# Patent

## Title
Structure-based peptide inhibitors of P53 aggregation as a new approach to cancer therapeutics

## Abstract

This invention relates, e.g., to an inhibitory peptide or CPP inhibitor which specifically binds to p53 having an aberrant conformation (e.g., is aggregated or misfolded) and inhibits p53 amyloidogenic aggregation or restores proper folding of the misfolded protein. Methods of using the inhibitory peptide or CPP inhibitor (e.g. to treat subjects having tumors that comprise aggregated p53) are described.

## Background
This application claims the benefit of the filing date of U.S. provisional application Ser. No. 61/821,157, filed May 8, 2013, which is incorporated by reference herein in its entirety.


This invention was made with Government support under Grant No. NSF MCB-0958111 awarded by the National Science Foundation. The Government has certain rights in the invention.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 8, 2014, is named 58086-364665_SL.txt and is 24,636 bytes in size.
BACKGROUND INFORMATION
Mutations in the tumor suppressor p53 are associated with 50% of all reported human cancers (Soussi et al., 2006). Structural instability of p53 mutants leads to partial unfolding (Bullock and Fersht, 2001) which in turn may cause p53 to form aggregates similar to those seen in amyloid diseases, such as Alzheimer's Disease (Xu et al, 2011; Levy et al, Eisenberg and Jucker, 2012). The process of p53 misfolding and aggregation results in protein inactivation, thereby removing the ‘guardian of the genome’ from its protective function (Xu et al, 2011).
In the past decades, it has been shown that several different p53 mutations, in particular those deemed to be “structural mutations,” affect p53 folding, lowering protein stability and inducing partial unfolding (Bullock and Fersht, 2001). These aberrant p53 conformations have been demonstrated in cancer biopsies by using the mutant-specific antibody PAb240 which recognizes an epitope buried in the protein core that gets solvent exposure only upon misfolding (Gannon et al, 1990). In addition, several lines of evidence have shown that fragments of p53 (Ishimaru et al, Biochemistry 2003; Silva et al, 2010; Ishimaru et al, 2009; Galea et al, 2005; Rigacci et al, 2008) as well as full-length mutant p53 (Wang et al, PNAS, 2012) undergo amyloidogenic aggregation in vitro. In addition, p53 was reported to be in the misfolded aggregated amyloid state in biopsies derived from breast cancer cases (Levy et al, 2011) as well as colon carcinomas (Xu et al, 2011) and basal cell carcinomas (Lasagna-Reeves et al, 2013).
There is a need for agents that can specifically destabilize p53 aggregates or prevent them from forming, in particular agents which are designed in a rational structure-based approach, for use in treating forms of cancer in which p53 is inactivated due to the fact that it is aberrantly folded and/or aggregated (is found inactive in fibrous form). Since about half of all diagnosed tumors present with p53 mutations, the potential for applicability of such a targeted therapeutic agent is great.



DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color.
FIG. 1 shows the X-ray high-resolution structures of two polymorphs of the amyloid aggregation prone segment of p53, namely p53 residues 253-258 (sequence: TIITLE (SEQ ID NO: 20)) and p53 residues 252-258 (sequence: LTIITLE (SEQ ID NO: 21)). In both cases, the protofilaments consist of two interdigitated beta sheets, with a tight, dry interface. Three layers of the sheets are shown; water molecules are shown as yellow spheres. The top view is down the fiber axis (indicated as a red diamond) while the bottom view is perpendicular to the axis (red arrow)
FIG. 2 shows INH-1R modeled on the p53252-258 crystal structure. Three adjacent sheets are represented. The cyan ball-and-stick inhibitor has a high surface complementarity to the LTIITLE (SEQ ID NO: 21) structure. The arginine (in yellow) of INH-1R collides with the opposing β-sheet, inhibiting further filament growth. The inhibitor can bind to the top and/or to the bottom (along the fiber axis) of the steric zipper template. The view is down the fiber axis.
FIG. 3 shows the in vitro inhibition of p53 aggregation (segment p53252-258) by INH-1R. The aggregation of p53 is monitored via Thioflavin T assay: an increase in Thioflavin T fluorescence is detected over time due to the formation of increasing amounts of amyloid to which the dye can specifically bind. INH-1R is added in solution at different concentrations and is able to delay the aggregation onset and lower the total amount of aggregates present, in a concentration dependent fashion. The sequence of this segment, LTIITLE, is SEQ ID NO: 21.
FIG. 4 shows that the cell-penetrating version of INH-1R (INH-1R CPP) is able to enter human cancer cells. INH-1R CPP was covalently linked to a FITC fluorescent label and added to different cancer cell lines or primary cells directly obtained from ovarian cancer patients (as depicted here). A. The peptide successfully penetrated into the cytosol and nucleus of the cells (in green) and co-localized with p53. The cell nuclei are stained with Hoechst 33342 (blue), while p53 is recognized by an anti-p53 antibody and stained in red. B. The peptide co-localizes with protein aggregates in the same cells. Protein aggregates are stained with the commercially available OC antibody (in red). This indicates that INH-1R CPP binds to aggregated p53 in these ovarian cancer cells.
FIG. 5 shows that INH-1R CPP causes re-localization of p53 and breaks up aggregation. In the top panels, p53 stain of primary cells from a serous ovarian carcinoma patient show punctate p53 cytosolic staining. Upon 24 hours of treatment with 10 μM of INH-1R CPP, all the puncta disappeared and p53 is now diffused and localized to the nucleus where it can exert its function of transcription factor and oncosuppressor as visible in the bottom panel. Again, INH-1R CPP co-localizes with p53 (INH-1R CPP in green, p53 in red, nuclei in blue).
FIG. 6 shows that INH-1R CPP causes misfolded p53 to acquire a wild type-like, functional fold. A stable cell line derived from an ovarian cancer tumor is stained with the commercially available DO-1 antibody which recognizes any p53, irrespective of its structural state. As shown in FIG. 5, treatment with the inhibitor but not with a scrambled peptide causes p53 to re-localize to the nucleus. In the bottom, the transition is accompanied by a refolding step: upon treatment with the inhibitor the antibody PAb240 does not bind to p53 in these cells, even though p53 is present in abundance (see DO-1 stain above). The PAb240 commercial antibody recognizes misfolded p53, therefore loss of PAb240 antigenicity reflects a change in the p53 population from misfolded to properly folded, functionally capable protein.
FIG. 7 shows that INH-1R CPP treated cells have a functional p53 that can respond to physiological regulation systems. p53 levels in OVCAR-3 cell lysates treated for 24 hours with 0, 1, 5 or 10 μM INH-1R were assessed by Western Blot and quantified using ImageJ. The total amount of p53 decreases upon INH-1R CPP treatment in a concentration dependent manner. The properly folded p53 (see FIG. 6) can now be degraded as is WT p53. The hyperstability due to misfolding/aggregation is lost, and the protein turnover is fast.
FIG. 8 shows that INH-1R CPP CPP effectively induces cell death in tumors cells bearing misfolded/aggregation-prone mutant p53. A. Dose dependent reduction of cell viability was detected in cells treated with 10 μM INH-1R CPP for 24 hours. Increased apoptosis (B.) and decreased proliferation (C.) was observed with increasing INH-1R CPP dosage. B. and C. show one representative case of primary cells from ovarian cancer. Similar results were observed with all sensitive primary cells as well as cell lines.
FIG. 9 shows that INH-1R CPP effectively induces cell death in cancer cells. Cells treated for 24 hours with either INH-1R or a control peptide were trypsinized and analyzed by FACS. Cell death is accompanied by a typical reduction of cell size and an increase in granularity in cells treated with INH-1R CPP as compared to vehicle treated cells (DMSO). Cancer cells treated with a control scrambled peptide sequence did not show any change in cell size or granularity.
FIG. 10 shows apoptosis and necrosis in cancer cells treated with INH-1R CPP. OVCAR-3 cells were treated for 24 hours with the indicated concentrations of either INH-1R CPP or a scrambled inhibitor sequence. Hoechst (blue) stains all nuclei while YO-PRO-1 (green) only stains apoptotic cells and propidium iodide (red) stains late apoptotic/necrotic cells. The samples treated with the inhibitor contain mostly dead cells, while the scrambled peptide has no effect, indicative of sequence specific effect.
FIG. 11 shows that INH-1R CPP induces up-regulation of p53 target genes p21 and Mdm2. Specificity and efficacy of INH-1R CPP was confirmed by up-regulation of p53 target genes only in tumor cells containing p53 misfolding/aggregating mutations.
FIG. 12 shows that INH-1R CPP limits tumor proliferation in vivo. A. Images of the xenografts from mice (n=3 for each group, one mouse had 2 xenografts) treated with INH-1R CPP, a scrambled peptide control, or vehicle for 14 days. B. Tumors of mice treated with INH-1R CPP were six times smaller compared to controls as evaluated by weight. C. Tumor volume was estimated daily. D. Residual tumors from INH-1R CPP treated animals showed up-regulation of the p53 target genes MDM2 and p21.
FIG. 13 shows serum concentration of INH-1R CPP as measured by MRM (Multiple Reaction Monitoring) mass spectrometry. Nude mice were injected in the flank with OVCAR-3 cells and the resulting xenografts were allowed to grow for two weeks. The mice were then treated for three days via TP with 15 mg/kg of INH-1R CPP peptide or scrambled control peptide. They were divided in groups of two and sacrificed at 1, 2, 4, 8, 12 and 24 hours post final peptide injection. Two naïve mice were sacrificed before treatment to obtain a reference value. Peak serum concentration of INH-1R CPP (as well as control peptide) was detected at 1 hour post injection. Average of two mice are shown.



DESCRIPTION
This application relates, e.g., to the design, synthesis and functional characterization of peptides which bind specifically (preferentially) to p53 protein molecules having an aberrant (e.g. pathological) conformation and which restore the conformation of the p53 molecules having the aberrant conformation. The aberrant conformation can be, for example, misfolding of the molecule resulting from a mutation in the molecule or other factors, or the formation of amyloid aggregates of wild type or mutant p53 molecules. As a result of the restoration of the conformation, biological or biochemical activities which were lost or inhibited as a result of the aberrant conformation are reactivated or restored. For example, the inhibitory peptides can inhibit (block) further aggregation of p53 amyloid aggregates and/or restore p53 functions such as, e.g., induction or initiation of apoptosis, inhibition of cell proliferation, and/or inducing shrinkage of a tumor. In some embodiments, the peptides are fused to cell penetrating peptides (CPP) which enhance their delivery into cells.
The present inventors recently showed that it is possible to efficiently arrest the aggregation of the Alzheimer's Disease related protein Tau and the semen-derived enhancer of HIV virus infection (SEVI) utilizing short amino-acid inhibitors designed to specifically “cap” the growing aggregates (Sievers et al., 2011). Accordingly, there is a new therapeutic window which targets a completely unexplored aspect of p53 biology that seems to have profound effects on cancer progression, i.e. p53 misfolding resulting in aggregation. The inventors hypothesized that mutations, overexpression or other cellular factors can destabilize the native p53 structure, exposing an adhesive, “steric-zipper” segment, proposed as the basic building block of amyloid aggregates (Nelson et al., Nature, 2005, Sawaya et al., Nature, 2007). As reported herein, the inventors therefore obtained high atomic resolution views of the amyloid spine of p53 aggregates, and used them as a template to develop structure based peptide inhibitors that can cap the aggregates, inhibit further p53 aggregation and therefore generate a pool of active p53 that can sensitize the cancer cells to treatment and induce or initiate apoptosis. These rational structure-based inhibitors of p53 aggregation provide a new chemotherapy efficient toward those tumors that have proven to be the most aggressive and resilient to standard treatment, due to p53 aggregation status (Xu et al., 2011; Levy et al. 2011).
This application relates, e.g., to such inhibitory peptides; molecules in which an inhibitory peptide of the invention is fused to a cell penetrating peptide (CPP), which fusion molecules are sometimes referred to herein as “CPP inhibitors”; pharmaceutical compositions comprising an inhibitory peptide or a CPP inhibitor of the invention and a pharmaceutically acceptable carrier; methods of using the inhibitory peptides or the CPP inhibitors to restore the structure and function of p53 molecules having an aberrant conformation, e.g. (a) to block or inhibit p53 aggregation (e.g., to delay the onset of aggregation and/or to lower the amount of aggregates, in solution, in a cell, or in a subject having a cancer or tumor that comprises p53 aggregates) and/or (b) to restore the folding of a misfolded p53, thereby re-activating a biological or biochemical activity of p53 due to the aberrant conformation; methods for treating a subject having a tumor which comprises aggregated p53 (e.g., either wild type or mutant aggregated p53), comprising administering to the subject or contacting the tumor with an effective amount of a CPP inhibitor of the invention; and computer-related embodiments, such as a method for designing and obtaining inhibitory peptides or small molecules based on the structural representation of the crystal structures described herein.
Advantages of the inhibitory peptides and CPP inhibitors of the invention include: (1) They are selectively active only on those cancer cells containing mutant or wild type aggregated p53 or misfolded p53; (2) They show no effect on folded and active p53 (no hyper-activation or increase of p53 concentration in normal cells); (3) They are conformation specific, rather than sequence (e.g. mutation) specific. A single inhibitor will work for different aggregating mutants as well as for wild type p53; (4) They can block co-aggregation of wild-type p53 as well as aggregation of p53 with homologues and other proteins including, for example, p63 and p73, the other members of the p53 family of proteins (Xu et al, 2011); (5) Cell penetration and protein stability are not challenging obstacles, thanks to their composition and small size; (6) They are unexpectedly stable: they are not proteolyzed and exhibit a sufficiently long half-life to function in vivo (e.g. in a body).
One aspect of the invention is an inhibitory peptide (e.g., an isolated peptide) represented by the consensus sequence [L,Y,E,W] T [R,K], I T [L,Y] E (SEQ ID NO: 1), or an active variant thereof. In one embodiment, the inhibitory peptide is represented by the consensus sequence [L,Y,E,W] T R I T [L,Y] E (SEQ ID NO: 3), or an active variant thereof. The inhibitory peptide may consist of the consensus sequence [L,Y,E,W] T [R,K], I T [L,Y] E (SEQ ID NO: 1), or it may consist of the consensus sequence [L,Y,E,W] T R I T [L,Y] E (SEQ ID NO: 3). In embodiments of the invention, the inhibitory peptide may consist of, or comprise, any of the inhibitory peptide sequences listed in Table 1. That is, the peptide may be LTRITLE (SEQ ID NO: 4), YTRITLE (SEQ ID NO: 5), ETRITLE (SEQ ID NO: 6), LTRIYLE (SEQ ID NO: 7), YTRIYLE (SEQ ID NO: 8), WTRITLE (SEQ ID NO: 9), WTRIYLE (SEQ ID NO: 10), ETRIYLE (SEQ ID NO: 11), LTKITLE (SEQ ID NO: 12), YTKITLE (SEQ ID NO: 13), WTKITLE (SEQ ID NO: 14), ETKITLE (SEQ ID NO: 15), LTKIYLE (SEQ ID NO: 16), YTKIYLE (SEQ ID NO: 17), WTRIYLE (SEQ ID NO: 10), ETKIYLE (SEQ ID NO: 18). Inhibitory peptides having the preceding sequences, including the active variants, are sometimes referred to herein as “inhibitory peptides of the invention.”
Another aspect of the invention is a CPP inhibitor which comprises an inhibitory peptide of the invention (including active variants) which is fused (linked, associated with, coupled), optionally via a linker sequence, to a cell penetrating peptide (CPP). The peptide can be fused the CPP in any of a variety of ways (e.g. chemically coupled, or fused via a peptide bond or other conventional means of chemical coupling).
In one embodiment, the CPP is represented by the consensus sequence (R1-16) P I [L,Y,E,W] T [R,K], I T [L,Y] E (SEQ ID NO: 19), or an active variant thereof. CPP inhibitors which comprise sequences encompassed by this consensus sequence or active variants thereof are sometimes referred to herein as “CPP inhibitors of the invention.”
Another aspect of the invention is a pharmaceutical composition comprising an inhibitory peptide or CPP of the invention and a pharmaceutically acceptable carrier. Such pharmaceutical compositions are sometimes referred to herein as “pharmaceutical compositions of the invention.”
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, in the preceding case, the pharmaceutical composition may comprise one or more inhibitory peptide molecules or CPPs of the invention, which can be the same or different.
Another aspect of the invention is a complex comprising a p53 protein molecule and an inhibitory peptide or CPP of the invention. They may be bound to, conjugated with, or otherwise associated with each other. The p53 and the inhibitory peptide or CPP may be covalently or non-covalently linked.
Another aspect of the invention is a method for restoring the conformation of a p53 protein molecule having an aberrant conformation (e.g. wherein the aberrant conformation is responsible, at least in part, for a loss of a function of the protein), comprising
contacting the p53 molecule having the aberrant conformation with an effective amount of an inhibitory peptide or a CPP inhibitor of the invention,
the contacted p53 having a restored conformation,
wherein the p53 molecule having the restored conformation exhibits an activity (e.g. a restored activity) selected from the induction of apoptosis, inhibition of cell proliferation and/or induction of shrinkage of a tumor.
In one embodiment of this method, the p53 protein molecule which is contacted is in a subject having cancer, and the p53 molecule having the restored conformation inhibits proliferation of cancer cells in the subject and/or induces shrinkage of a tumor in the subject.
Another aspect of the invention is a method for preventing and/or inhibiting cell proliferation (e.g. proliferation of cancer cells) resulting from (e.g. caused by) p53 with an aberrant conformation, comprising contacting the cell with an effective amount of an inhibitory peptide or a CPP inhibitor of the invention or with a pharmaceutical composition comprising an inhibitory peptide or a CPP inhibitor of the invention.
Another aspect of the invention is a method for treating a subject having a cancer associated with (e.g. mediated by) p53 having an aberrant conformation, comprising administering to the subject an effective amount of a pharmaceutical composition of the invention, thereby inhibiting proliferation of cancer cells in the subject and/or shrinking a tumor in the subject.
Another aspect of the invention is a method for treating a subject that has a mutant gene encoding p53 and therefore a susceptibility to develop cancers (e.g., Li-Fraumeni syndrome), comprising administering to the subject a dose (e.g. a plurality of doses, such as by a plurality of injections) comprising in total an effective amount of a pharmaceutical composition of the invention, thereby reducing or preventing the development of tumors in the subject.
Another aspect of the invention is a computer-implemented method for identifying an inhibitory peptide that inhibits aggregation of p53, comprising the steps of:
identifying a template peptide sequence comprising a zipper-forming sequence of the p53 segments TIITLE (SEQ ID NO: 20) or LTIITLE (SEQ ID NO: 21) or a mirror of the zipper forming sequence from the target polypeptide, wherein the zipper-forming sequence aggregates into a steric zipper;
designing on a computer at least one complementary peptide sequence that forms favorable steric and energetic intermolecular interactions with the template peptide sequence, wherein the interactions occur at one or both of the upper or lower ends of the steric zipper; and
identifying a candidate inhibitory peptidic compound selected from the group consisting of the complementary sequence, a mirror of the complementary sequence, a peptide mimetic of the complementary sequence and a peptide mimetic of the mirror of the complementary sequence.
Another aspect of the invention is a kit comprising an inhibitory peptide or CPP inhibitor of the invention, optionally packaged in a container.
Another aspect of the invention is a method for making an inhibitory peptide of the invention, comprising synthesizing it chemically or producing it recombinantly.
As described in the Examples herein, based on the determination of the atomic structure of a fiber forming segment of p53, the inventors have designed a series of peptide inhibitors which diminish p53 aggregation. The inventors designed peptidic inhibitors to specifically “cap” the growing aggregates of p53.
Using the ZipperDB algorithm (Goldschmidt et al. 2010), the inventors identified crystallizable amyloid-forming segments. The inventors chemically synthesized the p53252-258 and p53253-258 segments, crystallized them and determined their three-dimensional structures by micro-crystallography (FIG. 1). The structures displayed a typical steric zipper architecture, with parallel in-register β-strands and β-sheets interdigitating via hydrophobic side chains in a “face-to-back” (for p53252-258) and “face-to-face” (for p53253-258) orientation (Sawaya et al, Nature, 2007).
The inventors applied their Rosetta-based method (Sievers et al, Nature, 2011) to design inhibitors that disrupt p53 aggregation, using the p53252-258 structure as a template. In other embodiments of the invention, the p53253-258 structure is used as a template to design inhibitors.
Table 1 shows a list of 16 representative inhibitor sequences obtained by this method.







TABLE 1







List of designed inhibitors of amyloidogenic p53


aggregation including peptide sequences. INH-1R


is the first sequence designed. INH-1R and all


the other variants were synthesized fused to a


poly-Arginine tag as cell penetrating peptide


(CPP) and a short linker (sequence: RPI) derived


from the endogenous p53 protein sequence. The


inhibiting designed sequences are indicated with


bold type, while the poly-Arginine tag and linker


are in normal type.










Inhibitor



Name
sequence
CPP inhibitor





INH-1R
LTRITLE
RRRRRRRRRRPILTRITLE


 


INH-2R
YTRITLE
RRRRRRRRRRPIYTRITLE


 


INH-3R
ETRITLE
RRRRRRRRRRPIETRITLE


 


INH-4R
LTRIYLE
RRRRRRRRRRPILTRIYLE


 


INH-5R
YTRIYLE
RRRRRRRRRRPIYTRIYLE


 


INH-6R
WTRITLE
RRRRRRRRRRPIWTRITLE


 


INH-7R
WTRIYLE
RRRRRRRRRRPIWTRIYLE


 


INH-8R
ETRIYLE
RRRRRRRRRRPIETRIYLE


 


INH-1K
LTKITLE
RRRRRRRRRRPILTKITLE


 


INH-2K
YTKITLE
RRRRRRRRRRPIYTKITLE


 


INH-3K
WTKITLE
RRRRRRRRRRPIWTKITLE


 


INH-4K
ETKITLE
RRRRRRRRRRPIETKITLE


 


INH-5K
LTKIYLE
RRRRRRRRRRPILTKIYLE


 


INH-6K
YTKIYLE
RRRRRRRRRRPIYTKIYLE


 


INH-7R
WTRIYLE
RRRRRRRRRRPIWTRIYLE


 


INH-8K
ETKIYLE
RRRRRRRRRRPIETKIYLE









The inhibitor sequences in Table 1 are, reading from top to bottom of the second column of the table, SEQ ID NO: 4 to SEQ ID NO: 17, 10 and 18. The CPP inhibitor sequences are, reading from top to bottom of the third column of the table, SEQ ID NO: 22 to SEQ ID NO: 35, 28 and 36.

Peptide inhibitors of the invention bind specifically (selectively, preferentially) to p53 having an aberrant conformation (e.g. aggregated as amyloid fibrils or fibers, or partially or completely unfolded or misfolded), in comparison to binding to other protein targets (unintended targets), such as non-aggregated or folded p53 molecules which exhibit one or more of the p53-mediated functions described herein. In fact, no binding can be detected between the peptide inhibitors of the invention and non-aggregated or folded p53 molecules.
Other suitable peptide variants include, e.g., Leu-His-Arg-Ile-Tyr-Leu-Glu (SEQ ID NO: 37) and Leu-Tyr-Ile-Arg-Ile-Leu-Arg (SEQ ID NO: 38).
On the basis of this structural analysis, one consensus sequence, taking into account the 16 sequences shown in Table 1, is [L,Y,E,W] T [R,K], I T [L,Y] E (SEQ ID NO: 1). In another embodiment, the consensus sequence is [L,Y,E,W] T R I T [L,Y] E (SEQ ID NO: 3). Residues #1, 6 and, to a lesser extent, #3 have the least contact with the template structure and are thus the most variable of the 7 residues.
Active variants of the sequences described above are also included. These are variants which retain the properties of the inhibitory peptides described herein (e.g., the ability to bind specifically to aggregated p53 in a conformation-dependent, sequence-independent manner; to inhibit fibrillation of p53 to p53 or other proteins; to inhibit proliferation of cells, including cancer cells, e.g. in solution or in cells in culture or in a subject; the ability to induce or initiate apoptosis; or the ability to shrink a tumor). Fibrillation, as used herein, refers to the formation of fiber or fibrils, such as amyloid fibrils.
Suitable active variants include peptidomimetic compounds (any compound containing non-peptidic structural elements that is capable of mimicking the biochemical and/or biological action(s) of a natural mimicked peptide, including, for example, those designed to mimic the structure and/or binding activity (such as, for example, hydrogen bonds and hydrophobic packing interactions) of the peptides according to the methods disclosed herein). Inhibitory peptides or CPP inhibitors of the invention, including active variants thereof, are sometimes referred to herein as “peptidic compounds” or “compounds.”
In one embodiment, active variants of the inhibitory peptides are shortened by 1-3 (e.g., 1, 2 or 3) amino acids at either the N-terminus, the C-terminus, or both of the starting inhibitory peptide. In another embodiment, the active variants are lengthened (extended) by 1, 2, 3 or 4 amino acids at the C-terminal end of the starting inhibitory peptide.
A variety of other types of active variants are encompassed. In some embodiments, amino acids other than the ones noted above in the consensus sequence are substituted. These amino acids can help protect the peptide inhibitors against proteolysis or otherwise stabilize the peptides, and/or contribute to desirable pharmacodynamic properties in other ways. In some embodiments, the non-natural amino acids allow an inhibitor to bind more tightly to the target because the side chains optimize hydrogen bonding and/or apolar interactions with it. In addition, non-natural amino acids offer the opportunity of introducing detectable markers, such as strongly fluorescent markers which can be used, e.g., to measure values such as inhibition constants. Also included are peptide mimetics, such as, e.g., peptoids, beta amino acids, N-ethylated amino acids, and small molecule mimetics.
In one embodiment, non-natural amino acids are substituted for amino acids in the sequence. More than 100 non-natural amino acids are commercially available. These include, for example,
Non-Natural Amino Acids which can Substitute for LEU:


    
    
        Fmoc-L-cyclohexylglycine 161321-36-4
        Fmoc-L-phenylglycine 102410-65-1
        Fmoc-4-hydroxy-D-phenylglycine 178119-93-2
        Fmoc-L-α-t-butylglycine 132684-60-7
        Fmoc-cyclopentyl-Gly-OH 220497-61-0
        Fmoc-L-2-indanylglycine 205526-39-2

Non-Natural Amino Acids which can Substitute for THR:

        Fmoc-Thr(tBu)-OH 71989-35-0
        Fmoc-(RS)-2-amino-3-hydroxy-3-methylbutanoic acid 105504-72-1

Non-Natural Amino Acids which can Substitute for ILE:

        Fmoc-allo-Ilc-OH 251316-98-0
        Boc-N-Me-allo-Ile-OH 136092-80-3
        Fmoc-Homoleu-OH 180414-94-2

Non-Natural Amino Acids which can Substitute for GLU:

        Fmoc-γ-carboxy-L-glutamic acid 111662-64-7
        Fmoc-L-α-aminosuberic acid 218457-76-2

Non-Natural Amino Acids which can Substitute for ARG:

        Fmoc-Nω-nitro-L-arginine 58111-94-7
        Fmoc-L-citrulline 133174-15-9

Non-Natural Amino Acids which can Substitute for TYR:

        Fmoc-3-amino-L-tyrosine 726181-70-0
        Fmoc-3-nitro-L-tyrosine 136590-09-5
        Fmoc-3-methoxy-L-tyrosine
        Fmoc-3-iodo-L-tyrosine 134486-00-3
        Fmoc-3-chloro-L-tyrosine 478183-58-3
        Fmoc-3,5-dibrimo-L-tyrosine 201484-26-6

Non-Natural Amino Acids which can Substitute for LYS:

        Fmoc-Lys(retro-Abz-Boc)-OH 159322-59-5
        Fmoc-Lys(Mca)-OH 386213-32-7
        Fmoc-(Nδ-4-methyltrityl)-L-ornithine 343770-23-0
        N-α-Fmoc-N-ε-(d-Biotin)-L-lysine 146987-10-2
    
    


In another embodiment, one or more (e.g. 1, 2, 3, 4, 5, 6, or 7) of the L-amino acids are substituted with a D amino acid.
In another embodiment, one or more (e.g. 1, 2, 3, 4, 5, 6, or 7) N-methylated residues are included in the peptide. Some representative such peptides include, e.g.,









(SEQ ID NO: 39)



Leu-Thr-(Nme)Arg-Ile-Tyr-Leu-Glu



 



(SEQ ID NO: 40)



Leu-Thr-Arg-Ile-(Nme)Tyr-Leu-Glu



 



(SEQ ID NO: 41)



Leu-Thr-Arg-Ile-Tyr-(Nme)Leu-Glu



 



(SEQ ID NO: 42)



Leu-Thr-Arg-(Nme)Ile-Tyr-Leu-Glu






An inhibitory peptide of the invention can comprise, e.g., L-amino acids, D-amino acids, non-natural amino acids, or combinations thereof.
In one embodiment, the inhibitor is a small molecule which has been designed by the methods described by Jiang et al. eLife 2013 (which is incorporated herein by reference, particularly with regard to this method), using the atomic structure of one of the fiber forming segments of p53 described herein as the basis for designing the inhibitor. Suitable small molecules that can be identified by this method of Jiang et al. will be evident to a skilled worker.
In one embodiment of the invention, a peptide of the invention is modified so that 1, 2 or 3 of its amino acids are substituted with an amino acid having a non-naturally occurring side chain, such as the non-natural amino acids discussed above, or with an amino acid having a side chain modified by cross-linking (e.g., through the epsilon amino group of a Lys residue) of a small molecule which has been designed by Jiang et al. eLife 2013. Some representative fiber-binding molecules are shown below. These active variants not only cap growing aggregates of p53 but also, via the modified side chains, bind to (clamp against) the sides of the steric zipper, thereby enhancing the inhibitory activity of the peptide.
Fiber-binding compounds designed by Jiang et al. eLife 2013 include:










To enhance the cell permeability of inhibitory peptides of the invention, they can be fused to any of a variety of cell penetrating peptides (CPP's). CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively. A third class of CPP's are the hydrophobic peptides, containing only apolar residues, with low net charge or have hydrophobic amino acid groups that are crucial for cellular uptake. Some typical CPP's that can be fused to an inhibitory peptide of the invention are provided in Table 2.







TABLE 2





Name Sequence


Reference - original or review







polyARG nR where 4 < n < 17 (e.g., n = 5, 6, 7, 8,


9, 10, 11, 12, 13, 14, 15 or 16) (SEQ ID NO: 43)


 


Wender, P.A., Mitchell, D.J., Pattabiraman, K.,


Pelkey, E.T., Steinman, L., and Rothbard, J.B.


(2000). The design, synthesis, and evaluation of


molecules that enable or enhance cellular uptake:


peptoid molecular transporters. Proc. Natl. Acad.


Sci. U. S. A. 97, 13003-8.


 


polyLYS nK where 4 < K < 17 (e.g., K = 5, 6, 7, 8,


9, 10, 11, 12, 13, 14, 15 or 16) (SEQ ID NO: 44)


 


D-polyARG nR where 4 < n < 17 (e.g., n = 5, 6, 7,


8, 9, 10, 11, 12, 13, 14, 15 or 16)


 


D-polyLYSnK where 4 < K < 17 (e.g., K = 5, 6, 7, 8,


9, 10, 11, 12, 13, 14, 15 or 16)


 


SynB1 RGGRLSYSRRRFSTSTGR (SEQ ID NO: 45)


 


SynB3 RRLSYSRRRF (SEQ ID NO: 46)


 


Penetratin RQIKIWFQNRRIVIKWKK (SEQ ID NO: 47)


 


Derossi, D., Joliot, A.H., Chassaing, G., and


Prochiantz, A. (1994). The third helix of the


Antennapedia homeodomain translocates through


biological mem-branes. J. Biol. Chem. 269, 10444-


50.


 


PenArg RQIRIWFQNRRMRWRR (SEQ ID NO: 48)


 


PenLys KQIKIWFQNKKMKWKK (SEQ ID NO: 49)


 


TatP59W GRKKRRQRRRPWQ (SEQ ID NO: 50)


 


Tat (48-60) GRKKRRQRRRPPQ (SEQ ID NO: 51)


 


Vives, E., Brodin, P., and Lebleu, B. (1997). A


truncated HIV-1 Tat protein basic domain rapidly


translocates through the plasma membrane and


accumulates in the cell nucleus. J. Biol. Chem.


272, 16010-7.


 


R9-Tat GRRRRRRRRRPPQ (SEQ ID NO: 52)


 


Futaki, S. (2002) Arginine-rich peptides: potential


for intracellular delivery of macromolecules and


the mystery of the translocation mechanisms. Int.


J. Pharm. 245, 1-7.


 


Tat YGRKKRRQRRR (SEQ ID NO: 53)


 


Vives, E., Brodin, P., and Lebleu, B. (1997). A


truncated HIV-1 Tat protein basic domain rapidly


translocates through the plasma membrane and


accumulates in the cell nucleus. J. Biol. Chem.


272, 16010-7.


 


D-Tat GRKKRRQRRRPPQ (SEQ ID NO: 51)


 


Futaki, S. (2002) Arginine-rich peptides: potential


for intracellular delivery of macromolecules and


the mystery of the translocation mechanisms. Int.


J. Pharm. 245, 1-7.


 


BMVGag(7-25) KMTRAQRRAAARRNRWTAR (SEQ ID NO: 54)


 


Futaki, S. (2002) Arginine-rich peptides: potential


for intracellular delivery of macromolecules and


the mystery of the translocation mechanisms. Int.


J. Pharm. 245, 1-7.


 


FHVCoat(35-49) RRRRNRTRRNRRRVR (SEQ ID NO: 55)


 


Futaki, S. (2002) Arginine-rich peptides: potential


for intracellular delivery of macromolecules and


the mystery of the translocation mechanisms. Int.


J. Pharm. 245, 1-7.


 


HTLV-II Rex(4-16) TRRQRTRRARRNR (SEQ ID NO: 56)


 


Futaki, S. (2002) Arginine-rich peptides: potential


for intracellular delivery of macromolecules and


the mystery of the translocation mechanisms. Int.


J. Pharm. 245, 1-7.


 


P22 N-(14-30) NAKTRRHERRRKLAIER (SEQ ID NO: 57)


 


pVEC LLIILRRRIRKQAHAHSK (SEQ ID NO: 58)


 


Elmquist, A., Lindgren, M., Bartfai, T., and


Langel, Ü. (2001). VE-cadherin-derived cell-


penetrating peptide, pVEC, with carrier functions.


Exp. Cell Res. 269,237-44.


 


Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID


NO: 59)


 


Pooga, M., Hällbrink, M., Zorko, M., and Langel, Ü.


(1998). Cell penetration by transportan. FASEB J.


12,67-77.


 


TP10 AGYLLGKINLKALAALAKKIL (SEQ ID NO: 60)


 


Soomets, U., Lindgren, M., Gallet, X., Hällbrink,


M., Elmquist, A., Balaspiri, L., Zorko, M., Pooga,


M., Brasseur, R., and Langel, Ü. (2000). Dele-


tion analogues of transportan. Biochim. Biophys.


Acta 1467,165-76.


 


PTD-4 PIRRRKKLRRLK (SEQ ID NO: 61)


 


PTD-5 RRQRRTSKLMKR (SEQ ID NO: 62)


 


Pep-1 ac-KETWWETWWTEWSQPKKKRKV-cya (SEQ ID NO: 63)


 


Pep-2 ac-KETWFETWFTEWSQPKKKRKV-cya (SEQ ID NO: 64)


 


Morris, MC, Chaloin, L, Choob, M, Archdeacon, J,


Heitz, F and Divita, G (2004). Combination of a new


generation of PNAs with a peptide-based carrier


enables efficient targeting of cell cycle


progression. Gene Ther 11: 757-764.


 


Pep-3 ac-KWFETWFTEWPKKRK-cya (SEQ ID NO: 65)


 


Morris, MC, Gros, E, Aldrian-Herrada, G, Choob, M,


Archdeacon, J, Heitz, F et al. (2007). A non-


covalent peptide-based carrier for in vivo delivery


of DNA mimics. Nucleic Acids Res 35: e49.


 


E N(1-22) MDAQTRRRERRAEKQAQWKAAN (SEQ ID NO: 66)


 


B 21 N-(12-29) TAKTRYKARRAELIAERR (SEQ ID NO: 67)


 


U2AF(142-153) SQMTRQARRLYV (SEQ ID NO: 68)


 


PRP6(129-144) TRRNKRNRIQEQLNRK (SEQ ID NO: 69)


 


MAP KLALKLALKLALALKLA (SEQ ID NO: 70)


 


SBP MGLGLHLLVLAAALQGAWSQPKKKRKV (SEQ ID NO: 71)


 


FBP GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 72)


 


MPG ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya (SEQ ID NO:


73)


 


Morris, MC, Vidal, P, Chaloin, L, Heitz, F and


Divita, G (1997). A new peptide vector for


efficient delivery of oligonucleotides into


mammalian cells. Nucleic Acids Res 25: 2730-2736.


 


MPG(ΔNLS) ac- GALFLGFLGAAGSTMGAWSQPKSKRKV-cya (SEQ


ID NO: 74)


 


REV(34-50) TRQARRNRRRRWRERQR (SEQ ID NO: 75)


 


Futaki, S. (2002) Arginine-rich peptides: potential


for intracellular delivery of macromolecules and


the mystery of the translocation mechanisms. Int.


J. Pharm. 245,1-7.


 


ACPPs from Jiang et al., PNAS 2004-lower case


indicates D-aa. The symbol “_” in some of these


sequences indicates a position at which any of a


variety of art-recognized protease cleavage sites


can be inserted:


 


EEEEEDDDDK_AXRRRRRRRRRXC (SEQ ID NO: 76)


 


EEEEEDDDDK_ARRRRRRRRRXC (SEQ ID NO: 77)


 


EDDDDK_AXRRRRRRRRRXC (SEQ ID NO: 78)


 


EEDDDDK_ARXRRXRRXRRXRRXC (SEQ ID NO: 79)


 


DDDDDDK_ARRRRRRRRRXC (SEQ ID NO: 80)


 


EEDDDDK_AXrrarrrrrXC:


 


eeeeeeXPLG_LAGrn-rn-rrrXc


 


eeeeeeXPLG_LAGrrrrrrrrrXc


 


UeeeeeeeeXPLG_LAGrrrrrrrrrXk


 


eeeeeeXPLG_LAGrrrrrrrrrXc


 


UeeeeeeXPLG_LAGrrrrrrraXc


 


UeeeeeeeeXPLG_LAGrrrn-rn-rXk


 


[11-kDa PEG]XeeeeeeeeeXPLG_LAGrrrurrrrXk


 


[11-kDa PEG]XeeeeeeeeeXLALGPGrarrrraXk


 


F1-XrrrrrrrrrXPLG_LAGeeeeeeee


 


F1-XrrrrrrrrrXSGRS_Aeeeeeeee


 


eeeeeeXSGRS_AXrrrrrrrrrXc


 


F1-rrrrrrrrrc-_-ceeeeee









In another embodiment, the CPP is polyD(1-16).
In general, it is advisable that the length of the CPP is rather short, e.g. less than about 30 amino acids, in order to improve stability and pharmacodynamic properties once the molecule enters a cell.
In some embodiments, the CPP is directly attached (fused) to a peptide of the invention. In other embodiments, it is desirable to separate the highly charged CPP from the inhibitor peptide with a linker, to allow the inhibitor to retain its activity. Any of a variety of linkers can be used. The size of the linker can range, e.g., from 1-7 or even more amino acids (e.g., 1, 2, 3, 4, 5, 6 or 7 amino acids). In some embodiments, the linker has sequences from the endogenous p53 sequence. For example, the linker can be GGMNRRPI (SEQ ID NO: 81), or a truncated version thereof having 1, 2, 3, 4 or 5 of the contiguous amino acids N-terminal to RPI fused to the inhibitory peptide. The RPI linker used in the experiments described herein is one such linker.
In some embodiments of the invention, the CPP inhibitor is further modified in order to target specific cancer types specifically. For example,
(1) One embodiment is a modification of the approach described by Roger Tsien and coworkers (Olson et al, PNAS 2010) using ACPPs, activatable CPPs that can only enter a cell after a proteolytic cleavage by a cancer specific protease. In this embodiment, an inhibitor is targeted to a specific cancer type by utilizing a sequence that is specific for those proteases primarily expressed by the cancer of interest.
(2) Another embodiment is a modification of the strategy proposed by Hatakeyama and colleagues (Hatakeyama et al, PNAS 2011). These authors obtained targeted cancer cell delivery using the carbohydrate mimetic peptide IF7 (sequence IFLLWQR (SEQ ID NO: 82)), which binds annexin 1, a cancer vasculature marker. In this embodiment, a suitable tumor vasculature marker binding peptide is fused it to a CPP inhibitor of the invention.
(3) In another embodiment, an inhibitor is conjugated to nanoparticle. Any of a variety of suitable nanoparticles will be evident to a skilled worker. These include, e.g., empty vault shells, liposomes, polymeric nanoparticles, dendrimers or the like.
In one embodiment of the invention, an inhibitory peptide or CPP inhibitor of the invention is isolated or purified, using conventional techniques such as the methods described herein. By “isolated” is meant separated from components with which it is normally associated, e.g., components present after the peptide is synthesized. An isolated peptide can be a cleavage product of a protein which contains the peptide sequence. A “purified” inhibitory peptide can be, e.g., greater than 90%, 95%, 98% or 99% pure.
In embodiments of the invention, the inhibitory peptide or CPP inhibitor is detectably labeled. Labeled peptides can be used, e.g., to better understand the mechanism of action and/or the cellular location of the inhibitory peptide. Suitable labels which enable detection (e.g., provide a detectable signal, or can be detected) are conventional and well-known to those of skill in the art. Suitable detectable labels include, e.g., radioactive active agents, fluorescent labels, and the like. Methods for attaching such labels to a protein, or assays for detecting their presence and/or amount, are conventional and well-known.
An inhibitory peptide or CPP inhibitor of the invention can be synthesized (e.g., chemically or by recombinant expression in a suitable host cell) by any of a variety of art-recognized methods. In order to generate sufficient quantities of an inhibitory peptide for use in a method of the invention, a practitioner can, for example, using conventional techniques, generate nucleic acid (e.g., DNA) encoding the peptide and insert it into an expression vector, in which the sequence is under the control of an expression control sequence such as a promoter or an enhancer, which can then direct the synthesis of the peptide. For example, one can (a) synthesize the DNA de novo, with suitable linkers at the ends to clone it into the vector; (b) clone the entire DNA sequence into the vector; or (c) starting with overlapping oligonucleotides, join them by conventional PCR-based gene synthesis methods and insert the resulting DNA into the vector. Suitable expression vectors (e.g., plasmid vectors, viral, including phage, vectors, artificial vectors, yeast vectors, eukaryotic vectors, etc.) will be evident to skilled workers, as will methods for making the vectors, inserting sequences of interest, expressing the proteins encoded by the nucleic acid, and isolating or purifying the expressed proteins.
Another aspect of the invention is a pharmaceutical composition comprising one or more of the inhibitory peptides or CPP inhibitors and a pharmaceutically acceptable carrier. The components of the pharmaceutical composition may be detectably labeled, e.g. with a radioactive or fluorescent label, or with a label that is suitable for detection by positron emission spectroscopy (PET). In some embodiments, the inhibitory peptide or CPP inhibitor is present in an effective amount for the desired purpose.
“Pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use. For example, “pharmaceutically acceptable salts” of a compound means salts that are pharmaceutically acceptable, as defined herein, and that possess the desired pharmacological activity of the parent compound.
Other aspects of the invention include: a polynucleotide encoding an inhibitory peptide of the invention, optionally linked to a CPP sequence, which is optionally separated from the inhibitory peptide by a suitable linker. In embodiments of the invention, the polynucleotide is operably linked to a regulatory control sequence (e.g., a promoter or an enhancer) to facilitate production of the encoded protein following introduction (e.g. by transfection) into a suitable cell; a cell comprising the expression vector; and a method of making an inhibitory peptide of the invention comprising cultivating the cell and harvesting the polypeptide thus generated.
As used throughout this application, “about” means plus or minus 5% of a value.
Another aspect of the invention is a kit for carrying out any of the methods described herein. The kit may comprise a suitable amount of an inhibitory peptide or CPP inhibitor of the invention; reagents for generating the peptide or CPP inhibitor; reagents for assays to measure their functions or activities; or the like. Kits of the invention may comprise instructions for performing a method. Other optional elements of a kit of the invention include suitable buffers, media components, or the like; a computer or computer-readable medium providing the structural representation of one of the crystal structures described herein; containers; or packaging materials. Reagents for performing suitable controls may also be included. The reagents of the kit may be in containers in which the reagents are stable, e.g., in lyophilized form or stabilized liquids. The reagents may also be in single use form, e.g., in single reaction form for administering to a subject.
One aspect of the invention is a computer-implemented method for designing an inhibitory peptide which inhibits aggregation of p53, using a method as described herein. For example, the method can comprise identifying (e.g. with a computer) a template peptide sequence comprising a zipper-forming sequence of the p53 segments TIITLE (SEQ ID NO: 20) or LTIITLE (SEQ ID NO: 21) or a mirror of the zipper-forming sequence (wherein the zipper-forming sequence aggregates into a steric zipper); designing on a computer at least one complementary peptide sequence that forms favorable steric and energetic intermolecular interactions with the template peptide sequence, wherein the interactions occur at one or both of the upper or lower ends of the steric zipper; and identifying (e.g. with a computer) a candidate inhibitory peptidic compound selected form the group consisting of the complementary sequence, a mirror of the complementary sequence, a peptide mimetic of the complementary sequence and a peptide mimetic of the mirror of the complementary sequence. Details of this type of method are described in the patent which issued from U.S. Ser. No. 12/702,175, which is incorporated by reference herein in its entirety, particularly with regard to the method for designing an inhibitory peptidic compound which inhibits aggregation of an amyloid-forming target polypeptide of interest.
In embodiments of the invention, an inhibitory compound (e.g. a peptidic compound) designed by this method is synthesized and screened for the ability to bind to and/or to inhibit aggregation of p53, e.g., using one of the methods described herein.
Characterization of candidate inhibitory peptides or CPP inhibitors of the invention can be carried out by any of a variety of conventional methods. For example, the peptides or CPP inhibitors can be assayed for the ability to reduce or inhibit p53 aggregation or to re-activate p53. Functional p53 can then, for example, lead to apoptosis in cells. The assays can be carried out in vitro or in vivo.
One representative in vitro assay is the thioflavin T assay shown in FIG. 3 (Naiki, H., Higuchi, K., Hosokawa, M., and Takeda, T. (1989) Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavine T. Anal. Biochem., 177, 244-249). The assay can be performed using a target sequence peptide, such as LTIITLE (SEQ ID NO: 21), or it can be performed using full-length p53 (such as recombinant p53). In both cases, the peptide (e.g. LTIITLE) (SEQ ID NO: 21) or the p53 is placed in solution with thioflavin T and increasing concentrations of inhibitor. The assay is performed in a multiwall plate (e.g. a 384 well plate). This dye is amyloid specific and is only fluorescent when bound to amyloid aggregates. The fluorescence is measured over time, e.g. with a plate reader. Inhibition is detected as a delay in the onset of aggregation and/or less total amount of aggregates formed.
The following assays are among the conventional functional assays which can be performed in cells:
1. Reduction in p53 Aggregation
p53 aggregation is measured in cancer cells, as a measure of p53 inactivation (Xu et al, 2011; Lasagna-Reeves et al, 2013). Screens are performed for changes in the total amount of aggregates present with or without inhibitors using, e.g., one of the following conventional methods:

    
    
        Immunostain of the cells using amyloid conformation specific antibodies commercially available, e.g., A11 and OC
        Stain of cells with amyloid specific dyes, such as Thioflavin T and Congo Red
        Dot blot on cell lysates using OC or A11 antibodies
        Native page gels coupled to western blots on cell lysates using p53 specific antibodies to check for the presence/absence of high-molecular weight p53 aggregates
        Immunostain of the cells using different commercially available p53 antibodies that can discriminate between abnormally or normally folded p53
    
    


2. Re-Activation of p53 Function
As mentioned, p53 function is typically inhibited by aggregation (Xu et al, 2011; Lasagna-Reeves et al, 2013). p53 inactivation and re-activation are tested in the presence of different concentrations of inhibitors with the following conventional approaches:

    
    
        Restoration of p53 transcriptional activity measured by quantitating the transcripts of several p53 targets by RT-PCR or RNAseq
        Restoration of p53 transcriptional activity measured by detecting via Western blot several p53 targets at the protein level
        Ability of the inhibitor to arrest cell proliferation screening via soft agarose culture colony forming assay
        Ability of the inhibitor to arrest cell proliferation screening via BrdU incorporation or reduction in Ki67 stain
        Ability of the inhibitor to induce cell death measured via MTT or MTS assay
        Ability of the inhibitor to induce apoptosis measured via caspase apoptosis kit or Annexin V stain coupled to FACS
    
    


Cells are treated with the inhibitors alone or in combination with traditional chemotherapy as well as other chemotherapeutic molecules such as other targeted agents against kinases or other molecules. Any of the preceding methods can further comprise testing candidate inhibitory peptides for, e.g., their ability to bind to p53, to inhibit p53 fibrillation, or to sensitize cancer cells to chemotherapy, in vitro or in vivo.
One aspect of the invention is a method for reducing or inhibiting p53 aggregation, comprising contacting p53 amyloid protofilaments with an effective amount of one or more of the inhibitory peptides of CPP inhibitors of the invention. Such a method can be carried out in vitro (in solution) or in vivo (e.g. cells in culture or in a subject).
Another aspect of the invention is a method for restoring the conformation of a p53 protein molecule having an aberrant conformation. An “aberrant conformation,” as used herein, refers to a conformation which is different from the wild type conformation, and which results in a loss of function of the molecule. For example, p53 with an aberrant conformation can lose the ability to inhibit cell proliferation (e.g. of cancer cells), to induce or initiate apoptosis, or to shrink a tumor. Such aberrant conformation is sometimes referred to herein as pathological conformation. The aberrant conformation can take the form of amyloid aggregates or fibers (fibrils) of p53 molecules with other p53 molecules or with other proteins. Alternatively, the aberrant conformation can take the form of misfolding (e.g., partial or complete unfolding) of the p53 protein due to mutations or other factors. Without wishing to be bound by any particular mechanism, it is suggested that the misfolding-promoting mutations destabilize the native p53 structure causing the hydrophobic adhesive segment p53252-258 to get solvent exposure. The segments rapidly interact with other p53 molecules resulting in protein aggregation and inactivation. It is suggested that by generating an aggregation inhibitor which blocks these segments from interacting with each other, the aggregation process is halted and/or the inhibitor also chaperones the misfolded p53 into an active conformation, thereby potentially restoring a pool of functional and soluble p53, capable of driving a cell death response.
In this method for restoring the conformation of a p53 protein having an aberrant conformation, the p53 molecule having the aberrant conformation is contacted with an effective amount of an inhibitory peptide or a CPP inhibitor of the invention. The contacted p53 molecule has a restored conformation, and exhibits a restored or reactivated biological or biochemical activity selected from, e.g., induction or initiation of apoptosis, inhibition of cell proliferation and/or shrinkage of a tumor.
Another aspect of the invention is a method for reactivating or restoring a biological or biochemical activity (function) of a p53 protein which results from aberrant conformation of the p53 protein. The method comprises contacting the p53 protein molecule having an aberrant conformation with an effective amount of an inhibitor peptide or CPP inhibitor of the invention, wherein the biological or biochemical activity of the p53 molecule is induction or initiation of apoptosis and/or is inhibition of cell proliferation and/or is inducing shrinkage of a tumor. As a result of contacting the p53 protein having the aberrant conformation, the lost biological or biochemical activity of the p53 molecule is reactivated or restored.
Another aspect of the invention is a method for inhibiting or preventing a loss of a biological or biochemical activity (function), of a p53 protein which results from aberrant conformation of the p53 protein. The method comprises contacting the p53 protein molecule having an aberrant conformation with an effective amount of an inhibitor peptide or CPP inhibitor of the invention, wherein the biological or biochemical activity of the p53 molecule is induction or initiation of apoptosis and/or is inhibition of cell proliferation and/or is inducing shrinkage of a tumor. As a result of contacting the p53 protein having the aberrant conformation, the loss of activity of the p53 molecule is inhibited or prevented.
Another aspect of the invention is a method for treating a subject having a disease or condition which is mediated by loss of function of p53, such as a cancer or a tumor in which p53 has an abnormal conformation (e.g. is aggregated or misfolded). That is, the cancer is associated with p53 having an aberrant conformation. The method comprises administering to the subject an effective amount of one or more CPPs of the invention. In some embodiments, a cocktail of one of more of the CPP inhibitors is used. In some embodiments, the CPP inhibitor is used in conjunction with a conventional chemotherapeutic drug or regimen, in order to enhance the response of the subject the chemotherapeutic drug or regimen. Typical such chemotherapeutic drugs or regimens include, e.g., paclitaxel, taxol, gemcitabine, cisplatin, carboplatin, rapamycin, doxorubicin, 5-fluorouracil, trastuzumab, imatinib, sorafenib, vemurafenib, dasatinib, crizotinib, gefitinib, erlotinib, carfilzomib, PRIMA1-MET, MI-773, nutlin, and 17AAG.
An “effective amount” of a compound or pharmaceutical composition of the invention is an amount that can elicit a measurable amount of a desired outcome, e.g. inhibition of p53 aggregation; for a diagnostic assay, an amount that can detect a target of interest, such as a p53 aggregate; or in a method of treatment, an amount that can reduce or ameliorate, by a measurable amount, a symptom of the disease or condition that is being treated.
A “subject” can be any subject (patient) having p53 with an aberrant conformation (e.g., the p53 is aggregated or misfolded), in which the condition or disease can be treated by a method of the present invention. In one embodiment of the invention, the subject has a cancer, such as one of the cancers described in Soussi et al., 2006 that are associated with mutant p53. Typical subjects include vertebrates, such as mammals, including laboratory animals, dogs, cats, non-human primates and humans.
The inhibitors of the invention can be formulated as pharmaceutical compositions in a variety of forms adapted to the chosen route of administration, for example, orally, nasally, intraperitoneally, or parenterally, by intravenous, intramuscular, topical or subcutaneous routes, or by injection into tissue.
Suitable oral forms for administering the compounds include lozenges, troches, tablets, capsules, effervescent tablets, orally disintegrating tablets, floating tablets designed to increase gastric retention times, buccal patches, and sublingual tablets.
The compounds of the invention may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier, or by inhalation or insufflation. They may be enclosed in coated or uncoated hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the compounds may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. For compositions suitable for administration to humans, the term “excipient” is meant to include, but is not limited to, those ingredients described in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed. (2006) (hereinafter Remington's).
The compounds may be combined with a fine inert powdered carrier and inhaled by the subject or insufflated. Such compositions and preparations should contain at least 0.1% compounds. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of a given unit dosage form.
The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.
Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
In addition, the compounds may be incorporated into sustained-release preparations and devices. For example, the compounds may be incorporated into time release capsules, time release tablets, and time release pills. In some embodiments, the composition is administered using a dosage form selected from the group consisting of effervescent tablets, orally disintegrating tablets, floating tablets designed to increase gastric retention times, buccal patches, and sublingual tablets.
The compounds may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the compounds can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.
The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the compounds which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
Sterile injectable solutions are prepared by incorporating the compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
For topical administration, the compounds may be applied in pure form. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Other solid carriers include conventional nontoxic polymeric nanoparticles or microparticles. Useful liquid carriers include water, alcohols or glycols or water/alcohol/glycol blends, in which the compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
Useful dosages of the compounds of formula 1 can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.
For example, the concentration of the compounds in a liquid composition, such as a lotion, can be from about 0.1-25% by weight, or from about 0.5-10% by weight. The concentration in a semi-solid or solid composition such as a gel or a powder can be about 0.1-5% by weight, or about 0.5-2.5% by weight.
Effective dosages and routes of administration of agents of the invention are conventional. The exact amount (effective dose) of the agent will vary from subject to subject, depending on, for example, the species, age, weight and general or clinical condition of the subject, the severity or mechanism of any disorder being treated, the particular agent or vehicle used, the method and scheduling of administration, and the like. A therapeutically effective dose can be determined empirically, by conventional procedures known to those of skill in the art. See, e.g, The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds., Macmillan Publishing Co., New York. For example, an, effective dose can be estimated initially either in cell culture assays or in suitable animal models. The animal model may also be used to determine the appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans. A therapeutic dose can also be selected by analogy to dosages for comparable therapeutic agents.
The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is prophylactic). Treatment may involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years.
In general, however, a suitable dose will be in the range of from about 0.001 to about 100 mg/kg, e.g., from about 0.01 to about 100 mg/kg of body weight per day, such as above about 0.1 mg per kilogram, or in a range of from about 1 to about 10 mg per kilogram body weight of the recipient per day. For example, a suitable dose may be about 1 mg/kg, 10 mg/kg, or 50 mg/kg of body weight per day.
The compounds are conveniently administered in unit dosage form; for example, containing 0.05 to 10000 mg, 0.5 to 10000 mg, 5 to 1000 mg, or about 100 mg of active ingredient per unit dosage form. In some embodiments, the dosage unit contains about 1 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 750 mg, or about 1000 mg of active ingredient.
The invention also includes computer-related embodiments, such as a computer-readable medium, providing the structural representation of one of the crystal structures described herein, or for storing and/or evaluating the assay results described herein.
The storage medium (computer readable medium) in which the p53 structural representation is provided may be, e.g., random-access memory (RAM), read-only memory (ROM e.g. CDROM), a diskette, magnetic storage media, hybrids of these categories, etc. The storage medium may be local to the computer, or may be remote (e.g. a networked storage medium, including the internet). The present invention also provides methods of producing computer readable databases containing coordinates of 3-D structures of the invention; computer readable media embedded with or containing information regarding the 3-D structure of the invention; a computer programmed to carry out a method of the invention (e.g. for characterizing the structure of a p53 segment or for designing and/or selecting peptidic inhibitors), and data carriers having a program saved thereon for carrying out a method as described herein.
Any suitable computer can be used in the present invention.
An exemplary architecture for implementing a computing device in accordance with one or more embodiments, which may be used to implement any of the computing devices discussed herein, or any other computer system or computing device component thereof is described below. It will be appreciated that other devices that can be used with this computing device, such as a client or a server, may be similarly configured. The computing device may include a bus, a processor, a memory, a read only memory (ROM), a storage device, an input device, an output device, and a communication interface.
The bus may include one or more interconnects that permit communication among the components of the computing device. The processor may include any type of processor, microprocessor, or processing logic that may interpret and execute instructions (e.g., a field programmable gate array (FPGA)). The processor may include a single device (e.g., a single core) and/or a group of devices (e.g., multi-core). The memory may include a random access memory (RAM) or another type of dynamic storage device that may store information and instructions for execution by the processor. The memory may also be used to store temporary variables or other intermediate information during execution of instructions by the processor.
The ROM may include a ROM device and/or another type of static storage device that may store static information and instructions for the processor. The storage device may include a magnetic disk and/or optical disk and its corresponding drive for storing information and/or instructions. The storage device may include a single storage device or multiple storage devices, such as multiple storage devices operating in parallel. Moreover, the storage device may reside locally on the computing device and/or may be remote with respect to a server and connected thereto via network and/or another type of connection, such as a dedicated link or channel.
The input device may include any mechanism or combination of mechanisms that permit an operator to input information to the computing device, such as a keyboard, a mouse, a touch sensitive display device, a microphone, a pen-based pointing device, and/or a biometric input device, such as a voice recognition device and/or a finger print scanning device. The output device may include any mechanism or combination of mechanisms that outputs information to the operator, including a display, a printer, a speaker, etc.
The communication interface may include any transceiver-like mechanism that enables the computing device to communicate with other devices and/or systems, such as a client, a server, a license manager, a vendor, etc. For example, the communication interface may include one or more interfaces, such as a first interface coupled to a network and/or a second interface coupled to a license manager. Alternatively, the communication interface may include other mechanisms (e.g., a wireless interface) for communicating via a network, such as a wireless network. In one implementation, the communication interface may include logic to send code to a destination device, such as a target device that can include general purpose hardware (e.g., a personal computer form factor), dedicated hardware (e.g., a digital signal processing (DSP) device adapted to execute a compiled version of a model or a part of a model), etc.
The computing device may perform certain functions in response to the processor executing software instructions contained in a computer-readable medium, such as memory. In alternative embodiments, hardwired circuitry may be used in place of or in combination with software instructions to implement features consistent with principles of the disclosure. Thus, implementations consistent with principles of the disclosure are not limited to any specific combination of hardware circuitry and software.
Exemplary embodiments may be embodied in many different ways as a software component. For example, it may be a stand-alone software package, a combination of software packages, or it may be a software package incorporated as a “tool” in a larger software product. It may be downloadable from a network, for example, a website, as a stand-alone product or as an add-in package for installation in an existing software application. It may also be available as a client-server software application, or as a web-enabled software application. It may also be embodied as a software package installed on a hardware device.
EXAMPLES
Example I—Design and Characterization of Inhibitory Peptides and CPP Inhibitors
Based on the determination of the atomic structure of a fiber forming segment of p53, the inventors rationally designed a series of inhibitors that diminish aggregation in vitro. The inventors designed peptidic inhibitors to “cap” the growing aggregates of p53. Using the ZipperDB algorithm (Goldschmidt et al. 2010), the inventors identified the crystallizable amyloid-forming segments in the region that was also reported to be important for mutant p53 aggregation by Xu et al., 2011, who identified the sequence ILTIITL (SEQ ID NO: 2). The present inventors chemically synthesized the p53252-258 and p53253-258 segments, crystallized them and determined their three-dimensional structures by micro-crystallography (FIG. 1). The structures displayed a typical steric zipper architecture, with parallel in-register β-strands and β-sheets interdigitating via hydrophobic side chains in a “face-to-back” (for p53252-258) and “face-to-face” (for p53253-258) orientation (Sawaya et al, Nature, 2007).
The atomic coordinates of the p53253-258 (TIITLE) (SEQ ID NO: 20) and p53252-258 (LTIITLE) (SEQ ID NO: 21) structures are shown in Tables 3 and 4, respectively.







TABLE 3







REMARK Date 2012-02-17 Time 19:46:52 PST −0800 (1329536812.65 s)


REMARK PHENIX refinement


REMARK


REMARK ****************** INPUT FILES AND LABELS *******************


REMARK Reflections:


REMARK file name : t6e_all.mtz


REMARK labels: [‘IMEAN, SIGIMEAN’]


REMARK R-free flags:


REMARK file name: t6e_all.mtz


REMARK label: FreeR_flag


REMARK test_flag_value: 0


REMARK Model file name(s):


REMARK /home/absoriaga/APS/T6E/1.59A/build006_001-coot-0.pdb


REMARK


REMARK ******************** REFINEMENT SUMMARY: QUICK FACTS *********


REMARK Start: r_work = 0.1678 r_free = 0.1910 bonds = 0.009 angles =


1.861


REMARK Final: r_work = 0.1637 r_free = 0.1925 bonds = 0.009 angles =


1.861


REMARK


*********************************************************************


REMARK


REMARK Rigid body refinement target: auto


REMARK Information about total rigid body shift of selected groups:


REMARK rotation (deg) translation (A)


REMARK xyz total xyz total


REMARK group 1: −0.342 −0.301 0.287 0.54 −0.02 0.03 0.02 0.04


REMARK group 2: −0.000 0.000 0.000 0.00 0.01 −0.02 0.01 0.02


REMARK group 3: −0.029 −0.173 −0.204 0.27 0.01 −0.01 0.05 0.05


REMARK ****************** REFINEMENT STATISTICS STEP BY STEP *******


REMARK leading digit, like 1_, means number of macro-cycle


REMARK 0: statistics at the very beginning when nothing is done yet


REMARK 1_bss: bulk solvent correction and/or (anisotropic) scaling


REMARK 1_rbr: rigid body refinement


REMARK --------------------------------------------------------------


REMARK R-factors, x-ray target values and norm of gradient of x-ray


target


REMARK stage r-work r-free xray_target_w xray_target_t


REMARK 0: 0.1702 0.1913 1.200965e+00 1.236068e+00


REMARK 1_bss: 0.1678 0.1910 1.166180e+00 1.232305e+00


REMARK 1_ohs: 0.1678 0.1910 1.166180e+00 1.232305e+00


REMARK 1_rbr: 0.1678 0.1904 1.169224e+00 1.233750e+00


REMARK 1_adp: 0.1659 0.1935 1.164208e+00 1.257252e+00


REMARK 2_bss: 0.1657 0.1933 1.164333e+00 1.256966e+00


REMARK 2_ohs: 0.1657 0.1933 1.164333e+00 1.256966e+00


REMARK 2_adp: 0.1634 0.1913 1.153688e+00 1.227758e+00


REMARK 3_bss: 0.1634 0.1911 1.153695e+00 1.227577e+00


REMARK 3_ohs: 0.1634 0.1911 1.153695e+00 1.227577e+00


REMARK 3_adp: 0.1637 0.1925 1.158500e+00 1.245755e+00


REMARK 3_ohs: 0.1637 0.1925 1.158500e+00 1.245755e+00


REMARK --------------------------------------------------------------


REMARK stage k_sol b_sol b11 b22 b33 b12 b13 b23


REMARK 0: 0.400 80.000 −2.385 0.077 2.308 −0.000 −0.841 0.000


REMARK 1_bss: 0.444 141.213 −2.385 0.077 2.308 −0.000 −0.841 −0.000


REMARK 1_ohs: 0.444 141.213 −2.385 0.077 2.308 −0.000 −0.841 −0.000


REMARK 1_rbr: 0.400 80.000 −2.385 0.077 2.308 −0.000 −0.841 −0.000


REMARK 1_adp: 0.400 80.000 −2.385 0.077 2.308 −0.000 −0.841 −0.000


REMARK 2_bss: 0.400 80.000 −2.399 0.015 2.229 −0.000 −0.834 0.000


REMARK 2_ohs: 0.400 80.000 −2.399 0.015 2.229 −0.000 −0.834 0.000


REMARK 2_adp: 0.400 80.000 −2.399 0.015 2.229 −0.000 −0.834 0.000


REMARK 3_bss: 0.400 80.000 −2.407 −0.032 2.194 −0.000 −0.819 0.000


REMARK 3_ohs: 0.400 80.000 −2.407 −0.032 2.194 −0.000 −0.819 0.000


REMARK 3_adp: 0.400 80.000 −2.407 −0.032 2.194 −0.000 −0.819 0.000


REMARK 3_ohs: 0.400 80.000 −2.407 −0.032 2.194 −0.000 −0.819 0.000


REMARK ---------------------------------------------------------------


REMARK stage <pher> fom alpha beta


REMARK 0: 15.419 0.8841 0.0567 1.306


REMARK 1_bss: 15.363 0.8845 0.0567 1.295


REMARK 1_ohs: 15.363 0.8845 0.0567 1.295


REMARK 1_rbr: 15.338 0.8848 0.0568 1.298


REMARK 1_adp: 15.919 0.8793 0.0578 1.377


REMARK 2_bss: 15.892 0.8796 0.0577 1.375


REMARK 2_ohs: 15.892 0.8796 0.0577 1.375


REMARK 2_adp: 15.280 0.8856 0.0576 1.290


REMARK 3_bss: 15.263 0.8858 0.0575 1.289


REMARK 3_ohs: 15.263 0.8858 0.0575 1.289


REMARK 3_adp: 15.698 0.8817 0.0577 1.347


REMARK 3_ohs: 15.698 0.8817 0.0577 1.347


REMARK ---------------------------------------------------------------


REMARK stage angl bond chir dihe plan repu geom_target


REMARK 0: 1.861 0.009 0.072 10.557 0.007 4.221 3.0983e−01


REMARK 1_bss: 1.861 0.009 0.072 10.557 0.007 4.221 3.0983e−01


REMARK 1_ohs: 1.861 0.009 0.072 10.557 0.007 4.221 3.0983e−01


REMARK 1_rbr: 1.861 0.009 0.072 10.557 0.007 4.221 3.0983e−01


REMARK 1_adp: 1.861 0.009 0.072 10.557 0.007 4.221 3.0983e−01


REMARK 2_bss: 1.861 0.009 0.072 10.557 0.007 4.221 3.0983e−01


REMARK 2_ohs: 1.861 0.009 0.072 10.557 0.007 4.221 3.0983e−01


REMARK 2_adp: 1.861 0.009 0.072 10.557 0.007 4.221 3.0983e−01


REMARK 3_bss: 1.861 0.009 0.072 10.557 0.007 4.221 3.0983e−01


REMARK 3_ohs: 1.861 0.009 0.072 10.557 0.007 4.221 3.0983e−01


REMARK 3_adp: 1.861 0.009 0.072 10.557 0.007 4.221 3.0983e−01


REMARK 3_ohs: 1.861 0.009 0.072 10.557 0.007 4.221 3.0983e−01


REMARK ---------------------------------------------------------------


REMARK Maximal deviations:


REMARK stage angl bond chir dihe plan repu |grad|


REMARK 0: 9.207 0.052 0.183 22.762 0.017 1.935 7.3928e−01


REMARK 1_bss: 9.207 0.052 0.183 22.762 0.017 1.935 7.3928e−01


REMARK 1_ohs: 9.207 0.052 0.183 22.762 0.017 1.935 7.3928e−01


REMARK 1_rbr: 9.207 0.052 0.183 22.762 0.017 1.935 7.3928e−01


REMARK 1_adp: 9.207 0.052 0.183 22.762 0.017 1.935 7.3928e−01


REMARK 2_bss: 9.207 0.052 0.183 22.762 0.017 1.935 7.3928e−01


REMARK 2_ohs: 9.207 0.052 0.183 22.762 0.017 1.935 7.3928e−01


REMARK 2_adp: 9.207 0.052 0.183 22.762 0.017 1.935 7.3928e−01


REMARK 3_bss: 9.207 0.052 0.183 22.762 0.017 1.935 7.3928e−01


REMARK 3_ohs: 9.207 0.052 0.183 22.762 0.017 1.935 7.3928e−01


REMARK 3_adp: 9.207 0.052 0.183 22.762 0.017 1.935 7.3928e−01


REMARK 3_ohs: 9.207 0.052 0.183 22.762 0.017 1.935 7.3928e−01


REMARK ---------------------------------------------------------------


---------


REMARK |-----overall-----|---macromolecule----|------solvent-------|


REMARK stage b_max b_min b_ave b_max b_min b_ave b_max b_min b_ave


REMARK 0: 18.10 2.58 6.49 15.36 2.58 6.16 18.10 11.08 14.59


REMARK 1_bss: 18.10 2.58 6.49 15.36 2.58 6.16 18.10 11.08 14.59


REMARK 1_ohs: 18.10 2.58 6.49 15.36 2.58 6.16 18.10 11.08 14.59


REMARK 1_rbr: 18.10 2.58 6.49 15.36 2.58 6.16 18.10 11.08 14.59


REMARK 1_adp: 19.05 2.88 7.12 16.06 2.88 6.80 19.05 10.85 14.95


REMARK 2_bss: 19.05 2.88 7.12 16.06 2.88 6.80 19.05 10.85 14.95


REMARK 2_ohs: 19.05 2.88 7.12 16.06 2.88 6.80 19.05 10.85 14.95


REMARK 2_adp: 25.77 2.48 7.00 15.92 2.48 6.46 25.77 14.73 20.25


REMARK 3_bss: 25.77 2.48 7.00 15.92 2.48 6.46 25.77 14.73 20.25


REMARK 3_ohs: 25.77 2.48 7.00 15.92 2.48 6.46 25.77 14.73 20.25


REMARK 3_adp: 26.28 2.54 7.43 16.68 2.54 6.90 26.28 14.91 20.59


REMARK 3_ohs: 26.28 2.54 7.43 16.68 2.54 6.90 26.28 14.91 20.59


REMARK ---------------------------------------------------------------


---------


REMARK stage Deviation of refined


REMARK model from start model


REMARK max min mean


REMARK 0: 0.000 0.000 0.000


REMARK 1_bss: 0.000 0.000 0.000


REMARK 1_ohs: 0.000 0.000 0.000


REMARK 1_rbr: 0.000 0.000 0.000


REMARK 1_adp: 0.000 0.000 0.000


REMARK 2_bss: 0.000 0.000 0.000


REMARK 2_ohs: 0.000 0.000 0.000


REMARK 2_adp: 0.000 0.000 0.000


REMARK 3_bss: 0.000 0.000 0.000


REMARK 3_ohs: 0.000 0.000 0.000


REMARK 3_adp: 0.000 0.000 0.000


REMARK 3_ohs: 0.000 0.000 0.000


REMARK ---------------------------------------------------------------


---------


REMARK MODEL CONTENT.


REMARK ELEMENT ATOM RECORD COUNT OCCUPANCY SUM


REMARK C 31 31.00


REMARK Zn 1 1.00


REMARK O 13 13.00


REMARK N 6 6.00


REMARK TOTAL 51 51.00


REMARK ---------------------------------------------------------------


--------


REMARK r_free_flags.md5.hexdigest 250b0898748f0b3eeb9579b8c7c1d593


REMARK


REMARK IF THIS FILE IS FOR PDB DEPOSITION: REMOVE ALL FROM THIS LINE UP.


REMARK 3


REMARK 3 REFINEMENT.


REMARK 3 PROGRAM: PHENIX (phenix.refine: 1.7.3_928)


REMARK 3 AUTHORS: Adams, Afonine, Chen, Davis, Echols, Gildea, Gopal,


REMARK 3 : Grosse-Kunstleve, Headd, Hung, Immormino, Ioerger, McCoy,


REMARK 3 : McKee, Moriarty, Pai, Read, Richardson, Richardson, Romo,


REMARK 3 : Sacchettini, Sauter, Smith, Storoni, Terwilliger, Zwart


REMARK 3


REMARK 3 REFINEMENT TARGET: ML


REMARK 3


REMARK 3 DATA USED IN REFINEMENT.


REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS): 1.576


REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS): 21.494


REMARK 3 MIN (FOBS/SIGMA_FOBS): 1.43


REMARK 3 COMPLETENESS FOR RANGE (%): 93.38


REMARK 3 NUMBER OF REFLECTIONS: 635


REMARK 3 NUMBER OF REFLECTIONS (NON-ANOMALOUS): 635


REMARK 3


REMARK 3 FIT TO DATA USED IN REFINEMENT.


REMARK 3 R VALUE (WORKING + TEST SET): 0.1666


REMARK 3 R VALUE (WORKING SET): 0.1637


REMARK 3 FREE R VALUE: 0.1925


REMARK 3 FREE R VALUE TEST SET SIZE (%): 10.24


REMARK 3 FREE R VALUE TEST SET COUNT: 65


REMARK 3


REMARK 3 FIT TO DATA USED IN REFINEMENT (IN BINS).


REMARK 3 BIN RESOLUTION RANGE COMPL. NWORK NFREE RWORK RFREE


REMARK 3 1 21.4963-1.5756 0.93 570 65 0.1637 0.1925


REMARK 3


REMARK 3 BULK SOLVENT MODELLING.


REMARK 3 METHOD USED: FLAT BULK SOLVENT MODEL


REMARK 3 SOLVENT RADIUS: 1.00


REMARK 3 SHRINKAGE RADIUS: 0.73


REMARK 3 GRID STEP FACTOR: 4.00


REMARK 3 K_SOL: 0.400


REMARK 3 B_SOL: 80.000


REMARK 3


REMARK 3 ERROR ESTIMATES.


REMARK 3 COORDINATE ERROR (MAXIMUM-LIKELIHOOD BASED): 0.10


REMARK 3 PHASE ERROR (DEGREES, MAXIMUM-LIKELIHOOD BASED): 15.70


REMARK 3


REMARK 3 OVERALL SCALE FACTORS.


REMARK 3 SCALE = SUM(|F_OBS|*|F_MODEL|)/SUM(|F_MODEL|**2): 0.0589


REMARK 3 ANISOTROPIC SCALE MATRIX ELEMENTS (IN CARTESIAN BASIS).


REMARK 3 B11: −2.4068


REMARK 3 B22: −0.0323


REMARK 3 B33: 2.1936


REMARK 3 B12: −0.0000


REMARK 3 B13: −0.8190


REMARK 3 B23: 0.0000


REMARK 3


REMARK 3 R FACTOR FORMULA.


REMARK 3 R = SUM(||F_OBS| − SCALE*|F_MODEL||)/SUM(|F_OBS|)


REMARK 3


REMARK 3 TOTAL MODEL STRUCTURE FACTOR (F_MODEL).


REMARK 3 F_MODEL = FB_CART * (F_CALC_ATOMS + F_BULK)


REMARK 3 F_BULK = K_SOL * EXP(−B_SOL * S**2/4) * F_MASK


REMARK 3 F_CALC_ATOMS = ATOMIC MODEL STRUCTURE FACTORS


REMARK 3 FB_CART = EXP(−H(t) * A(−1) * B * A(−1t) * H)


REMARK 3 A = orthogonalization matrix, H = MILLER INDEX


REMARK 3 (t) = TRANSPOSE, (−1) = INVERSE


REMARK 3


REMARK 3 STRUCTURE FACTORS CALCULATION ALGORITHM: FFT


REMARK 3


REMARK 3 DEVIATIONS FROM IDEAL VALUES.


REMARK 3 RMSD MAX COUNT


REMARK 3 BOND: 0.009 0.052 47


REMARK 3 ANGLE: 1.861 9.207 64


REMARK 3 CHIRALITY: 0.072 0.183 11


REMARK 3 PLANARITY: 0.007 0.017 7


REMARK 3 DIHEDRAL: 10.557 22.762 17


REMARK 3 MIN NONBONDED DISTANCE: 1.935


REMARK 3


REMARK 3 MOLPROBITY STATISTICS.


REMARK 3 ALL-ATOM CLASHSCORE: 9.62


REMARK 3 RAMACHANDRAN PLOT:


REMARK 3 OUTLIERS: 0.00%


REMARK 3 ALLOWED: 0.00%


REMARK 3 FAVORED: 100.00%


REMARK 3 ROTAMER OUTLIERS: 0.00%


REMARK 3 CBETA DEVIATIONS: 0


REMARK 3


REMARK 3 ATOMIC DISPLACEMENT PARAMETERS.


REMARK 3 WILSON B: 3.10


REMARK 3 RMS(B_ISO_OR_EQUIVALENT_BONDED): 2.15


REMARK 3 ATOMS NUMBER OF ATOMS


REMARK 3 ISO. ANISO.


REMARK 3 ALL: 51 51


REMARK 3 ALL (NO H): 51 51


REMARK 3 SOLVENT: 2 2


REMARK 3 NON-SOLVENT: 49 49


REMARK 3 HYDROGENS: 0 0


REMARK 3


REMARK 3 TLS DETAILS.


REMARK 3 NUMBER OF TLS GROUPS: 1


REMARK 3 ORIGIN: CENTER OF MASS


REMARK 3 TLS GROUP: 1


REMARK 3 SELECTION: all


REMARK 3 ORIGIN FOR THE GROUP (A): 10.5250 −0.1582 3.9867


REMARK 3 T TENSOR


REMARK 3 T11: 0.0284 T22: 0.0494


REMARK 3 T33: 0.0290 T12: 0.0055


REMARK 3 T13: 0.0058 T23: −0.0102


REMARK 3 L TENSOR


REMARK 3 L11: 0.0034 L22: 0.2021


REMARK 3 L33: 0.0378 L12: 0.0063


REMARK 3 L13: 0.0031 L23: −0.0919


REMARK 3 S TENSOR


REMARK 3 S11: 0.0075 S12: −0.0003 S13: −0.0248


REMARK 3 S21: −0.0899 S22: 0.0437 S23: −0.0012


REMARK 3 S31: −0.0422 S32: 0.1216 S33: 0.0477


REMARK 3


CRYST1 43.018 4.849 19.774 90.00 92.12 90.00 C 1 2 1


SCALE1 0.023246 0.000000 0.000861 0.00000


SCALE2 0.000000 0.206228 0.000000 0.00000


SCALE3 0.000000 0.000000 0.050606 0.00000


ATOM 1 N THR Z 1 1.586 −0.480 4.758 1.00 5.90 N


ANISOU 1 N THR Z 1 761 678 805 101 −37 58 N


ATOM 2 CA THR Z 1 2.773 0.083 5.366 1.00 4.89 C


ANISOU 2 CA THR Z 1 618 556 685 109 −42 48 C


ATOM 3 C THR Z 1 4.028 −0.525 4.751 1.00 4.69 C


ANISOU 3 C THR Z 1 583 552 645 117 −45 21 C


ATOM 4 O THR Z 1 4.118 −1.734 4.602 1.00 4.25 O


ANISOU 4 O THR Z 1 514 503 596 129 −53 20 O


ATOM 5 CB THR Z 1 2.767 −0.177 6.879 1.00 10.67 C


ANISOU 5 CB THR Z 1 1337 1315 1404 117 −36 39 C


ATOM 6 OG1 THR Z 1 1.507 0.235 7.426 1.00 12.86 O


ANISOU 6 OG1 THR Z 1 1629 1577 1680 104 −37 45 O


ATOM 7 CG2 THR Z 1 3.887 0.582 7.558 1.00 6.79 C


ANISOU 7 CG2 THR Z 1 813 838 927 116 −24 34 C


ATOM 8 N ILE Z 2 4.974 0.322 4.359 1.00 3.60 N


ANISOU 8 N ILE Z 2 454 437 477 89 −22 0 N


ATOM 9 CA ILE Z 2 6.260 −0.149 3.852 1.00 4.86 C


ANISOU 9 CA ILE Z 2 602 629 613 86 −9 −27 C


ATOM 10 C ILE Z 2 7.394 0.432 4.683 1.00 5.86 C


ANISOU 10 C ILE Z 2 706 797 724 71 17 −44 C


ATOM 11 O ILE Z 2 7.493 1.644 4.841 1.00 4.81 O


ANISOU 11 O ILE Z 2 584 666 578 46 32 −46 O


ATOM 12 CB ILE Z 2 6.489 0.275 2.388 1.00 4.24 C


ANISOU 12 CB ILE Z 2 558 541 511 64 −4 −37 C


ATOM 13 CG1 ILE Z 2 5.349 −0.210 1.492 1.00 5.28 C


ANISOU 13 CG1 ILE Z 2 719 631 658 75 −31 −19 C


ATOM 14 CG2 ILE Z 2 7.836 −0.232 1.890 1.00 6.18 C


ANISOU 14 CG2 ILE Z 2 792 822 734 62 12 −64 C


ATOM 15 CD1 ILE Z 2 5.480 0.241 0.054 1.00 6.81 C


ANISOU 15 CD1 ILE Z 2 950 811 828 54 −28 −28 C


ATOM 16 N ILE Z 3 8.248 −0.436 5.205 1.00 3.28 N


ANISOU 16 N ILE Z 3 346 502 398 86 21 −58 N


ATOM 17 CA ILE Z 3 9.450 0.006 5.891 1.00 2.54 C


ANISOU 17 CA ILE Z 3 229 449 289 72 44 −76 C


ATOM 18 C ILE Z 3 10.639 −0.630 5.179 1.00 3.98 C


ANISOU 18 C ILE Z 3 400 659 453 72 55 −99 C


ATOM 19 O ILE Z 3 10.711 −1.845 5.063 1.00 2.86 O


ANISOU 19 O ILE Z 3 243 520 322 97 43 −100 O


ATOM 20 CB ILE Z 3 9.414 −0.400 7.376 1.00 4.84 C


ANISOU 20 CB ILE Z 3 487 754 599 91 40 −69 C


ATOM 21 CG1 ILE Z 3 8.220 0.261 8.067 1.00 4.33 C


ANISOU 21 CG1 ILE Z 3 436 660 551 92 31 −45 C


ATOM 22 CG2 ILE Z 3 10.697 0.002 8.080 1.00 2.84 C


ANISOU 22 CG2 ILE Z 3 221 529 331 70 56 −80 C


ATOM 23 CD1 ILE Z 3 7.800 −0.407 9.356 1.00 8.14 C


ANISOU 23 CD1 ILE Z 3 909 1126 1058 102 19 −27 C


ATOM 24 N THR Z 4 11.553 0.199 4.686 1.00 3.85 N


ANISOU 24 N THR Z 4 391 661 411 44 77 −116 N


ATOM 25 CA THR Z 4 12.701 −0.291 3.932 1.00 4.67 C


ANISOU 25 CA THR Z 4 487 790 498 42 91 −137 C


ATOM 26 C THR Z 4 14.018 0.243 4.471 1.00 6.53 C


ANISOU 26 C THR Z 4 710 1042 729 20 101 −138 C


ATOM 27 O THR Z 4 14.193 1.447 4.579 1.00 6.74 O


ANISOU 27 O THR Z 4 748 1068 746 −4 111 −138 O


ATOM 28 CB THR Z 4 12.604 0.142 2.459 1.00 6.65 C


ANISOU 28 CB THR Z 4 776 1022 729 25 96 −142 C


ATOM 29 OG1 THR Z 4 11.403 −0.379 1.887 1.00 7.91 O


ANISOU 29 OG1 THR Z 4 961 1142 903 41 71 −125 O


ATOM 30 CG2 THR Z 4 13.797 −0.363 1.668 1.00 9.32 C


ANISOU 30 CG2 THR Z 4 1109 1379 1053 22 109 −159 C


ATOM 31 N LEU Z 5 14.953 −0.649 4.775 1.00 4.74 N


ANISOU 31 N LEU Z 5 463 829 511 29 96 −140 N


ATOM 32 CA LEU Z 5 16.306 −0.228 5.093 1.00 7.05 C


ANISOU 32 CA LEU Z 5 745 1136 796 12 105 −146 C


ATOM 33 C LEU Z 5 17.251 −0.915 4.121 1.00 8.18 C


ANISOU 33 C LEU Z 5 887 1289 933 14 113 −156 C


ATOM 34 O LEU Z 5 17.302 −2.135 4.056 1.00 9.26 O


ANISOU 34 O LEU Z 5 1013 1426 1079 34 104 −156 O


ATOM 35 CB LEU Z 5 16.654 −0.594 6.532 1.00 5.48 C


ANISOU 35 CB LEU Z 5 527 942 611 19 93 −137 C


ATOM 36 CG LEU Z 5 15.814 0.143 7.577 1.00 10.92 C


ANISOU 36 CG LEU Z 5 1220 1622 1307 16 87 −126 C


ATOM 37 CD1 LEU Z 5 14.477 −0.548 7.783 1.00 11.06 C


ANISOU 37 CD1 LEU Z 5 1240 1621 1340 39 74 −114 C


ATOM 38 CD2 LEU Z 5 16.578 0.252 8.883 1.00 16.68 C


ANISOU 38 CD2 LEU Z 5 1938 2359 2040 12 83 −123 C


ATOM 39 N GLU Z 6 18.017 −0.137 3.371 1.00 6.37 N


ANISOU 39 N GLU Z 6 668 1065 689 −6 128 −164 N


ATOM 40 CA GLU Z 6 18.866 −0.744 2.353 1.00 7.06 C


ANISOU 40 CA GLU Z 6 755 1157 769 −3 139 −173 C


ATOM 41 C GLU Z 6 20.226 −1.181 2.865 1.00 10.47 C


ANISOU 41 C GLU Z 6 1163 1608 1206 −3 141 −176 C


ATOM 42 O GLU Z 6 20.778 −2.133 2.338 1.00 10.97 O


ANISOU 42 O GLU Z 6 1221 1676 1270 9 144 −181 O


ATOM 43 CB GLU Z 6 19.030 0.157 1.128 1.00 6.61 C


ANISOU 43 CB GLU Z 6 725 1094 693 −22 154 −180 C


ATOM 44 CG GLU Z 6 19.460 −0.607 −0.118 1.00 8.76 C


ANISOU 44 CG GLU Z 6 1009 1365 957 −14 164 −188 C


ATOM 45 CD GLU Z 6 19.538 0.284 −1.341 1.00 9.29 C


ANISOU 45 CD GLU Z 6 1106 1420 1005 −32 177 −192 C


ATOM 46 OE1 GLU Z 6 19.525 1.503 −1.155 1.00 9.75 O


ANISOU 46 OE1 GLU Z 6 1171 1476 1059 −53 180 −189 O


ATOM 47 OE2 GLU Z 6 19.597 −0.234 −2.473 1.00 8.35 O


ANISOU 47 OE2 GLU Z 6 1006 1293 875 −25 184 −197 O


ATOM 48 OXT GLU Z 6 20.577 −0.596 3.827 1.00 10.37 O


ANISOU 48 OXT GLU Z 6 1140 1602 1196 −13 137 −173 O


TER


HETATM 49 ZN ZN B 1 1.135 0.108 2.815 1.00 6.38 ZN


ANISOU 49 ZN ZN B 1 854 704 866 82 −52 60 ZN


TER


HETATM 50 O HOH A 1 −1.382 −1.029 5.472 1.00 14.91 O


ANISOU 50 O HOH A 1 1900 1800 1964 82 −56 65 O


HETATM 51 O HOH A 2 10.560 2.460 0.798 1.00 26.28 O


ANISOU 51 O HOH A 2 3357 3428 3199 −21 86 −116 O


TER


END
















TABLE 4







REMARK Date 2012-02-15 Time 20:23:19 PST −0800 (1329366199.79 s)


REMARK PHENIX refinement


REMARK


REMARK ****************** INPUT FILES AND LABELS ***************


REMARK Reflections:


REMARK file name: l7e.mtz


REMARK labels: [‘IMEAN, SIGIMEAN’]


REMARK R-free flags:


REMARK file name: l7e.mtz


REMARK label: FreeR_flag


REMARK test_flag_value: 0


REMARK Model file name(s):


REMARK /home/absoriaga/APS/L7E/1.72A/build009_001-coot-1.pdb


REMARK


REMARK ******************** REFINEMENT SUMMARY: QUICK FACTS **********


REMARK Start: r_work = 0.1662 r_free = 0.1917 bonds = 0.007 angles =


1.078


REMARK Final: r_work = 0.1626 r_free = 0.1919 bonds = 0.007 angles =


1.080


REMARK


*******************************************************************


REMARK


REMARK ****************** REFINEMENT STATISTICS STEP BY STEP *******


REMARK leading digit, like 1_, means number of macro-cycle


REMARK 0: statistics at the very beginning when nothing is done yet


REMARK 1 s: bulk solvent correction and/or (anisotropic) scaling


REMARK 1 z: refinement of coordinates


REMARK ---------------------------------------------------------------


REMARK R-factors, x-ray target values and norm of gradient of x-ray


target


REMARK stage r-work r-free xray_target_w xray_target_t


REMARK 0: 0.1771 0.1905 3.652480e−01 4.073357e−01


REMARK 1_bss: 0.1662 0.1917 2.117160e−01 3.975473e−01


REMARK 1_ohs: 0.1662 0.1917 2.117160e−01 3.975473e−01


REMARK 1_xyz: 0.1619 0.1890 1.754450e−01 3.580521e−01


REMARK 1_adp: 0.1674 0.1960 1.964973e−01 3.683984e−01


REMARK 2_bss: 0.1690 0.2055 2.335866e−01 4.382616e−01


REMARK 2_ohs: 0.1690 0.2055 2.335866e−01 4.382616e−01


REMARK 2_xyz: 0.1667 0.2061 2.401298e−01 4.654830e−01


REMARK 2_adp: 0.1654 0.2035 2.351643e−01 4.720430e−01


REMARK 3_bss: 0.1651 0.1920 2.011024e−01 3.900795e−01


REMARK 3_ohs: 0.1651 0.1920 2.011024e−01 3.900795e−01


REMARK 3_xyz: 0.1648 0.1901 1.963632e−01 3.805261e−01


REMARK 3_adp: 0.1648 0.1907 1.960629e−01 3.791067e−01


REMARK 3_bss: 0.1626 0.1919 1.911754e−01 3.915615e−01


REMARK 3_ohs: 0.1626 0.1919 1.911754e−01 3.915615e−01


REMARK ---------------------------------------------------------------


REMARK stage k_sol b_sol b11 b22 b33 b12 b13 b23


REMARK 0: 0.600 80.084 1.142 0.456 −1.597 −0.206 0.548 −3.160


REMARK 1_bss: 0.600 39.857 1.373 0.376 −1.749 −0.196 0.551 −3.115


REMARK 1_ohs: 0.600 39.857 1.373 0.376 −1.749 −0.196 0.551 −3.115


REMARK 1_xyz: 0.600 39.857 1.373 0.376 −1.749 −0.196 0.551 −3.115


REMARK 1_adp: 0.600 39.857 1.373 0.376 −1.749 −0.196 0.551 −3.115


REMARK 2_bss: 0.600 37.717 1.451 0.387 −1.837 −0.208 0.596 −3.087


REMARK 2_ohs: 0.600 37.717 1.451 0.387 −1.837 −0.208 0.596 −3.087


REMARK 2_xyz: 0.600 37.717 1.451 0.387 −1.837 −0.208 0.596 −3.087


REMARK 2_adp: 0.600 37.717 1.451 0.387 −1.837 −0.208 0.596 −3.087


REMARK 3_bss: 0.600 35.678 1.451 0.387 −1.837 −0.208 0.596 −3.087


REMARK 3_ohs: 0.600 35.678 1.451 0.387 −1.837 −0.208 0.596 −3.087


REMARK 3_xyz: 0.600 35.678 1.451 0.387 −1.837 −0.208 0.596 −3.087


REMARK 3_adp: 0.600 35.678 1.451 0.387 −1.837 −0.208 0.596 −3.087


REMARK 3_bss: 0.600 30.131 1.451 0.387 −1.837 −0.208 0.596 −3.087


REMARK 3_ohs: 0.600 30.131 1.451 0.387 −1.837 −0.208 0.596 −3.087


REMARK ---------------------------------------------------------------


REMARK stage <pher> fom alpha beta


REMARK 0: 26.144 0.8035 0.0979 0.313


REMARK 1_bss: 26.758 0.7974 0.0981 0.315


REMARK 1_ohs: 26.758 0.7974 0.0981 0.315


REMARK 1_xyz: 25.873 0.8067 0.0970 0.291


REMARK 1_adp: 25.849 0.8071 0.0970 0.294


REMARK 2_bss: 27.844 0.7856 0.0966 0.340


REMARK 2_ohs: 27.844 0.7856 0.0966 0.340


REMARK 2_xyz: 28.600 0.7770 0.0967 0.358


REMARK 2_adp: 28.733 0.7755 0.0978 0.362


REMARK 3_bss: 26.407 0.8007 0.0989 0.305


REMARK 3_ohs: 26.407 0.8007 0.0989 0.305


REMARK 3_xyz: 26.098 0.8041 0.0989 0.299


REMARK 3_adp: 25.979 0.8054 0.0988 0.297


REMARK 3_bss: 26.185 0.8034 0.0990 0.304


REMARK 3_ohs: 26.185 0.8034 0.0990 0.304


REMARK ---------------------------------------------------------------


REMARK stage angl bond chir dihe plan repu geom_target


REMARK 0: 1.078 0.007 0.059 18.680 0.003 4.264 6.7991e−02


REMARK 1_bss: 1.078 0.007 0.059 18.680 0.003 4.264 6.7991e−02


REMARK 1_ohs: 1.078 0.007 0.059 18.680 0.003 4.264 6.7991e−02


REMARK 1_xyz: 1.169 0.007 0.070 17.979 0.004 4.264 7.1164e−02


REMARK 1_adp: 1.169 0.007 0.070 17.979 0.004 4.264 7.1164e−02


REMARK 2_bss: 1.169 0.007 0.070 17.979 0.004 4.264 7.1164e−02


REMARK 2_ohs: 1.169 0.007 0.070 17.979 0.004 4.264 7.1164e−02


REMARK 2_xyz: 1.090 0.007 0.064 18.389 0.003 4.263 6.8180e−02


REMARK 2_adp: 1.090 0.007 0.064 18.389 0.003 4.263 6.8180e−02


REMARK 3_bss: 1.090 0.007 0.064 18.389 0.003 4.263 6.8180e−02


REMARK 3_ohs: 1.090 0.007 0.064 18.389 0.003 4.263 6.8180e−02


REMARK 3_xyz: 1.080 0.007 0.061 18.346 0.003 4.263 6.7329e−02


REMARK 3_adp: 1.080 0.007 0.061 18.346 0.003 4.263 6.7329e−02


REMARK 3_bss: 1.080 0.007 0.061 18.346 0.003 4.263 6.7329e−02


REMARK 3_ohs: 1.080 0.007 0.061 18.346 0.003 4.263 6.7329e−02


REMARK ---------------------------------------------------------------


REMARK Maximal deviations:


REMARK stage angl bond chir dihe plan repu |grad|


REMARK 0: 4.404 0.026 0.099 64.798 0.006 2.545 2.5455e−01


REMARK 1_bss: 4.404 0.026 0.099 64.798 0.006 2.545 2.5455e−01


REMARK 1_ohs: 4.404 0.026 0.099 64.798 0.006 2.545 2.5455e−01


REMARK 1_xyz: 3.519 0.024 0.127 61.369 0.007 2.579 2.8547e−01


REMARK 1_adp: 3.519 0.024 0.127 61.369 0.007 2.579 2.8547e−01


REMARK 2_bss: 3.519 0.024 0.127 61.369 0.007 2.579 2.8547e−01


REMARK 2_ohs: 3.519 0.024 0.127 61.369 0.007 2.579 2.8547e−01


REMARK 2_xyz: 2.977 0.027 0.123 64.073 0.006 2.591 2.4811e−01


REMARK 2_adp: 2.977 0.027 0.123 64.073 0.006 2.591 2.4811e−01


REMARK 3_bss: 2.977 0.027 0.123 64.073 0.006 2.591 2.4811e−01


REMARK 3_ohs: 2.977 0.027 0.123 64.073 0.006 2.591 2.4811e−01


REMARK 3_xyz: 3.213 0.022 0.113 63.776 0.005 2.591 2.4147e−01


REMARK 3_adp: 3.213 0.022 0.113 63.776 0.005 2.591 2.4147e−01


REMARK 3_bss: 3.213 0.022 0.113 63.776 0.005 2.591 2.4147e−01


REMARK 3_ohs: 3.213 0.022 0.113 63.776 0.005 2.591 2.4147e−01


REMARK --------------------------------------------------------------


REMARK |-----overall-----|---macromolecule----|------solvent-------|


REMARK stage b_max b_min b_ave b_max b_min b_ave b_max b_min b_ave


REMARK 0: 43.34 4.47 13 98 43.34 4.47 13.89 27.13 10.20 15.93


REMARK 1_bss: 43.73 4.86 14.37 43.73 4.86 14.27 27.52 10.59 16.31


REMARK 1_ohs: 43.73 4.86 14.37 43.73 4.86 14.27 27.52 10.59 16.31


REMARK 1_xyz: 43.73 4.86 14.37 43.73 4.86 14.27 27.52 10.59 16.31


REMARK 1_adp: 34.22 6.51 13.79 34.22 6.51 13.65 23.32 12.30 16.48


REMARK 2_bss: 34.28 6.57 13.85 34.28 6.57 13.71 23.39 12.36 16.54


REMARK 2_ohs: 34.28 6.57 13.85 34.28 6.57 13.71 23.39 12.36 16.54


REMARK 2_xyz: 34.28 6.57 13.85 34.28 6.57 13.71 23.39 12.36 16.54


REMARK 2_adp: 35.29 6.18 14.27 35.29 6.18 14.08 31.25 10.44 17.92


REMARK 3_bss: 35.29 6.18 14.27 35.29 6.18 14.08 31.25 10.44 17.92


REMARK 3_ohs: 35.29 6.18 14.27 35.29 6.18 14.08 31.25 10.44 17.92


REMARK 3_xyz: 35.29 6.18 14.27 35.29 6.18 14.08 31.25 10.44 17.92


REMARK 3_adp: 35.70 5.88 14.25 35.70 5.88 14.08 31.83 9.77 17.66


REMARK 3_bss: 35.70 5.88 14.25 35.70 5.88 14.08 31.83 9.77 17.66


REMARK 3_ohs: 35.70 5.88 14.25 35.70 5.88 14.08 31.83 9.77 17.66


REMARK ---------------------------------------------------------------


REMARK stage Deviation of refined


REMARK model from start model


REMARK max min mean


REMARK 0: 0.000 0.000 0.300


REMARK 1_bss: 0.000 0.000 0.000


REMARK 1_ohs: 0.000 0.000 0.000


REMARK 1_xyz: 0.204 0.016 0.055


REMARK 1_adp: 0.204 0.016 0.055


REMARK 2_bss: 0.204 0.016 0.055


REMARK 2_ohs: 0.204 0.016 0.055


REMARK 2_xyz: 0.351 0.010 0.060


REMARK 2_adp: 0.351 0.010 0.060


REMARK 3_bss: 0.351 0.010 0.060


REMARK 3_ohs: 0.351 0.010 0.060


REMARK 3_xyz: 0.336 0.009 0.059


REMARK 3_adp: 0.336 0.009 0.059


REMARK 3_bss: 0.336 0.009 0.059


REMARK 3_ohs: 0.336 0.009 0.059


REMARK ---------------------------------------------------------------


REMARK MODEL CONTENT.


REMARK ELEMENT ATOM RECORD COUNT OCCUPANCY SUM


REMARK C 40 37.00


REMARK O 15 14.00


REMARK N 7 7.00


REMARK TOTAL 62 58.00


REMARK ---------------------------------------------------------------


REMARK r_free_flags.md5.hexdigest 3bd517a07859c6c80cf6f8225f5dfe4a


REMARK


REMARK IF THIS FILE IS FOR PDB DEPOSITION: REMOVE ALL FROM THIS LINE


UP.


REMARK 3


REMARK 3 REFINEMENT.


REMARK 3 PROGRAM: PHENIX (phenix.refine: 1.7.3_928)


REMARK 3 AUTHORS: Adams, Afonine, Chen, Davis, Echols, Gildea, Gopal,


REMARK 3 : Grosse-Kunstleve, Headd, Hung, Immormino, Ioerger, McCoy,


REMARK 3 : McKee, Moriarty, Pai, Read, Richardson, Richardson, Romo,


REMARK 3 : Sacchettini, Sauter, Smith, Storoni, Terwilliger, Zwart


REMARK 3


REMARK 3 REFINEMENT TARGET: ML


REMARK 3


REMARK 3 DATA USED IN REFINEMENT.


REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS): 1.703


REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS): 21.302


REMARK 3 MIN (FOBS/SIGMA_FOBS): 2.14


REMARK 3 COMPLETENESS FOR RANGE (%): 93.72


REMARK 3 NUMBER OF REFLECTIONS: 507


REMARK 3 NUMBER OF REFLECTIONS (NON-ANOMALOUS) 507


REMARK 3


REMARK 3 FIT TO DATA USED IN REFINEMENT.


REMARK 3 R VALUE (WORKING + TEST SET): 0.1655


REMARK 3 R VALUE (WORKING SET): 0.1626


REMARK 3 FREE R VALUE: 0.1919


REMARK 3 FREE R VALUE TEST SET SIZE (%): 9.86


REMARK 3 FREE R VALUE TEST SET COUNT: 50


REMARK 3


REMARK 3 FIT TO DATA USED IN REFINEMENT (IN BINS).


REMARK 3 BIN RESOLUTION RANGE COMPL. NWORK NFREE RWORK RFREE


REMARK 3 1 21.3038-1.7025 0.94 457 50 0.1626 0.1919


REMARK 3


REMARK 3 BULK SOLVENT MODELLING.


REMARK 3 METHOD USED: FLAT BULK SOLVENT MODEL


REMARK 3 SOLVENT RADIUS: 1.30


REMARK 3 SHRINKAGE RADIUS: 1.11


REMARK 3 GRID STEP FACTOR: 4.00


REMARK 3 K_SOL: 0.600


REMARK 3 B_SOL: 30.131


REMARK 3


REMARK 3 ERROR ESTIMATES.


REMARK 3 COORDINATE ERROR (MAXIMUM-LIKELIHOOD BASED): 0.06


REMARK 3 PHASE ERROR (DEGREES, MAXIMUM-LIKELIHOOD BASED): 26.18


REMARK 3


REMARK 3 OVERALL SCALE FACTORS.


REMARK 3 SCALE = SUM (|F_OBS|*|F_MODEL|)/SUM(|F_MODEL|**2): 0.0995


REMARK 3 ANISOTROPIC SCALE MATRIX ELEMENTS (IN CARTESIAN BASIS).


REMARK 3 B11: 1.4505


REMARK 3 B22: 0.3869


REMARK 3 B33: −1.8374


REMARK 3 B12: −0.2078


REMARK 3 B13: 0.5956


REMARK 3 B23: −3.0866


REMARK 3


REMARK 3 R FACTOR FORMULA.


REMARK 3 R = SUM (||F_OBS| − SCALE*|F_MODEL||)/SUM(|F_OBS|)


REMARK 3


REMARK 3 TOTAL MODEL STRUCTURE FACTOR (F_MODEL).


REMARK 3 F_MODEL = FB_CART * ( F_CALC_ATOMS + F_BULK)


REMARK 3 F_BULK = K_SOL * EXP(−B_SOL * S**2/4) * F_MASK


REMARK 3 F_CALC_ATOMS = ATOMIC MODEL STRUCTURE FACTORS


REMARK 3 FB_CART = EXP(−H(t) * A(−1) * B * A(−1t) * H)


REMARK 3 A = orthogonalization matrix, H = MILLER INDEX


REMARK 3 (t) = TRANSPOSE, (−1) = INVERSE


REMARK 3


REMARK 3 STRUCTURE FACTORS CALCULATION ALGORITHM: FFT


REMARK 3


REMARK 3 DEVIATIONS FROM IDEAL VALUES.


REMARK 3 RMSD MAX COUNT


REMARK 3 BOND: 0.007 0.022 61


REMARK 3 ANGLE: 1.080 3.213 85


REMARK 3 CHIRALITY: 0.061 0.113 15


REMARK 3 PLANARITY: 0.003 0.005 9


REMARK 3 DIHEDRAL: 18.346 63.776 24


REMARK 3 MIN NONBONDED DISTANCE: 2.591


REMARK 3


REMARK 3 MOLPROBITY STATISTICS.


REMARK 3 ALL-ATOM CLASHSCORE: 0.00


REMARK 3 RAMACHANDRAN PLOT:


REMARK 3 OUTLIERS: 0.00%


REMARK 3 ALLOWED: 0.00%


REMARK 3 FAVORED: 100.00%


REMARK 3 ROTAMER OUTLIERS: 0.00%


REMARK 3 CBETA DEVIATIONS: 0


REMARK 3


REMARK 3 ATOMIC DISPLACEMENT PARAMETERS.


REMARK 3 WILSON B: 12.73


REMARK 3 RMS (B_ISO_OR_EQUIVALENT_BONDED): 2.28


REMARK 3 ATOMS NUMBER OF ATOMS


REMARK 3 ISO. ANISO.


REMARK 3 ALL: 62 62


REMARK 3 ALL (NO H): 62 62


REMARK 3 SOLVENT: 3 3


REMARK 3 NON-SOLVENT: 59 59


REMARK 3 HYDROGENS: 0 0


REMARK 3


REMARK 3 TLS DETAILS.


REMARK 3 NUMBER OF TLS GROUPS: 1


REMARK 3 ORIGIN: CENTER OF MASS


REMARK 3 TLS GROUP: 1


REMARK 3 SELECTION: all


REMARK 3 ORIGIN FOR THE GROUP (A): 0.1185 6.8484 9.0224


REMARK 3 T TENSOR


REMARK 3 T11: 0.0582 T22: 0.0750


REMARK 3 T33: 0.0849 T12: 0.0099


REMARK 3 T13: 0.0029 T23: 0.0090


REMARK 3 L TENSOR


REMARK 3 L11: 2.3157 L22: 4.0221


REMARK 3 L33: 4.8813 L12: −0.4063


REMARK 3 L13: −1.1355 L23: 1.8148


REMARK 3 S TENSOR


REMARK 3 S11: 0.0087 S12: −0.0689 S13: −0.0102


REMARK 3 S21: 0.1493 S22: 0.2397 S23: −0.0164


REMARK 3 S31: −0.1178 S32: 0.0395 S33: −0.1915


REMARK 3


CRYST1 4.811 12.599 21.340 86.59 89.29 79.15 P 1


SCALE1 0.207857 −0.039846 −0.000241 0.00000


SCALE2 0.000000 0.080817 −0.004715 0.00000


SCALE3 0.000000 0.000000 0.046944 0.00000


ATOM 1 N LEU Z 1 0.068 1.128 −2.181 1.00 15.58 N


ANISOU 1 N LEU Z 1 1869 2159 1891 −379 −166 −311 N


ATOM 2 CA LEU Z 1 −0.127 0.804 −0.768 1.00 13.08 C


ANISOU 2 CA LEU Z 1 1469 1787 1714 −344 −176 −260 C


ATOM 3 C LEU Z 1 0.557 1.846 0.111 1.00 12.34 C


ANISOU 3 C LEU Z 1 1366 1679 1644 −279 −114 −206 C


ATOM 4 O LEU Z 1 1.781 1.952 0.130 1.00 13.64 O


ANISOU 4 O LEU Z 1 1530 1824 1827 −273 −43 −284 O


ATOM 5 CB LEU Z 1 0.402 −0.599 −0.458 1.00 12.49 C


ANISOU 5 CB LEU Z 1 1398 1613 1735 −354 −160 −349 C


ATOM 6 CG LEU Z 1 −0.073 −1.318 0.805 1.00 16.04 C


ANISOU 6 CG LEU Z 1 1832 1974 2288 −335 −172 −291 C


ATOM 7 CD1 LEU Z 1 0.293 −2.794 0.696 1.00 16.64 C


ANISOU 7 CD1 LEU Z 1 1983 1918 2423 −348 −154 −382 C


ATOM 8 CD2 LEU Z 1 0.530 −0.711 2.067 1.00 13.96 C


ANISOU 8 CD2 LEU Z 1 1552 1694 2057 −229 −151 −224 C


ATOM 9 N THR Z 2 −0.245 2.601 0.848 1.00 9.66 N


ANISOU 9 N THR Z 2 1001 1359 1311 −237 −137 −97 N


ATOM 10 CA THR Z 2 0.258 3.731 1.615 1.00 9.85 C


ANISOU 10 CA THR Z 2 1039 1364 1338 −192 −78 −58 C


ATOM 11 C THR Z 2 −0.369 3.689 2.996 1.00 9.29 C


ANISOU 11 C THR Z 2 920 1277 1333 −139 −99 14 C


ATOM 12 O THR Z 2 −1.588 3.575 3.128 1.00 10.23 O


ANISOU 12 O THR Z 2 997 1428 1461 −132 −136 80 O


ATOM 13 CB THR Z 2 −0.098 5.087 0.930 1.00 12.90 C


ANISOU 13 CB THR Z 2 1518 1766 1616 −175 −48 11 C


ATOM 14 OG1 THR Z 2 0.500 5.144 −0.374 1.00 13.73 O


ANISOU 14 OG1 THR Z 2 1716 1876 1624 −232 0 −46 O


ATOM 15 CG2 THR Z 2 0.400 6.264 1.758 1.00 11.80 C


ANISOU 15 CG2 THR Z 2 1419 1573 1490 −154 36 33 C


ATOM 16 N ILE Z 3 0.474 3.750 4.022 1.00 6.76 N


ANISOU 16 N ILE Z 3 595 923 1051 −104 −75 −16 N


ATOM 17 C ILE Z 3 0.661 5.006 6.102 1.00 6.68 C


ANISOU 17 C ILE Z 3 605 896 1039 −19 −41 27 C


ATOM 18 O ILE Z 3 1.887 5.178 6.075 1.00 7.92 O


ANISOU 18 O ILE Z 3 742 1064 1205 −36 −33 −81 O


ATOM 19 CA ILE Z 3 0.020 3.798 5.405 1.00 7.02 C


ANISOU 19 CA ILE Z 3 625 937 1106 −48 −79 49 C


ATOM 20 CB ILE Z 3 0.398 2.499 6.141 1.00 5.88 C


ANISOU 20 CB ILE Z 3 498 739 998 −12 −112 30 C


ATOM 21 CG1 ILE Z 3 −0.197 1.292 5.414 1.00 7.70 C


ANISOU 21 CG1 ILE Z 3 733 931 1261 −74 −124 28 C


ATOM 22 CG2 ILE Z 3 −0.022 2.568 7.617 1.00 8.38 C


ANISOU 22 CG2 ILE Z 3 862 1029 1293 49 −99 106 C


ATOM 23 CD1 ILE Z 3 0.383 −0.041 5.862 1.00 10.12 C


ANISOU 23 CD1 ILE Z 3 1113 1135 1598 −27 −141 −2 C


ATOM 24 N ILE Z 4 −0.170 5.849 6.712 1.00 8.44 N


ANISOU 24 N ILE Z 4 855 1114 1239 17 −8 104 N


ATOM 25 CA ILE Z 4 0.310 7.046 7.404 1.00 7.11 C


ANISOU 25 CA ILE Z 4 735 924 1043 31 42 71 C


ATOM 26 C ILE Z 4 −0.366 7.145 8.763 1.00 9.16 C


ANISOU 26 C ILE Z 4 1019 1178 1284 100 54 127 C


ATOM 27 O ILE Z 4 −1.596 7.044 8.848 1.00 8.49 O


ANISOU 27 O ILE Z 4 916 1102 1207 133 78 218 O


ATOM 28 CB ILE Z 4 −0.014 8.320 6.591 1.00 8.14 C


ANISOU 28 CB ILE Z 4 940 1014 1138 18 112 105 C


ATOM 29 CG1 ILE Z 4 0.721 8.307 5.245 1.00 11.61 C


ANISOU 29 CG1 ILE 4 1404 1449 1560 −64 136 51 C


ATOM 30 CG2 ILE Z 4 0.332 9.585 7.377 1.00 8.29 C


ANISOU 30 CG2 ILE 4 1043 971 1135 18 190 66 C


ATOM 31 CD1 ILE Z 4 0.132 9.280 4.232 1.00 13.32 C


ANISOU 31 CD1 ILE Z 4 1748 1614 1700 −43 187 133 C


ATOM 32 N THR Z 5 0.410 7.339 9.826 1.00 6.75 N


ANISOU 32 N THR Z 5 745 875 946 121 39 57 N


ATOM 33 CA THR Z 5 −0.180 7.546 11.152 1.00 8.77 C


ANISOU 33 CA THR Z 5 1064 1122 1145 187 68 102 C


ATOM 34 C THR Z 5 0.418 8.797 11.800 1.00 10.69 C


ANISOU 34 C THR Z 5 1368 1352 1343 179 100 5 C


ATOM 35 O THR Z 5 1.642 8.947 11.834 1.00 11.07 O


ANISOU 35 O THR Z 5 1384 1431 1390 132 48 −131 O


ATOM 36 CB THR Z 5 0.030 6.323 12.090 1.00 10.48 C


ANISOU 36 CB THR Z 5 1320 1349 1315 244 5 121 C


ATOM 37 OG1 THR Z 5 1.406 6.224 12.461 1.00 13.87 O


ANISOU 37 OG1 THR Z 5 1741 1820 1710 274 −97 −3 O


ATOM 38 CG2 THR Z 5 −0.409 5.021 11.436 1.00 10.34 C


ANISOU 38 CG2 THR Z 5 1271 1306 1351 221 −8 189 C


ATOM 39 N LEU Z 6 −0.446 9.696 12.278 1.00 12.10 N


ANISOU 39 N LEU Z 6 1617 1488 1493 217 192 50 N


ATOM 40 C LEU 6 Z −0.524 10.931 14.393 1.00 14.83 C


ANISOU 40 C LEU Z 6 2136 1804 1694 281 272 −38 C


ATOM 41 O LEU Z 6 −1.656 10.521 14.660 1.00 14.90 O


ANISOU 41 O LEU Z 6 2144 1819 1699 347 331 79 O


ATOM 42 CD1 LEU Z 6 0.469 11.983 9.998 1.00 16.40 C


ANISOU 42 CD1 LEU Z 6 2268 1865 2100 55 338 −42 C


ATOM 43 CD2 LEU Z 6 −0.326 14.207 10.832 1.00 17.47 C


ANISOU 43 CD2 LEU Z 6 2662 1793 2182 133 528 −40 C


ATOM 44 CA ALEU Z 6 −0.015 10.912 12.963 0.50 14.01 C


ANISOU 44 CA ALEU Z 6 1950 1686 1686 202 244 −51 C


ATOM 45 CB ALEU Z 6 −0.561 12.158 12.261 0.50 14.80 C


ANISOU 45 CB ALEU Z 6 2124 1682 1819 202 359 −19 C


ATOM 46 CG ALEU Z 6 0.295 12.867 11.213 0.50 18.60 C


ANISOU 46 CG ALEU Z 6 2642 2089 2334 89 398 −91 C


ATOM 47 CA BLEU Z 6 −0.015 10.912 12.963 0.50 14.11 C


ANISOU 47 CA BLEU Z 6 1963 1699 1699 202 244 −51 C


ATOM 48 CB BLEU Z 6 −0.561 12.158 12.261 0.50 14.85 C


ANISOU 48 CB BLEU Z 6 2130 1688 1825 202 359 −19 C


ATOM 49 CG BLEU Z 6 0.295 12.867 11.213 0.50 18.60 C


ANISOU 49 CG BLEU Z 6 2642 2089 2334 89 398 −91 C


ATOM 50 N GLU Z 7 0.304 11.423 15.303 1.00 16.19 N


ANISOU 50 N GLU Z 7 2376 1992 1785 261 239 −177 N


ATOM 51 CA GLU Z 7 −0.131 11.710 16.667 1.00 21.28 C


ANISOU 51 CA GLU Z 7 3145 2639 2303 331 284 −189 C


ATOM 52 C GLU Z 7 0.706 12.866 17.251 1.00 26.95 C


ANISOU 52 C GLU Z 7 3942 3336 2963 264 284 −387 C


ATOM 53 O GLU Z 7 1.715 13.280 16.656 1.00 23.82 O


ANISOU 53 O GLU Z 7 3479 2938 2632 147 244 −521 O


ATOM 54 CB GLU Z 7 −0.080 10.461 17.558 1.00 21.58 C


ANISOU 54 CB GLU Z 7 3220 2754 2224 408 196 −134 C


ATOM 55 CG GLU Z 7 1.286 9.867 17.714 1.00 25.98 C


ANISOU 55 CG GLU Z 7 3733 3401 2738 411 3 −247 C


ATOM 56 CD GLU Z 7 1.279 8.604 18.550 1.00 30.97 C


ANISOU 56 CD GLU Z 7 4464 4072 3233 533 −84 −159 C


ATOM 57 OE1 GLU Z 7 0.937 8.687 19.747 1.00 30.45 O


ANISOU 57 OE1 GLU Z 7 4567 4010 2992 603 −53 −141 O


ATOM 58 OE2 GLU Z 7 1.617 7.528 18.011 1.00 35.70 O


ANISOU 58 OE2 GLU Z 7 5002 4678 3883 565 −171 −105 O


ATOM 59 OXT GLU Z 7 0.405 13.433 18.308 1.00 28.69 O


ANISOU 59 OXT GLU Z 7 4293 3537 3070 304 342 −438 O


TER 60 GLU Z 7


HETATM 60 O HOH A −2.703 7.785 14.692 1.00 9.77 O


ANISOU 60 O HOH A 1 1411 1229 1073 352 306 277 O


HETATM 62 O HOH A 2 1.726 3.935 14.170 1.00 31.83 O


ANISOU 62 O HOH A 2 4220 4052 3822 494 −238 95 O


TER 63 HOH A 2


END









Table 5 shows statistics of X-ray data collection and refinement for the crystal structures of the p53 segments. Table 5 discloses “LTIITLE” as SEQ ID NO: 21 and “TIITLE” as SEQ ID NO: 20.










TABLE 5







252-LTIITLE-258
253-TIITLE-258


















Crystal parameters




Space group
P1
C2


Cell dimensions


a, b, c (Å)
4.81, 12.60, 21.34
43.02, 4.85, 19.77


a, β, γ (°)
86.59, 89.29, 79.15
90, 92.12, 90


Molecules in Asymmetric
1
1


Unit


Data collection


Synchrotron beamline
APS (24-ID-E)
APS (24-ID-E)


Wavelength (Å)
0.9792
0.9792


Resolution range (Å)
1.70
1.58


Unique Reflections
507
635


Overall Redundancy
 3.1 (2.6)a
4.0 (3.0)


Completeness (%)
97.0 (91.4)
96.5 (87.7)


Rmerge (%)b
 9.6 (50.5)
11.4 (32.9)


<|/σ|>
10.6 (2.9) 
8.9 (4.1)


Refinement


Resolution (A)
21.30-1.70
21.49-1.58


Rwork (%)c
16.2
16.4


Rfree (%)d
19.2
19.3


No. atoms


Protein
59
48


Ligand/ion
0
1


Water
2
2


Overall B-factors
12.7
3.1


R.m.s. deviation


Bond length (Å)
0.007
0.009


Bond angle (°)
1.080
1.861





aValues in parentheses correspond to the highest resolution shell


bRmerge = Σ|/ − </>|/Σ/


cRwork = Σ|Fo − Fc|/ΣFo


dRfree = Σ|Fo − Fc|/ΣFo, calculated using a random set containing reflections that were not included throughout structure refinement






The inventors applied their Rosetta-based method (Sievers et al, Nature, 2011) to design inhibitors that disrupt p53 aggregation, using the p53252-258 structure as a template. In other embodiments of the invention, the p53253-258 structure is used as a template to design inhibitors.
Table 1, shown earlier in this application, shows a list of 16 representative sequences obtained by this method.
Table 6 shows the calculated properties of a selection of inhibitors.







TABLE 6







Aggregation propensities and capping energies of the designed inhibitors.









Capping energyb










Single Sheet
Triple Sheet













Zipper energya
absolute
relative
absolute
relative



(kcal/mol)
(kcal/mol)
to
(kcal/mol)
to
















LTIITLE
−26.2
−42.2
0.0
−86.2
0.0


(native)


LTRITLE
−18.2
−40.5
1.7
−84.6
1.6


LTRIYLE
−19.5
−41.0
1.1
−85.2
1.0


YTRITLE
−19.3
−40.3
1.9
−83.5
2.6


YTRIYLE
−19.8
−41.3
0.8
−85.3
0.9


ETRITLE
−19.0
−39.8
2.4
−83.0
3.1


LTKITLE
−25.5
−41.2
0.9
−84.0
2.2


WTKITLE
−24.3c
−40.1
2.1
−82.1
4.0


YTKITLE
−24.1
−40.8
1.3
−82.5
3.6









The peptides in Table 6, reading from top to bottom, are represented by SEQ ID NOS 21, 4, 7, 5, 8, 6, 12, 14 and 13.
FIG. 2 shows the INH-1R inhibitor modeled on the p53252-258 structure. As evident, the ARG substitution collides with the adjacent sheet efficiently inhibiting new monomers from attaching to the free fiber end and preventing further growth.
The designed inhibitors were synthesized and tested in an in vitro Thioflavin T aggregation assay (FIG. 3). The most effective design, INH-1R, delayed the onset of aggregation and lowered the amount of aggregates present at all concentrations tested, even at a molar ratio of 1 inhibitor to 10 p53 molecules (FIG. 3).
To render the inhibitors cell permeable, the inventors fused the peptide-inhibitor panel to a nine-residue poly arginine tag through a three-residue linker, of sequence RPI, derived from endogenous p53 sequence. To confirm their ability to enter cells, the cell penetrating INH-1R inhibitor was linked to a FITC moiety in order to detect the intracellular localization of the probe by fluorescence microscopy (FIG. 4). We treated different cancer cell lines, including but not limited to OVCAR-3, CAOV-3, WiDr, Detroit 562 as well as primary cells derived from cancer patients with the FITC labeled inhibitor for 24 hours. After extensive washes to remove unbound inhibitors, the cells were fixed in formaldehyde and p53 was stained using a commercially available p53 antibody. As visible in FIG. 4A, the inhibitor was not only able to penetrate the cell membrane, but also to actually enter the nucleus and co-localize with its target, p53. Additionally, the inhibitor was found co-localized with protein aggregates as stained by the conformation specific antibody OC (Kayed et al, 2007, FIG. 4B).
The structure-guided, rationally designed p53 amyloid inhibitors of the invention specifically target those cancer cells bearing p53 molecules having an aberrant conformation (e.g. aggregated or misfolded p53). We demonstrated the capability of the inhibitors to halt aggregation progression in vitro (FIG. 3). We then tested the inhibitor on established cancer cell lines as well as on primary cells derived from serous ovarian cancer patients in order to confirm the clinical relevance of our findings. Our inhibitor is designed to specifically target those tumor cells expressing an altered misfolded form of p53 and should have no effect on cells bearing functional, properly folded p53 or aggregation-incompetent p53 mutants. To confirm this, we included several controls. Three established cell lines, one bearing WT p53 (MCF-7) and two which were previously characterized for their p53 aggregation status, one with a non-aggregating mutation (WiDr) and one with a p53 aggregation prone mutation (Detroit 562), were used as negative and positive controls. One of the effects of the inhibitor was to cause re-localization of p53 from the cytosolic compartment to the nucleus (FIG. 5). This effect was not observed in case of the WT p53 bearing cells MCF 7 (FIG. 2a), suggesting that INH-1R is active only when p53 loses is structural integrity.
Another effect of the inhibitor was to cause re-folding of misfolded p53 into a WT-like, functional conformation. p53 treated with INH-1R in PA40 cells failed to be recognized by the antibody PAb240 which specifically targets partially unfolded, mutant p53 (FIG. 6), indicating that re-localization was accompanied by protein re-folding. As a consequence of p53 maintaining a physiological fold, the levels of p53 proteins in treated cells were decreasing in a dose dependent manner. Properly folded p53 is rapidly degraded in cells as a control mechanism. Misfolded/aggregated p53 cannot be efficiently targeted for degradation therefore protein levels are high in the absence of the inhibitor. This is additional evidence of the ability of the INH-1R to generate a population of folded p53. Without wishing to be bound by any particular mechanism, it is suggested that this is due to the inhibitor titrating out pre-existing p53 aggregates, by the inhibitor changing the equilibrium between aggregated/misfolded/folded p53, by the inhibitor chaperoning or masking the exposed aggregation prone segment.
INH-1R caused cell death in a dose dependent manner with less than 50% of cancer cells surviving after only 24 hours of treatment with a dose of 10 μM in a 2D culture system as detected by a standard MTS assay (FIG. 7A). The effect of INH-1R is specific since a poly-ARG alone or a scrambled inhibitor sequence did not elicit any effect. Similar results were obtained with both cancer cells or primary cells grown in a 3D culture system. In this case cells were plated in 1:1 mixture of PrEGM:matrigel. After cell layer solidified, cells were overlaid with warm PrEGM media containing either the inhibitor or a scrambled control peptide at concentration ranging from 0.1 to 10 μM for 5 to 7 days.
The inhibitor caused both an increase in apoptosis and a decrease in cell proliferation in a dose dependent manner (FIGS. 7B and 7C) as evidenced by an increase in AnnexinV stain and a decrease in Ki67 stain. Apoptosis as well as necrosis were induced specifically by treatment with INH-1R as opposed to a scrambled peptide sequence as evidence also by a combined Hoechst/PI/YO-PRO-1 stain (FIG. 8). Cell death can be monitored also by FACS methods as visible in FIG. 9, were OVCAR-3 cells treated for 24 hours with 10 μM of inhibitor showed a decrease in cell size and increase in granularity, typical manifestations of cell death.
Specificity and efficacy of INH-1R were confirmed by up-regulation of p53 target genes only in tumor cells bearing aggregation-prone mutations as tested by QPCR (FIG. 8) or RNAseq methods.
In our in vivo studies, we attempted to mimic a post-surgical debulking situation in which patients with minimal residual tumor mass are administered chemotherapy. NOD/SCID mice were injected subcutaneously with OVCAR-3 cells bearing the aggregation prone p53 R248Q mutation and treated intraperitoneally with 15 mg/kg of either INH-1R as a single therapeutic agent, a control scrambled peptide, or vehicle daily for 14 days (FIG. 9). Mice treated with INH-1R showed diminished tumor proliferation, with a xenograft mass reduced by 75% in weight (FIGS. 9B and 9C). The residual tumor tissue showed a pronounced p21 and MDM2 activation, indicative of p53 activation (FIG. 9D). The inhibitor was also tested in vivo on mice carrying pre-existing tumors with similar effect.
Pharmacokinetic profile of the intraperitoneally infused peptide showed a serum peak concentration of approx. 1.2 μM 1 hour after injection. The peptide concentration decreased to approximately 0.3 μM 2 hours post infusion, and remained stable for up to 12 hours (FIG. 10). Given the relative stability of the peptide in serum, IV administration is an acceptable alternative route of administration.
From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make changes and modifications of the invention to adapt it to various usage and conditions and to utilize the present invention to its fullest extent. The preceding preferred specific embodiments are to be construed as merely illustrative, and not limiting of the scope of the invention in any way whatsoever. The entire disclosure of all applications, patents, and publications cited herein, including U.S. provisional application Ser. No. 61/821,157, filed May 8, 2013 and in the figures are hereby incorporated in their entirety by reference, particularly with regard to the information for which they are cited.
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## Claims

1. A cell penetrating peptide (CPP) inhibitor represented by the sequence (R2-16) P I (L/Y/E/W) T (R/K) I T (L) E (SEQ ID NO: 19), provided the sequence does not comprise YTRITLE (SEQ ID NO: 5).

2. A pharmaceutical composition comprising the peptide of claim 1 and a pharmaceutically acceptable carrier.

3. A kit comprising the peptide of claim 1, packaged in a container.

4. A method of making the peptide of claim 1, comprising synthesizing it chemically or producing it recombinantly.

5. A peptide which comprises the amino acid sequence LTRITLE (SEQ ID NO: 4), wherein said amino acid sequence is coupled to 2 to 16 arginine (R) residues.

6. A pharmaceutical composition comprising the peptide of claim 5 and a pharmaceutically acceptable carrier.

7. A pharmaceutical composition comprising the peptide of claim 5 and a pharmaceutically acceptable carrier, wherein the peptide has been isolated.

8. A composition of matter comprising the peptide of claim 5;
wherein:
the peptide is coupled to 4 to 16 arginine residues.

9. The composition of claim 8, wherein the peptide is coupled to the arginine residues by a peptide linker comprising 1-7 amino acids.

10. The composition of claim 8, wherein the peptide forms a polycationic structure.

11. The composition of claim 8, wherein the peptide forms an amphipathic structure.

12. The composition of claim 8, wherein the peptide is less than 30 amino acids in length.

13. A composition of matter comprising:
the peptide of claim 5; and
a pharmaceutically acceptable carrier including a peptide stabilizing excipient.

14. The composition of claim 13, wherein the peptide stabilizing excipient is a preservative that inhibits the growth of microorganisms.

15. A composition of matter comprising a peptide comprising the sequence RRRRRRRRRRPILTRITLE (SEQ ID NO: 22).

16. The CPP inhibitor of claim 1 represented by the sequence (R2-16) P I L T R I T L E (SEQ ID NO: 19).