POLYPEPTIDES WITH THERAPEUTIC ACTIVITY AND METHODS OF USE

Abstract: Polypeptides and methods of use including treating bacterial infection and/or endotoxemia, decreasing the amount of TNF- alpha , inhibiting endothelial cell proliferation, inhibiting angiogenic-factor mediated inter-cellular adhesion molecule expression down-regulation, and inhibiting angiogenesis.

Description: Polypeptides with Therapeutic Activity and Methods of Use Cross-Reference to Related Applications The present application claims priority to Provisional Patent Application Serial Nos. 60/177,255, filed on January 20, 2000 and 60/210,297, filed June 8, 2000, both of which are incorporated herein by reference. Background A series of designed peptide 33mers (βpep peptides) has been reported to be bactericidal and to be capable of neutralizing the bacterial endotoxin lipopolysaccharide (LPS) (Mayo et al., Biochim. Biophys. Ada 1425, 81-92 (1998)). CD and NMR conformational analyses indicate that βpep peptides form β-sheets (Mayo et al., Protein Sci. 5, 1301-1315 (1996)), and one of these, βpep- 4, folds compactly as anti-parallel β-sheet sandwich (Ilyina et al., Biochemistry 36, 5245-5250 (1997)). βpep-19 is potently bactericidal in the 10 "8 M range ( Mayo et al., Biochim. Biophys. Ada 1425, 81-92 (1998)). βpep peptides function like Limulus anti-LPS factor (LALF) and the homologous bactericidal/permeability increasing protein (B/PI) (Hoess et al., EMBO J. 12, 3351-3356 (1993)) in that all appear to express activity through an amphipathic β-sheet structural motif having a cationic β-sheet face (Kelly et al., Surgery 114, 140-146 (1993); Siefferman et al., Infect. Immun. 59, 2152-2157 (1991); Beamer et al., Science 276, 1861-1864 (1997); and Gray et al., Biochim. Biophys. Ada 1244, 185-190 (1995)). Numerous studies on bactericidal peptides indicate the functional importance of a net positive charge and high hydrophobicity in the context of an amphipathic, usually helical, structure (Maloy et al., Biopolymers 37, 105-112 (1995)). The net positive charge promotes interaction with the negatively charged surface of bacterial membranes (Matsuzaki et al., Biochemistry 36, 2104-2111 (1997)), whereas structure-activity relationships demonstrate that the amphipathic conformation of the peptide promotes bacterial cell lysis (Andreu et al., Biochemistry 24, 1683-1688 (1985)). Bactericidal peptides such as cecropins (Lee et al., Proc. Natl. Acad. Sci. USA 86, 9159-9162 (1989)), magainins (Zasloff et al., J Biol. Chem. 264, 12826-12829 (1987)), prolme-arginine-rich peptides (Agerberth et al., Eur. J. Biochem. 202, 849-854 (1991)), and sapecin ( Matsuyama et al., J. Biol. Chem. 263, 11112-17116 (1988)), like βpep peptides, all have a net positive charge and considerable hydrophobic character. The cecropins and magainins are helix-forming peptides (Holak et al., Biochemistry 27, 7620-7629 (1988); and Marion et al., FEBs Lett. 227, 21-26 (1988)), whereas the sapacins contain both α-helix and β-sheet segments (Hanzawa et al., FEBs Lett. 269, 413-420 (1990)). Structures for the prolme-arginine-rich peptides are unknown. Tachyplesin, a bactericidal and endotoxin neutralizing peptide isolated from hemocytes of the horseshoe crab (Kawano et al., J Bio. Chem. 265, 15365-15367 (1990)), as well as anti-bacterial peptide defensins (Selsted et al., J. Biol. Chem. 264, 4003-4007 (1989); and Lehrer et al., Cell 64, 229-230 (1991)), form dimeric β-sheets which are stabilized by three intramolecular disulfide bridges (Hill et al., Science 251, 1481-1485 (1991)). In addition, a number of small, antibiotic peptides based on the structure of the anti-LPS, cyclic peptide polymyxin B (Morrison et al., Immunochem. 13, 813-818 (1976)) have been designed as short β-hairpins constrained by a disulfide bridge (Rustici et al., Science 259, 361-365 (1993)). Summary of the Invention The present invention provides a polypeptide selected from the group consisting of: ANIKLSVQMKLF (SEQ ID NO: 1); KLSVQMKLFKRH (SEQ ID NO:2); VQMKLFKRHLKW (SEQ ID NO:3); KLFKRHLKWKII (SEQ ID NO:4); KRHLKWKITVKL (SEQ ID NO:5); LKWKHVKLNDG (SEQ ID NO:6); KHVKLNDGREL (SEQ ED NO:7); VKLNDGRELSLD (SEQ ID NO:8); QMKLFKRHLKWK (SEQ ID NO:9); MKLFKRHLKWKI (SEQ ID NO: 10); MKLFKRHLKWKΗV (SEQ ID NO: 11); XLFKRHLKWKII (SEQ ID NO: 12); KLFXRHLKWKπ (SEQ ID NO:13); KLFKRHLXWKII (SEQ ID NO: 14); KLFKRHLKWXπ (SEQ ID NO:15); KLFKKHLKWKH (SEQ ID NO:16); KLFKHLKWKII (SEQ ID NO: 17); analogs thereof (preferably that are active for the treatment of bacterial infection and/or endotoxemia); and combinations thereof; wherein X is an amino acid. The present invention also provides a method for treating bacterial infection and/or endotoxemia. This includes administering to a patient an amount of a pharmaceutical composition effective to inhibit the bacterial infection and/or neutralize endotoxin, wherein the pharmaceutical composition includes a polypeptide (i.e., one or more polypeptide) listed above. A method for inhibiting bacterial infection and/or endotoxemia in vitro is also provided. The method includes contacting cells with an amount of a composition effective to inhibit the bacterial infection and/or to neutralize endotoxin, wherein the composition includes a polypeptide listed above. The present invention also provides a method for decreasing the amount of TNF-α in a patient. The method includes administering to the patient a therapeutically effective amount of a pharmaceutical composition including a polypeptide listed above. A method for decreasing the amount of TNF-α in vitro is also provided. The method includes incubating cells with an effective amount of a composition comprising a polypeptide selected listed above. Other polypeptides suitable for these methods include analogs of those listed above that are active for decreasing the amount of TNF-α. The present invention also provides a method for inhibiting endothelial cell proliferation in a patient. The method involves administering to the patient a therapeutically effective amount of a composition including a polypeptide listed above. A method for inhibiting endothelial cell proliferation in vitro is also provided. The method involves contracting cells with an effective amount of a composition including a polypeptide listed above. Other polypeptides suitable for these methods include analogs of those listed above that are active for inhibiting endothelial cell proliferation. The present invention also provides a method for inhibiting angiogenic- factor mediated inter-cellular adhesion molecule expression down-regulation in a patient. The method includes administering to the patient a therapeutically effective amount of a composition including a polypeptide listed above. A method for inhibiting angiogenic-factor mediated inter-cellular adhesion molecule expression down-regulation in vitro is also provided. The method includes contacting cells with an effective amount of a composition including a polypeptide listed above. Other polypeptides suitable for these methods include analogs of those listed above that are active for inhibiting antiogenic-factor mediated inter-cellular adhesion molecule expression down-regulation. The present invention also provides a method for inhibiting angiogenesis in a patient. The method includes administering to the patient a therapeutically effective amount of a composition including a polypeptide listed above. A method for inhibiting angiogenesis in vitro is also provided. The method includes contacting cells with an effective amount of a composition including a polypeptide listed above. Other polypeptides suitable for these methods include analogs of those listed above that are active for inhibiting angiogenesis. The present invention also provides a method for inhibiting tumorigenesis in a patient. The method includes admiriistering to the patient a therapeutically effective amount of a composition including a polypeptide listed above. Other polypeptides suitable for this method includes analogs of those listed above that are active for inhibiting tumorigenesis. The present invention also provides the three-dimensional structure of certain of the polypeptides using nuclear magnetic resonance (NMR) spectroscopy (e.g., one- and two-dimensional NMR) and circular dichroism (CD) spectroscopy. This information is of significant utility in fields such as drug discovery. Thus, the present invention also provides methods of using such structural information. The present invention provides a polypeptide having an amphipathic structure having one surface with positively charged amino acid residues and an opposing surface with hydrophobic amino acid residues, wherein these define a surface active domain. Preferably, the surface active domain includes amino acids Kl, K4, K8, and R5 shown in Figure 6, and more preferably, atoms 1-24, 64-109, and 146-167 listed in Table 5. More preferably, the polypeptide has the structure coordinates listed in Table 5 and most preferably has the sequence KLFKRHLKWKU (SEQ ID NO:4). One specific method of the present invention involves evaluating a candidate compound for structural similarity to that of KLFKRHLKWKII (SEQ ID NO:4) by: supplying a three-dimensional structure of KLFKRHLKWKII (SEQ ID NO:4) or a portion thereof; supplying a three-dimensional structure of a candidate compound; and comparing the three-dimensional structure of the candidate compound with the three-dimensional structure of KLFKRHLKWKII (SEQ ID NO:4) or a portion thereof. As used herein, "a" or "an" refers to one or more of the term modified. Thus, the compositions of the present invention include one or more polypeptides. "Amino acid" is used herein to refer to a chemical compound with the general formula: NH 2 -CRH-COOH, where R, the side chain, is H or an organic group. Where R is an organic group, R can vary and is either polar or nonpolar (i.e., hydrophobic). The amino acids of this invention can be naturally occurring or synthetic (often referred to as nonproteinogenic). As used herein, an organic group is a hydrocarbon group that is classified as an aliphatic group, a cyclic group or combination of aliphatic and cyclic groups. The term "aliphatic group" means a saturated or unsaturated linear or branched hydrocarbon group. This term is used to encompass alkyl, alkenyl, and alkynyl groups, for example. The term "cyclic group" means a closed ring hydrocarbon group that is classified as an alicyclic group, aromatic group, or heterocyclic group. The term "alicyclic group" means a cyclic hydrocarbon group having properties resembling those of aliphatic groups. The term "aromatic group" refers to mono- or polycyclic aromatic hydrocarbon groups. As used herein, an organic group can be substituted or unsubstituted. The terms "polypeptide" and "peptide" as used herein, are used interchangeably and refer to a polymer of amino acids. These terms do not connote a specific length of a polymer of amino acids. Thus, for example, the terms oligopeptide, protein, and enzyme are included within the definition of polypeptide or peptide, whether produced using recombinant techniques, chemical or enzymatic synthesis, or naturally occurring. This term also includes polypeptides that have been modified or derivatized, such as by glycosylation, acetylation, phosphorylation, and the like. The following abbreviations are used throughout the application: A = Ala = Alanine T = Thr = Threonine V = Val = Valine C = Cys = Cysteine L = Leu = Leucine Y = Tyr = Tyrosine I = He = Isoleucine N = Asn = Asparagine P = Pro = Proline Q = Gin = Glutamine F = Phe = Phenylalanine D = Asp = Aspartic Acid W = Trp = Tryptophan E = Glu = Glutamic Acid M = Met = Methionine K = Lys = Lysine G = Gly = Glycine R = Arg = Ajginine S = Ser = Serine H = His = Histidine Other abbreviations used throughout include: B/PI, bactericidal/permeability increasing protein; LALF, Limulus anti- lipopolysaccharide factor; LPS, lipopolysaccharide; PF4, platelet factor 4; NMR, nuclear magnetic resonance spectroscopy; NOE, nuclear Overhauser effect; rf, radio frequency; FID, free induction decay; CD, circular dichroism; HPLC, high performance liquid chromatography; PBS, phosphate buffered saline; FCS, fetal calf serum. Brief Description of the Drawings Figure 1. Peptide sequences: peptide sequences are shown for βpep-19 (SEQ ID NO: 18) and βpep-25 (SEQ ID NO: 19), along with those of eight dodecapeptides which "walk through" the sequence of βpep-25. The dodecapeptides are referred to as SC peptides. The -NH 2 at the right of each sequence indicates amidation of the C-terminal carboxylate group. Figure 2. Bactericidal Dose Resonse Curves for Peptides. A select number of dose response curves are shown in this figure as percent bacteria killed versus the log concentration of peptide. Data are provided for three strains of bacteria: (A) rough strain E. coli J5; (B) smooth strain E. coli IA2; and (C) a strain of Gram-positive S. aureus MNHO. Dose response curves were acquired as described in the Methods Section. Figure 3. Dose Response Curves for Endotoxin Neutralization. Dose response curves, as described in the Methods Section, are shown as percent LPS neutralization versus log concentration of peptide. The top panel illustrates results for SC peptides and for βpep-25, and the bottom panel illustrates results for various variants of SC-4 as described in the text. Figure 4. CD Spectra for SC Peptides. Far-ultraviolet circular dichroic spectra for two SC peptides, SC-5 and SC-7 are shown as mean residue ellipicity versus wavelength (nm). Peptide concentration was 40 μM in 20 mM potassium phosphate, pH 6.3. The temperature was set at 20°C. Other experimental conditions are discussed in the Methods Section. CD traces shown were acquired as a function of TFE concentration from 0% to 70% (volume/volume). The insert in the middle shows the change in (Θ)222 versus the concentration of trifluoroethanol (TFE). Figure 5. NOESY Spectra for SC-4. 600 MHz 1H NMR spectra are shown for SC-4 in the presence (A) and absence (B) of 30% trifluoroethanol/70% water. Peptide concentration was 6.3 mM in 10 mM potassium phosphate, pH 5.5 and 25°C. Spectra were accumulated with 8 k data points over 6000 Hz sweep width and were processed with 1 Hz line broadening. Only spectral regions downfield from the HDO resonance are shown and some resonance assignments are indicated. Figure 6. NOE-Derived Structures of SC-4. For dodecapeptide SC-4, 14 final NOE-derived structures have been superimposed at the left of the figure and structural statistics are given in Table 3. The average structure is shown at the right, and four positively charged residues, Kl, K4, R5, and K8, which lie on one surface of the amphipathic helix, are shown in the space-filling mode. Figure 7. Helical wheel projections for SC peptides. SC peptide sequences are shown in helical wheel projections as discussed in the text. Detailed Description of Preferred Embodiments This invention contributes to the development of agents in combating the ever-recurring problem of drug-resistant microorganisms. It involves the discovery of polypeptides effective in the treatment of bacterial infection and/or bacterial endotoxemia, as well as other disorders. The compositions comprising the polypeptides of this invention can be added to cells in culture or used to treat patients, such as mammals. Where the polypeptides are used to treat a patient, the polypeptide is preferably combined in a pharmaceutical composition with a pharmaceutically acceptible carrier such as a larger molecule to promote polypeptide stability or a pharmaceutically acceptible buffer that serves as a carrier for the polypeptide. Treatment can be prophylactic or therapeutic. Thus, treatment can be initiated before, during, or after the development of the condition (e.g., bacterial infection or endotoxemia). As such, the phrases "inhibition of or "effective to inhibit" a condition such as bacterial infection and/or endotoxemia, for example, includes both prophylactic and therapeutic treatment (i.e., prevention and/or reversal of the condition). The present invention provides a method for treating bacterial infection and/or endotoxemia in a patient (e.g., a mammal such as a human). This involves adrninistering to a patient an amount of a pharmaceutical composition effective to inhibit the bacterial infection and/or to neutralize endotoxin, wherein the pharmaceutical composition includes one or more polypeptides described herein. Analogously, the present invention provides a method for inhibiting bacterial infection and/or endotoxemia in vitro (e.g., in a cell culture). This method involves contacting cells with an amount of a composition effective to inhibit the bacterial infection and or to neutralize endotoxin, wherein the composition includes one or more polypeptides described herein. More than 100 years ago, it was determined that heat stable extracts of gram-negative enteric bacteria were highly toxic. Assuming that these toxins were released from the interior of the bacterium upon its death, the investigators termed these toxins "endotoxins." Subsequent chemical studies showed that these endotoxins are actually lipopolysaccharide (LPS) components of the outer membrane of enteric bacteria. Toxic LPS is composed of three structural units — an outer polysaccharide component, a core oligosacchari.de region, and the inner portion, lipid A, which affords the molecule its proinflammatory activities. Toxic LPS is released from the bacterium when it dies or during periods of rapid bacterial growth. In many species, for instance humans, cows, rabbits, mice, and rats, toxic LPS in serum binds rapidly to a plasma polypeptide named lipopolysaccharide binding protein (LBP). LBP, which is synthesized by hepatocytes as part of the acute phase response of inflammation, has a strong affinity for the lipid A portion of endotoxin. Activation of a cell by a toxic LPS-containing complex results in the synthesis, release, or activation of cell-derived proinflammatory mediators, which can include cytokines (e.g., interleukin-1, interleukin-6, interleukin-8, and tumor necrosis factor-α), platelet activating factor, nitric oxide, complement (e.g., C5a and C3a), prostagladins, leukotrienes, the kinin system, oxygen metabolites, catecholamines and endorphines. The mediators can impact organ systems including the heart, vascular system, coagulation system, lungs, liver, kidney and the central nervous system. These factors can lead to endotoxemia, also referred to as endotoxic shock, septic shock, circulatory shock, and septicemia, a progressive disease that can lead to death. Endotoxemia is typically caused by toxic LPS from Gram-negative bacteria, but can also be caused by Gram-positive bacteria, and occasionally, by fungi. Components released by Gram-positive bacteria that can cause endotoxemia include peptidoglycan and lipoteichoic acid, and lipoarabinomannan from the cell wall of Mycobacterium spp. Studies have shown that serum concentrations of the cytokine tumor necrosis factor (TNF) increase after onset of endotoxemia, and serum TNF activity is directly associated with the onset of signs of abdominal pain and fever, for example. Thus, endotoxin activity can also be measured by determining the amount of release of tumor necrosis factor alpha (TNF-α) from a macrophage cell line or by evaluating the symptoms of shock in animals. Production of TNF- α can be assayed as described by Mossman et ^.{Immunological Methods 65:55, 1983). In both the in vivo and in vitro methods, "inhibiting" a bacterial infection includes preventing as well as reversing or reducing the growth of bacteria in a patient or a cellular sample, and "neutralizing" endotoxin includes binding LPS and thereby removing it from the system of a patient or a cellular sample. The level of bacterial infection can be determined according to the bactericidal assay described in the Examples Section. The level of endotoxemia can be determined according to the LPS neutralization assay described in the Examples Section. These assays can be used to determine the effectiveness of a polypeptide, whether used in vivo or in vitro. To determine the effectiveness of the treatment of a patient having a bacterial infection, a blood sample can be taken, a culture developed, and the amount of live bacteria determined according to the bactericidal assay described in the Examples Section. To determine the effectiveness of the treatment of a patient having endotoxemia, a blood sample can be taken, a culture developed, and the amount of cytokines (e.g., TNF-α, JX- 1) can be determined using methods known to one of skill in the art. For example, the WEHI assay can be used for the detection of TNF-α (Battafarano et al., Surgery 118, 318-324 (1995)). The effective amount of a peptide for treating a bacterial infection will depend on the bacterial infection, the location of the infection and the peptide. An effective amount of the peptide for treating bacterial infection is that amount that diminishes the number of bacteria in the animal and that diminishes the symptoms associated with bacterial infection such as fever, pain and other associated symptoms of the bacterial infection. The effective amount of a peptide can be determined by standard dose response methods in vitro and an amount of peptide that is effective to kill at least about 50% to about 100% of the bacteria (LD 50 ) and more preferably about 60% to about 100% of the bacteria would be considered an effective amount. Preferably, the peptide has an effective dose at a concentration of about 1 x 10 "4 M to about 1 x 10 "10 M, and more preferably at a concentration of about 1 x 10 "7 M to about 1 x 10 "9 M. Peptides that are considered to be bactericidal kill at least one organism selected from the group of P. aeruginosa, P. cepacia, E. coli B, Salmonella, Proteus mirabilis, and Staphylococcus aureus at concentrations of about 10 "10 M or greater under physiological conditions (e.g., at a pH of 5.6). Alternatively, an effective amount of the peptide for treating a bacterial infection can be determined in an animal system such as a mouse. Acute peritonitis can be induced in mice such as outbred Swiss webster mice by intraperitoneal injection with bacteria such as P. aeruginosa as described by Dunn et al. {Surgery, 98:283, 1985); Cody et al. {Int. Surg. Res., 52:315, 1992). Different amounts of peptide can be injected at one hour intravenously prior to the injection of the bacteria. The percentage of viable bacteria in blood, spleen, and liver can be determined in the presence and absence of the peptide or other antibiotics. While not meant to limit the invention, it is believed that bactericidal peptide could also enhance the effectiveness of other antibiotics such as erythromycin, and the like. Bactericidal activity can be evaluated against a variety of bacteria, preferably Gram-negative bacteria, but the types of bacteria can include Pseudomonas spp including P. aeruginosa and P. cepacia, E. coli strains, including E. coli B, Salmonella, Proteus mirabilis and Staphylococcus strains such as Staphylococcus aureus. A preferred organism is Pseudomonas aeruginosa. Peptides with endotoxin neutralizing activity can be used to treat mammals infected with Gram-negative bacteria systemically and that exhibit symptoms of endotoxin shock such as fever, shock, and TNF-α release. The animals are typically infected with one or more Gram-negative bacteria such as Pseudomonas spp., rough strains of E. coli, encapsulated E. coli and smooth strain E. coli. The endotoxin neutralizing peptide can be combined with other agents that are known and used to treat endotoxin shock. Endotoxin neutralizing activity can be measure by deterrnining the molar concentration at which the peptide completely inhibits the action of lipopolysaccharide in an assay such as the Limulus amoebocyte lysate assay (LAL, Sigma Chemicals, St. Louis, MO) or the chromogenic LAL 1000 test (Biowhittacker, Walkersville, MD). Endotoxin neutralizing activity can also be measured by calculating an inhibitory dose 50 (LD 50 ) using standard dose response methods. An inhibitory dose 50 is that amount of peptide that can inhibit 50% of the activity of endotoxin. Peptides preferably neutralized endotoxin at a molar concentration of about 1 x 10 M to about 10 " M, more preferably about 10 "5 M to about 10 "6 M. Peptides considered to not have endotoxin neutralizing activity do not neutralize endotoxin at a molar concentration of about 10 "4 M or less. The present invention also provides a method for decreasing the amount of TNF-α in a patient (e.g., a mammal such as a human). This involves administering to a patient an amount of a pharmaceutical composition effective to decrease the amount of TNF-α in a patient's system as determined by evaluating serum levels of TNF-α, wherein the pharmaceutical composition includes one or more polypeptides described herein. Analogously, the present invention provides a method for decreasing the amount of TNF-α in vitro (e.g., in a cell culture). This method involves incubating cells with an amount of a composition effective to decrease TNF-α amounts in the cell culture, wherein the composition includes one or more polypeptides described herein. For both in vivo and in vitro methods, the WEFfl assay can be used for the detection of TNF- α (Battafarano et al., Surgery 118, 318-324 (1995)) in cell culture or in serum from a patient. Alternatively, the amount of TNF-α in a sample can be assayed using an anti-TNF-α antibody. A polypeptide "active" for decreasing TNF- α can be evaluated using an in vitro test, and preferably shows an at least 10% decrease in the amount of TNF-α. Angiogenesis is crucial to numerous biological functions in the body, from normal processes like embryogenesis and wound healing to abnormal processes like tumor growth, arthritis, restenosis and diabetic retinopathy. The use of agents that can inhibit angiogenesis in vitro and in vivo, particularly in anti-tumor research, has indicated that anti-angiogenic therapy will be a promising therapeutic modality in the future. The search for angiogenic inhibitors has been focused on controlling two of the processes that promote angiogenesis: endothelial cell (EC) growth and adhesion primarily because ECs are more accessible than are other cells to pharmacologic agents delivered via the blood and ECs are genetically stable and are not easily mutated into drug resistant variants. Most anti-angiogenic agents have been discovered by identifying endogenous molecules, primarily proteins, which inhibit EC growth. This traditional approach has produced a number of anti-angiogenics, such as platelet factor-4 (PF4), thrombospondin, tumor necrosis factor (TNF), interferon- γ inducible protein- 10, angiostatin, endostatin, vasostatin, and bactericidal- permeability increasing (BPI) protein. In toto, about forty anti-angiogenic agents, identified using various approaches, are currently known. It has also been postulated that tumor growth can be controlled by deprivation of vascularization (FolkmanJ natl. Cancer. Inst. 82, 4-6 (1990); Folkman et al., J biol. Chem. 267, 10931-10934 (1992)). A growing number of endogenous inhibitors of angiogenesis such as platelet factor-4 (PF4), interferon- γ inducible protein-10 (TP-10), thrombospondin-1 (TSP-1), angiostatin, as well as synthetic agents, e.g., thalidomide, TNP-470, and metalloproteinase inhibitors have been described. Some of these agents are currently being tested in phase I/π clinical trials. Previous research described in Griffioen et al., Blood 88, 667- 673 (1996), and Griffioen et al., Cancer Res. 56, 1111-1117 (1996) has shown that pro-angiogenic factors in tumors induce down-regulation of adhesion molecules on endothelial cells in the tumor vasculature and induce anergy to inflammatory signals such as tumor necrosis factor α (TNFα), mterleukin-1, and interferon-γ. EC exposed to vascular endothelial cell growth factor (VEGF) (Griffioen et al., Blood 88, 661-613 (1996)) and basic fibroblast growth factor (bFGF) (Griffioen et al., Blood 88, 667-673 (1996); and Melder et al., Nature Med. 2, 992-997 (1996)) have a severely hampered up-regulation of intercellular adhesion molecule- 1 (ICAM-1) and induction of vascular cell adhesion molecule- 1 (VCAM-1) and E-selectin. This phenomenon, which was named tumor-induced EC anergy, is one way in which tumors with an angiogenic phenotype may escape infiltration by cytotoxic leukocytes. Because angiogenesis-mediated down-regulation of endothelial adhesion molecules (EAM) may promote tumor outgrowh by avoiding the immune response (Griffioen et al., Blood 88, 661-613 (1996); Kitayama et al., Cancer. Res. 544729-4733 (1994); and Piali et al., J. exp. Med. 181, 811-816 (1995)), it is believed that inhibition of angiogenesis would overcome the down- regulation of adhesion molecules and the unresponsiveness to inflammatory signals. In support of this hypothesis, a relation between E-selectin up- regulation and the angiostatic agent AGM-1470 has been reported (Budson et al., Biochem. Biophys. Res. Comm. 225, 141-145 (1996)). It has also been shown that inhibition of angiogenesis by PF4 up-regulates ICAM-1 on bFGF-simulated EC. In addition, inhibition of angio-genesis by PF4 overcomes the angiogenesis- associated EC anergy to inflammatory signals. Thus, the present invention provides a method for inhibiting endothelial cell proliferation in a patient (e.g., a mammal such as a human). This involves administering to a patient an amount of a pharmaceutical composition effective to inhibit the growth or endothelial cells, wherein the pharmaceutical composition includes one or more polypeptides described herein. Analogously, the present invention provides a method for inhibiting endothelial cell proliferation in vitro (e.g., in a cell culture). This method involves contacting cells with an amount of a composition effective to prevent and/or reduce the growth of endothelial cells, wherein the composition includes one or more polypeptides described herein. For determining the amount of endothelial cell proliferation in vivo, various methods known to one of skill in the art could be used. For example, for evaluation of endothelial cell growth in tumors, tissue sections can be appropriately stained to quantify vessel density. For deterrnining the amount of endothelial cell proliferation in vitro, the EC Proliferation Assay described in the Examples Section can be used, which involves the uptake of tritiated thymidine by cells in cell culture. A polypeptide that is "active" for inhibiting endothelial cell proliferation is preferably one that causes an at least 10% reduction in endothelial cell proliferation at a concentration lower than 10 "4 M. The present invention also provides a method for inhibiting angiogenic- factor mediated inter-cellular adhesion molecule expression down-regulation in a patient (e.g., a mammal such as a human). This involves administering to a patient an amount of a pharmaceutical composition effective to prevent and/or reduce the amount of ICAM expression down-regulation, wherein the pharmaceutical composition includes one or more polypeptides described herein. Analogously, the present invention provides a method for inhibiting angiogenic- factor mediated inter-cellular adhesion molecule expression down-regulation in vitro (e.g., in a cell culture). This method involves contacting cells with an amount of a composition effective to prevent and/or reduce the amount of ICAM expression down-regulation, wherein the composition includes one or more polypeptides described herein. The present invention provides a method for inhibiting angiogenesis (i.e., new blood vessel formation) in a patient (e.g., a mammal such as a human). This involves administering to a patient an amount of a pharmaceutical composition effective to prevent and/or reduce angiogenesis, wherein the pharmaceutical composition includes one or more polypeptides described herein. Analogously, the present invention provides a method for inhibiting angiogenesis in vitro (e.g., in a cell culture). This method involves contacting cells with an amount of a composition effective to prevent and/or reduce angiogenesis, wherein the composition includes one or more polypeptides described herein. For determining the amount of angiogenesis in vivo, various methods known to one of skill in the art could be used. For example, for evaluation of angiogenesis in tumors, tissue sections can be appropriately stained to quantify vessel density. For determining the amount of angiogenesis in vitro, the In vitro Angiogenesis Assay (i.e., EC Tube Formation Assay) described in the Examples Section can be used, which involves the disappearance of EC sprouting in cell culture. A polypeptide that is "active" for angiogenesis inhibition is preferably one that causes an at least 10% reduction in endothelial cell sprouting at a concentration lower than 10 "4 M. The present invention provides a method for inhibiting tumorigenesis in a patient (e.g., a mammal such as a human). This involves acuTiimstering to a patient an amount of a pharmaceutical composition effective to prevent and/or reduce tumor growth, wherein the pharmaceutical composition includes one or more polypeptides described herein. Methods of determining the inhibition of tumorigenesis are well known to those of skill in the art, including evaluation of tumor shrinkage, survival, etc. A preferred polypeptide is selected from the group consisting of: ANIKLSVQMKLF (SEQ ID NO:l); KLSVQMKLFKRH (SEQ ID NO:2); VQMKLFKRHLKW (SEQ ID NO:3); KLFKRHLKWKII (SEQ ID NO:4); KRHLKWKHVKL (SEQ ID NO:5); LKWKHVKLNDG (SEQ ID NO:6); KHVKLNDGREL (SEQ ID NO:7); VKLNDGRELSLD (SEQ ID NO:8); and analogs thereof. An alternative polypeptide is selected from the group consisting of: QMKLFKRHLKWK (SEQ ID NO:9); MKLFKRHLKWKI (SEQ ID NO: 10); MKLFKRHLKWKIJV (SEQ ID NO: 11); XLFKRHLKWKΠ (SEQ ID NO: 12); KLFXRHLKWKπ (SEQ ID NO:13); KLFKRHLXWKΠ (SEQ ID NO:14); KLFKRHLKWXπ (SEQ ID NO: 15); KLFKKHLKWKΠ (SEQ ID NO: 16); KLFKHLKWKΠ (SEQ ID NO:17); and analogs thereof; wherein X is an amino acid, natural or synthetic. Preferably, X is norleucine. A more preferred polypeptide is selected from the group consisting of: ANIKLSVQMKLF (SEQ ID NO:l); KLSVQMKLFKRH (SEQ ID NO:2); VQMKLFKRHLKW (SEQ ID NO:3); KLFKRHLKWKII (SEQ ID NO:4); KRHLKWKHVKL (SEQ ID NO:5); LKWKJJVKLNDG (SEQ ID NO:6); KHVKLNDGREL (SEQ ID NO:7); and VKLNDGRELSLD (SEQ ID NO:8). The polypeptides of SEQ ID NOs: 1-17 can be in their free acid form or they can be amidated at the C-terminal carboxylate group. The present invention also includes analogs of the polypeptide of SEQ ID NOs: 1-17, which typically have structural similarity with SEQ ID NOs: 1-17. An "analog" of a polypeptide includes at least a portion of the polypeptide, wherein the portion contains deletions or additions of one or. more contiguous or noncontiguous amino acids, or containing one or more amino acid substitutions. Substitutes for an amino acid in the polypeptides of the invention are preferably conservative substitutions, which are selected from other members of the class to which the amino acid belongs. For example, it is well-known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity and hydrophilicity) can generally be substituted for another amino acid without substantially altering the structure of a polypeptide. For the purposes of this invention, conservative amino acid substitutions are defined to result from exchange of amino acids residues from v^thin one of the following classes of residues: Class I: Ala, Gly, Ser, Thr, and Pro (representing small aliphatic side chains and hydroxyl group side chains); Class II: Cys, Ser, Thr, and Tyr (representing side chains including an -OH or -SH group); Class HI: Glu, Asp, Asn, and Gin (carboxyl group containing side chains): Class TV: His, Arg, and Lys (representing basic side chains); Class V: He, Val, Leu, Phe, and Met (representing hydrophobic side chains); and Class VT: Phe, Tip, Tyr, and His (representing aromatic side chains). The classes also include related amino acids such as 3Hyp and 4Hyp in Class I; homocysteine in Class H; 2-aminoadipic acid, 2-aminopimelic acid, γ-carboxyglutamic acid, β- carboxyaspartic acid, and the corresponding amino acid amides in Class HI; ormthine, homoarginine, N-methyl lysine, dimethyl lysine, trimethyl lysine, 2,3- diaminopropionic acid, 2,4-diarninobutyric acid, homoarginine, sarcosine and hydroxylysine in Class IV; substituted phenylalanines, norleucine, norvaline, 2- arninooctanoic acid, 2-arninoheptanoic acid, statine and β-valine in Class V; and naphthylalanines, substituted phenylalanines, tetrahydroisoquinoline-3- carboxylic acid, and halogenated tyrosines in Class VI. Polypeptide analogs, as that term is used herein, also include modified polypeptides. Modifications of polypeptides of the invention include chemical and/or enzymatic derivatizations at one or more constituent amino acid, including side chain modifications, backbone modifications, and N- and C- terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like. A preferred polypeptide analog is characterized by having at least one of the biological activities described herein. Such an analog is referred to herein as a "biologically active analog" or simply "active analog." The biological activity of a polypeptide can be determined, for example, as described in the Examples Section. The polypeptides of the invention may be synthesized by the solid phase method using standard methods based on either t-butyloxycarbonyl (BOC) or 9- fluorenylmethoxy-carbonyl (FMOC) protecting groups. This methodology is described by G.B. Fields et al. in Synthetic Peptides: A User's Guide. W.M. Freeman & Company, New York, NY, pp. 77-183 (1992). The present peptides may also be synthesized via recombinant techniques well known to those skilled in the art. For example, U.S. Patent No. 5,595,887 describes methods of forming a variety of relatively small peptides through expression of a recombinant gene construct coding for a fusion protein which includes a binding protein and one or more copies of the desired target peptide. After expression, the fusion protein is isolated and cleaved using chemical and/or enzymatic methods to produce the desired target peptide. The polypeptides of this invention can be administered alone in a pharmaceutically acceptable carrier, as an antigen in association with another protein, such as an immunostimulatory protein or with a protein carrier such as, but not limited to, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, or the like. They may be employed in a monovalent state (i.e., free peptide or a single peptide fragment coupled to a carrier molecule). They may also be employed as conjugates having more than one (same or different) peptides bound to a single carrier molecule. The carrier may be a biological carrier molecule (e.g., a glycosaminoglycan, a proteoglycan, albumin or the like) or a synthetic polymer (e.g., a polyalkyleneglycol or a synthetic chromatography support). Typically, ovalbumin, human serum albumin, other proteins, polyethylene glycol, or the like are employed as the carrier. Such modifications may increase the apparent affinity and/or change the stability of a peptide. The number of peptides associated with or bound to each carrier can vary, but from about 4 to 8 peptides per carrier molecule are typically obtained under standard coupling conditions. The polypeptides can be conjugated to other polypeptides using standard methods such as activation of the carrier molecule with a heterobifunctional sulfosuccinimidyl 4-(n-maleimidomethyl) cyclohexane-1-carboxylate reagent. Cross-linking of an activated carrier to a peptide can occur by reaction of the maleimide group of the carrier with the sulfhydryl group of a peptide containing a cysteine residue. Conjugates can be separated from free peptide through the use of gel filtration column chromatography or other methods known in the art. For instance, peptide/carrier molecule conjugates may be prepared by treating a mixture of peptides and carrier molecules with a coupling agent, such as a carbodiimide. The coupling agent may activate a carboxyl group on either the peptide or the carrier molecule so that the carboxyl group can react with a nucleophile (e.g., an amino or hydroxyl group) on the other member of the peptide/carrier molecule, resulting in the covalent linkage of the peptide and the carrier molecule. For example, conjugates of a peptide coupled to ovalbiimin may be prepared by dissolving equal amounts of lyophilized peptide and ovalbumin in a small volume of water. In a second tube, l-ethyl-3-(3-dime ylamino-propyl)- carboiimide hydrochloride (EDC; ten times the amount of peptide) is dissolved in a small amount of water. The EDC solution is added to the peptide/ovalbumin mixture and allowed to react for a number of hours. The mixture may then be dialyzed (e.g., into phosphate buffered saline) to obtain a purified solution of peptide/ovalbumin conjugate. The present invention also provides a composition that includes one or more active agents (i.e., polypeptides) of the invention and one or more pharmaceutically acceptable carriers. One or more polypeptides with demonstrated biological activity can be administered to a patient in an amount alone or together with other active agents and with a pharmaceutically acceptable buffer. The polypeptides can be combined with a variety of physiological acceptable carriers for delivery to a patient including a variety of diluents or excipients known to those of ordinary skill in the art. For example, for parenteral administration, isotonic saline is preferred. For topical admimstration, a cream, including a carrier such as dimethylsulfoxide (DMSO), or other agents typically found in topical creams that do not block or inhibit activity of the peptide, can be used. Other suitable carriers include, but are not limited to, alcohol, phosphate buffered saline, and other balanced salt solutions. The formulations may be conveniently presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Preferably, such methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations. The methods of the invention include administering to a patient, preferably a mammal, and more preferably a human, the composition of the invention in an amount effective to produce the desired effect. The peptides can be administered as a single dose or in multiple doses. Useful dosages of the active agents can be determined by comparing their in vitro activity and the in vivo activity in animal models. Methods for extrapolation of effective dosages in mice, and other animals, to humans are known in the art; for example, see U.S. Patent No. 4,938,949. The agents of the present invention are preferably formulated in pharmaceutical compositions and then, in accordance with the methods of the invention, administered to a patient, such as a human patient, in a variety of forms adapted to the chosen route of administration. The formulations include, but are not limited to, those suitable for oral, rectal, vaginal, topical, nasal, ophthalmic, or parental (including subcutaneous, intramuscular, intraperitoneal, intratumoral, and intravenous) administration. Formulations suitable for parenteral administration conveniently include a sterile aqueous preparation of the active agent, or dispersions of sterile powders of the active agent, which are preferably isotonic with the blood of the recipient. Isotonic agents that can be included in the liquid preparation include sugars, buffers, and sodium chloride. Solutions of the active agent can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions of the active agent can be prepared in water, ethanol, a polyol (such as glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, glycerol esters, and mixtures thereof. The ultimate dosage form is sterile, fluid, and stable under the conditions of manufacture and storage. The necessary fluidity can be achieved, for example, by using liposomes, by employing the appropriate particle size in the case of dispersions, or by using surfactants. Sterilization of a liquid preparation can be achieved by any convenient method that preserves the bioactivity of the active agent, preferably by filter sterilization. Preferred methods for preparing powders include vacuum drying and freeze drying of the sterile injectible solutions. Subsequent microbial contamination can be prevented using various antimicrobial agents, for example, antibacterial, antiviral and antifungal agents including parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Absorption of the active agents over a prolonged period can be achieved by including agents for delaying, for example, aluminum monostearate and gelatin. Formulations of the present invention suitable for oral administration may be presented as discrete units such as tablets, troches, capsules, lozenges, wafers, or cachets, each containing a predetermined amount of the active agent as a powder or granules, as liposomes containing the chemopreventive agent, or as a solution or suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, an emulsion, or a draught. Such compositions and preparations typically contain at least about 0.1 wt-% of the active agent. The amount of polypeptide (i.e., active agent) is such that the dosage level will be effective to produce the desired result in the patient. Nasal spray formulations include purified aqueous solutions of the active agent with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucous membranes. Formulations for rectal or vaginal administration may be presented as a suppository with a suitable carrier such as cocoa butter, or hydrogenated fats or hydrogenated fatty carboxylic acids. Ophthalmic formulations are prepared by a similar method to the nasal spray, except that the pH and isotonic factors are preferably adjusted to match that of the eye. Topical formulations include the active agent dissolved or suspended in one or more media such as mineral oil, petroleum, polyhydroxy alcohols, or other bases used for topical pharmaceutical formulations. The tablets, troches, pills, capsules, and the like may also contain one or more of the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid, and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, fructose, lactose, or aspartame; and a natural or artificial flavoring agent. When the unit dosage form is a capsule, it may further contain a liquid carrier, such as a vegetable oil or a polyethylene glycol. 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, sugar, and the like. A syrup or elixir may contain one or more of a sweetening agent, a preservative such as methyl- or propylparaben, an agent to retard crystallization of the sugar, an agent to increase the solubility of any other ingredient, such as a polyhydric alcohol, for example glycerol or sorbitol, a dye, and flavoring agent. The material used in preparing any unit dosage form is substantially nontoxic in the amounts employed. The active agent may be incorporated into sustained- release preparations and devices. In the examples presented herein, a series of dodecapeptides (SC-1 through SC-8) which "walk through" the amino acid sequence of βpep-25 (SEQ ID NO: 19) was investigated for the ability to kill bacteria and to neutralize LPS, specifically to kill Gram-negative and positive bacteria and to neutralize endotoxin. One of these SC peptides, SC-4 (KLFKRHLKWKI I, SEQ ID NO:4), was identified as being exceptionally potent with an LD50 of 3 nanomolar against Gram-negative bacteria, even more potent than parent peptide βpep-25. Against Gram-positives, SC-4 also showed good activity with LD50s in the sub-micromolar range. To demonstrate broad spectrum bactericidal activity, several strains were investigated: a rough strain of Gram-negative bacteria, J5; four clinically- relevant Gram-negative smooth stains {Pseudomonas aeruginosa J96, H5 and IA2), and two Gram-positive strains (MN-8 and MNHO). Moreover, since peptides which form both β-sheet and helix conformations can be bactericidal and these dodecapeptides were derived from a β -sheet-forming βpep peptide, CD and NMR data were acquired in aqueous solution and in 30% trifluoroethanol (TFE) solution to investigate into which conformational type these 12mers fall. Leakage studies performed using fluorescence microscopy, indicated rapid bacterial membrane permeability with tl/2 values of about 10 minutes at 100%-kill doses of peptide. SC-4 also effectively neutralized endotoxin in the micromolar range and is not hemolytic below 10 "4 M. For all SC peptides, circular dichroic data strongly suggested the presence of both α-helix or 3 10 - helix. NOESY data acquired on SC peptides in the presence of 30% trifluoroethanol, also showNOEs characteristic of both α-helix or 3 10 -helix. Differences in activities among these helical dodecapeptides allowed some structure-function relations to be inferred. For SC-4 which is most 3 10 helix-like, NOE- based computational modeling yields an amphipathic 3 10 helical structure with Kl, K4, R5, K8 and K10 being arrayed pentagonally on one face of the helix. Several single-residue substituted variants of SC-4 were also investigated. Bactericidal activities in lysine/arginine-substituted norleucine variants of SC-4 vary, not surprisingly, from bacterial strain to strain. Against strain J5, any substitution shows no more than a 2-fold activity decrease, whereas against P. a., substitution at N-terminal positions 1, K4 or R5 drop activity significantly by about 20-fold. For endotoxin neutralization, substitution of charged groups has little effect, whereas removal of the C-terminal isoleucines drops activity by about 500-fold. Relative to other known bactericidal peptides in the linear peptide, helix-forming catagory, SC-4 appears to be the most potent, broad spectrum bactericidal agent identified to date. The present invention also provides the three-dimensional structure of certain of the polypeptides derived using nuclear magnetic resonance (NMR) spectroscopy (e.g., one- and two-dimensional) and optionally circular dichroism (CD) spectroscopy. This information is of significant utility in fields such as drug discovery. An understanding of the structure of, for example SC-4, allows the design of drugs having similar structure, and hence, similar activity. Such drugs can be peptides, peptidomimetics (e.g., peptidic compounds in which the peptide backbone is substituted with one or more benzodiazepine molecules, peptides in which all L-amino acids are substituted with the corresponding D- amino acids, and "retro-inverso" peptides), and nonpeptidic mimetics. Such drugs are preferably nonpeptidic mimetics. The structural data presented herein can be used (preferably in combination with Structure Activity Relationship (SAR) information and potential pharmacophore sites) to select a candidate compound (potential drug) from a database of three-dimensional structures of known compounds. The three-dimensional structures in the database can be either experimentally deteπnined or computationally generated. Alternatively, a candidate compound can also be designed de novo. The candidate compound will have a three- dimensional structure that is at least in part (and preferably, substantially) similar to the three-dimensional structure of one of the polypeptides presented herein, preferably SC-4. Various molecular modeling and/or compound database search techniques known to one of skill in the art can be used for selecting candidate compounds. Such compounds can be evaluated for their biological activity using the assays described herein. Specifically, the present invention provides detailed information about the shape and structure of the surface active domain of certain of the polypeptides described herein. Each of the constituent amino acids forming the "surface active domain" is defined by those shown in Fig. 6. The term "surface active domain" refers to a region of a molecule or molecular complex that, as a result of its shape, is active for the treatment of one or more conditions, such as those described herein. Such surface active domain is the part of the polypeptide that is believed to be important for imparting the desired function. Thus, the structural information of just this domain can be used for identifying candidate compounds as described above. The term "structure coordinates" refers to Cartesian coordinates (see Table 5) derived from computational modeling using internuclear distances obtained from NMR spectroscopic experiments, i.e., NOE's (see Table 6), as described in the Examples Section. The structure coordinates generate a unique configuration of points in space. It should be noted that these coordinates represent a statistical best fit representation of numerous structures for any one polypeptide, and that slight variations in individual structure coordinates would be expected. Also, similar or identical configurations can be defined by an entirely different set of coordinates, provided the distances and angles between coordinates remain essentially the same. Generally, herein the structure is an amphipathic structure, such as a helix (which can be viewed as a cylinder), wherein one surface includes positively charged amino acid residues (preferably, one surface is composed primarily of positively charged amino acid residues (i.e., hydrophilic amino acid residues)) and the opposing surface includes hydrophobic amino acid residues (preferably, the opposing surface is composed primarily of hydrophobic amino acid residues). The surface active domain is identified by the positively charged amino acid residues and the hydrophobic opposing surface. In the case of SC-4, which has a helical structure, one surface includes four positively charged amino acid residues, although there may be more or less for other structures, and the opposing surface includes hydrophobic amino acid residues. More specifically, for SC-4, the surface active domain includes the structure coordinates of the atoms of the amino acid residues Kl (atoms 1-24), K4 (atoms 64-85), R5 (86- 109), and K8 given in Table 5, as shown in Fig. 6. An alternative way of visualizing he surface active domains is in terms of spatial relationships among functionally key residues derived from SAR (see, for example, Fig. 6). Various computational analyses can be used to determine whether a compound is sufficiently similar to the three-dimensional structure desired. Such analyses can be carried out in current software applications, as known in the art. This can involve a comparison of three-dimensional structure, hydrophobicity, steric bulk, electrostatic properties, bond angles, size or molecular composition, etc. For example, Quanta's Molecular Similarity package (Molecular Simulations Inc., Waltham, MA) permits comparison between different structures, different conformations of the same structure, and different parts of the same structure. Typically, the structure of the compound being analyzed is translated and rotated to obtain an optimum fit with the structure of the active polypeptide. Preferred candidate structures are those having a set of structure coordinates with a root mean square deviation (i.e., the square root of the arithmetic mean of the squares of the deviations of the mean) of conserved residue atoms of less than 2.0 Angstroms when superimposed on the relevant structure coordinates. More preferably, the root mean square deviation is less than 1.0 Angstrom. Once a candidate compound is identified using molecular modeling or database comparison techniques, it can be synthesized by a variety of techniques known to those of skill in the art. One specific method of the present invention involves evaluating a candidate compound for structural similarity to that of KLFKRHLKWKII (SEQ ID NO:4) by: supplying a tliree-dimensional structure of KLFKRHLKWKH (SEQ ID NO:4) or a portion thereof; supplying a three-dimensional structure of a candidate compound; and comparing the three-dimensional structure of the candidate compound with the three-dimensional structure of KLFKRHLKWKH (SEQ ID NO:4) or a portion thereof. Examples Objects and advantages of this invention are further illustrated by the following examples, but the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed to unduly limit this invention. Methods & Materials Peptide preparation Peptides were synthesized using a Milligen/Biosearch 9600 peptide solid- phase synthesizer using fluorenylmethoxycarbonyl chemistry. Lyophilized crude peptides were purified by preparative reversed-phase HPLC on a CI 8 column with an elution gradient of 0-60% acetonitrile with 0.1% trifluoroacetic acid in water. Purity and composition of the peptides were verified by HPLC (Beckman Model 6300), amino acid analysis, and mass spectrometry. Bacterial strains Pseudomonas aeruginosa type 1 is a clinical, smooth strain isolate serotyped by using the scheme of Homma et al., Japan. J. Exp. Med. 46, 329-336 (1976) and maintained in the lab by monthly transfer on blood agar plates. E. coli J96, IA2, and H5 are smooth strain, uropathogenic clinical isolates kindly maintained and provided by J.R. Johnson and described in Johnson et al., J. Infect. Disease 173, 920-926 (1996) for J96 and IA2 and in Johnson et al., J Infect. Disease 173, 746-749 (1996) for H5. J5 is an E. coli rough strain initially referenced by G.R. Siber and discussed in Warren et al., Infect. Immunity 55, 1668-1673 (1987) and is analogous to the smooth strain E. coli 0111 :B4 used in the BioWhittaker LAL endotoxin detection and quantitation kit described below. Gram-positive MN8 and MNHO are two patient isolates of Staphylococcus aureus, which were kindly provided by P.M. Schlievert and described in Bohach et al., Rev. Infect. Diseases 11, 75-82 (1989) for MNHO and in Schlievert et al., J Infect. Diseases 147, 236-242 (1983) for MN8. All cultures are maintained on nutrient agar plates. Bactericidal assay Pyrogen-free solutions were used throughout the assay. Log phase bacteria were obtained by transferring an overnight culture or scraping crystals off -85°C glycerol stocks of overnight cultures. Bacteria were washed and resuspended in 0.9% sodium chloride with adjustment to an optical density at 650 nm which yields 3 x 10 s CFU/ml. Bacteria were then diluted 1 : 10 in 0.08 M citrate phosphate buffer, pH 7.0 (prepared by mixing 0.08 M citric acid with 0.08 M dibasic sodium phosphate). Bacteria (0.15 ml) were incubated with peptide in a final volume of 1.0 ml of buffer. The assay was done in 17x100 polypropylene tubes in a reciprocal water bath shaker at 37°C for 30 minutes. Following this 30 minute (min) incubation, 10-fold dilutions were made in 0.9% sodium chloride. Dilutions were done to 10 "4 and 20 μl of each dilution was streaked across an agar plate. Gram-positive organisms were plated on nutrient agar plates containing 2% agar and Gram-negative organisms were plated on MacConkey agar (2%). Plates were incubated overnight at 37°C and counted the next morning. The dilution containing 10-100 bacteria was counted and the number multiplied by 50 to adjust all counts to the number bacteria killed per milliliter. Peptide concentrations were converted to logarithm base ten and graphed. Bactericidal activity was determined by dose response where LD50 values were determined by best fits of a sigmoidal curve to the dose response data. Limulus amoebocyte lysate assay for LPS neutralization The ability of synthetic peptides to neutralize endotoxin was detected using the chromogenic QCL-1000 kit from BioWhittaker, Inc. (Walkersville, MD) as described in their protocol. This method is quantitative for Gram- negative bacterial endotoxin (lipopolysaccharide, LPS). In the Limulus amebocyte lysate (LAL) assay, peptides that are active inhibit the LPS-mediated activation of a proenzyme (Young et al., J Clin. Invest. 51, 1790-1798 (1972)) whose active form would release p-nitroaniline (pNA) from a colorless synthetic substrate (Ac-Ile-Glu-Ala-Arg-pNA), producing a yellow color (pNA) whose absorption is monitored spectrophotometrically at 405-410 nm. The initial rate of enzyme activation is proportional to the concentration of endotoxin present. The concentration of peptide required to bind to LPS and therefore to inhibit the Limulus amoebocyte lysate driven by 0.04 unit (or 0.01 ng) of E. coli 055:B5 LPS (from SIGMA) was deteπnined by dose response. Leakage Kinetics Bacterial cell membrane integrity was measured using the LIVE/DEAD BacLight bacterial viability kit (L-7007) from Molecular Probes, Inc. (Eugene, OR) which uses mixtures of red and green fluorescent stains to differentiate damaged and intact bacteria, respectively. Two E.coli strains (H5 and J5) and one Staphylococcus aureus strain (MN8) were investigated, and SC peptides were used at a dose that initiates 100% killing. Time points were taken between zero and 60 min. Fluorescence emission (excitation at 470 nm and emission at 490 nm to 700 nm) of each cell suspension was measured in a fluorescence spectrophotometer. 5 μL of each sample was placed on a microscope slide with a square cover slip and sealed to prevent movement. Hemolytic Activity Human red blood cells were washed three times using phosphate buffered saline (PBS: 35 mM phosphate buffer, 0.15 M NaCl, pH 7.0) prior to performing the assay. One hundred μL of human red blood cells suspended in 0.4% (v/v) PBS was placed in eppendorf tubes, and 100 μL of serially diluted peptides in PBS was added (peptide concentration ranged from 1 μM to 100 μM). Tubes were incubated for 1 hr at 37°C and centrifuged at 1000 g for 5 min. One hundred μL aliquots of the supernatant was then transferred to eppendorf tubes, and hemolysis was measured by absorbance at 414 nm. Zero percentage hemolysis and 100% hemolysis were determined in PBS and in 1% Triton-X 100, respectively. The hemolysis percentage was calculated using the following formula: % hemolysis = [( ^in the peptide solution - A414 in PBS)/(A414 in 1% Triton-X 100 - A414 in PBS)] x 100. Circular Dichroism Circular dichroic (CD) spectra were measured on a JASCO JA-710 automatic recording spectropolarimeter coupled with a data processor. Curves were recorded digitally and fed through the data processor for signal averaging and baseline subtraction. Peptides were dissolved (0.6 mg/mL) in 10 mM potassium phosphate buffer, pH 5.5, and CD spectra were recorded at 20°C over a 190 nm to 250 nm range using a 0.5 mm path-length, thermally-jacketed quartz cuvette. Temperature was controlled by using a NesLab water bath. The scan speed was 20 nm min. Spectra were signal-averaged 8 times, and an equally signal-averaged solvent baseline was subtracted. CD spectra on each peptide were acquired as a function of the concentration of trifluoroethanol (TFE) from 0% to 80% (volume/volume, v/v). CD spectra were deconvoluted as described by Sreerama et al., Anal. Biochem. 209, 32-44 (1993). NMR Measurements For NMR measurements, freeze-dried peptide was dissolved in H 2 O. Peptide concentration was usually about 5 to 6 mM. pH was adjusted to pH 5.5 by adding μL quantities of NaOD or DC1 to the peptide sample. NMR spectra were acquired on a Varian UNITY Plus-600 NMR spectrometer. The water resonance was suppressed by direct irradiation (0.8 s) at the water frequency during the relaxation delay between scans. 2D-homonuclear magnetization transfer (HOHAHA) spectra, obtained by spin- locking with a MLEV-17 sequence (Bax et al., J. Magn. Reson. 65, 355- 360. (1985)) with a mixing time of 60 ms, were used to identify spin systems. NOESY experiments (Wider et al., J Magn. Reson. 56, 207-234 (1984)) were performed for confbrmational analysis. All 2D-NMR spectra were acquired in the States-TPPI phase sensitive mode (States et al., J Magn. Reson. 48, 286-293 (1982); and Bodenhausen et al., J. Magn. Res. 37, 93-99 (1980)). The water resonance was suppressed by direct irradiation (0.8 s) at the water frequency during the relaxation delay between scans as well as during the mixing time in NOESY experiments. 2D-NMR spectra were collected as 256 to 512 tl experiments, each with 2k complex data points over a spectral width of 6 kHz in both dimensions with the carrier placed on the water resonance. For HOHAHA and NOESY spectra, 16 scans were time averaged per tl experiment. Data were processed directly on the spectrometer or offline using VNMR (Varian, Inc., Palo Alto, CA) or NMRPipe (Delagio et al., J Biomol. NMR 6, 277-293 (1995)) on an SGI workstation. Data sets were multiplied in both dimensions by a 30-60 degree shifted sine-bell function and zero-filled to Ik in the tl dimension prior to Fourier transformation. Since SC peptides are quite hydrophobic and later found to be amphipathic, pulsed field gradient (PFG) NMR self-diffusion measurements were performed as a check for peptide aggregation. PFG-NMR experiments were done as described by Mayo et al., Protein Sci. 5, 1301-1315 (1996) using a Varian Unity-Plus 500 NMR spectrometer. The maximum magnitude of the gradient was 6- G/cm, and the PFG longitudinal eddy-current delay pulse- sequence was used for all self-diffusion measurements which were performed in D20 temperatures of 5°C and 40°C. Peptide concentrations ranged from 0.1 mM to 15 mM. PFG NMR data were analyzed also as described by Mayo et al., Protein Sci. 5, 1301-1315 (1996). Structural Modeling Analysis of NOE growth curves indicated that backbone to backbone inter-proton NOEs were normally maximum at 300 ms to 400 ms. Interproton distance constraints were derived from NOEs assigned in 1H NOESY spectra acquired with mixing times of 200 ms and 400 ms. NOEs were classified as strong, medium, weak or very weak corresponding to upper bound distance constraints of 2.8, 3.3, 4.0, and 4.5 A, respectively. The lower bound restraint between non-bonded protons was set to 1.8 A. Pseudo-atom corrections were added to the upper bound distance constraints where appropriate, and a 0.5 A correction was added to the upper bound for NOEs invsolving methyl protons. Hydrogen bond constraints were identified from the pattern of sequential and interstrand NOEs involving NH and CαH protons, together with evidence of slow amide proton-solvent exchange. Each hydrogen bond identified was defined using two distance constraints; r NH . 0 = 1.8 to 2.5 A, and rN-o = 1.8 to 2.5 A. Derived internuclear distance constraints were used in calculating structures for dodecapeptide SC-4 by using X-PLOR (Brunger et al., X-plor Manual, Yale University Press, New Haven (1992)). SC-4 was created using parallhdg.pro force fields. A template coordinate set was generated by using the Template routine. The ab initio simulated annealing (SA) protocol was then used. The SA procedure ran high temperature dynamics (3000 K for 120 ps) and then cooled down to 100 K in 50 K steps with 1.5 ps molecular dynamis at each step. Powell minimization was performed at 100 K for 1000 steps. Structure refinement was done based on simulated annealing starting at 1000 K and ending at 100 K. Final structures were subjected to the X-PLOR Accept routine with the violation threshold for NOEs of 0.5 A and dihedral angles of 5°. Angles, bond lengths or impropers were not allowed to deviate from ideal geometry more than 5°, 0.05 A and 5°, respectively. Structures were superimposed using the BIOSYM INSIGHT viewer (Molecular Simulations, Inc.) and were analyzed using X-PLOR analysis routines. Cells, Cultures, and Reagents Human umbilical vein derived EC (HUVEC) can be harvested from normal human umbilical cords by perfusion with 0.125% trypsin/EDTA as described in Groenewegen et al., J Exp. Med. 164, 131-143 (1986). For determination of quiescent EC phenotype isolated ECs are immediately fixed in 1% paraformaldehyde. Human microvascular ECs (MVECs) are isolated. ECs are cultured in fibronectin coated tissue culture flasks in culture medium (RPMI- 1640 with 20% human serum (HS), supplemented with 2 mM glutamine and 100 U/ml penicillin and 0.1 mg/ml streptomycin). For isolation of recombinant PF4, the synthetic gene for human native PF4 is expressed as a non-fusion protein in E. coli (BL21) cells (Repligen Corp., Cambridge, MA). The protein is purified, cleaved, and refolded essentially as described in Yang et al., J.Biol. Chem. 269, 20110-20118 (1994). Purity was assessed by Coomassie staining of SDS PAGE, analytical C4 reverse phase HPLC, and amino acid analysis. Typically, several hundred milligrams of greater than 95% pure material is isolated from 100 grams (g) of starting material. EC Proliferation Assay EC proliferation is measured using a [ 3 H]thymidine incorporation assay. ECs are seeded at 5000 cells/well in flatbottomed tissue culture plates and grown for 3 days, in the absence or presence of regulators, in culture medium. During the last 6 hours of the assay, the culture is pulsed with 0.5 μCi [methyl- H]thymidine/well. In vitro Angiogenesis Assay A semi-natural matrix of collagen type I is prepared by mixing 8 volumes vitrogen 100 (Collagen Corporation, Fermont, CA, USA) with 1 volume lOx concentrated MEM (Life Technologies) and 1 volume of sodium bicarbonate (11.76 mg/ml). The matrix is dispensed into culture plates and allowed to gel at 37°C. Confluent BMEC (kindly provided by Dr. M. Furie, State University of New York, Stony Brook, USA) are trypsinized and seeded on top of this collagen matrix. When cells are grown to a confluent monolayer, medium is replaced by fresh medium, medium containing 25 ng/ml bFGF or medium containing 25 ng/ l bFGF and the peptides. In contrast to addition of the angiogenesis inducer on top of the matrix colon cell line (LS174T), spheroids are embedded in the collagen matrix before gelation. The peptides are added to the culture simultaneously with the EC. In both experiments, after 48 hours of incubation the (sprouting) endothelial monolayers are photographed with a Zeiss inverted phase-contrast photo microscope. The amount of sprouting in each well (i.e. the total length of the sprouts) is quantified by the computer program NIH image (Barendsz-Janson et al., J.Vasc.Res. 35, 109-114 (1998)). CAMAssay Fertile Lohman selected white leghorn eggs are incubated for three days at 37°C and 55% relative humidity and rotated once every hour. At day 3, a rectangular window (1x2 cm) is made in the eggshell. The window is covered with tape to prevent dehydration. The window allows undisturbed observation of the developing vasculature of the chorio-allantoic membrane (CAM). At day 7 a silastic ring (10 mm diameter) is placed on the CAM to allow local drug administration within the ring. Peptides dissolved in sterile saline (0.9% NaCl) are applied daily in aliquots of 65 μl from day 10 to day 13. At day 14 the CAMs are photographed. Immunofluorescence Fluorescence activated cell sorter (FACS) anaylsis using indirect phycoerythrin (PE)-conjugated reagents requirs 3 separate incubations. ECs (1 x 10 s ) are fixed for 1 hr in 1% paraformaldehyde, resuspended in 20 μl appropriately diluted MAb and incubated for 1 hour on ice. Subsequently, cells are washed 2 times in phosphate-buffered saline/bovine serum albumin (PGS/BSA) (0.1%) and incubated for another 30 minutes with biotinylated rabbit-anti-mouse Ig (Dako, Glostrup, Denmark). After another 2 washing, cells are incubated with sfreptavidm-phycoerythrin conjugate (Dako). Stained cells are analyzed on a FACScan flowcytometer. Data analysis is performed using PCLysys software (Becton Dickinson, Mountain View, CA). Inhibition of angiogenesis prevents bFGF induced ICAM-1 down-regulation To test the hypothesis that angiostatic factors can prevent angiogenesis- mediated down-regulation of EAM, a polypeptide can be tested for its effects on EAM expression. These studies concentrate on the expression of ICAM-1 because previous research using anti-ICAM-1 and anti-LFA-1 antibodies demonstrated that the ICAM-l/LFA-1 interaction is crucial, i.e., both essential and sufficient, in leukocyte EC adhesion and extravasation (Bevilacqua, Ann. Rev. Immunol. 11, 767-804 (1993); and Springer Cell 76, 301-314 (1994)). A 3- day incubation of EC with 10 ng/ml bFGF to evaluate the attenuation of ICAM-1 expression is compared with control cells. Results Figure 1 shows the amino acid sequences for βpep- 19 (SEQ ID NO: 18), βpep-25 (SEQ ID NO: 19), and peptide dodecamers (SC peptides) which "walk through" the sequence of βpep-25. The bactericidal activity of βpep-25 was observed to be equal to or better than that of βpep- 19, and these "walk throughs" were designed to identify segments of that amino acid sequence that contributed most to this activity. It was anticipated that bactericidal activity in these shorter SC peptides would be considerably less than that for parent peptide βpep-25. As reported below, this was not the case and is the basis for this work. Bactericidal Activities Chosen for investigation were seven bacterial strains, representing both Gram-negative and positive bacteria. For βpep peptides and βpep-derived dodecapeptides, bactericidal activities against these strains are given in Table 1 as the concentration which is effective at killing 50% of bacteria, i.e., LD50. LD50 values were determined from dose response curves like those shown in Figure 2 which plots percentage bacteria killed versus logarithm of the peptide concentration. Actual data points are shown with symbols as identified in the legend, and each curve represents the best fit using a sigmoidal function. LD50 values are read from the fitted curve at 50% mortality. Overall, SC-4 functions best at killing both rough and smooth strain Gram-negative bacteria. LD50s range from 3 nanomolar against J5 to 250 nanomolar against J96. Against strains of S. aureus, SC-4 (80 to 400 nanomolar) and SC-5 (180 to 350 nanomolar) are comparable in activity to βpep- 19 and βpep-25. BG-22, a peptide 28mer having the amino acid sequence from the β-sheet domain of bactericidal/ permeability increasing (B/PI) protein (residues 82-108) (Gray et al., Biochim. Biophys. Ada 1244, 185-190 (1995)) proposed as being that site which promotes bactericidal activity, is less bactericidal than βpep- 19 or βpep- 25 and considerably less active than SC-4. In general, SC-4 demonstrates the best broad spectrum bactericidal activity. Leakage Kinetic Bactericidal peptides are thought to function generally by integrating into the bacterial membrane and creating channels such that bacteria become "leaky" and die. To assess if this mechanism of action occurs with SC peptides, as well as with βpep peptides, membrane leakage kinetics were investigated against two E.coli strains (H5 and J5) and one S. aureus strain (MN8). In each case, bacteria became "leaky" within a few minutes after the addition of peptide. For any one of these strains, t 2 values are estimated to be in the range of 10 min. Hemolytic Activity Considering potential use of these peptides as antibiotic agents in mammals, it is important to know their potential to lyse eukaryotic cells. For these information, red blood cells were used as a model for all eukaryotic cells. Red blood cell lysis (hemolysis) was checked at peptide concentrations of 1 x 10 " 4 M, 1 x 10 -5 M, and 1 x 10 "6 M. At 10 "6 M, SC-1, SC-5 and BG-22 demonstrated 15% to 30% hemolysis which increased to around 70% at 10 "4 M. All other peptides showed less than 5% to 10% effect at 10 "6 M. Even at 10 "4 M, βpep-25 induced only about 5% hemolysis, while SC-3, SC-4, and SC-8 gave about a 10% effect, and SC-2, SC-6 and SC-7 gave about a 15% effect. For most SC peptides, 10 "4 M concentration is considerably greater than its bactericidal LD50 value. For example, for SC-4, LD50s against P. aeruginosa and MN-8 are 50,000-times and 500-times, respectively, less than this concentration. LPS neutralization Lysis of Gram-negative bacteria produces the endotoxin lipopolysaccharide (LPS) which triggers the pathologic disorder known as sepsis. In this respect, peptides which are not only bactericidal, but also effectively neutralize LPS, are of considerable importance in combating endotoxemia and sepsis. Therefore, these peptides were tested in the Limulus amebocyte assay for their ability to bind to and to neutralize LPS. IC50 values were determined from dose response curves shown in Figure 3 and are listed in units of micromolar at the right most column in Table 1. βpep-19, βpep-25 and SC-5 are essentially equi-potent (IC50 values of about 2 micromolar), with SC-4 being not far behind. SC-1 and SC-6 are slightly less effective having IC50s of 3.5 micromolar. SC-3 is about 10-fold less potent (IC50 = 25 x 10 "6 M) than either SC-4 or SC-5. SC-2, SC-7 and SC-8 show no LPS binding activity at or below 1 x 10 "4 M. This was not so surprising for SC-7 and SC-8 which are essentially bactericidally inactive, but SC-2 did demonstrate reasonable bactericidal activity. Table 1. SC series of peptides ~ LD50 values for bactericidal activity and IC50 values for neutralization of bacterial lipopolysaccharide (LPS). All values are given as micromolar quantities. Bacteria J5, J96, H5, and IA2 are strains of E. coli; bacteria MN8 and MNHO are strains of S. aureus. LD50 and IC50 values are in units of 10 "6 M. Standard deviations are about ± 30% of the values given. XXX means not active at concentrations below 1 x 10 "5 M in the bactericidal assay and below 1 10 M in the LPS neutralization assay. Circular Dichroism CD spectroscopy, used to assess confbrmational populations of SC peptides, indicates the presence of helical structure. This is demonstrated in Figure 4 which shows select CD traces for SC-5 and SC-7 acquired at various concentrations of TFE. In all cases, CD traces show a band of negative ellipticity at 222 nm which is characterisitc of helix conformation (Greenfield et al., Biochemistry 8, 4108-4116 (1969); Johnson et al., Proteins 7, 205-214 (1990); and Waterhous et al., Biochemistry 33, 2121-2128 (1994)). At lower concentrations of TFE, mostly random coil populations are present as evidenced by a negative ellipicity minimum at about 200 nm (rather than at 207/208 nm for helix) and a low [Θ] 2 22/[®] 2 oo ratio-- As the concentration of TFE is increased, helix population increases since the negative ellipicity band at 200 nm shifts closer to 207 nm and the 222 nm band becomes more negative. At about 20% to 30% TFE, both these observables have nearly plateaued as indicated in the insert to Figure 4 which plots [Θ] 222 versus % (v/v) TFE. These CD spectral characteristics, however, would result from the presence of either α-helix or 3 10 - helix or from a mixture of both. The right handed 3 10 helix displays a negative CD band at 207 nm (λ m i n ) and a shoulder centered near 222 nm with the ratio [Θ] 222 /[Θ] 207 being about 0.4 (Millhauser, Biochemistry 34, 3873-3877 (1995); and Toniolo et al., J. Am. Chem. Soc. 118, 2744-2745 (1996)). For an α-helix, the 207 nm and 222 nm bands are also present; however, the ratio [Θ] 22 2/[Θ] 2 o is closer to unity. When λ m j n is 207 nm and the ratio [Θ] 222 /[Θ] 20 is between 0.4 and 1.0, interconverting populations of 3 10 helix and α-helix are likely present. For SC peptides, Table 2 lists the wavelength for the far UV minimum, λmin. and the ratio [Θ] 2 22/[Θ] 2 o7 at 70% TFE. In cases where the [Θ]222/[©]207 ratio is closer to 0.4 than to 1.0 and λmj n is at 207 nm, the conformation of that SC peptide should have more 3 10 helix than α-helix character (Millhauser, Biochemistry 34, 3873-3877 (1995); and Toniolo et al., J. Am. Chem. Soc. 118, 2744-2745 (1996)). For the most active peptide, SC-4, λ min is 207 nm and [Θ] 2 22/[Θ]2Q 7 is 0.49, indicating the presence of mostly 3 10 helix. Even though peptides SC-1, -2, -3, -5 and -6 have λ m m values of 207 nm, larger [Θ] 222 [Θ] 207 ratios of 0.6 to 0.7 indicate less 3 10 helix character. Since SC-7 and SC-8 show λmi n values of 203 nm and 204 nm, respectively, significant populations of random coil should be present in then conformational ensembles. Table 2. Circular Dichroism Data Summary for peptides in the presence of 70% trifluoroethanol. λ minimum (nm) m > [Θ]?07 ratio SC-1 208 0.83 SC-2 207 0.65 SC-3 207 0.59 SC-4 207 0.46 SC-5 207 0.64 SC-6 207 0.73 SC-7 202 0.5 SC-8 204 0.48 SC-4 Variants: SC-401 207 0.63 SC-402 204 0.52 SC-403 208 0.79 SC-404 207 0.54 SC-405 207 0.71 SC-406 208 0.67 SC-407 207 0.58 SC-408 207 0.53 SC-409 208 0.65 NMR Conformational Analysis Figure 5 shows αH-NH andNH-NH regions from NOESY data for SC- 4 in the absence (panels A) and presence (panels B) of 30% (v/v) TFE. In either case, the series of NH-NH NOEs running from F3 to 112 alone indicates the presence of at least nascent helix conformation (Dyson et a., J Mol. Biol. 201, 201-217 (1988)). Moreover, even in the absence of TFE, all four αH-NH i,i+2 NOEs are easily distinguishable, supporting the presence of multiple turn or 3 10 helix (Wϋthrich et al. NMR of Proteins and Nucleic Acids, Wiley-Interscience, New York (1986); and Wishart et al., Biochemistry 31, 1647- 1651 (1992)). In the presence of TFE, these NOEs remain and other longer range NOEs appear. For example, two αH-NH i,i+3 (8/5, 10/7), three NH-NH i,i+2 (8/6,9/7, 10/8) and two NH-NH i,i+3 (10/7, 11/8) NOEs have been labeled in Figure 5. For pure 3 ιo helix, only i,i+2 and i,i+3 NOEs should be observed, whereas for pure α-helix, only i,i+3 and i,i+4 NOEs should be present (Wuthrich, NMR of Proteins and Nucleic Acids, Wiley-Interscience, New York (1986)). The presence of αH-NH i,i+2 NOEs characteristic of 3ιo helix is consistent with CD results as discussed above, α-helix conformation, however, must also contribute to the conformational ensemble as indicated by CD data. Since all SC peptides, to various extents, show similar NOE and CD trends, these peptides exist in aqueous solution in an equilibrium primarily among 3 10 helix, α-helix and random coil. Since some SC peptides are relatively hydrophobic and could tend to self-associate thereby affecting the presence and/or magnitude of NOEs, PFG NMR diffusion measurements (data not shown) were made. For all SC peptides, diffusion coefficients derived from these data remain unchanged over the peptide concentration range of 0.1 mM to 15mM, indicating the absence of aggregation. For peptide SC-4 which forms the most stable 3ι 0 helical structure and is generally the most bactericidal, conformational modeling was performed using NOE data acquired for the peptide in the presence of 30% TFE / 70% water. It should be emphasized that most of the same NOEs could be observed in the absence of TFE; however, 30% TFE did apparently stabilize the helix conformation and increase the overall tumbling correlation time, thereby generally increasing the magnitude of NOEs. A total of 140 NOE distance contraints were derived from analysis of NOESY spectra. These include 68 intraresidue, 24 sequential, 20 medium-range ( | i-j | < 5), and 27 long- range ( I i - j I > 5) constraints. In addition, a total of 8 hydrogen bonds could be identified inspection of initial SC-4 structures and from long lived backbone NHs, giving rise to 16 hydrogen bond distance constraints. The total number of experimentally derived constraints was therefore 156, giving an average of 13 contraints per residue. initially, 100 structures for SC-4 were calculated as described in the Methods Section. The best fit superpositions of backbone Cα atoms for the final 14 structures are shown in Figure 6 (left). These structures showed no NOE violations greater 0.5 A. Structural statistics are summarized in Table 3. The somewhat less stracturally-defined N-terminus is apparent. The structures satisfy experimental constraints quite well. For residues 3 through 12, atomic RMS differences with respect to the mean coordinate positions are 0.71 ± 0.08 A for backbone (N,C α , C) atoms and 1.4 ± 0.4 A for all heavy atoms. In addition, ψ and ψ angular order parameters are all > 0.8. Together, the above data indicate that these structures used to represent the solution conformation of SC-4 are well converged. The helical structure of SC-4 is, not surprisingly, between that of an α-helix and a 3 10 helix, with the average number of residues per turn being 3.3. From the N-terminus to W9, the helix is amphipathic with Kl, K4, R5 and K8 aligned on one face as illustrated in Figure 6 with these residues being highlighted. Table 3. Structural Statistics for the calculated structures of SC-4 from NMR data. RMS Deviations from experimental distance restraints (A) a NOE (140) 0.04 ± 0.0042 H-bond (16) Deviations from idealized geometry Bonds (A) 0.038 Angles (°) 0.65 ± 0.03 Impropers (°) 0.25 ± 0.03 Energies (kcaLmol "1 ) ENOE b - 9 ± 1 ECDIH 1.3 ± 0.6 ENCS ll.ό ± l EBOND 3.7 + 0.4 EANGLE 30 ± 5 EIMPROPER 16 ± 2 ETOTAL 72 ± 7 a None of the 14 final structures exhibited distance restraint violations greater than 0.5 A or dihedral angle violations greater than 5°. RMSD values represent the mean and standard deviations for the 14 structures. b The final values of the NOE (ENOE), torsion angle (ECDIH) and CS (ENCS) potentials have been calculated with force constants of 50 kcaLmol "1 . A "2 , 200 kcaLmoi rad "2 , and 300 kcaLmol "1 . "2 , respectively. Effects of Modifying the SC-4 Sequence Since SC-3 and SC-4 were the most bactericidal, least hemolytic and SC- 4 was one of the best at neutralizing endotoxin, additional dodecapeptide "walk throughs" were made to complete the single-residue shifts between SC-3 and SC-4: SC-401 QMKL FKRHLKWK-NH2 SC-402 MKL FKRHLKWKI-NH2 In addition, a longer variant which extends SC-4 by one residue on each side of the sequence was investigated: SC-403 MKL FKRHLKWKIIV-NH2 Since the presence of positively charged residues is known to be crucial to bactericidal and LPS neutralizing activities, a series of lysme/arginine- substituted SC-4 variants was investigated: SC-404 XLFKRHLKWKII-NH2 SC-405 KLFXRHLKWKII-NH2 SC-406 KLFKRHLXWKII-NH2 SC-407 KLFKRHLKWXII-NH2 SC-408 KLFKKHLKWKII-NH2 SC-409 KLFKXHLKWKII-H2 SC-404 thru SC-407 change each of the four lysines to norleucines (lysine without the side-chain amine group). Variants SC-408 and SC-409 change the arginine, R5, to lysine and norleucine, respectively. CD data indicate that these variants also fold helically, fluctuating between 3 10 and α-helical structure like parent peptides SC-3 and SC-4 (data not shown). Table 2 lists λmin and [Θ]222/[Θ]207 ratio values at 70% TFE. Whereas SC-401 and SC-403 both demonstrate λmin values of about 207 nm, then [Θ] 222 /[Θ]207 ratios are larger than those of either SC-3 or SC-4, indicating that these two SC-4 variants have more α-helix and less 3 10 helix character than parent peptide SC-4. SC-402, however, has a greater random coil population in its conformational ensemble since its λ m in value is 204 nm. The lysine/arginine variants, SC-404 through SC-409 behave conformationally like parent SC-4, with considerable 3 10 helix character. NMR data, acquhed in the presence of 30% TFE (data not shown), support the presence of significant populations of helical conformation as observed for SC-3 and SC-4. Bactericidal activities from these peptides were acquired against three of the strains used above, E. coli J5, P. aeruginosa and S. aureus MNHO. Results are given in Table 4. Against J5, SC-401 and SC-402 are more active than SC-3 and slightly less active than SC-4. Shifting the dodecapeptide unit from SC-3 towards SC-4, therefore, increases activity. On the other hand, addition of hydrophobic residues to both terminal positions in SC-4, i.e., SC-403, increases activity slightly. Using P. aeruginosa, bactericidal dose response curves indicate that either dodecapeptide, SC-401 or SC-402, diminishes activity. This is somewhat surprising since both SC-3 and SC-4 have the same activity against P. aeruginosa (see Table 1). This may have something to do with the way in which these peptides interact with the bacterial cell wall/membrane. Against S. aureus strain MNHO, shifting the dodecapeptide window from SC-3 towards SC-4 by only one residue, i.e., SC-401, essentially abolishes activity. Another one residue shift (SC-402) brings back some activity, and lengthening both termini of SC-4 has no effect. Table 4. SC-4 peptide variants SC401 thru SC-409 ~ LD50 values for bactericidal activity and IC50 values for neutralization of bacterial lipopolysaccharide (LPS). All values are given as micromolar quantities. *LD50 and IC50 values are in units of 10 M. Standard deviations are about ± 30% of the values given. XXX means not active at concentrations below 1 x 10 "5 M. With the lysine variants, substitution of Kl or K4 for norleucine reduces the bactericidal activity against J5 only slightly, whereas substitution of K8 and K10 for norleucine shows a somewhat more significant drop in activity. This is especially true for SC-407 (KlOX). Nonetheless, bactericidal activities remain respectable indicating that removal of a single positive charge alone does not abolish activity. Substitution of R5 for lysine, however, shows no change in activity, while substitution of R5 for norleucine causes a significant activity increase. The NRM solution structure of SC-4 shows that Kl, K4, R5 and K8 lie on one face of an amphipathic helix with K10 more to the side (Figure 5). This conformation of the peptide sandwiches K4 and R5 between Kl and K8. Removal of the charged group at position 5 may help to reduce charge-charge repulsion and to stabilize, via hydrophobic interactions, the remainder of the lysine core. Once again, any changes to the SC-4 amino acid sequence reduce bactericidal effects against P. aeruginosa (Table 4). In the lysme/arginine series, substitution of Kl, K4 and R5 for norleucine has the most detrimental effects. Against S. aureus strain MNHO, substitution of any one of these charged residues is more tolerated (Table 4). These data also indicate that while the mechanism of action of these peptides against various bacterial strains may be similar, there are discrete and significant differences related to the spatial orientation of charged and hydrophobic groups of the peptides. Moreover, even though CD and NMR data indicate helical conformation for these peptides, interaction with the bacterial cell wall/membrane may certainly modify this conformation making derivation of precise structure-activity relations impossible. The ability of these SC-4 variants to neutralize LPS was also investigated. Dose response curves are shown in Figure 3 and IC50 values are listed in Table 4. As shown above, SC-401 and SC-402 are dodecapeptides which shift one residue each from SC-3 to SC-4, respectively. SC-4 has an IC50 of 2 x 10 "6 M, whereas SC-3 has an IC50 more than ten-fold greater (25 x 10 "6 M). With a single residue shift from SC-3 towards SC-4, the activity of SC-401 (IC50 value of 126 x 10 "6 M) has dropped 5-fold relative to that of SC-3. Shifting one more residue to yield SC-402 brings the LPS neutralizing activity back to the level observed for SC-3 (IC50 of 25 x 10 "6 M). Comparison of these sequences indicates that it is apparently crucial to have at least one hydrophobic residue at the C-terminal position. SC-3 has Trp and SC-402 has He. Least active SC-401 ends in a Lys residue. The most active peptide SC-4 has the hydrophobic dipeptide He-He at its C-terminus. The exact functional significance is not clear since equipotent SC-5 has only one hydrophobic residue, Leu, at its C-terminus, whereas SC-1 has Phe, SC-2 has His, SC-6 has Gly, and inactive SC-7 also has a Leu. In the lysine-to-norleucine variants, substitution of the first two Lys residues (K1X and K4X) reduces the activity only about 3-fold. Substitution of the last two Lys residues (K8X and KlOX) has essentially no affect on the ability of the peptide to neutralize LPS. In this respect, no one of these lysines is crucial to this activity. A surprising finding, however, was observed with SC-408 and SC-409. SC-408 is the R5K variant. Here, the activity drops dramatically about 500-fold. Alone, this would suggest that the Arg residue is crucial to activity and charge conservation is insufficient to maintain activity. This conclusion, however, is challenged by results on SC-409 which has that Arg replaced with norleucine completely removing the positive charge, but maintain hydrophobic character from the side-chain methylenes. In SC-409, nearly full activity relative to that of SC-4, has been preserved. Therefore, a small positively charged arnine group in that position is detrimental to activity, but the charge itself plays no apparent role in activity. Discussion Against bacteria investigated in this study, SC-4, with LD50 values falling in the single-digit nanomolar to 100 nanomolar range, is, overall, the most effective, antibacterial of any of these peptides. Aside from SC-7 and SC-8 which are inactive at concentrations below 10 micromolar, the other SC peptides, particularly SC-3 and SC-5, are reasonably bactericidal as well. In fact, against S. aureus strains MN8 and MNHO, activities for peptides SC-1 through SC-6 are within about a factor of two of each other, but against E. coli strain IA2, SC-4 stands out again, being about 4- to 10-fold more active than any other SC peptide. The observation that bactericidal peptides like SC-4 are more effective against Gram-negative strains as opposed to Gram-positives, is generally observed with other known bactericidal peptides. Differences in bactericidal activities against various strains are most likely due to variability in the components of the bacterial membranes and how these peptides interact with those membranes. For instance, the outer membrane surface of Gram-negative bacteria includes lipopolysaccharides, whereas in Gram-positive bacteria there is no outer membrane and the cell wall includes the acidic polysaccharides, teichoic acids. SC-4 must capture the appropriate compositional and structural features necessary for interactions with these mostly negatively charged cell surfaces in order to exhibit such broad spectum bactericidal activity. Relative to bacterial membranes, normal mammalian cell membranes which are composed predominatly of zwitterionic phosphotidylcholine and sphingomyelin phospholipids, have more positive charge character. Given the significant drop in the net negative charge of eukaryotic cell membrane surfaces, bactericidal peptides generally interact less with mammalian cells. Nonetheless, bactericidal peptides can lyse mammalian cells at concentrations near then bactericidal LD50 concentrations (Maloy et al., Biopolymers 37, 105-112 (1995)). In this respect, one must check that these concentration thresholds are not near one another in order to have the most effective and least cytotoxic, therapeutic bactericidal agent. Red blood cells are usually chosen as models for eukaryotic cells and hemolysis often is used as an index of a peptide 's cytolytic activity against mammalian cells. For most SC peptides, significant hemolysis occurs only at concentrations above 1 x 10 "4 M, whereas LD50 values are all in the sub-micromolar to nanomolar ranges. This low cytotoxicity against mammalian cells is added advantage to using SC peptides as antibiotics. Antibacterial peptides have been classified in a number of ways. One classification is based on chemical-structural criteria and is broken down into two broad groups: linear peptides and cyclic peptides. Within the linear group, two subgroups can be distinguished: (1) linear peptides tending to adopt helical structure and (2) linear peptides of unusual composition, rich in amino acids such as Pro, Arg or even Trp. Given the observation that SC peptides are helical, they are classified in the first subgroup. A list of other linear antibacterial peptides with helical conformation have been tabulated by Andreu & Rivas (Andreu et al., Biopolymers (Pep. Sci.) 47, 415-433 (1998)) (see Table I in Andreu et al., Biopolymers (Pep. Sci.) 47, 415-433 (1998)) and include: andropin, BLP-1, bombinin, bombolitin, the cecropins, ceratotoxin, clavanin, CRAMP, dermaseptin, enbocin, FALL-39, lycotoxin I, magainin, melittin, misgurin, PGLa, pleurocidin, seπiinalplasmin, and styelin. Conformations of peptides within this group are most often discussed as being α-helical which is a specific type of helix having 13 atoms or 3.6 residues per helical turn. However, it has been observed that many short linear peptides form a mixture of α-helix and 3 10- helix in aqueous solution (Toniolo et al., J Am. Chem. Soc. 118, 2744- 2745 (1996); and Millhauser et al., Biochemistry 34, 3873-3877 (1995)). By reviewing structural data (mostly CD measurements) on other known helix- forming antibacterial peptides listed above, it is apparent that some of these peptides also have 3 10 helix character as well. In this respect, the term "α- helical" as used currently in the literature may actually refer to both types of helix, i.e., the 3 10 helix should not be excluded. Both CD and NMR conformational analyses of SC peptides, especially of SC-4, show them to exist in aqueous solution as a mixture of α-helix, 3 10 helix, and random coil. The degree of specific helix formation depends on the particular sequence and the presence or absence of TFE. With SC peptides, TFE does not appear to induce conformations that, to some extent, are already present in water. NOE patterns, for example, are essentially the same in water and in TFE. However, in TFE, NOEs are generally larger, indicating that TFE acts to stabilize existing conformations. Moreover, TFE also provides an environment of lower dielectric, which, in some ways, may mimic the low dielectric within a membrane. In terms of peptide folding, the presence of either type of helix is interesting because SC peptides were derived from βpep-25 which forms β-sheet structure in aqueous solution (Mayo et al., Protein Sci. 5, 1301-1315 (1996)). It appears, therefore, that when removed from the context of the entire βpep 33mer, SC peptides are free to form helical structure. It has also been observed that bactericidal activity in βpep peptides is negatively correlated with β-sheet stability (Mayo et al., Biochim. Biophys. Ada 1425, 81-92 (1998)), i.e., more stable β-sheet leads to decreased bactericidal activity. Within the bacterial membrane, this may make conformational rearrangements for some βpep peptides, easier, thereby promoting greater bactericidal activity. Based on NOE data, structural modeling shows SC-4 to be conformed as an amphipathic helix. For some time, amphipathic helical structure has been noted as being important for bactericidal activity. Even though the mechanism of action of bactericidal peptides is, for the most part, unclear, it has been shown that peptide-lipid, rather than receptor-mediated recognition processes, plays the major role in then function (Oren et al., Biopolymers (Pep. Sci.) 47, 451-463 (1998)). At least one antibacterial peptide: KLKLLLLLKLK-NH2 (SEQ HO NO:20) effective only at a much higher concentration (1 x 10 "4 M) than SC-4, has been shown to function by forming channels in bacterial membranes thereby increasing membrane permeability and interfering with function, ultimately resulting in cell death (Alvarez-Bravo et al., J. Biochem. 117, 1312- 1316 (1995)). SC peptides probably function similarly since leakage kinetics occur on the same time scale with a half-kill time of about 10 min. Two mechanisms of action have been proposed to explain membrane permeation by amphipathic, helical peptides: (1) transmembrane pore formation via a "barrel-stave" mechanism (Ehrenstain et al., Quart. Rev. Biophys. 10, 1-34 (1977)) or (2) membrane destruction/ solubilization via a "carpet-like" mechanism (Gazit et al., Biochemistry 34, 11479-11488 (1995); and Pouny et al., Biochemistry 31, 12416-12423 (1992)). In the generally favored "carpet-like" mechanism, amphipathic helical peptides interact with the bacterial membrane surface such that then hydrophobic surfaces face the membrane and then hydrophilic surfaces face the solvent. When a threshold concentration of peptide is reached, the membrane is disrupted and a transient pore is formed. From the present data, it is impossible to conclude definitively that SC peptides interact with cell membranes only when in a helical conformation. Nevertheless, when these peptides are conformed as amphipathic helices, then mechanism of action can be more readily explained. The membrane surface of both Gram-positive and negative bacteria has a net negative charge making it a favorable environment for interaction with polycationic peptides like SC-4. In fact, Gray et al. (Gray et al., Biochim. Biophys. Ada 1244, 185-190 (1995)) showed that much of the bactericidal activity of the protein B PI (bactericidal/permeability increasing protein) can be accounted for by the positively charged tripeptide sequence KWK. SC peptides, as well as the antibacterial peptide cecropins (Hanzawa et al., FEBs Lett. 269, 413-420 (1990)), also contain the KWK tripeptide motif. Relative to SC-4, however, then bactericidal activities against P. aeruginosa are much less with lethal concentrations, for example, of 6 micromolar for cecropin PI (Lee et al., Proc. Natl. Acad. Sci. USA 86, 9159-9162 (1989)) compared to 10 nanomolar for SC-4. It is important to emphasize that a high net positive charge alone does not guarantee optimal bactericidal activity. For example, SC-4 and SC-5 both have a net charge of +6, even though SC-4 is the more potent of the two against Gram-negative bacteria. Furthermore, whereas SC-6 and SC-1 have net charges of only +3, SC-6 demonstrates about the same general bactericidal activity as SC-5, and SC-1 is at least as active as SC-4 against E. coli strain J96. The presence of hydrophobic residues also modulates bactericidal activity; yet hydrophobicity alone can also not account for these activity differences since hydrophobic residue content in each SC peptide is about 50% to 60%. Another mostly positively charged and hydrophobic, bactericidal peptide, derived from C- terminal amphipathic α-helix of platelet factor-4 (Darveau et al., J. Clin. Invest. 90, 447-454 (1992)), for example, has four lysine residues arrayed in tandem on one face of its helix; yet its activity is about 100 times less than that of SC-4. Clearly, both composition and sequence contribute to function. Amino acid sequence, in turn, determines conformation which ultimately is the key to understanding bactericidal effects from SC peptides and from other bactericidal peptides in general, h the case of SC-4, Kl, K4, R5 and K8 are aligned on the same face of the helical molucule (see Figure 6). Since CD and NMR data indicate that the other SC peptides also have a propensity to form helical structure in aqueous solution and may as well promote then anti-bacterial activity when in a helical conformation, Figure 7 shows helical wheel projections for SC-1 through SC-8. Note that the most active SC peptides, SC-3 and SC-4, have four positively charged residues aligned in tandem on one face of the helix. Moreover, in both cases, a lysine residue lies at the top of a diamond shaped array with an R-K pair in the middle and another K at the bottom of this array. For SC-4, the diamond shaped array is Kl, R5-K4, K8, and for SC-3, the array is K4, R8-K7, Kl 1. hi SC-4, this array starts at the first turn of the helix from the N-terminus, whereas in SC-3, it starts from the second turn of the helix. This may be the reason why SC-4 is overall more active than SC-3. In the next most active peptide, SC-5, residues K1-R2 and K5 on the helix surface form a triangle shape which is part of a diamond shape. In less active peptides SC-1, -2 and -6, positively charged residues are positioned throughout the helix, and in the least active, or non-active, peptides SC-7 and -8, lysine residues are few and intersperced with negatively charged residues. Too much should not be made of this diamond shaped array in terms of anti-bacterial potency of SC peptides, since removal of any one of those positively charged residues form SC-4 does not reduce its activity to any great extent. Nevertheless, at least part of this charge distribution may be required for optimal activity. Disintegration of the Gram-negative bactericidal cell outer membrane liberates the endotoxin lipopolysaccharide (LPS) which in mammalian white blood cells triggers the over release of various cytokines like TNFα and interleukin-1. This in turn can lead to the pathologic disorder of sepsis and possibly eventual death. SC-4 is one such bactericidal peptides which has the added advantage of being able to neutralize LPS. While the LPS binding activity (IC50) of SC-4 (2 x 10 "6 M) is not as high as that of the 55 kD protein B/PI (2 x 10 "8 M) (Marion et al., FEBs Lett. 227, 21-26 (1988)), SC-4 is afterall only a dodecapeptide. Mechanistically, the neutralization of LPS is thought to be mediated primarily by interactions between the protein/peptide and the lipid A portion of any given LPS. The nature of LPS varies from bacterial strain to strain; however all LPS molecules contain a conserved core unit lipid A which is composed of a diphosphorylated diglycosaminoglycan with a number of attached polyphosphorylated oligosaccharides and several acyl chains. LPS neutralization has been proposed to occur via protein/peptide binding to Lipid A both via cationic amino acid interactions with the anionic phosphates and via hydrophobic intertactions with the acyl chains. In the SC series, positive charge does play a role in LPS neutralization. SC-4 and SC-5 which have the highest net positive charge of +6 also neutralize LPS best. However, as with bactericidal activity, a high net positive charge alone can not account for LPS neutralization. Consider for example SC-2 and SC-3, which both have a net charge of +5; yet SC-3 is considerably more active than SC-2. Moreover, in the Arg/Lys- substituted norleucine series (Table 4), removal of any one positively charged residue drops LPS binding activity only slightly, probably indicating that the positive charge field itself rather than any one individual charged residue, mediates activity. On the other hand, hydrophobic residues at the C-terminus appear to be more crucial to LPS binding. Removal of the two isoleucines from the C-terminus of SC-4 (peptide SC-401) drops activity 60-fold, whereas removal of only one of these isoleucines (peptide SC-402) drops activity by only 12-fold. One curious finding is that while removal of R5 (SC-408) does not affect LPS binding, replacing R5 with lysine reduces LPS binding by some 500- fold. Conclusion Identification of a dodecapeptide with potent bactericidal activity against both Gram-positive and negative bacteria may lead to the development of a highly effective, therapeutic antibiotic and bacterial endotoxin neutralizing agent. Production and purification of bactericidal proteins or peptides with longer sequences present problems which in part can be avoid by the identification of a small peptide like SC-4. As new strains of antibiotic resistant bacteria continually emerge, such novel bactericidal agents will be particularly useful. Table 5. REMARK overall,bonds,angles,vdw,noe,cdih REMARK energies: 80.52, 4.527, 30.51, 21.06, 10.3, 1.166, 12.96 REMARK= REMARK bonds, angles, impropers, noe, cdih REMARK rms-d: 4.2134E-03,0.6535,0.7938,4.3271E-02,0.9781 REMARK — = REMARK noe, cdih REMARK violations.: 0, 0 ATOM 1 CA LYS 1 13.611 -8.592 1.565 1.00 0.00 sc4 ATOM 2 HA LYS 1 14.428 -9.138 2.008 1.00 0.00 sc4 ATOM 3 CB LYS 1 13.338 -9.114 0.154 1.00 0.00 sc4 ATOM 4 HB1 LYS 1 14.076 -8.716 ■ -0.526 1.00 0.00 sc4 ATOM 5 HB2 LYS 1 12.352 -8.803 ■ -0.162 1.00 0.00 sc4 ATOM 6 CG LYS 1 13.419 - ■10.642 0.150 1.00 0.00 sc4 ATOM 7 HG1 LYS 1 12.672 ■ -11.040 -0.520 1.00 0.00 sc4 ATOM 8 HG2 LYS 1 13.242 - •11.013 1.149 1.00 0.00 sc4 ATOM 9 CD LYS 1 14.808 -11.079 -0.320 1.00 0.00 sc4 ATOM 10 HD1 LYS 1 15.520 -10.930 0.478 1.00 0.00 sc4 ATOM 11 HD2 LYS 1 15.098 -10.490 -1.178 1.00 0.00 sc4 ATOM 12 CE LYS 1 14.775 ■ -12.562 -0.703 1.00 0.00 sc4 ATOM 13 HE1 LYS 1 13.894 -12.780 -1.286 1.00 0.00 sc4 ATOM 14 HE2 LYS 1 14.801 -13.178 0.186 1.00 0.00 sc4 ATOM 15 NZ LYS 1 16.000 -12.784 -1.527 1.00 0.00 sc4 ATOM 16 HZ1 LYS 1 16.650 -13.416 -1.017 1.00 0.00 sc4 ATOM 17 HZ2 LYS 1 16.472 -11.874 -1.707 1.00 0.00 sc4 ATOM 18 HZ3 LYS 1 15.734 -13.222 -2.431 1.00 0.00 sc4 ATOM 19 C LYS 1 13.922 -7.094 1.533 1.00 0.00 sc4 ATOM 20 O LYS 1 13.110 -6.273 1.912 1.00 0.00 sc4 ATOM 21 N LYS 1 12.349 -8.841 2.321 1.00 0.00 sc4 ATOM 22 HT1 LYS 1 12.353 -9.810 2.696 1.00 0.00 sc4 ATOM 23 HT2 LYS 1 11.534 -8.717 1.686 1.00 0.00 sc4 ATOM 24 HT3 LYS 1 12.279 -8.167 3.111 1.00 0.00 sc4 ATOM 25 N LEU 2 15.093 -6.731 1.084 1.00 0.00 sc4 ATOM 26 HN LEU 2 15.734 -7.408 0.783 1.00 0.00 sc4 ATOM 27 CA LEU 2 15.455 -5.285 1.027 1.00 0.00 sc4 ATOM 28 HA LEU 2 15.225 ■ -4.809 1.962 1.00 0.00 sc4 ATOM 29 CB LEU 2 16.965 - ■ 5.261 0.787 1.00 0.00 sc4 ATOM 30 HB1 LEU 2 17.200 -4.521 0.037 1.00 0.00 sc4 ATOM 31 HB2 LEU 2 17.292 -6.234 0.450 1.00 0.00 sc4 ATOM 32 CG LEU 2 17.677 -4.904 2.093 1.00 0.00 sc4 ATOM 33 HG LEU 2 16.967 -4.469 2.781 1.00 0.00 sc4 ATOM 34 GDI LEU 2 18.273 -6.169 2.712 1.00 0.00 sc4 ATOM 35 HD11 LEU 2 17.502 -6.707 3.244 1.00 0.00 sc4 ATOM 36 HD12 LEU 2 19.061 -5.898 3.398 1.00 0.00 sc4 ATOM 37 HD13 LEU 2 18.675 -6.797 1.931 1.00 0.00 sc4 ATOM 38 CD2 LEU 2 18.797 -3.901 1.808 1.00 0.00 sc4 ATOM 39 HD21 LEU 2 19.638 -4.109 2.453 1.00 0.00 sc4 ATOM 40 HD22 LEU 2 18.439 -2.899 1.995 1.00 0.00 sc4 ATOM 41 HD23 LEU 2 19.104 -3.987 0.777 1.00 0.00 sc4 ATOM 42 C LEU 2 14.710 -4.604 -0.127 1.00 0.00 sc4 ATOM 43 O LEU 2 14.647 -3.394 -0.218 1.00 0.00 sc4 ATOM 44 N PHE 3 14.152 -5.384 -1.010 1.00 0.00 sc4 ATOM 45 HN PHE 3 14.226 -6.356 -0.909 1.00 0.00 sc4 ATOM 46 CA PHE 3 13.409 -4.818 -2.176 1.00 0.00 sc4 ATOM 47 HA PHE 3 14.088 -4.304 -2.836 1.00 0.00 sc4 ATOM 48 CB PHE 3 12.820 -6.034 -2.891 1.00 0.00 sc4 ATOM 49 HB1 PHE 3 11.884 -5.758 -3.355 1.00 0.00 sc4 ATOM 50 HB2 PHE 3 12.648 -6.824 -2.176 1.00 0.00 sc4 ATOM 51 CG PHE 3 13.781 -6.513 -3.951 1.00 0.00 sc4 ATOM 52 GDI PHE 3 14.143 -7.864 -4.006 1.00 0.00 sc4 ATOM 53 HD1 PHE 3 13.734 -8.562 -3.289 1.00 0.00 sc4 ATOM 54 CD2 PHE 3 14.310 -5.608 -4.880 1.00 0.00 sc4 ATOM 55 HD2 PHE 3 14.031 -4.566 -4.837 1.00 0.00 sc4 ATOM 56 CE1 PHE 3 15.033 -8.311 -4.989 1.00 0.00 sc4 ATOM 57 HE1 PHE 3 15.312 -9.353 -5.032 1.00 0.00 sc4 ATOM 58 CE2 PHE 3 15.200 -6.055 -5.863 1.00 0.00 sc4 ATOM 59 HE2 PHE 3 15.608 -5.358 -6.579 1.00 0.00 sc4 ATOM 60 CZ PHE 3 15.562 -7.406 -5.918 1.00 0.00 sc4 ATOM 61 HZ PHE 3 16.248 -7.751 -6.676 1.00 0.00 sc4 ATOM 62 C PHE 3 12.284 ■ -3.875 ■ -1.728 1.00 0.00 sc4 ATOM 63 O PHE 3 11.703 • -3.179 -2.533 1.00 0.00 sc4 ATOM 64 N LYS 4 11.940 ■ -3.865 • -0.470 1.00 0.00 sc4 ATOM 65 HN LYS 4 12.395 ■ -4.451 0.172 1.00 0.00 sc4 ATOM 66 CA LYS 4 10.822 - •2.978 - ■0.020 1.00 0.00 sc4 ATOM 67 HA LYS 4 10.265 ■ -2.626 ■ -0.873 1.00 0.00 sc4 ATOM 68 CB LYS 4 9.929 - 3.881 0.834 1.00 0.00 sc4 ATOM 69 HB1 LYS 4 9.816 - ■4.838 0.346 1.00 0.00 sc4 ATOM 70 HB2 LYS 4 8.960 - ■3.420 0.951 1.00 0.00 sc4 ATOM 71 CG LYS 4 10.567 -4.087 2.210 1.00 0.00 sc4 ATOM 72 HG1 LYS 4 10.237 -3.307 2.880 1.00 0.00 sc4 ATOM 73 HG2 LYS 4 11.643 -4.050 2.117 1.00 0.00 sc4 ATOM 74 CD LYS 4 10.149 -5.448 2.770 1.00 0.00 sc4 ATOM 75 HDl LYS 4 10.938 -5.839 3.393 1.00 0.00 sc4 ATOM 76 HD2 LYS 4 9.962 -6.130 1.953 1.00 0.00 sc4 ATOM 77 CE LYS 4 8.875 -5.289 3.605 1.00 0.00 sc4 ATOM 78 HE1 LYS 4 8.540 -6.249 3.966 1.00 0.00 sc4 ATOM 79 HE2 LYS 4 8.100 -4.816 3.019 1.00 0.00 sc4 ATOM 80 NZ LYS 4 9.263 -4.420 4.754 1.00 0.00 sc4 ATOM 81 HZ1 LYS 4 10.220 -4.042 4.601 1.00 0.00 sc4 ATOM 82 HZ2 LYS 4 9.247 -4.980 5.632 1.00 0.00 sc4 ATOM 83 HZ3 LYS 4 8.590 -3.632 4.834 1.00 0.00 sc4 ATOM 84 C LYS 4 11.329 -1.786 0.808 1.00 0.00 sc4 ATOM 85 O LYS 4 10.594 -0.854 1.070 1.00 0.00 sc4 ATOM 86 N ARG 5 12.555 -1.813 1.251 1.00 0.00 sc4 ATOM 87 HN ARG 5 13.131 -2.581 1.054 1.00 0.00 sc4 ATOM 88 CA ARG 5 13.067 -0.685 2.092 1.00 0.00 sc4 ATOM 89 HA ARG 5 12.383 -0.495 2.900 1.00 0.00 sc4 ATOM 90 CB ARG 5 14.384 -1.207 2.658 1.00 0.00 sc4 ATOM 91 HB1 ARG 5 14.723 -0.560 3.452 1.00 0.00 sc4 ATOM 92 HB2 ARG 5 15.128 -1.249 1.878 1.00 0.00 sc4 ATOM 93 CG ARG 5 14.151 -2.608 3.207 1.00 0.00 sc4 ATOM 94 HG1 ARG 5 14.374 -3.328 2.439 1.00 0.00 sc4 ATOM 95 HG2 ARG 5 13.115 -2.707 3.500 1.00 0.00 sc4 ATOM 96 CD ARG 5 15.055 -2.847 4.417 1.00 0.00 sc4 ATOM 97 HDl ARG 5 15.710 -2.004 4.571 1.00 0.00 sc4 ATOM 98 HD2 ARG 5 15.628 -3.755 4.284 1.00 0.00 sc4 ATOM 99 NE ARG 5 14.120 -2.983 5.569 1.00 0.00 sc4 ATOM 100 HE ARG 5 13.215 -2.612 5.510 1.00 0.00 sc4 ATOM 101 CZ ARG 5 14.505 -3.594 6.655 1.00 0.00 sc4 ATOM 102 NH1 ARG 5 14.788 -2.904 7.725 1.00 0.00 sc4 ATOM 103 HH11 ARG 5 14.709 -1.907 7.712 1.00 0.00 sc4 ATOM 104 HH12 ARG 5 15.081 -3.371 8.559 1.00 0.00 sc4 ATOM 105 NH2 ARG 5 14.607 -4.895 6.671 1.00 0.00 sc4 ATOM 106 HH21 ARG 5 14.391 -5.424 5.851 1.00 0.00 sc4 ATOM 107 HH22 ARG 5 14.901 -5.363 7.505 1.00 0.00 sc4 ATOM 108 C ARG 5 13.272 0.597 1.270 1.00 0.00 sc4 ATOM 109 O ARG 5 12.386 1.412 1.196 1.00 0.00 sc4 ATOM 110 N HIS 6 14.425 0.764 0.660 1.00 0.00 sc4 ATOM 111 HN HIS 6 15.110 0.074 0.745 1.00 0.00 sc4 ATOM 112 CA HIS 6 14.730 1.987 -0.178 1.00 0.00 sc4 ATOM 113 HA HIS 6 15.325 2.691 0.378 1.00 0.00 sc4 ATOM 114 CB HIS 6 15.548 1.424 -1.346 1.00 0.00 sc4 ATOM 115 HB1 HIS 6 16.549 1.192 -1.016 1.00 0.00 sc4 ATOM 116 HB2 HIS 6 15.583 2.145 -2.150 1.00 0.00 sc4 ATOM 117 CG HIS 6 14.865 0.172 -1.810 1.00 0.00 sc4 ATOM 118 ND1 HIS 6 13.686 0.218 -2.528 1.00 0.00 sc4 ATOM 119 HDl HIS 6 13.262 1.022 -2.896 1.00 0.00 sc4 ATOM 120 CD2 HIS 6 15.091 -1.151 -1.538 1.00 0.00 sc4 ATOM 121 HD2 HIS 6 15.930 -1.538 -0.981 1.00 0.00 sc4 ATOM 122 CE1 HIS 6 13.233 -1.037 -2.642 1.00 0.00 sc4 ATOM 123 HE1 HIS 6 12.279 -1.290 -3.065 1.00 0.00 sc4 ATOM 124 NE2 HIS 6 14.057 -1.917 -2.065 1.00 0.00 sc4 ATOM 125 C HIS 6 13.458 2.659 -0.725 1.00 0.00 sc4 ATOM 126 O HIS 6 13.390 3.863 -0.859 1.00 0.00 sc4 ATOM 127 N LEU 7 12.467 1.879 -1.047 1.00 0.00 sc4 ATOM 128 HN LEU 7 12.560 0.914 -0.922 1.00 0.00 sc4 ATOM 129 CA LEU 7 11.188 2.415 -1.599 1.00 0.00 sc4 ATOM 130 HA LEU 7 11.212 2.421 -2.672 1.00 0.00 sc4 ATOM 131 CB LEU 7 10.144 1.411 -1.113 1.00 0.00 sc4 ATOM 132 HB1 LEU 7 10.053 1.483 -0.039 1.00 0.00 sc4 ATOM 133 HB2 LEU 7 10.461 0.412 -1.376 1.00 0.00 sc4 ATOM 134 CG LEU 7 8.785 1.693 -1.753 1.00 0.00 sc4 ATOM 135 HG LEU 7 8.925 2.078 -2.753 1.00 0.00 sc4 ATOM 136 CD1 LEU 7 8.000 0.387 -1.811 1.00 0.00 sc4 ATOM 137 HDl 1 LEU 7 7.764 0.152 -2.836 1.00 0.00 sc4 ATOM 138 HD12 LEU 7 7.088 0.493 -1.242 1.00 0.00 sc4 ATOM 139 HD13 LEU 7 8.598 -0.407 -1.386 1.00 0.00 sc4 ATOM 140 CD2 LEU 7 8.001 2.705 -0.909 1.00 0.00 sc4 ATOM 141 HD21 LEU 7 7.719 2.248 0.029 1.00 0.00 sc4 ATOM 142 HD22 LEU 7 7.113 3.008 -1.440 1.00 0.00 sc4 ATOM 143 HD23 LEU 7 8.612 3.572 -0.714 1.00 0.00 sc4 ATOM 144 C LEU 7 10.847 3.806 -1.063 1.00 0.00 sc4 ATOM 145 O LEU 7 10.687 4.753 -1.807 1.00 0.00 sc4 ATOM 146 N LYS 8 10.679 3.914 0.218 1.00 0.00 sc4 ATOM 147 HN LYS 8 10.779 3.123 0.787 1.00 0.00 sc4 ATOM 148 CA LYS 8 10.281 5.216 0.826 1.00 0.00 sc4 ATOM 149 HA LYS 8 9.612 5.747 0.176 1.00 0.00 sc4 ATOM 150 CB LYS 8 9.546 4.833 2.119 1.00 0.00 sc4 ATOM 151 HB1 LYS 8 8.955 5.672 2.453 1.00 0.00 sc4 ATOM 152 HB2 LYS 8 10.270 4.583 2.879 1.00 0.00 sc4 ATOM 153 CG LYS 8 8.622 3.626 1.883 1.00 0.00 sc4 ATOM 154 HG1 LYS 8 8.542 3.052 2.793 1.00 0.00 sc4 ATOM 155 HG2 LYS 8 9.024 2.996 1.107 1.00 0.00 sc4 ATOM 156 CD LYS 8 7.234 4.123 1.476 1.00 0.00 sc4 ATOM 157 HDl LYS 8 6.568 3.282 1.363 1.00 0.00 sc4 ATOM 158 HD2 LYS 8 7.305 4.657 0.539 1.00 0.00 sc4 ATOM 159 CE LYS 8 6.693 5.059 2.559 1.00 0.00 sc4 ATOM 160 HE1 LYS 8 6.609 6.065 2.180 1.00 0.00 sc4 ATOM 161 HE2 LYS 8 7.334 5.034 3.430 1.00 0.00 sc4 ATOM 162 NZ LYS 8 5.343 4.524 2.892 1.00 0.00 sc4 ATOM 163 HZ1 LYS 8 5.418 3.870 3.696 1.00 0.00 sc4 ATOM 164 HZ2 LYS 8 4.958 4.019 2.067 1.00 0.00 sc4 ATOM 165 HZ3 LYS 8 4.711 5.309 3.145 1.00 0.00 sc4 t t o © » o HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH oooooooooooooooooooooooooooooooooooooooooooooo SJ W I W M N W t M W M W H M I-' H H M H Hl H H M I-' H K H H M I-' H H I-' H M H M h- M H H' H H O O O O O O O O O O Φ « Φ O « « Φ O I 0 \0 00 M M 00 M M 00 «1 » M I ^ -J V1 S) >.) SJ J S1 0 CM} (3 H O Φ Oo i oMΛ j- w t O Φ oo i σi υi - ω t H O Φ oo -j oi υi j- OJ W O O oo vi ^ ui ^ ω tJ O Φ OO vl ^ SSSooooSSo5SSoSo5SS5 « ^ « φ β β φ ' 0 φ φ φ φ * φ φ oφφ φ φ ' o *' 0 ' 000 M KM i— J ' O C W^ o σs tO o W k) όo lΛ i> lΛ -. >-' o ω ' 00 ^ l r' ^l < — ' o?^ o ATOM 212 C LYS 10 14.411 8.195 -1.519 1.00 0.00 sc4 ATOM 213 O LYS 10 15.268 9.028 -1.739 1.00 0.00 sc4 ATOM 214 N ΓLE 11 13.145 8.516 -1.469 1.00 0.00 sc4 ATOM 215 HN ILE 11 12.473 7.825 ■ -1.292 1.00 0.00 sc4 ATOM 216 CA ILE 11 12.720 9.929 ■ -1.683 1.00 0.00 sc4 ATOM 217 HA ILE 11 13.562 10.533 -1.982 1.00 0.00 sc4 ATOM 218 CB ELE 11 11.699 9.862 - ■2.819 1.00 0.00 sc4 ATOM 219 HB ILE 11 10.976 9.088 ■ -2.606 1.00 0.00 sc4 ATOM 220 CGI ILE 11 12.417 9.545 ■ -4.133 1.00 0.00 sc4 ATOM 221 HG11 H E 11 11.689 9.303 -4.892 1.00 0.00 sc4 ATOM 222 HG12 ILE 11 13.079 8.704 -3.987 1.00 0.00 sc4 ATOM 223 CG2 ILE 11 10.982 11.208 -2.943 1.00 0.00 sc4 ATOM 224 HG21 ILE 11 10.009 11.142 -2.478 1.00 0.00 sc4 ATOM 225 HG22 ILE 11 10.865 11.459 -3.987 1.00 0.00 sc4 ATOM 226 HG23 ILE 11 11.564 11.973 -2.451 1.00 0.00 sc4 ATOM 227 CDl ILE 11 13.230 10.762 -4.579 1.00 0.00 sc4 ATOM 228 HDl 1 ILE 11 13.810 10.507 -5.454 1.00 0.00 sc4 ATOM 229 HD12 ILE 11 13.893 11.064 -3.783 1.00 0.00 sc4 ATOM 230 HDl 3 ILE 11 12.560 11.575 -4.817 1.00 0.00 sc4 ATOM 231 C ILE 11 12.074 10.502 -0.415 1.00 0.00 sc4 ATOM 232 O ILE 11 11.955 11.701 -0.262 1.00 0.00 sc4 ATOM 233 N ILE 12 11.647 9.661 0.493 1.00 0.00 sc4 ATOM 234 HN ILE 12 11.746 8.695 0.355 1.00 0.00 sc4 ATOM 235 CA ILE 12 11.002 10.182 1.738 1.00 0.00 sc4 ATOM 236 HA ILE 12 10.216 10.875 1.483 1.00 0.00 sc4 ATOM 237 CB ELE 12 10.400 8.952 2.436 1.00 0.00 sc4 ATOM 238 HB ELE 12 10.048 8.255 1.691 1.00 0.00 sc4 ATOM 239 CGI ELE 12 9.225 9.389 3.313 1.00 0.00 sc4 ATOM 240 HG11 ILE 12 8.439 9.789 2.690 1.00 0.00 sc4 ATOM 241 HG12 ILE 12 9.556 10.147 4.008 1.00 0.00 sc4 ATOM 242 CG2 H E 12 11.450 8.267 3.315 1.00 0.00 sc4 ATOM 243 HG21 ILE 12 11.272 8.519 4.350 1.00 0.00 sc4 ATOM 244 HG22 ILE 12 12.435 8.602 3.028 1.00 0.00 sc4 ATOM 245 HG23 ILE 12 11.382 7.196 3.191 1.00 0.00 sc4 ATOM 246 CDl ILE 12 8.692 8.182 4.088 1.00 0.00 sc4 ATOM 247 HDl 1 ILE 12 9.097 7.275 3.665 1.00 0.00 sc4 ATOM 248 HD12 ILE 12 7.614 8.160 4.024 1.00 0.00 sc4 ATOM 249 HD13 ILE 12 8.989 8.260 5.124 1.00 0.00 sc4 ATOM 250 C ILE 12 12.043 10.874 2.627 1.00 0.00 sc4 ATOM 251 OT1 ILE 12 11.650 11.710 3.425 1.00 0.00 sc4 ATOM 252 OT2 ELE 12 13.213 10.557 2.492 1.00 0.00 sc4 Table 6. assign (resid 2 and name HN) (resid 1 and name HA) 1.8 04.0 assign (resid 3 and name HN) (resid 2 and name HA) 1.8 0 2.5 assign (resid 3 and name HN) (resid 4 and name HA) 1.8 0 4.0 assign (resid 3 and name HN) (resid 3 and name HB1) 1.8 0 3.0 assign (resid 3 and name HN) (resid 3 and name HB2) 1.8 0 3.0 assign (resid 3 and name HN) (resid 4 and name HG*) 1.8 0 5.0 assign (resid 3 and name HN) (resid 4 and name HD*) 1.8 0 5.0 assign (resid 3 and name HN) (resid 2 and name HB*) 1.8 0 3.5 assign (resid 3 and name HN) (resid 2 and name HD*) 1.8 04.5 assign (resid 4 and name HN) (resid 3 and name HA) 1.8 0 3.0 assign (resid 4 and name HN) (resid 2 and name HA) 1.8 04.0 assign (resid 4 and name HN) (resid 3 and name HB1) 1.8 0 3.0 assign (resid 4 and name HN) (resid 3 and name HB2) 1.8 0 3.0 assign (resid 4 and name HN) (resid 5 and name HB*) 1.8 0 3.5 assign (resid 4 and name HN) (resid 5 and name HG*) 1.8 0 3.5 assign (resid 4 and name HN) (resid 4 and name HB*) 1.8 0 3.0 assign (resid 4 and name HN) (resid 4 and name HG*) 1.8 03.5 assign (resid 4 and name HN) (resid 4 and name HD*) 1.8 0 3.5 assign (resid 5 and name HN) (resid 4 and name HA) 1.8 0 3.0 assign (resid 5 and name HN) (resid 5 and name HB*) 1.8 0 3.0 assign (resid 5 and name HN) (resid 5 and name HG*) 1.8 03.5 assign (resid 5 and name HN) (resid 5 and name HD*) 1.8 0 3.5 assign (resid 5 and name HN) (resid 2 and name HA) 1.8 04.0 assign (resid 5 and name HN) (resid 2 and name CO) 1.8 0 3.0 assign (resid 5 and name HN) (resid 1 and name CO) 1.8 0 3.0 assign (resid 6 and name HN) (resid 5 and name HA) 1.8 0 3.0 assign (resid 6 and name HN) (resid 6 and name HB*) 1.8 0 3.0 assign (resid 6 and name HN) (resid 5 and name HB*) 1.8 0 3.5 assign (resid 6 and name HN) (resid 5 and name HG*) 1.8 0 3.5 assign (resid 6 and name HEl) (resid 6 and name HA) 1.8 05.0 assign (resid 6 and name HEl) (resid 5 and name HA) 1.8 0 5.5 assign (resid 6 and name HEl) (resid 6 and name HB*) 1.8 0 5.5 assign (resid 6 and name HEl) (resid 5 and name HB*) 1.8 0 4.5 assign (resid 6 and name HEl) (resid 5 and name HG*) 1.8 04.5 assign (resid 6 and name HEl) (resid 8 and name HG*) 1.8 0 5.5 assign (resid 6 and name HD2) (resid 6 and name HB*) 1.8 0 3.3 assign (resid 6 and name HD2) (resid 5 and name HA) 1.8 0 3.5 assign (resid 6 and name HD2) (resid 3 and name HB*) 1.8 0 5.5 assign (resid 6 and name HN) (resid 2 and name CO) 1.8 0 3.0 assign (resid 6 and name HN) (resid 3 and name CO) 1.8 03.0 assign (resid 7 and name HN) (resid 6 and name HA) 1.8 03.0 assign (resid 7 and name HN) (resid 5 and name HA) 1.8 04.0 assign (resid 7 and name HN) (resid 6 and name HB*) 1.8 0 3.3 assign (resid 7 and name HN) (resid 5 and name HB*) 1.8 0 4.0 assign (resid 7 and name HN) (resid 5 and name HG*) 1.8 0 4.0 assign (resid 7 and name HN) (resid 8 and name HB*) 1.8 0 5.5 assign (resid 7 and name HN) (resid 7 and name HB*) 1.8 0 3.0 assign (resid 7 and name HN) (resid 7 and name HD*) 1.8 0 3.5 assign (resid 7 and name HN) (resid 4 and name HA) 1.8 04.5 assign (resid 7 and name HN) (resid 3 and name CO) 1.8 03.0 assign (resid 7 and name HN) (resid 4 and name CO) 1.8 0 3.0 assign (resid 8 and name HN) (resid 7 and name HA) 1.8 0 3.0 assign (resid 8 and name HN) (resid 6 and name HA) 1.8 0 3.5 assign (resid 8 and name HN) (resid 9 and name HB*) 1.8 0 5.0 assign (resid 8 and name HN) (resid 6 and name HB*) 1.8 04.5 assign (resid 8 and name HN) (resid 7 and name HD*) 1.8 04.0 assign (resid 8 and name HN) (resid 8 and name HB*) 1.8 0 3.0 assign (resid 8 and name HN) (resid 8 and name HG*) 1.8 0 3.5 assign (resid 8 and name HN) (resid 8 and name HD*) 1.8 0 3.5 assign (resid 8 and name HN) (resid 5 and name HA) 1.8 04.5 assign (resid 8 and name HN) (resid 5 and name HG*) 1.8 0 5.5 assign (resid 8 and name HN) (resid 5 and name CO) 1.8 0 3.0 assign (resid 8 and name HN) (resid 4 and name CO) 1.8 0 3.0 assign (resid 9 and name HN) (resid 6 and name HA) 1.8 0 4.0 assign (resid 9 and name HN) (resid 8 and name HA) 1.8 0 3.0 assign (resid 9 and name HN) (resid 7 and name HA) 1.8 0 3.5 assign (resid 9 and name HN) (resid 9 and name HB*) 1.8 0 3.0 assign (resid 9 and name HN) (resid 6 and name HB*) 1.8 0 5.0 assign (resid 9 and name HN) (resid 8 and name HB*) 1.8 0 3.5 assign (resid 9 and name HN) (resid 8 and name HG*) 1.8 0 3.5 assign (resid 9 and name HN) (resid 7 and name HB*) 1.8 04.5 assign (resid 9 and name HN) (resid 7 and name HD*) 1.8 0 5.0 assign (resid 9 and name HE3) (resid 9 and name HA) 1.8 02.8 assign (resid 9 and name HE3) (resid 5 and name HA) 1.8 04.5 assign (resid 9 and name HE3) (resid 8 and name HA) 1.8 04.0 assign (resid 9 and name HE3) (resid 9 and name HB*) 1.8 0 3.0 assign (resid 9 and name HE3) (resid 6 and name HB*) 1.8 0 5.0 assign (resid 9 and name HE3) (resid 7 and name HB*) 1.8 0 5.0 assign (resid 9 and name HE3) (resid 7 and name HD*) 1.8 0 5.0 assign (resid 9 and name HE3) (resid 10 and name HG*) 1.8 0 5.0 assign (resid 9 and name HE3) (resid 12 and name HD*) 1.8 0 5.5 assign (resid 9 and name HZ2) (resid 9 and name HA) 1.8 0 5.0 assign (resid 9 and name HZ2) (resid 12 and name HG*) 1.8 0 5.0 assign (resid 9 and name HZ2) (resid 12 and name HD*) 1.8 0 5.0 assign (resid 9 and name HZ3) (resid 9 and name HB*) 1.8 04.5 assign (resid 9 and name HZ3) (resid 8 and name HB*) 1.8 0 4.5 assign (resid 9 and name HZ3) (resid 8 and name HG*) 1.8 04.5 assign (resid 9 and name HZ3) (resid 7 and name HB*) 1.8 04.5 assign (resid 9 and name HZ3) (resid 7 and name HD*) 1.8 04.5 assign (resid 9 and name HH2) (resid 9 and name HA) 1.8 0 5.0 assign (resid 9 and name HH2) (resid 8 and name HA) 1.8 04.5 assign (resid 9 and name HH2) (resid 12 and name HG*) 2.5 0 5.0 assign (resid 9 and name HH2) (resid 12 and name HD*) 1.8 0 5.0 assign (resid 9 and name HH2) (resid 7 and name HB*) 1.8 04.5 assign (resid 9 and name HH2) (resid 7 and name HD*) 1.8 04.5 assign (resid 9 and name HN) (resid 5 and name CO) 1.8 0 3.0 assign (resid 9 and name HN) (resid 6 and name CO) 1.8 0 3.0 assign (resid 10 and name HN) (resid 9 and name HA) 1.8 0 3.0 assign (resid 10 and name HN) (resid 7 and name HA) 1.8 04.0 assign (resid 10 and name HN) (resid 10 and name HB*) 1.8 0 3.5 assign (resid 10 and name HN) (resid 10 and name HG*) 1.8 0 3.5 assign (resid 10 and name HN) (resid 10 and name HD*) 1.8 04.5 assign (resid 10 and name HN) (resid 9 and name HB*) 1.8 0 3.5 assign (resid 10 and name HN) (resid 7 and name HD*) 1.8 0 5.0 assign (resid 10 and name HN) (resid 8 and name HA) 1.8 04.0 assign (resid 11 and name HN) (resid 10 and name HA) 1.8 0 3.0 assign (resid 11 and name HN) (resid 8 and name HA) 1.8 0 4.0 assign (resid 11 and name HN) (resid 9 and name HA) 1.8 0 3.5 assign (resid 11 and name HN) (resid 11 and name HB) 1.8 0 3.5 assign (resid 11 and name HN) (resid 11 and name HG*) 1.8 0 3.5 assign (resid 11 and name HN) (resid 11 and name HD*) 1.8 04.5 assign (resid 11 and name HN) (resid 10 and name HB*) 1.8 0 3.5 assign (resid 11 and name HN) (resid 6 and name CO) 1.8 0 3.0 assign (resid 11 and name HN) (resid 7 and name CO) 1.8 0 3.0 assign (resid 12 and name HN) (resid 11 and name HA) 1.8 0 3.0 assign (resid 12 and name HN) (resid 11 and name HB) 1.8 0 4.5 assign (resid 12 and name HN) (resid 12 and name HB) 1.8 0 3.5 assign (resid 12 and name HN) (resid 12 and name HG*) 1.8 0 3.5 assign (resid 12 and name HN) (resid 12 and name HD*) 1.8 0 4.5 assign (resid 12 and name HN) (resid 9 and name HA) 1.8 04.0 assign (resid 12 and name HN) (resid 10 and name HA) 1.8 0 3.0 assign (resid 12 and name HN) (resid 9 and name HB*) 1.8 0 4.0 The complete disclosures of all patents, patent applications, publications, and nucleic acid and protein database entries, including for example GenBank accession numbers and EMBL accession numbers, that are cited herein are hereby incorporated by reference as if individually incorporated. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention, and it should be understood that this invention is not to be unduly limited to the illustrative embodiments set forth herein. Sequence Free Text The following are synthetic polypeptides: ANIKLSVQMKLF (SEQ HD NO:l) KLSVQMKLFKRH (SEQ ID NO:2) VQMKLFKRHLKW(SEQ ED NO:3) KLFKRHLKWKH (SEQ ID NO:4) KRHLKWKHVKL (SEQ HD NO:5) LKWKHVKLNDG (SEQ HD NO:6) KHVKLNDGREL (SEQ ID NO:7) VKLNDGRELSLD (SEQ ED NO:8) QMKLFKRHLKWK (SEQ ID NO:9) MKLFKRHLKWKI (SEQ ID NO:10) MKLFKRHLKWKHV (SEQ ID NO:l 1) XLFKRHLKWKH (SEQ ID NO:12) KLFXRHLKWKH (SEQ ID NO:13) KLFKRHLXWKH (SEQ ID NO: 14) KLFKRHLKWXπ (SEQ ID NO: 15) KLFKKHLKWKH (SEQ ED NO: 16) KLFKHLKWKΠ (SEQ ED NO: 17)

Claims: WHAT IS CLAIMED IS: 1. A polypeptide selected from the group consisting of those represented by SEQ ID NOs: 1 -8 and analogs thereof. 2. The polypeptide of claim 1 selected from the group consisting of those represented by SEQ ID NOs: 1-8. 3. A polypeptide selected from the group consisting of those represented by SEQ ID NOs:9-17, wherein X is an amino acid. 4. The polypeptide of claim 3 wherein X is norleucine. 5. The polypeptide of claim 3 which is active for the treatment of bacterial infection and/or endotoxemia. 6. A method for treating bacterial infection and/or endotoxemia comprising administering to a patient an amount of a pharmaceutical composition effective to inhibit the bacterial infection and/or neutralize endotoxin, wherein the pharmaceutical composition comprises a polypeptide selected from the group consisting of those represented by SEQ ID NOs: 1-17; analogs thereof active for the treatment of bacterial infection and/or endotoxemia; and combinations thereof; wherein X is an amino acid. 7. The method of claim 6 wherein the polypeptide neutralizes endotoxin. 8. The method of claim 6 wherein the polypeptide is bactericidal. 9. The method of claim 6 wherein the polypeptide is both bactericidal and capable of neutralizing endotoxin. 10. The method of claim 6 wherein the composition comprises a polypeptide having endotoxin neutralizing activity and is selected from the group consisting of those represented by SEQ ID NOs:l-8; and combinations thereof. 11. The method of claim 6 wherein the composition comprises a polypeptide having bactericidal activity and is selected from the group consisting of those represented by SEQ HD NOs: 1 -8; and combinations thereof. 12. The method of claim 6 wherein the composition comprises KLFKRHLKWKH (SEQ ED NO:4). 13. A method for inhibiting bacterial infection and/or endotoxemia in vitro, the method comprising contacting cells with an amount of a composition effective to inhibit the bacterial infection and/or to neutralize endotoxin, wherein the composition comprises a polypeptide selected from the group consisting of those represented by SEQ ED NOs: 1-17; analogs thereof active for the treatment of bacterial infection and/or endotoxemia; and combinations thereof; wherein X is an amino acid. 14. A method for decreasing the amount of TNF-α in a patient, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide selected from the group consisting of those represented by SEQ ID NOs: 1-17; active analogs thereof; and combinations thereof; wherein X is an amino acid. 15. The method of claim 14 wherein the composition comprises a polypeptide selected from the group consisting of those represented by SEQ ED NOs: 1-8: and combinations thereof. 16. The method of claim 14 wherein the composition comprises KLFKRHLKWKH (SEQ ID NO:4). 17. A method for decreasing the amount of TNF-α in vitro, the method comprising incubating cells with an effective amount of a composition comprising a polypeptide selected from the group consisting of those represented by SEQ ID NOs:l-17; active analogs thereof; and combinations thereof; wherein X is an amino acid. 18. A method for inhibiting endothelial cell proliferation in a patient, the method comprising adnnnistering to the patient a therapeutically effective amount of a composition comprising a polypeptide selected from the group consisting of those represented by SEQ ED NOs: 1-17; active analogs thereof; and combinations thereof; wherein X is an amino acid. 19. The method of claim 18 wherein the composition comprises a polypeptide selected from the group consisting of those represented by SEQ ID NOs: 1-8; and combinations thereof. 20. The method of claim 18 wherein the composition comprises KLFKRHLKWKH (SEQ HD NO:4). 21. A method for inhibiting endothelial cell proliferation in vitro, the method comprising contracting cells with an effective amount of a composition comprising a polypeptide selected from the group consisting of those represented by SEQ ED NOs:l-17; active analogs thereof; and combinations thereof; wherein X is an amino acid. 22. A method for inhibiting angiogenic-factor mediated inter-cellular adhesion molecule expression down-regulation in a patient, the method comprising administering to the patient a therapeutically effective amount of a composition comprising a polypeptide selected from the group consisting of those represented by SEQ ED NOs:l-17; active analogs thereof; and combinations thereof; wherein X is an amino acid. 23. The method of claim 22 wherein the composition comprises a polypeptide selected from the group consisting of those represented by SEQ ID NOs: 1-8; and combinations thereof. 24. The method of claim 23 wherein the composition comprises KLFKRHLKWKH (SEQ HD NO:4). 25. A method for inhibiting angiogenic-factor mediated inter-cellular adhesion molecule expression down-regulation in vitro, the method comprising contacting cells with an effective amount of a composition comprising a polypeptide selected from the group consisting of those represented by SEQ ID NOs:l-17; active analogs thereof; and combinations thereof; wherein X is an aniino acid. 26. A method for inhibiting angiogenesis in a patient, the method comprising administering to tlie patient a therapeutically effective amount of a composition comprising a polypeptide selected from the group consisting of those represented by SEQ ID NOs: 1-17; active analogs thereof; and combinations thereof; wherein X is an amino acid. 27. - The method of claim 26 wherein the composition comprises a polypeptide selected from the group consisting of those represented by SEQ ID NOs: 1-8: and combinations thereof. 28. The method of claim 27 wherein the composition comprises KLFKRHLKWKH (SEQ HD NO:4). 29. A method for inhibiting angiogenesis in vitro, the method comprising contacting cells with an effective amount of a composition comprising a polypeptide selected from the group consisting of those represented by SEQ HD NOs: 1-17; active analogs thereof; and combinations thereof; wherein X is an amino acid. 30. A method for inhibiting tumorigenesis in a patient, the method comprising administering to the patient a therapeutically effective amount of a composition comprising a polypeptide selected from the group consisting of those represented by SEQ ID NOs: 1-17; active analogs thereof; and combinations thereof; wherein X is an amino acid. 31. The method of claim 30 wherein the composition comprises a polypeptide selected from the group consisting of those represented by SEQ HD NOs: 1 -8; and combinations thereof. 32. The method of claim 31 wherein the composition comprises KLFKRHLKWKH (SEQ HD NO:4). 33. A polypeptide having an amphipathic structure having one surface comprising positively charged amino acid residues and an opposing surface comprising hydrophobic amino acid residues, wherein these define a surface active domain. 34. The polypeptide of claim 33 wherein the surface active domain comprises amino acids Kl, K4, K8, and R5 shown in Figure 6. 35. The polypeptide of claim 34 wherein the surface active domain comprises atoms 1-24, 64-109, and 146-167 listed in Table 5. 36. The polypeptide of claim 34 having the structure coordinates listed in Table 5. 37. The polypeptide of claim 33 having the sequence KLFKRHLKWKH (SEQ HD NO:4). 38. A method of evaluating a candidate compound for structural similarity to that of polypeptide KLFKRHLKWKH (SEQ HD NO:4), the method comprising: supplying a three-dimensional structure of KLFKRHLKWKH (SEQ ED NO:4) or a portion thereof; supplying a three-dimensional structure of a candidate compound; and comparing the three-dimensional structure of the candidate compound with the three-dimensional structure of KLFKRHLKWKH (SEQ ID NO:4) or a portion thereof. 39. The method of claim 38 wherein supplying a three-dimensional structure of KLFKRHLKWKH (SEQ ID NO:4) or a portion thereof comprises supplying the coordinates of atoms 1-24, 64-109, and 146-167 listed in Table 5. 40. The method of claim 38 wherein supplying a three-dimensional structure of KLFKRHLKWKH (SEQ ED NO:4) or a portion thereof comprises supplying the structure coordinates listed in Table 5.

Inventors: MAYO KEVIN H, MAYO, KEVIN, H.
Applicants: MAYO KEVIN H, UNIV MINNESOTA, MAYO, KEVIN, H., REGENTS OF THE UNIVERSITY OF MINNESOTA
Application year: 2001
Main classification: C07K14
IPC codes: A61K38, A61L2, C07K14