WO1998040091A1 - Microbicidal peptides and methods of use - Google Patents

Abstract

Microbicidal peptides are provided which represent bioactive domains, demonstrating antimicrobial activity against at least one microbial pathogen, of the amino acid sequences of naturally occurring defensins or bactenecins. The microbicidal peptide may also be an analog having at least 50 % identity with the bioactive domain, but further incorporates substitutions with one or more natural, or uncommon, amino acids or chemical moieties for improving structural and/or functional properties of the peptide. Also provided are methods of using the microbicidal peptides in formulations for antimicrobial therapy, or for incorporation onto or into surfaces which come into contact with microorganisms.

Description

MICROBICIDAL PEPTIDES AND METHODS OF USE

This invention was made in part with government support under grant number DE04898 awarded by the National Institutes of Dental Research. The government has certain rights in the invention. This application claims priority of earlier filed provisional application serial number

60/040,542, filed on March 13, 1997, which is incorporated herein by this reference.

FIELD OF THE INVENTION The present invention provides novel broad spectrum antimicrobial peptides which are derived from portions of naturally occurring antimicrobial cationic polypeptides, and also includes analogs of those portions. More particularly, the invention is directed to microbicidal peptides comprising either fragments, or analogs of the fragments, of defensins and bactenecins. The invention also provides methods of administering compositions containing the microbicidal peptides to inhibit the growth of a microorganism.

BACKGROUND OF THE INVENTION

Infectious diseases continue to be a major cause of morbidity and mortality in humans and animals despite the presence of naturally occurring antimicrobial compounds in the host, and despite the introduction of a wide variety of antimicrobial agents such as antibiotics. There have been repeated accounts of clinical disease due to the emergence of multi-drug resistant microorganisms, including bacteria, viruses, fungi, and parasites. For example, bacteria have developed a multitude of ways to deal with the presence of an antibiotic including production of chemical pumps to force out antibiotics that make it into the bacterial cell, production of enzymes that destroy or inactivate an antibiotic, or genetically evolve the bacterial factor or component that is the target of the antibiotic so that it is no longer sensitive. Thus, there is a continuing need to develop novel antimicrobial agents.

Leukocytes and epithelial cells play an important role in host defenses against infection by bacterial, viral, fungal, and parasitic pathogens. A number of cationic peptides having antimicrobial activity have been isolated from these cells including defensins, and bactenecins. Defensins are a family of arginine-rich peptides consisting of approximately 29 to approximately 42 amino acids, and containing six conserved cysteine residues that participate in intramolecular disulfide bonds. Amino acid sequences of various mammalian defensins have been reported previously (See, e.g., U.S. Patent Nos. 5,550,109; 5,459,235; 5,242,902; 5,210,027; and 5,032,574). Defensins have shown broad spectrum antimicrobial activity against gram negative bacteria, gram positive bacteria, fungi, and certain enveloped viruses (See, e.g., Cullor et al . , 1990, Arch. Opϊithalmol . 108:861-864; Daher et al . , 1986, J. Virol . 60:1068-1074; Lehrer et al . , 1985, J. Virol . 54:467-472; Lehrer et al., 1985, Infect . Immun . 49:207-11; Greenwald and Ganz, 1987, Infect . Im un . 55:1365-1368; Miyaski et al . , 1990, Infect. Immun. 58:3934-3940).

However, a problem in synthesizing active defensins is the requirement for proper disulfide bond formation since the active molecule contains three intramolecular disulfide bonds. Further, changing a single amino acid at the N- terminus of human defensins is sufficient to produce significant changes in microbicidal potency and selectivity (Lehrer et al., 1983, Infect . Immun. 42:10-14). Additionally, defensins present an arginine-rich substrate that may be susceptible to enzymes produced by microbial pathogens. For example, the oral pathogen Porphyromonas gingivalis has been shown to produce proteases (300 kilodaltons, Fujimura et al . , 1987, Infect . Immun . 55:716- 720; 120 kDa, Sojar et al . , 1993, Infect . Immun . 61:2369- 76) , and a hemagglutinin (44 kDa; Nishikata and Yoshimura, 1991, Biochem. Biophys . Res . Commun . 178:336-42) which hydrolyze proteins and arginine-containing synthetic substrates .

Bactenecins are a family of proline rich and arginine rich antimicrobial polypeptides of from about 42 amino acids to about 59 amino acids having antibacterial and antiviral activity against a wide variety of microorganisms (Stanfield et al., 1988, J. Biol . Che . 263:5933-35; Frank et al . , 1990, J. Biol . Chem. 265: 18871-74; Zerial et al . , 1987, Antiviral Res . 7:341-352) ; and having candidacidal activity (Raj et al . , 1996, Biochemistry 35:4314-25). Amino acid sequences of various mammalian bactenecins have been reported previously (Romeo et al . , 1988, J^". Biol . Chem. 263: 9573-75; Raj et al . , 1996, supra) . Bactenecins, like defensins, present an arginine-rich substrate that may be susceptible to enzymes produced by microbial pathogens. Additionally, both bactenecins and defensins, particularly isolated from another host species, are immunogenic and thus would limit clinical usefulness. Thus, there is a need for an effective microbicidal peptide that has broad spectrum antimicrobial activity, may be easily synthesized in active form, is substantially protease-resistant, and is substantially nonimmunogenic. Such novel microbicidal peptides may be used to inhibit the growth of micro-organisms, particularly those which have developed resistance to one or more synthetic antibiotics.

SUMMARY OF THE INVENTION

The invention provides for the design and production of novel microbicidal peptides representing defined portions ("bioactive domains") of the amino acid sequences of naturally occurring mammalian cationic antimicrobial polypeptides including defensins and bactenecins. The microbicidal peptides of the present invention may also be modified in structure and composition with one or more objects of: (a) enhancing resistance to proteolytic digestion; (b) stabilizing folded or extended conformations to maintain activity in various delivery formulations and in various physiologic environments; (c) minimizing the size necessary for bioactivity to diminish immunogenicity; and (d) improving the microbicidal activity by enhancing the interaction between the peptide and the microbial membrane and subsequent disruption of the membrane.

The microbicidal peptides of the present invention may be produced by known methods including, but not limited to, solid-phase synthesis or by recombinant DNA techniques. Also provided are various formulations (compositions) for delivering the microbicidal peptides to physiologic environments being treated for microbial infection. The formulation comprises one or more of the microbicidal peptides of the present invention with a carrier appropriate for the physiologic site to be treated, wherein the ionic strength, pH, and other properties of the carrier may be adjusted to maximize delivery and antimicrobial activity of the microbicidal peptide (s) to that site. Such formulations may include, but are not limited to a rinse, a topical agent (cream, ointment, gel, etc.), and a suppository. Additionally, the microbicidal peptides may be incorporated into or onto the surface of a product, thereby directing antimicrobial activity to microorganisms coming in proximity or in contact with the product. Such products include, but are not limited to, medical devices such as catheters, implants, stents, and dentures; and industrial products involved in food processing or preparation such as cutting/work surfaces in restaurants, and work surfaces and conveyor belts used in the meat-processing industry.

Methods of antimicrobial therapy (treatment) according to the present include administration of a formulation containing one or more of the microbicidal peptides to a physiologic environment in which infection is sought to be prevented or halted. The compositions and methods for antimicrobial therapy according to the present invention are not limited to use in humans, but also have veterinary applications.

Other objects, features, and advantages of the present invention will become apparent from the following drawings and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a three step process for selective disulfide formation in synthesizing a defensin or microbicidal peptide based thereon.

DETAILED DESCRIPTION

Definitions By the terms "antimicrobial activity" and "microbicidal" is meant, for the purposes of the specification and claims to refer to the ability of a peptide to inhibit growth of, kill, or irrevocably damage a target microorganism. By the term "expression vector" is meant, for the purposes of the specification and claims to refer to a DNA molecule which is operably linked to a nucleotide sequence that encodes a microbicidal peptide such that the production of the microbicidal peptide is effected in a suitable host. The vector may include, but is not limited to, a plasmid, phage, or a potential genomic insert.

By the terms "conservative substitution" in the amino acid sequence is meant, for the purposes of the specification and claims to refer to a substitution or modification of one or more amino acids such that the tertiary structure of the microbicidal peptide, and the antimicrobial activity, are substantially unchanged. "Conservative substitutions" includes substitutions of amino acids having substantially the same charge, size, hydrophilicity, and/or aromaticity as the amino acid replaced. Such substitutions, known to those of ordinary skill in the art, include glycine-alanine-valine; isoleucine-leucine; tryptophan- tyrosine; aspartic acid- glutamic acid; arginine-lysine; asparagine-glutamine; and serine-threonine. Particularly relevant to amino acid substitutions in an α-helix conformation, a helix- forming amino acid may be replaced with another helix- forming amino acid. Helix- forming amino acids include alanine, leucine, glutamine, and serine. Although only twenty amino acids are commonly used in vivo as building blocks for peptides, less common natural amino acids exist, as well as unnatural or uncommon amino acids, which can be used in making a conservative substitution in the amino acid sequences of the microbicidal peptides according to the present invention. Amino acids in these categories include enantiomers and diastereomers of the natural D-amino acids, hydroxyproline, norleucine, methionine sulfoxide, ornithine, citrulline, cyclohexylalanine, omega-amino acids such as 3 -amino propionic acid, and 4-amino butyric acid. Such amino acids can be synthesized, and incorporated into peptides using methods known in the art.

By the term "microbicidal peptide" is meant, for the purposes of the specification and claims to refer to a peptide of about 10 amino acids to about 24 amino acids that represents a defined portion, a bioactive domain which demonstrates antimicrobial activity against at least one microbial pathogen, of the amino acid sequences of naturally occurring defensins or bactenecins. The microbicidal peptide may also be an analog having at least 50% identity with the defined portion, but further incorporates substitutions with one or more natural or unnatural/ uncommon amino acids or chemical moieties with one or more objects of: (a) enhancing resistance to proteolytic digestion; (b) stabilizing folded or extended conformations to maintain activity in various delivery formulations and in various physiologic environments; (c) minimizing the size necessary for bioactivity to diminish immunogenicity; and (d) improving the microbicidal activity by enhancing the interaction between the peptide and the microbial membrane and subsequent disruption of the membrane.

By the term "physiologic site" is meant, for the purposes of the specification and claims to refer to tissues or organs which can become infected by microbial pathogens, including, but not limited to, the oral cavity, pharynx, skin, nasopharynx, vagina, and gastrointestinal tract.

For purposes of the description, the following embodiments illustrate the manner and process of making and using the invention and set forth the best mode contemplated by the inventor for carrying out the invention, but are not to be construed as limiting.

EXAMPLE 1 Production of the microbicidal peptides of the present invention can be achieved by methods known in the art including chemical synthesis, or recombinant DNA techniques. Peptides can be synthesized using one of the several methods of peptide synthesis known in the art including standard solid peptide synthesis using tert-butyloxycarbonyl amino acids (Mitchell et al . , 1978, J. Org. Chem. 43:2845-2852), using 9-fluorenyl-methyloxycarbonyl amino acids on a polyamide support (Dryland et al . , 1986, J^". Chem. So . Perkin Trans . I, 125-137); by pepscan synthesis (Geysen et al . , 1987, J^". Immunol . Methods 03:259; 1984, Proc. Natl . Acad. Sci . USA 81:3998); and by standard liquid phase peptide synthesis. Purification of the peptides can be accomplished by reverse phase high pressure liquid chromatography.

Purification can be monitored by analyzing for amino acid composition, amino acid sequencing, and/or spectral analysis.

Recombinant techniques may be used to produce the microbicidal peptide. In one illustration of this embodiment, a nucleotide sequence encoding the peptide can be inserted into, and expressed by various vectors including phage vectors and plasmids . Successful expression of the peptide requires that either the insert comprising the nucleotide sequence encoding the peptide, or the vector itself, contain the necessary elements for transcription and translation which is compatible with, and recognized by the particular host system used for expression. DNA encoding the peptides can be synthesized using methods for DNA amplification known to those skilled in the art. A variety of host systems may be utilized to express the peptide, which include, but are not limited to bacteria transformed with a bacteriophage vector, plasmid vector, or cosmid DNA; yeast containing yeast vectors; fungi containing fungal vectors; insect cell lines infected with virus (e.g. baculovirus) ; and mammalian cell lines transfected with plasmid or viral expression vectors, or infected with recombinant virus (e.g. vaccinia virus, adenovirus, adeno- associated virus, retrovirus, etc.). For example, expression in bacterial hosts may be accomplished using a method for producing cationic peptides as described in U.S. Patent No. 5,593,866. Briefly, a nucleotide sequence encoding a microbicidal peptide according to the present invention is inserted in an expression vector downstream from sequences encoding an anionic carrier peptide such that a fusion peptide is produced upon introduction of the recombinant expression vector into the host bacterial cell . After synthesis of the fusion peptide, the fusion peptide may be isolated and cleaved, followed by purification of the microbicidal peptide. Additionally, if the microbicidal peptides may be lethal or detrimental to the host cells, the host cell strain/line and expression vectors may be chosen such that the action of the promoter is inhibited until specifically induced. For example, in certain operons the addition of specific inducers is necessary for efficient transcription of the inserted DNA (e.g., the lac operon is induced by the addition of lactose or isopropylthio-beta-D-galactoside) . A variety of operons such as the trp operon, are under different control mechanisms. The trp operon is induced when tryptophan is absent in the growth media. The P_L promoter can be induced by an increase in temperature of host cells containing a temperature sensitive lambda repressor. In this way, greater than 95% of the promoter-directed transcription may be inhibited in uninduced cells. Thus, expression of recombinant microbicidal peptide may be controlled by culturing transformed or transfected cells under conditions such that the promoter controlling the expression from the inserted DNA encoding the peptide is not induced, and when the cells reach a suitable density in the growth medium, the promoter can be induced for expression from the inserted DNA. An example of an inducible plasmid system for expression in a bacterial host is the pET expression system (commercially available from Novagen) . When inserted into pET, the nucleotide sequence encoding a microbicidal peptide is expressed only after induction. Thus, the E. coli transformants are grown in the presence of isopropyl- -D-thiogalactopyranoside (IPTG) , and recombinant peptide is then harvested from the induced culture. Another example of a similar system (inducible by IPTG) which has been used to generate fusion proteins from which a cationic peptide has been cleaved and purified is the E. coli expression vector pGEX-2T (Tsai et al., 1996, Infect . Immun . 64:5000-07) .

Other control elements for efficient gene transcription or message translation include enhancers, and regulatory signals. Enhancer sequences are DNA elements that appear to increase transcriptional efficiency in a manner relatively independent of their position and orientation with respect to a nearby gene. Thus, depending on the host cell expression vector system used, an enhancer may be placed either upstream or downstream from the inserted DNA sequence encoding a microbicidal peptide to increase transcriptional efficiency.

EXAMPLE 2 In this illustrative embodiment of the present invention, provided are microbicidal peptides derived from known bactenecins. While other bactenecins may be used, for illustrative purposes, bactenecin 5 was selected for design and synthesis of defined portions thereof in producing microbicidal peptides of the present invention. Native bactenecin 5 was isolated from the extracts of neutrophils employing ion-exchange and reversed-phase high pressure liquid chromatography (HPLC) as described previously (Gennaro et al . , 1989, supra) . Three peptides were synthesized from the 43 amino acid sequence of bactenecin 5, including BN22 (SEQ ID NO: 6) comprising the first 22 amino acids (representing the N-terminus of bactenecin 5) , BN16 comprising amino acids 7 to 22 (SEQ ID NO:l) (representing the middle portion of bactenecin 5) , and BC24 comprising amino acids 20 to 43 (representing the C-terminus of bactenecin 5) . The peptides were synthesized by standard solid-phase procedures using 4-methylbenzhydrylamine and phenylacetamido-methyl resins and N- 1-butyloxycarbonyl (t- Boc) amino acids. The side-chain protecting groups were Ν- tosyl (arginine) and 0- (2 -bromobenzyl) oxycarbonyl

(tyrosine) . The coupling reactions were carried out with 3- fold excess of protected amino acids in a mixture (50% v/v) of N, N-dimethyl-formamide and dichloromethane using dicyclohexylcarbodiimide as the coupling reagent in the presence of 1-hydroxybenzotriazole. The t-Boc group on the Ν- terminus was deprotected in 30 minutes with a 25% solution of trifluoroacetic acid, and the aqueous solution was lyophilized. The resins containing the C-terminal t-Boc amino acid were used for synthesis of the peptides. The solid-phase synthesis yielded approximately 85-90% of the crude peptides. Peptides were then purified by HPLC on a C- 18 column employing an acetonitrile-water linear gradient elution (15-50% acetonitrile in water over a period of 60 minutes) with a flow rate of 2.0 mL min^"1.

The peptides were tested and compared with the intact bactenecin 5 for antimicrobial activity. Antifungal activity of bactenecin 5 and the peptides derived therefrom was assessed in vi tro against C. albicans . Candidacidal activity was measured by the loss of viability of the yeast cells, since the inability of the yeast to replicate following removal of the peptides indicates nonviability.

Candidacidal activity is influenced by peptide concentration and incubation time. C. albicans cells from a 48 hour culture were harvested, washed, and resuspended to a concentration of 5 x 10⁷ cells/mL in 0.01 M sodium phosphate buffer (pH 7.4) . Different concentrations of the peptides or bactenecin 5 in 0.01 M sodium phosphate buffer (100 μl ) were mixed with cells (100 μl; 5 x 10° cells) and incubated for 37°C for 1 hour with periodic shaking. A 100 mL aliquot was removed, diluted to 5 x 10² cells/mL in sodium phosphate buffer, and then vortexed. Aliquots of 0.5 mL of each suspension were spread onto agar plates, and the C. albicans was grown overnight at 37°C. Candidacidal activity was assessed as the ratio of colonies per test plate to the number of colonies on a control plate. Table 1 illustrates the candidacidal activity of bactenecin 5 (Bac5) , and fragment BN16 (SEQ ID N0:1), BN22 (SEQ ID NO:6), and BN24 as measured by % loss of viability.

Table 1

EI₅₀ is peptide concentration required to induce half-maximal loss in C. albicans cell viability as determined for the concentration effect curve.

While the cidal potency of each of the peptides BN16, BN22 and BC24 is comparable to native bactenecin 5, the activity of the C- terminal fragment diminishes significantly, as compared to the others, as the concentration diminishes. That the cidal activities of bactenecin 5, BN16 (SEQ ID N0:1) and BN22 (SEQ ID NO: 6) over the concentration range of 5-100μM appear almost identical, is evidence that BN16 and BN22 represent the bioactive domain of bactenecin 5.

The antimicrobial activity of cationic polypeptides is generally related to their efficacy in disrupting microbial membranes. An in vitro assay for the lytic effect on negatively charged lipid vesicles also correlates with cidal potency. Peptide- induced lysis of negatively charged liposomes

(dipalmitoylphosphatidylcholine:dioleylphosphatidylserine) encapsulating the fluorescent dye calcein (Staubinger and Papahadjopoulos, 1982, Methods Enzymol . 101:512-27) was monitored by measuring the dequenching of fluorescence caused by the leakage of calcein. Fluorescence intensity was measured at 520 nm by excitation at 495 nm as a function of time following incubation with the peptides and liposomes. Bactenecin 5, BN22 and BN16 each induced nearly 75% lysis of the negatively charged vesicles within 10 minutes after incubation. It appears that the high proline content of BN16 appears to stabilize the conformation of this peptide in solution. Thus, in making analogs, in some cases the poly proline backbone was conserved.

EXAMPLE 3

In this illustrative embodiment of the present invention, provided are microbicidal peptides synthesized as analogs from a bactenecin. While other bactenecins may be used, for illustrative purposes, BN16 was selected for design and synthesis of analogs in producing microbicidal peptides of the present invention. An analog incorporates substitutions of the native sequence with one or more natural or unnatural/ uncommon amino acids or chemical moieties with one or more objects of: (a) enhancing resistance to proteolytic digestion; (b) stabilizing folded or extended conformations to maintain activity in various delivery formulations and in various physiologic environments; (c) minimizing the size necessary for bioactivity to diminish immunogenicity; and (d) improving the microbicidal activity by enhancing the interaction between the peptide and the microbial membrane and subsequent disruption of the membrane.

For example, there have been many microbial proteases identified which cleave a peptide molecule at a cleavage site involving L-arginine. Thus, one method for improving the resistance of a microbicidal peptide representing a defined portion of the native sequence is to synthesize or recombinantly produce an analog wherein L-arginine is replaced with a natural or unnatural/ uncommon amino acid that can provide the strong basic charge provided by the L- arginine residues. In that regard, amino acids which may be used for the substitution of L-arginine include ornithine, D-arginine, lysine (D- or L-) and other basic diaminomonocarboxylic acids.

Additionally, organic molecules comprising heterocyclic compounds like cyclic aminocarboxylic acids containing both amino and carboxyl functions may be used for substitution in the proline backbone of the microbicidal peptide. Examples of such organic molecules include, but are not limited to, aminobenzoic acids and derivatives of pyridine, pyrrole, imidazole, triazole, quinoline, and cyclo carboxylic acids. In that regard, the organic molecules may include p-amino benzoic acid, o-amino benzoic acid, 3 -amino-pyridine-2- carboxylic acid, 3 -amino-pyridine-4 -carboxylic acid, 3- amino-quinilone-2- carboxylic acid, 3-amino-quinilone-4- carboxylic acid, imidazolidine-2-carboxylic acid, pyrrolidine-2 -acetic acid, imidazolidin-2-yl thio acetic acid, N-amino-inazol-3-yl thioacetic acid, 1- aminocyclopentanecarboxylic acid, and 1-aminoeyelohexane- carboxylic acid. These organic molecules may serve one or more purposes of stereochemically constraining the conformation, thereby making the constrained microbicidal peptide more resistant to enzymatic degradation; providing structural angles more conducive for helical conformations, thereby stabilizing the peptide for use in various formulations and environments; and providing more bulky/ hydrophobic molecules to enhance interaction with, and disruption of, microbial membranes.

As an illustration of such microbicidal peptides according to the present invention, four analogs of fragment 16 (BN16) of bactenecin 5 were synthesized using standard solid-phase procedures essentially as outlined in Example 2. BN16a (SEQ ID NO:2) is an analog identical to BN16, except bactenecin 16a contains ornithine substitutions in place of all L-arginine residues. BN16b (SEQ ID NO: 3) is an analog identical to BN16, except BNlδb contains D-arginine substitutions in place of all L-arginine residues. BN16c (SEQ ID NO: 4) is an analog identical to BN16, except BN16c contains 1-aminocyclopentanecarboxylic acid substitutions in place of all proline residues. BN16d (SEQ ID NO: 5) is an analog identical to BN16, except BN16d contained D-amino acid substitutions in place of the respective L- mino acid residues. BN16 and the BN16 analogs were then tested and compared for antimicrobial activity against C. albicans (antifungal) , and Actinobacillus actinomycetemcomi tans and p. gingivalis (antibacterial) . All of these microorganisms are known oral pathogens. A . actinomycetemcomi tans and P. gingivalis have been reported to be the major pathogens in advanced human periodontitis (Slots et al . , 1986, J^". Clin . Periodontol . 13:570-77; Slots and Listgarten. , 1988, J^". Clin . Periodontol . 15:85-93).

Candidacidal activity was assessed using the method as essentially described in Example 2. The bactericidal activity assay was performed using previously described methods (Miyaski et al . , 1990, supra, and Gennaro et al . , 1989, supra) . Briefly, A . actinomycetemcomi tans (grown under 5% C0₂) and P. gingivalis (grown anaerobically) were separately grown in broth cultures to early log phase (10⁹ cells/mL) and then subsequently adjusted to a concentration of 10⁶ cells per mL in 0.1% trypticase soy broth containing 10 rtiM sodium phosphate at pH 7.0. Bactericidal activity is measured by incubating 10⁶ cells with 100 μM of peptide at 37°C for 1.5 hours (P. gingivalis incubated in anaerobic conditions) , and then the samples were serially diluted with buffered saline, plated in appropriate agar medium, and incubated overnight to allow colony counts. Table 2 illustrates the antifungal and antibacterial activity of the N-terminal fragment of bactenecin 5, BN16 (SEQ ID NO:l), analog 16a (SEQ ID NO:2), analog 16b (SEQ ID NO:3), analog 16c (SEQ ID NO:4), and analog 16d (SEQ ID NO:5) as the minimum concentration (expressed in μM) of the peptide necessary to achieve 50% killing of the bacteria. Table 2

From the results illustrated in Table 2, microbicidal peptides, representing analogs containing substitutions to improve physical and functional properties thereof, retained substantial antimicrobial activity as compared to a microbicidal peptide representing a defined portion of a native bactenecin.

In another illustration of this embodiment, BN22 (SEQ ID NO: 6) was selected for design and synthesis of analogs in producing microbicidal peptides of the present invention. Four analogs of BN22 were synthesized using standard solid- phase procedures essentially as outlined in Example 2.

BN22a (SEQ ID NO: 7) is an analog identical to BN22, except BN22a contains ornithine substitutions in place of arginine residues at position 7,8,12, and 20 of BN22. BN22b (SEQ ID NO: 8) is an analog identical to BN22, except BN22b contains D-arginine substitutions in place of all the L-arginine residues. BN22c (SEQ ID NO: 9) is an analog identical to BN22, except BN22c contains 1-aminocyclopentane carboxylic acid in place of Proline residues at positions 9,10, 13,14, 17,18,21, and 22 of BN22. Bactericidal activity assays were performed for A. actinomycetemcomitans, P. gingivalis , S. gordonii, and S. mutans as follows. Briefly, S. gordonii and S. mutans were separately grown in broth cultures to early log phase (10⁹ cells/mL) and then diluted to a concentration of 10° cells/mL in lOmM sodium phosphate buffer at pH 7.0. Cells were incubated with various concentrations of the peptide in a total volume of 200μL at 370C in an anaerobic chamber for 90 minutes. The cells were then diluted to 10³ cells/mL and plated in appropriate agar plates. Colonies were counted after three days and compared with control plates. The cidal activity is calculated as [1- (cell survival after peptide incubation) - (cell survival in buffer alone)] x 100, which represents the percent killing of the bacterial cells. The results were calculated as percent loss of bacterial cell viability. EI₉₀ values are expressed as mean ± standard deviation for strains W50 and 381 of P. gingivalis and strains Y4 and 67 of A . actinomycetemcomi tans in Table 3, and for strain DLl of S. gordonii , and strain GS5 of S. mutans in Table 4.

Table 3

Table 4

These data indicate that microbial peptides of the present invention representing analogs of BN22 containing substitutions to improve physical and functional properties thereof, retained substantial microbicidal activity as compared to a microbicidal peptide representing a defined portion of a native bactenecin. EXAMPLE 4

In this illustrative embodiment of the present invention, provided are microbicidal peptides derived from known defensins. Sequences of known mammalian (human, rabbit, rat, guinea pig, and bovine) defensins have been published (See, e.g., U.S. Patent No. 5,242,902; Selsted et al., 1993, J. Biol . Chem. 268:6641-48). Additionally, avian defensin-like proteins have also been described (Evans et al., 1994, J. Leunkocyte Biol . 56:661-5). It is appreciated by those skilled in the art that the human defensins HNP1-, HNP-2, and HNP-3 differ in sequence only by the N-terminal amino acid. While other defensins may be used, for illustrative purposes, HNP-1 was selected for design and synthesis of defined portions thereof in producing microbicidal peptides. HNP-1 was synthesized by standard solid-phase procedures essentially as outlined in Example 2 herein. The side chain protecting groups were O-benzyl (Glu & Thr) , N-dibenzylcarbonyl (Arg) , O-2-bromobenzyloxycarbonyl (Tyr) , and N-formyl (Trp) . The coupling of glutamine to the growing peptide chain on the resin was achieved using N- (t-

Boc) -L-glutamine-p-nitrophenylester in dimethylformamide to avoid the dehydration of the amide side- chain by dicyclohexylcarbodiimide. Amino acid composition analysis and sequence analysis were used to confirm the sequence of the synthetic HNP-1.

Defensins contain six conserved cysteine residues that participate in intramolecular disulfide bonds. To achieve the synthesis of HNP-1 with the formation of the correct three disulfide bridges, three different protecting groups were used, and the pairs of cysteine residues were selectively deprotected and oxidized to form the disulfide linkages in three steps as shown in FIG. 1. These steps minimize the formation of undesired products. Additionally, polymerization during disulfide formation was also minimized by oxidizing the reduced cysteines at very low concentration (0.05 mM) of the peptide solution. Using similar standard solid-phase procedures for synthesis, three peptides were synthesized from the 30 amino acid sequence of HNP-1, including HNPF1 comprising the first 20 amino acids (SEQ ID NO: 10, representing the N- terminus and middle portion of defensin HNP-1, with a disulfide linkage between the Cysteine at amino acid position 2 and the Cysteine at amino acid position 19 of the peptide) ; HNPF2 comprising amino acids 7 to 30 (SEQ ID NO: 11, representing the middle and C-terminal portion of defensin HNP-1, with a disulfide linkage between the Cysteine at amino acid position 3 and the Cysteine at amino acid position 23 of the peptide) ; HNPF3 comprising amino acids 7 to 30 (SEQ ID NO: 12, with a disulfide linkage between the Cysteine at amino acid position 3 and the Cysteine at amino acid position 24 of the peptide) ; and HNPF4 comprising amino acids 7 to 25 (SEQ ID NO: 13, representing the middle portion of defensin HNP-1, with a disulfide linkage between the Cysteine at amino acid position 3 and the Cysteine at amino acid position 13 of the peptide) . HNP-1, and the microbicidal peptides representing a portion thereof (HNPF1, HNPF2, HNPF3 , and HNPF4) were then tested and compared for antimicrobial activity against C. albicans (antifungal) , and A . actinomycetemcomi tans and P . gingivalis (antibacterial) using the methods as essentially described in Example 3 herein, including use of peptides at a 100 μM concentration. Table 5 illustrates the antifungal activity and the antibacterial activity of HNP-1, HNPF1, HNPF2, HNPF3, and HNPF4 , as measured in μM. Table 5

As can be seen from the results illustrated in Table 5, HNP-1 and HNPF3 have comparable antimicrobial activity. Thus, microbicidal peptides of defensins should be derived from the central and C-terminal portions of the defensin. Further, it is noted that HNPF2 and HNPF3 have identical amino acid sequences, but differ in the placement of the disulfide linkage. Significant differences in potency of microbicidal activity are observed between HNPF2 and HNPF3. These results suggest that the final disulfide bond arrangement is particularly important for microbicidal potency. Linkage between the first cysteine residue (N- terminus of the peptide) and the last cysteine residue (C- terminus of the peptide) provides a cyclic peptide wherein the C- terminal folds back towards the N-terminal forming an optimal polar face for interaction with, and subsequent disruption of, the microbial membrane. These active microbicidal peptides appear to prefer to adopt an antiparallel β -sheet structure with a -turn, thereby acquiring amphiphilicity.

EXAMPLE 5

In this illustrative embodiment of the present invention, provided are microbicidal peptides synthesized as analogs from a defensin. While other defensins may be used, for illustrative purposes, HNPF3 is selected for design and synthesis of analogs in producing microbicidal peptides of the present invention. An analog incorporates substitutions of the native sequence with one or more natural or unnatural/ uncommon amino acids or chemical moieties with one or more objects of: (a) enhancing resistance to proteolytic digestion; (b) stabilizing folded or extended conformations to maintain activity in various delivery formulations and in various physiologic environments; (c) minimizing the size necessary for bioactivity to diminish immunogenicity; and (d) improving the microbicidal activity by enhancing the interaction between the peptide and the microbial membrane and subsequent disruption of the membrane .

For example, defensins, like bactenecins, are arginine- rich polypeptides. Thus, in view of microbial proteases identified which cleave a peptide molecule at a cleavage site involving L-arginine, one method for improving the resistance of a microbicidal peptide representing a defined portion of the native sequence of a defensin is to synthesize or recombinantly produce an analog wherein L- arginine is replaced with a natural or unnatural/uncommon amino acid that can provide the strong basic charge provided by the L-arginine residues. In that regard, amino acids which may be used for the substitution of L-arginine include ornithine, D-arginine, lysine (D- or L-) and other basic diaminomonocarboxylic acids. Using the methods according to Examples 2, 3 and 4 herein, a microbicidal peptide comprising an analog of HNPF3 (SEQ ID NO: 12) may be synthesized wherein the arginine residues of HNPF3 are substituted accordingly. Examples of such a microbicidal peptide are provided as SEQ ID NO: 14, wherein X is selected from the group consisting of ornithine, D-arginine, lysine (D- or L-), and other basic diaminomono-carboxylic acids. These microbicidal peptides may then be tested for antimicrobial activity using the methods, for example, according to Example 3 herein. EXAMPLE 6

In this illustrative embodiment of the present invention, provided are microbicidal peptides derived from a bovine defensin, BNPFS. The sequences of BNPFS has been published (Romeo, et al . , 1988, supra) . BNPFS was synthesized by standard solid-phase procedures essentially as outlined in Examples 2, 3 and 4 herein. BNPFS was selected for design and synthesis of analogs in producing microbicidal peptides of the present invention. An analog incorporates substitutions of the native sequence with one or more natural or unnatural/ uncommon amino acids or chemical moieties with one or more objects of: (a) enhancing resistance to proteolytic digestion; (b) stabilizing folded or extended conformations to maintain activity in various delivery formulations and in various physiologic environments; (c) minimizing the size necessary for bioactivity to diminish immunogenicity; and (d) improving the microbicidal activity by enhancing the interaction between the peptide and the microbial membrane and subsequent disruption of the membrane.

For example, BNPFS is arginine-rich, wherein arginine comprises more than 30% of the amino acid content. Thus, in view of microbial proteases identified which cleave a peptide molecule at a cleavage site involving L-arginine, one method for improving the resistance of a microbicidal peptide representing a defined portion of the native sequence of a defensin is to synthesize or recombinantly produce an analog wherein L-arginine is replace with a natural or unnatural/uncommon amino acid that can provide the strong basic charge provided by the L-arginine residues. In that regard, amino acids which may be used for the substitution of L-arginine include ornithine, D-arginine, lysine (D- or L-), and other basic diaminomonocarboxylic acids. Using the methods according to Examples 2, 3 and 4 herein, a microbicidal peptide comprising an analog of BNPFS may be synthesized wherein the arginine residues of BNPFS are substituted accordingly. Examples of such a microbicidal peptide are provided as SEQ ID NO: 15, wherein Xaa is ornithine, D-arginine, lysine (D- or L-), or another basic diaminomono-carboxylic acids. When microbicidal activity of BNPFS was tested with and without the disulfide bridge, the microbicidal activity was found to be significantly less for BNPFS without the disulfide bridge. Therefore, the microbicidal peptides are produced to include the disulfide bridge between the two cysteine residues within the sequence. In Analog 1 of BNPFS, "Xaa" is lysine. In Analog 2, "Xaa" is ornithine, and in Analog 3, all, arginines are D-arginines. These microbicidal peptides were then tested for antimicrobial activity using the methods, for example, according to Example 3 herein. Candidacidal activity was assessed using the method as described in Example 2. Bactericidal activity against P. gingivalis and A . actinomycetemcomi tans was assessed as described in Example 3, while bactericidal activity against S. mutans and S. gordonii was assayed as described in Example 3. The cidal activity is calculated as [1- (cell survival after peptide incubation) - (cell survival in buffer alone)] x 100, which represents the percent killing of the bacterial cells. Results are expressed as the peptide concentration necessary to achieve 90% killing in bacteria. The EI90 value for BNPFS and its analogs is shown in Table 6 for P. gingivalis and A . actinomycetemcomi tans and for S. mutans and S. gordonii in Table 7.

Table 6

Table 7

These data indicate that analogs of BNPFS of the present invention containing substitutions to improve physical and functional properties thereof, retained substantial microbicidal activity as compared to a microbicidal peptide representing a defined portion of a native bovine defensin.

EXAMPLE 7 In this illustrative embodiment of the present invention, formulations (compositions) are provided which comprise an effective amount of one or more of the microbicidal peptides of the present invention for treating a microbial infection, with a carrier appropriate for the physiologic site to be treated. Pharmaceutically acceptable carriers are generally known to include aqueous solutions such as water, various phosphate buffers, various buffered salines, alcoholic/aqueous solutions, and emulsions or suspensions; wherein the ionic strength, pH, and other properties of the pharmaceutically acceptable carrier may be adjusted to maximize delivery and antimicrobial activity of the microbicidal peptide (s) to that site. Regarding pH, generally optimal microbicidal activity is observed in a pH range of 6 to 8. Caution is warranted, as divalent cations such as calcium and magnesium have been reported to inhibit the antimicrobial activity of defensins, and may also inhibit some of microbicidal peptides. It will be appreciated by those skilled in the art that the carrier may comprise any suitable pharmaceutically acceptable liposome having incorporated therein one or more microbicidal peptides according to the present invention. Such liposomal compositions may be administered in any conventional mode for treating a microbial infection.

The pharmaceutically acceptable carrier may additionally comprise a detergent, preferably a nonionic detergent. The addition of a detergent in a concentration that does not inhibit microbicidal activity of the peptide (e.g. concentration of 1% or less) , may improve the solubility of the peptide in the formulation thereby enhancing activity. Nonionic detergents may include sodium lauryl sulfate, NP 40, or TWEEN 20. The nature of the carrier will depend on the intended area or physiologic site of application. For example, for topical application to the skin or mucous membranes a cream or ointment base is usually preferred. Suitable bases are known to those skilled in the art to include lanolin, polypropylene glycol, mineral oil, glycerin, and the like. Formulations containing one or more microbicidal peptides according to the present invention may include, but are not limited to a rinse, a topical agent (cream, ointment, gel, liquid, etc.), and a suppository. It will be appreciated by those skilled in the art that the effective concentration of the microbicidal peptide in the formulation will depend on other ingredients in the formulation, the mode of administration of the formulation, the physiologic site to be treated, and the particular microorganism that is the target of the treatment.

Depending on the physiologic site to be treated, the host, and the nature of the formulation, the microbicidal peptides can be administered in any one of the standard methods known in the art for administration of antimicrobial agents, including, but not limited to, topical, by injection (e.g., intravenously), aerosol spray, intranasal, etc. EXAMPLE 8

In this illustrative embodiment of the present invention, an effective amount of one or more of the microbicidal peptides of the present invention is incorporated into or onto the material comprising the surface of a product that, in use, comes in contact with microbial pathogens. As summarized above, such products include, but are not limited to, medical devices such as catheters, implants, stents, and dentures; and industrial products involved in food processing or preparation. Surface loading of antimicrobial proteins by either adsorption or chemical crosslinking has been described previously (See, e.g., Ito et al . , 1992, Biomaterials 13:789-94; Ito et al . , 1993, J^". BioiTie . Mater. .Res. 27:901- 7; Kang et al . , 1993, Biomaterials 14:787-92).

As an illustration, the microbicidal peptide according to the present invention may be surface adsorbed to denture acrylic, thereby permitting gradual release of the bioactive peptide. A denture of resin comprising poly (methyl methacrylate) (PMMA) may be surface modified by seed comonomer polymerization with methyl methacrylic acid as described previously (Edgarton et al . , 1995, J. Biomed. Mater. Res . 29:1277-86). A solution of the microbicidal peptide (e.g. 250 μM) is then allowed to adsorb for several hours at room temperature with the modified PMMA resin. The resulting denture resin may function as a controlled release device for the microbicidal peptide.

Having described the preferred embodiments of the present invention, it will be apparent to one of ordinary skill in the art that various modifications may be made to the disclosed embodiments, and that such modifications are intended to be within the scope of the present invention. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANTS: Periathamby, Antony R. Genco, Robert J.

(ii) TITLE OF INVENTION: Microbicidal Peptides and Methods of Use (iii) NUMBER OF SEQUENCES: 15 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Hodgson, Russ, Andrews, Woods & Goodyear

(B) STREET: 1800 One M&T Plaza

(C) CITY: Buffalo

(D) STATE: New York (E) COUNTRY: United States (F) ZIP: 14203-2391 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.5 inch

(B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: MS-DOS/ Microsoft Windows (D) SOFTWARE: Wordperfect for Windows (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 12 March 1998 (vii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Kadle, Ranjana

(B) REGISTRATION NUMBER: 40,041

(C) REFERENCE DOCKET NUMBER: 11520.0093 (viii) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (716) 856-4000 (B) TELEFAX: (716) 849-0349

(2) INFORMATION FOR SEQ ID NO:l :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16

(B) TYPE: amino acid

(C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Arg Arg Pro Pro lie Arg Pro Pro Phe Tyr Pro Pro Phe Arg Pro

5 10 15

Pro 16

(3) INFORMATION FOR SEQ ID NO: 2

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16

(B) TYPE: amino acid (C) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Orn Orn Pro Pro lie Orn Pro Pro Phe Tyr Pro Pro Phe Orn Pro

5 10 15 Pro 16

(4) INFORMATION FOR SEQ ID NO: 3 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16

(B) TYPE: amino acid

(C) TOPOLOGY: linear

(D) OTHER INFORMATION: all Arg are D-arginine (ii) MOLECULE TYPE: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

Arg Arg Pro Pro lie Arg Pro Pro Phe Tyr Pro Pro Phe Arg Pro

5 10 15

Pro 16

(5) INFORMATION FOR SEQ ID NO: 4 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 (B) TYPE: amino acid

(C) TOPOLOGY: linear

(D) OTHER INFORMATION: /note "Xaa = 1- aminocyclopentanecarboxylic acid"

(ii) MOLECULE TYPE: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Arg Arg Xaa Xaa lie Arg Xaa Xaa Phe Tyr Xaa Xaa Phe Arg Xaa

5 10 15

Xaa 16

(6) INFORMATION FOR SEQ ID NO: 5 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 (B) TYPE: amino acid

(C) TOPOLOGY: linear

(D) OTHER INFORMATION: all amino acids are D-amino acids

(ii) MOLECULE TYPE: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Arg Arg Pro Pro lie Arg Pro Pro Phe Tyr Pro Pro Phe Arg Pro

5 10 15

Pro 16

(7) INFORMATION FOR SEQ ID NO: 6 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 (B) TYPE: amino acid

(C) TOPOLOGY: linear

(D) OTHER INFORMATION: BN22 fragment of Bac5 (ii) MOLECULE TYPE: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Arg Phe Arg Pro Pro lie Arg Arg Pro Pro lie Arg Pro Pro Phe

5 10 15

Tyr Pro Pro Phe Arg Pro Pro 20 22

(8) INFORMATION FOR SEQ ID NO: 7 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 (B) TYPE: amino acid

(C) TOPOLOGY: linear

(D) OTHER INFORMATION: BN22a fragment of Bac5 (ii) MOLECULE TYPE: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Arg Phe Arg Pro Pro lie Orn Orn Pro Pro lie Orn Pro Pro Phe

5 10 15

Tyr Pro Pro Phe Orn Pro Pro 20 22

(9) INFORMATION FOR SEQ ID NO: 8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 (B) TYPE: amino acid

(C) TOPOLOGY: linear

(D) OTHER INFORMATION: BN22c fragment of Bac5 , all Arg are D-argining

(ii) MOLECULE TYPE: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Arg Phe Arg Pro Pro lie Arg Arg Pro Pro lie Arg Pro Pro Phe

5 10 15 Tyr Pro Pro Phe Arg Pro Pro

20 22

(10) INFORMATION FOR SEQ ID NO: 9 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22

(B) TYPE: amino acid

(C) TOPOLOGY: linear

(D) OTHER INFORMATION: BN22c fragment of Bac5 ; "Xaa" = 1-aminocyclopentanecarboxylic acid (ii) MOLECULE TYPE: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Arg Phe Arg Pro Pro lie Arg Arg Xaa Xaa lie Arg Xaa Xaa Phe

5 10 15 Tyr Xaa Xaa Phe Arg Xaa Xaa

20 22

(11) INFORMATION FOR SEQ ID NO: 10 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20

(B) TYPE: amino acid

(C) TOPOLOGY: cyclic

(D) OTHER INFORMATION: disulfide linkage between Cys at position 2 and Cys at position 19 (ii) MOLECULE TYPE: peptide

(iii) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Ala Cys Tyr Cys Arg lie Pro Ala Cys lie Ala Gly Glu Arg Arg

5 10 15 Tyr Gly Thr Cys lie

20

(12) INFORMATION FOR SEQ ID NO: 11 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24

(B) TYPE: amino acid

(C) TOPOLOGY: cyclic

(D) OTHER INFORMATION: disulfide linkage between Cys at position 3 and Cys at position 23 (ii) MOLECULE TYPE: peptide

(iii) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Pro Ala Cys lie Ala Gly Glu Arg Arg Tyr Gly Thr Cys lie Tyr

5 10 15 Gin Gly Arg Leu Trp Ala Phe Cys Cys

20 24

(13) INFORMATION FOR SEQ ID NO: 12 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24

(B) TYPE: amino acid

(C) TOPOLOGY: cyclic

(D) OTHER INFORMATION: disulfide linkage between Cys at position 3 and Cys at position 24 (ii) MOLECULE TYPE: peptide

(iii) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Pro Ala Cys lie Ala Gly Glu Arg Arg Tyr Gly Thr Cys lie Tyr 5 10 15

Gin Gly Arg Leu Trp Ala Phe Cys Cys

20 24 (14) INFORMATION FOR SEQ ID NO: 13 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19

(B) TYPE: amino acid

(C) TOPOLOGY: cyclic

(D) OTHER INFORMATION: disulfide linkage between Cys at position 3 and Cys at position 13

(ii) MOLECULE TYPE: peptide

(iii) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Pro Ala Cys lie Ala Gly Glu Arg Arg Tyr Gly Thr Cys lie Tyr

5 10 15

Gin Gly Arg Leu 19

(15) INFORMATION FOR SEQ ID NO: 14 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 (B) TYPE: amino acid

(C) TOPOLOGY: cyclic

(D) OTHER INFORMATION: disulfide linkage between Cys at position 3 and Cys at position 24; /note "Xaa is ornithine, D-arginine, L-lysine, D-lysine or other basic diaminomonocarboxylic acid

(ii) MOLECULE TYPE: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Pro Ala Cys lie Ala Gly Glu Xaa Xaa Tyr Gly Thr Cys lie Tyr 5 10 15

Gin Gly Xaa Leu Trp Ala Phe Cys Cys

20 24

(16) INFORMATION FOR SEQ ID NO: 15 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12

(B) TYPE: amino acid

(C) TOPOLOGY: cyclic

(D) OTHER INFORMATION: disulfide linkage between Cys at position 3 and Cys at position 11; /note "Xaa is ornithine, D-arginine, L-lysine, D-lysine or other basic diaminomonocarboxylic acid

(ii) MOLECULE TYPE: peptide

(iii) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Xaa Leu Cys Xaa lie Val Val lie Xaa Val Cys Xaa

5 10 12

Claims

What is claimed is :

1. A microbicidal peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 15, analogs thereof consisting of conservative substitutions therein, and analogs of SEQ ID NO:l, SEQ ID NO; 6, or SEQ ID NO: 10, wherein at least one proline is substituted with a cyclic aminocarboxylic acid.

2. The microbicidal peptide of claim 1, wherein the microbial peptide has the sequence of SEQ ID NO: 2.

3. The microbicidal peptide of claim 1, wherein the microbial peptide has the sequence of SEQ ID NO: 3.

4. The microbicidal peptide of claim 1, wherein the microbial peptide has the sequence of SEQ ID NO: 4.

5. The microbicidal peptide of claim 1, wherein the microbial peptide has the sequence of SEQ ID NO: 5.

6. The microbicidal peptide of claim 1, wherein the microbial peptide has the sequence of SEQ ID NO: 7.

7. The microbicidal peptide of claim 1, wherein the microbial peptide has the sequence of SEQ ID NO: 8.

8. The microbicidal peptide of claim 1, wherein the microbial peptide has the sequence of SEQ ID NO: 9.

9. The microbicidal peptide of claim 1, wherein the microbial peptide has the sequence of SEQ ID NO: 11.

10. The microbicidal peptide of claim 1, wherein the microbial peptide has the sequence of SEQ ID NO: 12.

11. The microbicidal peptide of claim 1, wherein the microbial peptide has the sequence of SEQ ID NO: 13.

12. The microbicidal peptide of claim 1, wherein the microbial peptide has the sequence of SEQ ID NO: 14.

13. A pharmaceutical composition comprising an microbicidally effective amount of the microbicidal peptide of claim l.

14. A method of microbicidal inhibition of survival or growth of a microorganism in a physiologic site capable of supporting survival or growth of the microorganism comprising administering to the site a microbicidally effective amount of the microbicidal peptide of claim 1.

15. The method of claim 14, wherein the physiologic site is in a human being.

16. The method of claim 14, wherein the physiologic site is in an animal .

17. A method of microbicidal inhibition of survival or growth of a microorganism on a material capable of contacting microbial pathogens comprising contacting the material with a microbicidally effective amount of the microbicidal peptide of claim 1.

18. The method of claim 17, wherein the microbicidal peptide is coated onto the surface of the material.

19. The method of claim 17, wherein the microbicidal peptide is incorporated into the material .