MXPA99007799A - Antipathogenic synthetic peptides and compositions comprising them - Google Patents

Abstract

Non-hemolytic cytolytic agents selected from peptides, complexes of bundled peptides, mixtures of peptides or random peptide copolymers have a selective cytolytic activity manifested in that they have a cytolytic activity on pathogenic cells, being cells which are non-naturally occurring within the body consisting of microbial pathogenic organisms and malignant cells;and are non-hemolytic, having no cytolytic effect on red blood cells. The peptides may be cyclic derivatives of natural peptides such as pardaxin and mellitin and fragments thereof in which L-amino acid residues are replaced by corresponding D-amino acid residues, or are diastereomers of linear peptides composed of varying ratios of at least one positively charged amino acid and at least one hydrophobic amino acid, and in which at least one of the amino acid residues is a D-amino acid. Pharmaceutical compositions comprising the non-hemolytic cytolytic agents can be used for the treatment of several diseases caused by pathogens including antibacterial, fungal, viral, mycoplasma and protozoan infections and for the treatment of cancer.

Description

SYNTHETIC PEPT1DOS ANT1PATOGENS AND COMPOSITIONS THAT UNDERSTAND THEM FIELD OF THE INVENTION The present invention relates to novel non-haemolytic cytolytic agents, to compositions comprising them and to their use in the treatment of diseases or disorders and in agriculture.

BACKGROUND OF THE INVENTION In the following text, reference is made to prior art documents, the full details of which can be found in the "References" section at the end of the specification, before the claims. The growing resistance of microorganisms to antimicrobial drugs has led to extensive studies focused on the development of alternative antimicrobial compounds. In addition to the highly specific cell-mediated immune response, or in addition to it, vertebrates and other organisms have a defense system composed of different groups of cytolytic peptides, eg, antibacterial, broad-spectrum.

Studies on the lipid-peptide interactions of such cytolytic peptides, also known as cytolysins, tend to emphasize the importance of the antipathic a-helical structure for their cytolytic activity. This conclusion is based mainly on studies with cytolysins that act alone, either on mammalian cells or bacteria, or on both types of cells. A major group of cytolytic peptides in this family are the short linear peptides (YAQ amino acids) of host defense, which are free of disuifide bridges (Boman, 1995). These peptides vary considerably in chain length, hydrophobic character and general charge distribution, but share a common structure after their association with lipid bilayers, namely, an antipathetic structure of alpha helix (Segrest et al., 1990). Examples of known cytolysins include: (i) antibacterial peptides that are cytolytic only on bacteria, eg, cecropins, isolated from the Cecropia moth (Steiner et al., 1981), magainins (Zasloff, 1987) and dermaseptins (Mor et al. , 1991), isolated from the skin of frogs; (ii) cytolysins that are selectively cytotoxic to mammalian cells, such as Ü-hemolysin, isolated from Staphylococcus aureus (Dhople and Nagaraj, 1993); and (iii) cytolysins that are non-cell selective, such as bee venom melitin (Haberman and Jentsch, 1967) and the parotoxin neurotoxin (Shai et al., 1988), which lyse both mammalian cells and bacteria.

Antibacterial peptides were initially discovered in invertebrates, and subsequently in vertebrates, including man. As a complementary or additional defense system, this secondary chemical inmuine system provides organisms with a repertoire of small peptides that are synthesized promptly after induction, and which act against the invasion of occasional and obligate pathogens, and also against proliferation. uncontrolled of commensal microorganisms (Boman, 1995). Up to now, more than 100 different antibacterial peptides have been isolated and characterized. The largest family, and probably the most studied one, includes the peptides that are positively charged and adopt an alpha-helical antipatic structure. Many studies on natural antibacterial peptides tend to emphasize the importance of alpha-helical antipatic structure and a positive net charge for cytolytic activity. The positive charge facilitates the interaction of the peptides with the negatively charged membranes (Andreu et al., 1985) which are found in higher concentrations in the pathogenic cell membrane, compared to normal eukaryotic cells, and the alpha-helical antipatic structure is essential for lithic activity (Chen et al., 1988). It has been proposed that these interactions destroy the energy metabolism of the target organism, increasing the permeability of the energy transducing membranes (Okada and Natori, 1984). Due to its antipathic structure, it has been suggested that these antibacterial peptides penetrate the membrane forming ionic channels / pores through a "Bottomless barrel" mechanism (Rizzo et al., 1987). In accordance with this model, the amphiphilic transmembrane alpha-helices form groups in which the hydrophobic surfaces directed outward interact with the lipid constituents of the membrane, while the hydrophilic surfaces facing inward produce a pore. Alternatively, the peptides are joined parallel to the surface of the membrane, cover the surface of the membrane in a manner similar to "mat" and dissolve it as a detergent (Shai, 1995). Despite extensive studies, the exact mode of action of short non-selective cell linear peptides, such as pardaxin and melitin, is not yet known; and it is not clear if similar structural characteristics are required for their cytotoxicity towards mammalian cells and bacteria. Pardaxin, a 33-element peptide, is an excitatory neurotoxin that has been purified from Soleide Moses of the Red Sea, Pardachirus marmoratus (Shai et al., 1988) and Soledad Budión of the western Pacific, Pardachirus pavoninus (Thompson et al., 1986). Pardaxin has a variety of biological activities, depending on its concentration (reviewed by Shai, 1994). At concentrations below 10-7 M, pardaxin induces the release of neurotransmitters in a calcium-dependent manner. At higher concentrations of 10"^ M to 10 ~ 8 M, the process is calcium independent, and above 10" 5 M, cytolysis is induced. Pardaxin also affects the activities of various physiological preparations in vitro Its biological functions have been attributed to its interference with the ion transport of the osmoregulator system in the epithelium and to the presynaptic activity forming ion channels that are voltage dependent and slightly selective of cations. A "bottomless keg" mechanism was proposed for the insertion of pardaxin in the membranes, based on its structure and several biophysical studies (reviewed by Shai, 1994). Pardaxin has a helix-helix-helix structure: The N-helix includes residues 1 -1 and the C-helix includes residues 14-26. The helices are separated by a proline residue located at position 13. This structural motif is found both in antibacterial peptides that can act specifically on bacteria (eg, cecropin), and in cytotoxic peptides that can lyse a variety of cells (eg, example, melittin). Melitin, an antipatic 26-element peptide, is the main component of the honey bee venom Apis mellifera (Hamann and Jentsch, 1967) and is one of the most studied membrane-seeking peptides (Dempsey, 1990). Melitin is highly cytotoxic for mammalian cells, but it is also a highly potent antibacterial agent (Steiner et al., 1981). Many studies have been undertaken to determine the nature of the interaction of melittin with membranes, both in order to understand the molecular mechanism of melitin-induced hemolysis, and as a model to study the general characteristics of protein structures. of membrane and the interactions of such proteins with the phospholipids of the membranes.

Much of the evidence currently described indicates that different molecular mechanisms can support different actions of melittin. However, it has been seen that the antipathic alpha-helical structure is a prerequisite for its different activities (Pérez et al., 1994). The structure of melittin has been investigated using several techniques. The results of X-ray crystallography and NMR in methanolic solutions indicate that the molecule consists of two alpha-helical segments (residues 1-10 and 13-26) that intersect at an angle of 120 °. These segments are joined by means of a hinge (1 1-12) to form an alpha-helical bending rod with the hydrophilic and hydrophobic sides facing opposite directions. Four of these monomeric melittin molecules are grouped together, by hydrophobic interactions, to form a tetramer (Anderson et al., 1980; Bazzo and others, 1988; Terwilliger and Eisenberg, 1982; Terwilliger and Eisenberg, 1982). After the initial interaction with membrane surfaces, it has been found that the tetramer dissociates into monomers, which retain the alpha-helical conformation before its insertion into the membrane (Altenbach and Hubbell, 1988). Melitin shares some similarities with pardaxin. Both pardaxin and melittin are composed of two helices with a proline hinge between them. In addition, they exhibit significant homology in their N-helices, which are mainly hydrophobic (Thompson et al., 1986). However, pardaxin (net charge +1) contains an additional residue of seven amino acids on its C-terminal side with a charge of -2, whereas the melittin (net charge +6) ends with an amide group and contains the positively charged tetrapeptide sequence Lys-Arg-Lys-Arg. There are several functional differences between pardaxin and melittin. Pardaxin binds similarly to both zwitterionic and negatively charged phospholipids (Rapaport and Shai, 1991), while melittin binds better to negatively charged phospholipids than to zwitterionics (Batenburg et al., 1987; Batenburg et al., 1987). Also, pardaxin binds to phospholipids with positive cooperativity (Rapaport and Shai, 1991), while melitin binds with negative cooperativity (Batenburg et al., 1987; Batenburg et al., 1987). Although the two, Pardaxin and Melitin, are potent antibacterial peptides against Gram-positive and Gram-negative bacteria, Pardaxin is 40 to 100 times less hemolytic than melittin to human erythrocytes (Oren and Shai, 1996). It has been shown that pardaxin analogs with L- to D- substitutions are capable of lysing human erythrocytes (Pouny and Shai, 1992). It was subsequently shown (see the results reported below) that two of the peptides described by Pouny and Shai, 1992, namely, D-Pro 7- pardaxin and D-Leul d Leu ^ -pardaxin, although they are hemolytic, have an antibacterial activity very low. It was also found that magainin analogues with L- to D- substitutions lack antibacterial activity (Chen et al., 1988).

GLOSSARY In the following, several coined terms will be used in order to simplify the reading of the text and better facilitate understanding of the invention. However, it should be noted that for full understanding of these terms, reference will sometimes be made to the following full description. These terms and their meanings herein are as follows: "Heterogeneous peptide" as used herein refers to a peptide comprising both D- and L- amino acid residues. "Homogeneous peptide" as used herein refers to a peptide comprising either only the natural L-amino acid residues, or only the D-amino acid residues. "Homogeneous L-peptide" and "D-homogeneous peptide" as used herein refers to the homogeneous peptide consisting entirely of amino acid residues L- or D-, respectively. "Heterogeneous peptide of base L-" and "Heterogeneous peptide of base D-", as used herein, refers to a heterogeneous peptide comprising mainly L-amino acid residues, for example, a peptide derived from a homogeneous L-peptide wherein one or more of the L-amino acid residues has been replaced with their equivalent D-enantiomers, and a heterogeneous peptide comprising mainly D-amino acid residues in which one or more of the D-residues has been replaced. amino acid with its equivalent L-enantiomers, respectively.

"Helical peptide" as used herein refers to a peptide having a continuous elongation of alpha-helix throughout the greater part of its length. The helical portion of a helical peptide consists completely of either L-amino acid or D-amino acid residues. "Non-helical peptide" as used herein refers to a peptide that has no alpha-helical structure, or that has non-continuous alpha-helical structures dispersed therealong. A non-helical peptide according to the invention can have an alpha-helical elongation which, if terminal, has a length that covers less than half a membrane width of a cell, for example, less than about 10-15 amino acid residues, and if it is a non-terminal alpha-helix, it has a length that is less than the full width of a cell membrane, for example, less than about 20-25 amino acid residues. A non-helical peptide can be a homogeneous peptide with alpha-helix breaker portions (see below) or it can be a heterogeneous peptide. "Breaker portion of alpha-helix" as used here refers to a portion that, if inserted into an alpha-helix structure, breaks its continuity. Said portion may be, for example, the amino acid residue proline or glycine, alpha-methyl-substituted amino acids, non-alpha amino acids both cyclic and acyclic, such as 6-amino-hexanoic acid, 3-amino-1-cyclohexanoic acid , 4-amino-1-cyclohexanoic acid, or it can be an L- or D-enantiomer inserted in an alpha-helix extension consisting of an extension of amino acid residues of the opposite enantiomer. "Pathogenic cells" as used herein refers to cells that appear unnaturally within the body, including cancer cells and pathogenic organisms such as bacteria, fungi, protozoa, viruses and mycoplasma, as well as mammalian cells infected with pathogenic organisms such as protozoan parasites, for example, Leishmania and Plasmodium. "Selective cytolytic activity" as used herein refers to the activity of an agent in the induction of cytolysis of a pathogenic cell, the selectivity being manifested in that the agent induces cytolysis of the pathogenic cells at a much lower concentration than required for the cytolysis of non-pathogenic normal cells such as red blood cells. "Non-haemolytic", as used herein, refers to agents that cause hemolysis of red blood cells at concentrations much higher than the concentration required to cause cytolysis of other cells, such as pathogenic cells such as microorganism cells, cancer cells , and similar. "Diastereomers" is used herein as a synonym for "heterogeneous peptide".

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a non-hemolytic cytolytic agent selected from a peptide, a complex of pooled peptides, a mixture of peptides or a random peptide copolymer, said agent having a selective cytolytic activity manifested because it has a cytolytic activity on pathogenic cells, being cells that do not occur naturally within the body, consisting of pathogenic microbial organisms and malignant cells; and is non-haemolytic, namely, has no cytolytic effects on erythrocytes or has a cytolytic effect on them at concentrations that are substantially greater than those in which said cytolytic activity manifests, said non-haemolytic cytolytic agent being selected from the group consisting of : (1) a cyclic derivative of a peptide having a net positive charge which is greater than + 1, and comprising residues of L-amino acids and residues of D-amino acids, or comprising one or both residues of L-amino acids and residues of D-amino acids, and comprising a disrupting portion of a-helix; (2) a peptide comprising residues of L-amino acids and residues of D-amino acids, having a net positive charge which is greater than + 1, and having an amino acid sequence such that a corresponding amino acid sequence comprising only L-amino acid residues are not found in nature, and cyclic derivatives thereof; (3) a complex consisting of a plurality of 2 or more non-haemolytic cytolytic peptides, each peptide having a net positive charge which is greater than +1, and comprising residues of L-amino acids and D-amino acid residues, or comprising one or both of L-amino acid residues and D-amino acid residues, and comprising a switch-a-helix portion, or cyclic derivatives of the foregoing, said peptides being pooled by the use of a linker molecule covalently linked to each of the same; (4) a mixture consisting of a plurality of two or more non-haemolytic cytolytic peptides, each peptide having a net positive charge which is greater than +1, and comprising residues of L-amino acids and D-amino acid residues, or comprising one or both of L-amino acid residues and D-amino acid residues, and comprising a-helical interrupter portion, or cyclic derivatives of the foregoing; and (5) a random copolymer consisting of different ratios of a hydrophobic amino acid, a positively charged amino acid and a D-amino acid. In one embodiment, the cyclic derivatives of subsection (1) above are derived from a non-selective natural cytolytic peptide such as, for example, pardaxin and melittin, or from a fragment thereof. These cyclic diastereomers are obtained by conventional cyclization methods for peptides. In one embodiment, the cyclic diastereomer is derived from the fragment 1-22 of pardaxin to which 1 to 3 Lys residues have been added to the N-terminus and cysteine residues have been added to the N and C termini for cyclization. The net positive charge of the peptides may be due to the native amino acid composition, the neutralization of the free carboxyl groups and / or the addition of positively charged amino acid residues or positively charged chemical groups. In another embodiment, the invention provides a non-haemolytic cytolytic peptide and cyclic derivatives thereof as defined in subsection (2) above, having the following characteristics: (a) it is an unnatural synthetic peptide composed of variable proportions of at least a hydrophobic amino acid and at least one positively charged amino acid, and in which sequence at least one of the amino acid residues is a D-amino acid; (b) the peptide has a positive net charge that is greater than +1; and (c) the proportion of hydrophobic amino acids to positively charged, is such that the peptide is cytolytic for pathogenic cells, but does not cause red blood cell cytolysis. Examples of positively charged amino acids are lysine, arginine and histidine, and the hydrophobic amino acids are leucine, isoleucine, glycine, alanine, valine, phenylalanine, proline, tyrosine and tryptophan. The net positive charge is due to the composition of the amino acid, but can be considered also the addition of positively charged chemical groups. In addition, polar amino acids such as serine, threonine, methionine, asparagine, glutamine and cysteine can be added to reduce the hydrophobicity and / or toxicity of the molecule. In a preferred embodiment, the peptide is composed of a hydrophobic amino acid such as leucine, alanine or valine, and a positively charged amino acid such as lysine or arginine. The non-natural synthetic peptide can have at least 6, particularly ten or more amino acid residues. In a preferred embodiment, the synthetic diastereomer is a 12-element peptide composed of leucine, alanine or valine and lysine, and at least one third of the sequence is composed of D-amino acids. In still another embodiment, the invention provides a non-hemolytic cytolytic complex as defined in the above (3), which consists of a plurality of 2 or more non-haemolytic cytolytic peptides of the invention, forming complexes or "groups" with each other, for example, using a linker molecule or "template" covalently linked to each of the peptides. The group may be composed of 2 or more, preferably 5 molecules of the same peptide or different peptides. The linker / template can be a peptide of the invention or a commonly used linker, for example, polymers such as polyesters, polyamides, polypeptides, polyamino acids (e.g., polylysine) bearing active groups such as OH, SH, COOH, NH2, CH2Br.

In still another embodiment, the invention provides a non-haemolytic cytolytic mixture as defined in subsection (4), obtained by adding a mixture composed of 1 eq of each of the D-amino acids, positively charged and hydrophobic desired in each coupling step of the solid phase method for the synthesis of peptides. In this way, a mixture of 3 ^ 2 different peptides was obtained with a mixture of lysine, Ieucine and D-leucine, and the hydrophilic mixture was obtained after breaking with HF, extraction with water and lyophilization. In a further embodiment, the invention provides a non-haemolytic cytolytic random copolymer as defined in item (5) above consisting of different ratios of a hydrophobic amino acid, a positively charged amino acid, and a D-amino acid, eg, copolymers 1 :eleven , 2: 1, 1, and 3: 1: 1 (Mol) of Lys: Leu: D-Leu. Preferably, the non-haemolytic cytolytic peptide has no alpha-helix structure or has an alpha-helix structure whose length is insufficient to span the width of a cell membrane. The peptide thus does not contain an uninterrupted extension of amino acid residues entirely D- or entirely L- of a length capable of being part of a transmembrane pore. Said length, typically, it is approximately 20-22 amino acids where the extension is in the non-terminal portion of the peptide, and approximately half, ie, 10-11 amino acids, where the extension is at the peptide end, that in such a case, two peptides can bind their ends together and encompass the cell membrane. The disruption of an extension of amino acid residues Do L- can be carried out by replacing one or more amino acids in the extension with the amino acid of the opposite enantiomer, or by placing in the continuous extension a portion of alpha-helix breaking such as proline, glycine, an alpha-methyl-alpha-amino acid or a non-alpha-amino acid. The peptides of the invention and the peptides comprised within the complexes, mixtures and copolymers of the invention have a net positive charge greater than +1. The positive net charge may be due to the native amino acid composition of the invention, to the neutralization of free COOH groups, for example by amidation, or it may be due to the addition of amino acids or positively charged chemical groups. It was found that selective cytolytic activity can sometimes be enhanced by increasing the positive net charge, for example, by attaching a positively charged amino acid and / or a positively charged group at any position in the molecule. For example, a polyamine group, an alkylamino group or an aminoalkylamino group, typically at their carboxyl terminus, can be attached at one of their ends. One of these preferred groups is the aminoethylamino group -NH-CH2-NH2, hereinafter referred to as "TA". The peptides that are derived from natural non-selective cytolytic peptides, for example, pardaxin and melittin, are unfriendly, that is, they have a surface that is composed mainly of residues of hydrophobic amino acids and an opposite surface that is composed mainly of hydrophilic amino acid residues. The antipathetic nature of the peptides that are within the scope of the invention can be verified in accordance with methods known in the art. An example of such methods is the use of the Shiffer wheel projection and Edmondson, where amino acid residues are written according to their sequence in a circle, such that each amino acid in the sequence is angularly displaced 100 ° from its neighboring amino acid residues (3.6 amino acids per circle). If most of the hydrophilic amino acids are concentrated on one side of the wheel and the hydrophobic amino acids are concentrated on the opposite side of the wheel, then the peptide can be considered unfriendly. Peptides of the invention that are not derived from natural non-selective cytolytic peptides, for example, synthetic diastereomers composed of hydrophobic amino acids, positively charged amino acids and D-amino acids, are not antipathetic. They have a net positive charge greater than +1, and an adequate proportion of hydrophobic amino acid to be positively charged, such that the resulting peptide is cytolytic for pathogenic cells, but is not hemolytic. These peptides can be very easily selected according to the invention using the antibacterial and hemolytic tests described herein. In one embodiment, for a peptide composed of leucine and lysine, an adequate ratio of Leu: Lys can be 64%: 36% for a diastereomer of 6 amino acid residues, and 66%: 34% for a diastereomer of 12 amino acid residues. Without intending to be limited by the theory, it is nevertheless believed that the cytolytic activity may be the result of the aggregation of several peptides on the surface of the membrane, and together said peptides cause the damage of the cell membrane. Consequently, as described above, it is contemplated in accordance with the invention to also use a plurality of the peptides of the invention forming a complex (or group) among them, for example, using a linker molecule covalently linked to each of the peptides. The individual peptide of the invention typically consists of at least six, and preferably ten or more amino acid residues. In a complex of the invention, each individual peptide can typically have a length of more than 5 amino acid residues. The present invention also provides a pharmaceutical composition comprising a non-hemolytic cytolytic peptide of the invention as the active agent, and a pharmaceutically acceptable carrier. The compositions are for use in the treatment of diseases or disorders caused by different pathogenic organisms such as Gram-positive and Gram-negative bacteria, viruses, fungi, mycoplasma, and protozoan parasites, for example, Leishmania. that causes leishmaniasis and Plasmodium that causes malaria. In a preferred embodiment, the anti-pathogenic composition is an antimicrobial composition, particularly antibacterial In addition, the compositions of the invention are useful against malignant cells and can be used in the treatment of cancer. A method of treatment comprising administering said non-haemolytic cytolytic peptide to a subject in need thereof is also provided with the present invention. The method of the invention, as well as the above composition, are applicable in both human and veterinary medicine. In addition, the use of said non-haemolytic cytolytic peptide is provided in accordance with the invention in the preparation of a pharmaceutical composition for the treatment of a disease or disorder in a human or a non-human animal, in particular antibacterial compositions. In a further embodiment, the selective peptides of the invention can be used as disinfectants for the destruction of microorganisms, that is, in solution for soaking contact lenses, they can be used as preservatives, for example in the cosmetics or food industry, and as pesticides in agriculture, that is, fungicides and bactericides, or for the conservation of agricultural products, for example fruits and vegetables.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows a spectrum of circular dichroism (DC) of peptides derived from aminoethylaminopardaxine (TApar). The spectra are taken at peptide concentrations of 0.8-2.0 x 10"5 M in 2,2,2-trifluoroethanol (TFE) / 40% water Symbols: TApar (); [D] P7-TApar (); [D] L18L 9-TApar () and [D] P? L18L 9_TApar (-); Figure 2 represents dose-response curves of the hemolytic activity of the TAPAR-derived peptides towards human erythrocytes (EH). The insert shows the results of the test at low concentration. Symbols: Squares in black, melitina; triangles in black, TApar; circles in black, [D] P7-TApar; circles in white, [D] L ^ 8L1 9-TApar; blank squares, [D] p7Ll 8Ll9-TApar; blank triangles, dermaseptine. Figures 3A-B show the maximum dissipation of the vesicle diffusion potential, induced by the TAPAR-derived peptides. The peptides were added to isotonic buffer and free of K + containing small unilamellar vesicles (PVU) composed of egg phosphatidylcholine / phosphatidylserine (FC / FS) (Figure 3A) or FC (Figure 3B), pre-equilibrated with fluorescent dye dS- C2-5 and valinomycin. The fluorescence recovery was measured 10 to 20 minutes after the peptides were mixed with the vesicles. Symbols: triangles in black, TApar; circles in black [D] P7-TApar; blank circles, [D] Ll 8Ll 9-TApar; blank squares, [D] p7L18L 9-TApar; Figures 4A-C show electron micrographs of negatively stained, untreated E. coli cell (Figure 4A) or treated with [D] P7Ll8L 9_tApar, at concentrations lower than the minimum inhibitory concentration (MIC) (4B) or at CIM concentrations (4C); Figure 5 shows DC spectra of melittin and diastereomers derived from melittin. The spectra were taken at peptide concentrations of 0.8-2.0 x 10 ~ 5 M in 40% TFE / water. Symbols: melitina, (); [D] -V5.8? | 17 ?? 21 -me | tina, (); [D] -V5.8, | 17 ^ 21 -.m? | Tina-COOH, (). Figure 6 represents dose-response curves of the hemolytic activity of the diastereomers derived from melittin towards EH. Symbols: circles in black, meltiina; circles in white, [DjV ^ .dj 17? 21 -melitin-COOH; triangles in black, [D] -V5'8, l1 7,? 21 -melitin. Figures 7A-C show electron micrographs of E. coli negatively stained, untreated (Figure 7A) or treated with [D] V5 > 8, | 17,? 21-melittin at concentrations lower than the CIM (7B), or at the MIC concentrations (7C). Figures 8A-B show the maximum dissipation of vesicle diffusion potential, induced by melittin and a diastereomer derived from melittin. The peptides were added to a K + free isotonic buffer containing PVU composed of FC (8A) or FC / FS (8B), pre-equilibrated with the fluorescent dye diS-C2-5 and valinomycin. The fluorescence recovery was measured 10 to 20 minutes after the peptides were mixed with the vesicles. Symbols: circles in black, melitina; triangles in black, [D] V5-8, I17, K21 -melitin. Figures 9A-B show the increase in fluorescence of [D] V5 > 8, I17, K21 -melitine (total concentration of 0.5μM) by titration with FC / FS vesicles (triangles in black) or CF vesicles (triangles in white), with excitation wavelength set at 280 nm and emission at 340 nm. The experiment was carried out at 25 ° C in 50 mM Na2S? 4, 25 mM HEPES-SO4-2, pH 6.8 (Figure 9A); and binding isotherm derived from Figure 9A plotting Xb * (molar ratio of peptide bound by 60% lipid) against Cf (equilibrium concentration of free peptide in the solution) (Figure 9B). Figures 10A-B show the extinction of environmentally sensitive tryptophan by means of brominated phospholipids. Melitin (Figure 10A) and [DjV5-8 i 7.K21 -melitin (10B) were added to the buffer containing FC / FS PVU (1: 1 w / w). The PVUs contained 25% of 6,7-Br-FC (-), or 9,10-Br-FC (), or 11, 12-Br-FC (). After 2 minutes of incubation, an emission spectrum of tryptophan was recorded using the spectrofluorometer with excitation set at 280 nm. For comparison, FCU FS (1: 1 w / w) without Br-FC () was used. Figure 11 shows the effect of the hydrophobic character of the diastereomers of Leu / Lys on the retention time in CLAR-FI. Figure 12 shows dose-response curves of the hemolytic activity of the diastereomers of Leu / Lys towards EH. The insert shows the test results at low concentrations. Symbols: blank squares, melitina; square in black, [D] -L3 > 4'8.10_K3Lg; circles in black, [D] -L3.4 > 8.10_? 4L8; blank triangles, [D] -L3 > .8.10_? 5L7; triangles in black, [D] -L3.4.8. 0-K7L5.

Figures 13A-B show the maximum dissipation of the vesicle diffusion potential, induced by the diastereomers of Leu / Lys. The peptides were added to a K + free isotonic buffer containing PVU composed of FC (Figure 13A) or FE / FG (13B), pre-equilibrated with the fluorescent dye diS-C2-5 and valinomycin. The fluorescence recovery was measured 3 to 10 minutes after the peptides were mixed with the vesicles. Symbols: squares in black, [D] -L3 > 4 > 8.10_K3L9; circles in black, [D] -L3'4'8.10_? 4L8; triangles in black, [D] -L3 '.8.10_? 5L7; crossed circles, [D] -L3 > 4'8 > '' U * -K7L5; Figures 14A-H show electron micrographs of E. coli negatively stained, untreated and treated with the different diastereomers of Leu / Lys at 80% of its MIC. Figure 14A, control; Figure 14B, E. coli treated with [D] -L3.4.8-10-K3L9; Figure 14C, E. coli treated with [D] -L3 '.8.10_? 4L8; Figure 14D, E. coli treated with [D] -L3-4.8'1 0-K5L7; Figure 14E, E. coli treated with [D] -L3'4-8 ^ ° -K7L5; Figure 14F, control; Figure 14G, E. coli treated with [D] - L3,4,8,10_? 4L8; Figure 14H. E. coli treated with [D] -L3.4.8.10_? 5L7.

DETAILED DESCRIPTION OF THE INVENTION The present inventors have found heterogeneous L- base peptides that possess selective cytolytic activity, manifested by selective destruction of pathogenic cells, eg, bacteria, with little or no effect on non-pathogenic cells, i.e., erythrocytes. East discovery is very surprising in view of the prevailing belief in the art that the cytolytic activity of the cytolytic peptides in cells, whether from pathogenic cells such as bacteria or from normal mammalian cells, arises from a single fundamental mechanism associated with the alpha-helix configuration. Functional and structural studies with pardaxin and melitin analogues (two known non-selective cell cytolysins) that have incorporated D-amino acids (diastereomers), carried out to understand the molecular mechanism that underpins cellular selectivity, revealed that the resulting diastereomers do not retain its alhelicoidal structure, which caused the cancellation of its cytotoxic effects on mammalian cells. However, the diastereomers retained a high antibacterial activity, which was excreted by the complete lysis of both Gram-positive and Gram-negative bacteria. In this way, it was shown that the alpha-helical structure of pardaxin and melittin is important for cytotoxicity against mammalian cells, but that is not a prerequisite for antibacterial activity. However, in another study, a single D-amino acid incorporated in the non-haemolytic antibacterial peptide magainin, almost completely abolished its antibacterial activity (Chen et al., 1988). The results obtained here with pardaxin and melitin diastereomers suggest that the hydrophobic character and a positive net charge confer selective antibacterial activity to non-selective cytolytic peptides, and that the antipathetic alpha-helical structure is not required. However, the diastereomers of pardaxin and melittin contained large extensions of L- amino acids (14-17 aa long), which evokes the possibility that the low helical character could be sufficient for the union and destabilization of the membrane. To examine whether the modulation of the hydrophobic character and the net positive charge of linear cytotoxic peptides is sufficient to confer selective antibacterial activity, we chose to investigate short model peptide diastereomers (12 aa in length), compounds of different proportions. of leucine and lysine, and a third of its sequence composed of D-amino acids. The length of the peptide and the position of D-amino acids were such that short peptides were constructed with short consecutive extensions of 1 -3 L-amino acids that can not form an alpha-helical structure. The diastereomers were evaluated for (1) their cytotoxicity against human bacteria and erythrocytes, (2) their structure, and (3) their ability to interact and disturb the morphology of the bacterial wall and membrane phospholipid model. The data show that the modulation of the hydrophobic character and the positive charge is sufficient to confer antibacterial activity and cytolytic selectivity. In addition, the resulting antibacterial peptides act synergistically at non-lethal concentrations with available antibacterial drugs such as tetracycline, and are fully resistant to inactivation of human serum which dramatically reduces the activity of natural antibacterial peptides. Shorter diastereomers (6 aa and 8 aa long) were prepared and analyzed and found to be cytolytic but not haemolytic.

The discovery that certain non-helical cytolytic peptides have an anti-pathogenic activity, paves the way for the preparation of anti-pathogenic agents comprising such non-helical polypeptides. In the case where the non-helical peptides are heterogeneous peptides composed of both L-amino acids and D-amino acids, the anti-pathogenic agents have the additional advantage of being more resistant to degradation, for example by proteases, than the L-amino acids. - homologous peptides, on the one hand, and on the other hand, are not completely resistant to degradation as the completely homogeneous D-peptides. The resistance to degradation can be inconvenient, in view of the slow elimination of the body with the possible associated toxic side effects. Anti-pathogenic non-alpha-helical peptides can be used in a variety of therapeutic methods. Since the homologous D-peptides are known to possess cytolytic activity essentially identical to that of the corresponding homologous L-peptides (Bessalle et al., 1990), it is evident that heterogeneous D- base peptides possess the same anti-pathogenic properties as heterogeneous peptides of base L. The discovery that certain non-alpha-helical peptides have a cytolytic activity against bacteria, without a cytolytic activity against erythrocytes, is a result of the fact that bacterial cells differ from erythrocytes in the composition of their cellular membrane. You can also find differences in the composition of the cell membrane between a variety of pathogenic cells, such as cancer cells, and normal cells. Thus, based on the discovery of the present invention, that certain non-alpha-helical peptides possess a specific cytolytic activity against a cell type, i.e., against bacterial cells, it facilitates the path for the development of a variety of drugs having a selective cytolytic activity against a class of cells within the body, such as bacterial cells, parasite cells, fungal cells, protozoan cells or cancer cells, with little or no activity against normal, non-pathogenic cells of the body. The non-haemolytic cytolytic peptides of the invention, which have a selective cytolytic activity against pathogenic cellsalthough they have no cytolytic activity against normal non-pathogenic cells, or if they have it is very low, they can be used for a variety of therapeutic applications with reduced toxic side effects or without them. A group of cyclic peptides according to the invention is derived from non-alpha-helical heterogeneous peptides derived from homogeneous peptides with an alpha-helical structure, which possess a wide range of cytolytic activity. Thus, the present invention provides in accordance with one embodiment, a heterogeneous cyclic peptide derivative comprising both D- and L- amino acid residues having such a sequence that a homogeneous peptide comprising only amino acid residues only L- or only D - and having the same amino acid sequence as said heterogeneous peptide, has an alpha-helical configuration and has a broad spectrum of cytolytic activity manifested on a variety of cells; said heterogeneous peptide has a cytolytic activity only on some of the cells on which said homogeneous peptide is cytolytically active. For example, a cytoiitic activity of the heterogeneous peptide is manifested only in pathogenic cells, while having no cytolytic activity on normal cells such as erythrocytes. Examples of non-haemolytic cytolytic peptides according to the invention are those derived from natural peptides having an alpha-helical structure and having a cytolytic activity. The non-alpha-helical cyclic peptides of the invention have a sequence that corresponds essentially to the complete or partial sequence of the natural peptide and to which D-amino acids are incorporated along the N- helices and C- of the molecule and have a positive net charge that is achieved by the addition of a positively charged amino acid residue, for example, lysine, arginine, histidine, for example at the N-terminus and / or a positively charged group, example, an aminoalkylamino group such as aminoethylamino, for example at the C-terminus of the molecule, or by neutralization of free carboxyl groups for example, by converting them to amide groups. Examples of such natural peptides are melittin and pardaxin, and fragments thereof. For example, the non-alpha-helical cyclic peptide can be derived from pardaxin, which is a 33-element peptide, or from melittin, which is a 26-element cyclic peptide; the non-alpha-helical cyclic peptide can be a 33-element or 26-element peptide comprising a sequence corresponding to the complete sequence of pardaxin or melittin, respectively, or it may be a non-alpha-helical cyclic peptide having a sequence corresponding to a partial sequence of pardaxin or melitina, for example, melitina sequence of 8-23 elements. In the case of a heterogeneous cyclic peptide derived from pardaxin, the heterogeneous peptide according to the invention may comprise a partial sequence corresponding to that of pardaxin, comprising as little as 10 amino acid residues and varying between 10 and 24 amino acid residues, Another group of peptides according to the invention are non-alpha-helical peptides having a sequence that has no natural homologs, and is composed of at least one hydrophobic amino acid and at least one positively charged amino acid, and in the sequence of which minus one amino acid residue is a D-amino acid. Previous studies with model peptides, used to elucidate the structure-activity study of antibacterial peptides, focused on three parameters: helical structure, hydrophobic character and charge (Anzai et al., 1991; Agawa et al., 1991). Each change in one of these parameters simultaneously caused changes in the other two, making it difficult to clarify the unique contribution of each parameter on the overall antibacterial activity. In accordance with the present invention, the effect of the helical structure was eliminated, which consequently permitted the study of only two parameters, namely the hydrophobic character and the net charge positive, varying the proportion of leucine and lysine. For this purpose, diastereomers of short model peptides (12 aa in length) containing extensions of only 1 -3 L-consecutive amino acids, which are too short to form an alpha-helical structure, were chosen in the investigation. DC spectroscopy revealed that these diastereomers of Leu / Lys are in fact totally lacking alpha-helical structure (data not shown), unlike the melittin and pardaxin diastereomers of the invention, which retain low alpha-helical structure. However, Leu / Lys diastereomers exhibit potent antibacterial activity similar to or greater than that of natural antibacterial peptides such as dermaseptine.

S or the tetracycline antibiotic drug. In addition, the more potent peptides [Dj-L3,4,8,10_ 4L3 and [D] -L3 '.8.10 -.? 5L7 (peptides 23 and 24, respectively of Example 3 hereof) were devoid of hemolytic activity against human erythrocytes highly susceptible to cytolysis. It should be noted that [D] -L3 > 4 < 8'10-- < 3Lg (peptide 22) lacks alpha-helical structure, but has considerable hemolytic activity that approximates that of the natural cytolytic peptide, pardaxin. This could indicate that the balance between hydrophobic character and positive charge compensates for the antipathic alpha-helical structure. However, the increase in the positive charge drastically reduced the hemolytic activity, while the antibacterial activity was preserved, demonstrating that the antipathic alpha-helical structure is not required for the antibacterial activity.

The interaction of Leu / Lys diastereomers with phospholipid membranes, both negatively charged and zwitterionic, was examined to elucidate the basis of their selective cytotoxicity against bacteria. Negatively charged FE / FG vesicles were used to mimic the lipid composition of E. coli (Shaw, 1974) and zwitterionic FC vesicles to mimic the outer leaflet of human erythrocytes (Verkleij et al., 1973). The biological activity of the Leu / Lys peptides on erythrocytes (Figure 12) and E. coli (Table 5) correlates well with their ability to penetrate model membranes. The only peptide that penetrated the CF vesicles was the only peptide with significant hemolytic activity. These results suggest that the phospholipid composition of the bacterial membrane has a role in its penetration by this family of antibacterial peptides. The ability of antibacterial and non-hemolytic peptides to bind and penetrate positively charged phospholipid vesicles, but not zwitterionics, is characteristic of natural antibacterial peptides (Gazit et al., 1994), and has been attributed to the fact that the bacterial surface contains lipopolysaccharides (LPS, in Gram-negative bacteria), and polysaccharides (teicoic acids, in Gram-positive bacteria), and their internal membranes contain phosphatidylglycerol (FG), all of which are negatively charged, while normal eukaryotic cells such as erythrocytes , predominantly express the zwitterionic phospholipid FC on its outer leaflet.

The antibacterial peptide magainin is a non-hemolytic peptide, while the melittin, pardaxin and a peptide model with a sequence similar to that of [D] -L3'4'8'10-K4L8, but composed entirely of L-amino acids, are hemolytic due mainly to its high hydrophobic character. When the alpha-helical structure of magainin is broken by the introduction of three D-amino acids, the resulting diastereomer has no antibacterial activity (Chen et al., 1988), even though its net positive charge is similar to that of magainin. natural. Thus, an optimal balance that already exists between the alpha-helical structure, hydrophobic character and positive net charge of the natural magainin, allows selective antibacterial activity, and any change in one of these properties can cause a lof the antibacterial activity of the magainin. Contrastingly, the hydrophobic character seems to have a greater function in compensating for the lof alpha-helical structure in the melittin, pardaxin and the Leu / Lys diastereomers of the invention. The results according to the invention suggest a new strategy for the design of a repertoire of short, simple and easily manipulated antibacterial peptides. Each of the diastereomeric peptides of Leu / Lys model has a unique spectrum of activity (Table 5). The existence of a repertoire of diastereomeric antibacterial peptides will make it pble to choose the most effective peptide with respect to the target cell. In addition, the simultaneous administration of multiple forms of diastereomeric peptides, acting separately or together, also it has a selective survival value, and provides better protection against a wider range of infectious microbes. All Leu / Lys diastereomers exhibited increased antibacterial activity against Gram-positive bacteria compared to Gram-negative. These results are important, considering the increasing resistance of Gram-positive bacteria such as Staphylococcus aureus, enterococci. and pneumococci to conventional antibiotics (Russell et al., 1995). In addition, unlike the natural antibacterial peptide dermaseptin S, [D] -l_3,4,8,10_Kg | _--, (peptide 24) retained its antibacterial activity in the presence of harvested human serum. Diastereomeric peptides must have several advantages over known antibacterial peptides: (1) Peptides must lack the various pathological and pharmacological effects induced by lytic cytokines of alpha-helix. For example, the staphylococcal delta toxin, the antibacterial peptide alamethicin, the direct lytic factor of the cobra and pardaxin, exert various histopathological effects on several cells, due to pore formation and activation of the arachidonic acid cascade. However, the pardaxin diastereomers do not exert these activities. In addition, many alpha-helical antipathetic peptides bind to calmodulin and evoke several cellular responses, even all D-amino acid alpha-helices, including melittin, have similar activity (Fisher et al., 1994). Dystereomers with cleaved alpha-helical structure are not expected to bind to calmodulin; (2) The local substitution of D- amino acid would result in the controlled elimination of antibacterial peptides by proteolytic enzymes, in contrast to the total protection acquired by the complete substitution of D-amino acids (Wade et al., 1990). The total resistance of a peptide to degradation is inconvenient for its therapeutic use. In addition, the antigenic character of short fragments containing amino acids D, L, is dramatically altered in comparison to their original fully-amino acid molecules L- or D- (Benkirane et al., 1993); (3) The total inhibition of bacterial growth induced by the diastereomers is associated with the total lysis of the bacterial wall, as shown by electron microscopy (Figure 14). Therefore, the bacteria might not easily develop resistance to the drugs that activate said destructive mechanism; (4) [D] - [_3,4,8,10_? < 5 - '7 (peptide 24) has the ability to disturb the cell wall of the bacterium at lower concentrations than its MIC, as seen by electron microscopy (Figure 14). The simultaneous administration of clinically used antibiotics, which have no activity due to their inability to penetrate the bacterial cell wall, together with peptide 24, may present a solution for this resistance mechanism of the bacterium. Next, the invention will be described with reference to some non-limiting drawings and examples.

EXPERIMENTAL PROCEDURES (i) Materials. Butyloxycarbonyl- (amino acid) - (phenylacetamido) -methyl resin was purchased from Applied Biosystems (Foster City, California), and butyloxycarbonyl- (Boc) -amino acids were obtained from Peninsula Laboratories (Belmont, California). Other reagents used for peptide synthesis include trifluoroacetic acid (TFA, Sigma), N, N-diisopropylethylamine (DIEA, Aldrich, distilled on ninhydrin), dicyclohexylcarbodiimide (DCC, Fluka), 1-hydroxybenzotriazole (HOBT, Pierce) and dimethylformamide ( peptide synthesis, Biolab). Egg phosphatidylcholine (FC) and bovine spinal cord phosphatidylserine (FS) (sodium salt grade I) were purchased from Lipid Products (South Nuttfield, United Kingdom). It was purchased phosphatidylglycerol (FG) from egg and phosphatidylethanolamine (FE) (type V, from Escherichia coli) from Sigma. Cholesterol (extrapure) was supplied by Merck (Darmstadt, Germany) and recrystallized twice from ethanol. 3,3'-Diethylthiodicarbocyanine iodide [diS-C2-5] was obtained from Molecular Probes (Eugene, Oregon). Natural methylline from Sigma was purchased. Commercially available melittin contains traces of phospholipase A2, which causes rapid hydrolysis of phospholipids. Therefore, special care was taken to remove all phospholipase A2 from melittin using CLAR-FI. All other reagents were analytical grade. Buffers were prepared in double distilled water. (ii) Synthesis and purification of peptides. Peptides were synthesized by means of a solid phase method on butyloxycarbonyl- (amino acid) - (phenylacetamide) methyl (0.05 meq) (Merrifield et al., 1982). The peptides bound to the resin were separated from the resins by means of hydrogen fluoride (HF), and after evaporation of the HF was extracted with dry ether. These crude peptide preparations contained a higher peak, revealed by CLAR-FI, which was peptide with a purity of 50 to 70% by weight.

The synthesized peptides were further purified by means of CLAR-FI on a C13 Bio-Rad reverse phase semipreparative column (pore size 300A). The column was eluted in 40 minutes, using a linear gradient of 10-60% acetonitrile in water, both containing 0.05% TFA (v / v) at a flow rate of 1.8 ml / min. The purified peptides, which were found to be homogeneous (-95%) according to analytical HPLC, were subjected to amino acid analysis and mass spectrometry to confirm their sequences. (iii) Transamination of the peptides. The peptides bound to the resin as in (ii) above, were transaminated with 30% ethylene diamine in DMF for 3 days, followed by filtration of the resin, precipitation of the protected peptides, ie aminoethylamino (TA) -peptides, with ether and removal of the protective groups with HF. The synthetic TA-peptides were purified (> 95% homogeneity) by means of reverse phase HPLC on a CJ S column using a linear gradient of 25-80% acetonitrile in 0.1% TFA, in 40 minutes, and then He underwent amino acid analysis to confirm its composition. (iv) Amidation of the peptides. The peptide bound to the resin (20 mg) was treated for 3 days with a mixture composed of saturated ammonia (30%) in methanol 1: 1 v / v and DMSO (1: 1 v / v), which resulted in the amidation of the carboxylate group of the glutamine residue located at the C-terminus of [D]? 5.8J ^ 1 -melitin. In this manner, peptides were obtained in which all the protecting groups remained attached, but whose C-terminal residues were modified by an amide group. The methanol and ammonia were evaporated under a stream of nitrogen, and the protected peptides were extracted from the resin with DMSO, and precipitated with dry ether. Then, the products were subjected to HF cleavage and further purification using CLAR-FI as described above. (v) Preparation of lipid vesicles. Small unilamellar vesicles (PVU) were prepared by sound treatment of FC / cholesterol dispersions (10: 1 w / w) or FC / FS (1: 1 w / w). Briefly, dry lipid and cholesterol (10: 1 w / w) were dissolved in a mixture of CHCl3 / MeOH (2: 1 v / v). The solvents were then evaporated under a stream of nitrogen and the lipids (at a concentration of 7.2 mg / ml) were subjected to a vacuum for 1 hour and then resuspended in the appropriate buffer by vortex formation. The resulting dispersions of lipid are • underwent sound treatment for 5-15 minutes in a bath-type sound treatment apparatus (Sonicator G1 125SP1, Laboratory Supplies Company Inc., New York) until clarification. The lipid concentrations of the resulting preparations were determined by phosphorus analysis (Bartlett, 1959). Vesicles were visualized using a JEOL JEM 100B electron microscope (Japan Electron Optics Laboratory Co., Tokyo, Japan), as follows. A drop of vesicles was deposited on a grid covered with carbon and negatively stained with uranyl acetate. Examination of the gratings showed that the vesicles were unilamellar with an average diameter of 20-50 nm (Papahadjopoulos and Miller, 1967). (vi) Preparation of serum. Blood was taken from 5 volunteers and allowed to clot at room temperature for 4 hours. Then, the blood was centrifuged for 15 minutes at 1, 500 x g, and the serum was removed and harvested. The serum complement was inactivated by heating at 56 ° C for 30 minutes. (vii) PC spectroscopy. The DC spectrum of the peptides was measured with a spectropolarimeter, after calibrating the instrument with (+) - 10-camphor sulfonic acid. The spectrum was swept at 23 ° C in a covered quartz optical cell, with a path length of 0.5 nm. Spectra were obtained at wavelengths of 250 to 190 nm. 8 scans were taken for each peptide at a scanning speed of 20 nm / min. Peptides were swept at concentrations of 1.5 x 10 ~ 5 - 2.0 x 10-5 in 40% trifluoroethanol (TFE), a solvent that strongly promotes the alpha-helical structure. Fractional helical characters were calculated (Greenfield and Fasman, 1969; Wu et al., 1981) as follows: [?] 222 - [?] ° 222 [?] '222 [?] ° 222 where [ÚJ222 is, at the average electicity of the residue observed experimentally at 222 nm, and it was estimated that the values for [Ú]? 222 and correspond to 0% and 100% helix content at 222 nm, were 2000 and 32000 grad-cm2 / dmoi, respectively (Wu et al., 1981). (viii) Antibacterial activity of the peptides. The antibacterial activity of the diastereomers was examined in sterile 96-well plates (microtiter plates Nunc F96) in a final volume of 100 μl, as follows: Aliquots (50μl) of a suspension containing bacteria at a concentration of 10 were added. ^ colony forming units (CFU) / ml of LB medium (Lauria broth), to 50 μl of water or 66% of normal human serum collected in PBS, containing the peptide in 2-fold serial dilutions. Growth inhibition was determined by measuring the absorption at 492 nm with an E1309 microplate auto-reader (Bio-tek Instruments), after incubation for 18-20 hours at 37 ° C. The antibacterial activity is expressed as the minimum inhibitory concentration (MIC), the concentration at which 100% inhibition of growth was observed after 18-20 hours of incubation. The bacteria used were Escherichia coli D21, Pseudomonas aeruqinosa ATCC 27853, Acinetobacter calcoaceticus Ac1 1, Salmonella tvphimurium LT2, Bacillus megaterium Bm1 1, Micrococcus luteus ATCC 9341, Bacillus subtilis ATCC 6051. (ix) Hemolysis of human erythrocytes. The peptides were tested for their hemolytic activities against human erythrocytes (EH). Fresh EH, with EDTA, were rinsed 3 times with PBS (35 mM phosphate / 0.15 M NaCl, pH 7.3) by centrifugation for 10 minutes at 800 x g, and resuspended in PBS. The peptides dissolved in PBS were then added to 50 μl of a solution of the EH supply solution to reach a final volume of 100 μl (final erythrocyte concentration, 5% v / v). The resulting suspension was incubated under stirring for 30 minutes at 37 ° C. Then, the samples were centrifuged at 800 x g for 10 minutes. The release of hemoglobin was monitored by measuring the absorption of the supernatant at 540 nm. The controls for zero hemolysis (white) and 100% hemolysis consisted of EH suspended in PBS and 1% Triton, respectively. (x) Visualization of the effects of peptides on bacteria by means of electron microscopy. Samples containing E. coli (106 CFU / ml) were incubated in LB medium, with the different peptides at their MIC, and at a dilution less than the MIC, for 16 hours, and then centrifuged for 10 minutes at 3000 x g. The pellets were resuspended, and one drop containing the bacteria was deposited on a carbon-coated grid, which was then stained negatively with 2% phosphotungstic acid (TFA), pH 6. 8. The gratings were examined using a JEOL JEM 100B electron microscope. (xi) Membrane penetration induced by the peptides. Membrane penetration was determined using the diffusion potential test (Loew et al., 1983; Sims et al., 1974) as described previously (Shai et al., 1991). In a typical experiment, in a glass tube, 4 μl of a liposome suspension (final phospholipid concentration of 33 μM) was diluted in a buffer containing K + (50 mM K2SO4, 25 mM HEPES-SO4-2, pH 6.8 ), in 1 ml of a free K + isotonic buffer (50 mM Na2S? 4, 25 mM HEPES-SO4- "2, pH 6.8), and then the potential-sensitive fluorescent dye, dS-C2, was added. -5. Valinomycin (1 μl of 10"7 M) was added to the suspension to slowly create a negative diffusion potential within the vesicles, which resulted in an extinction of the dye fluorescence. The peptides were added once the fluorescence stabilized, which took 3 to 10 minutes. The subsequent dissipation of the diffusion potential, reflected by an increase in fluorescence, was monitored in a Perkin Elmer LS-50B spectrofluorimeter, with the excitation set at 620 nm, the emission at 670 nm and the gain adjusted to 100%. The percentage of fluorescence recovery, F ^ was defined as: Ft = (it. | 0 / | f - | 0)? i oo where IQ = initial fluorescence, lf = total fluorescence observed before the addition of valinomycin, and \ x = the fluorescence observed after adding the peptide at time t. (xii) Binding of the peptides to vesicles. The interaction between [D] -V5,8 17 | < 21 -melitin and vesicles consisting of negatively charged phospholipids (FC / FS) or zwitterionic (FC), measuring changes in intrinsic tryptophan emission intensity of the peptides in PVU titration experiments. Briefly, PVU was added to a fixed amount of peptide (0.5 μM) dissolved in buffer containing 50 mM Na2S? 4, HEPES-SO42- 25 mM, pH 6.8, at 24 ° C. In all experiments a quartz cuvette with a path length of 1 cm containing a final reaction volume of 2 ml was used. The fluorescence intensity was measured as a function of the molar lipid / peptide ratio (4 separate experiments) in a Perkin-Elmer LS-5 spectrofluorimeter, with excitation set at 280 nm, using a 5 nm slot, and emission set at 340 nm, using a 2.5 nm slot. The binding isotherms were analyzed as a partition equilibrium using the following formula: b = p cf wherein Xβ is defined as the molar ratio of peptide attached (C) to total lipid (C? _), Kp corresponds to the partition coefficient, and Cf represents the equilibrium concentration of the free peptide in solution. For purposes In practice, it is assumed that the peptides were initially divided only on the outer leaflet (60%) of the PVU. Therefore, the partition equation is done: Xb * = p * Cf wherein Xb is defined as the molar ratio of bound peptide per 60% total lipid and Kp * is the estimated surface partition constant. The curve resulting from plotting Xb against free peptide, Cf, is referred to as the conventional binding isotherm. (xiii) Tryptophan extinction experiments. It has previously been used tryptophan which is sensitive to this environment, in combination with brominated phospholipids (Br-FC) to evaluate the location of peptide in the membrane (Bolen and Holloway, 1990, De Kroon et al., 1990). The Br-FC used as extinguishers of the fluorescence of tryptophan, are suitable to probe the insertion of peptides in the membrane, since they act in a short distance and do not drastically disturb the membrane. Melitin and its diastereomer, each of which contained a tryptophan residue (final concentration of 0.5 μM), were added to 2 ml of buffer (50 mM Na2S ?4, HEPES-SO42- 25 mM, pH 6.8) containing 20 μM. μl (50 μM) of PVU with Br-FC / FS (1: 1 w / w), thus establishing a lipid / peptide ratio of 100: 1. After 2 minutes of incubation at room temperature, an emission spectrum of tryptophan was recorded using a Perkin-Elmer LS-50B spectrofluorimeter, with excitation set at 280 nm (8 nm slot). PVUs composed of FC / FS (1: 1 w / w) and containing 25% of 6,7-Br-FC, or 9,10-Br-FC, or 11, 12-Br-FC were used. Three separate experiments were performed for each peptide. In control experiments, PVU with FC / FS (1: 1 w / w) without Br-FC was used.

EXAMPLE 1 SYNTHESIS AND BIOLOGICAL ACTIVITY OF DERIVED DIASTEREOMERS OF PARDAXINE 1. 1 Synthesis. To examine the function of the alpha-helical structure of a polycationic cytolysin in its cytotoxicity towards mammalian cells and bacteria, a series of peptides derived from pardaxin was synthesized, as described in sections (ii) and (iii) of the Procedures Experimental, and were characterized in terms of its structure, hemolytic activity on EH, antibacterial activity and effect on the morphology of the bacteria. Pardaxin (pair) is a 33-element peptide of the following sequence: Gly-Phe-Phe-Ala-Leu-lle-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Thr- Leu-Leu-Ser-Ala-Val-Glv-Ser-Ala-Leu-Ser-Ser-Ser-Gly-Gly-Gln-Glu Modification of pardaxin was made to introduce a positive charge, either by removing the C-acid end of the pardaxin or by converting the C-acid end of the pardaxin, or a fragment thereof, into a positive one by reacting both carboxyl groups of the Glu residue at the C-terminus with ethylenediamine (TA), and / or by adding positively charged amino acid residues, such as Lys, to the N- terminus in pardaxin diastereomers in which the N-helix and / or the C-helix was altered either by replacing the Pro residue in the position 7 of TApar, or of a fragment of pardaxin, with D-Pro (hereinafter indicated by [D] P7), or the two residues Leu in positions 18 and 19 of TApar, or of a fragment of pardaxin, with D -Leu (hereinafter [D] L18L '| 9)? 0 both (hereinafter [D] P L18L "19) OS D-amino acids were introduced at the centers of the N- and C- helices The following pardaxin derivatives were found to be non-hemolytic and exhibit selective cytolytic activity (the residues in bold and underlined they are D-amino acids.) The peptides will be represented by bold numbers in the following: 1. [D] P7L'l 8L'l 9_TApar of the sequence: Gly-Phe-Phe-Ala-Leu-Ile-JPro-Lys-Ile-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val-Gly-Ser-Ala- Leu-Ser-Ser-Ser-Gly-GIy-Gln-Glu- (NH-CH2-CH2-NH2) 2 2. [D] P L18Ll 9 [_22] -Follow the sequence: Gly-Phe-Phe-Ala-Leu-Ue-Pro-Lvs-Ile-Ue-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Lcu-Ser-Ala-Val-NH-CH2-CH2- NH2 l8Ll9 [l-22] -par of the sequence: Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lvs-Ile-Ue-Ser-Ser-Pro-Leu-Phe-Lys-T r-Lcu-Leu-Ser-Ala-Val 7L 8L19 [1-22] -Start of the sequence: Lys-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val-NH-CH2- CH2-NH2 D] P7L18L1 9 [1-22] -Follow the sequence: Lys-Lys-Gly-Phe-Phe-Ala-Leu-Ile-Lys-Ile-Ile-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val-NH-CH2- CH2-NH2 Lys-Lys-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-S-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val . [D] P7- [1-1 1] -Start the sequence: Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-NH-CH2-CH2-NH2 The following pardaxin derivatives were synthesized and found to be hemolytic, as shown in Table 1 below: 8. TApar 9. [D] P 13 -APP 10. [D] L5L19 -APP 11. [D] P7L19 -APP 12. [D] P7-TApar 13. [D] P7-par 14. [D] L18L19 -TApar 15. [D] L ^ L19 -par 16. [D] P7 L? s * Ll9 -par 17. [D] P7 [l-22] -TApar 1. 2 Determination of the secondary structure of the peptides.

Secondary structures of peptides 1, 8, 12, 14 were evaluated from their DC spectrum in 40% TFE, a solvent that strongly promotes an alpha-helical structure, as described in section (vii) of Experimental procedures, and in PBS (35 mM phosphate buffer / 0.15 M NaCl, pH 7.0). Figure 1 shows the DC spectrum of the diastereomers derived from pardaxin, where [8] (), [12] (•• -), [14] (- - -), and [1] (-) . As expected, a dramatic decrease in the alpha-helix content of the peptides was observed as more D-amino acids were incorporated, as reflected by the minimum at 208 and 222 nm in TFE 40%.

There was more than 90% reduction in alpha-helix content between 8 (TApar) (50% alpha-helix) and 1 ([D] P7L18L19-TApar) (4%). The alpha-helix contents of 12 ([D] P7-TApar) and 14 ([D] Ll8Ll 9-TApar) were 25% and 15%, respectively. It should be noted that the proline at position 7 does not introduce a ripple into the structure, but rather participates in the formation of the N-helix as revealed by NMR spectroscopy (Zagorski et al., 1991). In PBS, pardaxin gave a low value of approximately 12% alpha-helix content, while all analogs with D-amino acid residues gave very low signals that could not be attributed to specific structures (data not shown). 1.3 Hemolytic and antibacterial activity. Peptides 1-17 derived from pardaxin were examined for their hemolytic activity towards highly susceptible human erythrocytes, and for their potential to inhibit the growth of different species of bacteria, as described in sections (ix) and ( viii) of the Experimental Procedures, respectively. In addition, the cytotoxic melitin bee venom, the antibacterial peptide dermaseptin S, and the antibiotic tetracycline were used as controls. Figure 2 shows the dose-response curves of the hemolytic activity of peptides 1, 8, 12, and 14. It is shown that the D-amino acids introduced in TApar dramatically reduced their hemolytic activity, which correlates with the loss of content of alpha-helix in the corresponding analogues. Peptide 8, TApar, with the highest alpha-helix content, is the most hemolytic, while peptide 1, [D] P7Ll 8L ^ -TApar, with the lowest alpha-helix content, it practically lacks haemolytic activity up to the maximum tested concentration (50 μM). The inability to lyse EH is characteristic of most naturally occuring antibacterial peptides such as dermaseptin (see Figure 2), magainin and cecropins. Table 1 gives the MIC (in μM) of peptides 1-17 for a representative series of test bacteria, which includes two Gramnegative species, Escherichia coli and Acinetobacter calcoaceticus, and two Gram-positive species, Bacillus meqaterium and Bacillus subtilis, as well as% hemolysis to 50 μM of peptide. Table 2 gives the MIC (in μM) of peptides 1, 8, 12, and 14, and of melittin, dermaseptin S, and tetracycline, for some bacterial species. The data reveal that, despite the dramatic reduction in alpha-helix content and haemolytic activity of the diastereomeric analogs 1-7, all of them retained most of the potent antibacterial activity of the original pardaxin peptide, which is comparable with that of the known natural antibacterial peptides.

TABLE 1 Minimum inhibitory concentration (μM) and hemolytic activity of diastereomers of pardaxin analogues Minimum inhibitory concentration (μM) A. ß% hemolysis E. with M. luteus ohimurium P. aeruginosa Peptide calcoaceticus peptide a (D21) (Ac11) ml (TCC 9341) (LT2) (ATCC 27853) 50 μM 1. 6 6 0.9 12.5 N.D N.D 5 2. 12.5 12.5 2.5 N.Da N.D N.D 0 3. 130 > 130 30 N.D > 130 > 130 0 4. 7.5 7.5 1.5 N.D N.D N.D 0 . 3.5 3.5 0.75 N.D N.D N.D or 6. 15 6 6 N.D 120 60 or 7. > 120 > 120 30 N.D > 120 > 120 o 8. 3 3 0.8 5 15 8 100 9. 3 N.D 1.5 N.D N.D N.D 83 . 3 N.D 1.3 N.D N.D N.D 56 11. 3 N.D 1.5 N.D N.D N.D 82 12. 10 5 1.2 5 N.D N.D 49 13. 30 15 3.5 N.D > 100 > 100 23 14. 3.5 1.5 0.6 2.5 N.D N.D 100 . 15 3.5 1.7 N.D 60 60 44 16. 100 100 50 N.D. > 100 > 100 0 17. 10 ND 1 ND ND ND 17 a-ND, not determined.

TABLE 2 Minimum inhibitory concentration (uM) of the peptides Species Strain 12 14 Bacterial Dermaseptine Melitin S Tetracycline Escherichia coli D21 3 10 3.5 6 5 6 1.5 Acinetobacter Ac11 3 5 2.5 6 calcoaceticus 2 3 1.5 Bacillus megaterium Bm11 0.8 1.2 0.6 0.9 0.3 0.5 1.2 Bacillus subtilis ATCC-6051 1.5 2 1.5 3 0.6 4 6.5 to. The results are the average of 3 independent experiments each performed in duplicate, with standard deviation of 20%. 1 .4 membrane destabilization induced by the pardaxin-derived peptides. A common property of all naturally occurring, alpha-helical, positively charged antibacterial peptides, studied so far, is their ability to interact and penetrate more negatively charged phospholipids, than zwitterionic phospholipids. The relevance of these discoveries to their target biological membranes has been attributed to the fact that the surface of the bacterium contains lipopolysaccharides (LPS, in Gram-negative bacteria), and polysaccharides (teichoic acids, in Gram-negative bacteria), both of which are which are acids, while normal mammalian cells (e.g., erythrocytes) express the phospholipid FC, predominantly zwitterionic, on its outer leaflet. The dissipation of the diffusion potential was tested to determine the membrane penetration activity of the peptides on both phospholipid vesicles, FC and FC / FS (prepared in accordance with section v of the Experimental Procedures), as described in the section xi of the Experimental Procedures. The results shown in Figure 3 for peptides 1, 8, 12, 14 indicate that the D-amino acids introduced into pardaxin do not significantly affect the ability of the peptides to penetrate the phospholipid membranes. However, peptide 1, the only diastereomer lacking haemolytic activity but retaining antibacterial activity, better penetrates negatively charged phospholipids than zwitterionic phospholipids. As such, it behaves similarly to natural antibacterial peptides, although lacks alpha-helical structure. The lack of significant intermediate activities with peptides 12 and 14 can be explained by the fact that both have intact either the hydrophobic N-helix or the antipathetic C-helix, which is sufficient to promote strong binding with both types of vesicles through hydrophobic interactions. 1.5 Visualization of bacterial lysis using electron microscopy. The effect of the pardaxin-derived peptides on the morphology of intact and treated bacteria was visualized, using negative staining electron microscopy, as described in section x. The peptides were added to the bacteria at or below their MIC, under the same conditions used in the antibacterial test (see Example 1.3 above). The samples were taken after 18 hours of incubation and immediately fixed and examined by electron transmission microscopy. Figure 4 shows the photographs obtained with the non-haemolytic analog 1, [D] P7L ^ L ^ 9-TApar, as an example. It was found that at CIM, peptide 1 completely smooth to the bacterium, and only small fragments could be observed (figure 4C). However, at concentrations lower than the MIC, patches were observed on the bacterial wall (Figure 4B).

These patches could indicate the initial step involved in the lithic process.

EXAMPLE 2 SYNTHESIS AND BIOLOGICAL ACTIVITY OF DERIVED DIASTEREOMERS OF MELITINE 2. 1 Synthesis. To further examine the function of the alpha-helical structure of the cytolysins in their cytotoxicity against mammalian cells and bacteria, and to better understand the mechanism underlying this effect, four diastereomers of melittin (mel) were synthesized. Melitin is a peptide with 26 elements of the sequence: Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Lu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-lle-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln- Gln-NHi Modification of the melittin molecule was made to introduce a positive charge, converting the C-acid end of the melittin, or a fragment thereof, into a positive one by reacting the carboxyl group at the C-terminus with ethylenediamine, in diastereomers of melittin in which the N-helix and the C-helix were altered replacing the two Val residues at positions 5 and 8 of melittin, the residue lie at position 17 and the residue Lys at position 21, with D-Val, D-lle and D-Lys, respectively ([D] V5V8I 7K21 herein). It was found that the following diastereomers derived from melittin are not hemolytic and exhibit selective cytolytic activity (the bold and underlined residues are the D-amino acids): 8. [D] V5V8 | 1? 21-mel sequence: Gly-Ile-Glv-Ala-Val-Leu-Lvs-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-I] e-Ser-T -Ile-Lvs-Axg-Lys-Arg- Gln-Gln-NH2 9. [D] V5v8 | 17? 21-mel-COOH of the sequence: Gly-Ile-Gly-Ala-al-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-IIe-Ser-Tf-Ile-Lvs-Arg-Lys-Arg-Gln- Gln-COOH 0. [D] V5V8 | 17? 21- [1-22] -TAmel of the sequence: Glv-Ile-Glv-Ala-Val-Leu-Lvs-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lvs-Arg-NH-CH2-CH2- NH2 1. [D] V5v8 | 17? 21- [4-22] -TAmel of the sequence: Ala-Val-Leu-Lys-Val-Leu-T r-Thr-Gly-Leu-Pro-Ala-Leu-Ile; -Tf-Ile-Lvs-Arg-NH-CH2-CH2-NH2 Then, the peptides 18-21 were characterized with respect to their structure, biological function and interaction with bacteria and model membranes composed of zwitterionic phospholipids or negatively charged phospholipids. 2.2 PC spectroscopy. The magnitude of the alpha-helical structure of peptides 18 and 19 was determined from its DC spectrum at 40% TFE, a solvent that strongly promotes alpha-structure. helical. As expected, the alpha-helical content of the diastereomers was much lower (80% decrease) than that of the melittin, as reflected by the minimum at 208 and 222 nm (Figure 5). The alpha-helix content of melittin was 73%, compared to 15% and 7% of its diastereomers 18 and 19, respectively. 2.3 Antibacterial and hemolytic activity of the melittin diastereomers 18-21. The hemolytic activity of peptides 18-21 against EH was investigated, and its potential to inhibit the growth of different species of bacteria. The antibiotic tetracycline served as a control in the antibacterial test. A dose-response curve was obtained for the hemolytic activity of the peptides (Figure 6). Table 3 gives the MICs for a representative group of bacteria. It can be seen that the introduction of D-amino acids into melittin dramatically reduced its hemolytic activity, which was parallel with the loss of alpha-helical content in the corresponding analogs. Melitin, with the highest alpha-helical content, was the most haemolytic, while, up to the maximum concentration tested (50 μM), peptides 18 and 19, with the lowest alpha-helical content, practically lack hemolytic activity . However, despite the dramatic reduction in hemolytic activity of the melittin diastereomers 18 and 19, both retained most of the potent antibacterial activity of the original peptide. In addition, the antibacterial activity of peptide 19 was only slightly lower than that of 18, which indicates that the amide group at the C-terminus of melittin does not contribute significantly to the antibacterial activity. In contrast, it is known that cecropin, with a free C-carboxylic end, has a significantly lower antibacterial activity than that of cecropin, with a C-amidated end (Li et al., 1988).

TABLE 3 Minimum inhibitory concentration (μM) and hemolytic activity of diastereomers of melitin analogues Dermasepti 6 3 0.5 N.D 4 na S Tetracycline 1.5 1.5 1.2 N.D 6.5 2. 4 Electron microscopy study of bacterial lysis. The effect of peptide 18 on the morphology of intact and treated bacteria was visualized, using electron transmission microscopy. As shown in Figure 7, at CIM, peptide 18 caused total lysis of the bacteria (Figure 7C). However, at concentrations lower than the MIC, patches were observed on the bacterial wall (Figure 7B). These patches could represent an initial step in the lithic process. 2. 5 Mode of interaction with phospholipid membranes. Since the biological activities of peptides 18 and 19 were similar, only the mode of action of peptide 18 on model phospholipid membranes was compared with that of melittin, in order to elucidate the basis of the observed membrane selectivity. For that purpose, the ability of the peptides to dissipate the diffusion potential created in both vesicles, with FC and with FC / FS was measured, and the partition coefficients of the peptides with both types of vesicles were determined, and the location of the peptide when it is attached to the membranes. 2.5.1 Penetration of membrane induced by peptides. Several concentrations of melittin and peptide 18 were mixed with vesicles that had been pretreated with the fluorescent dye diS-C2-5 and valinomycin. The kinetics of the fluorescence recovery was monitored over time, and the maximum level reached as a function of the peptide concentration was determined. As shown in Figure 8, both melittin and peptide 18 had similar membrane penetration activity with FC / FS vesicles, which showed that the introduction of D-amino acids into melittin does not affect the capacity of the resulting diastereomer to penetrate negatively charged phospholipid membranes (FS / FC). However, although melitin was also highly active with FC vesicles, the diastereomer totally lacked membrane penetration activity with CF vesicles (up to the maximum concentration tested). 2. 5.2 Studies of union. The inability of the diastereomer 18 to penetrate the CF vesicles may be due to its inability to bind to FC, or alternatively, it may bind to the CF vesicles, but once attached it can not be organized into structures that induce membrane disruption. To differentiate between these two possibilities, a union study was carried out. The individual Trp residue at position 19 of peptide 18 was used as an intrinsic fluorescence probe to track its binding to FC and FC / FS vesicles. A fixed concentration (approximately 0.5 μM) of the peptide with the desired vesicles (FC or FC / FS) was titrated, and an increase in fluorescence intensity was observed if binding occurred. By plotting the resulting increases in the fluorescence intensities of Trp as a function of the lipid: peptide molar ratios, conventional binding curves were obtained (Figure 9A). The binding curve of peptide 18 with FC / FS reveals that almost all peptide molecules bound to the vesicles at a molar lipid: peptide ratio of 100: 1. However, with a CF vesicle, no net increase in Trp fluorescence was observed, even with the maximum molar ratio lipid: peptide tested, which indicates that the peptide does not bind to the CF vesicles. Binding isotherms were constructed by plotting X * b (the molar ratio of peptide bound by 60% of the total lipid) against Cf (the equilibrium concentration of the free peptide in the solution) (Figure 5B). The surface partition coefficients were estimated, extrapolating the initial slopes of the curves to Cf values of zero. The estimated surface partition coefficient, Kp *, of peptide 18, was 1.1 ± 0.2 x 10 ^ M " 1 (obtained from 4 measurements). The value is similar to the value reported for the binding of melittin to phosphatidylglycerol / phosphatidylcholine (4.5 ± 0.6 x 1? 4 M "1) (Beschiaschvili and Seelig, 1990) The shape of the peptide binding isotherm can provide information on the organization of the peptide within the membranes (Schwarz et al., 1987) The binding isotherm of peptide 18 slopes down indicating a negative cooperativity.A possible explanation for this negative cooperativity is that at low concentration, the binding of peptide 18 a FS / FC is reinforced by the negative charge of the phospholipid head groups, as compared to the partition equilibrium without loading effect.

In addition, after its binding to the membrane, the peptide partially neutralizes the negative charge on the surface of the membrane. However, once the surface loading of the membrane is neutralized, the additional binding of peptide 18 is difficult, since the repulsion of similar charges becomes the dominant factor. Similar results were obtained in melitin binding studies to negatively charged phospholipid membranes (Batenburg et al., 1987, Beschiaschvili and Seelig, 1990). Interestingly, unlike melittin that binds strongly also to CF vesicles (Kuchinka and Seelig, 1989), peptide 18 does not bind to CF vesicles. 2.6 Experiments of tryptophan extinction. A tryptophan residue naturally present in the sequence of a protein or a peptide, can serve as an intrinsic probe for the localization of the peptide within a membrane. Melitin contains a residue of tryptophan at position 19, the N-terminal side of the C-helix. With both, melittin and peptide 18, the greatest fluorescence extinction of tryptophan with 6,7-Br-FC / FS vesicles was observed (FIG. 10). Less extinction was observed with 9,10-Br-FC / FS, and the least with 1 1, 12-Br-FC / FS. These results indicate that upon binding to the vesicles, the peptides were located near the head groups of the phospholipids.

EXAMPLE 3 SYNTHESIS AND BIOLOGICAL ACTIVITY OF DIASTEREOMEROS MODEL OF LYS / LEU 3. 1 Design of diastereomers of Lys / Leu. Six diastereomers of 12-element model linear short peptides, compounds of varying proportions of lysine and leucine, were synthesized to (1) examine whether a balance between hydrophobicity and a positive net charge may be a sufficient criterion necessary for bacterial lysis selective, and (2) understand the mechanism that sustains this effect. In the first series of 12-element Lys / Leu model peptides, 22-25, one third of their sequence was composed of D-amino acid residues. The location of the D-amino acids remained constant in all the peptides that were constructed for maximum breaking of the alpha-helical structure. The D-amino acids were distributed throughout the peptide, leaving only very short extensions of 1 to 3 consecutive L-amino acids. The following peptides were synthesized: 22. [D] L3'4-8-10-K3L9 of the sequence: Lys-Leu-eu ^ Leu-Leu-Leu-Lys-Leu-Leu-Leu-Leu-Lvs-NH ^ 23. [D] L3'4 > 8'10-K4L8 of the sequence: Lys-Leu-Leu-Leu-Lys-Leu-Leu-eu-Lvs-Leu-Leu-Lvs-NH? 24. [D] L3'4.8-10-K5L7 of the sequence: Lys-Leu-Leu-Leu-Lys-Leu-Lys-Leu -Lvs-Leu-Leu-I .ys-NH.

. [D] L3,4,8,10.? 7L5 of the sequence: Lys-Lys-Leu-Leu-Lys-Leu-Lv .'.- Leu-Lvs-Leu-Lvs-Lys-NH? In the second series of 12-element Lys / Leu model peptides, 26-27, two-thirds of their sequence was composed of D-amino acid residues, at the exact positions of the L-amino acid residues of peptides 23 and 24 , as follows: 26. [D] K1 -5,9,12 2,6,7,1 1 _K4L8 of the sequence: Lvs-Leu-Leu-Leu-Lvs-Lcu-Lcu-Leu -T .vs-í .eu-Leu-Lvs-NH2 27. [D] Kl .5.7,9.12L2,6,11_? 5L7 of the sequence: Lvs-Leu-Leu-Leu-Lvs-Leu-Lvs-Leu-Lvs-Leu-Leu-Lvs-NH2 In a third series of Lys / Leu model peptides, a diastereomer of 6 elements and one of 8 elements (peptides 28 and 29, respectively) was synthesized. 28. [D] L1 '3K2L4, of the sequence: Lys-Leu-Leu- Leu -Leu-Lys 29. of the sequence: Lvs-Leu-Leu-Leu - Lvs-Leu-Leu-Lys Additional diastereomers of Lys / Leu were synthesized according to the invention: . Lys Leu Leu Leu Lvs Leu Lys Leu Lys Leu Leu Lys 31. Lys Leu Leu Leu Lys Leu Lys Leu Lvs Leu Leu Lys 32. Lys Leu Leu Leu Lvs Leu Lvs Leu Lvs Leu Leu Lys 3. 2 Synthesis of diastereomers of Lys / Leu. The peptides were synthesized as described in section (ii) of the Experimental Procedures. Afterwards, the peptides were characterized with respect to their structure, biological function and interaction with bacteria and with membranes model composed of zwitterionic phospholipids or negatively charged phospholipids. 3.3 Hydrophobic character. Table 4 lists the hydrophobic lacks and the positive net charges of the peptides 22-25. Mean values of hydrophobic character were calculated using consensus value of a hydrophobic scale (Eisenberg et al., 1984). As shown in Figure 11, a direct correlation was found between the hydrophobic character and the retention time of the peptides, suggesting that the structure does not contribute significantly to global hydrophobic interactions with the stationary phase.

TABLE 4 Hydrophobic character and net charge of the diastereomers of Leu / Lvs Peptide designation Net charge Hydrophobic character 22. +4 0.12 23. +5 -0.01 24. +6 -0.15 25. +8 -0.42 3. 4 PC spectroscopy. The magnitude of the alpha-helical structure of the diastereomers 22-25 was determined from their DC spectrum in 40% TFE. As expected, after the incorporation of D-amino acids, no signal was observed for any of the diastereomers, demonstrating the lack of any specific secondary structure (data not shown). It is to be noted that in a recent study, it was found that a peptide with a sequence identical to that of peptide 23, but composed only of L-amino acids, had approximately 40% alpha-helical structure in methanol and in DMPC vesicles (Cornut et al., 1994). 3.5 Antibacterial and hemolytic activity of peptides 22-29. The hemolytic activity of peptides 22-29 against EH was tested. Figure 12 shows a dose-response curve for the hemolytic activity of peptides 22-25, where the hemolytic activity of melittin served as control. A direct correlation was found between the hydrophobic character (Table 4) and the hemolytic activity of the diastereomers. Peptide 22, [D] L3'4'8'1 0K3Lg, which had the highest hydrophobic character, was the most haemolytic peptide. However, its haemolytic activity is very low compared to melittin (> 60 times less activity). All other peptides showed no significant haemolytic activity up to the maximum tested concentration (100 μM). Table 5 shows the hemolytic activity of the peptides 22-29. It should be noted that although peptide 23, [D] L3-4.8.10? I | _gj is not hemolytic at concentrations of > 100 times the required melittin for significant hemolysis, it has been seen in a recent study that its completely L-amino acid form has haemolytic activity similar to that of melittin (approximately 5 times less) (Cornut et al., 1994). Peptides 22-29 were also tested for their antibacterial activity against a representative group of bacteria, where tetracycline, dermaseptin S and melittin served as controls. The resulting CIM are shown in table 5. The data show that the antibacterial activity of the diastereomers 22-29 was modulated by the balance between hydrophobic character and positively charged amino acids. Both the more hydrophobic peptide, 22, and the more hydrophilic peptide, 25, exhibited the lowest scale of antibacterial activity (Table 5). However, peptides 23 and 24 exhibited high antibacterial activity against most of the tested bacteria, the latter being slightly more potent. further, each peptide had a unique spectrum of antibacterial activity, and each of them was more active against Gram-positive than Gram-negative bacteria. 3.6 Synergistic effects between tetracycline and the diastereomers of Lvs / Leu in serum. To investigate a possible synergistic relationship between the antibiotic tetracycline and the diastereomers, tetracycline was tested in 2-fold serial dilutions against Pseudomonas aeruqinosa (ATCC 27853) in the presence of a constant equimolar concentration (1 μM) of peptide 24, [D] L3 >; 4 '-' '0K5L7. The antibacterial activity of the mixtures was determined as described in section (xii) of the Experimental Procedures. A synergistic effect was observed between tetracycline and diastereomer 24. Tetracycline shows little activity against P. aeruginosa. However, when mixed with 1 μM solution of peptide 24, a concentration that is 10 times lower than that required for lytic activity against P. aeruginosa, an eightfold increase in tetracycline activity was observed (Table 6) . A possible explanation of the effect synergistic, is that the peptide slightly breaks the bacterial wall, which improves the separation of tetracycline towards the bacterium. It is supported by electron microscopy studies that show that below its MIC, peptide 24 causes morphological changes in the bacterial wall (Figure 14). Furthermore, it was found that the effect of human serum collected on the antibacterial activity of peptide 24 and the natural antibacterial peptide dermaseptin against P. aeruginosa and E. coli, differs considerably (Table 6). Although dermaseptin was 8-10 times less active in the presence of serum, peptide 24 retained its antibacterial activity.

TABLE 5 Minimum inhibitory concentration (μM) of the peptides 23 3.5 4 10 0.4 0.5 0 24 7 20 10 0.25 2 0 25 80 200 > 200 1 100 0 26 4 N.D N.D 0.5 N.D 0 27 7 N.D N.D 0.2 N.D 28 200 N.D N.D 50 N.D 29 3 N.D N.D 3 N.D Dermasepti 6 3 25 0.5 na S Metin 5 2 25 0.3 0.6 Tetracycline 1.5 1.5 50 1.2 6.5 to. The results are the average of 3 independent experiments each performed in duplicate, with standard deviation of 20%.

TABLE 6 Minimum inhibitory concentration (μM) a in the presence of human serum and synergistic activity of peptide 24 Minimum Inhibitory Concentration (μM) P. aeruginosa (ATCC-27853) E. coli (D21) Designation of 0% of serum 33% of 0% of 33% of peptide serum serum serum 24 10 10 7 7 Dermaseptin S 25 200 6 50 Tcb 50 Tc + 24 (1 μM) 6 to. The results are the average of 3 independent experiments each performed in duplicate, with standard deviation of 20%. b. Tc-tetracycline. 3.7 Penetration of membrane induced by peptide. Several concentrations of peptides were mixed with vesicles that had been pretreated with the fluorescent dye dS-C2-5, and valinomycin. The kinetics of the fluorescence recovery were monitored and the maximum fluorescence level was determined as a function of the peptide concentration (Figure 13). FC / cholesterol vesicles (10: 1) served as a model of the phospholipid composition of the outer erythrocyte leaflet (Verkleij et al., 1973), and FE / FG vesicles (7: 3) were used to mimic the composition of E. coli phospholipids (Shaw, 1974). A direct correlation was found between the potential of the peptides to penetrate model phospholipid membranes and their lytic activity against erythrocytes and E. coli. Only the hemolytic peptide 22 penetrated the zwitterionic phospholipid vesicles. In addition, the ability of the peptides to penetrate FE / FG vesicles correlates with the antibacterial activity of the peptides against E. coli (Table 5). Peptide 25, which had the lowest antibacterial activity, also had significantly reduced ability to penetrate FE / FG vesicles compared to the other three peptides, 22-24. 3.8 Study of electron microscopy of bacterial lysis. The effect of diastereomers 22-25 on the morphology of treated E. coli was visualized, using electron transmission microscopy. All the peptides caused total lysis of the bacteria at the MIC (data not shown). However, when the peptides were used at concentrations corresponding to 80% of their MIC, some differences in the morphology of the treated bacteria were observed, depending on the peptide used. The most hydrophobic peptide, 22, caused the greatest damage to the cell wall and membrane, while the less hydrophobic peptide, 25, only caused local perturbations (Figure 14).

EXAMPLE 4 SYNTHESIS AND BIOLOGICAL ACTIVITY OF MODEL DIASTEREOMERS DE LYS / ALA AND LYS / VAL 4. 1 Design of the diastereomer. To further examine whether the modulation of the hydrophobic character and the net positive charge of the linear cytotoxic peptides is sufficient to confer selective antibacterial activity, two additional 12-element model peptides, 33 and 34-37, composed of Lys / Ala residues were synthesized. or Lys? / al, respectively, with at least one third of their sequences being D-Ala or D-Val residues: 33. [D] A3-4-8'10K4A8 of the sequence: Lvs-Ala-Ala-Ala-Lys-Ala-Ala-Ala-Lys-Aia-Ala-Lys-NH2 34. [D] V3-4-8.1 ° K4V8 of the sequence: Lvs-Val-Val-Val-Lys-Val-Val-y_alrLys-Val-Val-Lys-NH2 . Lys Val Val Val Lys Val Val Val Val Val Val Val Val Val Val Val Val Val Val Lys 37 Val Val Val Val Val Val Val Val Val Val Val Val. Val Val Lys 4. 2 Synthesis. The diastereomers Lys / Ala and Lys / Val were synthesized as described in section (ii) of the Experimental Procedures. 4. 3 Antibacterial and hemolytic activity. Peptides 33 and 34 were tested against E. coli and B. meqaterium and hRBC. The results in table 7 show that both model diastereomers are antibacterial and non-hemolytic: TABLE 7 Minimum inhibitory concentration (uM) and hemolytic activity of peptides 28 and 29 Minimum Inhibitory Concentration (uM) Designation of E. coli B. megaterium% of peptide hemolysis (D21) (Bm1 1) at 100 μM 33 12 1 0 34 3.5 0.8 0 EXAMPLE 5 SYNTHESIS OF ADDITIONAL MODEL DIASTEREOMERS The following model diastereomers according to the invention were synthesized, consisting of sequences of 6, 8, 12, 14, 16, 19, 25, 26 and 30 residues of two, three or more different amino acids: 38. Lvs Leu He Leu Lys Leu 39. Lys Val Leu His Leu Leu 40. Leu Lvs Leu Arg Leu Leu 41. Lys Pro Leu His Leu Leu 42. Lys Leu He Leu Lvs Leu Val Arg 43. Lys Val Phe His Leu Leu His Leu 44. His Lvs Phe Arg He Leu Lvs Leu 45. Lys Pro Phe His lie Leu_His Leu 46. Lvs He He He Lys Jle Lys Jle Lys He He Lys 47. Lys lie He He Lys He Lys Jle Lvs He He Lys 48. Lvs He He He Lvs Lys He Lys JL Lvs He Jje Lys 49. Lys He Pro He Lys He Lvs He Lys He Pro Lys 50. Lys He Pro He Lys Jle Lys He Lys JJc Val Lys 51. Arg He He He Arg lie Arg Jle Arg He Jle Arg 52. Arg lie He He Arg He Arg He Arg He lie Arg 53. Arg He Jle He Arg He Arg Jle Arg He Jle Arg 54. Arg He Val He Arg Jle Arg Jle Arg Leu Jle Arg 55 Arg He He Val Arg He Arg Leu Arg He Jle Arg 56. Arg He Glv He Arg Leu Arg Val Arg He He Arg 57. Lys Jle Val He Arg Jle Arg Jle Arg Leu Jle Arg 58. Arg lie Wing Val Lys Tf Arg Leu Arg Phe He Lys 59. Lys He Glv Trp Lvs Leu Arg Val Arg_ He JJe Arg 60. Lvs Lvs He Glv Tf Leu Jle He Arg Val Arg Arg 1. Arg He Val He A g Jle Arg Jle Arg Leu Jle Arg Jle Arg 2. Arg He He Val Arg He Arg Leu Arg Jle Jle Arg Val Arg 3. Arg He Glv He Arg Leu Arg Val Arg He He Arg Arg Val 4. Lvs He Val He Arg He Arg He Arg Leu He Arg He Arg He Arg 5. A-rg He He Val Lys He Arg Leu Arg He He Lvs Lvs He Arg Leu 6. Lvs He Glv He Lvs Wing Arg Val Arg He He Arg Val Lvs lie He 7. Arg He He Val His He Arg Leu Arg Jle lie His His He Arg Leu 8. His He GJv He vs. Ala His Val Arg lle Jle Arg Vaj JHs_Ile JJe 69. Arg He Tvr Val Lys He His Leu Arg Tyr He Lvs Lvs He Arg Leu 70. Lvs He G] and His Lvs Aia Arg Val Jíis.Ile Jle Arg Tvr JLvs le JJe 71. Arg He Tyr Val Lys Pro His Pro Arg Tyr He Lys Lvs He Arg Leu 72. Lys Pro Glv His J ^ vs Wing Arg Pro Jis_lleJle Arg Tyr Lvs He He 73. Lys Jle Val He Arg He Arg He Arg Leu Jle Arg He Arg He Arg Lys He Val 74. Arg He He Val Lys He Arg Leu Arg He He Lvs Lvs He Arg Leu He Lys Lvs 75. Lys He Glv Trp Lvs Leu Arg Val Arg He He Arg Val Lvs He Glv Arg Leu Arg 76. Lys Jle Val He Arg He Arg He Arg Leu He Arg He Arg He Arg Lys He Val Lvs Val Lys Arg JJe Arg 77. Arg Phe Wing Val Lvs He Arg Leu Arg Jle Jle Lys Lvs He Arg Leu He Lys Lvs He Arg Lvs Arg Val He Lys 78. Lys Wing Glv Trp Lvs Leu Arg Val Arg He He Arg Val Lvs He Glv Arg Leu Arg Lys He Glv Trp Lvs Lys Arg Val Arg He Lvs 79. Arg lie Tyr Val Lvs Pro His Pro Arg Tyr He Lys Lvs He Arg Leu 80. Lvs Pro Glv His Lvs Wing Arg Pro His He He Arg TVr Lvs He Jle 81. Lys He Val He Arg He Arg He Arg Leu He Arg He Arg He Arg Lys He Val 82. Arg He He Val Lvs He Arg Leu Arg He Jle Lys Lvs He Arg Leu He Lys Lvs 83. Arg He Tyr Val Ser Lvs He Ser He Tyr He Lvs Lvs He Arg Leu 84. Lys He Val He Phe Thr Arg He Arg Leu Thr Ser He Arg He Arg Ser He Val 85. Lvs Pro He His Lvs Wing Arg Pro Thr He He Arg Tyr Lvs Met lie EXAMPLE 6 SYNTHESIS AND BIOLOGICAL ACTIVITY OF CYCLIC DIASTEREOMEROS 6. 1 Design The following cyclic derivatives of diastereomers of pardaxin fragments were synthesized, with cysteine residues at both the N- and C-terminus: 86. K [D] P7L18L 9 [1-22] -par cyclic, of the sequence: 87. K1K2 [D] P7L18Ll9 [1-22] -par cyclic, of the sequence: Cys-Lys-Lys-Gly-Phe-Phe-Ala-Leu-Ile ^ Prp-Lys-IIe-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Tl-r-jLeu ^ Leu-Ser-Ala- Val-Cvs 88. K? 2? 3 [D] P7L 8L19 [1-22] -par cyclic, of the sequence: Cys-Lys-Lys-Lys-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lvs-Ile-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-Ser-Ala Val-Cy The following cyclic derivatives of diastereomers of different amino acid residues were synthesized, with cysteine residues at both the N- and C-terminus: 89. Cyclic CysArgHeVallleArglIeArgNeArgLeulteArglIeArgCys 90. CvsLvsProGIvHisLvsAlaArgProHislleHeArqTyrLysIleíleCys Cyclic 91. CvsArqPheAlaValLvslleArgLeuArglIelleLvsLvslleArgLeulleLys LvslleArgLvsArgVallleLvsCvs Cyclic 92. Cys Lys Leu Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Cys cyclical 93. Cys Lys Leu Leu Leu Lys Leu Lys Leu Lys Leu Leu Lys Cyclic Cys. The following cyclic derivatives of diastereomers of different amino acid residues were synthesized without cysteine residues at the N and C termini: 94. HN-Lys Leu Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys - CO 95. HN-Lys Leu Leu Leu Lys Leu Lys Leu Lys Leu Leu Lys - CO 6. 2 Synthesis of the cyclic diastereomers. The cyclic peptides were synthesized by a solid phase method as described in the experimental procedures, section (ii), with and without cysteine residues at the N and C termini of the peptides. The cyclisation without cysteine was carried out by N-terminal protection, C-terminal activation, N-end deprotection and reaction of the terminal C and N groups while still bound to the resin. After cleavage with HF and purification with CLAR-FI, the peptides were solubilized at low concentration in PBS (pH 7.3), and the cyclization was completed after 12 hours. The cyclic peptides were further purified in CLAR-FI and subjected to amino acid analysis to confirm their composition and to SDS-PAGE to confirm their monomeric state. 6.3 Antibacterial and hemolytic activity. Peptides 86-88 were tested against E. coli and B. megaterium and EH. The results in table 8 show that the three cyclic diastereomers derived from pardaxin are antibacterial and non-hemolytic: TABLE 8 Minimum inhibitory concentration (μM) and hemolytic activity of the cyclic diastereomers derived from pardaxin Minimal Inhibitory Concentration (μM) Designation of E. coli B. megaterium% peptide hemolysis at 100 μM (D21) (Bm11) 86 30 10 0 87 15 6 0 88 7.5 2 0 6. 4 Antibacterial and hemolytic activity. Peptides 92 to 95 were tested against E. coli, B. subtilis and P. aeruginosa. The results in Table 8a show that the four cyclic diastereomers are antibacterial and non-hemolytic: TABLE 8a Minimum inhibitory concentration (μM) and hemolytic activity of the cyclic diastereomers Minimum inhibitory concentration (μM) Designation of E. coli B. subtilis P. aeruginosa% of peptide hemolysis at 50 μM 92 12.5 1.2 25 0 93 15 5 25 0 94 12.5 1.5 30 0 95 15 6 20 0 EXAMPLE 7 SYNTHESIS AND BIOLOGICAL ACTIVITY OF DIASTEREOMERS OF PEPTIDOS DE Lys / Leu GROUPED 7. 1 Design Using as peptide 23 template and as monomers peptide 23 or 24, with an additional cysteine residue at the C-terminus (23C and 24C, respectively), the following group sequences were produced: 96.

[D] L3 '.8.10K4L8 of the sequence: (Lys-Leu-Leu-Leu-Lvs-Leu-Leu-Leu-Lys-Leu-Leu-Lvs-Cys-NH?) S Lys-Leu-Le Leu-Lys-Leu-Leu-Leu -Lys-Leu Leu-Lys-NH? 97. ([D] L3A8. 0? 5L7C) 5 [D] L3. .8.10? 4L9 of the sequence: (Lys-Leu-I ^ eu ^ Leu-Lys-Leu-Lys-JLeu-Lvs-Leu-Leu-Lvs-Cvs-NH -.) ^ Lvs-Leu-Leu Leu-Lys-Leu-Leu-Leu -Lvs -Leu-Leu-T .vs-NT- 7. 2 Synthesis. To produce diastereomers attached to the template, a molar ratio of 1: 1 DCC and bromoacetic acid in DMSO was allowed to react at 25 ° C for 1 hour. The template (peptide 23) was added to the reaction mixture and left under stirring for 12 hours, after which the DMSO was lyophilized. The remaining bromoacetic acid was extracted with dry ether. Then, the mold was reacted with excess diastereomers 23C and 24C with cysteine residues at its C- end, in PBS pH 7.3, at 25 ° C for 1 hour. The template-bound diastereomers, 96 and 97, were further purified in CLAR-FI and examined in SDS-PAGE to confirm their aggregation status. 7.3 Antibacterial and hemolytic activity. The diastereomers attached to template 96 and 97 were tested against E. coli and B. meqaterium and hRBC. The results in Table 9 show that both group sequences are antibacterial and non-hemolytic.

TABLE 9 Minimum inhibitory concentration (uM) and hemolytic activity of the groups Minimum Inhibitory Concentration (μM) Designation of E. coli B. megaterium% of hemolysis peptide (D21) (Bm11) at 100 μM 96 0.2 0.05 0 97 0.1 0.02 00 EXAMPLE 8 SYNTHESIS AND BIOLOGICAL ACTIVITY OF MIXTURES OF DIASTEREOMEROS OF PEPTIDES OF 12 ELEMENTS OF Lvs / Leu HYDROSOLUBLES Peptides were synthesized by a solid phase method as described above in section (i) of the Experimental Procedures. In each coupling step, a mixture composed of 1 equivalent of each of lysine, leucine and D-leucine was added to the reaction. The synthesis resulted in a mixture of 31 different peptides. After cleavage with HF, water-soluble peptides were extracted with double distilled water (add) and lyophilized. The water-soluble mixture of the diastereomers of 12-element Lys / Leu peptides was tested against E. coli D21 (CIM: 15 μg / ml) and E ^ meqaterium Bm1 1 (MIC: 3 μg / ml) and EH (0 % hemolysis at 100 μM). As expected, the water soluble mixture had antibacterial activity but was not haemolytic.

EXAMPLE 9 SYNTHESIS AND BIOLOGICAL ACTIVITY OF RANDOM COPOLYMERS DE Lvs / Leu / D-Leu To produce diastereomers of polymers of different sizes, an excess of N-carboxyan hydride residues was allowed to polymerize on free amino acid initiators in DMF, at 25 ° C, for 4 hours (Katchalski and Sela, 1958). Polymers were produced consisting of different ratios of lysine, leucine and D-leucine, using different ratios of lysine-N-carboxyanhydride, leucine-N-carboxyanhydride and D-leucine-N-carboxyanhydride. Table 10 shows three of these polymers and their antibacterial and hemolytic activities.

TABLE 10 Minimum inhibitory concentration (uM) and hemolytic activity of Lvs / Leu / D-Leu copolymers Minimum Inhibitory Concentration (μg / ml) Relationship (molar) of E. coli B. megaterium% of amino acids (D21) (Bm11) hemolysis Lys: Leu: [D] -Leu at 100 μM 1 1: 1 90 15 0 2 1: 1 35 8 0 3 1: 1 80 20 0 EXAMPLE 10 ANTIMICTIC ACTIVITY OF DIASTEREOMERS The antifungal activity of the peptides derived from pardaxin 1 and 16 (see example 1 above) in sterile 96-well plates was examined (Nunc F96 microtiter plates) in a final volume of 100 μl, as follows: Fifty microliters of a supersension containing fungi at a concentration of 1 X 106 Colony Forming Units (CFU) / ml in culture medium (Sabouraud glucose broth medium), was added 50 μl of water containing the peptide in dilutions in 2 times in water series.

The inhibition of growth was determined by measuring the absorption at 492 nm with an E1309 Microplate auto-reader (Bio-tek Instruments), after an incubation time of 48 hours at 30 ° C. Activities were expressed as the minimum inhibitory concentration (MIC), the concentration at which 100% inhibition of growth was observed after 48 hours of incubation. The fungi used were: Candida albicans (IP886-65) and Crvptococcus neoformans (IP960-67). As shown in table 11, both peptides, 1 and 16, showed antifungal activity.

TABLE 11 Minimum inhibitory concentration (μM) of diastereomers 1 and 16 against fungi Minimum Inhibitory Concentration (μM) Designation of the peptide Candida albicans Cryptococcusneoform (IP886-65) ans (IP960-67) 1 35 50 16 120 150 EXAMPLE 11 ANTICANCEROUS ACTIVITY OF DIASTEREOMEROS The anticancer activity of the diastereomers of Lys / Leu 23 and 24 (see example 3 above) was examined against mouse adenocarcinoma. Cells were seeded at 5-10,000 / well in 96-well microtiter plates in Dulbecco's modified Eagle's medium. After the cells are bound, 20 μl of diluted peptide solution in normal saline was transferred to the well, to give final concentrations ranging from 20 to 150 μM. After 1 hour of incubation with the peptides, the viability of the cancer cells was measured by means of the Trypan blue vital staining test (0.1% w / v). In control experiments, only the peptide solvent was added to the cells. The anti-cancer activities are expressed as the minimum inhibitory concentration (MIC), the concentration at which 100% inhibition of growth was observed after 1 hour of incubation. The results in table 12 show that both peptides are active against malignant cells.

TABLE 12 Minimum inhibitory concentration (μM) of the diastereomers against mouse adenocarcinoma Minimum Inhibitory Concentration (μM) Designation of mouse Adenocarcinoma peptide 23 50 24 80 EXAMPLE 12 ACTIVITY OF DIASTEREOMERS AGAINST Leishmania mexicana The diastereomer peptide derived from melittin (see example 2 above) and the diastereomer peptide 23 from Leu / Lys (see example 3 above) against Leishmania were tested. Promastigotes of the NR strain of Leishmania mexicana were cultured for testing, at 27 ° C in RPMI 1640 medium supplemented with 10% fetal bovine serum. The parasites were harvested by centrifugation at 1200 x g for 10 minutes at 4 ° C, and washed twice with PBS (50 mM sodium phosphate, 150 mM NaCl, pH 7). The washed promastigotes were counted in a hemocytometer and adjusted to 1 x 10 ^ parasites / ml. Aliquots of this suspension were analyzed in a final volume of 100 μl, counting live (mobile) cells after 24 hours of incubation at 26 ° C, in the absence or in the presence of various concentrations of the diastereomers. The anti-Leishmania activities were expressed as the minimum inhibitory concentration (MIC), the concentration at which 100% death was observed after 24 hours of incubation. It was found that for peptide 23, the MIC is 17 μM, and for peptide 20, the MIC is 32 μM.

EXAMPLE 13 ANTIVIRAL ACTIVITY OF THE DIASTEREOMER 23 Sendai virus (strain Z) was developed in the allantoic sac of chicken embryo eggs for 10-11 days, harvested 48 hours after injection, and purified. The virus was resuspended in a buffer composed of 160 mM NaCl, 20 mM tricine, pH 7.4, and stored at -70 ° C. The hemagglutinin activity of virus was measured in haemagglutination units (UHA). One microliter contained approximately 60,000 UHA. Fresh human blood was obtained from a blood bank and stored for up to 1 month at 4 ° C. Before use, the erythrocytes were washed twice with PBS pH 7.2, and diluted to the desired concentration (% v / v) with the same buffer. Virions, erythrocytes and peptides were mixed in different orders of addition and in various quantities. The final incubation was always at 37 ° C for 60 minutes, followed by centrifugation at 5700 x g for 10 minutes to remove intact cells. In all cases, samples were used in duplicate and two aliquots of the supernatant of each sample were placed in two wells of a 96-well plate. The amount of hemoglobin release was monitored by measuring the absorption of the wells with the ELISA plate reader at 540 nm. The antiviral activity was expressed as the minimum inhibitory concentration (MIC), the concentration at which hemoglobin release was not observed after incubation. It was found that for the diastereomer peptide 23 of Lys / Leu, the MIC is 80 μM.

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The asymmetric distribution of phospholipids in the human network cell membrane. A combíned study using phospholipases and freeze-etch electron microscopy. Biochim. Biophys. Minutes 323: 178-193. 53. Wade, D., Boman, A., Wahlin, B., Drain, C.M., Andreu, D., Boman, H.G., and Merrifield, R.B. 1990. Proc. Nati Acad. Sci. USA. 87: 4761-4765. 54. Wu, C. S., Ikeda, K., and Yang, J. T. 1981. Ordered conformation of polypeptides and proteins in acidic dodecyl sulfate solution. Biochemistry 20: 566-570. 55. Zagorski, M.G., Norman, D.G., Barrow, C.J., Iwashita, T., Tachibana, K., and Patel, D.J. 1991. Solution structure of pardaxin P-2. Biochemistry 30: 8009-8017.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: YEDA RESEARCH AND DEVELOPMENT CO. LTD (B) STREET: at the Weizmann Institute of Science, PO Box 95 (C) CITY: Rehovot (E) COUNTRY: Israel (F) ZIP CODE: 76100 (G) TELEPHONE: 972-08-9470617 (H) TELEFAX: 972-08-9470739 (ii) TITLE OF THE INVENTION: ANTIPATHOGEN SYNTHETIC PEPTIDES AND COMPOSITIONS THAT COMPRISE THEM (iü) SEQUENCE NUMBER: 95 (iv) COMPUTER LEADABLE FORM: (A) TYPE OF MEDIUM: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Reléase # 1.0, Version # 1.30 (EPO) (v) CURRENT APPLICATION DATA: APPLICATION NUMBER: (vi) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: PCT / IL97 / 00066 (B) SUBMISSION DATE: FEB 20, 1997 ) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (i¡) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 1 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (7, 18, 19) (D) OTHER INFORMATION: / product = "D-amino acid residues" (ix) FEATURE: (A) ) NAME / FEATURE: Modified site (B) LOCATION: 33 (D) OTHER INFORMATION: / product = "OTHER" / note = "two aminoethylamino groups are attached at the C-terminus" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: Gly Phe Phe Ala Leu lie Pro Lys lie lie Ser Ser Pro Leu Phe Lys 1 5 10 15 Thr Leu Leu Ser Ala Val Gly Ser Ala Leu Ser Ser Gly Gly Gln 20 25 30 Glu (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) I: (B) CLON: peptide 2 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (7, 18, 19) (D) OTHER INFORMATION: / product = "D-amino acid residues" (X) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 22 (D) OTHER INFORMATION: / product = "OTHER" / note = "an aminoethylamino group is attached at the C-terminus" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Gly Phe Phe Ala Leu lie Pro Lys lie lie Ser Ser Pro Leu Phe Lys 1 5 10 15 Thr Leu Leu Ser Wing Val 20 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 3 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (7, 18, 19) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: Gly Phe Phe Ala Leu He Pro Lys He He Ser Ser Pro Leu Phe Lys 1 5 10 15 Thr Leu Leu Ser Wing Val 20 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 4 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (8, 19, 20) (D) OTHER INFORMATION: / product = "D-amino acid residues" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 23 (D) OTHER INFORMATION: / product = "OTHER" / note = "an aminoethylamino group is attached at the C-terminus" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: Lys Gly Phe Phe Ala Leu He Pro Lys He He Ser Ser Pro Leu Phe 1 5 10 15 Lys Thr Leu Leu Ser Wing Val 20 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 5 (¡x) CHARACTERISTIC: (A) NAME / FEATURE: Modified site (B) LOCATION: group (9, 20, 21) (D) OTHER INFORMATION: / product = "D-amino acid residues" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 24 (D) OTHER INFORMATION: / product = "OTHER" / note = "an aminoethylamino group is attached by the C terminus" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: Lys Lys Gly Phe Phe Wing Leu He Pro Lys He He Ser Ser Pro Leu 1 5 10 15 Phe Lys Thr Leu Leu Ser Wing Val 20 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 6 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (9, 20, 21) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Lys Lys Gly Phe Phe Wing Leu He Pro Lys He He Ser Ser Pro Leu Phe Lys Thr Leu Leu Ser Wing Val 20 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 7 (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 7 (D) OTHER INFORMATION: / product = "RESIDUALS OF D-AMINOACIDO" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 11 (D) OTHER INFORMATION: / product = "OTHER" / note = "A GROUP AMINOETILAMINO IS UNITED IN THE EXTREME C " (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: Gly Phe Phe Ala Leu He Pro Lys He He Ser 1 5 10 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (v) FRAGMENT TYPE: linear (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 8 (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 33 (D) OTHER INFORMATION: / product = "OTHER" / note = "TWO GROUPS AMINOETILAMINO ARE UNITED IN THE EXTREME C" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: Gly Phe Phe Ala Leu He Pro Lys He He Ser Ser Pro Leu Phe Lys 1 5 10 15 Thr Leu Leu Ser Ala Val Gly Ser Ala Leu Ser Ser Gly Gly Gln 20 25 30 Glu (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 9 (ix) CHARACTERISTIC: (A) NAME / FEATURE: Modified site (B) LOCATION: 13 (D) OTHER INFORMATION: / product = "RESIDUAL OF D-AMINOACIDO" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 33 (D) OTHER INFORMATION: / product = "OTHER" / note = "TWO GROUPS AMINOETILAMINO ARE UNITED IN THE EXTREME C" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: Gly Phe Phe Ala Leu He Pro Lys He He Ser Ser Pro Leu Phe Lys 1 5 10 15 Thr Leu Leu Ser Ala Val Gly Ser Ala Leu Ser Ser Gly Gly Gln 20 25 30 Glu (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (v) FRAGMENT TYPE: linear (vii) IMMEDIATE SOURCE: (B) CLON: peptide 10 (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: group (5, 19) (D) OTHER INFORMATION: / product = "RESIDUES OF D-AMINO ACIDS" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 33 (D) OTHER INFORMATION: / product = "OTHER" / note = "TWO GROUPS AMINOETILAMINO ARE UNITED IN THE EXTREME C" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: Gly Phe Phe Ala Leu He Pro Lys He He Ser Ser Pro Leu Phe Lys 1 5 10 15 Thr Leu Leu Ser Ala Val Gly Ser Ala Leu Ser Ser Gly Gly Gln 20 25 30 Glu (2) INFORMATION FOR SEQ ID NO: 1 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (v) FRAGMENT TYPE: linear (vii) IMMEDIATE SOURCE: (A) LIBRARY: peptide 1 1 (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: group (7, 19) (D) OTHER INFORMATION: / product = "RESIDUES OF D-AMINO ACIDS" (X) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 33 (D) OTHER INFORMATION: / product = "OTHER" / note = "TWO GROUPS AMINOETILAMINO ARE UNITED IN EXTREME C" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: Gly Phe Phe Ala Leu He Pro Lys He He Ser Ser Pro Leu Phe Lys 1 5 10 15 Thr Leu Leu Ser Ala Val Gly Ser Ala Leu Ser Ser Gly Gly Gln 20 25 30 Glu (2) INFORMA TION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (i¡) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 12 (¡X) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 7 (D) OTHER INFORMATION: / product = "RESIDUAL OF D-AMINOACIDO" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 33 (D) OTHER INFORMATION: / product = "OTHER" / note = "TWO AMINOETILAMINO GROUPS ARE UNITED IN THE EXTREME C " (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: Gly Phe Phe Ala Leu He Pro Lys He He Ser Ser Pro Leu Phe Lys 1 5 10 15 Thr Leu Leu Ser Ala Val Gly Ser Ala Leu Ser Ser Gly Gly Gln 20 25 30 Glu (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 13 (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 7 (D) OTHER INFORMATION: / product = "RESIDUAL OF D-AMINOACIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: Gly Phe Phe Ala Leu He Pro Lys He He Ser Ser Pro Leu Phe Lys 1 5 10 15 Thr Leu Leu Ser Ala Val Gly Ser Ala Leu Ser Ser Gly Gly Gln 20 25 30 Glu (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 14 (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: group (18, 19) (D) OTHER INFORMATION: / product = "RESIDUES OF D-AMINO ACIDS" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 33 (D) OTHER INFORMATION: / product = "OTHER" / note = "TWO AMINO-TYPE GROUPS ARE UNITED IN EXTREME C" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: Gly Phe Phe Ala Leu He Pro Lys He He Ser Ser Pro Leu Phe Lys 1 5 10 15 Thr Leu Leu Ser Ala Val Gly Ser Ala Leu Ser Ser Gly Gly Gln 20 25 30 Glu (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 15 (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: group (18, 19) (D) OTHER INFORMATION: / product = "RESIDUES OF D-AMINO ACIDS" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: Gly Phe Phe Ala Leu He Pro Lys He He Ser Ser Pro Leu Phe Lys 1 5 10 15 Thr Leu Leu Ser Ala Val Gly Ser Ala Leu Ser Ser Gly Gly Gln 20 25 30 Glu (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 16 (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: group (7, 18, 19) (D) OTHER INFORMATION: / product = "RESIDUALS OF D-AMINOACITIES" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: Gly Phe Phe Ala Leu He Pro Lys He He Ser Ser Pro Leu Phe Lys 1 5 10 15 Thr Leu Leu Ser Ala Val Gly Ser Ala Leu Ser Ser Gly Gly Gln 20 25 30 Glu (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 17 (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION-7 (D) OTHER INFORMATION: / product = "WASTE OF D-AMINOACIDO" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 22 (D) OTHER INFORMATION: / product = "OTHER" / note = "A GROUP AMINOETILAMINO IS UNITED IN THE EXTREME C" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: Gly Phe Phe Ala Leu He Pro Lys He He Ser Ser Pro Leu Phe Lys 1 5 10 15 Thr Leu Leu Ser Wing Val 20 (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vií) IMMEDIATE SOURCE: (B) CLON: peptide 18 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (5, 8, 17, 21) (D) OTHER INFORMATION: / product = "D-amino acid residues" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 26 (D) OTHER INFORMATION: / product = "OTHER" / note = "the carboxyl group at the C-terminus is replaced by an amino group" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: Gly He Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10 15 ' I Have Trp He Lys Arg Lys Arg Gln Gln 20 25 (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 19 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (5, 8, 17, 21) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19: Gly He Gly Wing Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10 15 I Have Trp He Lys Arg Lys Arg Gln Gln 20 25 (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 20 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (5, 8, 17, 21) (D) OTHER INFORMATION: / product = "D-amino acid residues" (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: 22 (D) OTHER INFORMATION: / product = "OTHER" / note = "an aminoethylamino group is attached at the C-terminus" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20: Gly He Gly Wing Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10 15 I Have Been Trp He Lys Arg 20 (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 21 (ix) CHARACTERISTIC: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 5, 14, 18) (D) OTHER INFORMATION: / product = "D-amino acid residues" (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: 19 (D) OTHER INFORMATION: / product = "OTHER" / note = "an aminoethylamino group is attached at the C-terminus" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21: Wing Val Leu Lys Val Leu Thr Thr Gly Leu Pro Wing Leu He Ser Trp 1 5 10 15 He Lys Arg (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 22 (ix) CHARACTERISTIC: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 4, 8, 10) (D) OTHER INFORMATION: / product = "D-amynoacid residues" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 12 (D) OTHER INFORMATION: / product = "OTHER" / note = "the carboxyl group at the C-terminus is replaced by an amino group" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22: Lys Leu Leu Leu Leu Leu Lys Leu Leu Leu Leu Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 23 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 4, 8, 10) (D) OTHER INFORMATION: / product = "D-amino acid residues" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 12 (D) OTHER INFORMATION: / product = "OTHER" / note = "the carboxyl group at the C-terminus is replaced by an amino group" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23: Lys Leu Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 24 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 4, 8, 10) (D) OTHER INFORMATION: / product = "D-amino acid residues" (X) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 12 (D) OTHER INFORMATION: / product = "OTHER" / note = "the carboxyl group at the C-terminus is replaced by an amino group " (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 24: Lys Leu Leu Leu Lys Leu Lys Leu Lys Leu Leu Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 25 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 4, 8, 10) (D) OTHER INFORMATION: / product = "D-amino acid residues" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 12 (D) OTHER INFORMATION: / product = "OTHER" / note = "the carboxyl group at the C-terminus is replaced by an amino group" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25: Lys Lys Leu Leu Lys Leu Lys Leu Lys Leu Lys Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 26 (ix) CHARACTERISTIC: (A) NAME / FEATURE: Modified site (B) LOCATION: group (1, 2, 5, 6, 7, 9, 11, 12) (D) OTHER INFORMATION: / product = "D-amino acid residues" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 12 (D) OTHER INFORMATION: / product = "OTHER" / note = "the carboxyl group at the C-terminus is replaced by an amino group" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 26: Lys Leu Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 27 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (1, 2, 5, 6, 7, 9, 11, 12) (D) OTHER INFORMATION: / product = "D waste" -amino acids" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 12 (D) OTHER INFORMATION: / product = "OTHER" / note = "the carboxyl group at the C-terminus is replaced by an amino group" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 27: Lys Leu Leu Leu Lys Leu Lys Leu Lys Leu Leu Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 28: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 28 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 4) (D) OTHER INFORMATION! ON: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 28: Lys Leu Leu Leu Leu Lys 1 5 (2) INFORMATION FOR SEQ ID NO: 29: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 29 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 4, 6) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 29: Lys Leu Leu Leu Lys Leu Leu Lys 1 5 (2) INFORMATION FOR SEQ ID NO: 30: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 30 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 6, 8, 11) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 30: Lys Leu Leu Leu Lys Leu Lys Leu Lys Leu Leu Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 31: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (i¡) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 31 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 3, 4, 8, 10, 1 1) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 31: Lys Leu Leu Leu Lys Leu Lys Leu Lys Leu Leu Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 32: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 32 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 5, 8, 9, 11) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 32: Lys Leu Leu Leu Lys Leu Lys Leu Lys Leu Leu Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 33: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 33 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 4, 8, 10) (D) OTHER INFORMATION: / product = "D-amino acid residues" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 12 (D) OTHER INFORMATION: / product = "OTHER" / note = "the carboxyl group at the C-terminus is replaced by an amino group" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 33: Lys Wing Wing Wing Lys Wing Wing Wing Lys Wing Wing Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 34: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 34 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 4, 8, 10) (D) OTHER INFORMATION: / product = "D-amino acid residues" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 12 (D) OTHER INFORMATION: / product = "OTHER" / note = "the carboxyl group at the C-terminus is replaced by an amino group" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 34: Lys Val Val Val Lys Val Val Val Lys Val Val Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 35: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 35 (ix) CHARACTERISTIC: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 6, 8, 11) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 35: Lys Val Val Val Lys Val Lys Val Lys Val Val Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 36: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 36 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (1..4, 8, 10, 11) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 36: Lys Val Val Val Lys Val Lys Val Lys Val Val Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 37: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 37 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 5, 8, 9, 1 1) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 37: Lys Val Val Val Lys Val Lys Val Lys Val Val Lys 10 (2) INFORMATION FOR SEQ ID NO: 38: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 38 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 6) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: Lys Leu He Leu Lys Leu 1 5 (2) INFORMATION FOR SEQ ID NO: 39: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 39 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 6) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: Lys Val Leu His Leu Leu 1 5 (2) INFORMATION FOR SEQ ID NO: 40: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 40 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 6) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: Leu Lys Leu Arg Leu Leu 1 5 (2) INFORMATION FOR SEQ ID NO: 41: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 41 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: 4 (D) OTHER INFORMATION: / product = "D-amino acid residue" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: Lye Pro Leu His Leu Leu 1 5 (2) INFORMATION FOR SEQ ID NO: 42: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 42 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 3, 4, 6) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 42: Lys Leu He Leu Lys Leu Val Arg 1 5 (2) INFORMATION FOR SEQ ID NO: 43: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 43 (¡X) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 4, 5, 6) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 43: Lys Val Phe His Leu Leu His Leu 1 5 (2) INFORMATION FOR SEQ ID NO: 44: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 44 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (1, 2, 4, 6, 7) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 44: His Lys Phe Arg He Leu Lys Leu 1 5 (2) INFORMATION FOR SEQ ID NO: 45: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 45 (X) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 5 (D) OTHER INFORMATION: / product = "D-amino acid residue" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 45 : Lys Pro Phe His He Leu His Leu 1 5 (2) INFORMATION FOR SEQ ID NO: 46: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 46 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (1, 2, 6, 8, 11) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 46: Lys He He He Lys He Lys He Lys He He Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 47: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 47 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 3, 4, 8, 10, 11) (D) OTHER INFORMATION: / product = "D-amino acid residues" ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 47: Lys He He He Lys He Lys He Lys He He Lys (2) INFORMATION FOR SEQ ID NO: 48: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 48 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (1, 3, 5, 8, 9, 11) (D) OTHER INFORMATION: / product = "D-amino acid residues" ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 48: Lys He He He Lys He Lys He Lys He He Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 49: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 49 (X) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 7 (D) OTHER INFORMATION: / product = "D-amino acid residue" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 49 : Lys He Pro He Lys He Lys He Lys He Pro Lys (2) INFORMATION FOR SEQ ID NO: 50: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 50 (X) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: group (6, 10) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 50: Lys He Pro He Lys He Lys He Lys He Val Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 51: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 51 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCALIZATION: group (2, 4, 6, 8, 11) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 51: Arg He He He Arg He Arg He Arg He He Arg 1 5 10 (2) INFORMATION FOR SEQ ID NO: 52: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 52 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 3, 4, 6, 7, 8, 10, 1 1) (D) OTHER INFORMATION: / product = "residues of D-amino acids "(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 52: Arg He He He Arg He Arg He Arg He He Arg 1 5 10 (2) INFORMATION FOR SEQ ID NO: 53: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 53 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCALIZATION: group (1, 3, 5, 8, 9, 1 1) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 53: Arg He He He Arg He Arg He Arg He He Arg 1 5 10 (2) INFORMATION FOR SEQ ID NO: 54: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 54 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 6, 8, 11) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 54: Arg He Val He Arg He Arg He Arg Leu He Arg 1 5 10 (2) INFORMATION FOR SEQ ID NO: 55: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 55 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 3, 4, 8, 10, 1 1) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 55: Arg He He Val Arg He Arg Leu Arg He He Arg 1 5 10 (2) INFORMATION FOR SEQ ID NO: 56: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids • (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 56 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 5, 8, 9, 11) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 56: Arg He Gly He Arg Leu Arg Val Arg He He Arg 1 5 10 (2) INFORMATION FOR SEQ ID NO: 57: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 57 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCALIZATION: group (2, 6, 8, 1 1) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 57: Lys He Val He Arg He Arg He Arg Leu He Arg 1 5 10 (2) INFORMATION FOR SEQ ID NO: 58: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 58 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 3, 4, 8, 10, 1 1) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 58: Arg He Wing Val Lys Trp Arg Leu Arg Phe He Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 59: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 59 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 5, 8, 9, 1 1) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 59: Lys He Gly Trp Lys Leu Arg Val Arg He He Arg 1 5 10 (2) INFORMATION FOR SEQ ID NO: 60: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 60 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 4, 7, 10, 1 1) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 60: Lys Lys He Gly Trp Leu He He Arg Val Arg Arg 1 5 10 (2) INFORMATION FOR SEQ ID NO: 61: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 61 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 6, 8, 1 1, 13) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 61: Arg He Val He Arg He Arg He Arg Leu He Arg He Arg 1 5 10 (2) INFORMATION FOR SEQ ID NO: 62: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 62 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (1..4, 8, 10, 1 1, 13, 14) (D) OTHER INFORMATION: / product = "D waste" -amino acids "(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 62: Arg He He Val Arg He Arg Leu Arg He He Arg Val Arg 1 5 10 (2) INFORMATION FOR SEQ ID NO: 63: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 63 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 5, 8, 9, 1 1, 14) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 63: Arg He Gly He Arg Leu Arg Val Arg He He Arg Arg Val 1 5 10 (2) INFORMATION FOR SEQ ID NO: 64: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 64 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (1, 2, 6, 7, 1 1, 15, 16) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 64: Lys He Val He Arg He Arg Ala Arg Leu He Arg He Arg He Arg 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 65: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 65 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 3, 4, 7, 8, 10, 1 1, 13, 14, 16) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 65: Arg He He Val Lys He Arg Leu Ar He He Lys Lys He Arg Leu 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 66: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 66 (ix) CHARACTERISTIC: (A) NAME / FEATURE: Modified site (B) LOCATION: group (1, 3, 5, 8, 9, 1 1, 13, 14 , 16) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 66: Lys He Gly He Lys Wing Arg Val Arg He He Arg Val Lys He He 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 67: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 67 (ix) CHARACTERISTIC: (A) NAME / FEATURE: Modified site (B) LOCALIZAC10N: group (2, 3, 4, 7, 8, 10, 11, 13, 14, 16) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 67: Arg He He Val His He Arg Leu Arg He He His His He Arg Leu 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 68: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (Ü) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 68 (ix) CHARACTERISTIC: (A) NAME / FEATURE: Modified site (B) LOCATION: group (1, 3, 5, 8, 9, 1 1, 13, 14 , 16) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 68: His He Gly He Lys Wing His Val Arg He He Arg Val His He He 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 69: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 69 (ix) CHARACTERISTIC: (A) NAME / CHARACTERISTIC: Modified site (B) LOCATION: group (2, 4, 7, 9, 10, 1 1, 13, 14, 16) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 69: Arg He Tyr Val Lys He His Leu Arg Tyr He Lys Lys He Arg Leu 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 70: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 70 (ix) CHARACTERISTIC: (A) NAME / FEATURE: Modified site (B) LOCATION: group (1, 3, 5, 8 , 9, 1 1, 13, 14, 16) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 70: Lys He Gly His Lys Wing Arg Val His He He Arg Tyr Lys He He 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 71: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 71 (ix) CHARACTERISTIC: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 3, 4, 7 , 8, 10, 1 1, 13, 14, 16) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 71: Arg He Tyr Val Lys Pro His Pro Arg Tyr He Lys Lys He Arg Leu 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 72: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 72 (ix) CHARACTERISTIC: (A) NAME / CHARACTERISTIC: Modified site (B) LOCATION: group (3, 5, 8, 9 , 11, 13, 14, 16) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 72: Lys Pro Gly His Lys Wing Arg Pro His He He Arg Tyr Lys He He 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 73: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 73 (¡x) CHARACTERISTIC: (A) NAME / CHARACTERISTIC: Modified site (B) LOCATION: group (2, 6, 7, 11 , 15, 16, 18) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 73: Lys He Val He Arg He Arg He Arg Leu He Arg He Arg He Arg 1 5 10 15 Lys He Val (2) INFORMATION FOR SEQ ID NO: 74: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 74 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 3, 4, 6, 7, 8, 10, 1 1, 13, 14, 16, 17, 19) (D) ) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 74: Arg He He Val Lys He Arg Leu Ar He He Lys Lys He Arg Leu 1 5 10 15 He Lys Lys (2) INFORMATION FOR SEQ ID NO: 75: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 75 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 4, 5, 8, 9, 1 1, 13, 14, 16, 17, 18) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 75: Lys He Gly Trp Lys Leu Arg Val Arg He He Arg Val Lys He Gly 1 5 10 15 Arg Leu Arg (2) INFORMATION FOR SEQ ID NO: 76: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 76 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCALlZAC10N: group (2, 6, 7, 1 1, 15, 16, 18, 20, 24, 25) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 76: Lys He Val He Arg He Arg He Arg Leu He Arg He Arg He Arg 1 5 10 15 Lys He Val Lys Val Lys Arg He Arg 20 25 (2) INFORMATION FOR SEQ ID NO: 77: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 77 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 3, 4, 6, 7, 8, 10, 11, 13, 14, 16, 17, 19..22, 24, 25) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 77: Arg Phe Wing Val Lys He Arg Leu Arg He He Lys Lys He Arg Leu 1 5 10 15 He Lys Lys He Arg Lys Arg He Lys 20 25 (2) INFORMATION FOR SEQ ID NO: 78: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 78 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 4, 5, 8, 9, 1 1, 13, 14, 16, 17, 18, 22, 23, 24, 27, 28, 30) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 78: Lys Wing Gly Trp Lys Leu Arg Val Arg He He Arg Val Lys He Gly 1 5 10 15 Arg Leu Arg Lys He Gly Trp Lys Lys Arg Val Arg He Lys 20 25 30 (2) INFORMATION FOR SEQ ID NO: 79: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 79 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2..5, 7, 8, 10, 11, 13, 14, 16) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 79: Arg He Tyr Val Lys Pro His Pro Arg Tyr He Lys Lys He Arg Leu 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 80: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 80 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (1, 2, 3, 5, 7..1 1, 13, 14, 16) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 80: Lys Pro Gly His Lys Wing Arg Pro His He He Arg Tyr Lys He He 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 81: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 81 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 4..8, 10..13, 15, 16, 18) (D) OTHER INFORMATION: / product = "waste of D-amino acids " (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 81: Lys He Val He Arg He Arg He Arg Leu He Arg He Arg He Arg 1 5 10 15 Lys He Val (2) INFORMATION FOR SEQ ID NO: 82: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 82 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 3, 4, 6..1 1, 13, 14, 16, 17, 19) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 82: Arg He He Val Lys He Arg Leu Ar He He Lys Lys He Arg Leu 1 5 10 15 He Lys Lys (2) INFORMATION FOR SEQ ID NO: 83: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 83 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2..5, 7, 8, 10, 11, 13, 14, 16) (D) OTHER INFORMATION: / product = " D-amino acid residues " (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 83: Arg He Tyr Val Ser Lys He Ser He Tyr He Lys Lys He Arg Leu 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 84: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Peptide 84 (ix) CHARACTERISTIC: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 4..8, 10..13, 15, 16, 18) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 84: Lys He Val He Phe Thr Arg He Arg Leu Thr Ser He Arg He Arg 1 5 10 15 Ser He Val (2) INFORMATION FOR SEQ ID NO: 85: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 85 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (1, 2, 3, 5, 7..1 1, 13, 14, 16) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 85: Lys Pro He His Lys Wing Arg Pro Thr He He Arg Tyr Lys Met He 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 86: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 86 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (9, 20, 21) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 86: Cys Lys Gly Phe Phe Wing Leu He Pro Lys He He Ser Ser Pro Leu 1 5 10 15 Phe Lys Thr Leu Leu Ser Wing Val Cys 20 25 (2) INFORMATION FOR SEQ ID NO: 87: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 87 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (10, 21, 22) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 87: Cys Lys Lys Gly Phe Phe Wing Leu He Pro Lys He He Ser Ser Ser 1 5 10 15 Leu Phe Lys Thr Leu Leu Ser Wing Val Cys 20 25 (2) INFORMATION FOR SEQ ID NO: 88: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 88 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (11, 22, 23) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 88: Cys Lys Lys Lys Gly Phe Phe Wing Leu He Pro Lys He He Ser Ser 1 5 10 15 Pro Leu Phe Lys Thr Leu Leu Ser Wing Val Cys 20 25 (2) INFORMATION FOR SEQ ID NO: 89: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 89 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 89: Cys Arg He Val He Arg He Arg He Arg Leu He Arg He Arg Cys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 90: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: circular (i¡) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 90 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (2, 3, 4, 6, 8..12, 14, 15, 17) (D) OTHER INFORMATION: / product = " D-amino acid residues " (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 90: Cys Lys Pro Gly His Lys Wing 'Arg Pro His He He Arg Tyr Lys He 1 5 10 15 He Cys (2) INFORMATION FOR SEQ ID NO: 91: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 91 (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 4, 5, 7, 8, 9, 1 1, 12, 14, 15, 17, 18, 20..23 , 25, 26) (D) OTHER INFORMATION: / product = "D-amino acid residues" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 91: Cys Arg Phe Wing Val Lys He Arg Leu Arg He He Lys Lys He Arg 1 5 10 15 Leu He Lys Lys He Arg Lys Arg He He Lys Cys 20 25 (2) INFORMATION FOR SEQ ID NO: 92: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: peptide 23C (part of peptide 92) (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 4, 8, 10) (D) OTHER INFORMATION: / product = "D-amino acid residues" (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: 13 (D) OTHER INFORMATION: / product = "OTHER" / note = "the carboxyl group at the C-terminus is replaced by an amino group " (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 92: Lys Leu Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Cys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 93: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: 24C peptide (part of peptide 93) (ix) CHARACTERISTICS: (A) NAME / FEATURE: Modified site (B) LOCATION: group (3, 4, 8, 10) (D) OTHER INFORMATION: / product = "D-amino acid residues" (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 13 (D) OTHER INFORMATION: / product = "OTHER" / note = "the carboxyl group at the C-terminus replaced by an amino group" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 93: Lys Leu Leu Leu Lys Leu Lys Leu Lys Leu Leu Lys Cys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 94: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: PARDAXIN (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 94: Gly Phe Phe Ala Leu He Pro Lys He He Ser Ser Pro Leu Phe Lys 1 5 10 15 Thr Leu Leu Ser Ala Val Gly Ser Ala Leu Ser Ser Gly Gly Gln 20 25 30 Glu (2) INFORMATION FOR SEQ ID NO: 95: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE: (B) CLON: Melittin (ix) FEATURE: (A) NAME / FEATURE: Modified site (B) LOCATION: 26 (D) OTHER INFORMATION: / product = "OTHER" / note = "the carboxyl group at the C-terminus is replaced by an amino group" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 95: Gly He Gly Wing Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10 15 I Have Trp He Lys Arg Lys Arg Gln Gln 20 25

Claims (2)

1. NOVELTY OF THE INVENTION CLAIMS 1. A non-hemolytic cytolytic agent selected from a peptide, a complex of pooled peptides, a mixture of peptides or a random peptide copolymer, said agent having a selective cytolytic activity manifested because it has a cytolytic activity on pathogenic cells, being cells that do not occur naturally within the body, consisting of pathogenic microbial organisms and malignant cells; and is non-haemolytic, namely, does not have cytolytic effects on erythrocytes or has a cytolytic effect on them at concentrations that are substantially greater than those in which said cytolytic activity manifests, said non-haemolytic cytolytic agent being selected from the group consisting of : (1) a cyclic derivative of a peptide having a net positive charge which is greater than +1, and comprising residues of L-amino acids and residues of D-amino acids, or comprising one or both residues of L-amino acids and residues of D-amino acids, and comprising a disrupting portion of a-helix; (2) a peptide comprising residues of L-amino acids and residues of D-amino acids, having a net positive charge which is greater than +1, and having an amino acid sequence such that a corresponding amino acid sequence comprising only residues of L-amino acids is not found in nature, and cyclic derivatives of the same; (3) a complex consisting of a plurality of 2 or more non-hemocytic cytolytic peptides, each peptide having a net positive charge which is greater than +1, and comprising residues of L-amino acids and D-amino acid residues, or comprising one or both of L-amino acid residues and D-amino acid residues, and comprising a switch-a-helix portion, or cyclic derivatives of the foregoing, said peptides being pooled by the use of a linker molecule covalently linked to each of the same; (4) a mixture consisting of a plurality of two or more non-haemolytic cytolytic peptides, each peptide having a net positive charge which is greater than +1, and comprising residues of L-amino acids and D-amino acid residues, or comprising one or both of L-amino acid residues and D-amino acid residues, and comprising a disrupting portion of a-helix, or cyclic derivatives of the foregoing; and (5) a random copolymer consisting of different ratios of a hydrophobic amino acid, a positively charged amino acid and a D-amino acid.
2. 2. The cyclic peptide according to claim 1 (1), characterized in that it comprises both D- and L- amino acid residues having such a sequence, that an open chain homogeneous peptide comprising only L-amino acid residues or only waste from D-amino acid and having the same amino acid sequence as said peptide, has an alpha-helix configuration and has a broad spectrum of cytolytic activity manifested on a variety of cells. 3. - The cyclic peptide according to claim 2, further characterized in that it is a cyclic diastereomer derived from pardaxin or melittin, or fragments thereof. 4. The cyclic peptide according to claim 3, further characterized in that the positive net charge greater than +1 is due to the natural composition of the amino acid, or is achieved by neutralization of free carboxyl groups or by the addition of amino acid residues positively charged and / or positively charged chemical groups. 5. The cyclic peptide according to claim 4, further characterized in that it is selected from a cyclic diastereomer of pardaxin, or a fragment thereof, to which residues of Lys have been added to the N- terminus and / or Aminoethylamino groups have been added to the C-terminus. 6. The cyclic peptides according to claim 5, further characterized in that it is selected from the cyclic peptides derived from pardaxin of the present designated as peptides 86 to 88, of the sequence:86. K1 [D] P7L1 8L1 9 [1-22] -par cyclic, of the sequence: 1 a p 104 87. K1? 2 [D] P7Ll8 | _19 [-? -.22] -par cyclic, from the sequence: 2nd p 104 88. K1 K2K3 [D] P7L18 19 [1_22] -P cyclic, of the sequence: 3a p 104 7. The peptide according to claim 1 (2), further characterized in that it comprises L-amino acid residues and D-amino acid residues, and having an amino acid sequence such that a corresponding amino acid sequence comprising only L-amino acid residues , it is not found in nature. 8. The peptide according to claim 7, further characterized in that it has the following characteristics: (a) it is an unnatural synthetic peptide formed of variable ratios of at least one hydrophobic amino acid, and at least one positively charged amino acid, and in which sequence at least one of the amino acid residues is a D-amino acid; (b) the peptide has a net positive charge which is greater than +1; and (c) the hydrophobic amino acid ratio: Positively charged amino acids are such that the peptide is cytolytic to pathogenic cells, but does not cause erythrocyte cytolysis. 9. The peptide according to claim 8, further characterized in that the positively charged amino acid is selected from lysine, arginine and histidine, and the hydrophobic amino acid is selected from leucine, isoleucine, glycine, alanine, valine, phenylalanine, proline, tyrosine and tryptophan. 10. The peptide according to claim 9, further characterized in that the net positive charge greater than +1 is due to the composition of amino acids or the addition of positively charged chemical groups, or whose hydrophobic character can be decreased by the addition of polar amino acids such as serine, threonine, methionine, asparagine, glutamine and cysteine. 1. The peptide according to claim 10, further characterized in that it has at least 6 amino acid residues, in which the hydrophobic amino acid is leucine, alanine or valine, and the positively charged amino acid is lysine. 12. The peptide according to claim 11, further characterized in that it is a diastereomer of a peptide of 6 elements, 8 elements or 12 elements, formed of leucine and lysine, in which at least one third of the sequence is formed of D-amino acids, except for the peptide of the present one designated as 23: SEQUENCE p 105 13.- A diastereomer of Leu / Lys according to claim 12, further characterized in that it is selected from the peptides herein designated as 24 to 29 (SEQ ID NO: 24-29, respectively) of the sequence: SEQUENCES 14. - The cyclic derivative of a non-natural synthetic peptide according to any of claims 7 to 13, further characterized in that it is selected from the peptides herein designated as 92 to 95 of the sequence: 92. Cys Lys Leu Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Cys cyclical 93. Cys Lys Leu Leu Leu Lys Leu Lys Leu Lys Leu Leu Lys Cyclic Cys. 94. HN-Lys Leu Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys - CO 95. HN-Lys Leu Leu Leu Lys Leu Lys Leu Lys Leu Leu Lys - CO 15. - A complex of grouped peptides according to claim 1 (3), characterized in that it consists of a plurality of 2 or more non-haemolytic cytolytic peptides according to any of claims 1 to 14, said peptides being grouped by means of a molecule linker covalently linked to each one of them. 16. The complex according to claim 15, further characterized in that the beam is formed of 2 or more, preferably 5, molecules of the same peptide or of different peptides, and the linker is a peptide according to any of the preceding claims , or a commonly used linker. 17. The complex according to claim 16, further characterized in that it is selected from the pooled Lys / Leu diastereomers of the present designated 96 and 97: 96. ([D] L3.4'8.10K4L8C) 5 [D ] L3 > 4.8,10 | < 4L8 of the sequence: p 106 97. ([D] L3A8.10? 5L7C) 5 [D] L3,4,8,10K4L9 of the sequence: p 106 18. - The mixture according to claim 1 (4), further characterized in that it consists of a plurality of two or more non-haemolytic cytolytic peptides, wherein the peptides are as defined in any of claims 1 to 14. 19.- The mixture according to claim 18, further characterized in that it comprises a mixture of peptide diastereomers of 12 elements of Lys / Leu. 20. The non-haemolytic cytolytic random copolymer according to claim 1 (5), characterized in that it consists of different ratios of a hydrophobic amino acid, a positively charged amino acid, and a D-amino acid. 21. The non-haemolytic cytolytic random copolymer according to claim 20, further characterized in that it is formed of lysine, leucine and D-leucine at a ratio of 1: 1: 1, 2: 1: 1 or 3: 1: 1 (Mol). 22. - A pharmaceutical composition, characterized in that it comprises a non-haemolytic cytolytic agent according to any of claims 1 to 21, and a pharmaceutically acceptable carrier. 23. The pharmaceutical composition according to claim 22, further characterized in that it is used for the treatment of infections caused by pathogenic organisms. 24. The pharmaceutical composition according to claim 23, further characterized in that the pathogenic organism is selected from bacteria, fungi, protozoa, microplasmas and viruses. 25. The pharmaceutical composition according to claim 24, further characterized in that the pathogenic organism is a bacterium. 26. The pharmaceutical composition according to claim 22, further characterized in that it is used for the treatment of cancer.