Antimicrobial peptides with alpha core helices

Abstract

To provide antimicrobial peptides having alpha core helices.SOLUTION: Novel computational systems and methods are described for identifying new α-helical antimicrobial peptides using a systemic consensus formula. Newly identified α-helical antimicrobial peptides are tested experimentally to show potential antimicrobial activities. The present disclosure describes the identification of a consensus formula representing α-helical antimicrobial peptides (AHAPs) from broad classes of higher eukaryotes. The disclosure describes the identification of a consensus formula representing α-helical antimicrobial peptides (AHAPs) from broad classes of higher eukaryotes.SELECTED DRAWING: None

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 505,013, filed May 11, 2017, the entire contents of which are incorporated herein by reference.

[0002] [Sequence table] This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web, the entirety of which is incorporated herein by reference. The ASCII copy generated on May 9, 2018 is named 254302WO_ST25.txt and is 2,033,153 bytes in size. [Background technology]

[0003] Antimicrobial host defense peptides (AHDPs) are an evolutionarily ancient weapon of host defense that first arose in prokaryotes as a means to neutralize competing microorganisms. Similar peptides have subsequently evolved in all classes of eukaryotes and continue to function as a first line of defense against microbial invaders. AHDPs from nearly all organisms are typically small in size, cationic, and amphipathic, properties that are believed to be essential for bactericidal activity. Given that the surface charge and membrane potential of microorganisms are relatively electronegative, the cationic character is thought to confer selectivity for microorganisms. Amphipathicity has been shown to be a key feature for AHDPs to successfully bind and permeabilize target microbial membranes.

[0004] Given that eukaryotic AHDPs act on rapidly evolving bacterial targets, they necessarily need to respond in a similar manner to maintain their efficacy. This arms race between the host and the microbe has provided a positive selection pressure that allows for a very high mutational tolerance in AHAPs, which have been shown to be some of the most rapidly evolving sequences studied to date. This process, when complicated over evolutionary timescales, has given rise to a highly diverse repertoire of eukaryotic sequences and structures capable of exerting bactericidal effects.

[0005] The inherent diversity in eukaryotic AHDPs has made the identification of common bactericidal motifs and SARs difficult. Many research groups have utilized computational and / or QSAR methods to attempt to identify such motifs, but they have largely focused on identifying improved drug candidates. As a result, although these investigations have identified many optimized or improved peptide-based therapeutics, the unifying physicochemical and three-dimensional features that confer bactericidal activity to native peptides have yet to be fully defined. Summary of the Invention

[0006] The present disclosure describes the identification of a consensus formula representing a broad class of higher eukaryotic alpha-helical antimicrobial peptides (AHAPs). When this formula is applied as a component of a logical search method against proteomic databases, a large proportion of the known AHAP family is consistently retrieved. Furthermore, this consensus formula has been useful in identifying many putative novel bactericidal peptides and in transferring antimicrobial activity in proteins to which such activity has not yet been assigned. Thus, according to one embodiment of the present disclosure, bactericidal peptides, compositions, methods and uses, as well as computer systems and methods for identifying consensus formulas and for searching bactericidal peptides are provided.

[0007] In one embodiment, the disclosure provides a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, and an amino acid derived from any one of SEQ ID NOs: 1-14 with one amino acid substitution, wherein the peptide is not more than 100 amino acid residues in length.

[0008] In some embodiments, the peptide has antimicrobial activity. In some embodiments, the peptide comprises the amino acid sequence of any one of SEQ ID NOS: 1-14. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NOS: 13 or 14.

[0009] In some embodiments, the peptide is no longer than 75 amino acid residues in length. In some embodiments, the peptide is no longer than 60 amino acid residues in length.

[0010] In some embodiments, a peptide is provided comprising an amino acid sequence of SEQ ID NO:19-6860, or an amino acid derived from a sequence of SEQ ID NO:19-6860 with one amino acid substitution, wherein the peptide is not more than 100 amino acid residues in length.

[0011] In some embodiments, a fusion peptide is also provided comprising a first fragment selected from the sequence of SEQ ID NO:19-6860 or an amino acid derived from the sequence of SEQ ID NO:19-6860 with one amino acid substitution, and a second fragment having antimicrobial activity, wherein the fusion peptide is not longer than 100 amino acid residues in length. In some embodiments, the second fragment comprises a gamma core motif comprising two antiparallel beta sheets flanked by a short turn region, one of the beta sheets incorporating a GXC or CXG sequence pattern. In some embodiments, the gamma core motif has a first amino acid sequence of CPTAQLIATLKNGRKICLDLQ (SEQ ID NO:15), or at least 85% sequence identity to SEQ ID NO:15.

[0012] In some embodiments, the peptide or fusion peptide comprises one or more unnatural amino acid residues.

[0013] In one embodiment, a composition is also provided comprising the peptide or fusion peptide and a pharma- ceutically acceptable carrier. In some embodiments, the composition further comprises an antimicrobial agent. In some embodiments, the antimicrobial agent is selected from the group consisting of imipenem, ceftazidime, colistin, chloroquine, artemisinin, vancomycin, and daptomycin.

[0014] In one embodiment, a method of treating an infection in a patient in need thereof is provided comprising administering to the patient an effective amount of a composition, hi some embodiments, the infection is caused by a gram-negative bacteria, a gram-positive bacteria, or a fungus.

[0015] A computer-implemented method is also provided. In one embodiment, a method is provided for identifying peptides with antimicrobial activity comprising the steps of identifying a consensus formula from aligned amino acid sequences known to have antimicrobial activity, adjusting the consensus formula with a test search against a plurality of proteins with known antimicrobial activity, and searching, using one or more processors, a protein database for amino acid fragments that match the consensus formula, the search taking as input one or more criteria selected from the group consisting of: position of the fragment in the protein, size of the protein, biology of the protein, and signal peptide of the protein.

[0016] In some embodiments, the adjustment involves shortening the length of the consensus formula or altering the substitution options at one or more amino acid residues.

[0017] In another embodiment, a computer-implemented method for identifying alpha-helical antimicrobial peptides is provided, comprising: using one or more processors, searching a protein database for amino acid fragments matching the following consensus formula: X-[VILMCFWYAG]-[KRHEDNQSTAG]-[KRHEDNQSTAG]-[VILMCFWYAG]-[VILMCFWYAG]-[KRHEDNQSTAG]-[KRHEDNQSTAG]-[VILMCFWYAG]-X-[KRHEDNQSTAG]-[VILMCFWYAG], where X represents any amino acid residue; filtering the retrieved fragments based on the presence of a signal peptide in each of said proteins; and evaluating the retrieved fragments with respect to one or more criteria selected from the group consisting of hydrophobic moment, average hydrophobicity, net charge, frequency or ratio of K and R, and isoelectric point. [Brief description of the drawings]

[0018] Particular features of various embodiments of the technology are set forth with particularity in the appended claims. A better understanding of the features and advantages of the technology will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the technology are utilized, and the accompanying drawings, in which:

[0019] [Figure 1A] A graphical representation of the alpha core sequence formula is shown. A helical wheel drawing of the 18 residue alpha core sequence formula. Hydrophobic residues (F, W, Y, V, I, L, M, C, A, G) are shown in various shades of green. More hydrophobicity is shown in darker colors. Hydrophilic residues (K, R, H, E, D, N, Q, S, T, A, G) are shown cationic in blue, anionic in red, and uncharged polar in orange. Alanine (yellow-green) and glycine (yellow) are included in both the hydrophilic and hydrophobic groups. H-hydrophobic, P-polar. [Figure 1B] FIG. 1 shows a graphical representation of the α-core sequence formula. Schematic representation of the linear formula. See legend to FIG. 1A.

[0020] [Figure 2A] Alignment of a typical AHAP with the α-core sequence formula is shown. Eighteen starting points are shown to scan the repeating α-core formula, and the process is illustrated by the ProSite pattern search tool being used to query the SwissProt database. Colors follow Figure 1A,B. [Figure 2B] Same as Figure 2A.

[0021] [Diagram 3] Iterative optimization of the alpha-core formula. Iterative refinement of the alpha-core formula was performed to evaluate the necessity of glycine and / or alanine as components of either the polar (hydrophilic) or non-polar (hydrophobic) residue sets. The percentage of sequences returned from the control AHAP dataset with more than 400 peptides is shown.

[0022] [Figure 4] 1 illustrates the process of identifying new AHAP sequences.

[0023] [Diagram 5] Positional and spatial amphipathic residue frequency by class. The percentage of individual residues on either polar or non-polar peptide faces of the studied peptides is represented by blocks of different colors. Residues above the x-axis are found on the polar faces of the obtained peptides, while residues below the axis are found on the non-polar faces. Panel A. Arthropods. Panel B. Amphibians. Panel C. Higher vertebrates.

[0024] [Figure 6]Comparison of NK / NK+NR ratios and hydrophobicity in AHAPs and toxin helices. The ratio of lysine (NK) to arginine (NR) is expressed by (NK / NK+NR) relative to hydrophobicity (H) in the studied AHAPs and toxins. As predicted by the saddle-spray rule, the preference for lysine compared to arginine is reflected in increased values ​​of H for peptides capable of generating NGCs in membranes.

[0025] [Figure 7] Mapping of net charge and hydrophobic moment for the studied peptides is shown. Values ​​of net charge (Q) versus hydrophobic moment (μH) are shown for the retrieved peptide dataset. All retrieved sequences are shown in grey. Groups of peptides selected for further characterization are shown in color. For comparison, a typical AHAP is shown in pink.

[0026] [Figure 8A] Helical wheel drawings of the research test peptides are shown. Hydrophobic moment (μH) and vector angle orientation are indicated. Color coding: cationic fully charged (KR)-blue, partially charged (H)-light blue, anionic-red, polar-yellow, small-grey, polar (NQ)-pink, (TS)-purple. [Figure 8B] Same as Figure 8A.

[0027] [Figure 9] Antimicrobial activity of the investigational test peptides is shown. Bactericidal activity of the investigational test peptides against a panel of typical Gram-positive (S. aureus), Gram-negative (S. typhimurium, P. aeruginosa, A. baumannii), and fungal (C. albicans) pathogens at two pHs representing the natural physiological (pH 7.5) or phagolysosomal (pH 5.5) environment is shown.

[0028] [Figure 10] 1 illustrates an example process flow chart of a method according to some implementations.

[0029] [Figure 11] FIG. 1 shows a block diagram of an example computer system on which any of the implementations described herein may be implemented. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] It is to be understood that the present disclosure is not limited to particular embodiments described, as such may, of course, vary, and the scope of the present disclosure will be limited only by the appended claims, and it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0031] It should be noted that as used in this specification and the appended claims, the singular forms "1," "one," and "a" include the plural forms unless the context clearly dictates otherwise. Thus, for example, the term "a peptide" includes a plurality of peptides.

[0032] [1.Definition] 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 disclosure pertains. As used herein, the following terms have the following meanings:

[0033] As used herein, the terms "comprising" or "including" are intended to mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements that have some essential importance to the combination for the purpose being described. Thus, a composition consisting essentially of the elements defined herein will not exclude other materials or steps that do not substantially affect the basic and novel properties claimed. "Consisting of" is intended to mean excluding more than trace amounts of other ingredients and substantial method steps. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0034] The term "about" when used before numerical designations, such as temperatures, times, amounts, and concentrations (including ranges), indicates approximations that may vary by ±10%, 5%, or 1%.

[0035] As used herein, the term "sequence identity" refers to the degree of amino acid residue or nucleotide identity between two peptides or two nucleic acid molecules. When a position in the compared sequence is occupied by the same base or amino acid, the molecules are identical at that position. A peptide (or polypeptide or peptide region) has a certain percentage (e.g., at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 83%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%) of sequence identity with another sequence means that a percentage of bases (or amino acids) is identical in the comparison of the two sequences when aligned. It should be noted that for any sequence disclosed in this application ("reference sequence"), sequences having at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 83%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% sequence identity to the reference sequence are also included within the present disclosure.

[0036] Similarly, the disclosure also includes sequences that have 1, 2, 3, 4 or 5 amino acid residue or nucleotide substitutions, deletions or additions compared to a reference sequence.

[0037] In any of the embodiments described herein, analogs of peptides comprising any of the amino acid sequences described herein are also provided.These have at least about 80%, or at least about 83%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% sequence identity with any of the reference amino acid sequences.In some embodiments, analogs comprise 1, 2, 3, 4, or 5 substitutions, deletions, or additions of amino acid residues compared to the reference sequence.In some embodiments, the substitutions are conservative substitutions.

[0038] As used herein, the term "amino acid" refers to either natural and / or unnatural or synthetic amino acids, including glycine, both the D and L optical isomers, amino acid analogs, and peptidomimetics. In some embodiments, unnatural amino acids are useful for tailoring or modifying helices or other secondary or tertiary structures of peptides or proteins for desired antimicrobial properties.

[0039] As is well known in the art, a "conservative substitution" of an amino acid or a "conservative substitution variant" of a peptide refers to an amino acid substitution that maintains 1) the secondary structure of the peptide, 2) the charge or hydrophobicity of the amino acid, and 3) the bulkiness of the side chain, or any one or more of these properties. By way of example, the well-known term "hydrophilic residue" refers to serine or threonine. A "hydrophobic residue" refers to leucine, isoleucine, phenylalanine, valine, or alanine, etc. A "positively charged residue" refers to lysine, arginine, ornithine, or histidine. A "negatively charged residue" refers to aspartic acid or glutamic acid. A residue with a "bulky side chain" refers to phenylalanine, tryptophan, or tyrosine, etc. A list of exemplary conservative amino acid substitutions is shown in Table A. [Table A] [Table 1]

[0040] Alternatively, non-limiting examples of conservative amino acid substitutions are provided below in Table B. A similarity score of 0 or greater indicates a conservative substitution between the two amino acids. [Table B] [Table 2]

[0041] Alternatively, non-limiting examples of conservative amino acid substitutions include replacing a polar amino acid with a different polar amino acid, or replacing a hydrophobic amino acid with a different hydrophobic amino acid, as shown below in Table C. In some embodiments, each of the polar or hydrophobic amino acids can be substituted with Ala or Gly. Table C [Table 3]

[0042] As used herein, the term "composition" refers to a formulation that contains at least one medicament active ingredient and is suitable for administration to a patient intended for therapeutic purposes, including any solid form thereof.The composition may contain at least one pharmaceutically acceptable ingredient, such as a suitable carrier, to provide improved formulation of the compound.In certain embodiments, the composition is formulated as a film, gel, patch, or solution.

[0043] As used herein, the term "pharmaceutical acceptable" indicates that the indicated material does not possess properties that would prevent a reasonably prudent medical practitioner, having regard to the disease or condition being treated and the respective route of administration, from administering the material to a patient. For example, such materials are generally required to be essentially sterile.

[0044] [2. Antimicrobial α-helical antimicrobial peptides] The present disclosure describes, in some embodiments, a computational approach for generating a systematic formula from known α-helical antimicrobial peptides and for using the formula to screen new α-helical antimicrobial peptides. The systematic formula integrates ideal amphipathic features and / or antimicrobial helices spanning up to 18 positions of a standard right-handed α-helix. Results show that nearly all families of known antimicrobial α-helical peptides align with the formula. In addition, many previously unidentified sequences were predicted to have direct antimicrobial activity. Synthesis of selected candidates and in vitro efficacy against human pathogens confirmed the veracity of the model predictions and validated the α-core formula and search strategy. As a result, novel protein and peptide families and their specific sequences were identified as having potent and direct bactericidal effects not previously attributed to them.

[0045] The identified protein and peptide families and their specific sequences are provided in Tables 2-4 and SEQ ID NOS: 518-6860. In some embodiments, isolated peptides are provided that include the amino acid sequences of Tables 2, 3 or 4, or any one of SEQ ID NOS: 518-6860, or derived therefrom with one, two or three amino acid substitutions. In some embodiments, the substitution is a conservative substitution. In some embodiments, the substitution is a replacement of a polar amino acid with a different polar amino acid (or A or G), or a replacement of a hydrophobic amino acid with a different hydrophobic amino acid (or A or G).

[0046] In some embodiments, isolated peptides are provided that comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, and amino acids derived from any one of SEQ ID NOs: 1-14 with one, two or three amino acid substitutions. In some embodiments, the substitution is a conservative substitution. In some embodiments, the substitution is a replacement of a polar amino acid with a different polar amino acid (or A or G), or a replacement of a hydrophobic amino acid with a different hydrophobic amino acid (or A or G). In some embodiments, the peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14.

[0047] In some embodiments, the peptide is a fragment or fusion peptide derived from a naturally occurring protein, hi some embodiments, the peptide differs from the naturally occurring protein by at least amino acid substitutions, additions, or deletions.

[0048] In some embodiments, the peptide is no longer than 100 amino acid residues in length. In some embodiments, the peptide is no longer than 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, or 25 amino acid residues in length. In some embodiments, the peptide has antimicrobial activity.

[0049] Fusion peptides are also described. In one embodiment, the disclosure provides a fusion peptide comprising a first fragment selected from a sequence of Table 2, 3 or 4, or any one of SEQ ID NOS: 518-6860, or an amino acid from a sequence of Table 2 or 3, or any one of SEQ ID NOS: 518-6860 with one, two or three amino acid substitutions, and a second fragment having antimicrobial activity. In some embodiments, the second fragment comprises a gamma core motif comprising two antiparallel beta sheets flanked by a short turn region, with one of the beta sheets incorporating a GXC or CXG sequence pattern.

[0050] As used herein, the term "gamma core motif" or "γ-core" and equivalents refer to a multidimensional protein signature, particularly a multidimensional antimicrobial signature, characterized in particular by two antiparallel β-sheets flanked by a short turn region, one β-sheet incorporating a conserved GXC (dextromeric) or CXG (levomeric) sequence pattern. Further features characterizing the γ-core motif include a hydrophobic bias toward the C-terminus, and cationic charges located at the inflection points and ends of the β-sheet domain, polarizing the charge along the longitudinal axis of the γ-core.

[0051] Kinocidin gamma core (γ KC The antimicrobial peptide γ core (γ AP ) repeats, which typically conform to an antiparallel β-hairpin containing a 13-17 amino acid pattern with a central hydrophobic region flanked by basic residues. KC The core motif may be characterized by the following consensus sequence formula: NH2[C]-[X 10-13 ]-[GX 2-3 C]-[X2]-[P]COOH

[0052] Kinocidin gamma core (γ KC Human IL-8 containing the core signature has the sequence NH2CANTEIIVKLSDGRELCLDP COOH.

[0053] This fragment of the IL-8 sequence is the consensus γ KC In addition, many kinocidins display a repeating amino acid position pattern, with the consensus γ KC Consistent with the core formula.

number

[0054] In one embodiment, the gamma core motif comprises CPTAQLIATLKNGRKICLDLQ (SEQ ID NO:15), or a first amino acid sequence having at least 85% sequence identity to SEQ ID NO:15. In one aspect, gamma core variants may include CPTAQLIATLKNGRKICLDLQP (SEQ ID NO:16), CPTAQLIATLKNGRKICLDLQAP (SEQ ID NO:17), and CPTAQLIATLKNGRKICLDLQA (SEQ ID NO:18).

[0055] A linker can be optionally included between the first and second fragments, which is preferably 10 amino acids or less in length. In some embodiments, the length of the spacer is 9, 8, 6, 5, 4, 3, 2 amino acids or less. The spacer can include any amino acid, such as Ala, Pro, Cys, and Gly.

[0056] In some embodiments, the fusion peptide has antimicrobial activity. In some embodiments, the peptide may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical, or PEG.

[0057] The peptides may be conjugated or fused to therapeutic agents, which may include detectable labels such as radioactive labels, immunomodulatory agents, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents, which may be drugs or toxins, ultrasound contrast agents, non-radioactive labels, combinations thereof, and other agents known in the art. The peptides may be detectably labeled by conjugation with a chemiluminescent compound. The presence of the chemiluminescently labeled antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0058] 3. Synthesis of antimicrobial peptides The peptides described herein can be ordered from commercial sources or can be partially or completely synthesized using methods well known in the art (e.g., chemical and / or biotechnological methods). In certain embodiments, the peptides are synthesized according to protocols of peptide solid-phase synthesis methods well known in the art. In another embodiment, the peptides are synthesized on a solid support according to the well-known Fmoc protocol, cleaved from the support with trifluoroacetic acid, and purified by chromatography according to methods well known to those skilled in the art. In other embodiments, the peptides are synthesized by utilizing biotechnology methods well known to those skilled in the art. In one embodiment, a DNA sequence encoding amino acid sequence information for a desired peptide is ligated into an expression plasmid (e.g., a plasmid incorporating an affinity tag for affinity purification of the peptide) by DNA recombination techniques well known to those skilled in the art, the plasmid is introduced into a host organism for expression, and the peptide is then separated from the host organism or growth medium, for example, by affinity purification.

[0059] Peptides can also be prepared using recombinant expression systems. This generally involves inserting a nucleic acid molecule into an expression system that is heterologous to the nucleic acid molecule (i.e., in which the nucleic acid molecule is not normally present). One or more desired nucleic acid molecules encoding the peptides of the present disclosure can be inserted into a vector. When multiple nucleic acid molecules are inserted, the multiple nucleic acid molecules can encode the same or different peptides. The heterologous nucleic acid molecule must be inserted into a vector that is properly aligned with the promoter and any other 5' regulatory molecules, as well as the proper reading frame for the correct reading frame. It is inserted into an expression system or vector in a (5'→3') orientation.

[0060] Purified peptides can be obtained by several methods. The peptides are preferably produced in purified form (preferably at least about 80% or 85% pure, more preferably at least about 90% or 95% pure) by conventional techniques. Depending on whether the recombinant host cells have been engineered to secrete the peptide into the growth medium (see U.S. Patent No. 6,596,509 to Bauer et al., incorporated herein by reference in its entirety), the peptides can be isolated and purified by centrifugation (to separate cellular components from the supernatant containing the secreted peptide), followed by sequential ammonium sulfate precipitation of the supernatant. To separate the peptides from other proteins, the fractions containing the peptides are subjected to gel filtration on appropriately sized dextran or polyacrylamide columns. If necessary, the peptide fractions can be further purified by HPLC.

[0061] 4. Antimicrobial Compositions and Formulations Compositions and formulations comprising any one or more peptides as disclosed herein are also provided. In one embodiment, a composition comprises any one or more peptides and a pharma- ceutically acceptable carrier.

[0062] "Pharmaceutically acceptable carrier" refers to any diluent, excipient, or carrier that can be used in the composition of the present disclosure. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol, and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceuti, Mack Publishing Company, a standard reference text in the field. Cal Sciences. Suitable pharmaceutical carriers are preferably selected having regard to the intended form of administration, i.e., oral tablets, capsules, elixirs, syrups, and the like, and consistent with conventional pharmaceutical practice.

[0063] The pharmaceutical compositions of the present disclosure can be manufactured by methods well known in the art, such as conventional granulation, mixing, dissolving, encapsulation, lyophilization, or emulsification processes, among others. The compositions can be produced in various forms, including granules, precipitates, particulates, powders (including freeze-dried, rotary-dried, or spray-dried powders), amorphous powders, injections, emulsions, elixirs, suspensions, or solutions. The formulations can optionally include stabilizers, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof.

[0064] Pharmaceutical preparations can be prepared as suspensions or solutions using sterile liquids such as oil, water, alcohol, and combinations thereof. For oral or parenteral administration, pharma- ceutical suitable surfactants, suspending agents, or emulsifying agents can be added. Suspensions can include oils such as peanut oil, sesame oil, cottonseed oil, corn oil, and olive oil. Suspensions can also include esters of fatty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides, and acetylated fatty acid glycerides. Suspensions can include alcohols such as ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol, and propylene glycol. Ethers such as poly(ethylene glycol), petroleum-based hydrocarbons such as mineral oil and petrolatum, and water can also be used in suspensions.

[0065] The compositions of the present disclosure are formulated for pharmaceutical administration to a mammal, preferably a human being. Such pharmaceutical compositions of the present disclosure can be administered by various routes, preferably parenterally.

[0066] The sterile injectable form of the composition of the present disclosure may be an aqueous or oily suspension. These suspensions may be formulated according to techniques well known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a 1,3-butanediol solution. Acceptable excipients and solvents that may be used may be water, Ringer's solution, or isotonic saline. In addition, sterile, fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any sterile, fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the formulation of injectables, as are pharmaceutically acceptable natural oils such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants, that are commonly used in the formulation of pharma- ceutically acceptable dosage forms, including emulsions and suspensions.Other commonly used surfactants, such as Tween, Span, and other emulsifiers, or bioavailability enhancers that are commonly used in the manufacture of pharma-ceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes.The compounds may be formulated for parenteral administration by injection, such as bolus injection or continuous infusion.The unit dosage form for injection may be an ampoule or a multi-dose container.

[0067] In addition to the above-mentioned dosage forms, pharma- ceutically acceptable excipients and carriers and dosage forms are generally known to those skilled in the art and are included in the present disclosure.It should be understood that the specific administration and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific peptide employed, age, weight, general health, sex and diet, the patient's renal and hepatic function, administration time, excretion rate, co-medication, the judgment of the treating physician or veterinarian, and the severity of the particular disease being treated.

[0068] In some embodiments, the composition may further comprise a secondary antimicrobial agent, non-limiting examples of which include imipenem, ceftazidime, colistin, chloroquine, artemisinin, vancomycin, and daptomycin.

[0069] [5. Treatment method] Methods of using the peptides, compositions and formulations of the present disclosure are also described. In one embodiment, the method is for preventing or treating microbial infection. The microbial organism may be a bacterium, such as a gram-negative or gram-positive organism, a fungus or a parasite.

[0070] The peptides, compositions and formulations are also useful for treating diseases or conditions associated with infection, such as wound abscesses, catheter biofilms, pneumonia, bacteremia, and the like.

[0071] In some embodiments, the method of treatment further comprises administering, either simultaneously or sequentially, a second, secondary antimicrobial agent, non-limiting examples of which include imipenem, ceftazidime, colistin, chloroquine, artemisinin, vancomycin, and daptomycin.

[0072] The peptides, compositions and formulations of the present disclosure can be administered into the systemic circulation via parenteral administration.The term "parenteral" used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.However, when infection is in a specific location (e.g., on the skin), the composition can be administered in a specific location, such as topical administration.

[0073] 6. Computer Systems and Methods The present disclosure provides, in some embodiments, a computer-implemented method for identifying antimicrobial sequences, and associated systems and non-transitory computer-readable media. In one embodiment, a computer-implemented method for identifying peptides with antimicrobial activity is provided, as shown in FIG. 10, which is a process flow chart of a method 1000. The various process steps and / or data flows depicted in FIG. 10 (and other figures) are described in more detail herein. The described steps may be realized using some or all of the system components described in detail above. In some implementations, the various steps may be performed in a different order, and various steps may be omitted. Additional steps may be performed with some or all of the steps depicted in the depicted flow diagram. One or more steps may be performed simultaneously. Thus, the steps depicted (described in more detail below) are exemplary in nature and should not be considered as limiting.

[0074] In step 1002, according to user commands, a system such as a personal computing device identifies a consensus formula from the aligned amino acid sequences that are known to have antimicrobial activity, such as known alpha-helical antimicrobial peptides. An example formula is X-[VILMCFWYAG]-[KRHEDNQSTAG]-[KRHEDNQSTAG]-[VILMCFWYAG]-[VILMCFWYAG]-[KRHEDNQSTAG]-[KRHEDNQSTAG]-[VILMCFWYAG]-X-[KRHEDNQSTAG]-[VILMCFWYAG], where X represents any amino acid residue. The formula can be tested with a data set that includes known antimicrobial peptides, and the formula can be further adjusted (step 1004). For example, the formula can be shortened or lengthened, or certain amino acid residues can include more or fewer substitutions.

[0075] The formula can then be used to search in a protein database for sequences or fragments that match the requirements of the formula (step 1006). In some embodiments, the search query includes one or more criteria, such as the location of the fragment in the protein, the size of the protein, and the biology of the protein (step 1008). Notably, in one embodiment, the search results are further evaluated for the presence of a signal peptide in the corresponding protein.

[0076] In some embodiments, the search results are further evaluated for biological, chemical, physical, or sequence properties. Examples of properties include, but are not limited to, hydrophobic moment, average hydrophobicity, net charge, K and R frequencies or ratios, and isoelectric point (PI). Each of these evaluation scores can be used to prioritize, rank, and filter the search results (step 1012). Optionally, a portion of the search results are synthesized and tested in the laboratory for antimicrobial activity.

[0077] 11 shows a block diagram of an example computer system 1100 in which any of the embodiments described herein may be implemented. The computer system 1100 includes a bus 1102 or other communication mechanism for communicating information, and one or more hardware processors 1104 coupled to the bus 1102 for processing information. The hardware processor 1104 may be, for example, one or more general-purpose microprocessors.

[0078] Computer system 1100 also includes a main memory 1106, such as a random access memory (RAM), cache, and / or other dynamic storage device, coupled to bus 1102 for storing information and instructions executed by processor 1104. Main memory 1106 may also be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 1104. Such instructions, when stored in storage media accessible to processor 1104, render computer system 1100 into a special-purpose machine customized to perform the operations specified in the instructions.

[0079] Computer system 1100 further includes a read-only memory (ROM) 1108 or other static storage device coupled to bus 1102 for storing static information and instructions for processor 1104. A storage device 1110, such as a magnetic disk, optical disk, or USB thumb drive (flash drive), is provided and coupled to bus 1102 for storing information and instructions.

[0080] The computer system 1100 may be coupled via a bus 1102 to a display 1112, such as a cathode ray tube (CRT) or LCD display (or touch screen), for displaying information to a computer user. An input device 1114, including alphanumeric and other keys, is coupled to the bus 1102 for communicating information and command selections to the processor 1104. Another type of user input device is a cursor control 1116, such as a mouse, trackball, or cursor direction keys, for communicating instructional information and command selections to the processor 1104 and for controlling the movement of a cursor on the display 1112. This input device typically has two degrees of freedom in two axes, a first axis (e.g., x) and a second axis (e.g., y), allowing the device to specify a position in a plane. In some embodiments, information and command selection in the same direction as the cursor control may be implemented via receiving touches on a touch screen without a cursor.

[0081] The computing system 1100 may include a user interface module to implement a GUI that may be stored in mass storage as executable software code executed by a computing device. This module and other modules may include, for example, software components, object-oriented software components, class components, task components, and other components, processes, functions, attributes, procedures, subroutines, segments of program code, drivers, firmware, microcode, circuits, data, databases, data structures, tables, arrays, and variables.

[0082] Generally, as used herein, the word "module" refers to logic embodied in hardware or firmware, or a collection of software instructions that may have entry and exit points, written in a programming language, such as Java, C, or C++. A software module may be compiled, linked into an executable program, installed in a dynamic link library, or written in an interpreted language, such as BASIC, Perl, or Python. It should be understood that a software module may be called from other modules, from itself, and / or in response to a detected event or interrupt. A software module configured to run on a computing device may be provided on a computer-readable medium, such as a compact disc, digital video disc, flash drive, magnetic disk, or any other tangible medium, or as a digital download (which may be initially stored in a compressed or installable format that requires installation, decompression, or decryption before execution). Such software code may be stored partially or completely in a memory device of the executing computing device for execution by the computing device. The software instructions may be embedded in firmware, such as an EPROM. It should be further understood that a hardware module may include connected logic units such as gates and flip-flops, and / or may include programmable units such as programmable gate arrays or processors. The functionality of the modules or computing devices described herein may be represented in hardware or firmware, although preferably implemented as software modules. In general, the modules described herein refer to logical modules that may be combined with other modules or divided into sub-modules without regard to physical organization or storage.

[0083] The computer system 1100 may implement the techniques described herein using customized hardwired logic, one or more ASICs or FPGAs, firmware and / or program logic that, in combination with the computer system, renders or programs the computer system 1100 into a special-purpose machine. According to one embodiment, the techniques described herein are executed by the computer system 1100 in response to the processor(s) 1104 executing one or more sequences of one or more instructions contained in the main memory 1106. Such instructions may be read into the main memory 1106 from another storage medium, such as the storage device 1110. Execution of the sequences of instructions contained in the main memory 1106 causes the processor(s) 1104 to perform the process steps described herein. In alternative embodiments, hardwired circuitry may be used in place of or in combination with software instructions.

[0084] As used herein, the term "non-transitory media" and similar terms refer to any medium that stores data and / or instructions that cause a machine to operate in a particular manner. Such non-transitory media may include non-volatile media and / or volatile media. Non-volatile media include, for example, optical or magnetic disks, such as storage device 1110. Volatile media include dynamic memory, such as main memory 1106. Common forms of non-transitory media include, for example, floppy disks, flexible disks, hard disks, solid state drives, magnetic tape, or any other magnetic data storage medium, CD-ROMs, any other optical data storage medium, any physical medium with a pattern of holes, RAM, PROMs, and EPROMs, FLASH-EPROMs, NVRAMs, any other memory chips or cartridges, and networked versions thereof.

[0085] Non-transitory media is distinct from, but may be used in conjunction with, transmission media, which involves the transmission of information between non-transitory media. For example, transmission media include coaxial cables, copper wire, and fiber optics, including the wires that comprise bus 1102. Transmission media may also take the form of acoustic or light waves, such as those generated during radio and infrared data communications.

[0086] Various forms of media may be involved in carrying one or more sequences of one or more instructions to the processor 1104 for execution. For example, the instructions may initially be carried on a magnetic disk or solid state drive of a remote computer. The remote computer can load the instructions into a dynamic memory and send the instructions over a telephone line using a modem. A modem local to the computer system 1100 can receive the data on the telephone line and use an infrared transmitter to convert the data to an infrared signal. An infrared detector can receive the data carried in the infrared signal and appropriate circuitry can place the data on the bus 1102. The bus 1102 carries the data to the main memory 1106, from which the processor 1104 retrieves and executes the instructions. Instructions received by the main memory 1106 may be retrieved and executed. The instructions received by the main memory 1106 may optionally be stored on storage device 1110 either before or after execution by the processor 1104.

[0087] The computer system 1100 may also include a communications interface 1118 coupled to the bus 1102. The communications interface 1118 provides a two-way data communication coupled to one or more network links that are connected to one or more local networks. For example, the communications interface 1118 may be an Integrated Services Digital Network (ISDN) card, a cable modem, a satellite modem, or a modem providing a data communication connection to a corresponding type of telephone line. As another example, the communications interface 1118 may be a local area network (LAN) card to provide a data communication connection to a corresponding LAN (or a WAN component in communication with a WAN). A wireless link may also be implemented. In any such implementation, the communications interface 1118 sends and receives electrical, electromagnetic or optical signals that carry digital data streams representing various types of information.

[0088] A network link typically provides data communication through one or more networks to other data devices. For example, a network link may provide a connection through a local network to a host computer or to data equipment operated by an Internet Service Provider (ISP). The ISP in turn provides data communication services through the world-wide packet data communication network now commonly referred to as the "Internet". Both local networks and the Internet use electrical, electromagnetic or optical signals that carry digital data streams. The signals through the various networks and the signals on the network link and through communication interface 1118 that carry the digital data to and from computer system 1100 are examples of forms of transmission media.

[0089] The computer system 1100 can send messages and receive data, including program code, through the network(s), network link and communication interface 1118. In the Internet example, a server might transmit a requested code for an application program through the Internet, an ISP, a local network and communication interface 1118.

[0090] The received code may be executed by processor 1104 as it is received, and / or stored in memory device 1110, or other non-volatile storage for later execution.

[0091] Each of the processes, methods, and algorithms described in the above sections may be embodied in, or fully or partially automated by, one or more computer systems or computer processors, including computer hardware. The processes and algorithms may be implemented partially or wholly in application specific circuitry.

[0092] The various features and processes described above may be used independently of each other or may be combined in various ways. All possible combinations or subcombinations are intended to fall within the scope of the present disclosure. In addition, certain method or process blocks may be omitted in some implementations. The methods and processes described herein are not limited to any particular order, and the blocks or states associated therewith may be performed in other orders as appropriate. For example, the described blocks or states may be performed in an order other than that specifically disclosed, or multiple blocks or states may be combined into a single block or state. The examples of blocks or states may be performed sequentially, in parallel, or in some other manner. Blocks or states may be added to or deleted from the example embodiments disclosed. The example systems and components described herein may be configured differently from the description. For example, elements may be added, removed, or rearranged compared to the example embodiments disclosed.

[0093] Conditional language, such as "may," "may," "well," or "good," among others, is generally intended to convey that certain embodiments include certain features, elements, and / or steps, but not other embodiments, unless otherwise specified or understood otherwise in the context in which it is used. Thus, conditional language is generally not intended to imply that features, elements, and / or steps are in any way required by one or more embodiments, or that one or more embodiments necessarily include logic, with or without user input or prompting, for determining whether those features, elements, and / or steps are included or performed in any particular embodiment.

[0094] Any process illustrations, elements, or blocks in the flow diagrams described herein and / or depicted in the accompanying figures should be understood to represent modules, segments, or portions of code that include one or more executable instructions for implementing specific logical functions or steps in the process. As will be appreciated by those skilled in the art, alternative implementations are within the scope of the embodiments described herein and, depending on the functionality involved, elements or functions may be omitted, shown, or performed in a different order than that described, including substantially simultaneously or in reverse order.

[0095] It should be emphasized that many variations and modifications can be made to the above-described embodiments. It is understood that those elements are included in other acceptable examples. All such modifications and variations are intended to be included within the scope of the present disclosure. The above description details certain embodiments of the present invention. However, no matter how detailed the above description may appear in text, it should be understood that the invention can be implemented in many ways. Also, as mentioned above, the use of a specific term when describing a particular feature or aspect of the present invention should not be construed as suggesting that the term is redefined herein to be limited to include any specific characteristics of the feature or aspect of the invention with which the term is associated. The scope of the present invention should therefore be construed according to the appended claims and any equivalents thereof.

[0096] [Engine, Components, Logic] Certain embodiments are described herein as including logic or a number of components, engines, or mechanisms. An engine may constitute either a software engine (e.g., code embodied in a machine-readable medium) or a hardware engine. A "hardware engine" is a tangible unit capable of performing certain operations and may be configured or arranged in a particular physical manner. In various exemplary embodiments, one or more computer systems (e.g., standalone computer systems, client computer systems, or server computer systems) or one or more hardware engines (e.g., processors or groups of processors) of a computer system may be configured by software (e.g., applications or application portions) as hardware engines that operate to perform certain operations described herein.

[0097] In some embodiments, the hardware engine may be implemented mechanically, electrically, or any suitable combination thereof. For example, the hardware engine may include dedicated circuitry or logic that is permanently configured to perform certain operations. For example, the hardware engine may be a dedicated processor, such as a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC). The hardware engine may also include programmable logic or circuitry that is temporarily configured by software to perform certain operations. For example, the hardware engine may include software executed by a general purpose processor or other programmable processor. Once configured by such software, the hardware engine becomes a dedicated machine (or a dedicated component of a machine) that is uniquely tailored to perform the function for which it is configured, and is no longer a general purpose processor. It should be understood that the decision as to whether to implement the hardware engine mechanically in a permanently configured dedicated circuit or in a temporarily configured circuit (e.g., configured by software) may be made based on cost and time considerations.

[0098] Thus, the phrase "hardware engine" should be understood to include a tangible entity that is physically constructed or permanently configured (e.g., hardwired) or temporarily configured (e.g., programmed) to operate in a particular manner or to perform particular steps described herein. As used herein, a "hardware-implemented engine" refers to a hardware engine. Given an embodiment in which the hardware engines are temporarily configured (e.g., programmed), each of the hardware engines need not be configured or embodied at any one time. For example, a hardware engine may include a general-purpose processor that is configured by software to be a special-purpose processor, and the general-purpose processor may be configured at different times as different special-purpose processors (e.g., including different hardware engines). Thus, software may configure a particular processor, or multiple processors, to configure a particular hardware engine at one time, and to configure different hardware engines at different times.

[0099] Hardware engines can provide information to and receive information from other hardware engines. Thus, the described hardware engines may be considered to be communicatively coupled. Multiple hardware engines may exist simultaneously, and communication may be achieved through signal transmission (e.g., over appropriate circuits or buses) between two or more hardware engines. In embodiments in which multiple hardware engines are configured or embodied at different times, communication between the hardware engines may be achieved, for example, through storage and retrieval of information in a memory structure accessible to the multiple hardware engines. For example, one hardware engine may perform an operation and store the output of the operation in a communicatively coupled memory device. An additional hardware engine may then later access the memory device and retrieve and process the stored output. Hardware engines may also initiate communication with input or output devices and act on resources (e.g., collecting information).

[0100] Various steps of the example methods described herein may be performed, at least in part, by one or more processors that are temporarily or permanently configured (e.g., by software) to perform the associated steps. Whether temporarily or permanently configured, such processors may constitute a processor-implemented engine that operates to perform one or more steps or functions described herein. As used herein, "processor-implemented engine" refers to a hardware engine that is implemented using one or more processors.

[0101] Similarly, the methods described herein may be implemented, at least in part, by a processor, with a particular processor or processors being examples of hardware. For example, at least some of the steps of the method may be performed by one or more processors or processor-implemented engines. Furthermore, one or more processors may also operate to support the execution of associated steps in a "cloud computing" environment or as "Software as a Service" (SaaS). For example, at least some of the steps may be performed by a collection of computers (as examples of machines including processors), making the steps accessible over a network (e.g., the Internet) and by one or more suitable interfaces (e.g., application program interfaces (APIs)).

[0102] The performance of a particular operation may be distributed among processors and spread across multiple machines rather than remaining confined to a single machine. In some exemplary embodiments, a processor or processor-implemented engine may be located in a single geographic location (e.g., a home environment, an office environment, or a server farm). In other exemplary embodiments, a processor or processor-implemented engine may be distributed across multiple geographic locations.

[0103] [Text] Throughout this specification, components, steps, or structures described as a single instance may be implemented in multiple instances. Although individual steps of one or more methods have been shown and described as separate steps, one or more of the individual steps may be performed simultaneously, and there is no requirement that the steps be performed in the order shown. Structures and functions presented as separate components in the example configurations may be implemented as a combined structure or component. Similarly, structures and functions presented as a single component may be implemented as separate components. These and other variations, modifications, additions, and improvements are within the scope of the subject matter of this specification.

[0104] Although the subject matter has been generally described with reference to specific exemplary embodiments, various modifications and changes may be made to these embodiments without departing from the broad scope of the disclosed embodiments. Such embodiments of the subject matter may be referred to herein individually or collectively under the term "invention," but this is for convenience only and is not intended to automatically limit the scope of this application to any single disclosure or concept, even if more than one is actually disclosed.

[0105] The embodiments shown herein are described in sufficient detail to enable those skilled in the art to practice the teachings of the disclosure. Other embodiments may be used or derived therefrom, and structural and logical substitutions and changes may be made without departing from the scope of the disclosure. Therefore, the detailed description is not to be taken in a limiting sense, and the scope of the various embodiments is defined solely by the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0106] It should be understood that the "engine," "system," "data store," and / or "database" may include software, hardware, firmware, and / or circuitry. In one example, one or more software programs including instructions executable by a processor may perform one or more functions of an engine, data store, database, or system described herein. In another example, circuitry may perform the same or similar functions. Alternative embodiments may include more, fewer, or functionally equivalent engine systems, data stores, or databases, and still be within the scope of the present embodiments. For example, the functionality of the various systems, engines, data stores, and / or databases may be combined or divided differently.

[0107] For the purposes of this specification, "open source" software is defined as source code that allows distribution, optionally with a license permitting modifications and derivative works, both as source code and in compiled form, using well-known, indexed means of obtaining the source.

[0108] The data stores described herein may be of any suitable structure (e.g., active databases, relational databases, self-referential databases, tables, matrices, arrays, flat files, document-oriented storage systems, non-relational NoSQL systems, etc.) and may be cloud-based, etc.

[0109] [example] [Example 1. Identification of a new antimicrobial α-core helix] This example describes an experiment in which a consensus sequence formula was created and used to search for new α-helical antimicrobial peptides (AHAPs) with antimicrobial activity.

[0110] The most effective natural antibiotics known to date include α-helical host defense peptides. These peptides function as a first line of defense against invading pathogens and have been isolated from species ranging from microbes to humans. Many previous investigations have analyzed the individual and class-specific properties through the function of these peptides delivery, but machine learning strategies that define unifying principles and underlying structure-activity relationships (SAR) have been limited. In this example, a systematic formula was designed that includes features of ideal amphipathic and / or antimicrobial helices spanning up to 18 positions of a canonical right-handed α-helix. The formula was then applied to search known protein databases for known or unknown proteins or peptides that satisfy the structural signature. The results demonstrated that nearly the entire family of known antimicrobial α-helical peptides matched this formula. Interestingly, a logical search algorithm using this formula found many previously uncharacterized sequences predicted to have direct antimicrobial activity. In laboratory studies, the veracity of the predictions was confirmed, validating the validity of the α-core formula and search strategy. As a result, novel protein and peptide families, and their specific sequences, have been identified as having potent and direct bactericidal effects not previously attributed to them.

[0111] Methods and Materials [Identification of Alpha Core Formula] To identify a consensus formula representative of nearly all classes of AHAPs, multiple sequence alignments with representative α-helical peptides were performed using CLUSTAL W (www.ebi.ac.uk / Tools / msa / clustalw2 / ). The alignments were then manually adjusted at specific positions using MEGA6. Through iterative refinement of this process, a generalized amphipathic formula of 18 residues emerged with sequence degeneracy at each position representative of nearly all classes of AHAPs. This formula could start at any position of a standardized 18-residue α-helical wheel.

[0112] [Assignment of residue polarity in α-core formula] In the formula, individual residues were categorized as either hydrophobic or hydrophilic according to the Wimley-White hydrophobicity scale, an empirically derived measure that includes the contribution of peptide bonds. One exception is alanine (A), which was also included with hydrophobic residues according to the Eisenberg and Kyte-Doolittle hydrophobicity scales. This assignment was made, in part, due to preliminary studies that showed that alanine is frequently located on the hydrophobic face of many antimicrobial peptides.

[0113] [Accuracy of the formula in obtaining helix sequences] To assess the fidelity of the formula in identifying helical domains, the above amphipathic helix consensus formula was queried against the PDB 3D database (www.wwpdb.org). The first 100 non-redundant retrieved structures were scored for the helicity of the target sequence. A protein was considered a positive hit if the target sequence was more than 75% helical.

[0114] [Using the Alpha Core Formula as a Database Query] The alpha core sequence formula was used with the ScanProsite (prosite.expasy.org / scanprosite / ) tool to perform an iterative pattern search of the UniProtKB Swiss-Prot database. In the first search, the database was queried with various lengths of amphipathic sequence formulas, but a relatively short query sequence of 12 residues was ultimately found to be the most efficient in retrieving the majority of antimicrobial peptide sequences. Iteration 1 of this query sequence is listed below: X-[VILMCFWYAG]-[KRHEDNQSTAG]-[KRHEDNQSTAG]-[VILMCFWYAG]-[VILMCFWYAG]-[KRHEDNQSTAG]-[KRHEDNQSTAG]-[VILMCFWYAG]-X-[KRHEDNQSTAG]-[VILMCFWYAG]

[0115] After this optimization process, the sequence formulas were used as queries against the UniProtKB Swiss-Prot and TrEMBL databases. To represent the entire 18-residue helical wheel coverage, the formulas were stepped one position at a time through 18 iterations. ScanProsite search results were further restricted by 1) protein size (<200 residues), 2) eukaryotes, and 3) localization of the pattern to the C-terminal region using the "X(0,50)>" logical operator.

[0116] [Determination of signal peptide and biophysical parameters] The acquired dataset was additionally screened for the presence of signal peptides using SignalP 4.1 (www.cbs.dtu.dk / services / SignalP / ). Hydrophobic moment (μH), average hydrophobicity (H), net charge (QK and R(+1); H(+0.5); D and E(-1)), K and R residue frequencies were determined in batches using a Python algorithm generated for this purpose. PI was determined using the ExPasy Compute PI tool (web.expasy.org / compute_pi / ).

[0117] [Candidate peptides and HDPs] Selected candidate bactericidal peptides were commercially synthesized by BioMatik (Biomatik USA, Wilmington, Delaware). Lyophilized peptides were reconstituted with ddIH20 ​​and stored in aliquots at −20° C. A prototypical human AHAP, LL-37 (Peptides International, Louisville, KY), was used for comparison in the bactericidal assay.

[0118] [Antimicrobial activity assay] Putative antimicrobial peptides were assayed for bactericidal activity using the well-established radial diffusion method adjusted to pH 5.5 or 7.5. A panel of microorganisms tested: Gram-positive Staphylococcus aureus (ISP 479C, ISP 479R), Gram-negative Salmonella typhimurium (MS 5996s, MS 14028), Pseudomonas aeruginosa (PA01), Acinetobacter baumanni (19606), and the fungus Candida albicans (36082S, 36082R). Organisms in logarithmic growth phase were inoculated (10 6 The cells were cultured at 37°C for 24 h and 48 h for 10 min at 37°C (CFU / ml) and plated. Peptides (10 μg) were introduced into the wells in the seeded matrix and incubated at 37°C for 3 h. Nutrient overlay medium was applied and the assay was incubated at 37°C for bacteria or 30°C for fungi. Zones of inhibition were measured after 24 h. Independent experiments were repeated a minimum of two times.

[0119] [result] [Derivation and iterative refinement of the α-core sequence formula] In a first study, alignments of representative archetypes of all of the major classes of AHAPs were performed to identify conserved sequence elements. This analysis revealed that almost all of these archetypes could be aligned with a degenerate sequence formula in which either polar or nonpolar residues were assigned to positions along the amphipathic helix. Peptides followed this formula, ranging from a minimum of 11 to a maximum of 16 residues, corresponding to 3-4.5 turns of the α-helix. Based on this analysis, consensus formulas representative of almost all of the major classes of AHAPs were extracted (Figure 1A, Figure 1B, Figure 2A, Figure 2B).

[0120] Once this preliminary consensus was defined, it was further refined by testing its efficiency in returning α-helical domains when used as a query against the PDB protein structure database. As a baseline, a version of the formula lacking the well-known helix breakers proline and glycine was run. The results of these analyses revealed that the efficiency of the formula in retrieving α-helical domains was very high, with these motifs (defined as spans that are at least 75% helical) being identified with a frequency of 92-94%.

[0121] Many AHAPs are known to contain proline and glycine residues, and subsequent studies were performed to evaluate the impact of these residues on the efficiency of the formula. When proline was included in the sequence formula, α-helical domains in the target sequence were obtained at a rate of less than 10%. These findings strongly support the theory that proline is not compatible with stable α-helix formation due to side chain and steric constraints. For this reason, proline was excluded from the formula.

[0122] In contrast, when glycine was allowed in the sequence formula, queries against structural databases revealed two different results, depending on whether the target was determined in an aqueous or non-polar environment. For structures determined in an aqueous environment, when a single glycine was present, the sequence formula obtained α-helical domains at a rate of about 74%. When two or more glycine residues were present, the value was reduced to 40%. In comparison, when the target structures were determined in a non-polar environment (high concentrations of TFE, SDS, or micellar components), the presence of glycine had a much smaller effect on the helicity of the peptide. These studies revealed that when as many as four glycine residues were present in the target sequence, the sequence formula obtained α-helical domains at a rate of about 92%. For this reason, glycine was included in the sequence formula, as this residue is highly prevalent in AHAPs. Furthermore, numerous biophysical studies have demonstrated that although many glycine-rich AHAPs are unstructured in aqueous environments, they almost always adopt an α-helical conformation when interacting with the hydrophobic membrane environment of target microorganisms.

[0123] [Alanine / glycine sides of the helix] While it was relatively easy to assign most residues to either polar or nonpolar residue groups in the sequence formula based on the initial AHAP consensus and well-known residue-specific hydrophobicity values, the placement of alanine and glycine was less clear due to their relatively neutral ΔG values ​​in polar and nonpolar environments. For this reason, the requirement of glycine and / or alanine for either the polar or nonpolar face of the helix was evaluated in an iterative manner via queries against a control dataset containing more than 400 AHAP peptides (Figure 3). Preliminary versions of the formula lacking alanine and glycine failed to retrieve a large number (>10%) of the control AHAP study set (Figure 3). The inclusion of alanine on the polar face of the amphipathic helix increased the retrieved fraction of peptides to 26%. When alanine was added to both polar and nonpolar search terms, the retrieved AHAPs increased to 48%. A similar relationship was found for glycine. Adding glycine to the polar face of the query sequence led to the identification of approximately 81% of the control AHAP data set. When glycine was included on both the polar and non-polar faces, the query retrieved close to 99% of the AHAP study set. As a result of this analysis, alanine and glycine were included as components of both polar and non-polar query search terms.

[0124] [α Core Sequence Formula] Based on the above considerations, a linear α-core formula was generated spanning all 18 positions of a canonical right-handed alpha helix, as shown below. When converted to three dimensions, the formula displays an idealized amphipathic helix with distinct hydrophobic and hydrophilic faces. Positions between the polar and nonpolar faces were assigned the value of "X" in the linear formula, with a maximum polar angle (θ) of 180° and a minimum of 140°. Due to the degenerate nature of the formula, each position along the helical span was represented by multiple polar or nonpolar residues (below, and Figures 1A, 1B, 2A, 2B).

number

[0125] [Primary database search using the alpha core sequence formula] Once refined, the α-core sequence formula was used as a query against the SwissProt and TrEMBL protein sequence databases (Figure 4). As described in the Methods, the database queries were further restricted to sequences of eukaryotic origin, less than 200 residues in length, and within 50 residues of the C-terminus.

[0126] The returned raw dataset consisted of over 70,000 sequences, often representing multiple target hits shifted along the amphipathic helical span of a single protein. Each of these returned hits was scored for hydrophobic moment and the sequences with the highest μH were extracted, generating a non-redundant dataset of approximately 13,000 unique sequences for downstream studies. The dataset proteins were also scored for the presence of a signal peptide and sequences lacking this motif were removed, generating a final dataset of approximately 3,800 sequences.

[0127] [Efficiency of Alpha Core Sequence Formula] The results of the above database queries showed that the amphipathic sequence algorithm was very robust, retrieving members from almost all of the known AHAP families (Table 1). Overall, the formula retrieved at least one member of more than 106 different helical peptide families, representing about 94% of all known AHAP classifications. Furthermore, the formula returned 827 individual antimicrobial peptide sequences that describe idealized amphipathic helical structures, but nevertheless represent about 88% of all known AHAPs in the SwissProt database. [Table 1. Alpha-helical peptide families obtained by alpha-core formula search] [Table 4] [Table 5] [Table 6]

[0128] Besides retrieving a high percentage of known AHAPs, the formula was also relatively specific in returning these sequences when restricted to sequences with a PI of 8.5 or higher. The search was performed in several stages, either from 0 to 50 or from 51 to 200 residues, depending on whether a signal peptide test was applied. For the set of 0 to 50 residues, about 71% of the retrieved sequences were known AHAPs. Meanwhile, for the set of 51 to 200 residues, about 27% of the identified sequences were α-helical antimicrobial proteins. Considering that there are about 940 (see Methods) named AHAPs in the 553,000-sequence SwissProt database (version 12-1-16), this represents an in-silico enrichment of about 130-fold.

[0129] The second most abundant group of other proteins retrieved by the sequence formula were various venom peptides, which comprised about 17% of the data set. Other protein families abundant in the retrieved data set included apovitellin, leptin, vasoactive intestinal proteins, and unidentified proteins.

[0130] [Residue frequency] Considering that the α-core sequence formula returns an aligned data set, it is possible to score the identified AHAP helices for the abundance of various residues at each position along the 18-residue amphipathic range. Furthermore, considering that the α-core formula identifies amphipathic patterns that may form helices, it is also possible to assume that specific residues are localized on either the polar or non-polar faces of these structures. Due to the significant evolutionary distance between the invertebrate and higher and lower vertebrate classes of AHAPs, the data set was partitioned into three groups representing arthropod, amphibian, and mammalian sequences (Figure 5).

[0131] Analysis of residue frequency revealed a number of findings regarding the composition of residues in the returned AHAP helices, as well as the distribution of hydrophilicity and hydrophobicity. One notable finding is that glycine is highly represented in AHAPs of lower organisms, comprising approximately 30-35% of all residues in the target amphipathic range in arthropods and amphibians. Glycine is still abundant in mammals, but less frequently, accounting for approximately 15% of the residues in the returned sequences.

[0132] It is also interesting to note that alanine is abundant in the amphipathic range of arthropods and amphibians, but is much less abundant in the identified sequences of mammals. Furthermore, alanine is found more frequently on the hydrophobic faces of peptides, but at frequencies of approximately 10-30% on the polar faces of arthropod and amphibian peptides.

[0133] With regard to charged residues, it is interesting to note that lysine is the most abundant polar residue in non-mammalian species (30%) and is strongly preferred over arginine, with a ratio of about 12:1. In contrast, in mammals, this preference is greatly reduced, with the ratio decreasing to a lysine to arginine abundance ratio of 3:1. It should also be noted that in the entire species group, negatively charged aspartic and glutamic acid residues were found at a low frequency of about 5% in the returned sequences.

[0134] [Biophysical signature] In addition to measuring the frequency of specific residues in the returned AHAP helices, sequences were scored for a number of biophysical parameters, including net charge (Q), isoelectric point (PI), hydrophobic moment (μH), hydrophobicity (H), and the ratio of lysines to arginines (K / K+R).

[0135] In this analysis, the average net charge for the AHAP subset of studied helices (n=803) was found to be +2.0. This value rises to +2.7 when considering only cationic sequences (Q≧0.5; n=707). In comparison, the average net charge for the studied toxin helices (n=717) was +1.3 and for the other studied helices +0.6. In terms of hydrophobic moment, the AHAP helices have an average μH of 0.52, whereas the toxins have an average μH of 0.42 and the other peptides are 0.39. Although some reports suggest that the average hydrophobicity may be greater for the toxins than for the AHAPs, in this study the hydrophobicity values ​​for the helices obtained were similar, with an average H of 0.42 for AHAPs and 0.34 for the toxins compared to the other peptides (0.39).

[0136] [Negative Gaussian curvature] The obtained sequences were also analyzed for the ratio of lysine to arginine residues (N K / N K +N R ) were scored for the NGC-N ratio. Previous studies have demonstrated that the relative abundance of these residues can significantly affect the tendency of a given peptide to induce negative Gaussian curvature (NGC) in membranes, a phenomenon related to pore formation and membrane permeabilization. Previous studies have demonstrated that in AHAPs, lysine residues are preferred over arginine. This substitution requires an increased level of peptide hydrophobicity to efficiently induce NGC. In the present example, the obtained AHAP helix largely follows this rule, with the NGC-N ratio being the most important. K / NK +N R We demonstrated a loose positive correlation between the ratio and peptide hydrophobicity (Figure 6). In comparison, the amphipathic helices of the toxins do not conform to this finding, and the N K / N K +N R A negative correlation between the ratio and peptide hydrophobicity was found.

[0137] [Application of the α-core sequence formula to obtain previously uncharacterized antimicrobial sequences] In addition to the well-known classes of antimicrobial proteins, the α-core formula also captured many unidentified proteins or proteins with alternative primary functions. Based on the efficiency of the formula to capture antimicrobial peptides, it was of interest to determine whether some of these additional sequences represented 1) unidentified antimicrobial proteins or 2) proteins with unidentified antimicrobial functions or assigned functions that also had antimicrobial domains in larger proteins.

[0138] To identify putative bactericidal sequences, peptides in the study set were scored for the biophysical parameters described above (μH, Q, H, K / K+R, PI) and analyzed to determine which factors and / or relationships were most effective in separating known AHAPs from non-bactericidal sequences. The results of these analyses demonstrated that average amphipathicity (μH) and either net charge (Q) (Figure 7) or PI were the most efficient means of distinguishing bona fide AHAPs from other sequences.

[0139] Based on the above analysis, the dataset proteins were scored for cationic or PI and either hydrophobic moment (Q*μH) or (PI*μH) to prioritize interesting sequences. This analysis retrieved many interesting peptides, but several biologically important families were consistently found among the top scoring sequences. These included members of the gamma-chain-dependent interleukin family, along with many interferon sequences. Furthermore, many consistently high scoring peptides were also derived from the plant pathogen Phytophthora parasitica, which has worldwide impact.

[0140] Taking the above considerations into account, 10 potential candidates representing these families were synthesized so that their antimicrobial properties could be determined (Table 2). As shown in Table 2, they include: Interleukins IL-5 – Meleagris gallopavo (turkey) IL-7 – Human IL-13 – Human IL-21 – Human interferon IFN-γ - Myotis davidii (little long-eared bat) IFN-γ – human Phytophthora parasitica unidentified sequence Pp-1 - P. parasitica Pp-2 - P. parasitica Pp-3 - P. parasitica Pp-4 - P. parasitica

[0141] When the localization of the pattern was relaxed to the C-terminal region, two additional candidates were identified. These two candidates, along with the two longer versions of each, are listed in Table 3. A further list of identified peptides is provided in Table 4, SEQ ID NOs: 518-6029 (localization required) and SEQ ID NOs: 6030-6860 (relaxed localization requirements). [Table 2 Peptides in the study set] [Table 7] [Table 3. Further peptides identified with relaxed localization requirements] [Table 8] [Table 4. Preferred alpha core peptide sequences] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17] [Table 18] [Table 19] [Table 20]

Table 21

Table 22

Table 23

Table 24

Table 25

Table 26

Table 27

Table 28

Table 29

Table 30

[0142] When evaluated against a panel of prototype and important human pathogens, the research test peptides were found to have potent activity against nearly all of the organisms considered (Figure 8A, Figure 8B). In particular, among the interleukins, sequences derived from turkey IL-5 (tIL-5) were remarkably effective against all organisms in physiologically representative pH (pH 7.5) and in the environment of abscesses (pH 5.5). Sequences derived from human interleukins (hIL-7, hIL-13, hIL-21) were somewhat less effective at pH 7.5, but maintained significant activity at the physiologically important pH of 5.5. Similarly, peptides derived from human and bat IFN-γ had significant activity against most organisms, and were somewhat more effective at pH 7.5 than at pH 5.5. Perhaps some of the most impressive research test peptides were derived from Phytophthora. Pp-1 and Pp-4 had significant activity against many organisms, while Pp-2 was particularly effective against bacteria and fungi and retained activity at pH 5.5.

[0143] This example utilized an alternative computational approach to screen for conserved elements common to a single class of AHAPs as a means to discover generic sequence and structural elements that may confer bactericidal activity. Herein, this example first identified a consensus sequence common to the majority of known AHAPs. This consensus was then subjected to iterative refinement based on alignments with both sequence and structural databases to generate a linear formula that exhibits an amphipathic helix span representative of most known AHAPs. In particular, this sequence formula consists of an 18 residue span that contains the following polar [EDKRHQNTS(AG)] and non-polar [VMCILFWY(AG)] residues. This α-core formula, when converted to a three-dimensional radial array, exhibits an idealized amphipathic helix that contains equivalent polar and non-polar faces (Figure 1A, Figure 1B, Figure 2A, Figure 2B).

[0144] One component of the iterative optimization of the involved α-core formulas involved queries against control AHAP data sets. These studies revealed that glycine and alanine found in the AHAP amphipathic domain were critical elements. Preliminary versions of the formulas lacking these residues were largely unsuccessful, retrieving less than 10% of the control AHAP sequences. Given the inherent amphipathic frequency of the α-core formulas, it was possible to evaluate the positional requirements of specific residues for either the polar or nonpolar face of the amphipathic helix. Initial studies with glycine showed that it was essential that this residue be included in both the polar and nonpolar residue search terms. Omission from either group dramatically reduced the number of sequences returned from the control data set. Similarly, a similar study with alanine revealed that inclusion of this residue in both the polar and nonpolar residue groups was important to efficiently retrieve the majority of AHAPs from the control data set. As the α-core formula is converted into a three-dimensional helical array, these studies further demonstrated that alanine and glycine can be found on either the polar or nonpolar faces of the AHAP amphipathic helices.

[0145] Similar to the sequence-based optimization studies above, structure-based alignments with the PDB database were also performed. These studies revealed that the inclusion of proline in the formula dramatically reduced the number of helices retrieved, whereas glycine and alanine were tolerated and commonly found in helical domains. These studies further revealed that the α-core sequence formula retrieved helical domains with high fidelity, correctly identifying such domains more than 90% of the time. Although the α-core formula was not designed as a secondary structure characterization tool, its efficiency in identifying helical domains compares favorably to such tools, correctly identifying helices with frequencies ranging from xx to 81.5%.

[0146] [Database Search] Once the α-core formulas were refined, they were used as queries against the SwissProt database. These studies revealed that they captured nearly all classes of AHAPs (94%) and approximately 89% of all known individual AHAPs, with relatively high specificity. Investigations revealed that these individual peptides or peptide families not captured by the formulas often contained relatively short stretches of amphipathic or interrupted helix domains, such as the amphibian "ranabox" peptides, which are characterized as having a C-terminal helix-turn-helix domain.

[0147] It should be noted that while the formula exhibits an ideal perfect amphipathic helix, we were successful in capturing almost the entire class and most of the individual AHAPs. This suggests that almost all AHAPs have a near perfect amphipathic range of at least 12 residues, representing the size of the domain queried in these studies. Given this finding, the current hypothesis that amphipathicity is a critical component of bactericidal activity and may be essential for the membrane-permeabilizing properties of AHAPs is supported.

[0148] [Residue frequency in AHAP helix] Because the α-core sequence formula returned an aligned data set, it was possible to assess the relative frequency of various residues in the α-helical domain of AHAPs. Furthermore, these data allowed some generalizations regarding generic features common to AHAP helices. Overall, besides the inherent amphipathicity found in AHAPs, these data revealed that some residues are strongly preferred in amphipathic helices from various classes of organisms.

[0149] [glycine] One notable finding of these studies is that glycine is highly prevalent in AHAP helices in arthropods and amphibians (30-35% of all residues) but is less frequent in mammals (15% of all residues).

[0150] [Glycine in arthropod and amphibian AHAP helices] The presence of glycine in most of the known AHAP helices was initially unexpected, since this residue is known to destabilize α-helical structures. However, as demonstrated by a large body of empirical evidence, many α-helical peptides are unstructured in aqueous environments and adopt the α-helical form only in hydrophobic environments. The mechanism by which this occurs is unknown, but it is possible that the peptides may be organized in the target, since the relatively electronegative microbial surface may help to orient cationic and other polar residues to one side of the amphipathic helical structure. In a similar manner, the hydrophobic interior of the microbial cell wall may help to organize the random coil structure by selectively interacting with hydrophobic residues in the peptide.

[0151] This lack of amphipathic mechanisms prior to target interaction may be an important mechanism behind the selective toxicity of AHAPs, allowing them to limit their activity against the host while optionally adopting a bactericidal form when interacting with target microorganisms.

[0152] [Glycine in the AHAP helix of higher eukaryotes] One note regarding the presence of glycine in the AHAP helix is ​​that it is common in short peptides found in lower organisms, but occurs less frequently in the longer AHAPs of higher eukaryotes, as well as in proteins with mixed domain architectures such as chemokines, granulysin, and NK lysin. If we speculate that glycine is an important means of maintaining AHAPs in an unstructured and inactive state prior to interacting with target microorganisms, the lack of glycine in these large peptides suggests that they may be used as an alternative means of bactericidal inhibition to limit toxicity to the host.

[0153] [Cationic residues in arthropod and amphibian AHAP helices] Besides demonstrating the abundance of glycine in AHAP helices, the residue frequency studies also revealed that lysine is highly preferred over arginine in the amphipathic helices of AHAPs considered in this study. Lysine is the most abundant cationic residue in the AHAP helices studied, but is strongly preferred over arginine in non-mammalian peptides, with a ratio of 12:1. In mammals, lysine is still preferred over arginine, but to a lesser extent, with a ratio of 3:1. In comparison, lysine is less abundant in toxins, but is preferred over arginine, with a ratio of 2:1.

[0154] It may be of some relevance that biophysical measurements demonstrated that lysine is less efficient at generating membrane destabilizing NGCs compared to arginine, since it can only interact with a single lipid head group at a time. For this reason, lysine-rich AHAPs often compensate for this lack of permeabilizing ability by increasing their average hydrophobicity, a phenomenon termed the "saddle-splay curvature selection" rule. In the current study, AHAP helices largely supported this rule, with the relative abundance of lysine (N K / N K +N R ) was found to increase with increasing mean peptide hydrophobicity.

[0155] [Biophysical properties of the AHAP helix in comparison with other α-helical domains] In addition to residue frequency measurements, many further properties of the helices acquired by the α-core formula were determined, including Q, μH, and H. When averaged across families, these quantitative data revealed many insights into the biophysical properties of the acquired AHAP helices compared to toxins and other groups.

[0156] One defining characteristic of AHAPs is that they are typically cationic in nature, a property that has been shown to enhance selectivity for the relatively electronegative surfaces of many microorganisms. The versatility of this biophysical property is supported by the finding in the current study that amphipathic helices derived from AHAPs have an average net charge of +2.0 over a 12-residue span. Furthermore, this value rises to +2.7 when only cationic AHAPs are considered (Q ≧0.5 for 707 of 803 retrieved sequences). By comparison, the average charges of the toxins and other helices retrieved in these studies were +1.3 and +0.6, respectively, suggesting that cationicity may be selectively favored in AHAPs compared to other amphipathic peptides.

[0157] Another feature of AHAPs is that they are frequently amphipathic in nature, a property that was deemed essential for their membrane permeabilizing activity against microorganisms. When α-core formula searches for amphipathic sequences, all of the helices returned by these queries were a priori amphipathic in nature. However, it is also interesting to compare the relative amphipathicity of the identified helices, quantified by μH, among various classes of peptides. The results of the current study support the theory that amphipathicity is essential in the biophysical properties of AHAPs. The average hydrophobic moment of the studied AHAP helices was 0.52, which is comparable to that of cecropin A (μH max =0.61) or LL-37(μH max = 0.78), which is relatively high compared to typical amphipathic helices. In comparison, the amphipathicity of helices derived from toxins and other proteins is somewhat lower, at μH = 0.42 and 0.39, respectively.

[0158] In addition, the average hydrophobicity (H) was somewhat greater for AHAPs (0.42) than for the toxins (0.34) or other peptides studied (0.39), a finding suggesting that a moderate degree of hydrophobicity may be essential for the membrane-permeabilizing properties of AHAPs.

[0159] [Identification of new antibacterial sequences] The α-core sequence formula also retrieved a large number of uncharacterized sequences and proteins with alternative primary functions. Given the fidelity of retrieving antimicrobial sequences, it is of interest to determine whether some of these alternative sequences also have antimicrobial properties. As described, putative bactericidal sequences were scored for the most characteristic properties (μH*Q or μH*PI) that separate AHAPs from other sequences, and promising candidates of biological interest were synthesized for further study.

[0160] Representative peptide helices from three distinct high-scoring groups, interleukins (tIL-5, hIL-7, hIL-13, and hIL-21), interferons (bIFN-γ, hIFN-γ), and an unidentified sequence from P. parasitica, were synthesized and tested for bactericidal activity. Notably, all of these peptides exhibited effective bactericidal activity, which in many cases was greater than that of conventional AHAPs such as LL-37. These data strongly suggest that the amphipathic sequence formula can successfully identify amphipathic helices with antimicrobial properties.

[0161] 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 disclosure pertains.

[0162] The disclosure illustratively described herein may be suitably implemented without any element or elements not specifically disclosed herein, without one or more limitations. Thus, for example, terms such as "include", "have", "comprise" and the like may be read broadly and without limitation. In addition, the terms and expressions employed herein are used as terms of description and not of limitation, and in the use of such terms and expressions, there is no intention to exclude any equivalents of the features shown and described, or portions thereof, and it is recognized that various modifications are possible within the scope of the disclosure claimed.

[0163] Thus, although the present disclosure has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications, improvements, and variations of the disclosure embodied herein may be made by those skilled in the art, and such modifications, improvements, and variations should be considered to be within the scope of the present disclosure. The materials, methods, and examples provided herein are representative of preferred embodiments, are for illustrative purposes, and are not intended to limit the scope of the present disclosure.

[0164] The disclosure has been described broadly and generically herein. Each of the narrower species and subgroupings falling within the generic disclosure also form part of this disclosure. This includes the generic description of the disclosure with a provisos or negative limitation that removes any subject matter from that species, regardless of whether the omitted material is specifically recited herein.

[0165] Additionally, features or aspects of the disclosure may be described in terms of Markush groups, and those skilled in the art will recognize that the disclosure may thereby be described in terms of individual members or subgroups of members of the Markush groups.

[0166] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In the case of conflict, the present specification, including definitions, will control.

[0167] While the present disclosure has been described in conjunction with the above embodiments, it should be understood that the above description and examples are intended to illustrate, rather than limit, the scope of the present disclosure. Other aspects, features, and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure pertains.

Claims

1. An isolated peptide having an amino acid sequence selected from the group consisting of sequence identification numbers 1-14 and an amino acid derived from any one of sequence identification numbers 1-14 having one amino acid substitution, wherein the isolated peptide is not more than 100 amino acid residues in length.

2. An isolated peptide described in claim 1, having antimicrobial activity.

3. An isolated peptide described in claim 1 or 2, comprising any one of the amino acid sequences of sequence identification numbers 1-14.

4. An isolated peptide described in claim 1 or 2, comprising an amino acid sequence of sequence identification number 13 or 14.

5. An isolated peptide according to any one of claims 1 to 4, having a length of no more than 75 amino acid residues.

6. An isolated peptide according to any one of claims 1 to 4, having a length of no more than 60 amino acid residues.

7. An isolated peptide having an amino acid sequence of SEQ ID NO: 19-6860, or an amino acid derived from the sequence of SEQ ID NO: 19-6860 having one amino acid substitution, wherein the isolated peptide is not more than 100 amino acid residues in length.

8. A fusion peptide comprising a first fragment selected from amino acids derived from sequence identification number 19-6860 or sequence identification number 19-6860 having one amino acid substitution, and a second fragment having antimicrobial activity, wherein the fusion peptide is not longer than 100 amino acid residues in length.

9. The second fragment has a gamma core motif containing two antiparallel beta sheets flanked by a short turn region, one of the beta sheets incorporating a GXC or CXG sequence pattern. The fusion peptide according to claim 8.

10. The fusion peptide described in claim 9, wherein the gamma core motif has a first amino acid sequence of CPTAQLIATLKNGRKICLDLQ (sequence identification number 15), or a sequence having at least 85% sequence identity to sequence identification number 15.

11. An isolated peptide described in any one of claims 1 to 7, or a fusion peptide described in any one of claims 8 to 10, comprising one or more unnatural amino acid residues.

12. A composition comprising an isolated peptide according to any one of claims 1 to 7, or a fusion peptide according to any one of claims 8 to 11, and a pharma- ceutically acceptable carrier.

13. The composition of claim 12, further comprising an antimicrobial agent.

14. The composition of claim 13, wherein the antimicrobial agent is selected from the group consisting of imipenem, ceftazidime, colistin, chloroquine, artemisinin, vancomycin and daptomycin.

15. A method for treating an infection in a patient in need of treatment, comprising administering to the patient an effective amount of a composition described in any one of claims 12 to 14.

16. The method of claim 15, wherein the infection is caused by a gram-negative bacterium, a gram-positive bacterium, or a fungus.

17. A computer-implemented method for identifying a peptide having antimicrobial activity, comprising: identifying a consensus formula from the aligned amino acid sequences known to have antimicrobial activity; adjusting the consensus formula by testing against a plurality of proteins with known antimicrobial activity; searching, with one or more processors, in a protein database for a fragment of amino acids that matches said consensus formula, said search taking as input one or more criteria selected from the group consisting of: location of said fragment in a protein, size of said protein, biology of said protein, and signal peptide of said protein; A method for providing the above.

18. The method of claim 17, wherein the adjustment comprises shortening the length of the consensus formula or changing the substitution options at one or more amino acid residues.

19. A computer-implemented method for identifying an alpha-helical antimicrobial peptide, comprising: searching, using one or more processors, for a fragment of amino acids in a protein database that matches the following consensus formula: X-[VILMCFWYAG]-[KRHEDNQSTAG]-[KRHEDNQSTAG]-[VILMCFWYAG]-[VILMCFWYAG]-[KRHEDNQSTAG]-[KRHEDNQSTAG]-[VILMCFWYAG]-X-[KRHEDNQSTAG]-[VILMCFWYAG], where X represents any amino acid residue; filtering the retrieved fragments based on the presence of a signal peptide in the respective protein; evaluating the retrieved fragments with respect to one or more criteria selected from the group consisting of hydrophobic moment, average hydrophobicity, net charge, K and R frequency or ratio, and isoelectric point; A method for providing the above.

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