TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates generally to methods and compositions used to treat bacterial infections and more specifically to Brevinin-1, Esculentin-2, Brevinin-2 and synthetic D-amino acid substituted synthetic peptides for treatment of a Gram-positive and Gram-negative bacteria as well as modifications including N-terminal fatty acid acylation, incorporation of fluorinated amino acids, peptoid residues, PEGylation of the peptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] None.

STATEMENT OF FEDERALLY FUNDED RESEARCH

[0003] This work was supported under a sanctioned project from, titled "Bioprospecting and Preclinical Development of peptide Antibiotics from frog skin secretions" funded by Department of Biotechnology, Govt, of India (BT/PR8835/NDB/52/61/2007).

INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

[0004] None.

BACKGROUND OF THE INVENTION [0005] Antimicrobial peptides (AMPs)/Cationic host defence peptides (CHDPs) have been recognized in prokaryotic cells since 1939 when Dubos identified gramicidin, the antimicrobial substance from a soil Bacillus strain; B. brevis. Gramicidin was found to exhibit activity both in vitro and in vivo against a wide range of Gram-positive bacteria. Gramicidins later showed their therapeutic potential for clinical use and turned out to be the first antimicrobial peptide to be commercially manufactured as antibiotics (Van Epps, 2006). In 1941 another AMP tyrocidin was discovered and it was found effective against Gram- positive and Gram-negative bacteria. Eukaryotic antimicrobial peptides first became a research focus in the middle decades of the twentieth century with the description of cecropins from moths and magainins from frogs. In 1896 Jago et.al showed that a substance that present in wheat is lethal to bread yeast. This molecule was found to be distributed across the plant kingdom, identified as a peptide which can inhibit the growth of a variety of phytopathogens (Fernandez et al., 1972) and was named as purothionin in the mid-1970s (Ohtani, 1977; Mak, 1976).

[0006] Another host defence molecule lysozyme was identified by Fleming in late 1920s, contributed to the organism's innate immunity that helped them to survive in their ecological niches laden with several pathogens. But with the discovery of penicillin in 1928, followed by streptomycin in 1943 (the golden age of antibiotics), researchers lost their interest in the defence strategy adopted by these natural host antibiotics and they remained unnoticed for more than two decades. Rise of multidrug-resistant microbial pathogens in early 1960s, led to the decline of the golden age of antibiotics and an awakened interest in the host defence molecules (Davies, 2006; Katz, 2006). This time point was considered to be the true origin of research into AMPs (Nakatsuji, 2012).

[0007] In 1950s certain cationic proteins were identified to be responsible for the ability of human neutrophils to kill bacteria via oxygen-independent mechanisms, which is not associated with adaptive immune system (Hirsch, 1956; Zeya and Spitznagel 1966). The first animal originated AMP reported is defensin, isolated from rabbit leukocyte (Hirsch, 1956), then bombinin from the epithelia of orange speckled frog, Bombina variegate (Kiss and Michl 1962), lactoferrin from cow's milk (Groves et al., 1965). Since then several groups reported a number of AMPs from leukocytes of humans (Ganz et al., 1985). Boman et al in 1981 isolated the first a-helical AMP cecropins from the hemolymph of silk moth. In 1987, Zasloff isolated and characterized cationic AMPs magainins from the African clawed frog, Xenopus laevis. A few years later, β-defensins and θ-defensins were characterized, from bovine granulocytes (Selsted et al., 1993) and leukocytes of the rhesus monkey (Tang et al., 1999). [0008] In animals, host defence peptides (HDPs) are mostly found in the tissues and organs that are exposed to pathogens (Conlon, 2010; Radek, 2007; Peters et al., 2010; Leippe, 1999). HDPs are produced in lymphs, epithelial cells in gastrointestinal and genitourinary systems, phagocytes and lymphocytes of the immune system (Ganz, 2003; Niyonsaba et al., 2002). From the above mentioned results, a number of investigators considered the possibility that CAMPs may play a role in the defence systems of organisms lacking an adaptive immune system (Ganz, 2003). In the mid-1990s, this was again confirmed on the fruit fly, Drosophila melanogaster, when it was shown that the deletion of a gene encoding an AMP rendered the insect susceptible to a massive fungal infection (Lemaitre et al., 1996). In total, more than 7,000 HDPs have been discovered or synthesised up to date (Zhao et al., 2013). [0009] Amphibians (frogs) enjoyed remarkable evolutionary success and emerged as one of the most successful clade of all organisms. Owing to this high diversity and the long evolutionary residence since their first appearance, amphibians have evolved into resilient organisms adapting to and thriving under all adverse conditions. Living and evolving closely with other dominant life forms such as microbes and insects, amphibians have developed an amazingly strong resistance to infectious microbes and in many cases established symbiotic associations with microbes. Interestingly, during 400 million years of existence, they have rarely succumbed to the evolution of microbial resistance against their potent antimicrobial immune defenses. Amphibians rely solely on their innate immune response for protection against infectious microbes. Their immune defence system is multilayered and a hallmark of their innate immunity is the synthesis and secretion of gene encoded AMPs -"a weapon that we all are born with", which are also seen in invertebrates, vertebrates and in humans to rapidly resist the attack of invading pathogens (Hoffmann et al., 1999; Kimbrell et al., 2001).

[0010] In anuran amphibians AMPs are synthesized and stored in the dermal glands, adrenergically innervated and secreted into the skin mucus upon alarm or injury (Duda et al., 2002; Conlon et al., 2004). These are short (12-50 amino acids), cationic, amphipathic peptides which are active against a wide spectrum of organisms including Gram-positive and -negative bacteria, enveloped viruses, fungi, parasites and even transformed and cancerous cells (Zasloff, 2002; Pukala, 2006). There is enough evidence to believe that these molecules kill the pathogens by disrupting their membrane. Some ABPs identified from frogs can detoxify LPS and thus can prevent septic shock in humans and animals that often associated with the antibiotic therapy. Their fast acting broad- spectrum antimicrobial activity with low induction of bacterial resistance and synergistic activity with classic antibiotics (Csordas et al., 1970) makes these small ABPs a most promising template to develop new generation anti-infective therapeutic agents (McPhee et al., 2005). 90 [0011] Unfortunately, our knowledge about the nature of the defence molecules that helps the amphibians of tropical climate to overcome the hurdles of invading pathogens is very limited. According to the report published by the Environmental Information System (ENVIS), Western Ghats (the mountain range that runs parallel to the western coast of Indian peninsula) is a biodiversity hotspot having 133 species of amphibians forming 11 families

95 and two orders. Since these regions are at entirely different climatic zones, it is very interesting to see the influence of the climatic conditions on the primary and secondary structure of AMPs. A detailed analysis of these vast pools of molecules associated with endemic anuran frogs may help to understand the influence of tropical climate on amino acid selectivity, effect of charge and their distribution in the primary sequence, amphipathic nature 100 and the effect of secondary structure on biological activity. A thorough understanding of the influence of above mentioned parameters on bacterial membrane and mechanisms of action may help to design novel anti-infective agents to control and eliminate threats associated with emerging pathogenic microorganisms especially against those showing resistance against the currently available antibiotics.

105 SUMMARY OF THE INVENTION

[0012] The present invention provides L-peptides and synthetic D-amino acid substituted peptides isolated, purified and characterized from frogs Clinotarsus curtipes and Hylarana temporalis of the Western Ghats, Kerala region of India. Peptides profiles are herein 110 described according to their killing kinetics of Gram-positive and gram-negative bacteria, red blood cell haemolysis and cytotoxic effect on cultured mammalian cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] For a more complete understanding of the features and advantages of the present 115 invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:

FIGURES 1A-1D are line charts showing killing kinetics of S. aureus (FIGURES 1A- 1B) and E. coli (FIGURES 1C-1D) by the B ICtcu peptides. FIGURES 2A-2C are line charts showing killing kinetics of S. aureus (FIGURE 2A), 120 E. coli (FIGURE 2B) and ETEC (FIGURE 2C) by the E2HLte peptides.

FIGURES 3 is a clustered column chart showing the hemolytic activity of E2HLte peptides on RBCs.

FIGURES 4 is a clustered column chart showing the cytotoxic effect of E2HLte peptides on HEK 293 cells.

125 FIGURES 5 is a clustered column chart showing the cytotoxic effect of E2HLte peptides on Hep G ₂ cells.

FIGURES 6A-C are line charts showing killing kinetics of S. aureus (FIGURE 6A), E. coli (FIGURE 6B) and ETEC (FIGURE 6C) by the B IHLte peptides.

FIGURE 7 is a clustered column chart showing the hemolytic activity of B IHLte 130 peptides on RBCs.

FIGURE 8 is a clustered column chart showing the cytotoxic effect of B IHLte peptides on HEK 293 cells.

FIGURE 9 is a clustered column chart showing the cytotoxic effect of B IHLte peptides on Hep G ₂ cells.

135 FIGURES lOA-lOC are line charts showing killing kinetics of S. aureus (FIGURE

10A), E. coli (FIGURE 10B) and ETEC (FIGURE IOC) by the B2HLte peptides.

FIGURE 11 is a clustered column chart showing the hemolytic activity of B2HLte peptides on RBCs.

FIGURE 12 is a clustered column chart showing the cytotoxic effect of B2HLte 140 peptides on HEK 293 cells.

FIGURE 13 is a clustered column chart showing the cytotoxic effect of B2HLte peptides on Hep G ₂ cells.

FIGURES 14A-14B are line charts showing the killing kinetics of ETEC and S. aureus by the RHLte peptides. 145 FIGURE 15 is a clustered column chart showing the hemolytic activity of RHLte peptides on RBCs.

FIGURE 16 is a clustered column chart showing the hemolytic activity of the RHLte 1 RHLte2, and RHLte3 cocktail of peptides on RBCs.

FIGURE 17 is a clustered column chart showing the cytotoxic effect of RHLte 150 peptides on HEK 293 cells .

FIGURE 18 is a clustered column chart showing the cytotoxic effect of RHLte peptides on Hep G ₂ cells.

DETAILED DESCRIPTION OF THE INVENTION

[0014] While the making and using of various embodiments of the present invention are 155 discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0015] To facilitate the understanding of this invention, a number of terms are defined below.

160 Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as

165 outlined in the claims.

[0016] The following abbreviations are used herein, HPLC: High Performance Liquid Chromatography; MALDI-TOF: Matrix- Assisted Laser Desorption Ionization Time of Flight; RACE: Rapid Amplification cDNA Ends; TFA: trifluoroacetic acid; MIC: Minimum Inhibitory Concentration; AMP: antimicrobial Peptide; HDP: Host Defence Peptides; CLSM: 170 Confocal Laser Scanning Microscopy; SEM: Scanning Electron Microscopy; AFM: Atomic Force Microscopy; FACS: Fluorescence-activated cell sorting; ATCC: American Type Culture Collection BIHLte: Brevininl Hylarana temporalis; CAMPAs: Cationic Anti- Bacterial Peptide Amides; CD: Circular Dichroism; E.coli: Escherichia coli; E2HLte: Esculentin2 Hylarana temporalis; RHLte :Ranacyclin Hylarana temporalis; Hep G ₂ : 175 Hepatocellular carcinoma (liver cancer cell line), HEK 293 cells; Human embryonic kidney cells; H. temporalis: Hylarana temporalis; MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5- Diphenyltetrazolium Bromide; μΜ: Micro molar; MIC: Minimum Inhibitory Concentration; MRSA: Multi drug resistant Staphylococcus aureus; μΐ: microliter; OD: Optical density; RBC: Red Blood Cells.

180 [0017] CABPAs were isolated skin secretion of frogs endemic to the Western Ghats region, Kerala, India. Five novel brevinin family Cationic antibacterial peptide (CABPs) were identified and categorised under brevinin-1 family (B lCTcul-B lCTcu5), were isolated from skin secretions of endemic bicoloured ranid frog Clinotarsus curtipes and fifteen CABPs were identified from the skin secretion of another frog Hylarana temporalis, categorised

185 under esculentin-2 family (four peptides :E2HLtel-E2HLte4), esculentin-1 family (three peptides: B 1 HLte 1 -B 1 HLte3 ) , brevinin-1 family (five peptides:B2HLtel-B2HLte5) and ranacyclin family (three peptides:RHLtel-RHLte3). All these peptides were chemically synthesized as peptides and as peptide amides. The CABPs and the Cationic antibacterial peptide amides (CABPAs) were named by giving emphasis to both the genus and species

190 from where they are isolated. In addition, five synthetic D-amino acid substituted peptides (DBlCTcul, -L DB lCTcul, 9DB lCTcu5, DB lCTcu5, DB lCTcu3) of the B lCTcul and B lCTcu5 brevinin-1 family peptides.

[0018] B lCTcul-B lCTcu4 are linear peptide with 18 to 21 amino acid residues while B lCTcu5 is a cyclic twenty-one residue peptide amide. It possess an N-terminal 195 hydrophobic region with a proline hinge middle region and a positively charged C-terminal seven member cyclic peptide domain named Rana box (SEQ ID NO: 1 Cys-Lys-Xaa4-Cys) formed by the cysteine intradisulphide bridge. All these peptides assume a-helical conformation and existed as non-interacting helices in membrane mimicking solvents. They assume alpha helical conformation upon binding with bacterial membrane.

200 [0019] These peptides exhibited antibacterial activity against broad spectrum of bacteria.

B lCTcul-B lCTcu3 showed high antibacterial activity against both Gram-positive and - negative strains with MICs ranging from 1.25-100 μg/ml. Since biofilim formation by the bacteria results pathogenesis and antibiotic resistance, these AMPs shows the ability to prevent its formation. B lCTcul is non-haemolytic while the other peptides are haemolytic at

205 higher concentrations. B lCTcu4 can arrest the multiplication of drug resistant superbug MRSA with an MIC of 12 μg/ml level for unknown mechanism. B lCTcu5 which is nontoxic to macrophages showed potent in vitro anti-M. tuberculosis activity and eliminates the intracellular mycobacterium growth within THP1 derived activated macrophages in a concentration dependent manner. B lCTcu5 penetrates into the host cell and localizes in the

210 cytoplasm. Its mode of action may involve several events including the lysis of the bacterial membrane which leads to the membrane rupture. B lCTcu5 can penetrate into the distinctive cell wall of M. tuberculosis which is the primary permeability barrier for drug molecules. Immune enhancing effect of the peptide may also play a major role in the elimination of intracellular mycobacterium. Anti-TB activity of B lCTcu5 is due to a multi-complex

215 mechanism involving several targets such as cell wall, membrane associated targets and intracellular targets.

[0020] Killing kinetic experiments showed that antibacterial peptides B ICTcul, B lCTcu2 and B lCTcu4 cannot induce complete membrane depolarization of the E. coli cells at their respective MICs even after 3 hours incubation. These data show that the membrane 220 depolarization cannot always lead to bacterial killing and could be one of the several events or an additional effect presumably after cell death.

[0021] Structure-activity analysis using several synthetic B lCTcu5 analogs designed by the Systematic and Modular Modification and Deletion (SMMD) approach showed that amino acid sequence present in the C-terminal rana box is responsible for its preferential interaction

225 with Gram-negative strains. Bactericidal nature of the B lCTcu5 is not affected by the enhancement in N-terminal cationicity or hydrophobicity by suitable amino acid substitution or deletion. The sequential removal of N-terminal amino acids had a negative effect on bactericidal potency of B lCTcu5. However, following removal of four N-terminal amino acids, the new peptide displayed bactericidal effect comparable to, or in excess of, the parent

230 peptide with reduced haemolytic character. The antibacterial pharmacophore N-terminal region of B lCTcu5 is the active core of B lCTcu5 and its higher activity resulted from its improved inner membrane permeabilisation capacity. The presence of the C-terminal disulphide bond is not necessary to display antibacterial activity. Moreover, this short cyclic region helped to promote hydrophobic character of the peptide, which in turn contributed to

235 its enhanced hemolytic nature. Removal of the C-terminal rana box region drastically reduced its antibacterial and haemolytic nature, showing that this region is important for membrane targeting. This amphiphilic region is also responsible for providing the required conformational stability to the peptide and reduced sensitivity to proteolytic cleavage.

[0022] Four novel Esculentin2 family cationic antibacterial peptide amides (E2CABPAs), 240 listed in Table 3, were identified from the skin secretion of frog Hylarana temporalis of the Western Ghats. These peptides are cyclic at the C-terminal region with Cys-Cys disulphide bond and are highly effective in arresting the bacterial growth of the tested panel of Gram- negative and Gram-positive bacteria strains. Though E2CABPAs are effective against both the Gram-positive and -negative bacteria, they showed comparatively higher preferential 245 activity against Gram-positive bacteria like Staphylococcus aureus, Streptococcus mutans, Streptococcus gordonii as depicted by their low MIC values (0.7-3 μΜ). Time dependent micro broth dilution assay showed that these peptides are bactericidal in nature. Haemolytic activity of E2CABPAs against red blood cells were tested up to ΙΟΟμΜ concentration. E2HLtel with an MIC of 0.7-25μΜ showed only negligible effect up to ΙμΜ. These peptides 250 are noncytotoxic to mammalian cells, human embryonic kidney cells (HEK 293) and human liver carcinoma cell line Hep G ₂ .

[0023] Peptides B lHLtel-3, listed in Table 5, showed antibacterial activity against both the Gram-positive and -negative bacteria with MIC ranging from 0.7 to 25 μΜ. These peptides are bactericidal in nature. Though the Brevininl peptides are highly haemolytic in nature. H. 255 temporalis derived Brevininl peptides are not that active against RBCs at their respective MICs. B lHLte3 showed low toxicity against all the tested bacterium and were also found to be nontoxic to RBCs. These peptides are nontoxic to HEK 293 cells and Hep G ₂ cells at their respective MICs.

[00241 Listed in Table 7. Brevinin2 peptides. B2HLte3 and B2HLte4 are the most potent 260 followed by B2HLte2, B2HLte5 and B2HLtel. These peptides are non-haemolytic in nature at their respective MIC. 30-50% of RBCs were destroyed at ΙΟΟμΜ concentration. B2HLte5 turned out to be the best among the B2CABPAs as it showed very good antibacterial activity and very low anti-erythrocyte activity. All these peptides are non-toxic to normal HEK 293 cells at lower concentration. B ICABPAs are found to be more toxic to B2CABPAs than to 265 Hep G ₂ cells.

[0025] Listed in Table 9, Ranacyclin peptides RHLtel and RHLte2 contain eighteen amino acid each in their primary sequence with a single amino acid mutation. These peptides lower in activity against the tested bacterial strains compared to other family peptides isolated from H. temporalis. Ranacyclin peptides are comparatively more active against Gram-positive 270 bacterial strains and showed good activity against P. aeruginosa even at low concentrations.

Bactericidal in nature these peptides showed a different type of killing curve compared to other family peptides. The H. temporalis Ranacyclin peptides are least toxic to RBCs and non-toxic to HEK 293 and Hep G ₂ cells at their respective MICs.

[0026] DB lCTcul, DB lCTcu3 and DB lCTcu5 are D-peptides. In -L DB lCTcul N-terminal 275 Leu is removed whereas in 9DB lCTcu5, N-terminal nine amino acids from the N-terminal region were replaced with corresponding D-amino acids. Antimicrobial activity of D peptides was tested against the below listed Gram-positive and -negative bacterial strains and the MICs were determined. These concentrations represent the minimum dosage required to kill the entire bacteria. All these peptides showed antimicrobial activity in the range of 8.25- 280 100μg/mL. Killing kinetic analysis showed the bactericidal nature of these frog derived peptides. These peptides are non-hemolytic and non-cytotoxic at their respective MICs. Hemolytic assay these peptides are nontoxic at their respective MICs.

[0027] The bactericidal potency of the D-peptide (DB lCTcu5) showed that there is no stereo-specific interaction between the peptide and the bacterial membrane. These data

285 suggest that both the C- and N-terminal regions of B lCTcu5 is necessary for bactericidal activity, even though the active core region is located near the N-terminal. It is important that there is an optimum balance between the cationicity and hydrophobicity of the peptide. This balance is necessary for its effective initial interaction with the bacterial membrane. An increase in the cationicity of the peptide can promote its interaction with anionic

290 phospholipids and the negatively charged lipopolysaccharides of the bacterial cell membrane.

Judicious modification at the N-terminal region can produce short SMMD analogs with enhanced bactericidal activity and low toxicity against eukaryotic cells.

[0028] Mass spectra were recorded on a MALDI-TOF mass spectrometer (Buker). Reagents, solutions used in study were sterile and vortexed prior to each use. Microorganisms were 295 collected from ATCC, MTCC. Clinical strains were collected from Disease Biology division, RGCB. Fmoc-amino acids, HBTU, HOBt, CLEAR amide resin, Rink linker were purchased from Novabiochem Ltd,UK., DIE A, Piperidine, TFA, Ethandithiol, Thioanisole, Triisopropyl silane, a-cyano-4-hydroxy cinnamic acid, Kanamycin, Ampicillin; Resazurin, EDTA, Sodium chloride, antimycotic solution, Sterile water, Triton X-100, LPS, Polymyxin B, 300 Dansyl polymyxin B, NPN, SYTOX green, FITC, POPC, and POPG were purchased from Sigma, USA. DiBAC ₄ (3) was from Invitrogen.

[0029] The secondary structure was analysed by CD spectroscopy as given in M4.7. As a measure of cytotoxicity, hemolysis (M4.5) and MTT assays were performed for all the tested peptides. The membrane destabilizing effect of the peptides were monitored flow

305 cytometrically as described in M4.10 and the results were compared. All solvents including DMF, Acetonitrile, Diethyl ether (specially dried) used were of HPLC grade, purchased from E-Merck (India). LB agar and broth were purchased from Hi-media, India RP-HPLC was done on a Shimadzu purifier using C-18 reverse phase analytical and semi preparative Phenomenex column of (150x 4.6mm and 250x25.2 mm). M. tuberculosis virulent laboratory

310 strain H37Rv was routinely subcultured on fresh Lowenstein- Jensen slants and incubated at 37°C for 4-6 weeks in a biosafety level three (BSL 3) facility. Broth cultures were prepared by inoculation of one loopful of bacterial colony to Middlebrook 7H9 broth (Difco, USA) containing Tween-80 (0.05%), glycerol (0.5%) and OADC (5%) supplement at 37°C for 7-10 days.

315 [0030] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

320 [0031] The following examples are included to demonstrate particular embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute specifically contemplated modes for its practice. However, those of skill in the art should, in light of the

325 present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0032] Example 1. Five peptides (B lCTcul-5) belong to brevinin-1 sub-family were identified from the frog Clinotarsus curtipes skin secretion. TABLE 1 illustrates the amino 330 acid sequences and their structural parameters (GRAVY: grand average of hydrophobicity, calculated as the sum of hydropathy values of all the amino acids, divided by the number of residues in the sequence.)

[0033] TABLE 2 depicts the Minimum Inhibitory Concentrations required to kill all microorganisms (MIC) for the B lCTcul-5 peptides against a panel of Gram-positive and Gram- negative bacterial strains. (ND = No Detectable activity tested up to 200μg/ml, IS = Isolated Strain).

MRSA ATCC 43300 ND 50 50 <6.25 ND

Gram-negative

E. coli MG1655 10 10 6.25 20 7

Vibrio cholerae (IS) 12.5 12.5 12.5 25 15

% Hemolysis 1 17.8 45 54.8 37.5

[0034] All the peptides were active in the range of 6.25-100 μg/mL. The MICs required to 340 eliminate different bacterial strains were found to be different. E. coli and V. cholerae were highly sensitive to these peptides and the MICs required was below 25 μg/mL. B ICTcul to B lCTcu3 were highly effective against S. aureus, while B lCTcu4 showed only a moderate effect. At the same time B lCTcu4 was highly active against MRSA at a concentration below 6.25μg/mL. Removal of the N-terminus phenylalanine residue from B lCTcu2 produced 345 B lCTcu3. Though this deletion helped the slight enhancement of antibacterial nature, its hemolytic potential also increased to about 2.5 fold excess. Overall, B ICTcul, B lCTcu2 and B lCTcu3 showed higher antibacterial activity against Gram-positive and -negative bacterial strains compared to B lCTcu4 and B lCTcu5.

[0035] About half of the Staphylococcus aureus strains isolated from various medical 350 institutions are found to be resistant to antibiotic methicillin. Emergence of the new enterococci, resistant to a highly potential antibiotic vancomycin may accelerate the spread of vancomycin-resistant genes, via plasmids, throughout the other species and eventually eliminate the efficacy of this drug. In this respect B lCTcu4 draw special interest because it can effectively arrest the multiplication of drug resistant super bug MRSA at very low 355 concentration, and the vancomycin resistant enterococcus at a moderate concentration.

[0036] Hemolytic assay can provide valid information regarding the cytotoxic nature of the peptides. In accordance with the previous reports of high hemolytic behavior of brevinin-1 peptides, B lCTcu2-5 were also hemolytic. In contrast, B ICTcul was a non-hemolytic peptide even at a higher concentration of 100μg/mL. The high antimicrobial activity of 360 B ICTcul along with its specificity towards prokaryotic over eukaryotic membrane makes it a promising candidate to design novel peptide antibiotics. B lCTcu4, though found to be an ideal antibacterial candidate against MRSA, is highly hemolytic. [0037] FIGURE 1 illustrates the Kinetics of killing of bacteria S. aureus (FIGURES 1A and IB) and E. coli (FIGURES 1C and ID). Cells in the mid-logarithmic phase of growth (10 ⁵ 365 colony forming units) were incubated with different concentration of peptides (FIGURES 1A and 1C, 3.25 mg/ml, FIGURES IB and ID, 6.5 mg/ml) at 37°C, aliquots were drawn out at different time intervals and plated onto a LB agar plate. The number of colony forming units were counted after incubating the plates at 37°C for 24 hours.

[0038] Killing kinetics experiment can provide the rate at which a peptide kill bacteria over 370 time and it indicates whether an antibacterial agent is bacteriostatic or bactericidal in nature.

Killing kinetics E. coli and S. aureus at their sub MICs showed that these peptides can initiate their action immediately after addition. About 50% reduction in CFUs were observed within a short period but their complete elimination required 60-90 minutes. When B lCTcu3 was added, a sudden decrease in S. aureus CFUs was observed in the first 5 minutes but in the 375 case of E. coli the entire cells were inhibited in about 15 minutes. BlCTcu3 was the rapid acting candidate against E. coli and S. aureus. Addition of B lCTcu2 produced an immediate drop in the viable S. aureus culture in 15 minutes. This immediate drop in CFUs reflects the peptide's bactericidal nature.

[0039] Example 2. TABLE 3. Shows the H. temporalis derived Esculentin2 family cationic 380 antibacterial peptide amides (E2CABPAs) consist of four members E2HLtel- E2HLte4.

[0040] The in vitro antibacterial analysis was carried out against a panel of Gram-negative and Gram-positive bacteria by micro both dilution assays. They are highly effective in arresting the bacterial growth of the tested strains. Effectiveness of the E2CABPAs can also 385 be clearly visualised from the reduction in turbidity and is quantitatively measured by noting the absorption at 600nm. The results were further confirmed by the resazurin assay where viable cells with active metabolism, reduces resazurin (7-Hydroxy-3H-phenoxazin-3-one 10- oxide) to pink coloured resorufin. E2HLte2 showed lowest antibacterial character among the E2CABPAs. Even though E2HLte2, E2HLte3 have the same percentage hydrophobicity, 390 E2HLte3 is a better antibacterial agent. This may be attributed to its higher cationicity. By virtue of its high hydrophobic nature, E2HLtel is the most potent antibacterial agent among the esculentins identified from the skin secretion of the frog H. temporalis.

[0041] Though E2CABPAs are effective against both the Gram-positive and -negative bacteria, they showed comparatively higher preferential activity against Gram-positive

395 bacteria like Staphylococcus aureus, Streptococcus mutans, Streptococcus gordonii as depicted by their low MIC values (0.7-3 μΜ). These peptides are highly effective against Bacillus subtilis, enterotoxigenic E. coli, McVo9-a clinical strain of Vibrio and P. aeruginosa. MIC of these peptides (except E2HLte2) against P. aeruginosa is 25 μΜ which is considered to be very good as most of the known E2CABPAs are reportedly inactive against

400 Pseudomonas. Ability of E2CABPAs to eliminate the bacteria decreases in the order E2HLtel > E2HLte4 > E2HLte3 > E2HLte2 which is in agreement with their net charge and

hydrophobicity.

[0042] TABLE 4 depicts the Antibacterial assays of Esculentin2 peptide amides. To provide 405 an effective chemical shield against external pathogenic microbial aggression, frog H. temporalis secrete all these family peptides at different concentrations. This cannot be analysed because it is impossible to know how much individual peptides are present in the secretion. Presence of a peptide or peptides may have a marked influence on the antibacterial activity of an individual peptide. This synergetic effect contributed by E2CABPAs 410 combination were tested by taking a cocktail containing 400μΜ of each peptides. This cocktail brought down MIC compared to any of the individual members showing that there was some synergetic effect.

[0043] Time dependent micro broth dilution assay of the E2CABPAs were carried out at their respective MICs and sub-MIC concentrations and plated against enterotoxigenic E. Coli,

415 S.aureus MTCC 9542 and E.coli 25922. FIGURE 2 depicts the Killing kinetics of E2 peptides against S. aureus, E. coli and ETEC. Killing kinetic was followed by counting the CFUs after 24 hr incubation as well as by measuring the OD values at 600nm. There is a drastic reduction of live cells immediately after the addition of E2CABPAs showing that they act very fast. Their addition eliminated about 50% of the cells within the first 15 minutes,

420 which was increased to about 99% within 30-60 minutes and after 3 hours OD values at 600nm remained same, showing that cell death was completed. This clearly demonstrated that the peptide induced bacterial killing is bactericidal in nature.

[0044] FIGURE 3 the shows effect of E2CABPAs on RBCs. Hemolytic activity of E2CABPAs against red blood cells were tested up to ΙΟΟμΜ concentration. E2HLtel with an

425 MIC of 0.7-25μΜ showed only negligible effect up to ΙμΜ. Percentage hemolysis increased as the peptide concentration increases. Addition of 12μΜ E2HLtel resulted about 20% hemolysis and increased to above 50% at ΙΟΟμΜ concentration. E2HLte2 is less toxic compared to E2HLtel and elicit about 20% hemolysis at 25μΜ and reached 30-35% at higher concentrations. E2HLte3 is the best among E2CABPAs as it induced only about 10%

430 hemolysis at ΙΟΟμΜ concentrations. Though E2HLte4 is less toxic at lower concentrations showed about 40% RBC lysis at ΙΟΟμΜ. Peptide cocktail elicited about 40% hemolysis at lower concentrations and went up to more than 50% at ΙΟΟμΜ which further established the hypothesis that esculentins are less toxic to RBCs compared to other family peptides. E2HLte3 is the best candidate that can serve as a template to design novel peptide based 435 antibacterial agents.

[0045] Concentration dependent cytotoxic effect of E2CABPAs on mammalian cells was further tested with HEK 293 and human liver carcinoma cell line Hep G ₂ (FIGURE 4). Almost all the H. temporalis E2CABPAs are non-toxic to normal HEK 293 cells up to ΙΟΟμΜ peptide concentrations and showed 90-100% cell viability.

440 [0046] FIGURE 5 illustrates the effect of E2CABPAs on Hep G ₂ cells. Liver cancer cells membrane consist of anionic PS, sialic acid linked to glycolipids and glycoproteins. Presence of these molecules in Hep G ₂ cell membrane contributed positively to enhance the overall negative charge on the membrane. This difference in membrane composition helped to provide membrane selectivity to the peptide. Though E2CABPAs maintained the Hep G ₂

445 cells viability at low concentration, it showed marked increase in toxicity at higher concentrations. This difference in toxicity compared to normal cell showed that E2CABPAs can selectively target the cancer cells compared to normal cells.

[0047] Example 3. TABLE 5 shows the three Brevininl family of cationic antibacterial peptide amides (B ICABPAs) identified from H. temporalis are named as B IHLtel, B lHLte2 450 and B lHLte3.

[0048] TABLE 6 illustrates the antibacterial activity of B ICABPAs. MICs are average triplicate values in three independent assays. Antibacterial activity wise B lHLte2 is the b among the B ICABPAs while B lHLte3 is the weakest. TABLE 6: Antimicrobial activities for B lHLte 1-3 (B l-1 to B l-3) peptides against Gram positive and Gram negative bacteria

455 [0049] The bacteriostatic or the bactericidal nature of the H. temporalis derived CABPAs- Brevinin peptides were analysed by the time dependent kinetic study using ETEC, E.coli 25922 and S .aureus MTCC 9542 (FIGURES 6A-6C). The kinetic analysis clearly demonstrated that like E2CABPAs, Brevinin peptides were also bactericidal in nature. Killing curves of B ICABPAs showed they are highly active. In the first 15 minutes there was

460 no considerable change in the number of colonies, rather there was an increase in colony forming units. But in 15-30 minutes there is a drastic reduction in the number of cells. About 50% cell death was observed in about 30 minutes that increased up to 99% in about 60-90 min. The rate of killing at their respective MICs clearly demonstrated the bactericidal nature of these peptides.

465 [0050] FIGURE 7 demonstrates the effect of B ICABPAs on red blood cells. Values are averages of triplicate values obtained in three independent assays. Brevinin 1 peptides are highly haemolytic in nature. H. temporalis derived B ICABPAs and B2CABPAs (FIGURE 11) are also found to be highly active against RBCs than the E2CABPAs even at lower concentrations. About 30-70% of the cells were destroyed by brevininl peptide addition.

470 B lHLtel is very active at lower concentration and destroyed about 30% of the RBCs soon after its addition. The percentage hemolysis increased steadily up to 70% as the peptide concentration increased from ΙμΜ to ΙΟΟμΜ. Potent antibacterial peptides B IHLtel and B lHLte2 are found to be equally potent against RBCs. B lHLte3 that showed low toxicity against all the tested bacterium was found to be nontoxic to RBCs. FIGURES 8 and 9 show the concentration dependent cytotoxic effect of B ICABPAs on mammalian HEK 293 and Hep G ₂ cells respectively.

[0051] Example 4. TABLE 7 depicts the Brevinin-2 family of cationic antibacterial peptide amides (B2CABPAs) identified from H. temporalis named as B2HLtel, B2HLte2, B2HLte3, B2HLte4 and B2HLte5.

[0052] Among B2CABPAs, antibacterial activity wise B2HLte3 is the most active while B2HLte5 is the least, followed by B2HLtel TABLE 8. The overall result showed that among the B2CABPAs, B2HLte3 and B2HLte4 with 53% hydrophobicity are most potent followed by B2HLte2, B2HLte5 and B2HLtel

TABLE 8: Antimicrobial activities for B2HLtel-5 (B2-1 to B2-5) peptides against Gram positive and Gram negative bacteria

[0053] FIGURES lOA-lOC are line charts showing killing kinetics of S. aureus (FIGURE 10A), E. coli (FIGURE 10B) and ETEC (FIGURE IOC) by the B2HLte peptides. The hemolytic nature of B2CABPAs are less compared to B lCABPAs and killed 30-50% of RBCs at ΙΟΟμΜ concentration. B2HLte5 turned out to be the best among in B2CABPAs as it showed very good antibacterial activity and very low anti-erythrocyte activity. Percentage hydrophobicity showed a marked influence on haemolytic nature, which is evident from the more anti-erythrocyte nature of B lCABPAs (FIGURE 7) and B2CABPAs (FIGURE 11) compared to E2CABPAs. Some of the peptides with good antimicrobial activity turned out to be toxic to RBCs also. [0054] The cytotoxic nature of B lCABPAs (FIGURE 8) and B2CABPAs (FIGURE 12) on mammalian cells were analysed using HEK 293 and B lCABPAs (FIGURE 9) and B2CABPAs (FIGURE 13) on Hep G ₂ cells. All these peptides are non-toxic to normal HEK 293 cells at lower concentration and at ΙΟΟμΜ concentrations more than 90% cell viability was observed. Compared to normal cells, membrane composition of cancer cells are different and this difference resulted higher interaction between the Hep G ₂ cell membrane and the peptides. B lCABPAs are found to be more effective than B2CABPAs in killing Hep G ₂ . B lCABPAs though maintained their Hep G ₂ cell viability at lower concentrations, they become toxic as the peptide concentration increased. B lCABPAs could reduce the cell viability to less than 50% while B2CABPAs which are non-toxic to Hep G ₂ cells at lower concentration can kill only 5-10% as the peptide concentration goes above 50μΜ.

[0055] Example 5. TABLE 7 depicts the Ranacyclin peptides identified from H. temporalis skin secretions are RHLtel to RHLte3.

[0056] Ranacyclin peptides RHLtel and RHLte2 contain eighteen amino acid each in their primary sequence. They are very similar with same hydrophobicity and the only difference is a single mutation in one amino acid residue.

TABLE 10: Antimicrobial activities for RHLtel-3 (R1-R3) peptides against Gram positive and Gram negative bacteria

[0057] TABLE 10 illustrates H temporalis derived Ranacyclin CABPAs are not that active against the tested bacterial strains compared to E2CABPAs, B l and B2CABPAs. Their percentage hydrophobicity is low and the helical wheel diagram indicates the presence of several hydrophobic residues distributed in the hydrophilic core region. These peptides are more active against Gram-positive bacterial strains. Both RHLtel and RHLte2 contain eighteen residues with almost the same percentage hydrophobicity. The only difference between them is a single amino acid mutation where one Gly is replace with Arg in RHLte2

520 which results in increased charge and its overall distribution. Normally this should enhance its interaction with the bacterial membrane and therefore its antibacterial nature. But RHLte2 showed no dramatic changes in its activity compared to RHLtel. RHLte3 with net four positive charges showed comparatively a better antibacterial activity. These peptides have good activity against P. aeruginosa even at low concentrations. RCABP cocktail did not

525 showed any notable change in activity except against P. aeruginosa where the mixture brought down MIC to 6μΜ.

[0058] RCABPAs showed a different type of killing curve when compared to E2CABPAs and BCABPAs. The killing kinetic analysis was carried out in ETEC and Staph aureus as the peptide was not showing any detectable activity against E. coli 25922 in the tested 530 concentrations (FIGURE 14). In the initial 30 min the number of cells in peptide treated plates and the growth control remained almost the same but after 60 min, about 30% reduction in cells were observed in RCABPAs treated cells. After 150 min incubation about 30-50% of the above mentioned cells were destroyed. Ranacyclin peptides are able to kill only about 60-70% of the cells even after 180 min of incubation.

535 [0059] Out of the four H. temporalis skin secreted peptide families E2CABPAs, B ICABPAs, B2CABPAs and RCABPAs, Ranacyclin peptides showed lowest hydrophobicity and toxicity against the bacterial cells. This result is in accordance with observations of Chou et ah, that peptide with high hydrophobicity and amphipathicity (hydrophobic moment) are haemolytic rather than good antibacterial agent. FIGURE 15 shows that H .temporalis Ranacyclin

540 peptides are least toxic to RBCs and only RHLtel induced about 60% RBC lysis at 200μΜ.

Their cocktail also showed only negligible RBC lysis, less than 10% at ΙΟΟμΜ concentration (FIGURE 16). All these results showed that hydrophobicity modulated the haemolytic activity and as the hydrophobicity increases RBC lytic activity also increases.

[0060] FIGURES 17 and 18 illustrates the toxic nature of these peptides were further tested 545 against HEK 293 and Hep G ₂ cells respectively. The results showed that RCABPAs are nontoxic to HEK 293 and Hep G ₂ cells at their respective MIC concentrations, but as their concentration increased from 50-100μΜ these peptides can kill only about 5-10% of cell. [0061] Example 6. TABLE 11 lists the Novel D-Peptides and their sequences determined as antibacterial drug leads for elemenating bacteria. DB lCTcul, DB lCTcu3 and DB lCTcu5 are D-peptides. In -L DB lCTcul N-terminal Leu is removed whereas in 9DB lCTcu5, N- terminal nine amino acids from the N-terminal region were replaced with corresponding D- amino acids.

[0062] As depicted in TABLES 12 and 13, antimicrobial activity of D peptides were tested against the listed Gram-positive and Gram-negative bacterial strains and the MICs were determined. These concentrations represent the minimum dosage required to kill the entire bacteria. All these peptides showed antimicrobial activity in the range of 8.25-100μg/mL.

[0063] Killing kinetic analysis showed the bactericidal nature of these frog derived peptides. These peptides are non-hemolytic and non-cytotoxic at their respective MICs. Hemolytic assay these peptides are nontoxic at their respective MICs.

[0064] TABLE 14 illustrates the details of biological resources and/ or associated knowledge used in the invention. Biological Resources Accessed

Common Scientific Isolate Source Village Town District State name name

Ranid frog Clinotarsus Skin Periyar Kumily Thekkad Idukki Kerala curtipes Secretio Wildlike y

n Sanctuary

Ranid frog Hylarana Skin Malakappa Athirappil Chalakku Thrissur Kerala temporalis Secretio ra iy dy

n

565 [0065] In summary, B lCTcu5 possess a C-terminal hepta peptide loop formed by an intra disulphide bridge whereas other members of this family B lCTcul-B lCTcu4 were acyclic. They were active against both Gram-positive and Gram-negative bacteria and the minimum dosage required to kill the entire bacteria which is termed as MIC is in the range 6.25- 10(Vg/mL. These molecules are bactericidal in nature. B lCTcu4 was highly active against

570 MRSA with the MIC 6.25μg/mL whereas B lCTcu5 is highly active against M. tuberculosis.

All these peptide amides are non-haemolytic and noncytotoxic to normal HEK 293 cells at their respective MICs. B lCTcu2-B lCTcu5 adopted a well-defined alpha helical structure in the membrane mimetic environment while B ICTcul exit as disordered conformation. The peptides can permeabilise the outer and inner membrane of the E. coli cells, after interacting

575 with LPS. Confocal and scanning microscopy analysis using E. coli cells confirmed the loss of membrane integrity and the leakage of cytoplasmic content leading to cell death. Flow cytometric analysis showed that membrane depolarization and cell death are two independent events.

[0066] The CABPAs isolated from H. temporalis possess alpha-helical conformation in 580 membrane mimicking environments and exhibited varied antibacterial activity against the tested bacteria. E2HLtel-E2HLte4 preferentially killed Gram-positive bacteria (MICs: 0.7- 3μΜ) to Gram-negative bacteria. They are non-toxic to RBC at their respective MICs, but their toxicity level increases with increase in peptide concentration. E2CABPAs cocktail (equal concentration of the peptides) showed very high antibacterial activity against both 585 Gram-positive and Gram-negative bacteria showing that synergism is playing an important role in deciding the activity. Ranacyclins are not that active compared to other family members but kills pseudomonas aerugenosa and their cocktail is found to be more effective (MIC: 6μΜ). Among the CABPAs E2HLtel, E2HLte3, E2HLte4, B IHLtel, B lHLte2, B2HLte3, B2HLte4 are the most potent peptides and their time dependent killing kinetic

590 analysis showed that they are bactericidal in nature as more than 90% cells were eliminated within 60 min at their MICs. Most of these peptides are nontoxic to RBCs at their respective MICs and the hemolytic nature of E2CABPAs are found to be less compared to B ICABPAs, B2CABPAs. Cytotoxicity measurements of H. temporalis CABPAs proved that they are safe to normal HEK 293 cells up to ΙΟΟμΜ but showed variations in their toxicity level against

595 liver cancer cell lines Hep G ₂ . Brevininl and Esculentin2 CABPAs required only less than 50% concentration of their respective MICs to eliminate Hep G ₂ cells.

[0067] Outer and inner membrane permeation studies showed that these peptides can permeabilise and damage both the outer and inner membrane which leads to the destruction of bacterial membrane integrity which results cell death. Peptide induced cytoplasmic

600 membrane damage takes place in a concentration dependent manner. As they penetrate into the cytoplasmic membrane, they can interfere and binds with the intercellular components like nucleic acids which can also leads to the cell death. FASC analysis showed that peptide induced depolarization of the inner membrane take place in the early hour of their addition. Membrane depolarization therefore is an independent event that allowed the peptide to enter

605 the cytoplasm and target other cellular components rather than limiting their action on bacterial membrane. These studies also showed that CABPAs can induce the formation of trans-membrane pores on the bacterial cell membrane to exert their action.

[0068] CABPAs associated with frogs living in the tropical climate region are unique, their antibacterial nature is quite different from those identified from the temperate region. The

610 tropical climatic conditions and environmental bacterial load might have been influenced the amino acid selection, the manner in which amino acid arranged in the sequence and their conformation. Hydrophobicity and amphipathicity are the two important parameters that influenced the antibacterial nature and membrane selectivity. Fatty acid acylation, incorporation of fluorinated amino acids or peptoid residues at the N-terminal region of

615 CABPAs can provide the required hydrophobic-hydrophilic balance to a peptide. N-terminal integrity of these peptide amides are very important for their activity. Deletions or substitution of amino acids from this region can drastically brought down the killing ability of the synthetic peptide. N-terminal fatty acid acylation helped to improve the hydrophobic character of CABPAs which helped to reduce their toxicity level without affecting much on 620 their MICs. PEGylation helped to provide the required hydrophilic-hydrophobic balance to CABPAs which helped to reduce the haemolytic nature without affecting their bioactivity. Preferential higher toxicity of Brevininl and Esculentin2 CABPAs towards liver cancer cell line Hep G ₂ can help to understand and design novel synthetic CABPAs that can selectively target the cancer cells. These peptides can be used as transporters to deliver anticancer drug

625 molecules to the specific target cells.

[0069] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

630 [0070] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such

635 equivalents are considered to be within the scope of this invention and are covered by the claims.

[0071] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent 640 as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0072] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term 645 "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

650 [0073] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In

655 embodiments of any of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of or "consisting of. As used herein, the phrase "consisting essentially of requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term "consisting" is used to indicate the presence of the recited integer (e.g., a feature, an

660 element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property(ies), method/process steps or limitation(s)) only.

[0074] The term "or combinations thereof as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations

665 thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limit on the number

670 of items or terms in any combination, unless otherwise apparent from the context.

[0075] As used herein, words of approximation such as, without limitation, "about", "substantial" or "substantially" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to 675 which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as "about" may vary from the stated value by at least +1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

680 [0076] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method 685 described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0077] To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of 690 the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words "means for" or "step for" are explicitly used in the particular claim.

[0078] For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the 695 prior claim provides a proper antecedent basis for a claim term or element.

References

1. Dubos, R.J. (1939) Studies on bactericidal agent extracted from a soil Bacillus: 700 Preparation of the agent. Its activity in vitro. The Journal of Experimental Medicine. 70,

1-10.

2. Van Epps, H. L. (2006) Rene Dubos: unearthing antibiotics. The Journal of Experimental Medicine. 203, 259- 259.

3. Fernandez de Caleya, R., Gonzalez- Pascual, B., Garcia-Olmedo, F., and Carbonero, P. 705 (1972) Susceptibility of phytopathogenic bacteria to wheat purothionins in vitro. Applied

Microbiology .23, 998 -1000.

4. Davies, J. (2006) Where have all the antibiotics gone? The Canadian Journal of Infectious Diseases & Medical Microbiology .17, 287 - 290.

5. Hirsch, J.G. (1956) Phagocytin: a bactericidal substance from polymorphonuclear 710 leucocytes. The Journal of Experimental Medicine. 103, 589 - 611.

6. Zeya, H. I., and Spitznagel, J. K. (1966) Cationic proteins of polymorphonuclear leukocyte lysosomes. II. Composition, properties, and mechanism of antibacterial action. Journal of bacteriology. 91, 755 - 762.

7. Groves, M. L., Peterson, R. F., and Kiddy, C. A. (1965) Polymorphism in the red protein 715 isolated from milk of individual cows. Nature. 207, 1007 - 1008

8. Ganz, T., Selsted, M. E., Szklarek, D., Harwig, S.S.L., Daher, K., Bainton, D.F., and Lehrer, R. I. (1985) Defensins - natural peptide antibiotics of human neutrophils . Journal of Clinical Investigation. 76, 1427-1435.

9. Kiss, G. and Michl, H. (1962) Uber das Giftsekret der Gelbbauchunke, Bombina 720 variegata L. Toxicon. 1, 33 - 34

10. Selsted, M. E., Novotny, M. J., Morris, W. L., Tang, Y. Q., Smith, W., and Cullen, J. S.

(1992) Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. . Biol. Chem. 267, 4292-4295.

11. Tang, Y.Q., Yuan, J., Osapay, G., Osapay, K., Tran, D., Miller, C.J., Ouellette , A.J., and 725 Selsted, M.E. (1999) A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins. Science. 286, 498-502.

12. Ganz, T. (2003) The role of antimicrobial peptides in innate immunity. Integrative and Comparative Biology. 43, 300 - 304.

13. Amiche, M., and Galanth, C. (2011) Dermaseptins as models for the elucidation of 730 membrane- acting helical amphipathic antimicrobial peptides. Current Pharmaceutical

Biotechnology. 12, 1184 - 1193.

14. Fernandez, D. I., Gehman, J. D., and Separovic, F. (2009) Membrane interactions of antimicrobial peptides from Australian frogs. Biochimica etBiophysica Acta - Biomembranes. 1788, 1630-1638. 735 15. Hancock, R. E. W., Bowdish, D. M. E., Davidson, D. J., and Scott, M.G.(2005) Immuno modulatory activities of small host defense peptides. Antimicrob .Agents Chemother. 49(5), 1727-1732

16. Zasloff, M. (2002) Antimicrobial peptides of multi-cellular organisms, Nature. 415, 389- 395.

740 17. Radek, K., and Gallo, R. (2007) Antimicrobial peptides: Natural effectors of the innate immune system. Semin.Immunopathol. 29, 27-43.

18. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J. M., and Hoffmann, J.A. (1996) The Dorsoventral Regulatory Gene Cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell. 86, 973 - 983.

745 19. Peters, B. M., Shirtliff, M. E., and Jabra-Rizk, M. A. (2010) Antimicrobial peptides:

Primeval molecules or future drugs? PLoSPathog. 6, el 001067.

20. Conlon, J. M., and Sonnevend, A. (2010) Antimicrobial peptides in frog skin secretions. Methods Mol. Biol.lS, 3-14.

21. Zhao, X., Wu, H., Lu, H., Li, G., and Huang, Q. (2013) Lamp: A database linking 750 antimicrobial peptides. PLoS One. 8, e66557.

22. T. Ganz, The role of antimicrobial peptides in innate immunity, Integr. Comp. Biol. 43 (2003) 300-304.

23. M.R. Yeaman, N.Y. Yount, Mechanisms of antimicrobial peptide action and resistance, Pharmacol. Rev. 55 (2003) 27-55.

755 24. H. Jenssen, P. Hamill, R.E. Hancock, Peptide antimicrobial agents, Clin. Microbiol. Rev.

19 (2006) 491-511.

25. M. Zasloff, Antimicrobial peptides of multicellular organisms, Nature 415 (2002) 389- 395.

26. W.E. Duellman, L. Trueb, Biology of Amphibians, The Johns Hopkins University Press, 760 Baltimore and London, 1994.

27. J.M. Park, J.E. Jung, B.J. Lee, Antimicrobial peptides from the skin of a Korean frog, Rana rugosa, Biochem. Biophys. Res. Commun. 205 (1994) 948-954.

28. S. Suzuki, Y. Ohe, T. Okubo, T. Kakegawa, K. Tatemoto, Isolation and characterization of novel antimicrobial peptides, rugosins A, B and C, from the skin of the frog, Rana

765 rugosa, Biochem. Biophys. Res. Commun. 212 (1995) 249-254.

29. N. Morikawa, K. Hagiwara, T. Nakajima, Brevinin-1 and -2, unique antimicrobial peptides from the skin of the frog, Rana brevipoda porsa, Biochem. Biophys. Res. Commun. 189 (1992) 184-190.

30. M. Simmaco, G. Mignogna, D. Barra, F. Bossa, Antimicrobial peptides from skin 770 secretions of Rana esculenta. Molecular cloning of cDNAs encoding esculentin and brevinins and isolation of new active peptides, J. Biol. Chem. 269 (1994) 11956-11961. 31. J.M. Conlon, T. Halverson, J. Dulka, J.E. Platz, F.C. Knoop, Peptides with antimicrobial activity of the brevinin-1 family isolated from skin secretions of the southern leopard frog, Rana sphenocephala, J. Pept. Res. 54 (1999) 522-527.

775 32. M. Simmaco, G. Mignogna, D. Barra, F. Bossa, Novel antimicrobial peptides from skin secretion of the European frog Rana esculenta, FEBS Lett. 324 (1993) 159-161.

33. D.P. Clark, S. Durell, W.L. Maloy, M. Zasloff, Ranalexin: a novel antimicrobial peptide from bullfrog (Rana catesbeiana) skin, structurally related to the bacterial antibiotic, polymyxin, J. Biol. Chem. 269 (1994) 10849-10855.

780 34. J. Goraya, F.C. Knoop, J.M. Conlon, Ranatuerins: antimicrobial peptides isolated from the skin of the American bullfrog, Rana catesbeiana, Biochem. Biophys. Res. Commun. 250 (1998) 589-592.

35. M. Simmaco, G. Mignogna, S. Canofeni, R. Miele, M.L. Mangoni, D. Barra, Temporins, antimicrobial peptides from the European red frog Rana temporaria, Eur. J. Biochem. 242

785 (1996) 788-792.

36. J.M.Conlon, J. Kolodziejek, N. Nowotny, Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of newtherapeutic agents, Biochim. Biophys. 1694 (2004) 1-14.

37. M.J. Tyler, D.J. Stone, J.H. Bowie, A novel method for the release and collection of 790 dermal, glandular secretions from the skin of frogs, J. Pharmacol. Toxicol. Methods 28

(1992) 199-200.

38. S. Leena, K.S. Kumar, Syntheses, characterization and application of crosslinked polystyrene-ethyleneglycol acrylate resin (CLPSER) as a novel polymer support for polypeptide syntheses, J. Pept. Res. 58 (2001) 117-128.

795 39. S. Nuding, K. Fellermann, J. Wehkamp, H.A. Mueller, E.F. Stange, A flow cytometric assay to monitor antimicrobial activity of defensins and cationic tissue extracts, J.Microbiol. Methods 65 (2006) 335-345.

40. P. Thomas, T.V. Kumar, V. Reshmy, K.S. Kumar, S. George, A mini review on the antimicrobial peptides isolated from the genus Hylarana (Amphibia: Anura) with a

800 proposed nomenclature for amphibian skin peptides, Mol. Biol. Rep. 39 (2012) 6943-

6947.

41. W. McGahee, F.D. Lowry, Staphylococcal infections in the intensive care unit, Semin.

Respir. Infect. 15 (2000) 308-313.

42. A. Christina, C. Nascimento, W. Fontes, A. Sebben, M.S. Castro, Antimicrobial peptides 805 from anurans skin secretion, Protein Pept. Lett. 10 (2003) 227-238.

43. J. Goraya, Y. Wang, Z. Li, M. O'Flaherty, F.C. Knoop, J.E. Platz, J.M. Conlon, Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs Rana luteiventris, Rana berlandieri and Rana pipiens, Eur. J. Biochem. 267 (2000) 894-900. 810 44. R.I. Jepras, F.E. Paul, S.C. Pearson, M.J. Wilkinson, Rapid assessment of antibiotic effects on Escherichia coli by bis-(l,3-dibutylbarbituric acid) trimethine oxonol and flow cytometry, Antimicrob. Agents Chemother. 41 (1997) 2001-2005.

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