Antimicrobial peptides and methods of use thereof

Abstract

Antimicrobial peptides and methods for their use are provided. Aspects of the present invention relate to peptides with antibacterial activity. In one aspect, the present invention relates to peptides with potent antibacterial activity, broad-spectrum antibacterial activity, and / or the ability to kill bacteria that are otherwise antibiotic-resistant or protected by biofilms.

Description

[Technical Field]

[0001] This invention was made with government support under contract 1R434EY024463-01 awarded by the National Institutes of Health and contract DM140274 awarded by the Department of Defense. The government has certain rights in this invention.

[0002] The present invention relates generally to peptides with antimicrobial activity, and more particularly to peptides with potent antimicrobial activity, broad spectrum antimicrobial activity, and / or the ability to kill bacteria that are otherwise antibiotic-resistant or protected by biofilms. [Background technology]

[0003] Antibiotic resistance is a major health problem, due in part to the widespread use of antibiotics in medicine as well as agriculture and animal husbandry. This overuse has created a strong selection bias for antibiotic-resistant bacteria while killing susceptible organisms. Antibiotic resistance is particularly problematic for immunocompromised individuals and is an increasingly serious problem for hospitalized patients. Acute bacterial skin and skin structure infections (ABSSSIs) cause 750,000 hospitalizations annually and incur significant costs. Broad-spectrum antibiotics for Gram-negative bacteria and multidrug-resistant Gram-positive bacteria, such as community-acquired methicillin-resistant Staphylococcus aureus (MRSA), are limited, leading to frequent outbreaks.

[0004] In addition to exhibiting genetic antibiotic resistance, many emerging bacterial strains can exist in complex communities called biofilms. The biofilm structure constitutes a physical barrier to antibiotic exposure. Biofilms are 20–1000 times more resistant to antibiotics than plankton and can form in and on tissues, particularly in chronic wounds and medical implants such as indwelling catheters and prosthetic devices, where they can cause systemic infections requiring aggressive treatment. Therefore, there is an urgent need for agents that are effective against antibiotic-resistant bacteria both in their free and biofilm forms.

[0005] Many organisms, including insects, amphibians, mammals, and humans, produce antimicrobial peptides as part of their innate defense against bacteria. These peptides are chemically diverse. Some are thought to act by penetrating and disrupting bacterial cell membranes. Others affect bacterial cellular processes. Many peptides exhibit selectivity, targeting bacteria over host cells. Unfortunately, host-produced antimicrobial peptides generally cannot effectively eliminate a wide range of microbial agents, including many antibiotic-resistant bacterial strains. Antimicrobial peptides with potent antimicrobial activity, broad-spectrum activity, and / or the ability to kill otherwise antibiotic-resistant bacteria are of particular interest. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides antimicrobial peptides and methods of using the same. The peptides of the present disclosure can have antibacterial, antifungal, and / or antiprotozoan activity. The peptides can kill microbial strains that are resistant to conventional antibiotics. The present disclosure provides antimicrobial peptides that can kill microorganisms (e.g., bacteria) that grow as microbial biofilms. The technology described and claimed below represents the first description of specific types of antimicrobial peptides that can be used to selectively kill microorganisms (e.g., bacteria) for various applications, such as treating conditions associated with microbial infections. [Means for solving the problem]

[0007] The present invention is set forth in the following description, drawings, and appended claims. [Brief explanation of the drawings]

[0008] [Figure 1] 1 shows an exemplary bubble region-containing peptide of the present disclosure. The peptide shown includes a bubble region having a disulfide bond flanked by amphipathic regions 1 and 2. [Figure 2A] Figures 2A and 2B show a peptide containing a cysteine-containing bubble region flanked by two amphipathic regions that themselves contain additional cysteine ​​residues. Figure 2A shows a monomeric version of the peptide in which the cysteine ​​residues located in the amphipathic regions may be linked via an intramolecular disulfide bond. [Figure 2B] FIG. 2B shows a dimeric version of the peptide in which the two cysteine ​​residues may be linked via an intermolecular disulfide bond. [Figure 3A] Figures 3A-3C show that RP557 rapidly eradicates P. aeruginosa and S. aureus without cytotoxicity to mammalian cells. In Figure 3A, viability assays were performed using the biosynthetic strain P. aeruginosa 19660. [Figure 3B] In Figure 3B, viability was measured using the S. aureus 49525 biosensor strain. [Figure 3C]In Figure 3C, viability assays were performed using a biosensor strain of L929 fibroblasts. [Figure 4A] Data are shown demonstrating that P. aeruginosa and S. aureus did not acquire resistance to RP557. Subinhibitory concentrations of RP557, gentamicin, and clindamycin were incubated for 24 hours with P. aeruginosa 19660 and S. aureus 49525. Bacteria that showed growth at the highest concentrations were serially re-passaged 30 times in fresh dilutions containing components below their respective minimum inhibitory concentration (MIC) levels. [Figure 4B] Data are shown demonstrating that P. aeruginosa and S. aureus did not acquire resistance to RP557. Subinhibitory concentrations of RP557, gentamicin, and clindamycin were incubated for 24 hours with P. aeruginosa 19660 and S. aureus 49525. Bacteria that showed growth at the highest concentrations were serially re-passaged 30 times in fresh dilutions containing components below their respective minimum inhibitory concentration (MIC) levels. [Figure 5A] Data are presented demonstrating that RP557 is a potent inhibitor of Candida albicans biofilms. Fluconazole was added to preformed Candida albicans 17-88 biofilms for 24 hours, and biofilm inhibition was assessed by metabolic assessment using XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)[phenyl-amino]carbonyl]-2H. [Figure 5B] Data are presented demonstrating that RP557 is a potent inhibitor of Candida albicans biofilms. RP557 was added to preformed Candida albicans 17-88 biofilms for 24 hours, and biofilm inhibition was assessed by metabolic assessment using XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)[phenyl-amino]carbonyl]-2H. [Figure 6A] We demonstrate that topical treatment with the exemplary peptide RP557 reduces polymicrobial infection in an infected porcine burn model. Full-thickness wounds were created on the backs of anesthetized pigs with a heated brass rod and trephine, followed by infection with a 2:2:1 mixture of Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa (a porcine isolate), and Fusobacterium ssp. [Figure 6B] We demonstrate that topical treatment with the exemplary peptide RP557 reduces polymicrobial infection in an infected porcine burn model. Full-thickness wounds were created on the backs of anesthetized pigs with a heated brass rod and trephine, followed by infection with a 2:2:1 mixture of Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa (a porcine isolate), and Fusobacterium ssp. [Figure 7A] Figure 1 shows that the exemplary peptide RP557 eradicates infection over a 24-hour period. Pharmacodynamic responses to RP557 are shown. Biofilm-associated wounds were treated with 2% RP557 24 hours after bacterial inoculation. Punch biopsies were taken, homogenized, cultured, and CFU counted 30 minutes, 180 minutes, and 24 hours after treatment. [Figure 7B] Figure 1 shows that the exemplary peptide RP557 eradicates infection over a 24-hour period. Pharmacodynamic responses to RP557 are shown. Biofilm-associated wounds were treated with 2% RP557 24 hours after bacterial inoculation. Punch biopsies were taken, homogenized, cultured, and CFU counted 30 minutes, 180 minutes, and 24 hours after treatment. [Figure 8A] Figure 8 shows that topical treatment with the exemplary peptide RP557 alleviates vaginal candidiasis in mice. Figure 8 shows the effects of RP557, miconazole, and oral fluconazole in a rodent model of vaginal Candida albicans infection. Rats were inoculated intravaginally with Candida albicans on day 0. RP557 and miconazole were administered twice daily (Figure 8A). A separate group received oral fluconazole (Figure 8B). Further details are provided in the experimental section. [Figure 8B] Figure 8 shows that topical treatment with the exemplary peptide RP557 alleviates vaginal candidiasis in mice. Figure 8 shows the effects of RP557, miconazole, and oral fluconazole in a rodent model of vaginal Candida albicans infection. Rats were inoculated intravaginally with Candida albicans on day 0. RP557 and miconazole were administered twice daily (Figure 8A). A separate group received oral fluconazole (Figure 8B). Further details are provided in the experimental section. DETAILED DESCRIPTION OF THE INVENTION

[0009] As noted above, the invention disclosed herein relates to antimicrobial peptides and methods of administering such antimicrobial peptides to a subject to prevent or treat microbial infections.

[0010] (antimicrobial peptides) Specific features of the antimicrobial peptides of the present disclosure are now described in more detail. Antimicrobial peptides generally comprise an amphipathic or striapathic region. In some embodiments, the peptide comprises one contiguous amphipathic or striapathic region. In some embodiments, the peptide comprises two amphipathic or striapathic regions separated by a linking or bubble region (e.g., as described herein).

[0011] "Amphipathic region" refers to a peptide region or segment that has both hydrophobic and hydrophilic structural elements or properties, e.g., a peptide region that can have a structure with both hydrophilic and hydrophobic surfaces. A peptide region is said to be in an amphipathic structure if it is amphipathic and exhibits amphipathic characteristics. The amphipathic properties of a peptide may depend in part on the environmental conditions to which the peptide is exposed and / or utilized, e.g., aqueous or physiological conditions. A peptide sequence (or a portion thereof) need not always be in an amphipathic structure to be considered or referred to as amphipathic. Rather, the presence of an amphipathic structure may be sufficient in some cases; e.g., a peptide can adopt an amphipathic structure under appropriate conditions, such as those under which the peptide is used.

[0012] The amphipathic region of a peptide is sometimes called a stripathic region. The term "stripathic region" refers to a region or portion of a peptide sequence that is composed of alternating hydrophobic and hydrophilic modules. A "hydrophobic module" is a peptide sequence consisting of 1 to 5 (e.g., 1 to 3 or 1 to 2) hydrophobic amino acid residues, e.g., 1, 2, 3, 4, or 5 hydrophobic amino acid residues. A "hydrophilic module" is a peptide sequence consisting of 1 to 5 (e.g., 1 to 3 or 1 to 2) hydrophilic amino acid residues, e.g., 1, 2, 3, 4, or 5 hydrophilic amino acid residues.

[0013] Hydrophobic amino acid residues are characterized by side chain groups that have predominantly non-polar chemical or physical properties in the environment in which the peptide is used, e.g., physiological conditions. Such hydrophobic amino acid residues may be naturally occurring or non-naturally occurring. Hydrophobic amino acid residues may be mimetics of naturally occurring amino acids that have predominantly non-polar chemical or physical properties in the environment in which the peptide is used, e.g., physiological conditions. Conversely, hydrophilic amino acid residues are characterized by side chain groups that are predominantly polar (e.g., charged or neutrally hydrophilic) in the environment in which the peptide is used, e.g., physiological conditions. Such hydrophilic amino acid residues may be naturally occurring or non-naturally occurring. Hydrophilic amino acid residues may be mimetics of naturally occurring amino acids that have predominantly hydrophilic (charged or neutrally polar) side chain groups. Examples of hydrophilic and hydrophobic amino acid residues are shown in Table 1 below. Hydrophobic and hydrophilic amino acid residues may be L-amino acid residues. Hydrophobic and hydrophilic amino acid residues may be D-amino acid residues. Suitable non-naturally occurring amino acid residues and amino acid mimetics that can find use in the subject peptides are readily available in the art. See, e.g., Liang et al. (2013), "An Index for Characterization of Natural and Non-Natural Amino Acids for Peptidomimetics," PLoS ONE 8(7):e67844.

[0014] Most amino acid residues can be considered either hydrophobic (non-polar) or hydrophilic (polar or charged), but some amino acid residues can behave as either hydrophobic or hydrophilic, depending on the context.

[0015] [Table 1]

[0016] As described in further detail below, aspects of the present disclosure include antimicrobial peptides having at least one amphipathic or stripathic region with a particular degree of cationic charge. In certain embodiments, the antimicrobial peptide comprises a tail region (e.g., a hydrophobic tail sequence). In certain embodiments, the antimicrobial peptide (or peptide agent) comprises two or more amphipathic or stripathic regions. In such embodiments, the two amphipathic regions of the antimicrobial peptide are in the form of a dimer, and the two amphipathic regions can have the same or different amino acid sequences (i.e., a homodimer or heterodimer). In certain embodiments, the two (or more) amphipathic or stripathic regions are connected via a linker or linking region. The linker can be a contiguous (or in-line) amino acid sequence or a non-amino acid moiety, as desired by the user. The linking region can be, for example, a bubble region or a β-turn region.

[0017] In certain embodiments, the amphipathic region of the peptide is called the stripathic region and is comprised of 1 to 5 hydrophobic residues (hydrophobic module J). 1-5 ) followed by 1 to 5 hydrophilic amino acid residues (hydrophilic module X 1-5 The stromal region contains an alternating sequence consisting of (X 1-5 J 1-5 ) n or (J 1-5 X 1-5 ) n where each X represents a hydrophilic amino acid residue and each J represents a hydrophobic amino acid residue. Each n is an integer from 1 to 15, e.g., 2 to 15, 2 to 10, 3 to 10, 4 to 10, or 5 to 10. For example, the amphipathic region can have a sequence according to Formula 1, Formula 2 (the reverse of Formula 1), or Formula 3. Formula 1:X 1 J 2 J 3 X 4 X 5 J 6 J 7 X 8 X 9 J 10 X 11X 12 J 13 J 14 X 15 X 16 J 17 J 18 Formula 2:J 1 J 2 X 3 X 4 J 5 J 6 X 7 X 8 J 9 X 10 X 11 J 12 J 13 X 14 X 15 J 16 J 17 X 18 Formula 3:J 1 X 2 J 3 X 4 J 5 X 6 J 7 X 8 J 9 X 10 J 11 X 12 J 13 X 14 J 15 X 16 J 17

[0018] Each hydrophobic amino acid residue J is selected from the group consisting of naturally occurring hydrophobic amino acids, non-naturally occurring hydrophobic amino acids, and hydrophobic amino acid mimetics. Each hydrophilic amino acid residue X is selected from the group consisting of naturally occurring hydrophilic amino acids, non-naturally occurring hydrophilic amino acids, and hydrophilic amino acid mimetics. In many cases, amphipathic structures are associated with specific secondary structures, such as helical structures. Thus, the amphipathic region of an antimicrobial polypeptide can be defined as an amphipathic triad. 10 The amphipathic region of the antimicrobial polypeptide can have an amphipathic-strand structure, an amphipathic-helical structure, an amphipathic-helical structure, or an amphipathic polyproline helical structure. Alternatively, the amphipathic region of the antimicrobial polypeptide can have an amphipathic-strand structure.

[0019] In certain embodiments, the amphipathic region of an antimicrobial peptide according to aspects of the present disclosure comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) large hydrophobic amino acid residues. Examples of large hydrophobic amino acid residues include tryptophan, phenylalanine, and tyrosine. Furthermore, under certain circumstances, histidine can be considered a large hydrophobic amino acid residue. In certain embodiments, the amphipathic region of an antimicrobial peptide according to aspects of the present disclosure comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) small hydrophobic amino acid residues. Examples of small hydrophobic residues include glycine, alanine, valine, leucine, threonine, and proline. Furthermore, under certain circumstances, serine or cysteine ​​can also be considered a small hydrophobic residue. In certain embodiments, the antimicrobial polypeptide has an amphipathic region comprising a combination of large and small hydrophobic residues.

[0020] (cationic charge / surface) Antimicrobial polypeptides according to aspects of the present disclosure can include an amphipathic region having a cationic surface. In certain embodiments, the amphipathic region has a cationic charge (i.e., a charge >0, e.g., +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, or more). Thus, in certain embodiments, the amphipathic region of the disclosed peptides includes one or more polar-cationic amino acid residues (i.e., residues having a positively charged side chain), such as two or more, three or more, four or more, or five or more polar-cationic amino acid residues, and in some cases, up to 10 polar-cationic amino acid residues. Examples of amino acid residues having a positively charged side chain (assuming physiological conditions) include lysine, ornithine, arginine, and in some cases, histidine. Thus, the antimicrobial polypeptide can have an amphipathic region containing 1 to 20 cationic amino acid residues, for example, 2 to 20, 3 to 30, 4 to 20, 5 to 20, 6 to 20, 8 to 20, or 10 to 20. Thus, the antimicrobial peptide of the present invention can contain at least 40% (e.g., 50%, 60%, 70%, 80%, 90%, or 100%) cationic polar amino acid residues (e.g., selected from arginine, lysine, and ornithine).

[0021] (tail region) In certain embodiments, the antimicrobial peptide comprises a tail region. The tail region of the antimicrobial peptide of the present invention can be 3 to 15 amino acid residues in length, and at least 50% (e.g., at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more) of the amino acid residues in the tail region are hydrophobic. The tail region can be located at either the N-terminus, C-terminus, or both termini of the antimicrobial peptide. The present disclosure provides peptides having a tail region comprising a sequence of 3, 4, or 5 hydrophobic amino acids. The present disclosure provides peptides having a tail region comprising a sequence of 3 to 6 amino acids lacking one or a hydrophilic amino acid residue. In certain embodiments, the tail region comprises one hydrophilic amino acid for every 6 amino acids in the sequence. In some aspects, the sequence of the tail region is represented by Formula 4, where each J represents a hydrophobic amino acid residue (e.g., in Table 1). Formula 4:J 1 J 2 J 3 J 4 J 5

[0022] In some embodiments of Formula 4, J 1 ,J 3 and J 5 are independently large hydrophobic residues (e.g., tryptophan, phenylalanine, and tyrosine), and J 2 and J 4 are independently small hydrophobic residues (e.g., glycine, alanine, valine, leucine, threonine, and proline). In some embodiments of Formula 4, each J is independently a hydrophobic amino acid residue selected from phenylalanine and alanine. In some embodiments of Formula 4, the tail region comprises the sequence FAFAF (SEQ ID NO: 19). In some embodiments of Formula 4, the tail region comprises the sequence AFAFA (SEQ ID NO: 20).

[0023] Specific examples of antimicrobial peptides according to embodiments of the present disclosure that include a tail region having the sequence FAFAF (SEQ ID NO: 19) include the following, with the tail region underlined: FIOKFAKOFKOFIOKFAK FAFAF (RP-551, SEQ ID NO:2), and FAFAF KAFKKAFKOFOOAFOOAF (RO-552, SEQ ID NO: 3)

[0024] (bubble area) In certain embodiments, the antimicrobial peptide comprises a bubble region. The "bubble" region of the antimicrobial peptides of the present disclosure is composed of a stretch of amino acid residues flanked by bridging residues (e.g., cysteine ​​residues (C) capable of disulfide formation) at or near each end of the region (see FIG. 1A). The sequence of amino acid residues located between the flanking bridging residues (e.g., cysteine ​​residues) may be 2 to 10 amino acid residues in length. In certain cases, the stretch of amino acid residues located between the bridging residues (e.g., cysteine ​​residues) is 3 to 6 amino acid residues in length, e.g., 3 to 5 amino acid residues in length, or particularly 3, 4, or 5 amino acid residues in length. The bubble region connects two amphipathic regions and can contribute to the formation of a desired hairpin secondary structure by the antimicrobial peptide. In some examples, the bubble region comprises an intramolecular bridge. Such intramolecular bridges, if present, may be covalent bonds (e.g., disulfides) or non-covalent bonds (e.g., salt bridges). In some cases, the bubble region contains a disulfide bond between two adjacent cysteine ​​residues (see Figure 1A). This region can therefore be classified as a type of "linker region" (as well as others described elsewhere herein). In certain embodiments, hairpin secondary structures can significantly improve antibacterial activity.

[0025] The bubble region may consist of two cysteine ​​residues linked via any sequence of four amino acids, e.g., the sequence C(AA 1 )(AA 2 )(AA 3 )(AA 4Optionally, the cysteine ​​residues of the bubble region may be linked to the adjacent stripathic region of the peptide by a linker, such as one or more linking residues. In some embodiments, one or more linking residues is a glycine residue, e.g., the sequence (G) n C(AA 1 )(AA 2 )(AA 3 )(AA 4 )C(G) m Here, n and m are independently 0 to 6, for example, 0, 1, 2, 3, or 4.

[0026] The bubble region can include, for example, a sequence as shown in Formula 5, where each J represents a hydrophobic amino acid residue and each X represents a hydrophilic amino acid residue. Formula 5:C(J / X)(J / X)(J / X)(J / X)C

[0027] In some embodiments of Formula 5, the bubble region has an array. Formula 5b: CJ(X / J)(X / J)JC

[0028] In some embodiments of Equation 5b, the bubble region has one of the following sequences: Formula 5c: CJGXJC Formula 5d:CJXGJC Formula 5e:CJGGJC

[0029] In some examples of formulas 5c-5e, each J is selected from F, L, I, V, and A; and in some examples of formulas 5c-5d, X is selected from K, R, and O.

[0030] Specific examples of antimicrobial peptides of the present disclosure that contain a bubble region having the sequence CLGRFC (SEQ ID NO: 21) or CYKGIC (SEQ ID NO: 22) include the following (bubble regions are underlined): FKIOARL CLGOFC IOARLK (RP-550, SEQ ID NO: 1), and RFCWKV CYKGIC FKKCK (RP-557, SEQ ID NO: 8)

[0031] In some embodiments, the antimicrobial peptides of the present invention comprise a bubble region and one or more additional linking regions. In certain embodiments, antimicrobial peptides of the present invention comprising a bubble region also comprise one or more additional stretches of amino acid residues flanked by cysteine ​​residues located on either side of the bubble region. For example, a subject antimicrobial peptide may comprise four cysteine ​​residues, with two cysteine ​​residues forming the bubble region and the remaining two cysteine ​​residues located on opposite sides of two amphipathic regions on either side of the bubble region, such that the bubble region is formed by the bubble region and the remaining two cysteine ​​residues form an additional linking region across the secondary hairpin structure (see FIG. 1B). Specific examples of antimicrobial peptides of the present invention comprising a bubble region having the sequence CYKGIC (SEQ ID NO: 22) and an additional linking region include the following (the bubble region and the additional linking region are underlined): RW C FKV CYKGIC YKK C K (RP-556, SEQ ID NO: 7) RF C WKV CYKGIC FKK C K (RP-557, SEQ ID NO: 8)

[0032] (dimerization) Without wishing to be limited by theory, in some cases, the effectiveness of the antimicrobial peptides of the present invention is due in part to peptide dimerization and clustering on the cytoplasmic membrane of target microorganisms (e.g., bacterial cells). Dimers are believed to be more efficient at penetrating and ultimately lysing the cytoplasmic membrane. The formation of such dimers is thermodynamically more favorable when the peptides are physically linked, for example, using a linker region or bridging amino acid residues. See Figure 2B. The linker region may contain additional amino acid residues (e.g., the bubble region described above) or may be a non-amino acid-containing linker moiety.

[0033] (β-turn region) A β-turn sequence can be used to physically link two regions, facilitating intramolecular interactions. This is thought to be particularly important for amphipathic lytic peptides, as it can shield their hydrophobic surfaces from the aqueous phase. A bubble region (e.g., as described herein) can provide a similar configuration. The β-turn sequence allows two intrachain amphipathic regions to form dimers in an antiparallel orientation. This region (along with others described elsewhere herein) can therefore be classified as a type of "linker region."

[0034] The β-turn sequence used to connect the two stripathic regions may be any β-turn sequence known in the art, e.g., the β-turn sequence may have the sequence shown in SEQ ID NO: 23, where J represents a hydrophobic amino acid residue and X represents a hydrophilic amino acid residue (i.e., any amino acid residue). (J / X)GPGR(J / X) (SEQ ID NO: 23)

[0035] Exemplary antimicrobial peptide sequences that include one or more of the above-described features are described below. Additional peptides of the present disclosure can be readily designed by one of skill in the art by combining different regions or features of the exemplary antimicrobial peptides in different ways, as described herein.

[0036] (Examples of antibacterial peptides) Specific antimicrobial peptides of interest, as well as fragments and variants thereof, for use in the subject pharmaceutical compositions and methods are now described in more detail. In certain embodiments, the subject antimicrobial peptides are 10-50 amino acid residues in length, e.g., 10-40, 10-30, 10-20, 12-30, 13-30, 14-30, 15-30, 15-25, 15-20, 15-19, 17-30, 17-25, or 17-20 amino acid residues in length. The subject peptides can comprise a stripathic region of alternating hydrophilic and hydrophobic modules that adopt an amphipathic structure under physiological conditions (e.g., as described herein). In particular examples, the striapathic region of the peptide is 5 to 30 amino acid residues in length, e.g., 5 to 25, 5 to 20, 5 to 19, 6 to 18, 6 to 17, 7 to 18 amino acid residues in length (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length). The striatopathic region can comprise two or more (e.g., three or more, four or more, five or more, or six or more) hydrophobic modules and one or more (e.g., three or more, four or more, five or more, or six or more) hydrophilic modules. In some examples, each hydrophilic module comprises at least one cationic residue. In some embodiments, the peptide has one and only one stripathic region 10-30 amino acid residues in length, e.g., 12-30, 15-25, or 10-20, or 15-20 amino acid residues in length (e.g., 15, 16, 17, 18, 19, or 20 amino acid residues in length). In some examples, the peptide has two stripathic regions linked via a bubble region or linking region, each stripathic region independently 5-15 amino acid residues in length, e.g., 5-12, 5-10, or 6-12 or 6-10 amino acid residues in length (e.g., 5, 6, 7, 8, 9, or 10 amino acid residues in length).

[0037] In particular examples, the striatomic region of the peptide is 5 to 30 amino acid residues in length (e.g., 6 to 20, 6 to 19, 7 to 19, 14, 15, 16, 17, 18, or 19 amino acids in length), and the peptide is optionally further modified (e.g., as described herein). The striatomic region can comprise: two or more (e.g., three or more, or four or more) hydrophobic modules and one or more (e.g., two or more, or three or more) hydrophilic modules, each comprising at least one cationic residue. In some examples, the striatomic region of the peptide has a length of 7 to 20 amino acid residues.

[0038] The hydrophobic module can be composed of any convenient residue. In certain embodiments, the hydrophobic module comprises an amino acid residue selected from phenylalanine, tryptophan, alanine, valine, glycine, isoleucine, leucine, cysteine, and tyrosine. The stripathic region can comprise a total of two or more cationic amino acid residues, for example, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or more. The antimicrobial peptide can comprise a stripathic region composed of three or more hydrophilic modules composed of any convenient hydrophilic residues. In some embodiments, the hydrophilic module comprises an amino acid residue selected from lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, and ornithine. In certain embodiments, the hydrophilic module comprises an amino acid residue selected from lysine, arginine, and ornithine.

[0039] In certain embodiments, the antimicrobial peptide consists of three or more hydrophilic modules (e.g., 3, 4, 5, or 6 or more) and three or more hydrophobic modules (e.g., 3, 4, 5, or 6 or more), and is a peptide of Formula 1 or 2. Formula 1 and Formula 2 can be represented as follows to show the hydrophilic and hydrophobic modules: Formula 1:[X 1 ]-[J 2 J 3 ]-[X 4 X5 ]-[J 6 J 7 ]-[X 8 X 9 ]-[J 10 ]-[X 11 X 12 ]-[J 13 J 14 ]-[X 15 X 16 ]-[J 17 J 18 ] Formula 2: [J 1 J 2 ]-[X 3 X 4 ]-[J 5 J 6 ]-[X 7 X 8 ]-[J 9 ]-[X 10 X 11 ]-[J 12 J 13 ]-[X 14 X 15 ]-[J 16 J 17 ]-[X 18 ] wherein each X is independently a hydrophilic amino acid residue (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, or ornithine), and each J is independently a hydrophobic residue (e.g., phenylalanine, tryptophan, alanine, valine, glycine, isoleucine, leucine, or tyrosine). It is understood that, in some cases, the antimicrobial peptide of Formula 1 has a sequence that can be reversed to provide an antimicrobial peptide having the sequence of Formula 2.

[0040] In some embodiments of Formula 1, each X is independently selected from arginine, lysine, and ornithine. In some embodiments of Formula 1, each X is independently selected from lysine and ornithine. In some embodiments of Formula 1, each J is independently selected from phenylalanine, alanine, isoleucine, leucine, and valine. In some embodiments of Formula 1, each J is independently selected from phenylalanine and alanine. In some embodiments of Formula 2, each X is independently selected from arginine, lysine, and ornithine. In some embodiments of Formula 2, each X is independently selected from lysine and ornithine. In some embodiments of Formula 2, each J is independently selected from phenylalanine, alanine, isoleucine, leucine, and valine. In some embodiments of Formula 2, each J is independently selected from phenylalanine, alanine, and isoleucine. In some embodiments, the antimicrobial polypeptide of Formula 1 or 2 comprises one or more additional hydrophilic or hydrophobic residues at the C-terminus or N-terminus. In some embodiments, the antimicrobial polypeptide of Formula 1 or 2 comprises a tail region (eg, as described herein) at the C-terminus or N-terminus.

[0041] (Peptide of Formula 3) In some embodiments, the antimicrobial polypeptide comprises a sequence defined by Formula 3, where each residue in the sequence alternates between a hydrophobic residue (J) and a hydrophilic residue (X). Formula 3:J 1 X 2 J 3 X 4 J 5 X 6 J 7 X 8 J 9 X 10 J 11 X 12 J 13 X 14 J 15 X 16 J 17 wherein each X is independently a hydrophilic amino acid residue and each J is independently a hydrophobic residue.

[0042] In some embodiments of Formula 3, each X is independently selected from lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, and ornithine. In some embodiments of Formula 3, each X is independently selected from ornithine, lysine, and arginine. In some embodiments of Formula 3, each X is independently selected from ornithine and lysine. In some embodiments of Formula 3, each J is independently selected from phenylalanine, tryptophan, alanine, valine, glycine, isoleucine, leucine, and tyrosine. In some embodiments of Formula 3, each J is independently selected from phenylalanine, alanine, isoleucine, leucine, and valine. In some embodiments of Formula 3, the antimicrobial polypeptide comprises one or more additional hydrophilic or hydrophobic residues at the C-terminus and / or N-terminus. In some embodiments of Formula 3, the antimicrobial peptide comprises a tail region (e.g., as described herein) at the C-terminus and / or N-terminus.

[0043] In some embodiments of the antimicrobial polypeptide of Formula 3, the sequence has the formula defined by Formula 3A. Formula 3A:FX 2 J 3 X 4 J 5 X 6 J 7 X 8 J 9 X 10 J 11 X 12 J 13 X 14 J 15 X 16 J 17 where: X 2 and X 14 are each independently selected from O and R; J 3 and J 17 are each independently selected from L and I; X 4 and X 16 are each independently selected from K and O; J 5is selected from A and I; X 6 , X 8 , X 10 and X 12 are each independently selected from R, K, and O; J 7 is selected from F, A and I; J 9 is selected from V and L; J 11 is selected from A, V and L; J 13 is selected from A, I, and L, and J 15 is selected from I, F and L.

[0044] In some embodiments of Formula 3A, the antimicrobial peptide comprises the peptide sequence FRLKIKARLKVKIRFKL (RP554) (SEQ ID NO: 5), or a variant or fragment thereof.

[0045] In some embodiments of the antimicrobial polypeptide of Formula 3, the sequence has the formula defined by Formula 3B. Formula 3B:FOJ 3 X 4 AX 6 J 7 X 8 VX 10 J 11 X 12 J 13 O.J. 15 X 16 J 17 (SEQ ID NO: 24) where: J 3 , J 15 and J 17 are each independently selected from I and L; X 4 , X 8 and X 16 are each independently selected from O and K; X 6 and X 12 are each independently selected from O and R; J 7is selected from I and F, X 10 are each independently selected from O, K, and R; and J 11 and J 13 are each independently selected from A and L.

[0046] In some embodiments of Formula 3A or 3B, the antimicrobial peptide comprises the peptide sequence of FOIKARFOVRARLOLKI (RP553) (SEQ ID NO: 4), or a variant or fragment thereof.

[0047] In some embodiments of Formula 3A or 3B, the antimicrobial peptide comprises the peptide sequence of FOLOAOIOVOLOAOIOL (RP555) (SEQ ID NO: 6), or a variant or fragment thereof.

[0048] In some embodiments of Formula 3A or 3B, the antimicrobial peptide comprises the peptide sequence of FOLOAOIKVKLOAOIOL (RP556) (SEQ ID NO: 7), or a variant or fragment thereof.

[0049] (Peptide of Formula 6) The antimicrobial polypeptide can include two striapathic domains linked via a bubble domain (eg, as described herein).

[0050] In some embodiments, the antimicrobial polypeptide comprises a sequence defined by Formula 6, in which two stripathic regions composed of alternating hydrophobic (J) and hydrophilic (X) residues are connected by a central bubble region (B). Formula 6:J 1 X 2 J 3 X 4 J 5 X 6 J 7 (X 8 ) m -B-(X 13 ) m J 14 X 15 J16 X 17 J 18 X 19 J 20 where: B is a sequence selected from the following formulae CZC and GGC-Z-CGG (SEQ ID NO: 25), where each C is a cysteine ​​residue, each G is a glycine residue, Z consists of 3 to 5 amino acid residues selected from hydrophobic amino acid residues (J) and hydrophilic amino acid residues (X) (e.g., as described herein), and m is 0 or 1.

[0051] In some embodiments of the antimicrobial polypeptide of formula 6, B has the formula CJ 9 J 10 X 11 J 12 C and (G) n CJ 9 J 10 X 11 J 12 C(G) n (SEQ ID NO:26), wherein each C is a cysteine ​​residue, each G is a glycine residue, each n is independently selected from 1 to 4 (e.g., 1, 2, or 3), each X is independently selected from hydrophilic amino acid residues (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, and ornithine), and each J is independently selected from hydrophobic residues (e.g., phenylalanine, tryptophan, alanine, valine, glycine, isoleucine, leucine, and tyrosine).

[0052] In some embodiments of the peptide, J 9 , J 10 and J 12 is independently selected from leucine, glycine, and phenylalanine. 11 is selected from ornithine and lysine.

[0053] In some embodiments of the antimicrobial polypeptide of Formula 6, the polypeptide comprises a sequence defined in Formula 6A. Formula 6A:FX2 IX 4 AX 6 L(X 8 ) m -B-(X 13 ) m IX 15 AX 17 LX 19 F (SEQ ID NO: 27) where: B is the following formula CLGX 11 FC (SEQ ID NO: 28), GCLGX 11 FCG (SEQ ID NO: 29) and GGCLGX 11 FCGG (SEQ ID NO: 30), wherein each C is a cysteine ​​residue, each G is a glycine residue, and X 11 is selected from O and K; X 2 , X 4 , each X 8 , each X 13 , X 15 and X 19 are each independently selected from O and K; X 6 and X 17 are each independently selected from R and O, and Each m is independently an integer selected from 0 and 1.

[0054] In some embodiments of Formula 6A, the antimicrobial peptide comprises the peptide sequence FOIOAOLGGCLGOFCGGIOAOLOF (RP564) (SEQ ID NO: 15), or a variant or fragment thereof.

[0055] In some embodiments of Formula 6A, the antimicrobial peptide comprises the peptide sequence FOIOAOLOGGCLGOFCGGOIOAOLOF (RP565) (SEQ ID NO: 16), or a variant or fragment thereof.

[0056] In some embodiments of Formula 6A, the antimicrobial peptide comprises the peptide sequence FOIKAOLGGCLGKFCGGIKAOLKF (RP566) (SEQ ID NO: 17), or a variant or fragment thereof.

[0057] In some embodiments of Formula 6A, the antimicrobial peptide comprises a peptide sequence.

[0058] FOIKAOLKGGCLGKFCGGKIKAOLKF (RP567) (SEQ ID NO: 18), or a variant or fragment thereof.

[0059] In some embodiments, the antimicrobial polypeptide comprises a sequence defined in Formula 6B, based on Formula 6. Formula 6B:J 1 X 2 J 3 X 4 J 5 X 6 J 7 -C.J. 9 J 10 X 11 J 12 CJ 14 X 15 J 16 X 17 J 18 X 19 wherein each C is a cysteine ​​residue, each X is independently selected from hydrophilic amino acid residues (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, and ornithine), and each J is independently selected from hydrophobic residues (e.g., phenylalanine, tryptophan, alanine, valine, glycine, isoleucine, leucine, and tyrosine). In some embodiments, each X is independently selected from ornithine, lysine, and arginine. In some embodiments, each J is independently selected from phenylalanine, alanine, isoleucine, leucine, and glycine.

[0060] In some embodiments of Formula 6B, the antimicrobial peptide comprises the peptide sequence of FKIOARLCLGOFCIOARLK(RP550) (SEQ ID NO: 1), or a variant or fragment thereof.

[0061] In some embodiments, the antimicrobial polypeptide comprises a sequence defined in Formula 6C, based on Formula 6. Formula 6C:J 1 X 2 J 3 X 4 J 5 X 6 J 7 -CGCJ 9 J 10 X 11 J 12 CGG-J 14 X 15 J 16 X 17 J 18 X 19 J 20 (SEQ ID NO: 31) wherein C is a cysteine ​​residue, G is a glycine residue, each X is independently a hydrophilic amino acid residue (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, or ornithine), and each J is independently a hydrophobic residue (e.g., phenylalanine, tryptophan, alanine, valine, glycine, isoleucine, leucine, or tyrosine). In some embodiments, each X is independently selected from ornithine, lysine, and arginine. In some embodiments, each J is independently selected from phenylalanine, alanine, isoleucine, leucine, and glycine.

[0062] In some embodiments of Formula 6C, the antimicrobial peptide comprises a peptide sequence comprising the sequence FOIOAOLGGCLGOFCGGIOAOLOF (RP564) (SEQ ID NO: 15), or a variant or fragment thereof.

[0063] In some embodiments of Formula 6C, the antimicrobial peptide comprises the peptide sequence FOIKAOLGGCLGKFCGGIKAOLKF (RP566) (SEQ ID NO: 17), or a variant or fragment thereof.

[0064] In some embodiments, the antimicrobial polypeptide comprises a sequence defined in Formula 6D, based on Formula 6. Formula 6D:J 1 X 2 J 3 X 4J 5 X 6 J 7 X 8 -GGCJ 9 J 10 X 11 J 12 CGG-X 13 J 14 X 15 J 16 X 17 J 18 X 19 J 20 (SEQ ID NO: 32) wherein C is a cysteine ​​residue, G is a glycine residue, each X is independently a hydrophilic amino acid residue (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, or ornithine), and each J is independently a hydrophobic residue (e.g., phenylalanine, tryptophan, alanine, valine, glycine, isoleucine, leucine, or tyrosine). In some embodiments, each X is independently selected from ornithine, lysine, and arginine. In some embodiments, each J is independently selected from phenylalanine, alanine, isoleucine, leucine, and glycine.

[0065] In certain embodiments of the antimicrobial polypeptide of any one of Formulas 6B-6D, J 1 X 2 J 3 X 4 J 5 X 6 J 7 FX 2 IX 4 AX 6 L (SEQ ID NO: 33), where X 2 and X 4 are each independently selected from O and K; 6 is selected from R and O. In one embodiment of any one of formulae 6B-6D, FX 2 IX 4 AX 6L (SEQ ID NO: 33) is selected from FKIOARL (SEQ ID NO: 34), FOIOAOL (SEQ ID NO: 35), and FOIKAOL (SEQ ID NO: 36). In any one of Formulas 6B to 6D, FX 2 IX 4 AX 6 L (SEQ ID NO: 33) is FKIOARL (SEQ ID NO: 34). In any one of Formulas 6B to 6D, FX 2 IX 4 AX 6 L (SEQ ID NO: 33) is FOIOAOL (SEQ ID NO: 35). In another embodiment of any one of Formulas 6B to 6D, FX 2 IX 4 AX 6 L (SEQ ID NO: 33) is FOIKAOL (SEQ ID NO: 36).

[0066] In some embodiments of Formula 6D, the antimicrobial peptide comprises the peptide sequence FOIOAOLOGGCLGOFCGGOIOAOLOF (RP565) (SEQ ID NO: 16), or a variant or fragment thereof.

[0067] In some embodiments of Formula 6D, the antimicrobial peptide comprises the peptide sequence FOIKAOLKGGCLGKFCGGKIKAOLKF (RP567) (SEQ ID NO: 18), or a variant or fragment thereof.

[0068] In certain embodiments of any of Formulas 6B through 6D, the sequence fragment J 14 X 15 J 16 X 17 J 18 X 19 J 20 IX 15 AX 17 LX 19 (F) p (SEQ ID NO: 37), wherein X 15 and X 19 are each independently selected from O and K; 17 is selected from R and O, and p is 1 or 0. In one embodiment of Formulae 6B to 6D, sequence fragment IX 15 AX17 LX 19 (F) p (SEQ ID NO: 37) is selected from IOARLK (SEQ ID NO: 38), IOAOLOF (SEQ ID NO: 39) and IKAOLKF (SEQ ID NO: 40). 15 AX 17 LX 19 (F) p (SEQ ID NO: 37) is IOARLK (SEQ ID NO: 38). In any one of Formulas 6B to 6D, sequence fragment IX 15 AX 17 LX 19 (F) p (SEQ ID NO: 37) is IOAOLOF (SEQ ID NO: 39). In any one of Formulas 6B to 6D, sequence fragment IX 15 AX 17 LX 19 (F) p is IKAOLKF (SEQ ID NO: 40).

[0069] In some embodiments of the antimicrobial polypeptide of Formula 6, the region having the sequence CZC or GGC-Z-CGG (SEQ ID NO: 25) is referred to as a bubble region as described herein. In certain embodiments, Z is a group of the formula J 9 J 10 X 11 J 12 wherein each J is independently selected from leucine, glycine, and phenylalanine, and X is selected from ornithine and lysine. In some embodiments, the bubble region in any of Formulas 6-6D has a sequence selected from CLGOFC (SEQ ID NO: 41), CLGKFC (SEQ ID NO: 42), GGCLGOFC (SEQ ID NO: 43), and GGCLGKFCGG (SEQ ID NO: 44).

[0070] (Peptide of Formula 7) In some embodiments, the antimicrobial polypeptide comprises a sequence defined by Formula 7, which includes four cysteine ​​residues. Formula 7:Z 1 -C 1 -Z 2 -C2 -Z 3 -C 3 -Z 4 -C 4 -Z 5 (SEQ ID NO: 45) where C 1 From C 4 are cysteine ​​residues, Z 1 ~Z 5 are each independently 1 to 5 amino acid residues. 1 , Z 2 , Z 4 and Z 5 Each of Z is a linear region consisting of a mixture of hydrophobic amino acid residues (J) and hydrophilic residues (X) (e.g., as described herein). 3 may be any convenient linked sequence of residues.

[0071] In some embodiments, Z 1 ~Z 5 Each of Z consists of a mixture of hydrophobic and hydrophilic amino acid residues. 1 From Z 4 Each of Z consists of a mixture of hydrophobic and hydrophilic residues, 5 is a single hydrophilic residue. In some embodiments, Z 2 From Z 5 Each of Z consists of a mixture of hydrophobic and hydrophilic residues, 1 is a single hydrophilic residue. In some embodiments, Z 1 , Z 3 and Z 5 each of which consists of a mixture of hydrophobic and hydrophilic residues, and Z 2 and Z 4 are each a single hydrophilic residue. 3 consists of a mixture of four amino acid residues selected from hydrophobic and hydrophilic residues. 2 and Z 4 consist of an equal number of amino acid residues.

[0072] In some embodiments, the antimicrobial polypeptide of Formula 7 comprises one or more additional hydrophilic or hydrophobic residues at the C-terminus or N-terminus.

[0073] In some embodiments, the antimicrobial polypeptide is based on Formula 7 and comprises a sequence defined by Formula 7A. Formula 7A:X 1 J 2 -C 1 -J 3 X 4 J 5 -C 2 -J 6 X 7 J 8 J 9 -C 3 -J 10 X 11 X 12 -C 4 -X 13 (SEQ ID NO: 46) where C 1 ~C 4 are each a cysteine ​​residue, each X is independently selected from a hydrophilic amino acid residue (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, and ornithine), and each J is independently selected from a hydrophobic residue (e.g., phenylalanine, tryptophan, alanine, valine, glycine, isoleucine, leucine, and tyrosine). In some embodiments, each X is independently selected from lysine, ornithine, and arginine. In some embodiments, each J is independently selected from phenylalanine, isoleucine, glycine, tryptophan, valine, and tyrosine.

[0074] In some embodiments, the antimicrobial polypeptide of Formula 7A is 1 It has. formula 7A 1 :X 1 J 2 -C 1 -J 3 X 4 VC 2 -YX 7 G.I.C. 3 -J 10 X11 X 12 -C 4 -X 13 (SEQ ID NO: 47) where X 1 is selected from O and R; J 2 and J 3 are each independently selected from F and W; X 4 , X 7 , X 11 , X 12 and X 13 are each independently selected from O and K, and J 10 is selected from Y and F.

[0075] formula 7A 1 In some embodiments, the antimicrobial peptide comprises the peptide sequence RFCWKVCYKGICFKKCK (RP557) (SEQ ID NO: 8), or a variant or fragment thereof.

[0076] formula 7A 1 In some embodiments, the antimicrobial peptide comprises the peptide sequence RWCFKVCYKGICYKKCK (RP560) (SEQ ID NO: 11), or a variant or fragment thereof.

[0077] formula 7A 1 In some embodiments, the antimicrobial peptide comprises the peptide sequence of OWCFOVCYOGICYOOCO (RP559) (SEQ ID NO: 10), or a variant or fragment thereof.

[0078] formula 7A 1 In some embodiments, the antimicrobial peptide comprises the peptide sequence of OFCWOVCYOGICFOOCO(RP661) (SEQ ID NO: 12), or a variant or fragment thereof.

[0079] In some embodiments, the antimicrobial polypeptide is based on Formula 7 and comprises a sequence defined in Formula 7B. Formula 7B:X 1 -C 1 -X2 X 3 J 4 -C 2 -J 5 J 6 X 7 J 8 -C 3 -J 9 X 10 J 11 -C 4 -J 12 X 13 (SEQ ID NO: 48) where C 1 ~C 4 are each a cysteine ​​residue, each X is independently selected from a hydrophilic amino acid residue (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, and ornithine), and each J is independently selected from a hydrophobic residue (e.g., phenylalanine, tryptophan, alanine, valine, glycine, isoleucine, leucine, and tyrosine). In some embodiments, each X is independently selected from lysine, ornithine, and arginine. In some embodiments, each J is independently selected from phenylalanine, isoleucine, glycine, tryptophan, valine, and tyrosine.

[0080] In some embodiments, the antimicrobial polypeptide of Formula 7B is 1 It has. Formula 7B 1 :X 1 -C 1 -X 2 X 3 J 4 -C 2 -IGX 7 YC 3 -VX 10 J 11 -C 4 -J 12 X 13 (SEQ ID NO: 49) where X 1 , X 2 , X 3 , X 7 and X 10 are each independently selected from O and R; J4 is selected from Y and F; J 11 and J 12 are each independently selected from F and W, and X 13 is selected from K and O.

[0081] Formula 7B 1 In some embodiments, the antimicrobial peptide comprises the peptide sequence RCRRYCIGRYCVRFCWK (RP558) (SEQ ID NO: 9), or a variant or fragment thereof.

[0082] Formula 7B 1 In some embodiments, the antimicrobial peptide comprises the peptide sequence OCOOFCIGOYCVOWCFO (RP562) (SEQ ID NO: 13), or a variant or fragment thereof.

[0083] In some embodiments, the antimicrobial polypeptide is based on Formula 7 and comprises a sequence defined in Formula 7C. Formula 7C:X 1 J 2 J 3 -C 1 -J 4 -C 2 -J 5 X 6 X 7 X 8 -C 3 -J 9 -C 4 -J 10 X 11 J 12 J 13 X 14 (SEQ ID NO: 50) where C 1 ~C 4are each cysteine ​​residue, each X is independently selected from hydrophilic amino acid residues (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, and ornithine), and each J is independently selected from hydrophobic residues (e.g., phenylalanine, tryptophan, alanine, valine, glycine, isoleucine, leucine, and tyrosine). In some embodiments, each X is arginine. In some embodiments, each J is independently selected from phenylalanine, glycine, leucine, valine, and tyrosine.

[0084] In some embodiments of the antimicrobial polypeptide of Formula 7, the sequence C 2 -Z 3 -C 3 is a bubble region as described herein. 3 consists of a mixture of 3-5 hydrophobic and hydrophilic amino acid residues (e.g., J and X as described herein). 3 consists of a mixture of four amino acid residues selected from tyrosine, glycine, phenylalanine, isoleucine, lysine, arginine, and ornithine. 3 is composed of three hydrophobic amino acid residues (J) and one hydrophilic amino acid residue (X). In certain cases, Z 3 consists of tyrosine, glycine, isoleucine, and lysine residues. 3 may consist of tyrosine, glycine, isoleucine, ornithine. In certain cases, Z 3 is composed of tyrosine, glycine, isoleucine, and lysine residues. In some embodiments, the bubble region of Formula 7 has the formula CYKGIC (SEQ ID NO: 22). In some embodiments, the bubble region in Formula 7 has the formula CYOGIC (SEQ ID NO: 51). In some embodiments, the bubble region in Formula 7 has the formula CIGRYC (SEQ ID NO: 52). In some embodiments, the bubble region in Formula 7 has the formula CIGOYC (SEQ ID NO: 53). In some embodiments, Z 3consists of one hydrophobic amino acid residue (J) and three hydrophilic amino acid residues (X). 3 consists of one phenylalanine residue and three arginine residues. In some embodiments, the bubble region of Formula 7 has the formula CFRRRC (SEQ ID NO: 54).

[0085] In some embodiments of the antimicrobial polypeptide of Formula 7, the sequence C 2 -Z 3 -C 3 The bubble region having 1 and C 4 are linked to form an additional linking region (see, for example, FIG. 1B). In certain embodiments, the antimicrobial peptide of formula 7 consists of a formula selected from 7A to 7C, each of which forms a hairpin secondary structure comprising two linking regions. Specific examples of antimicrobial peptides of the present invention according to 7A comprising a bubble region and an additional linking region include the following: RF C WKV CYKGIC FKK C K (RP557, SEQ ID NO: 8).

[0086] Examples of antimicrobial peptides according to embodiments of the present invention are shown in Table 2 below. These examples are representative and do not limit the scope of the invention. For example, fragments (e.g., as described herein) or variants (e.g., as described herein) are of interest. The "O" residue in the sequences shown below represents the amino acid ornithine.

[0087] [Table 2]

[0088] The exemplary antimicrobial polypeptide sequences described herein (e.g., Table 2) are merely exemplary and are not the only antimicrobial polypeptides provided herein. Indeed, fragments and variants of the disclosed peptide sequences are within the scope of the present disclosure.

[0089] "Fragments" of the present invention comprise at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive amino acid residues of a polypeptide disclosed herein (or up to one less than the number of amino acid residues in the subject polypeptide), and retain at least one antimicrobial property of the subject polypeptide. Thus, fragments of the present invention include polypeptides lacking 1, 2, 3, 4, or more amino acids from the N-terminus and / or C-terminus relative to the polypeptides disclosed herein.

[0090] "Variants" of the present invention are polypeptides that are substantially similar to the polypeptides disclosed herein and retain at least one antimicrobial property of the subject polypeptides. Variants can include deletions (i.e., truncations) of one or more amino acid residues at the N-terminus or C-terminus of the subject polypeptides disclosed herein, deletions and / or additions of one or more amino acid residues at one or more internal sites of the subject polypeptides disclosed herein, and / or substitutions of one or more amino acid residues at one or more positions of the subject polypeptides disclosed herein. For subject polypeptides 17 amino acid residues or shorter in length, the variant polypeptides can contain three or fewer (e.g., two, one, or none) deleted amino acid residues, whether located internally, at the N-terminus, and / or at the C-terminus.

[0091] Thus, in certain embodiments, the present invention provides polypeptides comprising an amino acid sequence having 1 to 10 amino acid differences (e.g., 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid difference) from any one of the antimicrobial polypeptides disclosed herein (e.g., as shown in Table 2), and which retain at least one antimicrobial property. As used herein, "amino acid difference" includes amino acid substitutions, amino acid insertions, terminal amino acid additions, amino acid deletions, terminal amino acid truncations, or any combination thereof. The substituted amino acid residue (or residues) may be unrelated to the amino acid residue being substituted (e.g., unrelated in terms of hydrophobicity / hydrophilicity, size, charge, polarity, etc.), or the substituted amino acid residue may constitute a similar, conservative, or highly conservative amino acid substitution. As used herein, "similar," "conservative," and "highly conservative" amino acid substitutions are defined as set forth in Table 3 below. The determination of whether an amino acid residue substitution is similar, conservative, or highly conservative is based solely on the side chain of the amino acid residue and not on the peptide backbone, which may be modified to enhance peptide stability as described below.

[0092] [Table 3]

[0093] Accordingly, the present invention further provides polypeptides comprising an amino acid sequence that is at least 50% identical (e.g., at least 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical) to any one of the antimicrobial polypeptides disclosed herein (e.g., those shown in Table 2), and that retains at least one antimicrobial property. In certain embodiments, such polypeptide sequences comprise a cationic-charged amphipathic region or a striapathic region, as described in detail above. Furthermore, such polypeptides may comprise additional structural features, such as a bubble region, a β-turn region, a polyproline helix structure, a tail, a dimer of an amphipathic region, or the like, as described herein.

[0094] In certain embodiments, the subject antimicrobial polypeptide comprises a sequence comprising: a) a peptide sequence selected from RP550 to RP567 (SEQ ID NOs: 1 to 18), b) a sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, or at least 95% sequence identity) with a sequence defined in a); or c) Sequences which have 5 or fewer (e.g., 4 or fewer, 3 or fewer, 2 or fewer, such as 1 or 2) amino acid substitutions relative to the sequence defined in a), wherein one or two of the amino acid substitutions are amino acid substitutions in accordance with Table 3 (e.g., similar amino acid substitutions, conservative amino acid substitutions, or highly conservative amino acid substitutions).

[0095] In certain embodiments, the subject antimicrobial polypeptide comprises a sequence comprising: a) a peptide sequence selected from RP550 to RP567 (SEQ ID NOs: 1 to 18), or b) sequences having five or fewer (e.g., four or fewer, three or fewer, two or fewer, such as one or two) amino acid substitutions relative to the sequences defined in a), wherein the five or fewer amino acid substitutions consist of substituting cationic amino acids of the sequence with alternative cationic amino acid residues (e.g., K for O, O for K, K for R, etc.).

[0096] In certain embodiments, the sequence defined in a) comprises at least one lysine residue substituted with an ornithine, histidine, or arginine residue. In some embodiments, the sequence defined in a) comprises at least one lysine residue substituted with an ornithine or arginine residue. In other embodiments, the sequence defined in a) consists of at least one ornithine residue substituted with a lysine, histidine, or arginine residue. In some cases, the sequence defined in a) consists of at least one ornithine residue substituted with a lysine or arginine residue. In other cases, the sequence defined in a) consists of at least one arginine residue substituted with an ornithine, histidine, or lysine residue. In some embodiments, the sequence defined in a) consists of at least one arginine residue substituted with an ornithine or lysine residue.

[0097] In certain embodiments, the sequence of interest consists of RP550 (SEQ ID NO: 1). In certain embodiments, the sequence of interest consists of RP551 (SEQ ID NO: 2). In certain embodiments, the sequence of interest consists of RP552 (SEQ ID NO: 3). In certain embodiments, the sequence of interest consists of RP553 (SEQ ID NO: 4). In certain embodiments, the sequence of interest consists of RP554 (SEQ ID NO: 5). In certain embodiments, the sequence of interest consists of RP555 (SEQ ID NO: 6). In certain embodiments, the sequence of interest consists of RP556 (SEQ ID NO: 7). In certain embodiments, the sequence of interest consists of RP557 (SEQ ID NO: 8). In certain embodiments, the sequence of interest consists of RP558 (SEQ ID NO: 9). In certain embodiments, the sequence of interest consists of RP559 (SEQ ID NO: 10). In certain embodiments, the sequence of interest consists of RP560 (SEQ ID NO: 11). In certain embodiments, the sequence of interest consists of RP561 (SEQ ID NO: 12). In certain embodiments, the sequence of interest consists of RP562 (SEQ ID NO: 13). In certain embodiments, the sequence of interest consists of RP563 (SEQ ID NO: 14). In certain embodiments, the sequence of interest consists of RP564 (SEQ ID NO: 15). In certain embodiments, the sequence of interest consists of RP565 (SEQ ID NO: 16). In certain embodiments, the sequence of interest consists of RP566 (SEQ ID NO: 17). In certain embodiments, the sequence of interest consists of RP567 (SEQ ID NO: 18).

[0098] In a particular embodiment, the sequence set forth in a) is RP550 (SEQ ID NO: 1). In a particular embodiment, the sequence set forth in a) is RP551 (SEQ ID NO: 2). In a particular embodiment, the sequence set forth in a) is RP552 (SEQ ID NO: 3). In a particular embodiment, the sequence set forth in a) is RP553 (SEQ ID NO: 4). In a particular embodiment, the sequence set forth in a) is RP554 (SEQ ID NO: 5). In a particular embodiment, the sequence set forth in a) is RP555 (SEQ ID NO: 6). In a particular embodiment, the sequence set forth in a) is RP556 (SEQ ID NO: 7). In a particular embodiment, the sequence set forth in a) is RP557 (SEQ ID NO: 8). In a particular embodiment, the sequence set forth in a) is RP558 (SEQ ID NO: 9). In a particular embodiment, the sequence set forth in a) is RP559 (SEQ ID NO: 10). In a particular embodiment, the sequence set forth in a) is RP560 (SEQ ID NO: 11). In a particular embodiment, the sequence set forth in a) is RP561 (SEQ ID NO: 12). In certain embodiments, the sequence set forth in a) is RP562 (SEQ ID NO: 13). In certain embodiments, the sequence set forth in a) is RP563 (SEQ ID NO: 14). In certain embodiments, the sequence set forth in a) is RP564 (SEQ ID NO: 15). In certain embodiments, the sequence set forth in a) is RP565 (SEQ ID NO: 16). In certain embodiments, the sequence set forth in a) is RP566 (SEQ ID NO: 17). In certain embodiments, the sequence set forth in a) is RP567 (SEQ ID NO: 18).

[0099] Particular examples of peptides (eg, described herein) and compositions of interest exclude one or more of the following peptide sequences in Table 4:

[0100] [Table 4]

[0101] (composition) The present disclosure provides compositions comprising the antimicrobial polypeptides described herein. For example, the antimicrobial polypeptide may be any of the polypeptides listed in Table 2, or a fragment or variant thereof that retains antimicrobial activity. In certain embodiments, the antimicrobial polypeptide included in a composition of the present disclosure is a synthetic polypeptide (e.g., chemically synthesized and / or recombinantly produced).

[0102] The compositions of the present invention can contain a single antimicrobial polypeptide or a combination of different antimicrobial polypeptides. The compositions can be substantially free of proteins and other polypeptides. As used herein, the term "substantially free of proteins and other polypeptides" means that less than 5% of the protein content of the composition is made up of proteins and other polypeptides that are not antimicrobial polypeptides of the present invention. A composition that is substantially free of non-antimicrobial polypeptides of the present invention can have 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less of other non-antimicrobial polypeptides.

[0103] In certain embodiments, the compositions of the invention comprise an antimicrobial polypeptide that does not naturally occur in humans or other mammals or animals.

[0104] The subject composition can be provided in any convenient form.In some embodiments, the composition is a solid composition.In certain embodiments, the composition is a liquid, for example, an aqueous composition.In certain embodiments, the solid composition is a lyophilized composition that can be provided for final use in a solid form suitable for reconstitution with a liquid, for example, an aqueous solution.

[0105] Compositions of the invention may comprise at least 0.1 mg (e.g., at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 1, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000 mg, or more) of antimicrobial polypeptide. Thus, for example, the composition may contain from about 0.1 mg to about 1 mg, or from about 1 mg to about 1000 mg (e.g., from about 5 mg to about 900 mg, from about 5 mg to about 800 mg, from about 5 mg to about 700 mg, from about 5 mg to about 600 mg, from about 10 mg to about 500 mg, from about 10 mg to about 400 mg, from about 10 mg to about 300 mg, from about 10 mg to about 250 mg, from about 10 mg to about 200 mg, from about 10 mg to about 150 mg, from about 10 mg to about 100 mg, from about 50 mg to about 500 mg, from about 50 mg to about 400 mg, from about 50 mg to about 300 mg, from about 50 mg to about 250 mg, from about 50 mg to about 500 mg, The composition may contain an amount of antimicrobial polypeptide equivalent to about 50 mg to about 150 mg, about 50 mg to about 100 mg, about 75 mg to about 500 mg, about 75 mg to about 400 mg, about 75 mg to about 300 mg, about 75 mg to about 250 mg, about 75 mg to about 200 mg, about 75 mg to about 150 mg, about 75 mg to about 100 mg, about 100 mg to about 500 mg, about 100 mg to about 400 mg, about 100 mg to about 300 mg, about 100 mg to about 250 mg, about 100 mg to about 200 mg, or another range inclusive of any two of the foregoing endpoints.

[0106] Compositions of the invention may comprise a solution comprising at least 0.1 mg / ml (e.g., at least 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / ml or more) of an antimicrobial polypeptide. Thus, for example, the composition can comprise a solution having an antimicrobial polypeptide concentration of from about 0.1 mg / ml to about 100 mg / ml (e.g., from about 5 mg / ml to about 90 mg / ml, from about 5 mg / ml to about 80 mg / ml, from about 5 mg / ml to about 70 mg / ml, from about 5 mg / ml to about 60 mg / ml, from about 5 mg / ml to about 50 mg / ml, from about 10 mg / ml to about 50 mg / ml, from about 10 mg / ml to about 40 mg / ml, from about 10 mg / ml to about 30 mg / ml, from about 10 mg / ml to about 25 mg / ml, from about 10 mg / ml to about 20 mg / ml, from about 10 mg / ml to about 15 mg / ml, or other range including two of the foregoing endpoints).

[0107] The compositions of the present invention include pharmaceutical compositions. Such pharmaceutical compositions can include one or more antimicrobial polypeptides and a pharmaceutically acceptable carrier. The pharmaceutical compositions can further include an additional bioactive agent other than the antimicrobial polypeptides of the present invention. The additional bioactive agent can be a therapeutic / antimicrobial agent, such as a conventional antibiotic. The conventional antibiotic can have antimicrobial or other properties that are enhanced or enhanced by the antimicrobial polypeptides of the present invention. In some embodiments, the additional bioactive agent is selected from antimicrobial agents, anti-inflammatory agents, anti-nausea agents, pain relievers, and combinations thereof. In certain embodiments, the pharmaceutical composition includes a carrier, e.g., a carrier protein such as serum albumin (e.g., HAS, BSA, etc.), which can be purified or recombinantly produced. Mixing the antimicrobial polypeptide(s) in the pharmaceutical composition with serum albumin can effectively "load" the serum albumin with the antimicrobial polypeptide, allowing for greater delivery of the antimicrobial polypeptide to the site of infection.

[0108] The pharmaceutical compositions of the present invention can be formulated for oral administration, parenteral administration, inhalation administration, topical administration, and the like. Compositions formulated for oral administration can include, for example, an enteric coating to ensure that the antimicrobial peptide contained therein reaches the intestine and beyond. Compositions for topical administration can be suspended in gels or creams, coated onto microneedles, or injected into bandages or topical patches to extend the duration of action of the antimicrobial peptide contained therein. Any inhalable formulation capable of providing an aerosolized form containing the peptide of interest for delivery to a patient via the pulmonary route can be used in conjunction with the present disclosure. In some embodiments, the inhalable composition comprises liposomes that are delivered to the lungs of a human patient via aerosol, the liposomes containing free and encapsulated peptides. The liposomes may be unilamellar or multilamellar and may have bioadhesive properties, including molecules such as hyaluronic acid. The composition may also include at least one therapeutic agent in addition to the free and liposomally encapsulated anti-infective agent. The therapeutic agent may be a free or encapsulated peptide present in a pharmaceutically acceptable carrier useful for direct inhalation into the human lungs.

[0109] In certain embodiments, it may be desirable to administer one or more compounds of the invention locally to the area in need of treatment. This may be achieved, for example, by local infusion during surgery, topical application, application after surgery, e.g., in conjunction with a wound dressing, injection, administration by catheter, administration by suppository, or administration by implant, which may be a porous, non-porous, or gelatinous material, including a membrane, e.g., a sialastic membrane, or a fiber.

[0110] In certain examples, the pharmaceutical composition is an ophthalmic composition formulated for the treatment of an ophthalmic disease or condition. The ophthalmic compositions described herein may be formulated in any applicable dosage form. Exemplary dosage forms include, but are not limited to, eye drops (liquids), ointments, oils, multi-phase systems (e.g., liposomes, micelles, homogenates or suspensions of liquid or semi-solid or solid particles), gels, creams, pads, or strips. In one embodiment, the active ingredient (peptide) is contained in an aqueous pharmaceutical preparation. In another embodiment, the active ingredient is in a petrolatum-based pharmaceutical product. One embodiment of the present invention is the use of a topical formulation of a peptide described herein to treat ocular infections caused by, but not limited to, bacteria, herpes simplex virus, cytomegalovirus, varicella-zoster virus, adenovirus, and / or combinations thereof. One embodiment of the present invention is the use of a topical formulation of a peptide described herein to treat bacterial keratitis. In some cases, bacterial keratitis is caused by infection with Staphylococcus aureus or Pseudomonas aeruginosa.

[0111] The present compositions can be applied topically to a patient in need of treatment. In some cases, the compositions are formulated as eye drops. In some cases, the compositions are formulated for subconjunctival administration. In some cases, the ophthalmic compositions are aqueous compositions containing the subject peptide or a pharmacologically acceptable salt, optionally in combination with any convenient additional excipient, such as a cellulose compound or a pharmacologically acceptable salt, in an amount effective to increase intraocular absorption into the aqueous humor. The ophthalmic compositions of the present invention can be applied discretely as eye drops one to four times daily. Alternatively, the antimicrobial peptide may be applied continuously over time by any topical means. Hydrophilic hydrogel contact lenses and ocular polymer inserts for topically applying the ophthalmic compositions to a patient's eye are also disclosed. Any convenient method and composition can be adapted for use in combination with the subject ophthalmic compositions. See, for example, U.S. Patent No. 9,044,425. The compositions can include any carrier or excipient suitable for intraocular administration known to those skilled in the art. Exemplary excipients include those described in US Patent Application Publication No. 2008 / 0241252 and WO 2004 / 043480 to Lyons et al., which are incorporated by reference in their entireties.

[0112] Although liposomes are described as a vehicle for providing encapsulation of therapeutic peptides, it is not intended that the formulations be limited to liposomal formulations. A combined immediate-release and sustained-release formulation or carrier of anti-infective peptides in the lungs can be achieved through a number of methods, including microspheres, polymers, gels, emulsions, microparticles, or suspensions, alone or in combination. Some formulations or carriers have properties that result in closer association with the biofilm matrix, which may prove more advantageous for increasing therapeutic levels of anti-infective peptides in proximity to biofilm bacteria.

[0113] Aspects of the present disclosure include antimicrobial compositions that find use in a variety of applications. In some examples, the subject compositions find use as antimicrobial preservatives, disinfectants, or aseptic preservatives. Applications of interest include use as disinfectants or preservatives for intraocular devices, such as contact lenses, intraocular lens implants, and drug-eluting intraocular devices. In certain examples, the subject compositions can be provided to a subject as solid or liquid compositions for use in solutions for storing, soaking, and / or rinsing contact lenses. This solution is well suited for ophthalmic use and is effective for cleaning, disinfecting, and sterilizing contact lenses upon exposure to the composition, without the need for physical or thermal treatment of the contact lenses. Any convenient carrier, excipient, and / or diluent used in sterilizing or disinfecting solutions can be adapted for inclusion in the subject antimicrobial compositions. See, for example, U.S. Patent No. 6,482,799.

[0114] Alternatively, the antimicrobial peptides of the present disclosure can be coated onto the surface of medical devices, such as implantable medical devices, surgical instruments, and indwelling medical devices (e.g., pacemakers, catheters, artificial joints, etc.) as a means of preventing infection.

[0115] (method) The polypeptides of the present disclosure provide powerful tools for treating or preventing a variety of conditions and diseases of interest. Method aspects include administering to a subject in need thereof a therapeutically effective amount of a subject peptide to treat the subject. A "therapeutically effective amount" refers to a concentration of peptide sufficient to elicit a desired biological effect (e.g., treatment of a condition or disease). "Treatment," "treating," and similar terms are intended to mean stabilizing a pathological condition, alleviating symptoms, preventing the onset, or curing. Thus, treatment also includes situations in which a pathological condition, or at least the symptoms associated therewith, are completely suppressed, e.g., prevented from occurring, or arrested, e.g., terminated, such that the host is no longer afflicted by the condition or at least the symptoms characterizing the condition. Thus, treatment includes (i) prevention, i.e., reducing the risk of developing clinical symptoms, including preventing clinical symptoms from developing, e.g., preventing the progression of the disease to a deleterious state; (ii) suppression, i.e., halting the onset or further development of clinical symptoms, e.g., reducing or completely suppressing active disease; and / or (iii) palliation, i.e., causing regression of clinical symptoms.

[0116] The antimicrobial polypeptides of the present invention provide a powerful tool for treating or preventing microbial infections in a subject caused by a variety of microorganisms (e.g., bacteria, viruses, fungi, and parasites). Accordingly, the present invention provides methods for eliminating, reducing the numbers of, or significantly reducing the replication of at least one microbial organism in a subject. The subject peptides can have broad-spectrum antifungal and antibacterial activity. In certain cases, the subject peptides and methods provide a reduced risk of developing pathogen resistance.

[0117] The subject may be any animal, such as a farm animal (e.g., horse, cow, pig, goat, sheep, rabbit, chicken, turkey, duck, etc.), a pet (e.g., dog, cat, rabbit, hamster, gerbil, bird, fish, etc.), a laboratory animal (e.g., mouse, rat, monkey, chimpanzee, owl, fish, etc.), a zoo animal (e.g., gorilla, orangutan, chimpanzee, monkey, elephant, camel, zebra, wild boar, lion, tiger, giraffe, bear, bird, etc.), a wild animal (e.g., deer, wolf, mountain lion, bird, etc.), or a human subject (e.g., a patient). In some cases, the subject is a human.

[0118] Subjects suitable for treatment with peptide compositions can be identified by well-established indicators of risk for developing disease or well-established characteristics of existing disease. For example, indicators of infection include fever, pus, positive cultures for microorganisms, inflammation, etc. Infectious diseases that can be treated with the peptides provided by the present invention include, without limitation, those caused by microorganisms, whether the infection is primary, secondary, opportunistic, etc. Examples of microorganisms include bacteria (e.g., gram-positive, gram-negative), fungi (e.g., yeast, mold), parasites (e.g., protozoa, nematodes, worms, echinoderms), viruses (e.g., HIV, HSV, VSV), algae, and prions. Specific organisms in these classes are well known (see, e.g., Davis et al., Microbiology, 3rd edition, Harper & Row, 1980, and Stanier et al., The Microbial World, 5th edition, Prentice Hall, 1986). Infectious diseases of interest that can be treated or prevented in accordance with the subject methods include, but are not limited to, toxic shock syndrome, diphtheria, cholera, typhoid, meningitis, whooping cough, botulism, tetanus, pyogenic infections, sinusitis, pneumonia, gingivitis, mucositis, folliculitis, cellulitis, acne and acne vulgaris, impetigo, osteomyelitis, and endocarditis. Ulcers, burns, dysentery, urinary tract infections, gastroenteritis, anthrax, Lyme disease, syphilis, rubella, sepsis, Buruli ulcer, mycosis, chromoblastomycosis, vaginal candidiasis, tuberculosis, otitis media, eczema (atopic dermatitis), diabetic ulcers, contagious impetigo, toenail fungus, venous ulcers, infected burns, infected wounds, infected ballistic wounds, plague. There are also primary, secondary, and opportunistic infections associated with trauma, surgery, endotracheal intubation, tracheotomy, and cystic fibrosis.

[0119] The subject may have other clinical indications associated with infection or inflammation that can be treated or prevented with the compositions and methods of the invention, including, but not limited to, those associated with implantable, indwelling, or similar medical devices, such as intravascular (e.g., intravenous and intraarterial) catheters, right heart flow directing catheters, Hickman catheters, arteriovenous fistulas, catheters used in hemodialysis and peritoneal dialysis (e.g., Silastic, central venous catheters, Tenckhoff, Teflon catheters), vascular access ports, indwelling urethral catheters, urethral catheters, silicone catheters, ventricular catheters, vascular prostheses (femoral, femoral peritoneal, etc.), artificial heart valves, artificial joints, orthopedic implants, penile implants, shunts (e.g., Scribner, Tarkuchen, central nervous system, portal venous system, ventricular system, cerebroventricular system), intrauterine devices, tampons, contact lenses, dental implants, ureteral stents, pacemakers, implantable cardioverter defibrillators, tubing, cannulae, probes, blood monitoring devices, needles, etc. Additionally, the term "medical device" refers to any device intended for use on animals, humans, or other subjects.

[0120] The amount of the subject peptide composition to be administered can be determined by any convenient method to be an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage forms of the present disclosure depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0121] The antimicrobial polypeptides can be administered at dosages and frequency depending on the species, size, and condition being treated of the animal. It should also be understood that the specific dosage and treatment regimen for a particular patient will depend on a variety of factors, including the activity of the particular compound employed, the method of administration, age, body weight, general health, sex, diet, excretion rate, drug combinations, the judgment of the treating physician, the condition being treated, and the severity of the condition. Such dosages are readily ascertainable by those skilled in the art. This dosage regimen may be adjusted to provide the optimal therapeutic response. In some examples, the antimicrobial polypeptide is administered daily (or every other day, or weekly) at a dose of about 1 mg to about 1000 mg (e.g., about 5 mg to about 900 mg, about 5 mg to about 800 mg, about 5 mg to about 700 mg, about 5 mg to about 600 mg, about 10 mg to about 500 mg, about 10 mg to about 400 mg, about 10 mg to about 300 mg, about 10 mg to about 250 mg, about 10 mg to about 200 mg, about 10 mg to about 150 mg, about 10 mg to about 100 mg, about 50 mg to about 500 mg, about 50 mg to about 400 mg, about 50 mg to about 300 mg, about 50 mg The daily dose may be administered in amounts ranging from about 50 mg to about 150 mg, about 50 mg to about 100 mg, about 75 mg to about 500 mg, about 75 mg to about 400 mg, about 75 mg to about 300 mg, about 75 mg to about 250 mg, about 75 mg to about 200 mg, about 75 mg to about 150 mg, about 75 mg to about 100 mg, about 100 mg to about 500 mg, about 100 mg to about 400 mg, about 100 mg to about 300 mg, about 100 mg to about 250 mg, about 100 mg to about 200 mg, or any other range including two of the aforementioned endpoints. The daily dose may be administered once during the day or divided into smaller doses administered at multiple times during the day. For humans (and mammals of similar size), a dose of 5 mg / kg may be administered every other day. The antimicrobial polypeptide can be administered for a period of time (e.g., 2-3 weeks), at intervals (e.g., administer the polypeptide for 2-3 weeks, wait 2-3 weeks, then repeat the cycle), or until the microbial organisms are eliminated or significantly reduced, the symptoms of the microbial infection are improved, or the risk of a potential microbial infection is reduced or eliminated (e.g., the wound is healed).

[0122] For pulmonary administration, patients may receive a dose of approximately 0.01 to 10 mg / kg / day of the peptide ±20% or ±10%. This dose may be administered via at least one, and preferably several, "puffs" from an aerosol device. The total daily dose is preferably administered at least once per day, but may be divided into two or more daily doses. Some patients may benefit from a period of "loading" the patient with the peptide of interest using higher or more frequent doses over several days or weeks, followed by a tapered or maintenance dose.

[0123] Administration of the antimicrobial polypeptide (or pharmaceutical composition comprising such polypeptide) in conjunction with any of the foregoing methods can be intravenous, intraperitoneal, parenteral, rectal, subcutaneous, by inhalation, nasal, buccal, sublingual, intraocular, via implantable depots, using nanoparticle-based delivery systems, microparticles, etc. Administration can be subcutaneous, topical, by inhalation, nasal, buccal, sublingual, intraocular, via implantable depots, using nanoparticle-based delivery systems, microneedle patches, microspheres, beads, osmotic or mechanical pumps, and / or other mechanical means. In some embodiments, the pharmaceutical compositions of the present invention can be administered to a subject by applying the composition to the surface of a medical device prior to inserting the medical device into the subject. Systemic administration can be achieved via intravenous, intramuscular, or subcutaneous injection or infusion. In certain examples, the subject peptide compositions are administered orally. In certain aspects, the subject peptide compositions are administered via inhalation. In some aspects, the subject peptide compositions are administered topically.

[0124] One aspect of the present disclosure provides methods for treating or preventing ocular infections caused by any pathogen of interest (e.g., as described herein), such as viral or bacterial infections. In some embodiments, the methods include administering a pharmaceutically acceptable composition to the ocular region of a subject. Any convenient ocular delivery route can be utilized with the subject methods and compositions, including, but not limited to, topical injection, subconjunctival, subtenon, intraocular, occipital, and intraocular administration. For example, the composition may be applied topically to the eye (e.g., as eye drops, via a hydrophilic hydrogel contact lens, or via an ocular polymer insert for topical application). The pharmaceutically acceptable composition comprises a pharmaceutically acceptable carrier and at least one peptide described herein. In another embodiment, the composition may be administered orally to a subject. Intraocular administration of the subject ophthalmic compositions can be achieved via intraconjunctival, intravitreous, under the retina, or behind the eyeball injection.

[0125] The eye infections described herein may be caused by any applicable pathogen that infects the eye, such as bacteria, fungi, and viruses. Exemplary infections include, but are not limited to, bacterial conjunctivitis (e.g., caused by Haemophilus influenzae, Streptococcus pneumoniae, or Staphylococcus aureus), gonococcal conjunctivitis, chlamydial conjunctivitis, fungal keratitis, trachoma, bacterial endophthalmitis, bacterial keratitis, and the like.

[0126] The viral infections described herein may be any applicable virus that infects the eye, such as viral keratitis. Exemplary viral infections include, but are not limited to, viral conjunctivitis, influenza, herpes simplex virus (HSV), human herpes virus, cytomegalovirus (CMV), and Epstein-Barr virus (EBV). Varicella-zoster virus (VZV), orthopoxvirus, variola major and minor, vaccinia, cowpox, camelpox, monkeypox, papillomavirus, adenovirus, polyomavirus including JC virus, BK virus, SV40, and combinations thereof. In some embodiments, the at least one viral infection is selected from cytomegalovirus, varicella-zoster virus, adenovirus, herpes simplex virus, and Epstein-Barr virus. In another embodiment, the viral infection is a localized viral infection.

[0127] Another aspect of the present invention is to provide a method for treating or preventing a posterior ocular condition. In some embodiments, the posterior ocular condition or retinal or neuroretinal degenerative condition is selected from macular degeneration, retinopathy, or retinitis pigmentosa. Furthermore, in one embodiment, the pharmaceutical composition for treating a posterior ocular disease is administered intraocularly (e.g., intraocular injection, including posterior, intravitreal, intraretinal, intraorbital, or subconjunctival injection).

[0128] Infection is a major determinant of wound healing, complication development, and outcome in burn patients. The primary causative organisms are Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, and various Gram-negative organisms. Frequent debridement and establishment of epidermis, grafts, or skin substitutes are essential to prevent infection. In some cases, the subject peptide compositions, alone or in combination with antibiotics and / or anti-inflammatory agents, are applied to the burn wound as a gel, ointment, or cream and / or administered systemically. Topical application can prevent systemic infection after superficial colonization or eradicate superficial infection. In some cases, the subject compositions, as described herein, can be administered as a 0.5%-5% gel, cream, or ointment, e.g., 0.5%-2%. Skin application can be performed once daily or frequently at dressing changes. Systemic administration can be achieved by intravenous, intramuscular, or subcutaneous injection or infusion. Other routes of administration can also be used.

[0129] The compositions and methods of the present disclosure also find use in treating hospital-acquired infections. For example, infections with Staphylococcus aureus can result in non-infectious lesions or infected wounds and are associated with increased infection rates following cardiac surgery, hemodialysis, orthopedic surgery, and both disease-induced and ectopic neutropenia. Intranasal and extranasal carriage of Staphylococcus spp. can lead to hospital-acquired infections of the same Staphylococcus strains that are carried intranasally and extranasally by patients and hospital personnel.

[0130] The disclosed compositions and methods can also be used to treat acute bacterial skin and skin structure infections (ABSSSIs). Also of interest are methods for treating Gram-negative pathogens and multidrug-resistant Gram-positive bacteria, such as community-acquired methicillin-resistant Staphylococcus aureus (MRSA). Gram-negative bacteria of interest that can be targeted according to the subject methods include, but are not limited to, A. baumannii and P. aerinosa, the Gram-positive bacteria Staphylococcus aureus and MRSA, and the fungal strains Candida albicans, Candida parapsilosis, Candida krusei, Aspergillus fumigatus, Aspergillus flavus, Absidia corymbifera, Fusarium solani, and Mucor.

[0131] In some cases, the subject being treated according to the subject methods is infected with an antibiotic-resistant microorganism selected from Gram-positive bacteria, Gram-negative bacteria, biofilm-forming bacteria, Streptococcus pneumoniae, Campylobacter, Neisseria gonorrhoeae, Salmonella, methicillin-resistant Staphylococcus aureus (MRSA), Shigella, vancomycin-resistant enterococci (VRE), vancomycin-resistant Staphylococcus aureus (VRSA), erythromycin-resistant group A streptococci, and clindamycin-resistant group B streptococci. including carbapenem-resistant Enterobacteriaceae (CRE), drug-resistant tuberculosis, extended-spectrum Enterobacteriaceae (ESBL), multidrug-resistant Acinetobacter (including MRAB), Clostridium difficile, enteropathogenic Escherichia coli (EPEC), Escherichia coli (EPEC), Pseudomonas aeruginosa, Helicobacter pylori, Streptococcus anginosus and uropathogenic Escherichia coli (UPEC), Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, Campylobacter, and Pseudomonas, or a combination thereof. The compositions and methods of the present disclosure also find use in treating or inhibiting biofilms. "Inhibiting" or "suppressing" biofilm formation refers to reducing the formation and / or growth of microorganisms associated with biofilms. The subject compositions are effective, alone or in combination, against biofilms produced by a wide range of microbial species, including, but not limited to, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, Campylobacter spp., Helicobacter pylori, and Pseudomonas. Biofilms are known to cause impetigo, boils, abscesses, folliculitis, cellulitis, necrotizing fasciitis, pyotomy, surgical / traumatic wound infections, and infected ulcers and burns, osteomyelitis, device-related osteoarticular infections, impetigo, secondary infected skin lesions, meningitis, brain abscess, subdural empyema, spinal epidural abscess, arterial injury, gastritis, urinary tract infection, biliary tract infection, pyelonephritis, cystitis, sinus infection, external ear canal infection, otitis media, otitis externa, leprosy, tuberculosis, and conjunctivitis. The bacterial infection may be associated with a bacterial infection selected from bloodstream infections, benign prostatic hyperplasia, chronic prostatitis, pulmonary infections, osteomyelitis, catheter infections, bloodstream infections, skin infections, acne, rosacea, dental caries, periodontitis, gingivitis, hospital-acquired infections, arterial injuries, endocarditis, periprosthetic joint infections, open or chronic wound infections, venous stasis ulcers, diabetic ulcers, arterial leg ulcers, pressure ulcers, endocarditis, pneumonia, orthopedic prosthesis and orthopedic implant infections, peritoneal dialysis peritonitis, liver cirrhosis, and other acute or chronic infections involving or harboring biofilms.

[0132] In some embodiments, the subject antimicrobial peptides have anti-inflammatory activity. The present disclosure also provides methods for treating cancer-related conditions. Cancers of interest include solid tumor cancers. In such methods, a pharmaceutical composition comprising a subject peptide (e.g., as described herein) can be administered locally to a site of interest, for example, intratumorally via injection.

[0133] In conjunction with any of the foregoing methods, the antimicrobial polypeptide (or pharmaceutical composition comprising such a polypeptide) can be administered in combination with another agent, such as an antibiotic, antiviral, antifungal, antiprotozoal, antimalarial, or agent for treating a non-infectious disease or other condition. In certain embodiments, the other agent can alleviate symptoms of the disease / microbial infection (e.g., reduce or prevent fever, treat or prevent nausea, etc.). In any case, the antimicrobial polypeptide can be administered before, simultaneously with, or after the administration of the other agent.

[0134] As discussed above, the subject antimicrobial peptides can be used synergistically in combination with additional antimicrobial agents, including, but not limited to, penicillins, cephalosporins, carbapenems, cephamycins, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones. Examples of antibiotics include penicillin G (CAS Registry Number: 61-33-6); methicillin (CAS Registry Number: 61-32-5); nafcillin (CAS Registry Number: 147-52-4); oxacillin (CAS Registry Number: 66-79-5); cloxacillin (CAS Registry Number: 61-72-3); dicloxacillin (CAS Registry Number: 3116-76-5); ampicillin (CAS Registry Number: 69-53-4); amoxicillin (CAS Registry Number: 26787-78-0); ticarcillin (CAS Registry Number: 34787-01-4); Carbenicillin (CAS Registry Number: 4697-36-3); Mezlocillin (CAS Registry Number: 51481-65-3); Azlocillin (CAS Registry Number: 37091-66-0); Piperacillin (CAS Registry Number: 61477-96-1); Imipenem (CAS Registry Number: 74431-23-5); Aztreonam (CAS Registry Number: 78110-38-0); Cafalotin (CAS Registry Number: 153-61-7); Cefazolin (CAS Registry Number: 25953-19-9); Cefaclor (CAS Registry Number: 70356-03-5); cefamandole sodium formate (CAS Registry Number: 42540-40-9); cefoxitin (CAS Registry Number: 35607-66-0); cefuroxime (CAS Registry Number: 55268-75-2); cefonicid (CAS Registry Number: 61270-58-4); cefmetazole (CAS Registry Number: 56796-2 0-4); cefotetan (CAS Registry Number: 69172-56-7); cefprozil (CAS Registry Number: 92665-29-7); loracarbef (CAS Registry Number: 121961-22-6); cefetamet (CAS Registry Number: 65052-63-3); cefoperazone (CAS Registry Number: 62893-19-0); cefotaxime (CAS Registry Number: 63527-52-6);Ceftizoxime (CAS Registry Number: 68401-81-0), ceftriaxone (CAS Registry Number: 73384-59-5), ceftazidime (CAS Registry Number: 72558-82-8), cefepime (CAS Registry Number: 88040-23-7), cefixime (CAS Registry Number: 79350-37-1), cefpodoxime (CAS Registry Number: 80210-62-4), cefsulodin (CAS Registry Number: 62587-73-9), fleroxacin (CAS Registry Number: 79960-72-3), and nalidixic acid (CAS Registry Number: 389-08-2). norfloxacin (CAS Registry Number: 70458-96-7); ciprofloxacin (CAS Registry Number: 85721-33-1); ofloxacin (CAS Registry Number: 82419-36-1); enoxacin (CAS Registry Number: 74011-58-8); lomefloxacin (CAS Registry Number: 98079-51-7); cinoxacin (CAS Registry Number: 28657-80-9); doxycycline (CAS Registry Number: 564-25-0); minocycline (CAS Registry Number: 10118-90-8); tetracycline (CAS Registry Number: 6 0-54-8); amikacin (CAS Registry Number: 37517-28-5); gentamicin (CAS Registry Number: 1403-66-3); kanamycin (CAS Registry Number: 8063-07-8); netilmicin (CAS Registry Number: 56391-56-1); tobramycin (CAS Registry Number: 32986-56-4); streptomycin (CAS Registry Number: 57-92-1); azithromycin (CAS Registry Number: 83905-01-5); clarithromycin (CAS Registry Number: 81103-11-9); erythromycin (CAS Registry Number: No.: 114-07-8); erythromycin estolate (CAS Registry No.: 3521-62-8); erythromycin ethylsuccinate (CAS Registry No.: 41342-53-4); erythromycin glucoheptonate (CAS Registry No.: 23067-13-2); erythromycin lactobinate (CAS Registry No.: 3847-29-8); erythromycin stearate (CAS Registry No.: 643-22-1); vannomycin (CAS Registry No.: 1404-90-6); teicoplanin (CAS Registry No.: 61036-64-4);Chloramphenicol (CAS Registry Number: 56-75-7); clindamycin (CAS Registry Number: 18323-44-9); trimethoprim (CAS Registry Number: 738-70-5); sulfamethoxazole (CAS Registry Number: 723-46-6); nitrofurantoin (CAS Registry Number: 67-20-9); rifampin (CAS Registry Number: 13292-46-1); mupirocin (CAS Registry Number: 12650-69-0); metronidazole (CAS Registry Number: 443-48-1); cephalexin (CAS Registry Number: 15686-71-2); roxithromycin (CAS Registry Number: 80214-83-1); co-amoxiclavate, combinations of piperacillin and tazobactam, and various salts, acids, bases, and other derivatives thereof, and combinations thereof;

[0135] The subject antimicrobial peptides can also be used in combination with antifungal agents, including, but not limited to, terbinafine hydrochloride, nystatin, amphotericin B, griscofulvin, ketoconazole, miconazole nitrate, flucytosine, fluconazole, itraconazole, clotriamzole, benzoic acid, salicylic acid, and selenium sulfide.

[0136] The subject antimicrobial peptides can also be used in combination with antiviral agents, including, but not limited to, amantadine hydrochloride, rimantadine, acyclovir, famciclovir, foscarnet, ganciclovir sodium, idoxuridine, ribavirin, sorivudine, trifluridine, valacyclovir, vidarabine, didanosine, stavudine, zalcitabine, zidovudine, interferon alpha, edoxuridine, and the like.

[0137] The subject antimicrobial peptides can also be used in combination with antiparasitic agents. Exemplary antiparasitic agents of interest include pyrethrins / piperonyl butoxide, permethrin, iodoquinol, metronidazole, diethylcarbamazine citrate, piperazine, pyrantel pamoate, mebendazole, thiabendazole, praziquantel, albendazole, proguanil, quinidine gluconate injection, quinine sulfate, chloroquine phosphate, mefloquine hydrochloride, primaquine phosphate, atovaquone, cotrimoxazole (sulfamethoxazole / trimethoprim), pentamidine isethionate, and the like.

[0138] The subject compounds can be used in a variety of research applications, including identifying and testing candidate compounds (e.g., for drug development) and conducting research on disease states of interest that involve the subject microorganisms. For research applications, the subject compounds can be used in a variety of in vivo assays, such as high-throughput screening assays, titer assays, competitive inhibition assays, etc., and the subject peptides are useful as control compounds or as tools in investigating subject samples.

[0139] (Definition explanation) Before describing the present invention in more detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0140] When a range of values ​​is provided, each intervening value between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and may also be encompassed within the invention, subject to any specifically excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0141] Certain ranges are presented herein by preceding the numerical values ​​with the term "about." The term "about" is used herein to literally support the exact number it precedes, as well as numbers that are near or approximately equal to the number it precedes. In determining whether a number is near or approximately a particular recited number, a near or approximately unrecited number may be a number that is substantially equivalent to the specifically recited number in the context in which it is presented.

[0142] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative examples of methods and materials are described below.

[0143] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference to disclose and describe the methods and / or materials for which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0144] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should further be noted that the claims may be drafted to exclude any element. Accordingly, this statement is intended to serve as a premise for using exclusive terminology such as "solely," "merely," etc. in connection with the recitation of claim elements or the use of a "negative" limitation, or as a prior basis for using a "negative" limitation.

[0145] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that may be readily separated from or combined with any of the features of any of the other embodiments without departing from the scope or spirit of the invention. Any method described can be carried out in the order of events recited or in any other order that is logically practicable.

[0146] The present disclosure provides antimicrobial polypeptides (sometimes referred to as "RP peptides") that satisfy one or more of the structural formulas or sequences described herein. The present disclosure also provides antimicrobial polypeptides that share minimal homology (e.g., as described herein) with any of the exemplary RP peptides disclosed herein. Thus, a peptide or polypeptide of the present disclosure is an antimicrobial polypeptide that satisfies any of the formulas described herein or shares a minimal degree of homology with any of the exemplary RP peptides disclosed herein.

[0147] The term "antimicrobial" refers to the ability of a compound, i.e., a subject peptide, to reduce the population of microscopic flora and / or fauna in an environment or sample. Antimicrobial activity includes bacteriostatic, antibacterial, antifungal, and antialgal activity. An antimicrobial agent need not eliminate all microorganisms; it need only reduce the viable population on the treated surface. Similarly, "antimicrobial activity" refers to the ability of a compound to inhibit or irreversibly prevent the growth of microorganisms. Such inhibition or prevention can occur via microbicidal action or microbiocidal inhibition. "Antimicrobial inhibition" refers to the ability of an antimicrobial agent to kill or irreversibly damage a targeted organism. "Microbiological inhibition" refers to the ability of a microorganism or antimicrobial compound to inhibit or slow the growth of a target organism without causing death. Microbial or microbiological inhibition can be applied to either environments currently exhibiting microbial growth (i.e., therapeutic treatment) or environments at risk of supporting such growth (i.e., prophylaxis or prevention).

[0148] "Biofilm" refers to any group of microorganisms in which the cells are attached to each other on a surface.

[0149] The terms "peptide" and "polypeptide" are used interchangeably to refer to a polymer composed of amino acid residues.

[0150] As used herein, the term "amino acid residue" refers to any naturally occurring amino acid, non-naturally occurring amino acid, or amino acid mimetic (such as a peptoid monomer). Amino acid residues may be in the L- or D-form.

[0151] The "length" of a polypeptide refers to the number of amino acid residues connected end-to-end that make up the polypeptide, excluding non-peptide linkers and / or modifications that the polypeptide may contain.

[0152] In certain embodiments of the antimicrobial polypeptides described herein, a numbering scheme is utilized for convenience and simplicity to refer to particular positions in the structure and / or sequence of the compound, e.g., positions where a particular variant amino acid residue of interest is incorporated into a polypeptide motif. This numbering scheme is based on the sequential order of amino acid residues to assign numbered positions to amino acid residues of interest, e.g., positions in the motifs or structural models described herein. By way of example, an antimicrobial peptide of the sequence FIOKFAKOFKOFIOKFAK (SEQ ID NO: 55) may be described and numbered according to the following scheme: F 1 I 2 O 3 K 4 F 5 A 6 K 7 O 8 F 9 K 10 O 11 F 12 I 13 O 14 K 15 F 16 A 17 K 18 (SEQ ID NO: 55). It is understood that the numbering of the sequences is not intended to limit the length of the subject antimicrobial peptides, and that the numbered sequences may include one or more additional amino acid residues and / or terminal modifications at the N-terminus and / or C-terminus.

[0153] In certain instances, antimicrobial peptides of interest may include bridging residues (e.g., cysteine ​​residues capable of disulfide formation) and / or bubble regions, e.g., regions described herein flanked by linking residue(s) such as cysteine ​​and / or glycine residues. In such instances, the bridging residues are generally not included in the sequential numbering of the polypeptide motifs, but may include their own separate numbering. By way of example, an antimicrobial peptide of the sequence RFCWKVCYKGICFKKCK (RP557, SEQ ID NO: 8), which contains four cysteine ​​residues, may be numbered according to the following scheme: R 1 F 2 -C1 -W 3 K 4 V 5 -C 2 -Y 6 K 7 G 8 I 9 -C 3 -F 10 K 11 K 12 -C 4 -K 13 (RP557, SEQ ID NO:8). It is understood that bridging residues (e.g., cysteine ​​residues) contained in the subject peptides are capable of forming intramolecular bonds. In some cases, the bridging residue is a cysteine ​​residue that can form a disulfide bond with another.

[0154] A "linker" or "linker sequence" may be any moiety that links two peptide sequences together. In some embodiments, the linker is an amino acid sequence that is colinear with the peptide sequences being linked together, while in other embodiments, the linker is a separate moiety that is attached to the two peptide sequences, e.g., via a covalent bond. The linker may be an amino acid sequence or a non-amino acid moiety. In certain embodiments, the linker is used to promote dimerization of two amphipathic regions.

[0155] As used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and animals (such as one or more of the animal "patients" or "subjects" described above) without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio. "Pharmaceutically acceptable salts," as used herein, refer to derivatives of the compounds defined herein, wherein the parent compound is modified by making acid or base salts thereof.

[0156] (Example) The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0157] (General method for evaluating peptides) Example 1: Activity against planktonic Gram-negative and Gram-positive bacteria Peptides are tested against the following challenge organisms according to the M11-A8 ECLSI standard for antimicrobial susceptibility testing of anaerobic bacteria: Enterococcus faecium ATCC 700221; Enterobacter aerogenes ATCC 13048; Staphylococcus aureus MRSA ATCC 33591; Streptococcus pneumoniae ATCC 49619; Pseudomonas aeruginosa ATCC 27853; Acinetobacter baumannii ATCC 17978D-5; Pseudomonas aeruginosa ATCC 19660; and Staphylococcus epidermidis ATCC 51625. Sample dilutions range from the initial sample to 1:2048. MQA Laboratories tests 11 concentrations in duplicate in 96-well plates. Results are presented as minimum bactericidal concentrations (MBCs), which are the concentrations of each peptide required to produce 99.9% lethality against each of the eight challenge organisms.

[0158] Example 2: Activity against biofilm bacteria A minimum biofilm eradication concentration (MBEC) test is used. The MBEC value provides an estimate of the concentration of antimicrobial product required to kill biofilm bacteria. Calgary Biofilm Device (CBD) plates are used to form biofilms on lids containing 96 pegs. Bacterial cultures are grown in tryptic soy broth (TSB) to a concentration of approximately 1 x 10 before inoculating the CBD plates. 7The cells are diluted to CFU / mL and incubated at 35°C for 24 hours on a shaker at 125 rpm.

[0159] The peg lids containing the biofilm are first washed with PBS to remove planktonic cells before being treated with two-fold serial dilutions of the test and control products overnight at 35°C. The peg lids are washed twice with PBS before being sonicated in fresh medium to disrupt the biofilm attached to the pegs. The plates are then incubated overnight to assess growth. Bacterial quantification is performed by measuring the absorbance at 650 nm (A650). An A650 value of less than 0.1 is defined as the disappearance of the biofilm.

[0160] Example 3: Activity against bacterial biochemical treatment (Burkholderia thailandensis) The in vivo activity of the test article and the comparator antibiotic (ceftazidime) is tested as follows: 1 x 10 per well in a sterile 96-well plate. 5 CFU of bacteria were incubated with serial dilutions of antibiotic (control) and peptide in 10 mM phosphate buffer (3 h, 37°C). Bacterial viability was determined by serially diluting each peptide concentration in sterile PBS. The dilutions were plated in triplicate on nutrient agar plates and incubated at 37°C for 24 h. Colony counts were then performed to determine viability. Bacterial viability was calculated as the ratio of the number of colonies on each experimental plate to the mean number of colonies on control plates without antimicrobial peptide. The concentration of antimicrobial peptide required to kill 50% of Burkholderia thailandensis (EC50) was determined by graphing the viability versus the logarithm of peptide concentration. EC50 was determined by fitting the data to a standard sigmoidal dose-response curve. Each experiment was repeated three times.

[0161] Example 4: Antibacterial and antifungal activity of selected peptides (IC50 values) Measurement of antibacterial and antifungal activity is determined by standard micrometer dilution methods. Briefly, cells are grown overnight in the medium designated for each strain and diluted in the same medium. Serial dilutions of peptides are added to microtiter plates in a volume of 50 μl, followed by 50 μl, 5 × 10, or 10 μl of bacteria or fungi. 5 CFU / ml are added. Plates are incubated at 37°C for 24 hours and the minimum inhibitory concentration (MIC) is determined as the lowest peptide concentration that inhibited bacterial growth by 50%.

[0162] Example 5: Screening of peptides for in vivo bactericidal activity The bacteria tested included Burkholderia cepacia Toronto strain (Bc), Porphyromonas gingivalis strains A7436 and HG405, Actinobacillus actinomycetemcomitans strain A7154 (Aa), Fusobacterium nucleatum strain 1594 (Fn), Escherichia coli strain (Ec), Staphylococcus aureus strain ATCC 29213 (Sa), and Pseudomonas aeruginosa strain (Pa). All bacteria were grown to the early exponential phase of growth in the appropriate medium under an appropriate atmosphere. The medium was inoculated with a minimum of fivefold bacterial volume before harvest. Cultures were washed twice by centrifugation with saline and resuspended in the appropriate saline solution. For the initial screening, all peptides were used at a final concentration of 10 μM in saline, with an estimated optical density at 660 nm of 10. 6 CFU / ml of target bacteria were used. A control group was treated with an equal volume of saline. The suspension was incubated at 37°C and aliquoted temporally (0-2 hours) for quantitative recovery of colony-forming units. This allowed for determination of the killing kinetics of individual peptides with different bacterial strains. A significant killing effect was considered if the peptide-treated control group showed a 1-log or greater reduction in recoverable CFU compared to the saline-treated control group. Peptides that did not kill at 10 μM were considered inactive. Peptides that showed a 2-log or greater reduction were considered inactive at 10 μM. 6Prior to testing with CFU / ml of target bacteria, titrations are performed at 2-, 5-, or 10-fold dilutions. The endpoint titration is determined as the final concentration (μM) of peptide that produces a 2-log or greater reduction in recoverable CFU compared to a saline-treated control group (the "2-log reduction concentration").

[0163] Example 6: Killing antibiotic-resistant bacteria Staphylococcus aureus, Pseudomonas aeruginosa, and Clostridium difficile are exemplary organisms that are associated with hospital-acquired infections and are tested for sensitivity to the antimicrobial polypeptides. Experiments are performed as described in Example 5.

[0164] (Evaluation of antimicrobial peptide activity) Example 7: Peptide synthesis and evaluation Antimicrobial peptides (AMPs) were designed based on sequences found in naturally occurring AMPs. The targeted AMPs are amphipathic cationic peptides that kill microorganisms by disrupting their membrane function. This mechanism of action allows rapid killing of antibiotic-resistant microorganisms, even in biofilms. In general, the tested bacteria did not develop resistance to the targeted AMPs.

[0165] Thirty-four AMPs were synthesized and evaluated in three replicate rounds. Each AMP was assessed for its antibacterial activity against 11 bacterial and 7 fungal strains, and its cytotoxicity was measured using L929 fibroblasts and human keratinocytes. AMPs were evaluated for their ability to eradicate biofilms and induce antimicrobial resistance.

[0166] Three AMPs were selected from the in vivo screening test and further evaluated in a porcine burn wound model infected with Pseudomonas aeruginosa and Staphylococcus aureus. Full-thickness wounds were created using a heated brass rod and a 2-cm trephine. After approximately 10 minutes of homeostasis, the wounds were infected with a 2:2:1 mixture of Staphylococcus aureus, Pseudomonas aeruginosa, and Fusobacterium ssp. Treatment of the infected wounds began 24 hours after infection to allow for biofilm formation.

[0167] The exemplary AMP RP557 is an amphipathic α-helical molecule with an NP (nonpolar, hydrophobic face) and a P (polar, hydrophilic face). It has 17 amino acids and a hairpin structure formed by two disulfide bonds. The positive charge on the polar face can interact with and disrupt negatively charged phospholipids on microbial cell membranes.

[0168] The exemplary AMP RP557 was prepared in 1-gram lots with a purity of >98%. RP557 possesses appropriate physicochemical properties, including hydrophilicity, high solubility, stability at extreme pH (<4), resistance to proteases, and stability in serum (human serum at 37°C for 72 hours as monitored via LC-MS / MS). Furthermore, advances in solid-phase and solid-phase peptide chemistry have enabled the rapid and economical production of high-yield, high-purity materials in accordance with Good Manufacturing Practice (GMP).

[0169] Example 8: Activity results of RP557 RP557 has broad-spectrum antibacterial activity against the Gram-negative bacteria Acinetobacter baumannii and Pseudomonas aeruginosa, the Gram-positive bacteria Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), and multiple clinical isolates of the fungal strains Candida albicans, Candida parapsilosis, Candida krusei, Aspergillus fumigatus, Aspergillus flavus, Absidia corymbifera, Fusarium solani, and Mucor.

[0170] Artificial time-kill assays demonstrated that RP557 rapidly killed both Gram-positive and Gram-negative bacteria at low doses (1–2 μg / mL) without cytotoxicity to mammalian cells (Figure 3). Rapid destruction of pathogenic cells by a potent, broad-spectrum topical anti-infective agent theoretically reduces the likelihood of bacterial resistance. Figures 3A–3C show that RP557 rapidly eradicates Pseudomonas aeruginosa and Staphylococcus aureus without cytotoxicity to mammalian cells. Cell viability was measured using bioluminescent strains of Pseudomonas aeruginosa 19660 (Figure 3A), Staphylococcus aureus 49525 (Figure 3B), and L929 fibroblasts (Figure 3C) and imaged using an IVIS Lumina system. Data represent the average of triplicate measurements.

[0171] Pathogens do not develop resistance to RP557.

[0172] Although P. aeruginosa and S. aureus bacteria did not develop resistance to subinhibitory concentrations of RP557 after 30 rounds of selection, resistance to standard antibiotics emerged, as evidenced by growth of gentamicin and clindamycin at 4096- and 256-fold minimum inhibitory concentrations (MICs), respectively, against P. aeruginosa and S. aureus after 30 days (Figures 4A-4B). When the resistant strains were subsequently treated with RP557 after 30 passages, both strains were found to be readily susceptible, indicating no cross-resistance exists between RP557 and gentamicin-resistant P. aeruginosa or clindamycin-resistant S. aureus.

[0173] Figures 4A-4B. Pseudomonas aeruginosa and Staphylococcus aureus did not acquire resistance to RP557. Subinhibitory concentrations of RP557, gentamicin, and clindamycin were incubated with Pseudomonas aeruginosa 27853 and Staphylococcus aureus 29213 for 24 hours. Bacteria grown at the highest concentration were serially re-passaged 30 times in fresh dilutions containing subminimal inhibitory concentration (MIC) levels of each component, and the average values ​​are shown.

[0174] (Biofilm inactivation) RP557 prevents and treats biofilms of various bacterial and fungal species found in combat wounds, thereby mitigating the effects of infection on healing and regeneration. A representative study of RP557 against Candida albicans is shown in Figures 5A-5B.

[0175] Figures 5A-5B: RP557 is a potent inhibitor of Candida biofilms. Fluconazole or RP557 was added to preformed Candida 17-88 biofilms for 24 hours, and biofilm inhibition was assessed by metabolic assessment using XTT and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)[phenyl-amino]carbonyl]-2H. Data represent the mean ± SD of triplicate determinations. Statistical significance was determined by one-way analysis of variance followed by Dunnett's test compared to the solvent control group (*p < 0.05, **p < 0.01, ***p < 0.001). These results demonstrate that biofilms are not resistant to antimicrobial peptides such as RP557.

[0176] (Bactericidal activity in vivo) RP557 dose-dependently reduced both P. aeruginosa and S. aureus in a polymicrobially infected, full-thickness porcine burn model (Figures 6A-6B). A single topical application of RP557 reduced S. aureus CFU counts from the solvent control level of 4.81 ± 0.724 log CFU / g tissue to 4.20 ± 1.19, 2.11 ± 1.75, and 1.46 ± 2.05 log CFU / g tissue for 0.1%, 0.2% (p < 0.01), and 1% (p < 0.0001) RP557, respectively. Furthermore, a 2% RP557 dose completely eradicated the S. aureus infection. Furthermore, a single dose of RP557 reduced the number of viable P. aeruginosa from 5.87 ± 0.477 log CFU / g tissue to 2.34 ± 2.10 and 1.96 ± 1.64 log CFU / g tissue at 1% (p < 0.0001) and 2% (p < 0.0001), respectively.

[0177] Figures 6A-6B show that topical treatment with the exemplary peptide RP557 reduces polymicrobial infection in a dose-response manner in an infected porcine burn model. Full-thickness wounds were made on the backs of anesthetized pigs with a heated brass rod and trephine, followed by infection with a 2:2:1 mixture of Staphylococcus aureus ATCC 6538 (Figure 6B) and Pseudomonas aeruginosa (a porcine isolate) (Figure 6A) and Fusobacterium ssp. After 3 hours, 0, 0.1, 0.2, 1, or 2% RP557 was applied. After 24 hours, punch biopsies were taken from the wounds, and bacterial counts were assessed, expressed as log CFU (colony-forming units) / g. Data are presented as the mean ± SE of eight replicates. Statistical significance compared to vehicle was determined by position-way analysis of variance followed by Dunnett's test (**p < 0.01, ****p < 0.0001).

[0178] (RP557 eliminates infection over a 24-hour period) Figures 7A-7B show the pharmacodynamic response to RP557. Biofilm-associated wounds were treated with 2% RP557 24 hours after bacterial inoculation. Punch biopsies were taken, homogenized, cultured, and CFU counted at 30, 180, and 24 hours after treatment. Statistical significance was calculated using the Holm-Sidak method. *p<0.05, ***p<0.001, ****p<0.0001, dAMP vs. control group.

[0179] (Bactericidal activity in vivo) RP557 was effective in eradicating Candida albicans in a murine model of vulvovaginal candidiasis (Figures 8A-8B). RP557 significantly reduced fungal counts compared to the vehicle control (****p < 0.0001). Furthermore, it was more effective than oral fluconazole, which showed minimal activity.

[0180] Figures 8A-8B. Topical treatment with RP557 reduces murine vaginal candidiasis. Effect of RP557, miconazole, and oral fluconazole in a murine model of vaginal infection with Candida albicans (ATCC 44858). On day 0, 1.46 x 10 7Mice were inoculated with 100 CFU / rat of Candida albicans. RP557 and 20 mg / mL miconazole were administered intravenously at 0.1 mL / rat twice daily, 8-hour intervals, for 3 days, starting 48 hours postinfection. CFU were assessed on day 5, with a limit of detection (LOD) of 0.7 CFU / rat. Significant differences from vehicle were determined by one-way analysis of variance followed by Dunnett's test (*p < 0.05, ****p < 0.0001). *p < 0.05 vs. a separate group receiving oral fluconazole at 0.1 and 10 mg / kg.

[0181] The results provided in the Examples demonstrate the effectiveness of the antimicrobial peptides of the invention in killing a wide range of microorganisms, including those causing medically important human infections.

[0182] (item) Without limiting the scope of the appended claims, the following items are provided to describe aspects of the present disclosure.

[0183] [Item 1] Antimicrobial peptides, including: a) a peptide sequence selected from RP550-567 (SEQ ID NOs: 1-18); b) a sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, or at least 95% sequence identity) with a sequence defined in a); or c) Sequences which have five or fewer (e.g., four or fewer, three or fewer, two or fewer, such as one or two) amino acid substitutions relative to a sequence defined in a), wherein five or fewer (e.g., four or fewer, three or fewer, two or fewer, such as one or two) amino acid substitutions are amino acid substitutions in accordance with Table 2 (e.g., similar amino acid substitutions, conservative amino acid substitutions, or highly conservative amino acid substitutions).

[0184] [Item 2] The antimicrobial peptide according to item 1, comprising: a) a peptide sequence selected from RP550-567 (SEQ ID NOs: 1-18), or b) Sequences having no more than five (e.g., no more than four, no more than three, no more than two, such as one or two) amino acid substitutions relative to the sequences defined in a), wherein no more than five (e.g., no more than four, no more than three, no more than two, such as one or two) amino acid substitutions are substitutions of cationic amino acids of the sequence with alternative cationic amino acid residues (e.g., K for O, O for K, K for R, etc.).

[0185] [Item 3] The antimicrobial peptide according to Item 1, comprising a peptide sequence selected from RP550-567 (SEQ ID NOs: 1-18).

[0186] [Item 4] The antimicrobial peptide according to Item 1, consisting of a peptide sequence selected from RP550-567 (SEQ ID NOs: 1-18).

[0187] [Item 5] Formula 7A 1 2. The antimicrobial peptide according to item 1, comprising the peptide sequence: X 1 J 2 -C 1 -J 3 X 4 VC 2 -YX 7 G.I.C. 3 -J 10 X 11 X 12 -C 4 -X 13 (7A 1 ) (SEQ ID NO: 47) Here, as follows: X 1 is selected from O and R; J 2 and J 3 are each independently selected from F and W; X 4 , X 7 , X 11 , X 12 and X 13 are each independently selected from O and K, and J 10 is selected from Y and F.

[0188] [Item 6] Formula 7A 1 6. The antimicrobial peptide according to item 5, wherein the peptide sequence is selected from the group consisting of: RFCWKVCYKGICFKKCK (RP557) (SEQ ID NO: 8), RWCFKVCYKGICYKKCK (RP560) (SEQ ID NO: 11), OWCFOVCYOGICYOOCO (RP559) (SEQ ID NO: 10), and OFCWOVCYOGICFOOCO (RP561) (SEQ ID NO: 12).

[0189] [Item 7] Formula 7B 1 2. The antimicrobial peptide according to item 1, comprising the peptide sequence: X 1 -C 1 -X 2 X 3 J 4 -C 2 -IGX 7 YC 3 -VX 10 J 11 -C 4 -J 12 X 13 (7B 1 ) (SEQ ID NO: 49) where X 1 , X 2 , X 3 , X 7 and X 10 are each independently selected from O and R; J 4 is selected from Y and F; J 11 and J 12 are each independently selected from F and W, and X 13 is selected from K and O.

[0190] [Item 8] Formula 7B 1 8. The antimicrobial peptide according to item 7, wherein the peptide sequence is selected from the group consisting of: RCRRYCIGRYCVRFCWK (RP558) (SEQ ID NO: 9) and OCOFCIGOYCVOWCFO (RP562) (SEQ ID NO: 13).

[0191] [Item 9] The antimicrobial peptide according to Item 1, comprising the peptide sequence of Formula 6A. FX 2 IX 4 AX 6 L(X 8 ) m -B-(X 13 ) m IX 15 AX 17 LX 19 F(6A) (SEQ ID NO: 27) where: B is for CLGX 11 FC (SEQ ID NO: 28), GCLGX 11 FCG (SEQ ID NO: 29) and GGCLGX 11 FCGG (SEQ ID NO: 30), wherein each C is a cysteine ​​residue, each G is a glycine residue, and X 11 is selected from O and K; X 2 , X 4 , X 8 , X 13 , X 15 and X 19 are each independently selected from O and K; X 6 and X 17 are each independently selected from R and O, and m is an integer selected from 0 or 1.

[0192] [Item 10] The antimicrobial peptide according to Item 9, wherein the peptide sequence of Formula 6A is selected from the group consisting of: FKIOARLCLGOFCIOARLK (RP550) (SEQ ID NO: 1), FOIOAOLGGCLGOFCGGIOAOLOF (RP564) (SEQ ID NO: 15), FOIOAOLOGGCLGOFCGGOIOAOLOF (RP565) (SEQ ID NO: 16), FOIKAOLGGCLGKFCGGIKAOLKF (RP566) (SEQ ID NO: 17), and FOIKAOLKGGCLGKFCGGKIKAOLKF (RP567) (SEQ ID NO: 18).

[0193] [Item 11] The antimicrobial peptide according to Item 1, comprising the peptide sequence of Formula 3A. FX 2 J 3 X 4 J 5 X 6 J 7 X 8 J 9 X 10 J 11 X 12 J 13 X 14 J 15 X 16 J 17 (3A) where: X 2 and X 14 are each independently selected from O and R; J 3 and J 17 are each independently selected from L and I; X 4 and X 16 are each independently selected from K and O; J 5 is selected from A and I; X 6 , X 8 , X 10 and X 12 are each independently selected from R, K, and O; J 7 is selected from F, A and I; J 9 is selected from V and L; J 11 is selected from A, V and L; J 13 is selected from A, I, and L, and J 15 is selected from I, F and L.

[0194] [Item 12] The antimicrobial peptide according to Item 11, wherein the peptide sequence of Formula 3A is selected from the group consisting of FOIKARFOVRARLOLKI (RP553) (SEQ ID NO: 4), FOLOAOIOVOLOAOIOL (RP555) (SEQ ID NO: 6), FOLOAOIKVKLOAOIOL (RP556) (SEQ ID NO: 7), and FRLKIKARLKVKIRFKL (RP554) (SEQ ID NO: 5).

[0195] [Item 13] The antimicrobial peptide according to any one of Items 1 to 4, wherein the peptide sequence selected from RP550-567 is RFCWKVCYKGICFKKCK (RP557) (SEQ ID NO: 8).

[0196] [Item 14] The antimicrobial peptide according to any one of Items 1 to 4, wherein the peptide sequence selected from RP550-567 is FKIOARLCLGOFCIOARLK (RP550) (SEQ ID NO: 1).

[0197] [Item 15] An antimicrobial peptide according to any one of Items 1 to 4, wherein the peptide sequence selected from RP550-567 is FIOKFAKOFKOFIOKFAKFAFAF (RP551) (SEQ ID NO: 2).

[0198] [Item 16] An antimicrobial peptide according to any one of Items 1 to 4, wherein the peptide sequence selected from RP550-567 is FAFAFKAFKKAFKOFOOAFOOAF (RP552) (SEQ ID NO: 3).

[0199] [Item 17] An antimicrobial peptide according to any one of items 1 to 4, wherein the peptide sequence selected from RP550-567 is FOIKARFOVRARLOLKI (RP553) (SEQ ID NO: 4).

[0200] [Item 18] An antimicrobial peptide according to any one of Items 1 to 4, wherein the peptide sequence selected from RP550-567 is FRLKIKARLKVKIRFKL (RP554) (SEQ ID NO: 5).

[0201] [Item 19] An antibacterial peptide according to any one of items 1 to 4, wherein the peptide sequence selected from RP550-567 is FOLOAOIOVOLOAOIOL (RP555) (SEQ ID NO: 6).

[0202] [Item 20] An antimicrobial peptide according to any one of items 1 to 4, wherein the peptide sequence selected from RP550-557 is FOLOAOIKVKLOAOIOL (RP556) (SEQ ID NO: 7).

[0203] [Item 21] An antimicrobial peptide according to any one of Items 1 to 4, wherein the peptide sequence selected from RP550-567 is RCRRYCIGRYCVRFCWK (RP558) (SEQ ID NO: 9).

[0204] [Item 22] An antimicrobial peptide according to any one of Items 1 to 4, wherein the peptide sequence selected from RP550-567 is OWCFOVCYOGICYOOCO (RP559) (SEQ ID NO: 10).

[0205] [Item 23] An antimicrobial peptide according to any one of Items 1 to 4, wherein the peptide sequence selected from RP550-567 is RWCFKVCYKGICYKKCK (RP560) (SEQ ID NO: 11).

[0206] [Item 24] An antimicrobial peptide according to any one of Items 1 to 4, wherein the peptide sequence selected from RP550-567 is OFCWOVCYOGICFOOCO (RP561) (SEQ ID NO: 12).

[0207] [Item 25] An antimicrobial peptide according to any one of Items 1 to 4, wherein the peptide sequence selected from RP550-567 is OCOOFCIGOYCVOWCFO (RP562) (SEQ ID NO: 13).

[0208] [Item 26] An antimicrobial peptide according to any one of Items 1 to 4, wherein the peptide sequence selected from RP550-567 is RGVCVCFRRRCYCLRGGR (RP563) (SEQ ID NO: 14).

[0209] [Item 27] ​​An antimicrobial peptide described in any one of Items 1 to 4, wherein the peptide sequence selected from RP550-557 is FOIOAOLGGCLGOFCGGIOAOLOF (RP564) (SEQ ID NO: 15).

[0210] [Item 28] An antimicrobial peptide according to any one of Items 1 to 4, wherein the peptide sequence selected from RP550-557 is FOIOAOLOGGCLGOFCGGOIOAOLOF (RP565) (SEQ ID NO: 16).

[0211] [Item 29] An antimicrobial peptide according to any one of Items 1 to 4, wherein the peptide sequence selected from RP550-557 is FOIKAOLGGCLGKFCGGIKAOLKF (RP566) (SEQ ID NO: 17).

[0212] [Item 30] An antimicrobial peptide according to any one of Items 1 to 4, wherein the peptide sequence selected from RP550-557 is FOIKAOLKGGCLGKFCGGKIKAOLKF (RP567) (SEQ ID NO: 18).

[0213] [Item 31] An antimicrobial peptide having the formula (7A 1 ) an antibacterial peptide comprising the peptide sequence X 1 J 2 -C 1 -J 3 X 4 VC 2 -YX 7 G.I.C. 3 -J 10 X 11 X 12 -C 4 -X 13 (7A 1 ) (SEQ ID NO: 47) Here, as follows: X 1 is selected from O and R; J 2 and J 3 are each independently selected from F and W; X 4 , X 7 , X 11 , X 12 and X 13 are each independently selected from O and K, and J 10 is selected from Y and F.

[0214] [Item 32] Formula 7A 1 32. The antimicrobial peptide according to item 31, wherein the peptide sequence is selected from the group consisting of: RFCWKVCYKGICFKKCK (RP557) (SEQ ID NO: 8), RWCFKVCYKGICYKKCK (RP560) (SEQ ID NO: 11), OWCFOVCYOGICYOOCO (RP559) (SEQ ID NO: 10), OFCWOVCYOGICFOOCO (RP661) (SEQ ID NO: 12).

[0215] [Item 33] An antimicrobial peptide having the formula (7B 1 ) an antibacterial peptide comprising the peptide sequence X 1 -C 1 -X 2 X 3 J 4 -C 2 -IGX 7 YC 3 -VX 10 J 11 -C 4 -J 12 X 13 (7B 1 ) (SEQ ID NO: 49) where X 1 , X 2 , X 3 , X 7 and X 10 are each independently selected from O and R; J 4 is selected from Y and F; J 11 and J12 are each independently selected from F and W, and X 13 is selected from K and O.

[0216] [Item 34] The antibacterial peptide according to Item 33, wherein the peptide sequence of formula 7B1 is selected from the group consisting of RCRRYCIGRYCVRFCWK (RP558) (SEQ ID NO: 9), OCOFCIGOYCVOWCFO (RP562) (SEQ ID NO: 13).

[0217] [Item 35] An antibacterial peptide comprising a peptide sequence of formula (6A). FX 2 IX 4 AX 6 L(X 8 ) m -B-(X 13 ) m IX 15 AX 17 LX 19 F(6A) (SEQ ID NO: 27) where: B is for CLGX 11 FC (SEQ ID NO: 28), GCLGX 11 FCG (SEQ ID NO: 29) and GGCLGX 11 FCGG (SEQ ID NO: 30), wherein each C is a cysteine ​​residue, each G is a glycine residue, and X 11 is selected from O and K, and X 2 , X 4 , X 8 , X 13 , X 15 and X 19 are each independently selected from O and K; X 6 and X 17 are each independently selected from R and O, and m is an integer selected from 0 or 1.

[0218] [Item 36] The antibacterial peptide according to Item 35, wherein the peptide sequence of Formula 6A is selected from the group consisting of FKIOARLCLGOFCIOARLK (RP550) (SEQ ID NO: 1), FOIOAOLGGCLGOFCGGIOAOLOF (RP564) (SEQ ID NO: 15), FOIOAOLOGGCLGOFCGGOIOAOLOF (RP565) (SEQ ID NO: 16), FOIKAOLGGCLGKFCGGIKAOLKF (RP566) (SEQ ID NO: 17), and FOIKAOLKGGCLGKFCGGKIKAOLKF (RP567) (SEQ ID NO: 18).

[0219] [Item 37] An antibacterial peptide comprising a peptide sequence of formula (3A). FX 2 J 3 X 4 J 5 X 6 J 7 X 8 J 9 X 10 J 11 X 12 J 13 X 14 J 15 X 16 J 17 (3A) where: X 2 and X 14 are each independently selected from O and R; J 3 and J 17 are each independently selected from L and I; X 4 and X 16 are each independently selected from K and O; J 5 is selected from A and I; X 6 , X 8 , X 10 and X 12 are each independently selected from R, K, and O; J 7 is selected from F, A and I; J 9 is selected from V and L; J 11 is selected from A, V and L; J 13 is selected from A, I, and L, and J 15 is selected from I, F and L.

[0220] [Item 38] The antibacterial peptide according to Item 37, wherein the peptide sequence of Formula 3A is selected from the group consisting of: FOIKARFOVRARLOLKI (RP553) (SEQ ID NO: 4), FOLOAOIOVOLOAOIOL (RP555) (SEQ ID NO: 6), FOLOAOIKVKLOAOIOL (RP556) (SEQ ID NO: 7), FRLKIKARLKVKIRFKL (RP554) (SEQ ID NO: 5).

[0221] [Item 39] A pharmaceutical composition comprising the antimicrobial peptide according to any one of Items 1 to 38 and a pharmaceutically acceptable carrier.

[0222] [Item 40] The pharmaceutical composition according to Item 39, wherein the composition is formulated for oral administration, parenteral administration, or topical administration.

[0223] [Item 41] The pharmaceutical composition according to Item 39, wherein the composition is formulated for oral administration and further comprises an enteric coating.

[0224] [Item 42] The pharmaceutical composition of Item 39, wherein the composition is formulated for local delivery in a form selected from the group consisting of a gel suspension, a cream, a microneedle, and injected into a bandage or topical patch.

[0225] [Item 43] The pharmaceutical composition according to Item 39, wherein the composition is formulated for inhalation.

[0226] [Item 44] The pharmaceutical composition of Item 39, wherein the composition is an ophthalmic composition formulated for delivery to the subject's eye, for example, via local injection, subconjunctival, sub-Tenon, intraocular, posterior segment, or intraocular administration.

[0227] [Item 45] The pharmaceutical composition according to Item 43, wherein the composition further comprises liposomes containing free and / or encapsulated antimicrobial peptides.

[0228] [Item 46] The pharmaceutical composition according to any one of Items 39-45, further comprising an additional bioactive agent.

[0229] [Item 47] The pharmaceutical composition of Item 46, wherein the additional bioactive agent is selected from the group consisting of antibacterial agents, anti-inflammatory agents, anti-nausea agents, anti-pain agents, and combinations thereof.

[0230] [Item 48] The pharmaceutical composition according to Item 39, wherein the composition is formulated to be coated on the surface of an implantable medical device.

[0231] [Item 49] The pharmaceutical composition according to Item 48, wherein the medical device is selected from the group consisting of surgical instruments and implantable medical devices.

[0232] [Item 50] A method for treating or preventing a microbial infection (e.g., as described herein) in a subject in need thereof, comprising administering to the subject a pharmaceutical composition described in any one of Items 39-49.

[0233] [Item 51] The method according to Item 50, wherein the pharmaceutical composition is administered to the subject orally, parenterally, via inhalation, or topically.

[0234] [Item 52] The method of Item 50, wherein the pharmaceutical composition is an ophthalmic composition administered to the subject's eye (e.g., via topical injection, subconjunctival, subtenon, intravitreal, retrothoracic, or intracavitary routes of administration, as described herein).

[0235] [Item 53] The method of Item 50, wherein the pharmaceutical composition is administered to the subject by applying the composition to the surface of the medical device before inserting the medical device into the subject.

[0236] [Item 54] The method according to any one of Items 50-53, wherein the subject is selected from the group consisting of humans, livestock, farm animals, and zoo animals.

[0237] [Item 55] The method according to any one of Items 50-54, wherein the pharmaceutical composition is administered in combination with an antibacterial agent and / or an antibiotic.

[0238] [Item 56] A method for treating a microbial infection in an animal, comprising administering to an infected or at-risk animal a pharmaceutical or veterinary product, medical device, or feed product comprising a peptide (e.g., as described herein) in an amount effective to promote growth and weight gain in the animal.

[0239] [Item 57] A method for inhibiting microbial growth, colony (e.g., as described herein), or infection in a sample containing a microorganism, the method comprising contacting the sample with an antimicrobial peptide described in any one of Items 1-38 to inhibit microbial growth, colony, or infection.

[0240] [Item 58] The method according to Item 57, wherein the sample is a cell sample.

[0241] Additionally, while the present invention has been described above in terms of preferred embodiments, those skilled in the art will recognize that it is not limited thereto. The various features and aspects of the present invention described above can be used individually or jointly. Moreover, while the present invention has been described in the context of implementation in particular environments and for particular applications, those skilled in the art will recognize that its usefulness is not limited thereto, and the present invention can be beneficially utilized in any number of environments and implementations. Accordingly, the claims set forth below should be construed in view of the full breadth and spirit of the invention as disclosed herein.

Claims

1. a) a peptide sequence selected from RP557, 559-562 (SEQ ID NOs: 8, 10-13), or b) a sequence having five or fewer amino acid substitutions relative to the sequence defined in a); Including, The five or fewer amino acid substitutions are c) G to A substitution, d) substitution of I, L, or M for V; e) substitution of I with V, L, M, or F; f) substitution of F with W or L; g) Y replacement with F or W; h) W to F substitution; i) substitution of K with R or O; j) substitution of R with K or O, and k) Substitution of O with R or K One or more substitutions are selected from Antimicrobial peptides.

2. a) a peptide sequence selected from RP557, 559-562 (SEQ ID NOs: 8, 10-13), or b) a sequence having five or fewer amino acid substitutions relative to the sequence defined in a); Including, The five or fewer amino acid substitutions are substitutions of cationic amino acids of the peptide sequence with alternative cationic amino acid residues. The antimicrobial peptide of claim 1.

3. a) The peptide sequence selected from RP557, RP559-562 defined in a) is RFCWKVCYKGICFKKCK (RP557) (SEQ ID NO: 8). The antimicrobial peptide of claim 1.

4. a) The peptide sequence selected from RP557, RP559-RP562 defined in a) is RWCFKVCYKGICYKKCK (RP560) (SEQ ID NO: 11). The antimicrobial peptide of claim 1.

5. RFCWKVCYKGICFKKCK (RP557) (SEQ ID NO: 8), OFCWOVCYOGICFOOCO (RP561) (SEQ ID NO: 12), RWCFKVCYKGICYKKCK (RP560) (SEQ ID NO: 11), and OWCFOVCYOGICYOOCO (RP559) (SEQ ID NO: 10) comprising a peptide sequence selected from The antimicrobial peptide of claim 1.

6. An antimicrobial peptide according to any one of claims 1 to 5; a pharmaceutically acceptable carrier; 10. A pharmaceutical composition comprising:

7. Formulated for oral, parenteral, inhaled, or topical administration The pharmaceutical composition according to claim 6.

8. Formulated for oral administration and further comprising an enteric coating The pharmaceutical composition according to claim 6.

9. Formulated for topical administration in a form selected from gel suspension, cream, microneedle, and injection into a bandage or topical patch. The pharmaceutical composition according to claim 6.

10. Formulated for inhalation The pharmaceutical composition according to claim 6.

11. Further comprising an additional bioactive agent The pharmaceutical composition according to claim 6.

12. The additional bioactive agent is selected from antimicrobial agents, anti-inflammatory agents, anti-nausea agents, pain relievers, and combinations thereof. The pharmaceutical composition of claim 11.

13. Formulated to be coated on the surface of an implantable medical device The pharmaceutical composition of claim 12.

14. Use of the pharmaceutical composition according to any one of claims 6 to 13 in the manufacture of a medicament for the treatment or prevention of microbial infections.

15. The medicament may be prepared in oral, parenteral, inhaled, or topical dosage forms. Use of the pharmaceutical composition according to claim 14.

16. The medicament is applied to the surface of the medical device prior to insertion of the medical device into a subject for treatment or prevention of the microbial infection. Use of the pharmaceutical composition according to claim 14.

17. The subject of the treatment or prevention of microbial infections may be any animal, including humans, domestic animals, farm animals, and zoo animals. Use of a pharmaceutical composition according to any one of claims 14 to 16.

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