification of Fc-L10-OSK1 from E. coli paste is described in Example 40 herein below.
Example 11
Fc-L-OSK1(E16K, K20D) Bacterial Expression
Bacterial expression of Fc-L-OSK1(E16K, K20D). The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-OSK1(E16K,K20D).
Oligos used to form duplex are shown below:







//SEQ ID NO: 733







TGGTTCCGGTGGTGGTGGTTCCGGTGTTATCATCAACGTTAAATGCAAAA


 


TCTCCCGTCAGTGCCTGAAACCGTGCAAAGACG;


 







//SEQ ID NO: 734







CTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATGCCACTGCACCCCG


 


AAA;


 







//SEQ ID NO: 735







CTTATTTCGGGGTGCAGTGGCATTTACCGTTCATGCATTTACCGAAACGC


 


ATACCAGCGTCTTTGCACGGTTTCAGGCACTGA;


 







//SEQ ID NO: 736







CGGGAGATTTTGCATTTAACGTTGATGATAACACCGGAACCACCACCACC


 


GGA;






The oligos shown above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen for later use.
Example 12
Fc-L-Anuroctoxin Bacterial Expression
Bacterial expression of Fc-L-Anuroctoxin. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-Anuroctoxin.
Oligos used to form duplex are shown below:







//SEQ ID NO: 740







TGGTTCCGGTGGTGGTGGTTCCAAAGAATGCACCGGTCCGCAGCACTGCA


 


CCAACTTCTGCCGTAAAAACAAATGCACCCACG;


 







//SEQ ID NO: 741







GTAAATGCATGAACCGTAAATGCAAATGCTTCAACTGCAAA;


 







//SEQ ID NO: 742







CTTATTTGCAGTTGAAGCATTTGCATTTACGGTTCATGCATTTACCGTGG


 


GTGCATTTGTTTTTACGGCAGAAGTTGGTGCAG;


 







//SEQ ID NO: 743







TGCTGCGGACCGGTGCATTCTTTGGAACCACCACCACCGGA;






The oligos shown above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 13
Fc-L-Noxiustoxin Bacterial Expression
Bacterial expression of Fc-L-Noxiustoxin or Fc-L-NTX. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-NTX.
Oligos used to form duplex are shown below:







//SEQ ID NO: 747







TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGTTAAATGCACCTCCC


 


CGAAACAGTGCTCCAAACCGTGCAAAGAACTGT;


 







//SEQ ID NO: 748







ACGGTTCCTCCGCTGGTGCTAAATGCATGAACGGTAAATGCAAATGCTAC


 


AACAAC;


 







//SEQ ID NO: 749







CTTAGTTGTTGTAGCATTTGCATTTACCGTTCATGCATTTAGCACCAGCG


 


GAGGAACCGTACAGTTCTTTGCACGGTTTGGAG;


 







//SEQ ID NO: 750







CACTGTTTCGGGGAGGTGCATTTAACGTTGATGATGGTGGAACCACCACC


 


ACCGGA;






The oligos shown above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 14
Fc-L-Pi2 Bacterial Expression
Bacterial expression of Fc-L-Pi2. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-Pi2.
Oligos used to form duplex are shown below:







//SEQ ID NO: 754







TGGTTCCGGTGGTGGTGGTTCCACCATCTCCTGCACCAACCCG;


 







//SEQ ID NO: 755







AAACAGTGCTACCCGCACTGCAAAAAAGAAACCGGTTACCCGAACGCTAA


 


ATGCATGAACCGTAAATGCAAATGCTTCGGTCGT;


 







//SEQ ID NO: 756







CTTAACGACCGAAGCATTTGCATTTACGGTTCATGCATTTAGCG;


 







//SEQ ID NO: 757







TTCGGGTAACCGGTTTCTTTTTTGCAGTGCGGGTAGCACTGTTTCGGGTT


 


GGTGCAGGAGATGGTGGAACCACCACCACCGGA;






The oligos above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 15
ShK[1-35]-L-Fc Bacterial Expression
Bacterial expression of ShK[1-35]-L-Fc. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Pep-Fc and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of ShK[1-35]-L-Fc.
Oligos used to form duplex are shown below:







//SEQ ID NO: 761







TATGCGTTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTC


 


AATGTAAACATTCTATGAAATATCGTCTTTCTT;


 







//SEQ ID NO: 762







TTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGGTGGTTCT;


 







//SEQ ID NO: 763







CACCAGAACCACCACCACCAGAACAAGTACCACAAGTTTTACGACAAAAA


 


GAAAGACGATATTTCATAGAATGTTTACATTGA;


 







//SEQ ID NO: 764







AAAGCAGTACAACGAGATTTTGGAATAGTATCAATACAAGAACG;






The oligos shown above were used to form the duplex shown below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen. Purification of met-ShK[1-35]-Fc was as described in Example 51 herein below.
Example 16
ShK[2-35]-L-Fc Bacterial Expression
Bacterial expression of ShK[2-35]-L-Fc. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Pep-Fc and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of ShK[2-35]-L-Fc.
Oligos used to form duplex are shown below:







//SEQ ID NO: 768







TATGTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTCAAT


GTAAACATTCTATGAAATATCGTCTTTCTT;


 







//SEQ ID NO: 769







TTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGGTGGTTCT;


 







//SEQ ID NO: 770







CACCAGAACCACCACCACCAGAACAAGTACCACAAGTTTTACGACAAAAA


GAAAGACGATATTTCATAGAATGTTTACATTGA;


 







SEQ ID NO: 771







AAAGCAGTACAACGAGATTTTGGAATAGTATCAATACAAGA;






The oligos above were used to form the duplex shown below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen. Purification of the ShK[2-35]-Fc was as described in Example 50 herein below.
Example 17
Fc-L-ChTx Bacterial Expression
Bacterial expression of Fc-L-ChTx. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ChTx.
Oligos used to form duplex are shown below:







//SEQ ID NO: 775







TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCAACGTT;


 







//SEQ ID NO: 776







TCCTGCACCACCTCCAAAGAATGCTGGTCCGTTTGCCAGCGTCTGCACAA


CACCTCCCGTGGTAAATGCATGAACAAAAAATGCCGTTGCTACTCC;


 







//SEQ ID NO: 777







CTTAGGAGTAGCAACGGCATTTTTTGTTCATGCATTTA;


 







//SEQ ID NO: 778







CCACGGGAGGTGTTGTGCAGACGCTGGCAAACGGACCAGCATTCTTTGGA


GGTGGTGCAGGAAACGTTGGTGAACTGGGAACCACCACCACCGGA;










Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 18
Fc-L-MTX Bacterial Expression
Bacterial expression of Fc-L-MTX. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-MTX.
Oligos used to form duplex are shown below:







//SEQ ID NO: 782







TGGTTCCGGTGGTGGTGGTTCCGTTTCCTGCACCGGT;


 







//SEQ ID NO: 783







TCCAAAGACTGCTACGCTCCGTGCCGTAAACAGACCGGTTGCCCGAACGC


TAAATGCATCAACAAATCCTGCAAATGCTACGGTTGC;


 







//SEQ ID NO: 784







CTTAGCAACCGTAGCATTTGCAGGATTTGTTGATGCAT;


 







//SEQ ID NO: 785







TTAGCGTTCGGGCAACCGGTCTGTTTACGGCACGGAGCGTAGCAGTCTTT


GGAACCGGTGCAGGAAACGGAACCACCACCACCGGA;






The oligos above were used to form the duplex shown below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 19
Fc-L-ChTx (K32E) Bacterial Expression
Bacterial expression of Fc-L-ChTx (K32E). The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ChTx (K32E).
Oligos used to form duplex are shown below:







//SEQ ID NO: 789







TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCAACGTTTCCTG;


 







//SEQ ID NO: 790







CACCACCTCCAAAGAATGCTGGTCCGTTTGCCAGCGTCTGCACAACACCT


CCCGTGGTAAATGCATGAACAAAGAATGCCGTTGCTACTCC;


 







//SEQ ID NO: 791







CTTAGGAGTAGCAACGGCATTCTTTGTTCATGCATTTACCACG;


 







//SEQ ID NO: 792







GGAGGTGTTGTGCAGACGCTGGCAAACGGACCAGCATTCTTTGGAGGTGG


TGCAGGAAACGTTGGTGAACTGGGAACCACCACCACCGGA;






The oligos shown above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 20
Fc-L-Apamin Bacterial Expression
Bacterial expression of Fc-L-Apamin. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-Apamin.
Oligos used to form duplex are shown below:







//SEQ ID NO: 796







TGGTTCCGGTGGTGGTGGTTCCTGCAACTGCAAAGCTCCGGAAACCGCTC


TGTGCGCTCGTCGTTGCCAGCAGCACGGT;


 







//SEQ ID NO: 797







CTTAACCGTGCTGCTGGCAACGACGAGCGCACAGAGCGGTTTCCGGAGCT


TTGCAGTTGCAGGAACCACCACCACCGGA;






The oligos above were used to form the duplex shown below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 21
Fc-L-Scyllatoxin Bacterial Expression
Bacterial expression of Fc-L-Scyllatoxin or Fc-L-ScyTx. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ScyTx.
Oligos used to form duplex are shown below:







//SEQ ID NO: 801







TGGTTCCGGTGGTGGTGGTTCCGCTTTCTGCAACCTGCG;


 







//SEQ ID NO: 802







TATGTGCCAGCTGTCCTGCCGTTCCCTGGGTCTGCTGGGTAAATGCATCG


GTGACAAATGCGAATGCGTTAAACAC;


 







//SEQ ID NO: 803







CTTAGTGTTTAACGCATTCGCATTTGTCACCGATGCATTT;


 







//SEQ ID NO: 804







ACCCAGCAGACCCAGGGAACGGCAGGACAGCTGGCACATACGCAGGTTGC


AGAAAGCGGAACCACCACCACCGGA;






The oligos above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 22
Fc-L-IbTx Bacterial Expression
Bacterial expression of Fc-L-lbTx. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-lbTx.
Oligos used to form duplex are shown below:







//SEQ ID NO: 808







TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCGACGTTGACTGCTCCGT;


 







//SEQ ID NO: 809







TTCCAAAGAATGCTGGTCCGTTTGCAAAGACCTGTTCGGTGTTGACCGTG


GTAAATGCATGGGTAAAAAATGCCGTTGCTACCAG;


 







//SEQ ID NO: 810







CTTACTGGTAGCAACGGCATTTTTTACCCATGCATTTACCACGGTCAA;


 







//SEQ ID NO: 811







CACCGAACAGGTCTTTGCAAACGGACCAGCATTCTTTGGAAACGGAGCAG


TCAACGTCGGTGAACTGGGAACCACCACCACCGGA;






The oligos above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 23
Fc-L-HaTx1 Bacterial Expression
Bacterial expression of Fc-L-HaTx1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-HaTx1.
Oligos used to form duplex are shown below:







//SEQ ID NO: 815







TGGTTCCGGTGGTGGTGGTTCCGAATGCCGTTACCTGTTCGGTGGTTG;


 







//SEQ ID NO: 816







CAAAACCACCTCCGACTGCTGCAAACACCTGGGTTGCAAATTCCGTGACA


AATACTGCGCTTGGGACTTCACCTTCTCC;


 







//SEQ ID NO: 817







CTTAGGAGAAGGTGAAGTCCCAAGCGCAGTATTTGTCACGGAATTTGC;


 


 







//SEQ ID NO: 818







AACCCAGGTGTTTGCAGCAGTCGGAGGTGGTTTTGCAACCACCGAACAGG


TAACGGCATTCGGAACCACCACCACCGGA;






The oligos above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Refolding and purification of Fc-L-HaTx1 expressed in bacteria. Frozen, E. coli paste (13 g) was combined with 100 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml water using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet (2.6 g) was then dissolved in 26 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The dissolved pellet was then reduced by adding 30 μl 1 M dithiothreitol to 3 ml of the solution and incubating at 37° C. for 30 minutes. The reduced pellet solution was then centrifuged at 14,000 g for 5 min at room temperature, and then 2.5 ml of the supernatant was transferred to 250 ml of the refolding buffer (2 M urea, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl, 4 mM cysteine, pH 8.5) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 2 days at 4° C. The refolding solution was then filtered through a 0.22 μm cellulose acetate filter and stored at −70° C.
The stored refold was defrosted and then diluted with 1 L of water and the pH was adjusted to 7.5 using 1 M H3PO4. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 10 ml Amersham SP-HP HiTrap column at 10 ml/min in S-Buffer A (20 mM NaH2PO4, pH 7.3) at 7° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3) followed by a step to 100% S-Buffer B at 5 ml/min 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data (15 ml). The pool was then loaded on to a 1 ml Amersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was then washed with several column volumes of 20 mM NaH2PO4 pH 6.5, 1 M NaCl and eluted with 100 mM glycine pH 3.0. To the elution peak (1.4 ml), 70 μl 1 M tris HCl pH 8.5 was added, and then the pH adjusted material was filtered though a 0.22 μm cellulose acetate filter.
A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 29F). The concentration of the filtered material was determined to be 1.44 mg/ml using a calculated molecular mass of 30,469 g/mol and extinction coefficient of 43,890 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 29B). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 33-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <4 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 20 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 29G). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 29H) and these studies confirmed the integrity of the purified peptibody, within experimental error. The product was then stored at −80° C.
Example 24
Fc-L-PaTx2 Bacterial Expression
Bacterial expression of Fc-L-PaTx2. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-PaTx2.
Oligos used to form duplex are shown below:







//SEQ ID NO: 822







TGGTTCCGGTGGTGGTGGTTCCTACTGCCAGAAATGGA;


 







//SEQ ID NO: 823







TGTGGACCTGCGACGAAGAACGTAAATGCTGCGAAGGTCTGGTTTGCCGT


 


CTGTGGTGCAAACGTATCATCAACATG;


 







//SEQ ID NO: 824







CTTACATGTTGATGATACGTTTGCACCACAGACGGCAAA;


 







//SEQ ID NO: 825







CCAGACCTTCGCAGCATTTACGTTCTTCGTCGCAGGTCCACATCCATTTC


 


TGGCAGTAGGAACCACCACCACCGGA;






The oligos above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 25
Fc-L-wGVIA Bacterial Expression
Bacterial expression of Fc-L-wGVIA. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-wGVIA.
Oligos used to form duplex are shown below:







//SEQ ID NO: 829







TGGTTCCGGTGGTGGTGGTTCCTGCAAATCCCCGGGTT;


 







SEQ ID NO: 830







CCTCCTGCTCCCCGACCTCCTACAACTGCTGCCGTTCCTGCAACCCGTAC


 


ACCAAACGTTGCTACGGT;


 







//SEQ ID NO: 831







CTTAACCGTAGCAACGTTTGGTGTACGGGTTGCAGGAA;


 







//SEQ ID NO: 832







CGGCAGCAGTTGTAGGAGGTCGGGGAGCAGGAGGAACCCGGGGATTTGCA


 


GGAACCACCACCACCGGA;






The oligos above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 26
Fc-L-ωMVIIA Bacterial Expression
Bacterial expression of Fc-L-ωMVIIA. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ωMVIIA.
Oligos used to form duplex are shown below:







//SEQ ID NO: 836







TGGTTCCGGTGGTGGTGGTTCCTGCAAAGGTAAA;


 







//SEQ ID NO: 837







GGTGCTAAATGCTCCCGTCTGATGTACGACTGCTGCACCGGTTCCTGCCG


 


TTCCGGTAAATGCGGT;


 







//SEQ ID NO: 838







CTTAACCGCATTTACCGGAACGGCAGGAACCGGT;


 







//SEQ ID NO: 839







GCAGCAGTCGTACATCAGACGGGAGCATTTAGCACCTTTACCTTTGCAGG


 


AACCACCACCACCGGA;






The oligos above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 27
Fc-L-PtuI Bacterial Expression
Bacterial expression of Fc-L-Ptu1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-Ptu1.
Oligos used to form duplex are shown below:







//SEQ ID NO: 843







TGGTTCCGGTGGTGGTGGTTCCGCTGAAAAAGACTGCATC;


 







//SEQ ID NO: 844







GCTCCGGGTGCTCCGTGCTTCGGTACCGACAAACCGTGCTGCAACCCGCG


 


TGCTTGGTGCTCCTCCTACGCTAACAAATGCCTG;


 







//SEQ ID NO: 845







CTTACAGGCATTTGTTAGCGTAGGAGGAGCACCAAGCACG;


 







//SEQ ID NO: 846







CGGGTTGCAGCACGGTTTGTCGGTACCGAAGCACGGAGCACCCGGAGCGA


 


TGCAGTCTTTTTCAGCGGAACCACCACCACCGGA;






The oligos above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 28
Fc-L-ProTx1 Bacterial Expression
Bacterial expression of Fc-L-ProTx1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ProTx1.
Oligos used to form duplex are shown below:







//SEQ ID NO: 850







TGGTTCCGGTGGTGGTGGTTCCGAATGCCGTTACTGGCTGG;


 







//SEQ ID NO: 851







GTGGTTGCTCCGCTGGTCAGACCTGCTGCAAACACCTGGTTTGCTCCCGT


 


CGTCACGGTTGGTGCGTTTGGGACGGTACCTTCTCC;


 







//SEQ ID NO: 852







CTTAGGAGAAGGTACCGTCCCAAACGCACCAACCGTGACGA;


 







//SEQ ID NO: 853







CGGGAGCAAACCAGGTGTTTGCAGCAGGTCTGACCAGCGGAGCAACCACC


 


CAGCCAGTAACGGCATTCGGAACCACCACCACCGGA;






The oligos above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 29
Fc-L-BeKM1 Bacterial Expression
Bacterial expression of Fc-L-BeKM1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-BeKM1.
Oligos used to form duplex are shown below:







//SEQ ID NO: 857







TGGTTCCGGTGGTGGTGGTTCCCGTCCGACCGACATCAAATG;


 







//SEQ ID NO: 858







CTCCGAATCCTACCAGTGCTTCCCGGTTTGCAAATCCCGTTTCGGTAAAA


 


CCAACGGTCGTTGCGTTAACGGTTTCTGCGACTGCTTC;


 







//SEQ ID NO: 859







CTTAGAAGCAGTCGCAGAAACCGTTAACGCAACGACCGTTGG;


 







//SEQ ID NO: 860







TTTTACCGAAACGGGATTTGCAAACCGGGAAGCACTGGTAGGATTCGGAG


 


CATTTGATGTCGGTCGGACGGGAACCACCACCACCGGA;






The oligos above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 30
Fc-L-CTX Bacterial Expression
Bacterial expression of Fc-L-CTX. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-CTX.
Oligos used to form duplex are shown below:







//SEQ ID NO: 864







TGGTTCCGGTGGTGGTGGTTCCATGTGCATGCCGTGCTTCAC;


 







//SEQ ID NO: 865







CACCGACCACCAGATGGCTCGTAAATGCGACGACTGCTGCGGTGGTAAAG


 


GTCGTGGTAAATGCTACGGTCCGCAGTGCCTGTGCCGT;


 







//SEQ ID NO: 866







CTTAACGGCACAGGCACTGCGGACCGTAGCATTTACCACGAC;


 







//SEQ ID NO: 867







CTTTACCACCGCAGCAGTCGTCGCATTTACGAGCCATCTGGTGGTCGGTG


 


GTGAAGCACGGCATGCACATGGAACCACCACCACCGGA;






The oligos above were used to form the duplex below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 31
N-Terminally PEGylated-Des-Arg1-ShK
Peptide Synthesis of reduced Des-Arq1-ShK. Des-Arg1-ShK, having the sequence







(Peptide 1, SEQ ID NO: 92)









SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC






was synthesized in a stepwise manner on a SymphonyTM multi-peptide synthesizer by solid-phase peptide synthesis (SPPS) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) coupling chemistry at 0.1 mmol equivalent resin scale on TentagelTM-S PHB Fmoc-Cys(Trt)-resin. N-alpha-(9-fluorenylmethyloxycarbonyl)- and side-chain protected amino acids were purchased from Midwest Biotech Incorporated. Fmoc-Cys(Trt)-TentagelTM resin was purchased from Fluka. The following side-chain protection strategy was employed: Asp(OtBu), Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(Nε-Boc), Ser(OtBu), Thr(OtBu) and Tyr(OtBu). Two Oxazolidine dipeptides, Fmoc-Gly-Thr(ψMe,MePro)-OH and Fmoc-Leu-Ser(ψMe,MePro)-OH, were used in the chain assembly and were obtained from NovaBiochem and used in the synthesis of the sequence. The protected amino acid derivatives (20 mmol) were dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF (v/v). Protected amino acids were activated with 20 mM HBTU, 400 mM NMM in 20% DMSO in DMF, and coupling were carried out using two treatments with 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in 20% DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotection reactions were carried out with two treatments using a 20% piperidine in DMF (v/v) solution for 10 minutes and then 15 minutes. Following synthesis, the resin was then drained, and washed with DCM, DMF, DCM, and then dried in vacuo. The peptide-resin was deprotected and released from the resin by treatment with a TFA/EDT/TIS/H2O (92.5:2.5:2.5:2.5 (v/v)) solution at room temperature for 1 hour. The volatiles were then removed with a stream of nitrogen gas, the crude peptide precipitated twice with cold diethyl ether and collected by centrifugation. The crude peptide was then analyzed on a Waters 2795 analytical RP-HPLC system using a linear gradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile) on a Jupiter 4 μm ProteoTM 90 Å column. A PE-SciexTM API Electro-spray mass spectrometer was used to confirm correct peptide product mass. Crude peptide was obtained in 143 mg yield at approximately 70% pure based as estimated by analytical RP-HPLC analysis. Reduced Des-Arg1-ShK (Peptide 1) Retention time (Rt)=5.31 minutes, calculated molecular weight=3904.6917 Da (average); Experimental observed molecular weight 3907.0 Da.

Folding of Des-Arq1-ShK (Disulphide bond formation). Following TFA cleavage and peptide precipitation, reduced Des-Arg1-ShK was then air-oxidized to give the folded peptide. The crude cleaved peptide was extracted using 20% AcOH in water (v/v) and then diluted with water to a concentration of approximately 0.15 mg reduced Des-Arg1-ShK per mL, the pH adjusted to about 8.0 using NH4OH (28-30%), and gently stirred at room temperature for 36 hours. Folding process was monitored by LC-MS analysis. Following this, folded Des-Arg1-ShK peptide was purified using reversed phase HPLC using a 1′′ Luna 5 μm C18 100 Å ProteoTM column with a linear gradient 0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1% TFA in acetonitrile). Folded Des-Arg1-ShK crude peptide eluted earlier (when compared to the elution time in its reduced form) at approximately 25% buffer B. Folded Des-Arg1-ShK (Peptide 2) was obtained in 23.2 mg yield in >97% purity as estimated by analytical RP-HPLC analysis (FIG. 20A). Calculated molecular weight=3895.7693 Da (monoisotopic), experimental observed molecular weight=3896.5 Da(analyzed on a Waters LCT Premier Micromass MS Technologies). (FIG. 20B). Des-Arg1-ShK disulfide connectivity was C1-C6, C2-C4, C3-C5.




N-terminal PEGylation of Folded Des-Arq1-ShK. Folded Des-Arg1-ShK, (Peptide 2) was dissolved in water at 1 mg/ml concentration. A 2 M MeO-PEG-Aldehyde, CH3O—[CH2CH2O]n-CH2CH2CHO (average molecular weight 20 kDa), solution in 50 mM NaOAc, pH 4.5, and a separate 1 M solution of NaCNBH3 were freshly prepared. The peptide solution was then added to the MeO-PEG-Aldehyde containing solution and was followed by the addition of the NaCNBH3 solution. The reaction stoichiometry was peptide:PEG:NaCNBH3 (1:2:0.02), respectively. The reaction was left for 48 hours, and was analyzed on an Agilent 1100 RP-HPLC system using ZorbaxTM 300SB-C8 5 μm column at 40° C. with a linear gradient (6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN in water). Mono-pegylated folded Des-Arg1-ShK constituted approximately 58% of the crude product by analytical RP-HPLC. Mono Pegylated Des-Arg1-ShK was then isolated using a HiTrapTM 5 ml SP HP cation exchange column on AKTA FPLC system at 4° C. at 1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers: A=20 mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH 4.0). The fractions were analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC (as described for the crude). SDS-PAGE gels were run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product was then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM NaOAc, 5% sorbitol, pH 4.0). The dialyzed product was then concentrated in 10 K microcentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μM syringe filter to give the final product. N-Terminally PEGylated-Des-Arg1-ShK (Peptide 3) was isolated in 1.7 mg yield with 85% purity as estimated by analytical RP-HPLC analysis (FIG. 23).
The N-Terminally PEGylated-Des-Arg1-ShK, also referred to as “PEG-ShK[2-35]”, was active in blocking human Kv1.3 (FIG. 38A and FIG. 38B) as determined by patch clamp electrophysiology (Example 36).
Example 32
N-Terminally PEGylated ShK
The experimental procedures of this working example correspond to the results shown in FIG. 17.
Peptide Synthesis of reduced ShK. ShK, having the amino acid sequence









(Peptide 4, SEQ ID NO: 5)









RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC






was synthesized in a stepwise manner on a SymphonyTM multi-peptide synthesizer by solid-phase peptide synthesis (SPPS) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) coupling chemistry at 0.1 mmol equivalent resin scale on TentagelTM-S PHB Fmoc-Cys(Trt)-resin. N-alpha-9-fluorenylmethyloxycarbonyl) and side-chain protected amino acids were purchased from Midwest Biotech Incorporated. Fmoc-Cys(Trt)-TentagelTM resin was purchased from Fluka. The following side-chain protection strategy was employed: Asp(OtBu), Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(Nε-Boc), Ser(OtBu), Thr(OtBu) and Tyr(OtBu). Two Oxazolidine dipeptides, Fmoc-Gly-Thr(ψMe,MePro)-OH and Fmoc-Leu-Ser(ψMe,MePro)-OH, were used in the chain assembly and were obtained from NovaBiochem and used in the synthesis of the sequence. The protected amino acid derivatives (20 mmol) were dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF (v/v). Protected amino acids were activated with 200 mM HBTU, 400 mM NMM in 20% DMSO in DMF, and coupling were carried out using two treatments with 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in 20% DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotections were carried out with two treatments using a 20% piperidine in DMF (v/v) solution for 10 minutes and then 15 minutes. Following synthesis, the resin was then drained, and washed with DCM, DMF, DCM, and then dried in vacuo. The peptide-resin was deprotected and released from the resin by treatment with a TFA/EDT/TIS/H2O (92.5:2.5:2.5:2.5 (v/v)) solution at room temperature for 1 hour. The volatiles were then removed with a stream of nitrogen gas, the crude peptide precipitated twice with cold diethyl ether and collected by centrifugation. The crude peptide was then analyzed on a Waters 2795 analytical RP-HPLC system using a linear gradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile) on a Jupiter 4 μm ProteoTM 90 Å column. A PE-Sciex API Electro-spray mass spectrometer was used to confirm correct peptide product mass. Crude peptide was approximately was obtained 170 mg yield at about 45% purity as estimated by analytical RP-HPLC analysis. Reduced ShK (Peptide 4) Retention time (Rt)=5.054 minutes, calculated molecular weight=4060.8793 Da (average); experimental observed molecular weight=4063.0 Da.

Folding of ShK (Disulphide bond formation). Following TFA cleavage and peptide precipitation, reduced ShK was then air oxidized to give the folded peptide. The crude cleaved peptide was extracted using 20% AcOH in water (v/v) and then diluted with water to a concentration of approximately 0.15 mg reduced ShK per mL, the pH adjusted to about 8.0 using NH4OH (28-30%), and gently stirred at room temperature for 36 hours. Folding process was monitored by LC-MS analysis. Following this, folded ShK peptide was purified by reversed phase HPLC using a 1′′ Luna 5 μm C18 100 Å ProteoTM column with a linear gradient 0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1% TFA in acetonitrile). Folded ShK crude peptide eluted earlier (when compared to the elution time in its reduced form) at approximately 25% buffer B. Folded ShK (Peptide 5) was obtained in 25.5 mg yield in >97% purity as estimated by analytical RP-HPLC analysis. See FIG. 60. Calculated molecular weight=4051.8764 Da (monoisotopic); experimental observed molecular weight=4052.5 Da (analyzed on Waters LCT Premier
Micromass MS Technologies). ShK disulfide connectivity was C1-C6, C2-C4, and C3-C5.




N-terminal PEGylation of Folded ShK. Folded ShK, having the amino acid sequence








RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
(SEQ ID NO: 5)






can be dissolved in water at 1 mg/ml concentration. A 2 M MeO-PEG-Aldehyde, CH3O—CH2CH2O]n-CH2CH2CHO (average molecular weight 20 kDa), solution in 50 mM NaOAc, pH 4.5 and a separate 1 M solution of NaCNBH3 can be freshly prepared. The peptide solution can be then added to the MeO-PEG-Aldehyde containing solution and can be followed by the addition of the NaCNBH3 solution. The reaction stoichiometry can be peptide:PEG:NaCNBH3 (1:2:0.02), respectively. The reaction can be left for 48 hours, and can be analyzed on an AgilentTM 1100 RP-HPLC system using ZorbaxTM 300SB-C8 5 μm column at 40° C. with a linear gradient (6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN in water). Mono-pegylated Shk (Peptide 6) can be then isolated using a HiTrapTM 5 mL SP HP cation exchange column on AKTA FPLC system at 4° C. at 1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers: A=20 mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH 4.0). The fractions can be analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product can be then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM sodium acetate, 5% sorbitol, pH 4.0). The dialyzed product can be then concentrated in 10 K microcentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μM syringe filter to give the final product.

Example 33
N-Terminally PEGylated ShK by Oxime Formation
Peptide Synthesis of reduced ShK. ShK, having the sequence








RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
(SEQ ID NO: 5)






can be synthesized in a stepwise manner on a SymphonyTM multi-peptide synthesizer by solid-phase peptide synthesis (SPPS) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) coupling chemistry at 0.1 mmol equivalent resin scale on TentagelTM-S PHB Fmoc-Cys(Trt)-resin. N-alpha-(9-fluorenylmethyloxycarbonyl)- and side-chain protected amino acids can be purchased from Midwest Biotech Incorporated. Fmoc-Cys(Trt)-TentagelTM resin can be purchased from Fluka. The following side-chain protection strategy can be employed: Asp(OtBu), Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(Nε-Boc), Ser(OtBu), Thr(OtBu) and Tyr(OtBu). Two Oxazolidine dipeptides, Fmoc-Gly-Thr(ΨMe,MePro)-OH and Fmoc-Leu-Ser(ψMe,MePro)-OH, can be used in the chain assembly and can be obtained from NovaBiochem and used in the synthesis of the sequence. The protected amino acid derivatives (20 mmol) can be dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF (v/v). Protected amino acids can be activated with 200 mM HBTU, 400 mM NMM in 20% DMSO in DMF, and coupling can be carried out using two treatments with 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in 20% DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotection reactions can be carried out with two treatments using a 20% piperidine in DMF (v/v) solution for 10 minutes and then 15 minutes. Following the chain-assembly of the Shk peptide, Boc-amionooxyacetic acid (1.2 equiv) can be coupled at the N-terminus using 0.5 M HBTU in DMF with 4 equiv collidine for 5 minutes. Following synthesis, the resin can be then drained, and washed with DCM, DMF, DCM, and then dried in vacuo. The peptide-resin can be deprotected and released from the resin by treatment with a TFA/amionooxyacetic acid/TIS/EDT/H2O (90:2.5:2.5:2.5:2.5) solution at room temperature for 1 hour. The volatiles can be then removed with a stream of nitrogen gas, the crude peptide precipitated twice with cold diethyl ether and collected by centrifugation. The aminooxy-Shk peptide (Peptide 7) can be then analyzed on a Waters 2795 analytical RP-HPLC system using a linear gradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water also containing 0.1% aminooxyacetic acid, B: 0.1% TFA in acetonitrile) on a Jupiter 4 μm ProteoTM 90 Å column.

Reversed-Phase HPLC Purification. Preparative Reversed-phase high-performance liquid chromatography can be performed on C18, 5 μm, 2.2 cm×25 cm) column. Chromatographic separations can be achieved using linear gradients of buffer B in A (A=0.1% aqueous TFA; B=90% aq. ACN containing 0.09% TFA and 0.1% aminooxyacetic acid), typically 5-95% over 90 minutes at 15 mL/min. Preparative HPLC fractions can be characterized by ESMS and photodiode array (PDA) HPLC, combined and lyophilized.
N-Terminal PEGylation of Shk by Oxime Formation. Lyophilized aminooxy-Shk (Peptide 7) can be dissolved in 50% HPLC buffer A/B (5 mg/mL) and added to a two-fold molar excess of MeO-PEG-Aldehyde, CH3O—[CH2CH2O]n—CH2CH2CHO (average molecular weight 20 kDa). The reaction can be left for 24 hours, and can be analyzed on an AgilentTM 1100 RP-HPLC system using ZorbaxTM 300SB-C8 5 μm column at 40° C. with a linear gradient (6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN in water). Mono-pegylated reduced Shk constituted approximately 58% of the crude product by analytical RP-HPLC. Mono PEGylated (oximated) Shk (Peptide 8) can be then isolated using a HiTrapTM 5 mL SP HP cation exchange column on AKTA FPLC system at 4° C. at 1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers: A 20 mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH 4.0). The fractions can be analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product can be then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM NaOAc, 5% sorbitol, pH 4.0). The dialyzed product can be then concentrated in 10 K microcentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μM syringe filter to give the final product.
Folding of ShK (Disulphide bond formation). The mono-PEGylated (oximated) Shk can be dissolved in 20% AcOH in water (v/v) and can be then diluted with water to a concentration of approximately 0.15 mg peptide mL, the pH adjusted to about 8.0 using NH4OH (28-30%), and gently stirred at room temperature for 36 hours. Folding process can be monitored by LC-MS analysis. Following this, folded mono-PEGylated (oximated) Shk (Peptide 9) can be purified using by reversed phase HPLC using a 1′ Luna 5 μm C18 100 Å ProteoTM column with a linear gradient 0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1% TFA in acetonitrile). Mono-PEGylated (oximated) ShK disulfide connectivity can be C1-C6, C2-C4, and C3-C5.




Example 34
N-Terminally PEGylated ShK (Amidation)
The experimental procedures of this working example correspond to the results shown in FIG. 18.
N-Terminal PEGylation of Shk by Amide Formation. A 10 mg/mL solution of folded Shk (Peptide 5), in 100 mM Bicine pH 8.0, can be added to solid succinimidyl ester of 20 kDa PEG propionic acid (mPEG-SPA; CH3O—[CH2CH2O]n-CH2CH2CO—NHS) at room temperature using a 1.5 molar excess of the mPEG-SPA to Shk. After one hour with gentle stirring, the mixture can be diluted to 2 mg/mL with water, and the pH can be adjusted to 4.0 with dilute HCl. The extent of mono-pegylated Shk (Peptide 10), some di-PEGylated Shk or tri-PEGylated Shk, unmodified Shk and succinimidyl ester hydrolysis can be determined by SEC HPLC using a SuperdexTM 75 HR 10/30 column (Amersham) eluted with 0.05 M NaH2PO4, 0.05 M Na2HPO4, 0.15 M NaCl, 0.01 M NaN3, pH 6.8, at 1 mL/min. The fractions can be analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product can be then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM NaOAc, 5% sorbitol, pH 4.0). The dialyzed N-terminally PEGylated (amidated) ShK (Peptide 10) can be then concentrated in 10 K microcentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μM syringe filter to give the final product.




Example 35
Fc-L-SmIIIA
Fc-SmIIIA expression vector. A 104 bp BamHI-NotI fragment containing partial linker sequence and SmIIIA peptide encoded with human high frequency codons was assembled by PCR with overlapping primers 3654-50 and 3654-51 and cloned into to the 7.1 kb NotI-BamHI back bone to generate pcDNA3.1(+) CMVi-hFc-SmIIIA as described in Example 1.








  BamHI



5′GGATCCGGAGGAGGAGGAAGCTGCTGCAACGGCCGCCGCGGCTGCAGCAGCCGCTGG


                       C  C  N  G  R  R  G  C  S  S  R  W


 


TGCCGCGACCACAGCCGCTGCTGCTGAGCGGCCGC3′ //SEQ ID NO: 872


C  R  D  H  S  R  C  C     NotI


SEQ ID NO: 873


 


Forward 5′-3′:


GGAGGAGGATCCGGAGGAGGAGGAAGCTGCTGCAACGGCCGCCGCGGCTGCAGCAGC CGC //SEQ ID NO: 874


 


Reverse 5′-3′:


ATTATTGCGGCCGCTCAGCAGCAGCGGCTGTGGTCGCGGCACCAGCGGCTGCTGCAG CCGC SEQ ID NO: 875






The sequences of the BamHI to NotI fragments in the final constructs were verified by sequencing.

Transient expression of Fc-L-SmIIIa. 7.5 ug of the toxin peptide Fc fusion construct pcDNA3.1(+) CMVi-hFc-SmIIIA were transfected into 293-T cells in 10 cm tissue culture plate with FuGENE 6 as transfection reagent. Culture medium was replaced with serum-free medium at 24 hours post-transfection and the conditioned medium was harvested at day 5 post-transfection. Transient expression of Fc-SmIIIA from 293-T cells was analyzed by Western blot probed with anti-hFc antibody (FIG. 25A and FIG. 25B). Single band of expressed protein with estimated MW was shown in both reduced and non-reduced samples. Transient expression level of Fc-SmIIIA was further determined to be 73.4 μg/ml according to ELISA.
Example 36
Electrophysiology Experiments
Cell Culture. Stable cell line expressing human Kv1.3 channel was licensed from Biofocus. Cells were kept at 37° C. in 5% CO2 environment. Culture medium contains DMEM with GlutaMaxTM (Invitrogen), 1× non-essential amino acid, 10% fetal bovine serum and 500 μg/mL geneticin. Cells were plated and grown at low confluence on 35 mm culture dishes for at least 24 hours prior to electrophysiology experiments.
Electrophysiology Recording by Patch Clamping. Whole-cell currents were recorded from single cells by using tight seal configuration of the patch-clamp technique. A 35 mm culture dish was transferred to the recording stage after rinsing and replacing the culture medium with recording buffer containing 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, and 5 mM Glucose. pH was adjusted to 7.4 with NaOH and the osmolarity was set at 300 mOsm. Cells were perfused continuously with the recording buffer via one of the glass capillaries arranged in parallel and attached to a motorized rod, which places the glass capillary directly on top of the cell being recorded. Recording pipette solution contained 90 mM K-gluconate, 20 mM KF, 10 mM NaCl, 1 mM MgCl2-6H2O, 10 mM EGTA, 5 mM K2-ATP, and 10 mM HEPES. The pH for the internal solution was adjusted to 7.4 with KOH and the osmolarity was set at 280 mOsm. Experiments were performed at room temperature (20-22° C.) and recorded using MulticlampTM 700 A amplifier (Molecular Devices Inc.). Pipette resistances were typically 2-3 MΩ.
Protein toxin potency determination on Kv1.3 current: HEK293 cells stably expressing human Kv1.3 channel were voltage clamped at −80 mV holding potential. Outward Kv1.3 currents were activated by giving 200 msec long depolarizing steps to +30 mV from the holding potential of −80 mV and filtered at 3 kHz. Each depolarizing step was separated from the subsequent one with a 10 s interval. Analogue signals were digitized by DigidataTM 1322A digitizer (Molecular Devices) subsequently stored on computer disk for offline analyses using ClampfitTM 9 (Molecular Devices Inc.). In all studies, stable baseline Kv1.3 current amplitudes were established for 4 minutes before starting the perfusion of the protein toxin at incremental concentrations. A steady state block was always achieved before starting the perfusion of the subsequent concentration of the protein toxin.
Data analysis. Percent of control (POC) is calculated based on the following equation: (Kv1.3 current after protein toxin addition/Kv1.3 current in control)*100. At least 5 concentrations of the protein toxin (e.g. 0.003, 0.01, 0.03, 0.1, 0.3, 100 nM) were used to calculate the IC50 value. IC50 values and curve fits were estimated using the four parameter logistic fit of XLfit software (Microsoft Corp.). IC50 values are presented as mean value±s.e.m. (standard error of the mean).
Drug preparations. Protein toxins (typically 10-100 μM) were dissolved in distilled water and kept frozen at −80° C. Serial dilutions of the stock protein toxins were mixed into the recording buffer containing 0.1% bovine serum albumin (BSA) and subsequently transferred to glass perfusion reservoirs. Electronic pinch valves controlled the flow of the protein toxin from the reservoirs onto the cell being recorded.
Example 37
Immunobiology and Channel Binding
Inhibition of T cell cytokine Production following PMA and anti-CD3 antibody stimulation of PBMCs. PBMC's were previously isolated from normal human donor Leukophoresis packs, purified by density gradient centrifugation (Ficoll Hypaque), cryopreserved in CPZ Cryopreservation Medium Complete (INCELL, MCPZF-100 plus 10% DMSO final). PBMC's were thawed (95% viability), washed, and seeded at 2×105 cells per well in culture medium (RPMI medium 1640; GIBCO) supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin 2 mM L-glutamine, 100 uM non-essential amino acids, and 20 uM 2-ME) in 96-well flat-bottom tissue culture plates. Cells were pre-incubated with serially diluted (100 nM-0.001 nM final) ShK[1-35], Fc-L10-ShK[1-35] or fc control for 90 min before stimulating for 48 hr with PMA/anti-CD3 (1 ng/ml and 50 ng/ml, respectively) in a final assay volume of 200 ul. Analysis of the assay samples was performed using the Meso Scale Discovery (MSD) SECTORTM Imager 6000 (Meso Scale Discovery, Gaithersbury, Md.) to measure the IL-2 and IFNg protein levels by utilizing electrochemiluminescence (ECL). The conditioned medium (50 ul) was added to the MSD Multi-spot 96-well plates (each well containing three capture antibodies; IL-2, TNF, IFNγ). The plates were sealed, wrapped in tin foil, and incubated at room temperature on a plate shaker for 2 hr. The wells were washed 1× with 200 ul PBST (BIOTEK, Elx405 Auto Plate Washer). For each well, 20 ul of Ruthenium-labeled detection antibodies (1 μg/ml final in Antibody Dilution Buffer; IL-1, TNF, IFNγ) and 130 ul of 2×MSD Read Buffer added, final volume 150 ul. The plates were sealed, wrapped in tin foil, and incubated at room temperature on a plate shaker for 1 hr. The plates were then read on the SECTORTM Imager 6000. FIGS. 35A & 35B shows the CHO-derived Fc-L10-ShK[1-35] peptibody potently inhibits IL-2 and IFNg production from T cells in a dose-dependent manner. Compared to native ShK[1-35] peptide, the peptibody produces a greater extent of inhibition (POC=Percent Of Control of response in the absence of inhibitor).
Inhibition of T cell cytokine production following anti-CD3 and anti-CD28 antibody stimulation of PBMCs. PBMCs were previously isolated from normal human donor Leukopheresis packs, purified by density gradient centrifugation (Ficoll Hypaque), and cryopreserved using INCELL Freezing Medium. PBMCs were thawed (95% viability), washed, and seeded (in RPMI complete medium containing serum replacement, PSG) at 2×105 cells per well into 96-well flat bottom plates. Cells were pre-incubated with serially diluted (100 nM-0.003 nM final) ShK[1-35], Fc-L10-ShK[1-35], or Fc control for 1 hour before the addition of aCD3 and aCD28 (2.5 ng/mL and 100 ng/mL respectively) in a final assay volume of 200 mL. Supernatants were collected after 48 hours, and analyzed using the Meso Scale Discovery (MSD) SECTORTM Imager 6000 (Meso Scale Discovery, Gaithersbury, Md.) to measure the IL-2 and IFNg protein levels by utilizing electrochemiluminescence (ECL). 20 mL of supernatant was added to the MSD multi-spot 96-well plates (each well containing IL-2, TNFa, and IFNg capture antibodies). The plates were sealed and incubated at room temperature on a plate shaker for 1 hour. Then 20 mL of Ruthenium-labeled detection antibodies (1 μg/ml final of IL-2, TNFα, and IFNγ in Antibody Dilution Buffer) and 110 mL of 2×MSD Read Buffer were added. The plates were sealed, covered with tin foil, and incubated at room temperature on a plate shaker for 1 hour. The plates were then read on the SECTORTM Imager 6000. FIGS. 37A & 37B shows the CHO-derived Fc-L10-ShK[1-35] peptibody potently inhibits IL-2 and IFNg production from T cells in a dose-dependent manner. Compared to native ShK[1-35] peptide which shows only partial inhibition, the peptibody produces nearly complete inhibition of the inflammatory cytokine response. (POC=Percent Of Control of response in the absence of inhibitor).
Inhibition of T cell proliferation following anti-CD3 and anti-CD28 antibody stimulation of PBMCs. PBMC's were previously isolated from normal human donor Leukophoresis packs, purified by density gradient centrifugation (Ficoll Hypaque), cryopreserved in CPZ Cryopreservation Medium Complete (INCELL, MCPZF-100 plus 10% DMSO final). PBMC's were thawed (95% viability), washed, and seeded at 2×105 cells per well in culture medium (RPMI medium 1640; GIBCO) supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 100 μM non-essential amino acids, and 20 μM 2-ME) in 96-well flat-bottom tissue culture plates. Cells were pre-incubated with either anti-human CD32 (FcyRII) blocking antibody (per manufacturers instructions EASY SEP Human Biotin Selection Kit #18553, StemCell Technologies Vancouver, BC) or Fc-L10-ShK (100 nM-0.001 nM final) for 45 min. Fc-L10-ShK (100 nM-0.001 nM final) was then added to the cells containing anti-human CD32 blocking antibody while medium was added to the cells containing Fc-L10-ShK. Both sets were incubated for an additional 45 min before stimulating for 48 hr with aCD3/aCD28 (0.2 ng/ml and 100 ng/ml, respectively). Final assay volume was 200 ul. [3H]TdR (1 uCi per well) was added and the plates were incubated for an additional 16 hrs. Cells were then harvested onto glass fiber filters and radioactivity was measured in a B-scintillation counter. FIGS. 36A & 36B shows the CHO-derived Fc-L10-ShK[1-35] peptibody potently inhibits proliferation of T cells in a dose-dependent manner. Pre-blocking with the anti-CD32 (FcR) blocking antibody has little effect on the peptibodies ability to inhibit T cell proliferation suggesting Kv1.3 inhibition and not FcR binding is the mechanism for the inhibition observed (POC=Percent Of Control of response in the absence of inhibitor).
Immunohistochemistry analysis of Fc-L10-ShK[1-35] binding to HEK 293 cells overexpressing human Kv1.3. HEK 293 cells overexpressing human Kv1.3 (HEK Kv1.3) were obtained from BioFocus plc (Cambridge, UK) and maintained per manufacturer's recommendation. The parental HEK 293 cell line was used as a control. Cells were plated on Poly-D-Lysine 24 well plates (#35-4414; Becton-Dickinson, Bedford, Mass.) and allowed to grow to approximately 70% confluence. HEK KV1.3 were plated at 0.5×10e5 cells/well in 1 ml/well of medium. HEK 293 cells were plated at a density of 1.5×10e5 cells/well in 1 ml/well of medium. Before staining, cells were fixed with formalin (Sigma HT50-1-1 Formalin solution, diluted 1:1 with PBS/0.5% BSA before use) by removing cell growth medium, adding 0.2 ml/well formalin solution and incubating at room temperature for ten minutes. Cells were stained by incubating with 0.2 ml/well of 5 μg/ml Fc-L10-ShK[1-35] in PBS/BSA for 30′ at room temperature. Fc-L10-ShK[1-35] was aspirated and then the cells were washed one time with PBS/0.5% BSA. Detection antibody (Goat F(ab)2 anti-human IgG-phycoerythrin; Southern Biotech Associates, Birmingham, Ala.) was added to the wells at 5 μg/ml in PBS/0.5% BSA and incubated for 30′ at room temperature. Wash cells once with PBS/0.5% BSA and examine using confocal microscopy (LSM 510 Meta Confocal Microscope; Carl Zeiss AG, Germany). FIG. 33B shows the Fc-L10-ShK[1-35] peptibody retains binds to Kv1.3 overexpressing HEK 293 cells but shows little binding to untransfected cells (FIG. 33A) indicating the Fc-L10-ShK[1-35] peptibody can be used as a reagent to detect cells overexpressing the Kv1.3 channel. In disease settings where activated T effector memory cells have been reported to overproduce Kv1.3, this reagent can find utility in both targeting these cells and in their detection.
An ELISA assay demonstrating Fc-L10-ShK[1-35] binding to fixed HEK 293 cells overexpressing Kv1.3. FIG. 34A shows a dose-dependent increase in the peptibody binding to fixed cells that overexpress Kv1.3, demonstrating that the peptibody shows high affinity binding to its target and the utility of the Fc-L10-ShK[1-35] molecule in detection of cells expressing the channel. Antigen specific T cells that cause disease in patients with multiple sclerosis have been shown to overexpress Kv1.3 by whole cell patch clamp electrophysiology,—a laborius approach. Our peptibody reagent can be a useful and convenient tool for monitoring Kv1.3 channel expression in patients and have utility in diagnostic applications. The procedure shown in FIG. 34A and FIG. 34B follows.
FIG. 34A. A whole cell immunoassay was performed to show binding of intact Fc-L10-ShK[1-35] to Kv1.3 transfected HEK 293 cells (BioFocus plc, Cambridge, UK). Parent HEK 293 cells or HEK Kv1.3 cells were plated at 3×10e4 cells/well in poly-D-Lysine coated ninety-six well plates (#35-4461; Becton-Dickinson, Bedford, Mass.). Cells were fixed with formalin (Sigma HT50-1-1 Formalin solution, diluted 1:1 with PBS/0.5% BSA before use) by removing cell growth medium, adding 0.2 ml/well formalin solution and incubating at room temperature for 25 minutes and then washing one time with 100 μl/well of PBS/0.5% BSA. Wells were blocked by addition of 0.3 ml/well of BSA blocker (50-61-00; KPL 10% BSA Diluent/Blocking Solution, diluted 1:1 with PBS; KPL, Gaithersburg, Md.) followed by incubation at room temperature, with shaking, for 3 hr. Plates were washed 2 times with 1×KP Wash Buffer (50-63-00; KPL). Samples were diluted in Dilution Buffer (PBS/0.5% Tween-20) or Dilution Buffer with 1% Male Lewis Rat Serum (RATSRM-M; Bioreclamation Inc., Hicksville, N.Y.) and 0.1 ml/well was added to blocked plates, incubating for 1 hr at room temperature with shaking. Plates were washed 3 times with 1xKP Wash Buffer and then incubated with HRP-Goat anti-human IgG Fc (#31416; Pierce, Rockford, Ill.) diluted 1:5000 in PBS/0.1% Tween-20 for 1 hr at room temperature, with shaking. Plates were washed plates 3 times with 1xKP Wash Buffer, and then 0.1 ml/well TMB substrate (52-00-01; KPL) was added. The reactions were stopped by addition of 0.1 ml/well 2 N Sulfuric Acid. Absorbance was read at 450 nm on a Molecular Devices SpectroMax 340 (Sunnyvale, Calif.).
FIG. 34B. Whole cell immunoassay was performed as above with the following modifications. HEK 293 cells were plated at 1×10e5 cells/well and HEK Kv1.3 cells were plated at 6×10e4 cells/well in poly-D-Lysine coated 96 well plates. Fc Control was added at 500 ng/ml in a volume of 0.05 ml/well. HRP-Goat anti-human IgG Fc (#31416; Pierce, Rockford, Ill.) was diluted 1:10,000 in PBS/0.1% Tween-20. ABTS (50-66-00, KPL) was used as the substrate. Absorbances were read at 405 nm after stopping reactions by addition of 0.1 ml/well of 1% SDS.
Example 38
Purification of Fc-L10-ShK(1-35)
Expression of Fc-L10-ShK[1-35] was as described in Example 3 herein above. Frozen, E. coli paste (18 g) was combined with 200 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 22,000 g for 15 min at 4° C. The pellet was then resuspended in 200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 22,000 g for 15 min at 4° C. The pellet was then resuspended in 200 ml water using a tissue grinder and then centrifuged at 22,000 g for 15 min at 4° C. The pellet (3.2 g) was then dissolved in 32 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The pellet solution was then centrifuged at 27,000 g for 15 min at room temperature, and then 5 ml of the supernatant was transferred to 500 ml of the refolding buffer (3 M urea, 20% glycerol, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl, 4 mM cysteine, pH 9.5) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 2 days at 4° C. The refolding solution was then stored at −70° C.
The stored refold was defrosted and then diluted with 2 L of water and the pH was adjusted to 7.3 using 1 M H3PO4. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 60 ml Amersham SP-FF (2.6 cm I.D.) column at 20 ml/min in S-Buffer A (20 mM NaH2PO4, pH 7.3) at 7° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3) followed by a step to 100% S-Buffer B at 10 ml/min 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pool was then loaded on to a 1 ml Amersham rProtein A HiTrap column in PBS at 1 ml/min 7° C. Then column was then washed with several column volumes of 20 mM NaH2PO4 pH 6.5, 1 M NaCl and eluted with 100 mM glycine pH 3.0. To the elution peak, 0.0125 volumes (25 ml) of 3 M sodium acetate was added.
A spectral scan was then conducted on 50 μl of the combined pool diluted in 700 μl water using a Hewlett Packard 8453 spectrophotometer (FIG. 46A). The concentration of the filtered material was determined to be 2.56 mg/ml using a calculated molecular mass of 30,410 g/mol and extinction coefficient of 36,900 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 46B). The macromolecular state of the product was then determined using size exclusion chromatography on 20 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 46C). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). One milliliter of the resultant solution was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses. The product was then stored at −80° C.
The IC50 for blockade of human Kv1.3 by purified E. coli-derived Fc-L10-ShK[1-35], also referred to as “Fc-L-ShK[1-35]”, is shown in Table 35 (in Example 50 herein below).
Example 39
Purification of Bacterially Expressed Fc-L10-ShK(2-35)
Expression of Fc-L10-ShK[2-35] was as described in Example 4 herein above. Frozen, E. coli paste (16.5 g) was combined with 200 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 22,000 g for 15 min at 4° C. The pellet was then resuspended in 200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 22,000 g for 15 min at 4° C. The pellet was then resuspended in 200 ml water using a tissue grinder and then centrifuged at 22,000 g for 15 min at 4° C. The pellet (3.9 g) was then dissolved in 39 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The pellet solution was then centrifuged at 27,000 g for 15 min at room temperature, and then 5 ml of the supernatant was transferred to 500 ml of the refolding buffer (3 M urea, 20% glycerol, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl, 4 mM cysteine, pH 9.5) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 2 days at 4° C. The refolding solution was then stored at −70° C.
The stored refold was defrosted and then diluted with 2 L of water and the pH was adjusted to 7.3 using 1 M H3PO4. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 60 ml Amersham SP-FF (2.6 cm I.D.) column at 20 ml/min in S-Buffer A (20 mM NaH2PO4, pH 7.3) at 7° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3) followed by a step to 100% S-Buffer B at 10 ml/min 7° C. The fractions containing the desired product were pooled and filtered through a 0.22 μm cellulose acetate filter. The pool was then loaded on to a 1 ml Amersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was then washed with several column volumes of 20 mM NaH2PO4 pH 6.5, 1 M NaCl and eluted with 100 mM glycine pH 3.0. To the elution peak, 0.0125 volumes (18 ml) of 3 M sodium acetate was added, and the sample was filtered through a 0.22 μm cellulose acetate filter.
A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl water using a Hewlett Packard 8453 spectrophotometer (FIG. 40A). The concentration of the filtered material was determined to be 3.20 mg/ml using a calculated molecular mass of 29,282 g/mol and extinction coefficient of 36,900 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 40B). The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 40C). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). One milliliter of the resultant solution was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 40D). The product was then stored at −80° C.
The IC50 for blockade of human Kv1.3 by purified E. coli-derived Fc-L10-ShK[2-35], also referred to as “Fc-L-ShK[2-35]”, is shown in Table 35 (in Example 50 herein below).
Example 40
Purification of Bacterially Expressed Fc-L10-OsK1
Frozen, E. coli paste (129 g; see Example 10) was combined with 1290 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 7.8 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 17,700 g for 15 min at 4° C. The pellet was then resuspended in 1290 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 17,700 g for 15 min at 4° C. The pellet was then resuspended in 1290 ml water using a tissue grinder and then centrifuged at 17,700 g for 15 min at 4° C. 8 g of the pellet (16.3 g total) was then dissolved in 160 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. 100 ml of the pellet solution was then incubated with 1 ml of 1 M DTT for 60 min at 37° C. The reduced material was transferred to 5000 ml of the refolding buffer (1 M urea, 50 mM tris, 160 mM arginine HCl, 2.5 mM EDTA, 1.2 mM cystamine HCl, 4 mM cysteine, pH 10.5) at 2 ml/min, 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 3 days at 4° C.
The pH of the refold was adjusted to 8.0 using acetic acid. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 50 ml Amersham Q Sepharose-FF (2.6 cm I.D.) column at 10 ml/min in Q-Buffer A (20 mM Tris, pH 8.5) at 8° C. with an inline 50 Amersham Protein A column (2.6 cm I.D.). After loading, the Q Sepharose column was removed from the circuit, and the remaining chromatography was carried out on the protein A column. The column was washed with several column volumes of Q-Buffer A, followed by elution using a step to 100 mM glycine pH 3.0. The fractions containing the desired product were pooled and immediately loaded on to a 50 ml Amersham SP-Sepharose HP column (2.6 cm I.D.) at 20 ml/min in S-Buffer A (20 mM NaH2PO4, pH 7.0) at 8° C. The column was then washed with several column volumes of S-Buffer A followed by a linear gradient from 5% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.0) followed by a step to 100% S-Buffer B. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE. The fractions containing the bulk of the desired product were pooled and then applied to a 75 ml MEP Hypercel column (2.6 cm I.D.) at 5 ml/min in MEP Buffer A (20 mM tris, 200 mM NaCl, pH 8.0) at 8° C. Column was eluted with a linear gradient from 5% to 50% MEP Buffer B (50 mM sodium citrate pH 4.0) followed by a step to 100% MEP Buffer B. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the bulk of the desired product were pooled.
The MEP pool was then concentrated to about 20 ml using a Pall Jumbo-Sep with a 10 kDa membrane followed by buffer exchange with Formulation Buffer (20 mM NaH2PO4, 200 mM NaCl, pH 7.0) using the same membrane. A spectral scan was then conducted on 50 μl of the combined pool diluted in 700 μl Formulation Buffer using a Hewlett Packard 8453 spectrophotometer (FIG. 41A). The concentration of the material was determined to be 4.12 mg/ml using a calculated molecular mass of 30,558 g/mol and extinction coefficient of 35,720 M−1 cm−1. The purity of the material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 41B). The macromolecular state of the product was then determined using size exclusion chromatography on 123 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 41C). The product was then subject to mass spectral analysis by chromatographing approximately 4 μg of the sample through a RP-HPLC column (Vydac C4, 1×150 mm). Solvent A was 0.1% trifluoroacetic acid in water and solvent B was 0.1% trifluoroacetic acid in 90% acetonitrile, 10% water. The column was pre-equilibrated in 10% solvent B at a flow rate of 80 μl per min. The protein was eluted using a linear gradient of 10% to 90% solvent B over 30 min. Part of the effluent was directed into a LCQ ion trap mass spectrometer. The mass spectrum was deconvoluted using the Bioworks software provided by the mass spectrometer manufacturer. (FIG. 41D). The product was filtered through a 0.22 μm cellulose acetate filter and then stored at −80° C.
The yield for the E. coli-expressed Fc-L10-OSK1 prep was 81 mg from 40 g of cell paste (129 g×(8 g/16.3 g)×(100 ml/160 ml)=39.6 g which was rounded to 40 g), the purity was greater than 80% judging by SDS-PAGE, it is running as the expected dimer judging by SEC-HPLC, and the mass was within the expected molecular weight range judging by MS.
The IC50 for blockade of human Kv1.3 by purified E. coli-derived Fc-L10-OSK1, also referred to as “Fc-L-OSK1”, is shown in Table 35 (in Example 50 herein below).
Example 41
Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1 [K7S,E16K,K20D] Expressed by Mammalian Cells
Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1 [K7S,E16K,K20D], inhibitors of Kv1.3, were expressed in mammalian cells. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a linker sequence and a monomer of the Kv1.3 inhibitor peptide OSK1, OSK1[K7S], OSK1[E16K,K20D], or OSK1[K7S,E16K,K20D] was constructed as described below. Methods for expressing and purifying the peptibody from mammalian cells (HEK 293 and Chinese Hamster Ovary cells) are disclosed herein.
For construction of Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] expression vectors, a PCR strategy was employed to generate the full length genes, OSK1, OSK1[K7S], OSK1[E16K,K20D], and OSK1[K7S,E16K,K20D], each linked to a four glycine and one serine amino acid linker with two stop codons and flanked by BamHI and NotI restriction sites as shown below.
Two oligos for each of OSK1, OSK1[K7S], OSK1[E16K,K20D], and [K7S,E16K,K20D]OSK1 with the sequence as depicted below were used in a PCR reaction with PfuTurbo HotStart DNA polymerase (Stratagene) at 95° C.-30 sec, 55° C.-30 sec, 75° C.-45 sec for 35 cycles; BamHI (ggatcc) and NotI (gcggccgc) restriction sites are underlined.








OSK1:



Forward primer: cat gga tcc gga gga gga
(SEQ ID


gga agc ggc gtg atc atc aac gtg aag tgc
NO: 876)


aag atc agc cgc cag tgc ctg gag ccc tgc



aag aag gcc g;



 


Reverse primer: cat gcg gcc gct tac tac
(SEQ ID


ttg ggg gtg cag tgg cac ttg ccg ttc atg
NO: 877)


cac ttg ccg aag cgc atg ccg gcc ttc ttg



cag ggc tcc a;



 


OSK1[K7S]:



Forward primer: cat gga tcc gga gga gga
(SEQ ID


gga agc ggc gtg atc atc aac gtg agc tgc
NO: 878)


aag atc agc cgc cag tgc ctg gag ccc tgc



aag aag gcc g;



 


Reverse primer: cat gcg gcc gct tac tac
(SEQ ID


ttg ggg gtg cag tgg cac ttg ccg ttc atg
NO: 879)


cac ttg ccg aag cgc atg ccg gcc ttc ttg



cag ggc tcc a;



 


OSK1[E16K, K20D]:



Forward primer: cat gga tcc gga gga gga
(SEQ ID


gga agc ggc gtg atc atc aac gtg aag tgc
NO: 880)


aag atc agc cgc cag tgc ctg aag ccc tgc



aag gac gcc g;



 


Reverse primer: cat gcg gcc gct tac tac
(SEQ ID


ttg ggg gtg cag tgg cac ttg ccg ttc atg
NO: 881)


cac ttg ccg aag cgc atg ccg gcg tcc ttg



cag ggc ttc a;



 


OSK1[K7S, E16K, K20D]:



Forward primer: cat gga tcc gga gga gga
(SEQ ID


gga agc ggc gtg atc atc aac gtg agc tgc
NO: 882)


aag atc agc cgc cag tgc ctg aag ccc tgc



aag gac gcc g;



 


Reverse primer: cat gcg gcc gct tac tac
(SEQ ID


ttg ggg gtg cag tgg cac ttg ccg ttc atg
NO: 883)


cac ttg ccg aag cgc atg ccg gcg tcc ttg



cag ggc ttc a.







The resulting PCR products were resolved as the 155 bp bands on a four percent agarose gel. The 155 bp PCR product was purified using PCR Purification Kit (Qiagen), then digested with BamHI and NotI (Roche) restriction enzymes, and agarose gel was purified by Gel Extraction Kit (Qiagen). At the same time, the pcDNA3.1(+) CMVi-hFc-Shk[2-35] vector was digested with BamHI and NotI restriction enzymes and the large fragment was purified by Gel Extraction Kit. The gel purified PCR fragment was ligated to the purified large fragment and transformed into One Shot® Top10F′ (Invitrogen). DNAs from transformed bacterial colonies were isolated and digested with BamHI and NotI restriction enzymes and resolved on a two percent agarose gel. DNAs resulting in an expected pattern were submitted for sequencing. Although, analysis of several sequences of clones yielded a 100% percent match with the above sequences, only one clone from each gene was selected for large scaled plasmid purification. The DNA of Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] in pCMVi vector was resequenced to confirm the Fc and linker regions and the sequence was 100% identical to the above sequences. The sequences and pictorial representations of Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] are depicted in FIG. 42A-B, FIG. 43A-B, FIG. 44A-B and FIG. 45A-B, respectively.
HEK-293 cells used in transient transfection expression of Fc-L110-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] in pCMVi protein were cultured in growth medium containing DMEM High Glucose (Gibco), 10% fetal bovine serum (FBS from Gibco), 1× non-essential amino acid (NEAA from Gibco) and 1× Penicillin/Streptomycine/Glutamine (Pen/Strep/Glu from Gibco). 5.6 μg each of Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] in pCMVi plasmid that had been phenol/chloroform extracted was transfected into HEK-293 cells using FuGENE 6 (Roche). The cells were recovered for 24 hours, and then placed in DMEM High Glucose, 1x NEAA and 1× Pen/Strep/Glu medium for 48 hours. Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] were purified from medium conditioned by these transfected HEK-293 cells using a protocol described in Example 50 herein below.
Fifteen μl of conditioned medium was mixed with an in-house 4× Loading Buffer (without β-mercaptoethanol) and electrophoresed on a Novex 4-20% tris-glycine gel using a Novex Xcell II apparatus at 101V/46 mA for 2 hours in a 1× Gel Running solution (25 mM Tris Base, 192 mM Glycine, 3.5 mM SDS) along with 20 μl of BenchMark Pre-Stained Protein ladder (Invitrogen). The gel was then soaked in Electroblot buffer (25 mM Tris base, 192 mM glycine, 20% methanol,) for 5 minutes. -A nitrocellulose membrane from Invitrogen (Cat. No. LC200, 0.2 μm pores size) was soaked in Electroblot buffer. The pre-soaked gel was blotted to the nitrocellulose membrane using the Mini Trans-Blot Cell module according to the manufacturer instructions (Bio-Rad Laboratories) at 300 mA for 2 hours. The blot was rinsed in Tris buffered saline solution pH7.5 with 0.1% Tween20 (TBST). Then, the blot was first soaked in a 5% milk (Camation) in TBST for 1 hour at room temperature, followed by washing three times in TBST for 10 minutes per wash. Then, incubated with 1:1000 dilution of the HRP-conjugated Goat anti-human IgG, (Fcγ) antibody (Pierece Biotechnology Cat. no. 31413) in TBST with 5% milk buffer for 1 hour with shaking at room temperature. The blot was then washed three times in TBST for 15 minutes per wash at room temperature. The primary antibody was detected using Amersham Pharmacia Biotech's ECL western blotting detection reagents according to manufacturers instructions. Upon ECL detection, the western blot analysis displayed the expected size of 66 kDa under non-reducing gel conditions (FIG. 46).
Plasmids containing the Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] inserts in pCMVi vector were digested with XbaI and NotI (Roche) restriction enzymes and gel purified. The inserts were individually ligated into SpeI and NotI (Roche) digested pDSRα24 (Amgen Proprietary) expression vector. Integrity of the resulting constructs were confirmed by DNA sequencing. Although, analysis of several sequences of clones yielded a 100% percent match with the above sequence, only one clone was selected for large scaled plasmid purification.
AM1 CHOd- (Amgen Proprietary) cells used in the stable expression of Fc-L10-OSK1 protein were cultured in AM1 CHOd- growth medium containing DMEM High Glucose, 10% fetal bovine serum, 1× hypoxantine/thymidine (HT from Gibco), 1×NEAA and 1× Pen/Strep/Glu. 5.6 μg of pDSRα-24-Fc-L10-OSK1 plasmid was transfected into AM1 CHOd- cells using FuGene 6. Twenty-four hours post transfection, the cells were split 1:11 into DHFR selection medium (DMEM High Glucose plus 10% Dialyzed Fetal Bovine Serum (dFBS), 1×NEAA and 1× Pen/Strep/Glu) at 1:50 dilution for colony selection. The cells were selected in DHFR selection medium for thirteen days. The ten 10-cm2 pools of the resulting colonies were expanded to ten T-175 flasks, then were scaled up ten roller bottles and cultured under AM1 CHOd- production medium (DMEM/F12 (1:1), 1×NEAA, 1× Sodium Pyruvate (Na Pyruvate), 1× Pen/Strep/Glu and 1.5% DMSO). The conditioned medium was harvested and replaced at one-week intervals. The resulting six liters of conditioned medium were filtered through a 0.45 μm cellulose acetate filter (Corning, Acton, Mass.), and characterized by SDS-PAGE analysis as shown in FIG. 47. Then, transferred to Protein Chemistry for purification.
Twelve colonies were selected after 13 days on DHFR selection medium and picked into one 24-well plate. The plate was allowed to grow up for one week, and then was transferred to AM1 CHOd- production medium for 48-72 hours and the conditioned medium was harvested. The expression levels were evaluated by Western blotting similar to the transient Western blot analysis with detection by the same HRP-conjugated Goat anti-human IgG, (Fcγ) antibody to screen 5 μl of conditioned medium. All 12 stable clones exhibited expression at the expected size of 66 kDa. Two clones, A3 and C2 were selected and expanded to T175 flask for freezing with A3 as a backup to the primary clone C2 (FIG. 48).
The C2 clone was scaled up into fifty roller bottles (Corning) using selection medium and grown to confluency. Then, the medium was exchanged with a production medium, and let incubate for one week. The conditioned medium was harvested and replaced at the one-week interval. The resulting fifty liters of conditioned medium were filtered through a 0.45 μm cellulose acetate filter (Corning, Acton, Mass.), and characterized by SDS-PAGE analysis (data not shown). Further purification was accomplished as described in Example 42 herein below.
Example 42
Purification of Fc-L10-OSK1, Fc-L10-OSK1(K7S), Fc-L10-OSK1(E16K,K20D), and Fc-L10-OSK1(K7S,E16K,K20D) Expressed by Mammalian Cells
Purification of Fc-L10-OSK1. Approximately 6 L of CHO (AM1 CHOd-) cell-conditioned medium (see, Example 41 above) was loaded on to a 35 ml MAb Select column (GE Healthcare) at 10 ml/min 7° C., and the column was washed with several column volumes of Dulbecco's phosphate buffered saline without divalent cations (PBS) and sample was eluted with a step to 100 mM glycine pH 3.0. The MAb Select elution was directly loaded on to an inline 65 ml SP-HP column (GE Healthcare) in S-Buffer A (20 mM NaH2PO4, pH 7.0) at 10 ml/min 7° C. After disconnecting the MAb select column, the SP-HP column was then washed with several column volumes S-Buffer A, and then developed using a linear gradient from 5% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.0) at 10 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pooled material was then concentrated to about 20 ml using a Pall Life Sciences Jumbosep 10K Omega centrifugal ultra-filtration device. The concentrated material was then buffer exchanged by diluting with 20 ml of 20 mM NaH2PO4, pH 7.0 and reconcentrated to 20 ml using the Jumbosep 10K Omega filter. The material was then diluted with 20 ml 20 mM NaH2PO4, 200 mM NaCl, pH 7.0 and then reconcentrated to 22 ml. The buffer exchanged material was then filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang E membrane at 1 ml/min room temperature. A spectral scan was then conducted on 50 μl of the filtered material diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 49A, black trace). The concentration of the filtered material was determined to be 4.96 mg/ml using a calculated molecular mass of 30,371 g/mol and extinction coefficient of 35,410 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 49B). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 30-fold dilution of the sample in Charles Rivers Laboratories Endotoxin Specific Buffer yielding a result of 1.8 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 149 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 49C). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). One milliliter of the resultant solution was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from about 200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses. (FIG. 49D). The product was then stored at −80° C.
The yield for the mammalian Fc-L10-OSK1 prep was 115 mg from 6 L, the purity was >90% judging by SDS-PAGE; Fc-L10-OSK1 ran as the expected dimer judging by SEC-HPLC, and the mass is with the expected range judging by MS.
The activity of purified Fc-L10-OSK1 in blocking human Kv1.3 and human Kv1.1 is described in Example 43 herein below.
Purification of Fc-L10-OSK1(K7S). Fc-L10-OSK1(E16K,K20D), and Fc-L10-OSK1(K7S,E16K,K20D). Approximately 500 mL of medium conditioned by transfected HEK-293 (see, Example 41 above) was combined with a 65% slurry of MAb Select resin (1.5 ml) (GE Healthcare) and 500 μl 20% NaN3. The slurry was then gently agitated for 3 days at 4° C. followed by centrifugation at 1000 g for 5 minutes at 4° C. using no brake. The majority of the supernatant was then aspirated and the remaining slurry in the pellet was transferred to a 14 ml conical tube and combined with 12 ml of Dulbecco's phosphate buffered saline without divalent cations (PBS). The slurry was centrifuged at 2000 g for 1 minute at 4° C. using a low brake and the supernatant was aspirated. The PBS wash cycle was repeated an additional 3 times. The bound protein was then eluted by adding 1 ml of 100 mM glycine pH 3.0 and gently agitating for 5 min at room temperature. The slurry was then centrifuged at 2000 g for 1 minute at 4° C. using a low brake and the supernatant was aspirated as the first elution. The elution cycle was repeated 2 more times, and all 3 supernatants were combined into a single pool. Sodium acetate (37.5 μl of a 3 M solution) was added to the elution pool to raise the pH, which was then dialyzed against 10 mM acetic acid, 5% sorbitol, pH 5.0 for 2 hours at room temperature using a 10 kDa SlideAlyzer (Pierce). The dialysis buffer was changed, and the dialysis continued over night at 4° C. The dialyzed material was then filtered through a 0.22 μm cellulose acetate filter syringe filter. Then concentration of the filtered material was determined to be 1.27 mg/ml using a calculated molecular mass of 30,330 and extinction coefficient of 35,410 M−1 cm−1 (FIG. 50A). The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 50B). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 25-fold dilution of the sample in Charles Rivers Laboratories Endotoxin Specific Buffer yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 50C). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). One milliter of the resultant solution was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses. (FIG. 50D). The product was then stored at −80° C.
FIGS. 51A-D show results from the purification and analysis for Fc-L10-OsK1(E16K, K20D), which was conducted using the same protocol as that for the Fc-L110-OsK1 (K7S) molecule (described above) with the following exceptions: the concentration was found to be 1.59 mg/ml using a calculated molecular mass of 30,357 g/mol and a calculated extinction coefficient of 35,410; the pyrogen level was found to be <1 EU/mg using a 32-fold dilution.
FIGS. 52A-D show results from the purification and analysis for Fc-L10-OsK1(K7S,E16K, K20D), which was conducted using the same protocol as that for the Fc-L10-OsK1(K7S) molecule (described above) with the following exceptions: the concentration was found to be 0.81 mg/ml using a calculated molecular mass of 30,316 g/mol and a calculated extinction coefficient of 35,410; the pyrogen level was found to be <1 EU/mg using a 16-fold dilution.
The activity of purified Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K, K20D] and Fc-L10-OSK1[K7S, E16K, K20D] in blocking human Kv1.3 and human Kv1.1 is described in Example 43 herein below.
Example 43
Electrophysiology of OSK1 and OSK1 Peptibody Analogs
A 38-residue peptide toxin of the Asian scorpion Orthochirus scrobiculosus venom (OSK1) was synthesized (see, Examples 41) to evaluate its impact on the human Kv1.1 and Kv1.3 channels, subtypes of the potassium channel family. The potency and selectivity of synthetic OSK1 in inhibiting the human Kv1.1 and Kv1.3 channels was evaluated by the use of HEK293 cell expression system and electrophysiology (FIG. 53). Whole cell patch clamp recording of stably expressed Kv1.3 channels revealed that the synthetic OSK1 peptide is more potent in inhibiting human Kv1.3 when compared to Kv1.1 (Table 33).
Fusion of OSK1 peptide toxin to antibody to generate OSK1 peptibody. To improve plasma half-life and prevent OSK1 peptide toxin from penetrating the CNS, the OSK1 peptide toxin was fused to the Fc-fragment of a human antibody IgG1 via a linker chain length of 10 amino acid residues (Fc-L10-OSK1), as described in Example 41 herein. This fusion resulted in a decrease in the potency of Kv1.3 by 5-fold when compared to the synthetic OSK1 peptide. However, it significantly improved the selectivity of OSK1 against Kv1.1 by 210-fold when compared to that of the synthetic peptide alone (4-fold; Table 33 and FIG. 54).
Modification of OSK1-peptibody (Fc-L10-OSK1). OSK1 shares 60 to 80% sequence homology to other members of scorpion toxins, which are collectively termed α-KTx3. Sequence alignment of OSK1 and other members of α-KTx3 family revealed 4 distinct structural differences at positions 12, 16, 20, and 36. These structural differences of OSK1 have been postulated to play an important role in its wide range of activities against other potassium channels, which is not observed with other members of α-KTx3 family. Hence, two amino acid residues at position 16 and 20 were restored to the more conserved amino acid residues within the OSK1 sequence in order to evaluate their impact on selectivity against other potassium channels such as Kv1.1, which is predominantly found in the CNS as a heterotetromer with Kv1.2. By substituting for glutamic acid at position 16, and for lysine at position 20, the conserved lysine and aspartic acid residues, respectively (i.e., Fc-L10-OSK1[E16K, K20D]), we did not observe a significant change in potency when compared to that of Fc-L10-OSK1 (1.3-fold difference; FIG. 56 and Table 33). However, this double mutation removed the blocking activity against Kv1.1. The selectivity ratio of Kv1.1/Kv1.3 was 403-fold, which was a significant improvement over the selectivity ratio for Fc-L10-OSK1 (210-fold). A single amino acid mutation at position 7 from lysine to serine (Fc-L10-OSK1[K7S]) produced a slight change in potency and selectivity by 2- and 1.3-fold, respectively, when compared to those of Fc-L10-OSK1 (FIG. 55 and Table 33). There was a significant decrease in potency as well as selectivity when all three residues were mutated to generate Fc-L10-OSK1[K7S, E16K, K20D] (FIG. 57 and Table 33).
As demonstrated by the results in Table 33, we dramatically improved selectivity against Kv1.1 by fusing the OSK1 peptide toxin to the Fc-fragment of the human antibody IgG1, but reduced target potency against Kv1.3. The selectivity against Kv1.1 was further improved when 2 residues at two key positions were restored to the conserved residues found in other members of the α-KTx3 family.
Table 33 shows a summary of IC50 values for OSK1 and OSK1 analogs against hKv1.3 and hKv1.1 channels. All analogues are ranked based on their potency against hKv1.3. Also shown in the table is the selectivity ratio of hKv1.1/hKv1.3 for all OSK1 analogs.














hKv1.3:
hKv1.1:




IC50
IC50
hKv1.1/


Compound
[pM]
[pM]
hKv1.3


















Synthetic OSK1
39
160
4


Fc-L10-OSK1
198
41600
210


Fc-L10-OSK1[E16K, K20D]
248
100000
403


Fc-L10-OSK1[K7S]
372
100000
269


Fc-L10-OSK1[K7S, E16K, K20D]
812
10000
12









Example 44
Pharmacokinetic Study of PEG-ShK[1-35] Molecule in Rats
The intravenous (IV) pharmacokinetic profile was determined of a about 24-kDa 20K PEG-ShK[1-35] molecule and the about 4-kDa small native ShK peptide was determined in Spraque Dawley rats. The IV dose for the native ShK peptide and our novel 20K PEG-ShK[1-35] molecule was 1 mg/kg. This dose represented equal molar amounts of these two molecules. The average weight of the rats was about 0.3 kg and two rats were used for each dose & molecule. At various times following IV injection, blood was drawn and about 0.1 ml of serum was collected. Serum samples were stored frozen at −80° C. until analysis.
Assay Plate preparation for electrophysiology. Rat serum samples containing the 20K PEG-ShK[1-35] molecule or the native ShK peptide from pharmacokinetic studies were received frozen. Before experiments, each sample was thawed at room temperature and an aliquot (70 to 80 μl) was transferred to a well in a 96-well polypropylene plate. In order to prepare the Assay Plate, several dilutions were made from the pharmacokinetic serum samples to give rise to Test Solutions. Dilutions of serum samples from the pharmacokinetic study were into 10% Phosphate Buffered Saline (PBS, with Ca2, and Mg2+). For determination of the amount of our novel 20K PEG-ShK[1-35] molecule in serum samples from the pharmacokinetic study, the final serum concentrations in the Test Solutions were 90%, 30%, 10%, 3.3% and 1.1%. Purified 20K PEG-Shk[1-35] Standard inhibition curves were also prepared in the Assay Plate. To do this, 8-point serial dilutions of the purified 20K PEG-ShK[1-35] molecule (Standard) were prepared in either 90%, 30%, 10%, 3.3% or 1.1% rat serum and the final concentration of standard was 50, 16.7, 5.5, 1.85, 0.62, 0.21, 0.068 and 0.023 nM.
Cell preparation for electrophysiology. CHO cells stably expressing the voltage-activated K+ channel, KV1.3 were plated in T-175 tissue culture flasks (at a density of 5×106) 2 days before experimentation and allowed to grow to around 95% confluence. Immediately prior to the experiment, the cells were washed with PBS and then detached with a 2 ml mixture (1:1 volume ratio) of trypsin (0.25%) and versene (1:5000) at 37° C. (for 3 minutes). Subsequently, the cells were re-suspended in the flask in 10 ml of tissue culture medium (HAM's F-12 with Glutamax, Invitrogen, Cat#31765) with 10% FBS, 1×NEAA and 750 μg/ml of G418) and centrifuged at about 1000 rpm for 11⁄2 minutes. The resultant cell pellet was re-suspended in PBS at 3-5×106 cells/ml.
IonWorks electrophysiology and data analysis. The ability of Test solutions or Standards in serum to inhibit K+ currents in the CHO-Kv1.3 cells was investigated using the automated electrophysiology system, IonWorks Quattro. Re-suspended cells, the Assay Plate, a Population Patch Clamp (PPC) PatchPlate as well as appropriate intracellular (90 mMK-Gluconate, 20 mMKF, 2 mM NaCl, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.35) and extracellular (PBS, with Ca2+ and Mg2+) buffers were positioned on IonWorks Quattro. Electrophysiology recordings were made from the CHO-Kv1.3 cells using an amphotericin-based perforated patch-clamp method. Using the voltage-clamp circuitry of the IonWorks Quattro, cells were held at a membrane potential of −80 mV and voltage-activated K+ currents were evoked by stepping the membrane potential to +30 mV for 400 ms. K+ currents were evoked under control conditions i.e., in the absence of inhibitor at the beginning of the experiment and after 10-minute incubation in the presence of the Test Solution or Standard. The mean K+ current amplitude was measured between 430 and 440 ms and the data were exported to a Microsoft Excel spreadsheet. The amplitude of the K+ current in the presence of each concentration of the Test Solution or Standard was expressed as a percentage of the K+ current in control conditions in the same well.
Standard inhibition curves were generated for each standard in various levels of rat serum and expressed as current percent of control (POC) versus log of nM concentration. Percent of control (POC) is inversely related to inhibition, where 100 POC is no inhibition and 0 POC is 100% inhibition. Linear regression over a selected region of the curve was used to derive an equation to enable calculation of drug concentrations within Test solutions. Only current values within the linear portion of the Standard curve were used to calculate the concentration of drug in Test solutions. The corresponding Standard curve in a given level of serum, was always compared to the same level of serum of Test solution when calculating drug level. The Standard curves for ShK and 20K PEG-ShK[1-35] are shown in FIG. 58A and FIG. 58B, respectively, and each figure contains linear regression equations for each Standard at a given percentage of serum. For the 20K PEG-ShK[1-35] standard curve the linear portion of the Standard curve was from 20 POC to 70 POC and only current values derived from the Test solution which fell within this range were used to calculate drug concentration within the Test solution.
The pharmacokinetic profile of our novel 20K PEG ShK[1-35] molecule after IV injection is shown in FIG. 59. The terminal half-life (t1/2 b) of this molecule is estimated from this curve to be between 6 to 12 hours long. Beyond 48 hours, the level of drug falls outside the linear range of the Standard curve and is not calculated. The calculated 6 to 12 hour half-life of our novel 20K PEG-ShK[1-35] molecule was substantially longer than the approximately 0.33 hour (or 20 min) half-life of the native ShK molecule reported earlier by C. Beeton et al. [C. Beeton et al. (2001) Proc. Natl. Acad. Sci. 98, 13942-13947], and is a desirable feature of a therapeutic molecule. A comparison of the relative levels of Kv1.3 inhibitor after an equal molar IV injection of ShK versus 20K PEG-ShK[1-35] is shown in FIG. 60. As can be seen from this figure examining 5% serum Test solutions, the 20K PEG-ShK[1-35] molecule showed significant suppression of Kv1.3 current (<70 POC) for more than 24 hours, whereas the native ShK peptide only showed a significant level of inhibition of Kv1.3 current for the first hour and beyond 1 hour showed no significant blockade. These data again demonstrate a desirable feature of the 20K PEG ShK[1-35] molecule as a therapeutic for treatment of autoimmune disease.
Example 45
PEGylated Toxin Peptide Suppressed Severe Autoimmune Encephalomyelitis in Animal Model
The 20KPEG-ShK inhibitor of Kv1.3 shows improved efficacy in suppressing severe autoimmune encephalomyelitis in rats. Using an adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE) model of multiple sclerosis described earlier [C. Beeton et al. (2001) J. Immunol. 166, 936], we examined the activity in vivo of our novel 20KPEG-ShK molecule and compared its efficacy to that of the ShK toxin peptide alone. The study design is illustrated in FIG. 61. The results from this in vivo study are provided in FIG. 62 and FIG. 63. The 20KPEG-ShK molecule delivered subcutaneously (SC) at 10 μg/kg daily from day −1 to day 3 significantly reduced disease severity and increased survival, whereas animals treated with an equal molar dose (10 μg/kg) of the small ShK peptide developed severe disease and died.
The 35-amino acid toxin peptide ShK (Stichodactyla helianthus neurotoxin) was purchased from Bachem Bioscience Inc and confirmed by electrophysiology to potently block Kv1.3 (see Example 36 herein). The synthesis, PEGylation and purification of the 20KPEG ShK molecule was as described herein above. The encephalomyelogenic CD4+ rat T cell line, PAS, specific for myelin-basic protein (MBP) originated from Dr. Evelyne Beraud. The maintenance of these cells in vitro and their use in the AT-EAE model has been described earlier [C. Beeton et al. (2001) PNAS 98, 13942]. PAS T cells were maintained in vitro by alternating rounds of antigen stimulation or activation with MBP and irradiated thymocytes (2 days), and propagation with T cell growth factors (5 days). Activation of PAS T cells (3×105/ml) involved incubating the cells for 2 days with 10 μg/ml MBP and 15×106/ml syngeneic irradiated (3500 rad) thymocytes. On day 2 after in vitro activation, 10-15×106 viable PAS T cells were injected into 6-12 week old female Lewis rats (Charles River Laboratories) by tail IV. Daily subcutaneous injections of vehicle (2% Lewis rat serum in PBS), 20KPEG-ShK or ShK were given from days −1 to 3 (FIG. 61), where day −1 represent 1 day prior to injection of PAS T cells (day 0). In vehicle treated rats, acute EAE developed 4 to 5 days after injection of PAS T cells (FIG. 62). Serum was collected by retro-orbital bleeding at day 4 and by cardiac puncture at day 8 (end of the study) for analysis of levels of inhibitor. Rats were weighed on days −1, 4, 6, and 8. Animals were scored blinded once a day from the day of cell transfer (day 0) to day 3, and twice a day from day 4 to day 8. Clinical signs were evaluated as the total score of the degree of paresis of each limb and tail. Clinical scoring: 0=No signs, 0.5=distal limp tail, 1.0=limp tail, 2.0=mild paraparesis, ataxia, 3.0=moderate paraparesis, 3.5=one hind leg paralysis, 4.0=complete hind leg paralysis, 5.0=complete hind leg paralysis and incontinence, 5.5=tetraplegia, 6.0=moribund state or death. Rats reaching a score of 5.5 were euthanized.
Treatment of rats with the Kv1.3 blocker PEG-ShK prior to the onset of EAE caused a lag in the onset of disease, inhibited the progression of disease, and prevented death in a dose-dependent manner (FIG. 62). Onset of disease in rats that were treated with the vehicle alone, 10 μg/kg ShK or 1 μg/kg of PEG-ShK was observed on day 4, compared to day 4.5 in rats treated with 10 μg/kg PEG-ShK or 100 μg/kg PEG-ShK. In addition, rats treated with vehicle alone, 10 μg/kg ShK or 1 μg/kg of PEG-ShK all developed severe disease by the end of the study with an EAE score of 5.5 or above. In contrast, rats treated with 10 μg/kg PEG-ShK or 100 μg/kg PEG-ShK, reached a peak clinical severity score average of <2, and all but one rat survived to the end of the study. Furthermore, we found that rat body weight correlated with disease severity (FIG. 63). Rats treated with vehicle alone, 10 μg/kg ShK or 1 μg/kg of PEG-ShK all lost an average of 31 g, 30 g, and 30 g, respectively, while rats treated with 10 μg/kg PEG-ShK or 100 μg/kg PEG-ShK lost 18 g and 11 g, respectively. Rats in the latter two groups also appeared to be gaining weight by the end of the study, a sign of recovery. It should be noted that rats treated with 10 μg/kg ShK and 10 μg/kg PEG-ShK received molar equivalents of the ShK peptide. The significantly greater efficacy of the PEG-ShK molecule relative to unconjugated ShK, is likely due to the PEG-ShK molecule's greater stability and prolonged half-life in vivo (see, Example 44).
Example 46
Compositions Including Kv1.3 Antagonist Peptides Block Inflammation in Human Whole Blood
Ex vivo assay to examine impact of Kv1.3 inhibitors on secretion of IL-2 and IFN-g. Human whole blood was obtained from healthy, non-medicated donors in a heparin vacutainer. DMEM complete media was Iscoves DMEM (with L-glutamine and 25 mM Hepes buffer) containg 0.1% human albumin (Bayer #68471), 55 μM 2-mercaptoethanol (Gibco), and 1× Pen-Strep-Gln (PSG, Gibco, Cat#10378-016). Thapsigargin was obtained from Alomone Labs (Israel). A 10 mM stock solution of thapsigargin in 100% DMSO was diluted with DMEM complete media to a 40 μM, 4× solution to provide the 4× thapsigargin stimulus for calcium mobilization. The Kv1.3 inhibitor peptide ShK (Stichodacytla helianthus toxin, Cat# H2358) and the BKCal inhibitor peptide IbTx (Iberiotoxin, Cat# H9940) were purchased from Bachem Biosciences, whereas the Kv1.1 inhibitor peptide DTX-k (Dendrotoxin-K) was from Alomone Labs (Israel). The CHO-derived Fc-L10-ShK[2-35] peptibody inhibitor of Kv1.3 was obtained as described herein at Example 4 and Example 39. The calcineurin inhibitor cyclosporin A was obtained from the Amgen sample bank, but is also available commercially from a variety of vendors. Ten 3-fold serial dilutions of inhibitors were prepared in DMEM complete media at 4× final concentration and 50 μl of each were added to wells of a 96-well Falcon 3075 flat-bottom microtiter plate. Whereas columns 1-5 and 7-11 of the microtiter plate contained inhibitors (each row with a separate inhibitor dilution series), 50 μof DMEM complete media alone was added to the 8 wells in column 6 and 100 μof DMEM complete media alone was added to the 8 wells in column 12. To initiate the experiment, 100 μof whole blood was added to each well of the microtiter plate. The plate was then incubated at 37° C, 5% CO2 for one hour. After one hour, the plate was removed and 50 μof the 4× thapsigargin stimulus (40 μM) was added to all wells of the plate, except the 8 wells in column 12. The plates were placed back at 37° C, 5% CO2 for 48 hours. To determine the amount of IL-2 and IFN-g secreted in whole blood, 100 μl of the supernatant (conditioned media) from each well of the 96-well plate was transferred to a storage plate. For MSD electrochemilluminesence analysis of cytokine production, 20 μl of the supernatants (conditioned media) were added to MSD Multi-Spot Custom Coated plates (meso-scale.com). The working electrodes on these plates were coated with four Capture Antibodies (hIL-5, hIL-2, hIFNg and hIL-4) in advance. After addition of 20 μl of conditioned media to the MSD plate, 150 μof a cocktail of Detection Antibodies and P4 Buffer were added to each well. The 150 μcocktail contained 20 μl of four Detection Antibodies (hIL-5, hIFNg and hIL-4) at 1 μg/ml each and 130 μl of 2× P4 Buffer. The plates were covered and placed on a shaking platform overnight (in the dark). The next morning the plates were read on the MSD Sector Imager. Since the 8 wells in column 6 of each plate received only the thapsigargin stimulus and no inhibitor, the average MSD response here was used to calculate the “High” value for a plate. The calculate “Low” value for the plate was derived from the average MSD response from the 8 wells in column 12 which contained no thapsigargin stimulus and no inhibitor. Percent of control (POC) is a measure of the response relative to the unstimulated versus stimulated controls, where 100 POC is equivalent to the average response of thapsigargin stimulus alone or the “High” value. Therefore, 100 POC represents 0% inhibition of the response. In contrast, 0 POC represents 100% inhibition of the response and would be equivalent to the response where no stimulus is given or the “Low” value. To calculate percent of control (POC), the following formula is used: [(MSD response of well)−(“Low”)]/[(“High”)−(“Low”)]×100. The potency of the molecules in whole blood was calculated after curve fitting from the inhibition curve (IC) and IC50 was derived using standard curve fitting software. Although we describe here measurement of cytokine production using a high throughput MSD electrochemillumenescence assay, one of skill in the art can readily envision lower throughput ELISA assays are equally applicable for measuring cytokine production.
Ex vivo assay demonstrating Kv1.3 inhibitors block cell surface activation of CD40L & IL-2R. Human whole blood was obtained from healthy, non-medicated donors in a heparin vacutainer. DMEM complete media was Iscoves DMEM (with L-glutamine and 25 mM Hepes buffer) containing 0.1% human albumin (Bayer #68471), 55 μM 2-mercaptoethanol (Gibco), and 1× Pen-Strep-Gln (PSG, Gibco, Cat#10378-016). Thapsigargin was obtained from Alomone Labs (Israel). A 10 mM stock solution of thapsigargin in 100% DMSO was diluted with DMEM complete media to a 40 μM, 4× solution to provide the 4× thapsigargin stimulus for calcium mobilization. The Kv1.3 inhibitor peptide ShK (Stichodacytla helianthus toxin, Cat# H2358) and the BKCa1 inhibitor peptide IbTx (Iberiotoxin, Cat# H9940) were purchased from Bachem Biosciences, whereas the Kv1.1 inhibitor peptide DTX-k (Dendrotoxin-K) was from Alomone Labs (Israel). The CHO-derived Fc-L110-ShK[2-35] peptibody inhibitor of Kv1.3 was obtained as described in Example 4 and Example 39. The calcineurin inhibitor cyclosporin A was obtained from the Amgen sample bank, but is also available commercially from a variety of vendors. The ion channel inhibitors ShK, IbTx or DTK-k were diluted into DMEM complete media to 4× of the final concentration desired (final=50 or 100 nM). The calcineurin inhibitor cyclosporin A was also diluted into DMEM complete media to 4× final concentration (final=10 μM). To appropriate wells of a 96-well Falcon 3075 flat-bottom microtiter plate, 50 μl of either DMEM complete media or the 4× inhibitor solutions were added. Then, 100 μl of human whole blood was added and the plate was incubated for 1 hour at 37° C., 5% CO2. After one hour, the plate was removed and 50 μl of the 4× thapsigargin stimulus (40 μM) was added to all wells of the plate containing inhibitor. To some wells containing no inhibitor but just DMEM complete media, thapsigargin was also added whereas others wells with just DMEM complete media had an additional 50 μl of DMEM complete media added. The wells with no inhibitor and no thapsigargin stimulus represented the untreated “Low” control. The wells with no inhibitor but which received thapsigargin stimulus represented the control for maximum stimulation or “High” control. Plates were placed back at 37° C., 5% CO2 for 24 hours. After 24 hours, plates were removed and wells were process for FACS analysis. Cells were removed from the wells and washed in staining buffer (phosphate buffered saline containing 2% heat-inactivated fetal calf serum). Red blood cells were lysed using BD FACS Lysing Solution containing 1.5% formaldehyde (BD Biosciences) as directed by the manufacturer. Cells were distributed at a concentration of 1 million cells per 100 microliters of staining buffer per tube. Cells were first stained with 1 microliter of biotin-labeled anti-human CD4, washed, then stained simultaneously 1 microliter each of streptavidin-APC, FITC-labeled anti-human CD45RA, and phycoerythrin (PE)-labeled anti-human CD25 (IL-2Ra) or PE-labeled anti-human CD40L. Cells were washed with staining buffer between antibody addition steps. All antibodies were obtained from BD Biosciences (San Diego, Calif.). Twenty to fifty thousand live events were collected for each sample on a Becton Dickinson FACSCaliber (Mountain View, Calif.) flow cytometer and analyzed using FlowJo software (Tree Star Inc., San Carlos, Calif.). Dead cells, monocytes, and granulocytes were excluded from the analysis on the basis of forward and side scatter properties.
FIG. 64 and FIG. 67 demonstrate that Kv1.3 inhibitors ShK and Fc-L10-ShK[2-35] potently blocked IL-2 secretion in human whole blood, in addition to suppressing activation of the IL-2R on CD4+ T cells. The Kv1.3 inhibitor Fc-L10-ShK[2-35] was more than 200 times more potent in blocking IL-2 production in human whole blood than cyclosporine A (FIG. 64) as reflected by the IC50. FIG. 65 shows that Kv1.3 inhibitors also potently blocked secretion of IFNg in human whole blood, and FIG. 66 demonstrates that upregulation of CD40L on T cells was additionally blocked. The data in FIGS. 64-67 show that the Fc-L10-ShK[2-35] molecule was stable in whole blood at 37° C. for up to 48 hours, providing potent blockade of inflammatory responses. Toxin peptide therapeutic agents that target Kv1.3 and have prolonged half-life, are sought to provide sustained blockade of these responses in vivo over time. In contrast, despite the fact the Kv1.3 inhibitor peptide ShK also showed potent blockade in whole blood, the ShK peptide has a short ( ̃20 min) half-life in vivo (C. Beeton et al. (2001) Proc. Natl. Acad. Sci. 98, 13942), and cannot, therefore, provide prolonged blockade. Whole blood represents a physiologically relevant assay to predict the response in animals. The whole blood assays described here can also be used as a pharmacodynamic (PD) assay to measure target coverage and drug exposure following dosing of patients. These human whole blood data support the therapeutic usefulness of the compositions of the present invention for treatment of a variety immune disorders, such as multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, and lupus.
Example 47
PEGylated Peptibodies
By way of example, PEGylated peptibodies of the present invention were made by the following method. CHO-expressed FcL10-OsK1 (19.2 mg; MW 30,371 Da, 0.63 micromole) in 19.2 ml A5S, 20 mM NaBH3CN, pH 5, was treated with 38 mg PEG aldehyde (MW 20 kDa; 3×, Lot 104086). The sealed reaction mixture was stirred in a cold room overnight. The extent of the protein modification during the course of the reaction was monitored by SEC HPLC using a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech) eluted with a 0.05 M phosphate buffer, 0.5 M NaCl, pH 7.0 at 0.4 ml/min. The reaction mixture was dialyzed with A5S, pH 5 overnight. The dialyzed material was then loaded onto an SP HP FPLC column (16/10) in A5S pH 5 and eluted with a 1 M NaCl gradient. The collected fractions were analyzed by SEC HPLC, pooled into 3 pools, exchanged into DPBS, concentrated and submitted for functional testing (Table 34).
In another example, FcL10-ShK1 (16.5 mg; MW 30,065 Da, 0.55 micro mole) in 16.5 ml A5S, 20 mM NaBH3CN, pH 5 was treated with 44 mg PEG aldehyde (MW 20 kDa; 4×, Lot 104086). The sealed reaction mixture was stirred in a cold room overnight. The extent of the protein modification during the course of the reaction was monitored by SEC HPLC using a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech) eluted with a 0.05 M phosphate buffer, 0.5 M NaCl, pH 7.0 at 0.4 ml/min. The reaction mixture was dialyzed with A5S, pH 5 overnight. The dialyzed material was loaded onto an SP HP FPLC column (16/10) in A5S pH 5 and was eluted with a 1 M NaCl gradient. The collected fractions were analyzed by SEC HPLC, pooled into 3 pools, exchanged into DPBS, concentrated and submitted for functional testing (Table 34).
The data in Table 34 demonstrate potency of the PEGylated peptibody molecules as Kv1.3 inhibitors.
Table 34 shows determinations of IC50 made by whole cell patch clamp electrophysiology with HEK 293 as described in Example 36 herein above. The sustained IC50 was derived from the current 400 msecs after voltage ramp from −80 mV to +30 mV. Pool #2 samples comprised di-PEGylated peptibodies and Pool #3 samples comprised mono-PEGylated peptibodies.














PEGylated Peptibody
Pool #
IC50 Sustained (nM)








PEG-Fc-L10-SHK(2-35)
3
0.175(n = 4)



PEG-Fc-L10-SHK(2-35)
2
0.158(n = 4)



PEG-Fc-L10-OSK1
3
0.256(n = 3)



PEG-Fc-L10-OSK1
2
0.332(n = 3)









Example 48
PEGylated Toxin Peptides
Shk and Osk-1 PEGylation, purification and analysis. Synthetic Shk or OSK1-1 toxin peptides were selectively PEGylated by reductive alkylation at their N-termini. Conjugation was achieved, with either Shk or OSK-1 toxin peptides, at 2 mg/ml in 50 mM NaH2PO4, pH 4.5 reaction buffer containing 20 mM sodium cyanoborohydride and a 2 molar excess of 20 kDa monomethoxy-PEG-aldehyde (Nektar Therapeutics, Huntsville, Ala.). Conjugation reactions were stirred overnight at room temperature, and their progress was monitored by RP-HPLC. Completed reactions were quenched by 4-fold dilution with 20 mM NaOAc, pH 4, adjusted to pH 3.5 and chilled to 4° C. The PEG-peptides were then purified chromatographically at 4° C.; using SP Sepharose HP columns (GE Healthcare, Piscataway, N.J.) eluted with linear 0-1M NaCl gradients in 20 mM NaOAc, pH 4.0. (FIG. 68A and FIG. 68B) Eluted peak fractions were analyzed by SDS-PAGE and RP-HPLC and pooling determined by purity >97%. Principle contaminants observed were di-PEGylated toxin peptide and unmodified toxin peptide. Selected pools were concentrated to 2-5 mg/ml by centrifugal filtration against 3 kDa MWCO membranes and dialyzed into 10 mM NaOAc, pH 4 with 5% sorbitol. Dialyzed pools were then sterile filtered through 0.2 micron filters and purity determined to be >97% by SDS-PAGE and RP-HPLC (FIG. 69A and FIG. 69B). Reverse-phase HPLC was performed on an Agilent 1100 model HPLC running a Zorbax 5 μm 300SB-C8 4.6×50 mm column (Phenomenex) in 0.1% TFA/H20 at 1 ml/min and column temperature maintained at 40° C. Samples of PEG-peptide (20 μg) were injected and eluted in a linear 6-60% gradient while monitoring wavelengths 215 nm and 280 nm.
Electrophysiology performed by patch clamp on whole cells (see, Example 36) yielded a peak IC50 of 1.285 nM for PEG-OSK1 and 0.169 nM for PEG-ShK[1-35] (FIG. 74), in a concentration dependent block of the outward potassium current recorded from HEK293 cells stably expressing human Kv1.3 channel. The purified PEG-ShK[1-35] molecule, also referred to as “20K PEG-ShK[1-35]” and “PEG-ShK”, had a much longer half-life in vivo than the small ShK peptide (FIG. 59 and FIG. 60). PEG-ShK[1-35] suppressed severe autoimmune encephalomyelitis in rats (Example 45, FIGS. 61-63) and showed greater efficacy than the small native ShK peptide.
PEG conjugates of OSK1 peptide analogs were also generated and tested for activity in blocking T cell inflammation in the human whole blood assay (Example 46). As shown in Table 43, OSK1[Ala12], OSK1[Ala29], OSK1[Nal34] and OSK1[Ala29, 1Nal34] analogs containing an N-terminal 20K PEG conjugate, all provided potent blockade of the whole blood cytokine response in this assay. The 20K PEG-ShK was also highly active (Table 43).
Example 49
Fc Loop Insertions of ShK and OSK1 Toxin Peptides
As exemplified in FIG. 70, FIG. 71, FIG. 72, and FIG. 73, disulphide-constrained toxin peptides were inserted into the human IgG1 Fc-loop domain, defined as the sequence D137E138T139T140K141, according to the method published in Example 1 in Gegg et al., Modified Fc molecules, WO 2006/036834 A2 [PCT/US2005/034273]). Exemplary FcLoop-L2-OsK1-L2, FcLoop-L2-ShK-L2, FcLoop-L2-ShK-L4, and FcLoop-L4-OsK1-L2 were made having three linked domains. These were collected, purified and submitted for functional testing.
The peptide insertion for these examples was between Fc residues Leu139 and Thr140 and included 2-4 Gly residues as linkers flanking either side of the inserted peptide. However, alternate insertion sites for the human IgG1 Fc sequence, or different linkers, are also useful in the practice of the present invention, as is known in the art, e.g., as described in Example 13 of Gegg et al., Modified Fc molecules, WO 2006/036834 A2 [PCT/US2005/034273]).
Purified FcLoop OSK1 and FcLoop ShK1 molecules were tested in the whole blood assay of inflammation (see, Example 46). FcLoop-L2-OsK1-L2, FcLoop-L4-OsK1-L4 and FcLoop-L2-ShK-L2 toxin conjugates all provided potent blockade of the whole blood cytokine response in this assay, with IC50 values in the pM range (Table 43).
Example 50
Purification of ShK(2-35)-L-Fc from E. coli 
Frozen, E. coli paste (117 g), obtained as described in Example 16 herein above, was combined with 1200 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 7.5 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 17,700 g for 30 min at 4° C. The pellet was then resuspended in 1200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 17,700 g for 30 min at 4° C. The pellet was then resuspended in 1200 ml water using a tissue grinder and then centrifuged at 17,700 g for 30 min at 4° C. 6.4 g of the pellet (total 14.2 g) was then dissolved in 128 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. 120 ml of the pellet solution was then incubated with 0.67 ml of 1 M DTT for 60 min at 37° C. The reduced material was transferred to 5500 ml of the refolding buffer (3 M urea, 50 mM tris, 160 mM arginine HCl, 2.5 mM EDTA, 2.5 mM cystamine HCl, 4 mM cysteine, pH 9.5) at 2 ml/min, 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 3 days at 4° C.
The refold was diluted with 5.5 L of water, and the pH was adjusted to 8.0 using acetic acid, then the solution was filtered through a 0.22 μm cellulose acetate filter and loaded on to a 35 ml Amersham Q Sepharose-FF (2.6 cm I.D.) column at 10 ml/min in Q-Buffer A (20 mM Tris, pH 8.5) at 8° C. with an inline 35 ml Amersham Mab Select column (2.6 cm I.D.). After loading, the Q Sepharose column was removed from the circuit, and the remaining chromatography was carried out on the Mab Select column. The column was washed with several column volumes of Q-Buffer A, followed by elution using a step to 100 mM glycine pH 3.0. The fractions containing the desired product immediately loaded on to a 5.0 ml Amersham SP-Sepharose HP column at 5.0 ml/min in S-Buffer A (10 mM NaH2PO4, pH 7.0) at 8° C. The column was then washed with several column volumes of S-Buffer A followed by a linear gradient from 5% to 60% S-Buffer B (10 mM NaH2PO4, 1 M NaCl, pH 7.0) followed by a step to 100% S-Buffer B. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE. The fractions containing the bulk of the desired product were pooled and then applied to a 50 ml MEP Hypercel column (2.6 cm I.D.) at 10 ml/min in MEP Buffer A (20 mM tris, 200 mM NaCl, pH 8.0) at 8° C. Column was eluted with a linear gradient from 5% to 50% MEP Buffer B (50 mM sodium citrate pH 4.0) followed by a step to 100% MEP Buffer B. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the bulk of the desired product were pooled.
The MEP-pool was then concentrated to about 10 ml using a Pall Jumbo-Sep with a 10 kDa membrane. A spectral scan was then conducted on 50 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 76A). Then concentration of the material was determined to be 3.7 mg/ml using a calculated molecular mass of 30,253 and extinction coefficient of 36,900 M−1 cm−1. The purity of the material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 76B). The macromolecular state of the product was then determined using size exclusion chromatography on 70 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 76C). The product was then subject to mass spectral analysis by chromatographing approximately 4 μg of the sample through a RP-HPLC column (Vydac C4, 1×150 mm). Solvent A was 0.1% trifluoroacetic acid in water and solvent B was 0.1% trifluoroacetic acid in 90% acetonitrile, 10% water. The column was pre-equilibrated in 10% solvent B at a flow rate of 80 μl per min. The protein was eluted using a linear gradient of 10% to 90% solvent B over 30 min. Part of the effluent was directed into a LCQ ion trap mass spectrometer. The mass spectrum was deconvoluted using the Bioworks software provided by the mass spectrometer manufacturer. (FIG. 76D). The product was filtered through a 0.22 μm cellulose acetate filter and then stored at −80° C.
In Table 35, IC50 data for the purified E. coli-derived ShK[2-35]-L-Fc are compared to some other embodiments of the inventive composition of matter.







TABLE 35







E. coli-derived recombinant Fc-L-ShK[1-35], Fc-L-ShK[2-35], Fc-L-OSK1,


Shk[1-35]-L- Fc and ShK[2-35]-L-Fc peptibodies containing Fc at either the 


N-terminus or C-terminus show potent blockade of human Kv1.3. The


activity of the CHO-derived Fc-L10-ShK[1-35] R1Q mutant is also shown.


Whole cell patch clamp electrophysiology (WCVC), by methods described in 


Example 36, was performed using HEK293/Kvl.3 cells and the IC50 shown


is the average from dose-response curves from 3 or more cells.


IonWorks TM (IWQ) planar patch clamp electrophysiology by methods


described in Example 44 was on CHO/Kv1.3 cells and the average


IC50 is shown. The inventive molecules were obtained by methods as


described in the indicated Example: E. coli-derived Fc-L-ShK[1-35]


(Example 3 and Example 38), E. coli-derived Fc-L-ShK[2-35] (Example 4 


and Example 39), E. coli Fc-L-OSK1 (Example 10 and Example 40),


ShK[1-35]-L-35 Fc (Example 15 and Example 51), and ShK[2-35]-L-Fc


(Example 16 and this Example 50). CHO-derived Fc-L10-ShK[1-35] R1Q


molecule was generated using methods similar to those described for


CHO-derived Fc-L10-ShK[1-35].










Kv1.3 IC50
Kv1.3 IC50


Molecule
by WCVC (nM)
by IWQ (nM)





E. coli-derived Fc-L-ShK[1-35]
1.4



E. coli-derived Fc-L-ShK[2-35]
1.3
2.8


E. coli-derived Fc-L-OSK1
3.2



E. coli-derived Shk[1-35]-L-Fc

2.4


E. coli-derived ShK[2-35]-L-Fc

4.9


CHO-derived Fc-L10-ShK[1-35]

2.2


R1Q









Example 51
Purification of Met-ShK(1-35)-Fc from E. coli 
Frozen, E. coli paste (65 g), obtained as described in Example 15 herein above was combined with 660 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 7.5 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 17,700 g for 30 min at 4° C. The pellet was then resuspended in 660 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 17,700 g for 30 min at 4° C. The pellet was then resuspended in 660 ml water using a tissue grinder and then centrifuged at 17,700 g for 30 min at 4° C. 13 g of the pellet was then dissolved in 130 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. 10 ml of the pellet solution was then incubated with 0.1 ml of 1 M DTT for 60 min at 37° C. The reduced material was transferred to 1000 ml of the refolding buffer (2 M urea, 50 mM tris, 160 mM arginine HCl, 2.5 mM EDTA, 1.2 mM cystamine HCl, 4 mM cysteine, pH 8.5) at 2 ml/min, 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 3 days at 4° C.
The refold was diluted with 1 L of water, and filtered through a 0.22 μm cellulose acetate filter then loaded on to a 35 ml Amersham Q Sepharose-FF (2.6 cm I.D.) column at 10 ml/min in Q-Buffer A (20 mM Tris, pH 8.5) at 8° C. with an inline 35 ml Amersham Mab Select column (2.6 cm I.D.). After loading, the Q Sepharose column was removed from the circuit, and the remaining chromatography was carried out on the Mab Select column. The column was washed with several column volumes of Q-Buffer A, followed by elution using a step to 100 mM glycine pH 3.0. The fractions containing the desired product immediately loaded on to a 5.0 ml Amersham SP-Sepharose HP column at 5.0 ml/min in S-Buffer A (20 mM NaH2PO4, pH 7.0) at 8° C. The column was then washed with several column volumes of S-Buffer A followed by a linear gradient from 5% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.0) followed by a step to 100% S-Buffer B. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE. The fractions containing the bulk of the desired product were pooled.
The S-pool was then concentrated to about 10 ml using a Pall Jumbo-Sep with a 10 kDa membrane. A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 77A). Then concentration of the material was determined to be 3.1 mg/ml using a calculated molecular mass of 30,409 and extinction coefficient of 36,900 M−1 cm−1. The purity of the material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 77B). The macromolecular state of the product was then determined using size exclusion chromatography on 93 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 77C). The product was then subject to mass spectral analysis by MALDI mass spectrometry.
An aliquot of the sample was spotted with the MALDI matrix sinapinic acid on sample plate. A Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse) was used to collect spectra. The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from  ̃200 laser shots (FIG. 77D). External mass calibration was accomplished using purified proteins of known molecular masses.
The IC50 for blockade of human Kv1.3 by purified E. coli-derived Met-ShK(1-35)-Fc, also referred to as “ShK[1-35]-L-Fc”, is shown in Table 35 herein above.
Example 52
Bacterial Expression of OsK1-L-Fc Inhibitor of Kv1.3
The methods to clone and express the peptibody in bacteria were as described in Example 3. The vector used was pAMG21amgR-pep-Fc and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of OsK1-L-Fc. Oligos used to form duplex are shown below:







//SEQ ID NO: 1347







GGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCAGTGCCTGGAAC


 


CGTGCAAAAAAGCTGGTATGCGT;


 







//SEQ ID NO: 1348







TTCGGTAAATGCATGAACGGTAAATGCCACTGCACCCCGAAATCTGGTGG


 


TGGTGGTTCT;


 







//SEQ ID NO: 1349







CACCAGAACCACCACCACCACCAGATTTCGGGGTGCAGTGGCATTTACCG


 


TTCATGCATTTACCGAAACGCAT;


 







//SEQ ID NO: 1310







ACCAGCTTTTTTGCACGGTTCCAGGCACTGACGGGAGATTTTGCATTTAA


 


CGTTGATGATAAC;






The oligos shown above were used to form the duplex shown below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 53
Bacterial Expression of Gly-ShK(1-35)-L-Fc Inhibitor of Kv1.3
The methods to clone and express the peptibody in bacteria were as described in Example 3. The vector used was pAMG21amgR-pep-Fc and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Gly-ShK(1-35)-L-Fc. Oligos used to form duplex are shown below:







//SEQ ID NO: 1313







GGGTCGTTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTC


 


AATGTAAACATTCTATGAAATATCGTCTTTCTT;


 







//SEQ ID NO: 1314







TTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGGTGGTTCT;


 







//SEQ ID NO: 1353







CACCAGAACCACCACCACCAGAACAAGTACCACAAGTTTTACGACAAAAA


 


GAAAGACGATATTTCATAGAATGTTTACATTGA;


 







//SEQ ID NO: 1354







AAAGCAGTACAACGAGATTTTGGAATAGTATCAATACAAGAACG






The oligos shown above were used to form the duplex shown below:




Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.
Example 54
Bacterial Expression of CH2-OSK1 Inhibitor of Kv1.3
The methods to clone and express the fusion of a CH2 domain of an Fc with OSK1 in bacteria were generally as described in Example 3. The vector used was pAMG21.G2.H6.G3.CH2.(G4S)2.OSK. Briefly, the pAMG21 vector was modified to remove the multi-cloning site's BamHI. This allowed the BamHI in front of the OSK as a site to swap out different sequences for fusion with the OSK. The sequence upstream of the OSK1 coding sequence was ligated between the NdeI and BamHI sites.
The sequence of the entire vector, including the insert was the following:







//SEQ ID NO: 4914







gtcgtcaacgaccccccattcaagaacagcaagcagcattgagaacttt


 


ggaatccagtccctcttccacctgctgaccggatcagcagtccccggaa


 


catcgtagctgacgccttcgcgttgctcagttgtccaaccccggaaacg


 


ggaaaaagcaagttttccccgctcccggcgtttcaataactgaaaacca


 


tactatttcacagtttaaatcacattaaacgacagtaatccccgttgat


 


ttgtgcgccaacacagatcttcgtcacaattctcaagtcgctgatttca


 


aaaaactgtagtatcctctgcgaaacgatccctgtttgagtattgagga


 


ggcgagatgtcgcagacagaaaatgcagtgacttcctcattgagtcaaa


 


agcggtttgtgcgcagaggtaagcctatgactgactctgagaaacaaat


 


ggccgttgttgcaagaaaacgtcttacacacaaagagataaaagttttt


 


gtcaaaaatcctctgaaggatctcatggttgagtactgcgagagagagg


 


ggataacacaggctcagttcgttgagaaaatcatcaaagatgaactgca


 


aagactggatatactaaagtaaagactttactttgtggcgtagcatgct


 


agattactgatcgtttaaggaattttgtggctggccacgccgtaaggtg


 


gcaaggaactggttctgatgtggatttacaggagccagaaaagcaaaaa


 


ccccgataatcttcttcaacttttgcgagtacgaaaagattaccggggc


 


ccacttaaaccgtatagccaacaattcagctatgcggggagtatagtta


 


tatgcccggaaaagttcaagacttctttctgtgctcgctccttctgcgc


 


attgtaagtgcaggatggtgtgactgatcttcaccaaacgtattaccgc


 


caggtaaagaacccgaatccggtgtttacaccccgtgaaggtgcaggaa


 


cgctgaagttctgcgaaaaactgatggaaaaggcggtgggcttcacttc


 


ccgttttgatttcgccattcatgtggcgcacgcccgttcgcgtgatctg


 


cgtcgccgtatgccaccagtgctgcgtcgtcgggctattgatgcgctct


 


tgcaggggctgtgtttccactatgacccgctggccaaccgcgtccagtg


 


ctccatcaccacgctggccattgagtgcggactggcgacggagtctgct


 


gccggaaaactctccatcacccgtgccacccgtgccctgacgttcctgt


 


cagagctgggactgattacctaccagacggaatatgacccgcttatcgg


 


gtgctacattccgaccgatatcacgttcacatctgcactgtttgctgcc


 


ctcgatgtatcagaggaggcagtggccgccgcgcgccgcagccgtgtgg


 


tatgggaaaacaaacaacgcaaaaagcaggggctggataccctgggcat


 


ggatgaactgatagcgaaagcctggcgttttgttcgtgagcgttttcgc


 


agttatcagacagagcttaagtcccgtggaataaagcgtgcccgtgcgc


 


gtcgtgatgcggacagggaacgtcaggatattgtcaccctggtgaaacg


 


gcagctgacgcgcgaaatcgcggaagggcgcttcactgccaatcgtgag


 


gcggtaaaacgcgaagttgagcgtcgtgtgaaggagcgcatgattctgt


 


cacgtaaccgtaattacagccggctggccacagcttccccctgaaagtg


 


acctcctctgaataatccggcctgcgccggaggcttccgcacgtctgaa


 


gcccgacagcgcacaaaaaatcagcaccacatacaaaaaacaacctcat


 


catccagcttctggtgcatccggccccccctgttttcgatacaaaacac


 


gcctcacagacggggaattttgcttatccacattaaactgcaagggact


 


tccccataaggttacaaccgttcatgtcataaagcgccatccgccagcg


 


ttacagggtgcaatgtatcttttaaacacctgtttatatctcctttaaa


 


ctacttaattacattcatttaaaaagaaaacctattcactgcctgtcct


 


tggacagacagatatgcacctcccaccgcaagcggcgggcccctaccgg


 


agccgctttagttacaacactcagacacaaccaccagaaaaaccccggt


 


ccagcgcagaactgaaaccacaaagcccctccctcataactgaaaagcg


 


gccccgccccggtccgaagggccggaacagagtcgcttttaattatgaa


 


tgttgtaactacttcatcatcgctgtcagtcttctcgctggaagttctc


 


agtacacgctcgtaagcggccctgacggcccgctaacgcggagatacgc


 


cccgacttcgggtaaaccctcgtcgggaccactccgaccgcgcacagaa


 


gctctctcatggctgaaagcgggtatggtctggcagggctggggatggg


 


taaggtgaaatctatcaatcagtaccggcttacgccgggcttcggcggt


 


tttactcctgtttcatatatgaaacaacaggtcaccgccttccatgccg


 


ctgatgcggcatatcctggtaacgatatctgaattgttatacatgtgta


 


tatacgtggtaatgacaaaaataggacaagttaaaaatttacaggcgat


 


gcaatgattcaaacacgtaatcaatatcgggggtgggcgaagaactcca


 


gcatgagatccccgcgctggaggatcatccagccggcgtcccggaaaac


 


gattccgaagcccaacctttcatagaaggcggcggtggaatcgaaatct


 


cgtgatggcaggttgggcgtcgcttggtcggtcatttcgaaccccagag


 


tcccgctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgc


 


gaatcgggagcggcgataccgtaaagcacgaggaagcggtcagcccatt


 


cgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctg


 


atagcggtccgccacacccagccggccacagtcgatgaatccagaaaag


 


cggccattttccaccatgatattcggcaagcaggcatcgccatgagtca


 


cgacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacag


 


ttcggctggcgcgagcccctgatgctcttcgtccagatcatcctgatcg


 


acaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcg


 


cttggtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccg


 


cattgcatcagccatgatggatactttctcggcaggagcaaggtgagat


 


gacaggagatcctgccccggcacttcgcccaatagcagccagtcccttc


 


ccgcttcagtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgt


 


ggccagccacgatagccgcgctgcctcgtcctgcaattcattcaggaca


 


ccggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgaca


 


gccggaacacggcggcatcagagcagccgattgtctgttgtgcccagtc


 


atagccgaatagcctctccacccaagcggccggagaacctgcgtgcaat


 


ccatcttgttcaatcatgcgaaacgatcctcatcctgtctcttgatctg


 


atcttgatcccctgcgccatcagatccttggcggcaagaaagccatcca


 


gtttactttgcagggcttcccaaccttaccagagggcgccccagctggc


 


aattccggttcgcttgctgtccataaaaccgcccagtctagctatcgcc


 


atgtaagcccactgcaagctacctgctttctctttgcgcttgcgttttc


 


ccttgtccagatagcccagtagctgacattcatccggggtcagcaccgt


 


ttctgcggactggctttctacgtgttccgcttcctttagcagcccttgc


 


gccctgagtgcttgcggcagcgtgaagctacatatatgtgatccgggca


 


aatcgctgaatattccttttgtctccgaccatcaggcacctgagtcgct


 


gtctttttcgtgacattcagttcgctgcgctcacggctctggcagtgaa


 


tgggggtaaatggcactacaggcgccttttatggattcatgcaaggaaa


 


ctacccataatacaagaaaagcccgtcacgggcttctcagggcgtttta


 


tggcgggtctgctatgtggtgctatctgactttttgctgttcagcagtt


 


cctgccctctgattttccagtctgaccacttcggattatcccgtgacag


 


gtcattcagactggctaatgcacccagtaaggcagcggtatcatcaaca


 


ggcttacccgtcttactgtcgaagacgtgcgtaacgtatgcatggtctc


 


cccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctc


 


agtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgc


 


tctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaag


 


caacggcccggagggtggcgggcaggacgcccgccataaactgccaggc


 


atcaaattaagcagaaggccatcctgacggatggcctttttgcgtttct


 


acaaactcttttgtttatttttctaaatacattcaaatatggacgtcgt


 


acttaacttttaaagtatgggcaatcaattgctcctgttaaaattgctt


 


tagaaatactttggcagcggtttgttgtattgagtttcatttgcgcatt


 


ggttaaatggaaagtgaccgtgcgcttactacagcctaatatttttgaa


 


atatcccaagagctttttccttcgcatgcccacgctaaacattcttttt


 


ctcttttggttaaatcgttgtttgatttattatttgctatatttatttt


 


tcgataattatcaactagagaaggaacaattaatggtatgttcatacac


 


gcatgtaaaaataaactatctatatagttgtctttctctgaatgtgcaa


 


aactaagcattccgaagccattattagcagtatgaatagggaaactaaa


 


cccagtgataagacctgatgatttcgcttctttaattacatttggagat


 


tttttatttacagcattgttttcaaatatattccaattaatcggtgaat


 


gattggagttagaataatctactataggatcatattttattaaattagc


 


gtcatcataatattgcctccattttttagggtaattatccagaattgaa


 


atatcagatttaaccatagaatgaggataaatgatcgcgagtaaataat


 


attcacaatgtaccattttagtcatatcagataagcattgattaatatc


 


attattgcttctacaggctttaattttattaattattctgtaagtgtcg


 


tcggcatttatgtctttcatacccatctctttatccttacctattgttt


 


gtcgcaagttttgcgtgttatatatcattaaaacggtaatagattgaca


 


tttgattctaataaattggatttttgtcacactattatatcgcttgaaa


 


tacaattgtttaacataagtacctgtaggatcgtacaggtttacgcaag


 


aaaatggtttgttatagtcgattaatcgatttgattctagatttgtttt


 


aactaattaaaggaggaataacatatgggcggccatcatcatcatcatc


 


atggcgggggaccgtcagttttcctcttccccccaaaacccaaggacac


 


cctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtg


 


agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtgg


 


aggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcac


 


gtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaat


 


ggcaaggagtacaagtgcaaggtctccaacaaagccctcccagccccca


 


tcgagaaaaccatctccggcggcggcggcagcggcggcggcggatccgg


 


tgttatcatcaacgttaaatgcaaaatctcccgtcagtgcctggaaccg


 


tgcaaaaaagctggtatgcgtttcggtaaatgcatgaacggtaaatgcc


 


actgcaccccgaaataatgaattcgagctcactagtgtcgacctgcagg


 


gtaccatggaagcttactcgaagatccgcggaaagaagaagaagaagaa


 


gaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataa


 


ctagcataaccccttggggcctctaaacgggtcttgaggggttttttgc


 


tgaaaggaggaaccgctcttcacgctcttcacgcggataaataagtaac


 


gatccggtccagtaatgacctcagaactccatctggatttgttcagaac


 


gctcggttgccgccgggcgttttttattggtgagaatcgcagcaacttg


 


tcgcgccaatcgagccatgtc.






The insert DNA sequence was the following:







//SEQ ID NO: 4915







atgggcggccatcatcatcatcatcatggcgggggaccgtcagttttcct


 


cttccccccaaaacccaaggacaccctcatgatctcccggacccctgagg


 


tcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc


 


aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcg


 


ggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcc


 


tgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaac


 


aaagccctcccagcccccatcgagaaaaccatctccggcggcggcggcag


 


cggcggcggcggatccggtgttatcatcaacgttaaatgcaaaatctccc


 


gtcagtgcctggaaccgtgcaaaaaagctggtatgcgtttcggtaaatgc


 


atgaacggtaaatgccactgcaccccgaaa.






The amino acid sequence of the CH2-OSK1 fusion protein product was the following:







//SEQ ID NO: 4917







MGGHHHHHHGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF


 


NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN


 


KALPAPIEKTISGGGGSGGGGSGVIINVKVKISRQCLEPCKKAGMRFGKC


 


MNGKCHCTPK.






SEQ ID NO:4917 includes the OSK1 sequence







(SEQ ID NO: 25)









GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK.






Purification and refolding of CH2-OSK1 expressed in bacteria. Frozen, E. coli paste (13.8 g) was combined with 180 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 17,700 g for 50 min at 4° C. The pellet was then resuspended in 90 ml 1% deoxycholate using a tissue grinder and then centrifuged at 15,300 g for 40 min at 4° C. The pellet was then resuspended in 90 ml water using a tissue grinder and then centrifuged at 15,300 g for 40 min at 4° C. The pellet (3.2 g) was then dissolved in 64 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The suspension was then incubated at room temperature (about 23° C.) for 30 min with gentle agitation followed by centrifugation at 15,300 g for 30 min at 4° C. The supernatant (22 ml) was then reduced by adding 220 μl 1 M dithiothreitol and incubating at 37° C. for 30 minutes. The reduced suspension (20 ml) was transferred to 2000 ml of the refolding buffer (1 M urea, 50 mM ethanolamine, 160 mM arginine HCl, 0.02% NaN3, 1.2 mM cystamine HCl, 4 mM cysteine, pH 9.8) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for approximately 2.5 days at 4° C.
Ten milliliters of 500 mM imidazole was added to the refolding solution and the pH was adjusted to pH to 8.0 with 5 M acetic acid. The refold was then filtered through a 0.45 μm cellulose acetate filter with two pre-filters. This material was then loaded on to a 50 ml Qiagen Ni-NTA Superflow column (2.6 cm ID) in Ni-Buffer A (50 mM NaH2PO4, 300 mM NaCl, pH 7.5) at 15 ml/min 13° C. The column was then washed with 10 column volumes of Ni-Buffer A followed by 8% Ni-Buffer B (250 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 7.5) at 25 ml/min. The column was then eluted with 60% Ni-Buffer B followed by 100% Ni-Buffer B at 10 ml/min. The peak fractions were collected and dialyzed against S-Buffer A (10 mM NaH2PO4, pH 7.1)
The dialyzed sample was then loaded on to a 5 ml Amersham SP-HP HiTrap column at 5 ml/min in S-Buffer A at 13° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (10 mM NaH2PO4, 1 M NaCl, pH 7.1) followed by a step to 100% S-Buffer B at 1.5 ml/min 13° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pool was then concentrated to about 1.6 ml using a Pall Macrosep with a 10 kDa membrane at 4° C. The concentrated sample was then filtered through a 0.22 μm cellulose acetate centrifugal filter.
A spectral scan was then conducted on 10 μl of the combined pool diluted in 150 μl water using a Hewlett Packard 8453 spectrophotometer (FIG. 78). The concentration of the filtered material was determined to be 3.35 mg/ml using a calculated molecular mass of 17,373 g/mol and extinction coefficient of 17,460 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 79). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 67-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 80). The product was then subject to mass spectral analysis by chromatographing approximately 4 μg of the sample through a RP-HPLC column (Vydac C4, 1×150 mm). Solvent A was 0.1% trifluoroacetic acid in water and solvent B was 0.1% trifluoroacetic acid in 90% acetonitrile, 10% water. The column was pre-equilibrated in 10% solvent B at a flow rate of 80 μl per min. The protein was eluted using a linear gradient of 10% to 90% solvent B over 30 min. Part of the effluent was directed into a LCQ ion trap mass spectrometer. The mass spectrum was deconvoluted using the Bioworks software provided by the mass spectrometer manufacturer. (FIG. 81). The product was then stored at −80° C.
PEGylation of CH2-OSK1. The CH2-OSK1 fusion protein was diluted to 2 mg/ml in 50 mM sodium acetate, 10 mM sodium cyanoborohydride, pH 4.8 with a 4-fold molar excess of 20 kD methoxy-PEG-aldehyde (Nektar Therapeutics, Huntsville, Ala.). The reaction was allowed to proceed overnight ( ̃18 hrs) at 4° C. Upon completion, reaction was quenched with 4 volumes of 10 mM sodium acetate, 50 mM NaCl, pH 5, then loaded at 0.7 mg protein/ml resin to an SP Sepharose HP column (GE Healthcare, Piscataway, N.J.) equilibrated in 10 mM sodium acetate, 50 mM NaCl, pH 5. The mono-PEGylated CH2-Osk fusion was eluted with a linear 50 mM-1 M NaCl gradient (FIG. 82). Peak fractions were evaluated by SDS-PAGE and the mono-PEG-CH2-OSK1 fractions pooled, concentrated and dialyzed into Dulbecco's Phosphate Buffered Saline. The final product was analyzed by SDS-PAGE (FIG. 83).
As shown in Table 43, the purified CH2-OSK1 (“L2-6H-L3-CH2-L10-OsK1(1-38)”) and PEGylated CH2-OSK1 (“20 k PEG-L2-6H-L3-CH2-L10-OSK1(1-38)”) molecules were active in blocking inflammation in the human whole blood assay (See, Example 46).
Example 55
Bioactivity of OSK1 Peptide Analogs
The activity of OSK1 peptide analogs in blocking human Kv1.3 versus human Kv1.1 current is shown in FIGS. 84 through 86 and Tables 37-41. Three electrophysiology techniques were used (See, e.g., Example 36 and Example 44). Whole cell patch clamp (FIGS. 84 & 85 and Table 41) represents a low throughput technique which is well established in the field and has been available for many years. We also used two new planar patch clamp techniques, PatchXpress and IonWorks Quattro, with improved throughput which facilitate assessment of potency and selectivity of novel OSK1 analogs described in this application. The PatchXpress technique is of moderate throughput and the novel OSK1[Ala-12] analog (SEQ ID No:1410) had similar Kv1.3 potency and selectivity over Kv1.1 to that observed by whole cell patch clamp (FIG. 84 and Table 41). IonWorks Quattro represents a 384-well planar patch clamp electrophysiology system of high throughput. Using this IonWorks system, the novel OSK1[Ala-29] analog (SEQ ID No:1424) showed potent inhibition of the Kv1.3 current and improved selectivity over Kv1.1 (FIG. 86). The OSK1[Ala-29] analog (SEQ ID No:1424) showed similar Kv1.3 activity and selectivity over Kv1.1 by whole cell patch clamp electrophysiology (FIG. 85) to that observed by IonWorks (FIG. 86). The Kv1.3 and Kv1.1 activities of Alanine, Arginine, Glutamic acid and 1-Naphthylalanine analogs of OSK1 were determined by IonWorks electrophysiology and is reported in Tables 37-40. OSK1 peptide analogs identified by IonWorks to have good potency or Kv1.3 selectivity, were tested further in whole cell patch clamp studies (see, Table 41). The Kv1.3 IC50 of the His34Ala analog of OSK1 (SEQ ID No:1428) was 797 fold lower than its IC50 against Kv1.1 (Table 41), demonstrating that this analog is a highly selective Kv1.3 inhibitor. In this same assay, native OSK1 (SEQ ID NO: 25) showed only slight Kv1.3 selectivity, with the Kv1.1 IC50 being only 5 fold higher than Kv1.3.
The novel OSK1 peptide analogs described in this application which inhibit Kv1.3 are useful in the treatment of autoimmune disease and inflammation. Kv1.3 is expressed on T cells and Kv1.3 inhibitors suppress inflammation by these cells. As one measure of inflammation mediated by T cells, we examined the impact of OSK1 analogs on IL-2 and IFN-g production in human whole blood following addition of a pro-inflammatory stimulus (Tables 36-40 and Table 42). “WB/IL-2” in these tables refers to the assay measuring IL-2 response of whole blood (see, Example 46), whereas “WB/IFNg” refers to the assay measuring IFNg response of whole blood (see, Example 46). The IC50 values listed in Tables 36-40 and 42, represent the average IC50 value determined from experiments done with two or more blood donors. The whole blood assay (see Example 46) allows for a combined measurement of the potency of the analogs in blocking inflammation and Kv1.3, as well as an assessment of the stability of the molecules in a complex biological fluid. Using this assay, several OSK1 analogs were examined and found to potently suppress inflammation (FIGS. 90C & 90D, Tables 36-40). Some of these analogs showed reduced activity in this whole blood assay, which may indicate that these residues play an important role in binding Kv1.3. Relative to the immunosuppressive agent cyclosporin A, Kv1.3 peptide inhibitors ShK-Ala22, OSK1-Ala29, and OSK1-Ala12 were several orders of magnitude more potent in blocking the cytokine response in human whole blood (FIG. 90).
The solution NMR structure of OSK1 has been solved and is provided as pdb accession number “1SCO” in Entrez's Molecular Modeling Database (MMDB) [J. Chen et al. (2003) Nucleic Acids Res. 31, 474-7]. FIG. 89 shows space filling (FIG. 89A, 89B, 89D) and worm (FIG. 89C) Cn3D rendering of the OSK1 structure. Light colored OSK1 amino acid residues Phe25, Gly26, Lys27, Met29 and Asn30 are shown in FIG. 89B. Some analogs of these residues were found to significantly reduce Kv1.3 activity (Tables 37-40), implying that these residues may make important contacts with the Kv1.3 channel. The molecular structure shown in FIG. 89A indicates these amino acids reside on a common surface of the OSK1 three-dimensional (3D) structure. FIG. 89D shows OSK1 residues (light shading) Ser11, Met29 and His34. These residues when converted to some amino acid analogs, provide improved Kv1.3 selectivity over Kv1.1 (Table 41). Although about 23 amino acids are between residues Ser11 and His34 in the contiguous polypeptide chain, the structure shown in FIG. 89D illustrates that in the 3D structure of the folded molecule these residues are relatively close to one another. Upon comparing FIGS. 89D and 89B, one can see that residues His34 and Ser11 (FIG. 89D) are on the left and right side, respectively, and adjacent to the major Kv1.3 contact surface displayed in FIG. 89B. It is envisioned that molecular modeling can be used to identify OSK1 analogs with improved Kv1.3 activity and selectivity, upon considering the Kv1.3 and Kv1.1 bioactivity information provided in Tables 37 through 42 and the solution NMR structure of OSK1 described above. FIG. 89C shows a worm rendering of the OSK1 structure with secondary structure elements (beta strands and alpha helices) depicted. The primary amino acid sequence of OSK1 is provided in FIG. 89E and amino acid residues comprising the beta strands & alpha helix are underlined. Wiggly lines in FIG. 89C indicate amino acid residues between or beyond these secondary structure elements, whereas straight lines depict the three disulfide bridges in OSK1. The first beta strand (β1) shown in FIGS. 89C and 89E contains no disulfide bridges to link it covalently to other secondary structure elements of the OSK1 molecule, unlike beta strand 3 (β3) that has two disulfide bridges with the alpha helix (α1). As shown in Table 42, OSK1 analogs without beta strand 1 (labeled “des 1-7”) still retain activity in blocking inflammation (see SEQ ID No: 4989 of Table 42) suggesting that this region of OSK1 is not essential for the molecules Kv1.3 bioactivity.
OSK1 analogs containing multiple amino acid changes were generated and their activity in the human whole blood assay of inflammation is provided in Table 42. Several analogs retain high potency in this assay despite as many as 12 amino acid changes. Based on the improved Kv1.3 selectivity of analogs with single amino acid changes, anologs with multiple amino acid changes may result in additional improvements in selectivity. It is also envisioned that analogs with multiple amino acid changes may have improved activity or stability in vivo, alone or in the context of a peptide conjugate to a half-life prolonging moiety.
Kv1.3 peptide toxins conjugated to half-life prolonging moieties are provided within this application. The bioactivity of several toxin conjugates is described in Table 43. OSK1 analogs with a N-terminal half-life prolonging 20K PEG moiety (see Example 48) were found to provide potent suppression of the whole blood IL-2 (“WB/IL-2”) and IFNg (“WB/IFNg”) response (Table 43). The 20K PEG-ShK conjugate, shown earlier to have prolonged half-life in vivo (see Examples 44 and 48), was also highly active in this whole blood assay. The FcLoop-OSK1 conjugates (see Example 49) were highly active in blocking inflammation (Table 43), and the CH2-OSK1 or PEG-CH2-OSK1 conjugates (see Example 54) provided modest blockade of the whole blood cytokine response (Table 43). The IL-2 and IFNg cytokine response measured in this whole blood assay results from T cell activation. Since this cytokine response is Kv1.3 dependent and potently blocked by the Kv1.3 peptide and peptide-conjugate inhibitors described herein, these whole blood studies illustrate the therapeutic utility of these molecules in treatment of immune disorders.







TABLE 36







Activity of OSK1 analogs in blocking thapsigargin-induced IL-2 and IFNg


production in 50% human whole blood as described in Example 46.









Thapsigargin Induced IL-2 & IFNg in



Human Whole Blood













Average
Std Dev
Average
Std Dev
Analog IC50 Divided



IC50 (nM)
IC50 (nM)
IC50 (nM)
IC50 (nM)
by Ala-1 IC50













OSK1 Analog
IL-2
IL-2
IFNg
IFNg
IL2
IFNg
















Ala-1 (SEQ ID No: 1400)
0.1220
0.0791
0.1194
0.0802
1.00
1.00


Ala-2 (SEQ ID No: 1401)
0.0884
0.0733
0.1035
0.0776
0.72
0.87


Ala-3 (SEQ ID No: 1402)
0.0883
0.0558
0.0992
0.1007
0.72
0.83


Ala-4 (SEQ ID No: 1403)
0.1109
0.1098
0.0873
0.0993
0.91
0.73


Ala-5 (SEQ ID No: 1404)
0.0679
0.0566
0.0670
0.0446
0.56
0.56


Ala-6 (SEQ ID No: 1405)
0.0733
0.0477
0.0805
0.0696
0.60
0.67


Ala-7 (SEQ ID No: 1406)
0.0675
0.0383
0.0591
0.0260
0.55
0.49


Ala-9 (SEQ ID No: 1407)
0.0796
0.0761
0.0711
0.0627
0.65
0.60


Ala-10 (SEQ ID No: 1408)
0.0500
0.0425
0.0296
0.0084
0.41
0.25


Ala-12 (SEQ ID No: 1410)
0.1235
0.0823
0.1551
0.0666
1.01
1.30


Ala-13 (SEQ ID No: 1411)
0.1481
0.0040
0.1328
0.0153
1.21
1.11


Ala-15 (SEQ ID No: 1412)
0.1075

0.1075

0.88
0.90


Ala-16 (SEQ ID No: 1413)
0.1009

0.1009

0.83
0.84


Ala-17 (SEQ ID No: 1414)
0.1730

0.1730

1.42
1.45


Ala-19 (SEQ ID No: 1415)
0.1625

0.1625

1.33
1.36


Ala-20 (SEQ ID No: 1416)
0.3790

0.3790

3.11
3.17


Ala-22 (SEQ ID No: 1418)
7.0860

7.0860

58.07
59.33


Ala-23 (SEQ ID No: 1419)
0.2747

0.2747

2.25
2.30


Ala-25 (SEQ ID No: 1421)
3.0800

3.0800

25.24
25.79


Ala-27 (SEQ ID No: 1423)
3.4510
2.5781
1.5792
1.9217
20.28
13.22


Ala-29 (SEQ ID No: 1424)
0.4469
0.1727
0.2919
0.2422
3.66
2.44


Ala-30 (SEQ ID No: 1425)
0.9710
0.7533
0.6370
0.2674
7.96
5.33


Ala-34 (SEQ ID No: 1428)
0.0725
0.0275
0.0573
0.0341
0.59
0.48


Pro-12, Lys-16, Asp-20, Ile-
0.5138
0.4064
0.5127
0.1597
4.21
4.29


23, Ile-29, Ala-34 (SEQ ID


No: 1393)
















TABLE 37







OSK1 Alanine Analogs.









SEQ ID

Analogue Activity (IC50, pM)












NO:
Analogue
Kv1.3
Kv1.1
WB/IL-2
WB/IFNg















1400
G1A
41.11
13.89
122.035
119.425


1401
V2A
81.78
9.94
88.395
103.515


1402
I3A
96.59
10.64
88.255
99.16


1403
I4A
195.30
16.92
110.865
87.255


1404
N5A
159.98
14.01
67.91
66.985


1405
V6A
173.75
12.84
73.26
80.465


1406
K7A
181.04
21.88
67.5
59.075


1407
K9A
166.27
40.59
79.58
71.065


1408
I10A
91.23
4.46
49.97
29.63


1409
S11A
40.79
113.15
90
110


1410
R12A
389.90
55.89
123.49
155.1


1411
Q13A
249.46
21.65
148.05
132.75


1412
L15A
43.07
15.04
107.5
107.5


1413
E16A
21.55
6.87
100.9
100.9


1414
P17A
33.89
9.08
173
173


1415
K19A
210.48
16.85
162.5
162.5


1416
K20A
1036.08
185.01
379
379


1417


1418
G22A
>3000
>3000
7086
7086


1419
M23A
71.39
38.63
274.7
274.7


1420
R24A
>3000
1890.78


1421
F25A
1486.97
47.30
3080
3080


1422
G26A
710.98
733.36
12075
10730


1423
K27A
232.44
>3000
1232
1579.15


1424
M29A
59.47
>3333
446.9
291.85


1425
N30A
692.54
>3000
971
637


1426
G31A
70.17
61.78


1427
K32A


41.3
34


1428
H34A
19.36
368.41
72.54
57.29


1429
T36A


728.4
723.5


1430
P37A


956
849.7


1431
K38A


221
343
















TABLE 38







OSK1 Arginine Analogs.









SEQ ID

Analogue Activity (IC50, pM)












NO:
Analogue
Kv1.3
Kv1.1
WB/IL-2
WB/IFNg















1432
G1R
68.75
9.91
554
991


1433
V2R
133.34
25.79
775
986


1434
I3R
19.90
2.47
148
180


1435
I4R
10.41
1.92
168
175


1436
N5R
13.62
2.15
95
120


1437
V6R
8.65
2.40
84
115


1438
K7R
13.17
<1.52401
78
71


1439
K9R
11.99
2.01
107
77


1440
I10R
11.68
1.73
307
474


1441
S11R
16.72
210.05
2118
4070


1442
Q13R
15.34
<1.52401
160
172


1443
L15R
13.73
2.16
93
116


1444
E16R
10.36
<1.52401
556
454


1445
P17R
10.42
<1.52401
202
355


1446
K19R
12.57
2.41
44
62


1447
K20R
9.85
<1.52401
67
83


1448
A21R
14.92
2.61
90
149


1449
G22R
23.74
3.49
292
349


1450
M23R
12.34
2.01
182
148


1451
F25R
>3333
817.42
25027
30963


1452
G26R
>3333
>3333
100000
100000


1453
K27R
1492.94
>3333
15088
10659


1454
M29R
200.39
1872.11
11680
7677


1455
N30R
18.90
45.71
405
445


1456
G31R
22.16
1.59
314
343


1457
K32R
30.83
7.24
28
34


1458
H34R
13.57
4.49
92
108


1459
T36R
1308.07
26.55
9697
10050


1460
P37R
13.32
2.01
229
253


1461
K38R
14.99
1.84
39
40
















TABLE 39







OSK1 Glutamic Acid Analogs.









SEQ ID

Analogue Activity (IC50, pM)












NO:
Analogue
Kv1.3
Kv1.1
WB/IL-2
WB/IFNg















1462
G1E
185.78
50.97
1217
1252


1463
V2E
36.23
35.01
97
184


1464
I3E
22.00
42.99
120
160


1465
I4E
15.65
3.19
218
191


1466
N5E
23.38
4.44
100
65


1467
V6E
17.73
2.43
48
68


1468
K7E
14.16
<1.52401
58
68


1469
K9E
31.76
110.67
179
171


1470
I10E
120.35
33.50
2573
2736


1471
S11E
>3333
>3333
39878
16927


1472
R12E
89.71
193.25
1787
2001


1473
Q13E
45.87
6.28
1063
799


1474
L15E
47.48
436.05
785
1059


1475
P17E
14.47
1.81
520
947


1476
K19E
23.51
13.71


1477
K20E
25.45
5.76


1478
A21E
7.37
<1.52401
117
138


1479
G22E
13.88
2.56
109
164


1480
M23E
24.28
10.44
606
666


1481
R24E


7161
9543


1482
F25E
>3333
>3333
100000
100000


1483
G26E
>3333
>3333
100000
100000


1484
K27E
>3333
548.55
5548
7144


1485
M29E
>3333
>3333
27099
24646


1486
N30E


14024
24372


1487
G31E
12.01
2.37
95
111


1488
K32E
15.56
17.31
62
63


1489
H34E
330.15
1689.82
1618
2378


1490
T36E
161.06
>3333
1742
2420


1491
P37E
62.67
622.18
239
1604


1492
K38E
25.76
34.33
526
713
















TABLE 40







OSK1 Naphthylalanine Analogs.









SEQ ID

Analogue Activity (IC50, pM)












NO:
Analogue
Kv1.3
Kv1.1
WB/IL-2
WB/IFNg















1493
G1Nal
20.66
33.93
2793
2565


1494
V2Nal
11.55
2.46
750
524


1495
I3Nal
10.31
2.34
907
739


1496
I4Nal
15.03
<1.52401
1094
1014


1497
N5Nal
21.78
<1.52401
760
431


1498
V6Nal
20.97
<1.52401
1776
2465


1499
K7Nal
23.61
<1.52401
222
246


1500
K9Nal
65.82
2.92
1070
1217


1501
I10Nal
45.44
<1.52401
184
257


1502
S11Nal
95.87
>3333
23915
17939


1503
R12Nal
37.66
24.99
460
387


1504
Q13Nal
13.44
<1.52401
140
198


1505
L15Nal
17.84
<1.52401
358
370


1506
E16Nal
9.58
<1.52401
1025
1511


1507
P17Nal
16.19
<1.52401
193
357


1508
K19Nal
17.22
<1.52401
58
99


1509
K20Nal
13.53
<1.52401
74
125


1510
A21Nal
26.10
<1.52401
315
434


1511
G22Nal
>3333
426.27
10328
10627


1512
M23Nal
35.96
64.37
581
1113


1513
R24Nal
45.26
2.85
293
818


1514
F25Nal
28.63
51.75
1733
1686


1515
G26Nal
>3333
1573.75
9898
10651


1516
K27Nal
>3333
1042.84
14971
27025


1517
M29Nal
93.46
46.88
100000
100000


1518
N30Nal
>3333
1283.88
100000
37043


1519
G31Nal
33.76
<1.52401
331
467


1520
K32Nal
26.13
1.91
134
196


1521
H34Nal
60.31
>3333
3323
6186


1522
T36Nal


100000
37811


1523
P37Nal
70.80
6.74
1762
3037


1524
K38Nal


308
409
















TABLE 41







OSK1 Analogues with Improved Selectivity at Kv1.3


over Kv1.1 (whole cell patch clamp ePhys).











SEQ

Kv1.3
Kv1.1
Kv1.3 Selectivity


ID NO:
Analogue
(IC50, pM)
(IC50, pM)
(= Kv1.1/Kv.3 IC50)














25
wild-type
39
 202
5


1441
S11R
40
 9130
228


1502
S11Nal
1490
85324
57


1410
R12A
25
 440
17


1474
L15E
190
65014
342


1423
K27A
289
 10085*
35


1424
M29A
33
 3472
105


1454
M29R
760
23028
30


1425
N30A
766
10168
14


1428
H34A
16
12754
797


1521
H34Nal
215
29178
136


1489
H34E
1322
39352
30


1490
T36E
1921
83914
44


1491
P37E
241
15699
65





*PatchXpress data













TABLE 42







OSK1 Analogues with Multiple Amino Acid Substitutions.












# Amino






Acid

WB/IL-2
WB/IFNg


SEQ ID NO:
Changes
Amino Acid Changes
(IC50, nM)
(IC50, nM)














4988
2
M29A, H34Nal
0.087
0.102


296
2
E16K, K20D
1.579
1.32


4986
2
I4K(Gly), H34A
0.470
0.451


4987
2
H34A, K38K(Gly)
1.249
2.380


4985
2
K19K(Gly), H34A
1.514
1.633


1392
3
R12A, E16K, K20D
0.041
0.092


4990
3
E16K, K20D, H34A
65.462
26.629


298
3
E16K, K20D, T36Y
0.639
0.923


4991
4
S11Nal, R12A, M29A, H34Nal
12.665
14.151


1396
5
E16K, K20D, des36-38
3.941
6.988


1395
5
GGGGS-Osk1
1.357
2.204


1274
5
E16K, K20D, T36G, P37G, K38G
2.636
3.639


4992
5
S11Nal, R12A, M29A, H34Nal, P37E
3.511
5.728


4994
5
S11Nal, R12A, M23F, M29A, H34Nal
8.136
22.727


1398
5
R12P, E16K, K20D, T37Y, K38Ne
0.527
0.736


1397
5
R12P, E16Om, K20E, T37Y, K38Ne
6.611
18.454


4995
6
S11Nal, R12A M23Ne, M29A, H34Nal, P37E
14.32
68.158


4916
7
des1, V2G, R12A, E16K, K19R, K20D, H34A
1.499
2.244


4993
7
S11Nal, R12A, L15E, M29A, H34Nal, T36E, P37E
>100
>100


4989
12
des1-7, E16K, K20D, des36-38
8.179
8.341
















TABLE 43







Bioactivity of OSK1 and OSK1 peptide analog conjugates with


half-life-extending moieties as indicated. Fcloop structures


G2-OSK1-G2 (SEQ ID NO:976), G4-OSK1-G2 (SEQ ID NO:979),


and G2-ShK-G2 (SEQ ID NO:977) are described in Example 49,


and CH2-L10-OSK1(1-38) SEQ ID NO:4917 is described in


Example 54.










F1 (and

WB/IL-2
WB/IFNg


F2, if

(IC50,
(IC50,


present)
Short-hand Designation
nM)
nM)













PEG
20k PEG-OSK1[Ala12]
0.270
0.137


PEG
20k PEG-OSK1[Ala29]
5.756
5.577


PEG
20k PEG-OSK1[Ala29, 1Nal34]
0.049
0.081


PEG
20k PEG-OSK1[1Nal11]
0.019
0.027


PEG
20k PEG-ShK
0.046
0.065


FcLoop
FcLoop-G2-OSK-G2
0.028
0.056


FcLoop
FcLoop-G4-OSK-G2
0.150
0.195


FcLoop
FcLoop-G2-ShK-G2
0.109
0.119


PEG-
20k PEG-L2-6H-L3-CH2-L10-
8.325
50.144


CH2
OsK1(1-38)




CH2
L2-6H-L3-CH2-L10-OsK1(1-38)
38.491
55.162









Example 56
Design and Expression of Monovalent Fc-Fusion Molecules
There may be pharmacokinetic or other reasons, in some cases, to prefer a monovalent dimeric Fc-toxin peptide fusion (as represented schematically in FIG. 2B) to a (“bivalent”) dimer (as represented schematically in FIG. 2C). However, conventional Fc fusion constructs typically result in a mixture containing predominantly dimeric molecules, both monovalent and bivalent. Monovalent dimeric Fc-toxin peptide fusions (or “peptibodies”), including monovalent dimeric Fc-OSK1 peptide analog fusions and Fc-ShK peptide analog fusions, can be isolated from conditioned media which also contains bivalent dimeric Fc-toxin peptide, and dimeric Fc lacking the toxin peptide fusion. Separation of all three species can be accomplished using ion exchange chromatography, for example, as described in Examples 1, 2, and 41 herein.
A number of other exemplary ways that a monovalent dimeric Fc-toxin peptide fusion can be produced with greater efficiency are provided here, including for the production of monovalent dimeric Fc-OSK1 peptide analog fusions:
(1) Co-expressing equal amounts of Fc and Fc-toxin peptide in the same cells (e.g. mammalian cells). With the appropriate design, a mixture of bivalent dimeric Fc-toxin peptide fusion, monovalent dimeric Fc-toxin peptide fusion and dimeric Fc will be produced and released into the conditioned media. The monovalent dimeric Fc-toxin peptide can be purified from the mixture using conventional purification methods, for example, methods described in Examples 1, 2, and 41 herein.
(2) Engineering and recombinantly expressing in mammalian cells a single polypeptide construct represented by the following schematic:

Signal peptide-Fc-furin cleavage site-linker-furin cleavage site-Fc-toxin peptide

Furin cleavage occurs as the molecule travels through the endoplasmic reticulum and the intra-molecular Fc pairing (resulting in monovalent dimeric Fc-toxin peptide fusion) can occur preferentially to intermolecular Fc pairing (resulting in dimeric Fc-toxin peptide being expressed into conditioned medium; FIG. 87A-B).

By way of example of method (2) above, a DNA construct was produced for recombinant expression in mammalian cells of the following schematic polypeptide construct:

Signal peptide-Fc-furin cleavage site-linker-furin cleavage site-Fc-ShK(2-35)

The DNA construct had the following nucleotide coding sequence:







//SEQ ID NO: 5007







atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt


 


ccactccgacaaaactcacacatgcccaccgtgcccagcacctgaactcc


 


tggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctc


 


atgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagcca


 


cgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgc


 


ataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgt


 


gtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagga


 


gtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaa


 


ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctg


 


cccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcct


 


ggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatg


 


ggcagccggagaacaactacaagaccacgcctcccgtgctggactccgac


 


ggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggca


 


gcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaacc


 


actacacgcagaagagcctctccctgtctccgggtaaacgaggcaagagg


 


gctgtggggggcggtgggagcggcggcgggggctcaggtggcgggggaag


 


tggcgggggagggagtggagggggagggagtggaggcgggggatccggcg


 


gggggggtagcaagcgtcgcgagaagcgggataagacccatacctgcccc


 


ccctgtcccgcgcccgagttgctcgggggccccagcgtgtttttgtttcc


 


tcccaagcctaaagatacattgatgattagtagaacacccgaagtgacct


 


gtgtcgtcgtcgatgtctctcatgaggatcccgaagtgaaattcaattgg


 


tatgtcgatggggtcgaagtccacaacgctaaaaccaaacccagagaaga


 


acagtataattctacctatagggtcgtgtctgtgttgacagtgctccatc


 


aagattggctcaacgggaaagaatacaaatgtaaagtgagtaataaggct


 


ttgcccgctcctattgaaaagacaattagtaaggctaagggccaacctag


 


ggagccccaagtctatacactccctcccagtagagacgagctgaccaaga


 


accaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatc


 


gccgtggagtgggagagcaatgggcagccggagaacaactacaagaccac


 


gcctcccgtgctggactccgacggctccttcttcctctacagcaagctca


 


ccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg


 


atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtc


 


tccgggtaaaggaggaggaggatccggaggaggaggaagcagctgcatcg


 


acaccatccccaagagccgctgcaccgccttccagtgcaagcacagcatg


 


aagtaccgcctgagcttctgccgcaagacctgcggcacctgctaa.






The resulting expressed polypeptide (from vector pTT5-Fc-Fc-L10-Shk(2-35)) had the following amino acid sequence before furin cleavage (the first 19 residues are a signal peptide sequence; furin cleavage sites are underlined):







//SEQ ID NO: 5008







mewswvflfflsvttgvhsdkthtcppcpapellggpsvflfppkpkdtl


 


misrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyr


 


vvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytl


 


ppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsd


 


gsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkrgkra


 


vggggsggggsggggsggggsggggsggggsggggskrrekrdkthtcpp


 


cpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwy


 


vdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkal


 


papiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdia


 


vewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvm


 


healhnhytqkslslspgkggggsggggsscidtipksrctafqckhsmk


 


yrlsfcrktcgtc.






FIG. 87A-B demonstrates recombinant expression of a monovalent dimeric Fc-L-ShK(2-35) molecule product expressed by and released into the conditioned media from transiently transfected mammalian cells. FIG. 88 shows results from a pharmacokinetic study on the monovalent dimeric Fc-ShK(1-35) in SD rats. Serum samples were added to microtiter plates coated with an anti-human Fc antibody to enable affinity capture. Plates were then washed, captured samples were released by SDS and run on a polyacrylamide gel. Samples were then visualized by western blot using an anti-human Fc-specific antibody and secondary-HRP conjugate. The MW of bands from serum samples is roughly identical to the original purified material, suggesting little, if any, degradation of the protein occurred in vivo over a pro-longed half-life, in spite of the presence of Arg at position 1 of the ShK(1-35) sequence.
(3) Similar to (2) above, a Fc-toxin peptide fusion monomer can be conjugated with an immunoglobulin light chain and heavy chain resulting in a monovalent chimeric immunoglobulin-Fc-toxin peptide molecule. We have termed an immunoglobulin (light chain+heavy chain)-Fc construct a “hemibody”; such “hemibodies” containing a dimeric Fc portion can provide the long half-life typical of a dimeric antibody. The schematic representation in FIG. 92A-C illustrates an embodiment of a hemibody-toxin peptide fusion protein and its recombinant expression by mammalian cells.
If the antibody chosen is a target specific antibody (e.g., an anti-Kv1.3 or anti-IKCa1 antibody), the chimeric molecule may also enhance the targeting efficiency of the toxin peptide. FIG. 91A-B demonstrates that such chimeric molecules, in this example Fc-L10-ShK(2-35) dimerized with human IgG1 or human IgG2 light and heavy chains, can be expressed and released into the conditioned media from transfected mammalian cells.
Example 57
Osk1 PEGylated at Residue 4 by Oxime Formation
[Dp(AOA)-PEG)4]Osk1 Peptide Synthesis of reduced [Dpr(AOA)4]Osk1. [Dpr(AOA)4]Osk1, having the sequence:







(SEQ ID NO: 5009)







GVI[Dpr(AOA)]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK






can be synthesized in a stepwise manner on a SymphonyTM multi-peptide synthesizer by solid-phase peptide synthesis (SPPS) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) coupling chemistry at 0.1 mmol equivalent resin scale on Fmoc-Lys(Boc)-Wang resin (Novabiochem). N-alpha-(9-fluorenylmethyloxycarbonyl)- and side-chain protected amino acids can be purchased from Novabiochem. The following side-chain protection strategy can be employed: Asp(OtBu), Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(Nε-Boc), Ser(OtBu), Thr(OtBu) and Tyr(OtBu). Dpr(AOA), i.e., N-α-Fmoc-N-b-(N-t.-Boc-amino-oxyacetyl)-L-diaminopropionic acid, can be purchased from Novabiochem (Cat. No. 04-12-1185). The protected amino acid derivatives (20 mmol) can be dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF (v/v). Protected amino acids can be activated with 200 mM HBTU, 400 mM NMM in 20% DMSO in DMF, and coupling can be carried out using two treatments with 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in 20% DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotection reactions can be carried out with two treatments using a 20% piperidine in DMF (v/v) solution for 10 minutes then 15 minutes. Following synthesis and removal of the N-terminal Fmoc group, the resin can be then drained, and washed with DCM, DMF, DCM, and then dried in vacuo. The peptide-resin can be deprotected and released from the resin by treatment with a TFA/amionooxyacetic acid/TIS/EDT/H2O (90:2.5:2.5:2.5:2.5) solution at room temperature for 1 hour. The volatiles can be then removed with a stream of nitrogen gas, the crude peptide precipitated twice with cold diethyl ether and collected by centrifugation. The [Dpr(AOA)4]Osk1 peptide can be then analyzed on a Waters 2795 analytical RP-HPLC system using a linear gradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water also containing 0.1% aminooxyacetic acid, B: 0.1% TFA in acetonitrile) on a Jupiter 4 μm ProteoTM 90 Å column.

Reversed-Phase HPLC Purification. Preparative Reversed-phase high-performance liquid chromatography can be performed on C18, 5 μm, 2.2 cm×25 cm) column. The [Dpr(AOA)4]Osk1 peptide is dissolved in 50% aqueous acetronitrile containing acetic acid and amionooxyacetic acid and loaded onto a preparative HPLC column. Chromatographic separations can be achieved using linear gradients of buffer B in A (A=0.1% aqueous TFA; B=90% aq. ACN containing 0.09% TFA), typically 5-95% over 90 minutes at 15 mL/min. Preparative HPLC fractions can be characterized by ESMS and photodiode array (PDA) HPLC, combined and lyophilized.
Osk1 peptide analog PEGylated at residue 4 by oxime formation: [Dpr(AOA)-PEG)4]Osk1 (i.e., GVI[Dpr(AOA-PEG)]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK//SEQ ID NO:5010) can be made as follows. Lyophilized [Dpr(AOA)4]Osk1 peptide can be dissolved in 50% HPLC buffer A/B (5 mg/mL) and added to a two-fold molar excess of MeO-PEG-aldehyde, CH3O—[CH2CH2O]n—CH2CH2CHO (average molecular weight 20 kDa). The aminoxyacetyl group within the peptide at residue 4 reacts with the aldehyde group of the PEG to form a covalent oxime linkage. The reaction can be left for 24 hours, and can be analyzed on an AgilentTM 1100 RP-HPLC system using ZorbaxTM 300SB-C8 5 μm column at 40° C. with a linear gradient (6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN in water). Mono PEGylated [Dpr(AOA)-PEG)4]Osk1 peptide can be then isolated using a HiTrapTM 5 mL SP HP cation exchange column on AKTA FPLC system at 4° C. at 1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers: A=20 mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH 4.0). The fractions can be analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product, mono-PEGylated [Dpr(AOA)-PEG)4]Osk1 peptide, can be then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM NaOAc, 5% sorbitol, pH 4.0). The dialyzed product can be then concentrated in 10 K microcentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μM syringe filter to give the final product.
Folding of [Dpr(AOA)-PEG)4]Osk1 (Disulphide bond formation). The mono-PEGylated [Dpr(AOA)-PEG)4]Osk1 peptide can be dissolved in 20% AcOH in water (v/v) and can be then diluted with water to a concentration of approximately 0.15 mg peptide mL, the pH adjusted to about 8.0 using NH4OH (28-30%), and gently stirred at room temperature for 36 hours. Folding process can be monitored by LC-MS analysis. Following this, folded mono-PEGylated [Dpr(AOA)-PEG)4]Osk1 can be purified using by reversed phase HPLC using a 1′′ Luna 5 μm C18 100 Å ProteoTM column with a linear gradient 0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1% TFA in acetonitrile). Mono-PEGylated (oximated) [Dpr(AOA)-PEG)4]Osk1 peptide disulfide connectivity can be C1-C4, C2-C5, and C3-C6.
ABBREVIATIONS
Abbreviations used throughout this specification are as defined below, unless otherwise defined in specific circumstances.
Ac acetyl (used to refer to acetylated residues)
AcBpa acetylated p-benzoyl-L-phenylalanine
ADCC antibody-dependent cellular cytotoxicity
Aib aminoisobutyric acid
bA beta-alanine
Bpa p-benzoyl-L-phenylalanine
BrAc bromoacetyl (BrCH2C(O)
BSA Bovine serum albumin
Bzl Benzyl
Cap Caproic acid
COPD Chronic obstructive pulmonary disease
CTL Cytotoxic T lymphocytes
DCC Dicylcohexylcarbodiimide
Dde 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)ethyl
ESI-MS Electron spray ionization mass spectrometry
Fmoc fluorenylmethoxycarbonyl
HOBt 1-Hydroxybenzotriazole
HPLC high performance liquid chromatography
HSL homoserine lactone
IB inclusion bodies
KCa calcium-activated potassium channel (including IKCa, BKCa, SKCa)
Kv voltage-gated potassium channel
Lau Lauric acid
LPS lipopolysaccharide
LYMPH lymphocytes
MALDI-MS Matrix-assisted laser desorption ionization mass spectrometry
Me methyl
MeO methoxy
MHC major histocompatibility complex
MMP matrix metalloproteinase
1-Nap 1-napthylalanine
NEUT neutrophils
Nle norleucine
NMP N-methyl-2-pyrrolidinone
PAGE polyacrylamide gel electrophoresis
PBMC peripheral blood mononuclear cell
PBS Phosphate-buffered saline
Pbf 2,2,4,6,7-pendamethyldihydrobenzofuran-5-sulfonyl
PCR polymerase chain reaction
Pec pipecolic acid
PEG Poly(ethylene glycol)
pGlu pyroglutamic acid
Pic picolinic acid
pY phosphotyrosine
RBS ribosome binding site
RT room temperature (25° C.)
Sar sarcosine
SDS sodium dodecyl sulfate
STK serine-threonine kinases
t-Boc tert-Butoxycarbonyl
tBu tert-Butyl
THF thymic humoral factor
Trt trityl

## Claims

1. A composition of matter of the formula


(X1)a—(F1)d—(X2)b—(F2)e—(X3)c 


and multimers thereof, wherein:
F1 and F2 are half-life extending moieties, wherein F1 or F2, or both is a polyethylene glycol, a copolymer of ethylene glycol, a polypropylene glycol, a copolymer of propylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, a poly-1,3-dioxolane , a poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid, a dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinyl alcohol, a linear or branched glycosylated chain, a polyacetal, a long chain fatty acid, a long chain hydrophobic aliphatic group, an immunoglobulin light chain and heavy chain, an immunoglobulin Fc domain or portion thereof, a CH2 domain of Fc, an Fc domain loop, an albumin, an albumin-binding protein, a transthyretin, a thyroxine-binding globulin, or a ligand that has an affinity for a long half-life serum protein, said ligand being selected from the group consisting of peptide ligands and small molecule ligands; or a combination of any of these members; and d and e are each independently 0 or 1, provided that at least one of d and e is 1;
X1, X2, and X3 are each independently -(L)f-P-(L)g, and f and g are each independently 0 or 1;
P is a toxin peptide of no more than about 80 amino acid residues in length, comprising at least two intrapeptide disulfide bonds, and at least one P is an OSK1 peptide analog comprising the amino acid sequence of SEQ ID NO:4916;
L is a linker; and
a, b, and c are each independently 0 or 1, provided that at least one of a, b and c is 1.

2. The composition of matter of claim 1 of the formula P-(L)g-F1.

3. The composition of matter of claim 1 of the formula F1-(L)f-P.

4. The composition of matter of claim 1 of the formula P-(L)g-F1-(L) f-P.

5. The composition of matter of claim 1 of the formula F1-(L)f-P-(L) g-F2.

6. The composition of matter of claim 1 of the formula F1-(L)f-P-(L)gF 2-(L)f-P.

7. The composition of matter of claim 1 of the formula F1-F2-(L)f-P.

8. The composition of matter of claim 1 of the formula P-(L)g-F1-F2.

9. The composition of matter of claim 1 of the formula P-(L)g-F1-F2-(L) f-P.

10. The composition of matter of claim 1 wherein F1 or F2, or both, comprises a human IgG Fc domain or a portion thereof.

11. The composition of matter of claim 1, wherein F1 and F2 are different half-life extending moieties.

12. The composition of matter of claim 1, wherein F1 or F2, or both, comprises a sequence selected from SEQ ID NOS: 2, 4, 70, 71, 72, 74, 75, 76, 1340 through 1342, and 1359 through 1363 as set forth in FIGS. 3, 4, 11A-C, 12A-C, and 12E-F.

13. The composition of matter of claim 1, wherein F1 or F2, or both, comprises a biologically suitable polymer or copolymer.

14. The composition of matter of claim 1, in which the toxin peptide is inserted into a human IgG1 Fc domain loop.

15. The composition of matter of claim 1, wherein the C-terminal carboxylic acid moiety of the OSK1 peptide analog is replaced with a moiety selected from
(A) —COOR, where R is independently (C1-C8)alkyl, haloalkyl, aryl or heteroaryl;
(B) —C(═O)NRR, where R is independently hydrogen, (C1-C8)alkyl, haloalkyl, aryl or heteroaryl; and
(C) —CH2OR where R is hydrogen, (C1-C8) alkyl, aryl or heteroaryl.

16. The composition of matter of claim 1, wherein the OSK1 peptide analog is conjugated to a polyethylene glycol (PEG) via:
(a) 1, 2, 3 or 4 amino functionalized sites of the PEG;
(b) 1, 2, 3 or 4 thiol functionalized sites of the PEG;
(c) 1, 2, 3 or 4 maleimido functionalized sites of the PEG;
(d) 1, 2, 3 or 4 N-succinimidyl functionalized sites of the PEG;
(e) 1, 2, 3 or 4 carboxyl functionalized sites of the PEG; or
(f) 1, 2, 3 or 4 p-nitrophenyloxycarbonyl functionalized sites of the PEG.

17. The composition of matter of claim 1, wherein the OSK1 peptide analog is conjugated to an acyl, aryl, fatty acid, or polyethylene glycol (PEG) via:
(a) 1, 2, 3 or 4 amino functionalized sites in the OSK1 peptide analog;
(b) 1, 2, 3 or 4 thiol functionalized sites in the OSK1 peptide analog;
(c) 1 or 2 ketone functionalized sites in the OSK1 peptide analog;
(d) 1 or 2 azido functionalized sites in the OSK1 peptide analog;
(e) 1 or 2 carboxyl functionalized sites in the OSK1 peptide analog;
(f) 1 or 2 aminooxy functionalized sites in the OSK1 peptide analog; or
(g) 1 or 2 seleno functionalized sites in the OSK1 peptide analog.

18. The composition of matter of claim 1, wherein the OSK1 peptide analog is covalently linked at its N-terminal to a moiety selected from acyl, aryl, fatty acid, or polyethylene glycol.

19. The composition of matter of claim 1, wherein the OSK1 peptide analog is covalently linked at its N-terminal to a moiety selected from benzyl, dibenzyl, benzoyl, benzyloxycarbonyl, N,N-dimethylglycine, creatine, formyl, acetyl, propanoyl, butanyl, heptanyl, hexanoyl, octanoyl, nonanoyl, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, and stearic acid.

20. The composition of matter claim 1, further comprising, covalently bound to F1, F2, or to P, an additional agonistic peptide or an antagonistic peptide, in relation to the activity of the OSK1 peptide analog, or a targeting peptide.

21. A pharmaceutical composition, comprising the composition of claim 1 and a pharmaceutically acceptable carrier.

22. A composition of matter of the formula


(X1)a—(F1)d—(X2)b—(F2)e—(X3)c 

and multimers thereof, wherein:
F1 and F2 are half-life extending moieties selected from an acyl, aryl, fatty acid, and polyethylene glycol (PEG), and d and e are each independently 0 or 1, provided that at least one of d and e is 1;
X1, X2, and X3 are each independently -(L)f-P-(L)g-, and f and g are each independently 0 or 1;
P is an OSK1 peptide analog of no more than about 80 amino acid residues in length, comprising at least two intrapeptide disulfide bonds, comprising the amino acid sequence of SEQ ID NO:4916;
L is a linker;
a, b, and c are each independently 0 or 1, provided that at least one of a, b and c is 1; and the toxin peptide is conjugated to F1 or F2, or both, via:

(a) 1, 2, 3 or 4 amino functionalized sites in the toxin peptide;
(b) 1, 2, 3 or 4 thiol functionalized sites in the toxin peptide;
(c) 1 or 2 ketone functionalized sites in the toxin peptide;
(d) 1 or 2 azido functionalized sites in the toxin peptide;
(e) 1 or 2 carboxyl functionalized sites in the toxin peptide;
(f) 1 or 2 aminooxy functionalized sites in the toxin peptide; or
(g) 1 or 2 seleno functionalized sites in the toxin peptide.

23. A pharmaceutical composition, comprising the composition of claim 22 and a pharmaceutically acceptable carrier.