WO2001058942A1 - Antimicrobial peptides isolated from the skin of the hyperoliid frog, kassina senegalensis - Google Patents

Abstract

The present invention provides antimicrobial peptides derived from the skin of the Hyperoliid Frog, Kassina senegalensis. The novel peptides inhibit the growth of a variety of Gram-negative bacteria, Gram-positive bacteria and yeast. Pharmaceutical compositions and methods of treating microbial infections also are provided.

Description

ANTIMICROBIAL PEPTIDES ISOLATED FROM THE SKIN OF THE HYPEROLHD FROG, Kassina senegalensis

This application claims the benefit of U.S. Provisional Application No. 60/181,934, filed February 11, 2000, which is herein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions and methods for treating bacterial infections.

2. Description of the Related Art

The indiscriminate use of antibiotics in hospitals has created drug- resistant pathogens. For example, data collected by the Synercid Microbiology Assessment of Resistance Trends surveillance project revealed that more than 31 % of 17,000 bacterial isolates of Streptococcus pneumoniae obtained from patients at U.S. hospitals were intermediately or completely resistant to penicillin. The project further showed that 29% of Staphylococcus aureus strains were methicillin-resistant. Tuberculosis, which was supposedly eradicated in the United States, has reappeared in form^'s that are resistant to traditional isoniazid therapy. Also, bacterial infections in animals have displayed similar drug resistance.

Drug-resistant pathogens are particularly problematic for immunocompromised patients and patients infected with HIV or tuberculosis. In addition to causing health-care problems, drug-resistant pathogens exact a substantial economic toll. It has been estimated that drug-resistant infections acquired in hospitals nearly triple the cost and duration of an average hospital stay. Hoffert, S.P., "Companies seeking solutions to emerging drug resistance," The Scientist, 12(8): 1-6 (1998).

Following the development of successful antimicrobial agents during the 1980s and early 1990s, many large pharmaceutical companies cut back or eliminated research programs to develop new antimicrobial compounds, believing the battle against infectious diseases had been won. However, the emergence of large numbers of drug-resistant pathogens has once again stimulated the search for novel antimicrobial agents.

Amphibians are a promising source of antimicrobial agents. By necessity, amphibians live in a warm, moist environment, which is particularly conducive to the growth of microorganisms. As a result, Anurans (frogs and toads), over the course of evolution, have developed effective strategies for combating microbial infections. Anurans synthesize and secrete, through granular glands present in their skin, polypeptides with a broad spectrum of antimicrobial activity. The bioactive peptides are released into skin secretions in a holocrine fashion upon stress or injury and protect against invasion by pathogenic microorganisms. The amphibian antimicrobial peptides are generally synthesized as members of structurally related families. Examples include magainins from Xenopus laevis, bombinins from Bombina variegata and Bombina oήentalis, dermaseptins from Phyllomedusa sauvagii and Phyllomedusa bicolor, buforins from Bufo bufo gargarizans, and caerins from Litoήa chioris and Litoria splendida. Despite structural similarities among peptide families, each member of a particular family has a distinct spectrum of antimicrobial activity. It is speculated that this molecular diversity is important in protecting the animal from invasion by a wide variety of microorganisms.

A variety of antimicrobial peptides have been obtained from North American frogs of the genus Rana. See PCT/US99/ 18575. The peptides were categorized into seven families based upon structural similarities. Some of the peptides possessed broad antimicrobial activity, inhibiting the growth of Gram-negative bacteria (Escherichia colϊ), Gram-positive bacteria (Staphylococcus aureus), and yeast (Candida albicans), while others were effective only against particular pathogens.

As drug-resistant pathogens present a myriad of health concerns and exert economic hardships on society, there is a need for a variety of effective antimicrobial agents. Furthermore, practitioners require a wide-selection of antimicrobial agents to adequately treat multiple infections and immunocompromised patients.

SUMMARY OF THE INVENTION

It is therefore one object of the present invention to provide antimicrobial peptides. It is another object of the invention to provide methods of treating microbial infections. In accomplishing these and other objects of the invention, there are provided, in accordance with one aspect of the present invention, isolated peptides possessing antimicrobial activity. In other embodiments, functional variants of the antimicrobial peptides are provided. In still other embodiments, modified peptides, with enhanced antimicrobial activity, are provided.

In another embodiment, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a peptide of the present invention and a suitable carrier. Pharmaceutical compositions comprising a mixture of two or more antimicrobial peptides also are provided. In another embodiment, there is provided a method of treating a microbial infection in a patient, comprising administering to the patient a pharmaceutical composition of the present invention. There also is provided a method of treating a bacterial infection in a patient, comprising administering to the patient a pharmaceutical composition of the present invention. In one embodiment, the patient is a human. In other embodiments, the patient is a mammal or a bird.

In another embodiment, there is provided a kit useful for treating an antimicrobial infection, comprising a pharmaceutical composition comprising a therapeutically effective amount of a peptide of the present invention and ancillary reagents to effect administration of the peptide. Other objects, features and advantages of the present invention will become apparent from the following detailed description. The detailed description and specific examples, while indicating preferred embodiments, are given for illustration only as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Further, the examples demonstrate the principle of the invention and cannot be expected to specifically illustrate the application of this invention to all examples of infections where it will be obviously useful to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the partial purification of an extract of the skin of Kassina senegalensis, after purification on Sep-Pak cartridges, using reversed-phase HPLC on a semi-preparative Vydac C-18 column. The dashed line shows the concentration of acetonitrile in the eluting solvent. The fractions denoted by the bar contained growth- inhibitory activity towards Escherichia coli, Staphylococcus aureus, and Candida albicans.

Figure 2 illustrates the chromatographic separation of peptide kassinatuerin-1 (peak 1) and peptide kassinatuerin-2 (peak 2) on an analytical Vydac C-4 column. The arrows indicate where peak collection began and ended.

Figure 3 provides a comparison of the primary structures of kassinatuerin-1 and kassinatuerin-2 with linear, cationic antimicrobial peptides identified in the skins of other Anuran species. The suffix (^a) denotes that the peptide contains a C-terminal, α-amidated amino acid. Figure 4 is an Edmundson wheel diagram of kassinatuerin-1 , illustrating the amphipathic character of the putative helix. Hydrophobic amino acids are shaded.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention provides antimicrobial peptides derived from the skin of the African running frog, Kassina senegalensis. One peptide, termed kassinatuerin-1, effectively inhibited the growth of the Gram-negative bacterium Escherichia coli, the Gram-positive bacterium Staphylococcus aureus, and the yeast Candida albicans. The invention encompasses isolated kassinatuerin-1 and isolated functional variants of kassinatuerin-1. As used herein, an "isolated" peptide is one that is separated from one or more components with which it is naturally associated and is at least 90% pure, based on weight. In preferred embodiments, an isolated peptide of the invention is at least 95% , 98% , or 99% pure. Techniques are well-known in the art for determining purity, such as SDS PAGE, high performance liquid chromatography (HPLC), amino acid composition analysis, amino acid sequence analysis, mass spectrometry, and peptide fingerprinting. Pharmaceutical compositions comprising the antimicrobial peptides of the present invention are provided. Methods of treating microbial infections also are provided.

Kassina senegalensis belongs to the Hyperoliidae family of African frogs, which is divided into three sub-families: Hyperoliinae (small reed and lily frogs frequenting sedges and rushes), Kassininae (medium-sized species that prefer to walk or run rather than hop) and Leptopelinae (true tree frogs). See Duellman and Trueb, Biology of Amphibians, The Johns Hopkins University Press, Baltimore and London (1994). A comprehensive phylogeny of the Hyperoliidae family, based upon the nucleotide sequences of mitochondrial 12S rRNA, has been presented. See Richards, CM. and Moore, W.S. , Mol. Phylogenet. Evol. , 5:522-532 (1996). An earlier study described the isolation of the tachykinin kassinin from a methanolic extract of the skin of K. senegalensis. See Anastasi et al. , Experientia, 33:857-858 (1977).

Purification of the Peptides

The inventive peptides were isolated from the skin of pithed adult frogs of the species K. senegalensis. The skin was removed and immediately frozen on dry ice. The frozen tissue was cut into small pieces and extracted by homogenization. Following centrifugation, the supernatant was passed through three Sep-Pak C-18 cartridges (Waters Associates) connected in series. Bound material was eluted with acetonitrile/water/trifluoroacetic acid and freeze-dried. Alternative methods of purifying peptides are well-known in the art. See Ausubel et al. , 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY. After partial purification on the Sep-Pak cartridges, the frog skin extract was redissolved in 1 % (v/v) trifluoroacetic acid/water (4 ml) and injected onto a (25 x 1 cm) Vydac 218TP510 (C-18) reversed-phase HPLC column (Separations Group) equilibrated with 0.1 % trifluoroacetic acid/water at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 21 % over 10 min and to 63% over 60 min using linear gradients. Absorbance was monitored at 214 nm and 280 nm and fractions (1 min) were collected. Fractions containing antimicrobial activity were successively rechromatographed on (250 x 4.6 mm) Vydac 214TP54 (C-4) and Vydac 219TP54 (phenyl) columns. The concentration of acetonitrile in the eluting solvent was raised from 21 % to 56% over 50 min, and the flow rate was 1.5 ml/min. Rechromatography of the fraction displaying antimicrobial activity against E. coli and S. aureus led to the elution of two prominent and well-resolved peaks, designated 1 (containing kassinatuerin-1) and 2 (containing kassinatuerin-2) (Figure 2). The peptides also were eluted from an analytical Vydac phenyl column as sharp, symmetrical peaks, indicative of near homogeneity (chromatograms not shown). Antimicrobial Assays

Activity of the peptides was monitored by incubating lyophilized aliquots of chromatographic effluent (50 μl) in Mueller-Hinton broth (50 μl) with an inoculum (50 μl of 10⁴ CFU/ml) from an overnight culture of either Escherichia coli (ATCC 25922) or Staphylococcus aureus (NCTC 8325) in 96-well microtiter cell-culture plates for 18 hours at 37°C in a humidified atmosphere of 5% CO in air. See Goraya et al., J. Biol. Chem. , 250:589-592 (1998). Incubations with Candida albicans (ATCC 90028) were carried out in RPMI 1640 medium for 48 hours at 35 °C. After incubation, the absorbance at 550 nm of each well was determined using a M.A. Bioproducts model MA308 microtiter plate reader. Minimal inhibitory concentrations (MICs) were measured by a standard microdilution method (Barchiesi et al. , J. Clinical Microbiol. , 32:2494-2500 (1994)) and taken as the lowest concentration of peptide at which no visible growth was observed. To monitor the validity of the assay, incubations with E. coli and S. aureus were carried out in parallel with increasing concentrations of the broad-spectrum antibiotic, bacitracin. Incubations with C. albicans were carried out in parallel with amphotercin B.

The endogenous peptide kassinatuerin-1 possessed broad antimicrobial activity. For example, the minimal inhibitory concentration of purified, endogenous kassinatuerin-1 was 4 μM against E. coli, 8 μM against S. aureus, and 70 μM against C. albicans. In contrast, kassinatuerin-2 was inactive against E. coli, S. aureus, and C. albicans at concentrations up to 100 μM.

Structural Analysis

The amino acid compositions of kassinatuerins-1 and -2 were determined by precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, using a Waters AccQ Tag system with fluorescence detection. The amino acid derivatives were separated by reversed-phase HPLC. The primary structures of kassinatuerins-1 and -2 were determined using automated Εdman degradation. The primary structures of the peptides are shown below and in Figure 3. kassinatuerin-1 GFMKYIGPLIPHAVKAISDLF (SΕQ ID NO: 1) kassinatuerin-2 FIQYLAPLIPHAVKAISDLf (SΕQ ID NO: 2)

The amino acid compositions of the peptides were consistent with the amino acid sequences determined by automated Εdman degradation. Εlectrospray mass spectrometry demonstrated that both peptides contained a C-terminal, α-amidated amino acid residue (kassinatuerin-1: observed Mr 2282.5, calculated Mr 2282.8; kassinatuerin-2: observed Mr 2221.3, calculated Mr 2221.7). The presence of the C-terminal, α-amidated residue was confirmed by analyzing the products of a tryptic digestion. Cleavage of kassinatuerin-1 with trypsin generated three peptides, identified by Edman degradation as the (1-4), (5-15) and (16-21) fragments. The observed molecular mass of the C-terminal fragment Ala-Ile- Ser-Asp-Leu-Ile.NH2 was 629.5 Daltons, compared with a calculated mass of 629.4 Dal tons.

The primary structure of kassinatuerin-1 is distinct from those of antimicrobial peptides isolated from the skins of other frogs. See Figure 3. The peptide is the same length as PGLa, a peptide isolated from skin secretions of the S. African clawed frog Xenopus laevis (Soravia et al , FEBS Lett. 228:337-340 (1988)), and contains the same N- terminal amino acid. However, other than these similarities, structural homology of kassinatuerin-1 to other frog peptides is minimal or nonexistent. Similarly, kassinatuerin-1 contains no significant amino acid sequence identity with the bombinins, magainins, dermaseptins, caerins, uperins, or temporins.

Kassinatuerin-1 contains a net positive charge and has the capacity to form an amphipathic α-helical conformation. The preferred conformation of kassinatuerin-1 in the environment of the bacterial cell membrane is not yet known, but analysis of the secondary structure of the peptide by the method of Gamier et al. (J.Mol. BioL , 120:97-121 (1978)) predicts an α-helical segment between residues 11 and 19. Alignment of the amino acid residues of kassinatuerin-1 in an Edmundson wheel diagram (See Figure 4) demonstrates the amphipathic nature of such a putative helix with the Lys⁴ and Lys¹⁵ residues forming part of a hydrophilic face and the hydrophobic residues segregating together in a hydrophobic face. Although identical to kassinatuerin-1 in its C-terminal region, kassinatuerin-2, contains the substitution Lys⁴ — Gin. Thus, kassinatuerin-2 is not a cationic peptide. This may explain why kassinatuerin-2 is inactive against E. coli, S. aureus, and C. albicans.

It has been demonstrated that cationic antimicrobial peptides, such as magainins, cecropins and dermaseptins, frequently adopt an amphipathic α-helical conformation upon interaction with the phospholipid bilayer of cytoplasmic membranes of microorganisms. See Oren, Z. and Shai, Y. , Biopolymers , 70:451-463 (1998); Boman, H.G., Annu. Rev. Immunol , 13:61-92 (1995); Hancock, R.E.W. and Lehrer, R. , Trends Biotechnol. , 16:82- 88 (1998). While the mechanism of action of these peptides is unclear and the inventors do not wish to be bound by a single theory, it has been suggested that cationic residues, particularly lysine, destroy the ionic gradient across cell membranes by forming ion channels, and that hydrophobic residues disrupt and permeabilize the inner membrane. See Cruciani et al. Eur. J. Pharmacol. , 226:287-296 (1992); Hancock et al. , Adv. Microb. Physiol. , 37:135-175 (1995). For example, a recent study employing Fourier-transform infrared spectroscopy has shown that the action of the human antimicrobial peptide LL-37 involves a 'carpet-like mechanism' with the peptide in a predominantly α-helical conformation aligning parallel with the surface of the zwitterionic-lipid bacterial membrane. See Oren et al , Biochem. J., 341:501-513 (1999).

Synthesis of Peptides

In one embodiment, the antimicrobial peptides of the present invention can be prepared synthetically. A variety of procedures and devices for synthesizing peptides are well-known in the art. As one example, a synthetic version of kassinatuerin-1 was synthesized by solid-phase methodology on a 0.025 mmol scale on an Perkin-Elmer model 432A peptide synthesizer, according to the manufacturer's standard protocols. The peptide, referred to as kassinatuerin-I, was cleaved from the resin and purified to near homogeneity by chromatography on a 25 x 1 cm Vydac 218TP510 C-18 reversed-phase HPLC column. Kassinatuerin-I was prepared with a C-terminal, α-amidated carboxy residue. The identity of the peptide was confirmed by automated Edman degradation and electrospray mass spectrometry.

The chromatographic retention time of the synthetic kassinatuerin-I was the same as that of the endogenous peptide. See Figure 2. The synthetic peptide possessed antimicrobial activity similar to its endogenous counterpart. The minimal inhibitory concentration for synthetic kassinatuerin-I was 5μM against E. coli; 6 μM against S. aureus and 40 μM against C. albicans.

The peptides of the present invention also can be prepared using standard recombinant techniques well-known in the art. See Sambrook et al. , 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY; Ausubel et al , 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY. The invention encompasses an isolated polynucleotide encoding an antimicrobial peptide of the invention. As used herein, an "isolated" polynucleotide is one that is separated from one or more components with which it is naturally associated and is at least 90% pure, based on weight. In preferred embodiments, an isolated polynucleotide of the invention is at least 95% , 98% , or 99% pure. Poly(A)-rich RNA from a frog's skin can be isolated by affinity chromatography and used to construct a cDNA library. A pool of mixed oligonucleotides corresponding to appropriate regions of the target peptide can be synthesized for use as a primer in the RACE (rapid amplification of cDNA 3' end) protocol using the polymerase chain reaction. Amplification products can be cloned into the XlhoI/EcoRV restriction site of the BlueScript vector (Stratagene). The clone can be labeled by random priming and then used to screen the cDNA library to obtain a full-length clone. Positive clones can be selected and analyzed by nucleotide sequencing. This approach has been used successfully to clone the cDNAs encoding esculentin and brevinin IE from the frog Rana esculenta (J. Biol. Chem., 269: 11956-11961 (1994)). An isolated cDNA clone can be expressed using known recombinant techniques. When the peptides are expressed in bacterial hosts, such as E. coli, a preferred technique involves inducing the culture to express the peptide after the culture has reached maturity, as the peptides are antimicrobial.

Alternatively, as the genetic code is universal, one skilled in the art can easily deduce one or more nucleotide sequences which will encode a peptide of the present invention. Also, one of skill in the art will recognize that so-called "preferred codons" can be used to make such a sequence, depending on the host organism to be used for expression. The derived polynucleotide can then be synthesized chemically using the solid-phase phosphoramidite triester method (Beaucage and Caruthers, Tetra. Letts. 22(20): 1859-1862 (1981)); an automated synthesizer (VanDevanter et al. , Nucleic Acids Res. , 12: 6159-6168 (1984)); or the solid support method of U.S. Patent No. 4,458,066. Chemical synthesis generally produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. Functional Variants

One skilled in the art will recognize that modest changes to the composition of the peptides of the present invention will not disrupt their antimicrobial function. Functional variants of the disclosed regulatory region are preferably identified using antimicrobial assays. Functional variants can be identified by comparing the variant's MIC for a particular pathogen to that achieved by the disclosed peptides. Any variant which achieves a MIC against one or more microorganisms, that is greater than 50% of the MIC achieved by one of the disclosed peptides is considered a functional variant within the present invention. Other functional variants within the present invention are those which achieve MIC's at a level greater than 60% or 70% or 80% or 90% or 95% or 98% or 99% of the MIC achieved by one of the disclosed peptides.

Functional variants also can be identified by comparing their structural similarity, or homology, to the disclosed peptides. A peptide possessing 75% or more sequence homology, especially at least 85-95% , to the disclosed peptides is considered a functional variant and is encompassed by the present invention. Mathematical algorithms, for example the Smith-Waterman algorithm, also can be used to determine sequence homology. See Smith and Waterman, J. Mol. BioL, 147: 195-197 (1981); Pearson, Genomics, 11:635-650 (1991). Although any sequence algorithm can be used to identify functional variants, the present invention defines functional variants with reference to the Smith-Waterman algorithm, where kassinatuerin-1 is used as the reference sequence to define the percentage of homology of peptide homologues over its length. The choice of parameter values for matches, mismatches, and inserts or deletions is arbitrary, although some parameter values have been found to yield more biologically realistic results than others. One preferred set of parameter values for the Smith-Waterman algorithm is set forth in the "maximum similarity segments" approach, which uses values of 1 for a matched residue and -1/3 for a mismatched residue (a residue being a single amino acid) (Waterman, Bulletin of Mathematical Biology 46:473-500 (1984)). Insertions and deletions x, are weighted as X = 1 + k/3, where k is the number of residues in a given insert or deletion (Id.). Preferred variant peptides are those having about 75% sequence homology to kassinatuerin-1 using the Smith-Waterman algorithm. Particularly preferred variant peptides have at least about 90% sequence homology. Even more preferred variant peptides have at least about 95% sequence homology, and most preferred variant peptides have at least 98% sequence homology.

Functional variants of the inventive peptides include peptides that are modified forms of the inventive peptides. For example, the antimicrobial activity of the peptides of the present invention can be improved by modifying the amino acid composition of the peptides or by modifying the side chains of the amino acids. Also, variant peptides with antimicrobial activities similar to those of the inventive peptides can be made.

For example, the cationic character of kassinatuerin-1 can be increased by replacing one or more lysine residues with arginine, by replacing the aspartic acid residue with asparagine or alanine, or by replacing any residue with lysine. Such substitutions are likely to promote the peptide's ability to affect the ionic gradient of the cellular membrane. In other examples, the α-helical character of the peptide can be promoted by replacing one or more glycine or proline residues with alanine or serine. In still other examples, the peptide's resistance to degradation by proteolytic enzymes can be increased by replacing one or more amino acids with its D-amino acid isomer.

In other embodiments, the antimicrobial activity of the inventive peptides can be enhanced or maintained by modifying the side chains of the amino acids. For example, the epsilon amino group of a lysine residue can be coupled to a fatty acid such a palmitate or glycosylated sugar. In other examples, the epsilon carboxy group of aspartic or glutamic acid residues or a hydrolyzed glutamine residue can be coupled to fatty alcohols or glycosylated sugars. In still other examples, the epsilon hydroxyl group of threonine, tyrosine, or serine residues can be coupled to fatty acids or glycosylated sugars.

The peptides of the present invention also can be modified to form functional variants by truncating the peptides or by deleting particular residues within the peptides. Examples of preferred functional variants include, but are not limited to, truncated fragments of kassinatuerin-1 , comprising residues 2-21, 3-21, 4-21, 5-21, 6-21, 7-21 , 8-21 and 9-21. In addition, the truncated peptides can be synthetically condensed with a non- natural peptide or peptide sequence optionally containing heterocyclic organic moieties such as a β- or γ-amino acid, an aliphatic diamine, an aliphatic or aromatic dicarboxylic acid, pyridine carboxylic acid, aromatic diols, and the like.

Functional variants also can be made by modifying the amino acid at the C-terminus of the peptide. Such modifications can be used to affect peptide activity. In a preferred embodiment, the peptides of the present invention possess a C-terminal, α-amidated amino acid. In other embodiments, the C-terminal residue can be in the form of a C-terminus carboxylic acid or C-terminus ester. The amide can be a simple amide (CONH2) or an amide derived from a Cl to CIO primary, secondary or tertiary aliphatic or aromatic amine. Similarly, the ester can be derived from Cl to CIO aliphatic or aromatic alcohols. In addition, functional variants of the inventive peptides can be formed by modification with conservative substitutions. The term "conservative substitution" means substitution of an amino acid residue by another that has the same side chain ionicity, basicity, acidity, lipophilicity or hydrogen bonding character as the residue being replaced. Examples include isoleucine, leucine, alanine, valine, phenylalanine, proline and glycine as an interchangeable group, lysine, histidine and arginine as an interchangeable group, serine, tyrosine and threonine as an interchangeable group, cysteine and methionine as an interchangeable group, asparagine, glutamine and tryptophan as an interchangeable group, and aspartic acid and glutamic acid as an interchangeable group.

Therapeutic Uses

For effective prophylactic and anti-infectious use, the inventive peptides and their modified and truncated forms can be administered either alone or in combination with a pharmaceutically-acceptable carrier, by topical, oral, anal, ocular, buccal, nasal, intramuscular, subcutaneous, intravenous or parenteral routes. The ultimate choice of route, formulation and dose is made by the attending physician or veterinarian and is based upon the patient's or animal's unique condition. The inventive peptides can be administered individually or as a composition comprising a mixture of two or more antimicrobial peptides. In addition, the peptides of the present invention can be administered along with one or more additional antimicrobial agents.

A "therapeutically effective" amount of a peptide of the invention may determined by prevention or amelioration of adverse conditions or symptoms of diseases, injuries, or disorders being treated. In particular, the inventive peptides of the present invention are effective for preventing and treating organisms suffering from microbial infections. However, the usual dosage of a therapeutically effective amount, for administration to humans, lies in the range of approximately 50-2000 mg peroral per day, and preferably in about one to four doses where the dose is based upon the activity of the pure peptide. The usual dosage of a therapeutically effective amount, for administration to small animals, is approximately that of humans. For large animals, the usual dosage is higher per unit of body weight, so that the dose given lies in the range of about 20 to 20,000 mg peroral per day. This dosage may vary somewhat with the weight of the subject (human or animal) being treated; in general, about 1 to 40 mg/kg of body weight per day can be employed for humans and small animals while about 1 to 400 mg/kg of body weight per day can be employed for large animals. The peptides of the invention can be used to treat humans, other mammals, such as cattle and other livestock, and other animals, such as birds, including but not limited to chickens, turkeys, ducks, quail, squab, geese, pheasants, and ostriches.

The peptides of this invention can be combined with inert pharmaceutical excipients such as lactose, oil, mannitol, and starch to form pharmaceutical compositions. Such compositions can be formulated into dosage forms such as elixirs, liquids, ointments, lotions, IV fluids, alcohol, tablets, capsules, and the like. For parenteral, intramuscular, subcutaneous and intravenous administration, these peptides can be formulated with an inert, parenterally acceptable vehicle such as water, saline, sesame oil, ethanol buffered aqueous medium, propylene glycol and the like. For topical and oral administration, the inventive peptides can be formulated with waxes, oils, buffered aqueous medium, and the like. These various pharmaceutical dosage forms are compounded by methods well-known in the art.

The peptides of the present invention also can be prepared as pharmaceutically acceptable salts. A pharmaceutically acceptable salt of a peptide of the invention means a salt formed between any one or more of the charged groups in the peptide and any one or more pharmaceutically acceptable, non-toxic cations or anions. Organic and inorganic salts include, for example, those prepared from acids such as hydrochloric, sulfuric, sulfonic, tartaric, fumaric, hydrobromic, glycolic, citric, maleic, phosphoric, succinic, acetic, nitric, benzoic, ascorbic, p-toluenesulfonic, benzenesulfonic, naphthalenesulfonic, propionic, carbonic, and the like. Pharmaceutically acceptable salts may also contain cations including, but not limited to, ammonium, sodium, potassium, calcium, or magnesium.

By following the treatment regimen described above, the skilled practitioner can use the peptides of the present invention to treat a variety of microbial infections, including, but not limited to, Escherichia coli, Enterococcus sp. , Bactereoides fragilis , Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens, Mycobacterium tuberculosis, Streptococcus pneumoniae, Streptococcus pyrogenes, Haemophilus influenzae, Staphylococcus saprophyticus and Candida albicans.

In another embodiment, there is provided a kit useful for treating an antimicrobial infection, comprising a pharmaceutical composition comprising a therapeutically effective amount of a peptide of the present invention and ancillary reagents to effect administration of the peptide. Examples of ancillary reagents include, but are not limited to, buffered solutions and application devices, such as syringes.

Examples

1. Tissue Extraction of Peptides

Adult specimens of K. senegalensis were anaesthetized by immersion in crushed ice and sacrificed by pithing. The skin was immediately removed, frozen on dry ice and stored at -55°C until time of extraction. The frozen tissue was cut into small pieces and extracted by homogenization in ethanol / 0.7 M HCl (3: 1 v/v; 100 ml) at 0°C using a PowerGen rotor/stator-type homogenizer. The homogenate was stirred for 2 hours at 0°C and centrifuged (4000χg, 30 min, 4°C). Ethanol was removed from the supernatant under reduced pressure. After further centrifugation (4000χg, 30 min, 4°C), the extract was pumped onto three Sep-Pak C-18 cartridges (Waters Associates) connected in series at a flow rate of 2 ml/min. Bound material was eluted with acetonitrile/water/trifluoroacetic acid (70.0:29.9:0.1, v/v/v) and freeze-dried.

2. Purification of the Peptides

The frog skin extract, after partial purification on Sep-Pak cartridges, was redissolved in 1 % (v/v) trifluoroacetic acid/water (4 ml) and injected onto a (25x1 cm) Vydac 218TP510 (C-18) reversed-phase HPLC column (Separations Group) equilibrated with 0.1 % trifluoroacetic acid/water at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 21 % over 10 min and to 63% over 60 min using linear gradients. Absorbance was monitored at 214 nm and 280 nm and fractions (1 min) were collected. The fractions containing antimicrobial activity were successively rechromatographed on (250x4.6 mm) Vydac 214TP54 (C-4) and Vydac 219TP54 (phenyl) columns. The concentration of acetonitrile in the eluting solvent was raised from 21 % to 56% over 50 min and the flow rate was 1.5 ml/min. Figure 1 shows that the antimicrobial activity in the extract of K. senegalensis skin, measured against both E. coli and S. aureus, eluted in a single fraction after partial purification on Sep-Pak cartridges. Figure 2 illustrates that rechromatography of this fraction on an analytical Vydac C-4 column led to the elution of two prominent and well- resolved peaks, designated 1 (containing kassinatuerin-1) and 2 (containing kassinatuerin- 2). The peptides were also eluted from an analytical Vydac phenyl column as sharp, symmetrical peaks, indicative of near homogeneity (chromatograms not shown). The approximate yields of the pure peptides were kassinatuerin-1, 103 nmol and kassinatuerin-

2, 133 nmol. Both isolated peptides were greater than 95% pure, as measured by analysis on the analytical Vydac phenyl column.

3. Structural Analysis of Purified Peptides

The amino acid compositions of the peptides were determined by precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using a Waters AccQ Tag system with fluorescence detection. Amino acid derivatives were separated by reversed-phase HPLC. Approximately 1 nmol of peptide was hydrolyzed in 5.7 M HCl for 24 hours at 110°C. The primary structures of the peptides were determined by automated Εdman degradation using a Perkin Elmer Procise model 491 sequenator. Electrospray mass spectrometry was carried out using a Perkin Elmer Sciex API 15OEX single quadrupole instrument. The accuracy of mass determinations was + 0.02% . Kassinatuerin-1 (approximately 5 nmol) in 0.1 M ammonium bicarbonate (100 μl) was digested for 3 hours at 37°C with l-tosylainide-2-phenylethylchloromethylketone (TPCK)-treated trypsin (Sigma) at a substrate/enzyme ratio of 50: 1 (w/w). The reaction mixture was injected onto a (250x4.6 mm) Vydac 214TP54 (C-4) column equilibrated with 0.1 % trifluoroacetic acid/water at a flow rate of 1.5 ml/min. The concentration of acetonitrile was raised to 49% over 60 min using a linear gradient.

The amino acid sequences of the kassinatuerins were determined by automated Edman degradation. The primary structures of kassinatuerin-1 and kassinatuerin-2 are shown in Figure 3. Electrospray mass spectrometry showed that both peptides contained a C-terminal, α-amidated amino acid (kassinatuerin-1: observed Mr 2282.5, calculated Mr 2282.8; kassinatuerin-2: observed Mr 2221.3, calculated Mr 2221.7). The presence of the C-terminal, α-amidated residues was confirmed by tryptic digestion. Cleavage of kassinatuerin-1 with trypsin generated three peptides identified by Edman degradation as the (1-4), (5-15) and (16-21) fragments. The observed molecular mass of the C-terminal fragment Ala-Ile-Ser-Asp-Leu-Ile.NH₂ was 629.5, compared with a calculated mass of 629.4.

4. Synthesis of the Kassinatuerin-I Peptide A synthetic version of the kassinatuerin-1 peptide, entitled kassinatuerin-I, was prepared by solid-phase methodology on a 0.025 mmol scale using a Perkin-Elmer model 432 A peptide synthesizer according to the manufacturer's standard protocols. The peptide was cleaved from the resin with trifluoroacetic acid/water/thioanisole/l,2-ethanedithiol (90.0/2.5/5.0/2.5, by vol.) at 25°C for 2 hours. The crude synthetic peptide was purified to near homogeneity by chromatography on a 25x1 cm Vydac 218TP510 C-18 reversed- phase HPLC column equilibrated with 0.1 % (v/v) trifluoroacetic acid/water at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised from 24.5 % to 52.5% over 50 min. Identity of the peptide was confirmed by automated Edman degradation and electrospray mass spectrometry (observed Mr 2282.5, calculated Mr2282.8). The retention time of synthetic kassinatuerin-I was the same as that of the endogenous peptide under the conditions of chromatography shown in Figure 2.

5. Evaluation of Antimicrobial Activity

Activity of the peptides was monitored by incubating lyophilized aliquots of chromatographic effluent (50 μl) in Mueller-Hinton broth (50 μl) with an inoculum (50 μl of 10⁴ CFU/ml) from an overnight culture of either Escherichia coli (ATCC 25922) or Staphylococcus aureus (NCTC 8325) in 96-well microtiter cell-culture plates for 18 hours at 37°C in a humidified atmosphere of 5% CO2 in air. See Goraya et al. , J. BioL Chem. , 250:589-592 (1998). Incubations with Candida albicans (ATCC 90028) were carried out in RPMI 1640 medium for 48 hours at 35 °C. After incubation, the absorbance at 550 nm of each well was determined using a M.A. Bioproducts model MA308 microtiter plate reader. Minimal inhibitory concentrations (MICs) were measured by a standard microdilution method (Barchiesi et al. , J. Clinical MicrobioL , 32:2494-2500 (1994)) and were taken as the lowest concentration of peptide at which no visible growth was observed. To monitor the validity of the assay, incubations with E. coli and S. aureus were carried out in parallel with increasing concentrations of the broad-spectrum antibiotic, bacitracin. Incubations with C. albicans were carried out in parallel with amphotercin B. The minimal inhibitory concentration of endogenous kassinatuerin-1 was 4 μM against E. coli, 8 μM against S. aureus, and 70 μM against C. albicans. The corresponding MIC values for synthetic kassinatuerin-I were: 5 μM against E. coli, 6 μM against S. aureus and 40 μM against C. albicans. Kassinatuerin-2 was inactive against E. coli, S. aureus and C. albicans at concentrations up to 100 μM. The MIC values for a synthetic kassinatuerin-1 analog containing a carboxyl group at its C-terminus were: 16 μM against E. coli, 55 μM against S. aureus and 160 μM against C. albicans. 6. Evaluation of Antimicrobial Activity Using Clinical Isolates

To further evaluate the antimicrobial activity of kassinatuerin-1, 19 clinical isolates were tested. The clinical isolates were obtained from the Clinical Microbiology Laboratory of St. Joseph's Hospital (Omaha, NE). Peptide activity was monitored by incubating lyophilized aliquots of chromatographic effluent (50 μl) in Mueller-Hinton broth (50 μl) with an inoculum (50 μl of 10⁴ CFU/ml) from an overnight culture of each isolate in 96-well microtiter cell-culture plates for 18 hours at 37°C in a humidified atmosphere of 5% CO2 in air. See Goraya et al., J. BioL Chem. , 250:589-592 (1998). For the H. influenza isolate, the Mueller-Hinton broth was supplemented with 10% (v/v) fetal calf serum. After incubation, the absorbance at 550 nm of each well was determined using a M.A. Bioproducts model MA308 microtiter plate reader. Minimal inhibitory concentrations (MICs) were measured by a standard microdilution method (Barchiesi et al. , J. Clinical Microbiol. , 32:2494-2500 (1994)) and were taken as the lowest concentration of peptide at which no visible growth was observed. To monitor the validity of the assay, incubations were carried out in parallel with increasing concentrations of the broad- spectrum antibiotic, bacitracin. The results are provided in Table 1.

7. Evaluation of Functional Variants

Several functional variants of kassinatuerin-1 were prepared and evaluated. The antimicrobial activity of each variant was compared to its hemolytic activity against human erythrocytes. Such a comparison enables the practitioner to identify antimicrobial peptides which possess both a high HCso value, indicative of minimal damage to host cells, and a low MIC value, indicative of high antimicrobial potency. The cultures were prepared as described in Example 5. To determine the HCso values, peptides in the concentration range 1-140 μM were incubated with washed human erythrocytes (2χl0⁷ cells) from a single donor in Dulbecco's phosphate-buffered saline, pH 7.4 (100 ml) for 1 hr at 37°C. After centrifugation (900χg, 10 min), the absorbance at 541 nm of the supernatant was measured. A parallel incubation in the presence of 1 % v/v Tween-20 was performed to determine the absorbance associated with 100% hemolysis.

The results are provided in Table 2. Kassinatuerin-1. COHN2 and kassinatuerin- l .COOH refer, respectively, to a kassinatuerin-1 peptide possessing a C-terminally α- amidated isoleucine residue or a C-terminally isoleucine in the free acid form. The term "NA" indicates there was no activity up to 200 μM. The HCso value is the concentration (in μM) that produces 50% hemolysis. The values in parenthesis denote the hemolysis at a concentration of 200 μM.

8. Evaluation of Mixed Compositions To evaluate the efficacy of using combinations of two or more antimicrobial peptides, MIC values for kassinatuerin-1 were compared to those obtained from compositions comprising kassinatuerin-1 and kassinatuerin-2. The MIC of kassinatuerin-1 against E. coli and S. aureus was measured in the presence of kassinatuerin-2 at various concentrations, e.g. 0, 10, 22.5, 45, and 65 μM, under standard incubation conditions. MIC values were determined as described above. The results are provided in Table 3. While kassinatuerin-2 was inactive against E. coli and S. aureus at concentrations up to 200 μM, it potentiated the antimicrobial activity of kassinatuerin-1 up to 16-fold against S. aureus and up to 12-fold against E. coli. These results demonstrate that administering compositions comprising a mixture of two or more peptides may be more effective than administering kassinatuerin-1 alone.

Claims

WE CLAIM:

1. An isolated peptide having the sequence of SEQ ID NO: 1.

2. An isolated peptide having the sequence of SEQ ID NO: 2.

3. A functional variant of a peptide of the sequence of SEQ ID NO: 1.

4. A functional variant of a peptide of the sequence of SEQ ID NO: 2.

5. A pharmaceutical composition comprising a therapeutically effective amount of the peptide of SEQ ID NO: 1 and a suitable carrier.

6. A pharmaceutical composition comprising a therapeutically effective amount of a functional variant of the peptide of SEQ ID NO: 1 and a suitable carrier.

7. A pharmaceutical composition comprising therapeutically effective amounts of the peptides of SEQ ID NO: 1 and SEQ ID NO: 2 and a suitable carrier.

8. A pharmaceutical composition of any of claims 5-7, for use in treating an antimicrobial infection in a patient.

9. The use of a pharmaceutical composition of any of claims 5-7, for treating an antimicrobial infection in a patient.

10. The pharmaceutical composition of claim 8, wherein said infection is a bacterial infection.

11. The pharmaceutical composition of claim 8, wherein said patient is a human.

12. The pharmaceutical composition of claim 8, wherein said patient is a mammal.

13. The pharmaceutical composition of claim 8, wherein said patient is a bird.

14. An isolated polynucleotide encoding a peptide having the sequence of SEQ ID NO: 1.

15. An isolated polynucleotide encoding a peptide having the sequence of SEQ ID NO: 2.

16. A kit useful for treating an antimicrobial infection, comprising a pharmaceutical composition of any of claims 5-7 and ancillary reagents to effect administration of said composition.