WO2019136139A2 - Antimicrobial peptides and use thereof - Google Patents

Description

DESCRIPTION

ANTIMICROBIAL PEPTIDES AND USE THEREOF

[0001] This application claims the benefit of United States Provisional Patent Application No. 62/613,176, filed January 3, 2018, the entirety of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The invention was made with government support under Grant No. R01 AI125337 awarded by the National Institutes of Health. The government has certain rights in the invention.

1. Field of the Invention

[0003] The present invention relates generally to the fields of microbiology and medicine. More particularly, it concerns isolated peptides with antimicrobial activity.

2. Description of Related Art

[0004] Antibiotic resistant bacteria are projected to kill 30 million people by 2050 (O'Neill, 2016). As emphasized by recent World Health Organization reports, antibiotics to treat Gram-negative bacterial infections are needed most (WHO, 2017). The path from antibiotic discovery to clinical therapy has a high attrition rate, with the last new class of antibiotics to combat Gram-negative bacteria being discovered over 40 years ago (Clatworthy etal, 2007; Payne el a/. 2007). Most antibiotic screening methods have not evolved far from the innovation of Waksman’s approach developed in the l930s, and are no longer able to quickly identify new lead compounds (Lewis, 2013; Woodruff, 2014). Necessitated by the lack of new leads and sources for natural products, companies are attempting to resurrect previously unsuccessful drug candidates (Lewis, 2013). Reliable and robust antibiotic discovery platforms are urgently needed to discover new leads against new microbial targets in our arms race against resistance.

[0005] Antimicrobial peptides are a potent class of antimicrobials with potential to combat multi-drug resistant bacteria. The ability to screen comprehensive libraries based on natural and synthetic peptides scaffolds dramatically increases the understanding and development of lead sequences with therapeutic potential. Current approaches allow routine peptide screening of a few thousand short, linear, sequences at a time, but require combinatorial chemistry and robotics for scale up that is beyond the reach of most research programs. While marking an important advance in peptide screening, this capacity has not facilitated antimicrobial peptide exploration beyond naturally available templates leaving the vast majority of therapeutically valuable peptide chemical space undiscovered. Most antimicrobial peptide studies focus on a single dominant class of naturally occurring cationic antimicrobial peptides (CAMPs). The research glimpse outside of CAMPs has identified sequences of diverse length, chemistry, and structure acting on a wide range of molecular targets and underscores that no single peptide sequence has evolved as singularly effective against all pathogens in all setting. Thus, there is an unmet need for a peptide screening platform to identify antimicrobial peptides from the undiscovered chemical space.

SUMMARY OF THE INVENTION

[0006] Certain embodiments of the present disclosure provide a screening platform for identifying antimicrobial peptides. Additional embodiments provide isolated peptides or polypeptides, such as comprising SEQ ID NOs: 1-7968 or provide in Table A, and compositions ( e.g pharmaceutical compositions and disinfecting compositions) comprising one or more of the antimicrobial peptides. Further embodiments provide methods of treating microbial infections comprising administering the antimicrobial peptides provided herein as well as methods of disinfecting surfaces. SEQ ID NOs: 1-7968 (provide in Table A) in certain aspects can refer to any of the open reading frames (ORFs) provided in the sequences of Table A. In preferred aspects, a polypeptides of the embodiments comprises the amino acid sequence before any encoded stop codon of SEQ ID NOs: 1-7968 or listed in Table A. In further aspects, polypeptides of the embodiments comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous amino acids of SEQ ID NOs: 1-7968 or listed in Table A.

[0007] In one embodiment, there is provided an isolated peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1-7968. In some aspects, the peptide essentially consists of an amino acid sequence of any one of SEQ ID NOs: 1-7968. In some aspects, the peptide consists of an amino acid sequence of any one of SEQ ID NOs: 1-7968. In additional aspects, the peptide further comprises a cell penetrating peptide (CPP).

[0008] In certain aspects, the peptide further comprises no more than 5 (e.g., no more than 6, 7, 8, 9, or 10) additional amino acids on either end of the peptide. In certain aspects, the peptide is no more than 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids in length. In some aspects, the peptide is less than 50 (e.g., less than 40, 30, 20, or 10) amino acids in length. In certain aspects, the peptide is 15-25 amino acids in length, such as 19, 20, or 21 amino acids in length. In particular aspects, the peptide is 20 amino acids in length.

[0009] In particular aspects, the peptide exhibits antimicrobial activity. In some aspects, the antimicrobial activity is further defined as antimicrobial activity against a gram negative bacterial strain. In certain aspects, the antimicrobial activity is further defined as antimicrobial activity against a multi-drug resistant bacterial strain.

[0010] In some aspects, the peptide is further defined as a linear peptide. In other aspects, the peptide is further defined as a cyclic peptide. In specific aspects, the cyclic peptide comprises one or more disulfide bonds.

[0011] In certain aspects, the N terminus or the C terminus has been chemically modified. In some aspects, the chemical modification results in the peptide having a reduced susceptibility of enzymatic cleavage. In particular aspects, both the N terminus and the C terminus have been chemically modified.

[0012] In another embodiment, there is provided a polypeptide multimer comprising at least two peptides according to the embodiments (e.g., comprising SEQ ID NOs: 1-7968). In some aspects, a first peptide of the at least two peptides is essentially identical to a second peptide. In other aspects, a first peptide of the at least two peptides is not identical to a second peptide.

[0013] Further provided herein is a pharmaceutical composition comprising a peptide of the embodiments ( e.g ., comprising SEQ ID NOs: 1-7968) and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition is formulated for oral, intravenous, intraarticular, parenteral, enteral, topical, subcutaneous, intramuscular, buccal, sublingual, rectal, intravaginal, intrapenile, intraocular, epidural, intracranial, or inhalational administration. Also provided herein is a polynucleotide comprising a nucleic acid sequence encoding a peptide of the embodiments (e.g., comprising SEQ ID NOs: 1-7968).

[0014] A further embodiment provides a method of treating a microbial infection in a subject comprising administering to the subject an effective amount of a peptide of the embodiments (e.g., comprising SEQ ID NOs: 1-7968). [0015] In some aspects, the microbial infection was caused by a bacterium, a fungus, a virus, or a protozoan. In particular aspects, the microbial infection was caused by a bacterium. In some aspects, the bacterium is a gram-negative bacterium, such as Acinetobacter baumannii or Pseudomonas aeruginosa. In certain aspects, the bacterium is a multi-drug resistant bacterium.

[0016] In some aspects, the subject is a human. In specific aspects, the subject has shown resistance to one or more antibiotics.

[0017] In certain aspects, the peptide is administered orally, enterically ( e.g ., to the small intestine or to the colon by rectal suppository or enema), topically, intravenously, intraperitoneally, intramuscularly, endoscopically, percutaneously, subcutaneously, regionally, or by direct injection. In some aspects, the orally administered peptide is a capsule or tablet, such as an enterically-coated capsule or tablet.

[0018] In additional aspects, the method further comprises administering at least a second therapeutic agent. In some aspects, the second therapeutic agent is an antibiotic or a protease inhibitor. In particular aspects, the antibiotic is a b-lactam antibiotic, amoxicillin, bacitracin, chloramphenicol, clindamycin, capreomycin, colistimethate, ciprofloxacin, doxy cy cline, erythromycin, fusidic acid, fosfomycin, fusidate sodium, gramicidin, gentamycin, lincomycin, minocycline, macrolides, monobactams, nalidixic acid, novobiocin, ofloxcin, rifamycins, tetracyclines, vancomycin, tobramycin, and/or trimethoprim. The peptide and second therapeutic agent may be administered sequentially or simultaneously. The peptide and second therapeutic agent may be administered by the same route or by distinct routes.

[0019] In another embodiment, there is provided a method of disinfecting a surface comprising applying to said surface an effective amount of a composition comprising at least one peptide of the embodiments (e.g., comprising SEQ ID NOs: 1-7968). Also provided herein is an antimicrobial disinfecting solution comprising a peptide according to the embodiments (e.g., comprising SEQ ID NOs: 1-7968) and an acceptable carrier.

[0020] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0022] FIGS. 1A-1G: (1A) Diagram of surface display system. Antimicrobial peptide surface display system composed of (1) Lpp signal sequence, (2) OmpA (46-159) transmembrane protein, (3) flexible tether, (4) C-terminal peptide. The Lpp signal sequence is shown for clarity, but is removed prior to insertion into the outer membrane. (IB) Optical density plot over a period of 6 hours of a control peptide, tandem influenza hemagglutinin peptide 2xHA (top), and an antimicrobial peptide, cecropin Pl (bottom) expressed in the surface display system induced with 0 mM, 0.1 mM, and 1 mM IPTG. (1C) Surface display expression of cecropin Pl as in (B) reported as colony forming units (cfu/mL) over time. (ID) Expression of cecropin Pl at 0.1 mM IPTG in the parent strain W3110 (blue) CAMP resistant W3110 strain WD101 (purple), and ep/A deletion in WD101 (red). (IE) The surface display is amenable to disulfide-forming peptides. Expression of protegrin 1 (top) and defensin HNP-l (middle), and a defensin cysteine mutant (bottom) plotted as optical density versus time in the E. coli strain W3110. (IF) The surface display system functions across many Gram-negative species such as Acinetobacter baumannii and Pseudomonas aeruginosa. Each strain is displaying protegrin 1 at 0 mM, 0.1 mM and 1 mM IPTG. Plotted are recorded as optical density over 6 hours. (1G) Neighboring cells are unaffected by surface expression of antimicrobial peptides. White and blue cells with empty plasmid and cecropin Pl respectively. Input cultures (left) were collected, serial diluted, and spotted before induction of 1 mM IPTG. Cells were induced at a total starting OD 600nm of 0.01. After 3 hours of surface expression, cells were collected, serial diluted, and spotted (right). All growth curves were performed in triplicate. Data are represented as mean ± SEM.

[0023] FIG. 2: Schematic of peptide batch screening workflow. Batch screening of peptides using our surface display system can be achieved by first constructing a random library using random PCR primers that flank the peptide region (i), followed by collection of transformants, plasmid isolation, and subsequent transformation into a bacterial strain of interest. Next, the library is grown in culture and induced (ii). Peptides with antimicrobial activity (colored red) will drop out of the population (iii). Next-generation sequencing of the initial input at time zero and output (iv) at a pre-defmed number of hours provides a read out of sequencing counts (v). From this information, top hits can be identified and tested. Further libraries can be constructed based on the identified top hits and the process can be repeated. A more detailed explanation of our workflow can be found in the methods section.

[0024] FIG. 3: Graph showing that intracellular expression of surface display system with cecropin Pl does not kill W3110 cells. [0025] FIG. 4: Graph depicting Cecropin Pl activity increases with tether length.

Cecropin Pl is displayed with no tether, 1 ^c tether and a 2* at 0 mM and 0.1 mM IPTG. Growth curves were performed in triplicate over 6 hours. Data are represented as mean ± SEM.

[0026] FIG. 5: Graph showing altered Cecropin Pl activity with the addition of Mg²⁺ (top) and Trypsin (bottom). Cecropin Pl is displayed with increasing concentrations of magnesium and trypsin. Controls were cecropin Pl expressed with no added magnesium or trypsin enzyme. Growth curves were performed in triplicate over 6 hours. Data are represented as mean ± SEM.

[0027] FIG. 6: Graph showing Dermaseptin surface display activity expressed in E. coli W3110 cells. [0028] FIG. 7: A defined set of 5 peptides (2xHA, Cecropin Pl, Protegrin 1, and

Defensin HNP-l, and Defensin HNP-l (w/ no cysteines)) were cloned and pooled into a small library. The library was tested as described in Fig. 2 and methods over a period of 4 hours with plasmids isolation at 0, 2, 3 and 4 hour time points in duplicate. Reads were normalized to the input counts and plotted as a function of time. [0029] FIG. 8: Mean normalized input and output counts of total peptide library.

Manually tested synthesized peptides are marked and labeled.

[0030] FIGS. 9A-9D: Computational analysis of the random 20-mer peptide screen results (9A) Mean normalized input and output counts of total peptide library. Peptides considered active with lfcMLE < or = -1 are plotted in green. Peptides with lfcMLE > -1 were considered inactive are plotted in orange. Peptides removed from further analysis contained initial reads in either replicate of less than 50 and are plotted in yellow. (9B) Screened peptides are plotted according to their hydrophobicity and charge properties. Active peptides are colored in green and inactive peptides are colored in orange. Ellipses represent a 95% confidence interval assuming a t-distribution. (9C) A charge vs hydrophobicity plot comparing SLAY active peptides and known active peptides. Known antimicrobial peptides complied from six available online databases are colored in black, active peptides from our screen are colored green. Ellipses represent a 95% confidence interval assuming a t-distribution. (9D) Plot of amino acid frequencies of known, active and inactive peptides from our screen. The error bars represent the SEM (standard error of the mean) and the asterisks correspond to Bonferroni adjusted p-values (*, **, and *** denote p-value <0.05, <0.01, and <0.001 respectively) derived from Tukey's range test performed in conjunction with an ANOVA.

[0031] FIG. 10: Logo sequences of a random 20-mer library, the total library, and the 7,968 top hits. Sequence logo comparison between a randomly generated 20 amino acid library, our library, and the top hits generated from our library. Sequence logos were generated from either the entire set of possible killing peptides (7,968 sequences), 10,000 randomly sampled sequences of the total library, or an amino acid translation of 10,000 randomly generated nucleotide sequences of a repeated“NNB” motif. All logos are plotted in units of probability.

[0032] FIG. 11: Dendrogram of clustered top killing peptides.

[0033] FIGS. 12A-12C: Mechanism of action of select peptides. (12A) The membrane damage of E. coli treated by peptides, as measured by an increase in fluorescence intensity of PI. E. coli was treated with 25mM peptide. Controls were processed without peptides. (12B) Time-kill analysis of selected active peptides from our screen and cecropin Pl. Hemolytic activity of selected peptides at 50 mM. (12C) Hemolytic activity of selected peptides at 50 mM.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0034] The study of antimicrobial peptides is greatly improved by accessible in vivo tools to navigate the vast combinatorial landscape of amino acid space. The present disclosure provides a platform for Gram-negative antimicrobial screening of massive numbers of peptides of any length, composition, and structure in a single tube. The present platform facilitates physiologically relevant interactions of individual bacteria and peptide sequences to be decoded using next-generation sequencing for rapid and batch screening of large populations. The screening platform provided herein can be used for the identification of antimicrobial peptides with activity against a wide range of microbes. The screening platform comprises an Lpp-OmpA localization module and tether to display peptides on the surface of microbes, such as Gram-negative bacterial cells. Accordingly, expression of the peptide can be induced and cell growth and viability is measured to identify antimicrobial peptides.

[0035] Demonstrating the approach, the present studies tested over 800,000 twenty amino acid long peptides against E. coli and identified 7,968 peptides covering a wide range of physiochemical properties that dramatically increases the landscape of recognized amino acid sequences with antimicrobial activity. In addition, synthesis of select linear and cyclic hits shows that at least 80% of sequences are active in synthetic form, many with broad-spectrum activity, no hemolytic activity, and non-pore forming mechanism of actions. Thus, the present disclosure provides antimicrobial peptides as well as methods of their use including treating microbial infections and disinfecting surfaces.

I. Definitions

[0036] As used herein,“essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0037] As used herein the specification,“a” or“an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word“comprising,” the words“a” or “an” may mean one or more than one.

[0038] The use of the term“or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and“and/or.” As used herein “another” may mean at least a second or more.

[0039] Throughout this application, the term“about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. [0040] The term "peptide" as used herein typically refers to a sequence of amino acids made up of a single chain of amino acids joined by peptide bonds. Generally, peptides contain at least two amino acid residues and are less than about 50 amino acids in length, unless otherwise defined.

[0041] The“antimicrobial” peptide according to the present disclosure is a peptide capable of killing a microbial organism or inhibiting its growth. The antimicrobial activities of the antimicrobial peptides can include, without limitation, antibacterial, antiviral, or antifungal activities. In particular aspects, the present disclosure provides antimicrobial peptides with activity against gram-negative bacteria.

[0042] The term "identity" or "homology" shall be construed to mean the percentage of amino acid residues in the candidate sequence that are identical with the residue of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" or“homology” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. Neither N- or C-terminal extensions nor insertions shall be construed as reducing identity or homology. Alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols In Molecular Biology (F. M. Ausubel et al, eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=l0; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,

GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR.

[0043] The term "polypeptide" or“protein” is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g. ester, ether, etc. As used herein the term "amino acid" refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. The term "peptidomimetic" or "peptide mimic" means that a peptide according to the present disclosure is modified in such a way that it includes at least one non-peptidic bond such as, for example, urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.

[0044] The terms "subject" and "individual" and "patient" are used interchangeably herein, and refer to an animal, for example a human or non-human animal ( e.g a mammal), to whom treatment, including prophylactic treatment, with a pharmaceutical composition as disclosed herein, is provided. The term "subject" as used herein refers to human and non-human animals. The term "non-human animals" includes all vertebrates, e.g., mammals, such as non human primates (particularly higher primates), sheep, dogs, rodents (e.g. mouse or rat), guinea pigs, goats, pigs, cats, rabbits, cows, and non-mammals such as chickens, amphibians, reptiles etc. In one embodiment, the subject is human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. Non-human mammals include mammals such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g. mouse or rat), guinea pigs, goats, pigs, cats, rabbits and cows.

[0045]“Treating” or treatment of a disease or condition refers to executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus,“treating” or“treatment” may include “preventing” or “prevention” of disease or undesirable condition. In addition, “treating” or“treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient. By way of example, the administration of one or more antimicrobial peptides provided herein may be used to prevent, treat or relieve the symptoms of a microbial infection.

[0046] "Administering" and/or "administer" as used herein refer to any route for delivering a pharmaceutical composition to a patient. In one embodiment, the compositions described herein are administered enterically to the small intestine. Routes of delivery may include non-invasive peroral (through the mouth), topical (skin), transmucosal (nasal, buccal/sublingual, vaginal, ocular and rectal) and inhalation routes, as well as parenteral routes, and other methods known in the art. Parenteral refers to a route of delivery that is generally associated with injection, including intraorbital, infusion, intraarterial, intracarotid, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrastemal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.

[0047] As used herein, the term "antimicrobial" is meant to include prevention, inhibition or termination of a microbe. In some aspects, "prevention" can be considered to be the obstruction or hindrance of any potential microbial growth, and "inhibition" can be considered to be a reduction in microbial growth. This may occur via, but is not limited to, a microbiostatic mechanism such as interference in the synthesis of the cell wall or binding to ribosomal subunits to prevent production of microbial proteins. "Termination" can be considered to be actual killing of the microbes by the presence of the composition. This may occur via, but is not limited to, a microbiocidal mechanism such as a change in osmotic pressure leading to bursting of the cell or formation of leaky channels in the cell wall and membrane causing loss of cellular material.

[0048] As used herein, the term "microbe(s)" is meant to include any organism comprised of the phylogenetic domains bacteria and archaea, as well as unicellular and filamentous fungi ( e.g yeasts and molds), unicellular and filamentous algae, unicellular and multicellular parasites, and viruses.

[0049] By "isolated" it is meant that the peptide or polypeptide has been separated from any natural environment, such as a body fluid, e.g., blood, and separated from the components that naturally accompany the peptide.

[0050] By isolated and "substantially pure" is meant a peptide or polypeptide that has been separated and purified to at least some degree from the components that naturally accompany it. Typically, a peptide or polypeptide is substantially pure when it is at least about 60%, or at least about 70%, at least about 80%, at least about 90%, at least about 95%, or even at least about 99%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. For example, a substantially pure peptide or polypeptide may be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in a cell that does not normally express that protein, or by chemical synthesis. [0051] The term "variant" as used herein refers to a peptide, polypeptide or nucleic acid that differs from the peptide, polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, additions, substitutions or side-chain modifications, yet retains one or more specific functions or biological activities of the molecule. Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring or a non- conventional amino acid residue. Such substitutions may be classified as "conservative", in which case an amino acid residue contained in a peptide or polypeptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present disclosure may also be "non-conservative", in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties, such as naturally-occurring amino acid from a different group (e.g., substituting a charged or hydrophobic amino; acid with alanine), or alternatively, in which a naturally- occurring amino acid is substituted with a non- conventional amino acid. In some embodiments, amino acid substitutions are conservative. Also encompassed within the term variant when used with reference to a polynucleotide or polypeptide, refers to a polynucleotide or polypeptide that can vary in primary, secondary, or tertiary structure, as compared to a reference polynucleotide or polypeptide, respectively (e.g., as compared to a wild- type polynucleotide or polypeptide).

[0052] The term "insertions" or "deletions" are typically in the range of about 1 to 5 amino acids. The variation allowed can be experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.

[0053] The term "substitution" when referring to a peptide, refers to a change in an amino acid for a different entity, for example another amino acid or amino-acid moiety. Substitutions can be conservative or non-conservative substitutions.

[0054] An "analog" of a molecule such as a peptide refers to a molecule similar in function to either the entire molecule or to a fragment thereof. The term "analog" is also intended to include allelic species and induced variants. Analogs typically differ from naturally occurring peptides at one or a few positions, often by virtue of conservative substitutions. Analogs typically exhibit at least 80 or 90% sequence identity with natural peptides. Some analogs also include unnatural amino acids or modifications of N or C terminal amino acids. Examples of unnatural amino acids are, for example but not limited to, disubstituted amino acids, /V- alkyl amino acids, lactic acid, 4-hydroxyproline, y-carboxygl utamate. e-NNN- trimethyllysine, e-/V-acetyllysine, O-phosphoserine, /V-acetylserine, /V-formyl methionine. 3- methylhistidine, 5-hydroxylysine, and s-N- methylarginine. Fragments and analogs can be screened for prophylactic or therapeutic efficacy in transgenic animal models.

[0055] By "covalently bonded" is meant joined either directly or indirectly ( e.g through a linker) by a covalent chemical bond.

[0056] When used in the context of a chemical group: “hydrogen” means -H; “hydroxy” means -OH;“oxo” means =0;“carbonyl” means -C(=0)-;“carboxy” means -C(=0)OH (also written as -COOH or -CO2H);“halo” means independently -F, -Cl, -Br or -I;“amino” means -NH2;“hydroxyamino” means -NHOH;“nitro” means -NO2; imino means =NH;“cyano” means -CN;“isocyanate” means -N=C=0;“azido” means -N3; in a monovalent context“phosphate” means -OP(0)(OH)2 or a deprotonated form thereof; in a divalent context “phosphate” means -0P(0)(0H)0- or a deprotonated form thereof; “mercapto” means -SH; and“thio” means =S;“sulfonyl” means -S(0)2-; and“sulfmyl” means -S(O)-.

[0057] For the chemical groups and compound classes, the number of carbon atoms in the group or class is as indicated as follows:“Cn” defines the exact number (n) of carbon atoms in the group/class. “C<n” defines the maximum number (n) of carbon atoms that can be in the group/class, with the minimum number as small as possible for the group/class in question, e.g., it is understood that the minimum number of carbon atoms in the group“alkenyl(c_<8)” or the class“alkene(c_<8)” is two. Compare with“alkoxycc_<io)”, which designates alkoxy groups having from 1 to 10 carbon atoms. “Cn-n'” defines both the minimum (n) and maximum number (h') of carbon atoms in the group. Thus,“alkyl(_C2-io)” designates those alkyl groups having from 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical groups or class it modifies and it may or may not be enclosed in parenthesis, without signifying any change in meaning. Thus, the terms“C5 olefin”,“C5 -olefin”,“olefm(C5)”, and “olefines” are all synonymous. When any of the chemical groups or compound classes defined herein is modified by the term“substituted”, any carbon atom(s) in the moiety replacing a hydrogen atom is not counted. Thus methoxyhexyl, which has a total of seven carbon atoms, is an example of a substituted alkyl(ci-6). Unless specified otherwise, any chemical group or compound class described within the context of this disclosure without a carbon atom limit has a carbon atom limit of less than or equal to twelve.

[0058] The term“saturated” when used to modify a compound or chemical group means the compound or chemical group has no carbon-carbon double and no carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond. In the case of substituted versions of saturated groups, one or more carbon oxygen double bond or a carbon nitrogen double bond may be present. And when such a bond is present, then carbon-carbon double bonds that may occur as part of keto-enol tautomerism or imine/enamine tautomerism are not precluded. When the term “saturated” is used to modify a solution of a substance, it means that no more of that substance can dissolve in that solution.

[0059] The term“aliphatic” when used without the“substituted” modifier signifies that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic hydrocarbon compound or group. In aliphatic compounds/groups, the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic). Aliphatic compounds/groups can be saturated, that is joined by single carbon-carbon bonds (alkanes/alkyl), or unsaturated, with one or more carbon-carbon double bonds (alkenes/alkenyl) or with one or more carbon-carbon triple bonds (alkynes/alkynyl).

[0060] The term“aromatic” when used to modify a compound or a chemical group refers to a planar unsaturated ring of atoms with An +2 electrons in a fully conjugated cyclic p system.

[0061] The term“alkyl” when used without the“substituted” modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen. The groups -CH3 (Me), -CH2CH₃ (Et), -CH2CH2CH3 (n- Pr or propyl), -CH(CH3)2 (z-Pr, 'Pr or isopropyl), -CH2CH2CH2CH₃ (n-Bu). -CH(CH3)CH₂CH₃ (sec-butyl). -CH₂CH(CH₃)2 (isobutyl), -C(CH3)3 (tert- butyl, /-butyl, /-Bu or 'Bu). and -CH2C(CH3)3 (neo -pentyl) are non-limiting examples of alkyl groups. The term“alkanediyl” when used without the“substituted” modifier refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-limiting examples of alkanediyl groups. The term“alkylidene” when used without the“substituted” modifier refers to the divalent group =CRR' in which R and R' are independently hydrogen or alkyl. Non-limiting examples of alkylidene groups include: =CH2, =CH(CH2CH3), and =C(CH3)2. An“alkane” refers to the class of compounds having the formula H-R, wherein R is alkyl as this term is defined above. Similarly, the term“alkanoyl” refers to the class of compounds having the formula: -C(0)-R, wherein R is alkyl as this term is defined above. When any of these terms is used with the“substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(0)CH₃, -NHCH3, -NHCH2CH3, -N(CH₃)₂, -C(0)NH₂, -C(0)NHCH₃, -C(0)N(CH₃)2, -0C(0)CH₃, -NHC(0)CH₃, -S(0)₂0H, or -S(0)₂NH₂. The following groups are non-limiting examples of substituted alkyl groups: -CH2OH, -CH2CI, -CF3, -CH2CN, -CH₂C(0)0H, -CH₂C(0)0CH₃, -CH₂C(0)NH₂, -CH₂C(0)CH₃, -CH2OCH3, -CH₂0C(0)CH₃, -CH2NH2, -CH₂N(CH₃)2, and -CH2CH2CI. The term“haloalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to halo (i.e. -F, -Cl, -Br, or -I) such that no other atoms aside from carbon, hydrogen and halogen are present. The group, -CH2CI is a non-limiting example of a haloalkyl. The term“fluoroalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to fluoro such that no other atoms aside from carbon, hydrogen and fluorine are present. The groups -CH2F, -CF3, and -CH2CF3 are non-limiting examples of fluoroalkyl groups.

[0062] The term“cycloalkyl” when used without the“substituted” modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, said carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to a carbon atom of the non-aromatic ring structure. The term “cycloalkanediyl” when used without the“substituted” modifier refers to a divalent saturated aliphatic group with two carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The group

is a non- limiting example of cycloalkanediyl group. A“cycloalkane” refers to the class of compounds having the formula H-R, wherein R is cycloalkyl as this term is defined above. Similarly, the term“cycloalkanoyl” refers to the class of compounds having the formula: -C(0)-R, wherein R is cycloalkyl as this term is defined above. When any of these terms is used with the “substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH₂, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(0)CH₃, -NHCH3, -NHCH2CH3, -N(CH₃)2, -C(0)NH₂, -C(0)NHCH₃, -C(0)N(CH₃)₂, -OC(0)CH₃, -NHC(0)CH₃, -S(0)₂OH, or -S(0)₂NH₂.

[0063] The term“alkenyl” when used without the“substituted” modifier refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon- carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH=CH₂ (vinyl), -CH=CHCH₃, -CH=CHCH₂CH3, -CH₂CH=CH₂ (allyl), -CH2CH=CHCH3, and -CH=CHCH=CH2. The term“alkenediyl” when used without the “substituted” modifier refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched, a linear or branched acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and -CH2CH=CHCH2- are non-limiting examples of alkenediyl groups. It is noted that while the alkenediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure. The terms“alkene” and“olefin” are synonymous and refer to the class of compounds having the formula H-R, wherein R is alkenyl as this term is defined above. Similarly, the terms“terminal alkene” and“a-olefin” are synonymous and refer to an alkene having just one carbon-carbon double bond, wherein that bond is part of a vinyl group at an end of the molecule. Furthermore, the term“alkenoyl” refers to the class of compounds having the formula: -C(0)-R, wherein R is alkenyl as this term is defined above. When any of these terms are used with the“substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH₂, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(0)CH₃, -NHCH3, -NHCH2CH3, -N(CH₃)2, -C(0)NH₂, -C(0)NHCH₃, -C(0)N(CH₃)₂, -OC(0)CH₃, -NHC(0)CH₃, -S(0)₂OH, or -S(0)₂NH₂. The groups -CH=CHF, -CH=CHCl and -CH=CHBr are non-limiting examples of substituted alkenyl groups.

[0064] The term“alkynyl” when used without the“substituted” modifier refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -CºCH, -CºCCH3, and -CH2CºCCH3 are non-limiting examples of alkynyl groups. An“alkyne” refers to the class of compounds having the formula H-R, wherein R is alkynyl. Similarly, the term“alkynoyl” refers to the class of compounds having the formula: -C(0)-R, wherein R is alkynyl as this term is defined above. When any of these terms are used with the“substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH₂, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(0)CH3, -NHCH3, -NHCH2CH3, -N(CH₃)2, -C(0)NH₂, -C(0)NHCH₃, -C(0)N(CH₃)2, -0C(0)CH₃, -NHC(0)CH₃, -S(0)₂0H, or -S(0)₂NH₂.

[0065] The term“aryl” when used without the“substituted” modifier refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of a one or more aromatic ring structure, wherein the ring atoms are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. As used herein, the term aryl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C6H4CH2CH₃ (ethylphenyl), naphthyl, and a monovalent group derived from biphenyl (e.g., 4-phenylphenyl). The term “arenediyl” when used without the“substituted” modifier refers to a divalent aromatic group with two aromatic carbon atoms as points of attachment, said carbon atoms forming part of one or more six-membered aromatic ring structure(s) wherein the ring atoms are all carbon, and wherein the monovalent group consists of no atoms other than carbon and hydrogen. As used herein, the term arenediyl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. Non-limiting examples of arenediyl groups include:

An“arene” refers to the class of compounds having the formula H-R, wherein R is aryl as that term is defined above. Benzene and toluene are non-limiting examples of arenes. Similarly, the term“aroyl” refers to the class of compounds having the formula: -C(0)-R, wherein R is aryl as this term is defined above. When any of these terms are used with the“substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(0)CH₃, -NHCH3, -NHCH2CH3, -N(CH₃)2, -C(0)NH₂, -C(0)NHCH₃, -C(0)N(CH₃)₂, -OC(0)CH₃, -NHC(0)CH3, -S(0)₂OH, or -S(0)₂NH₂.

[0066] The term“aralkyl” when used without the“substituted” modifier refers to the monovalent group -alkanediyl-aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term aralkyl is used with the “substituted” modifier one or more hydrogen atom from the alkanediyl and/or the aryl group has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH₂, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(0)CH3, -NHCH3, -NHCH2CH3, -N(CH₃)2, -C(0)NH₂, -C(0)NHCH₃, -C(0)N(CH₃)₂, -0C(0)CH₃, -NHC(0)CH₃, -S(0)₂0H, or -S(0)₂NH₂. Non- limiting examples of substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl- eth-l-yl.

[0067] The term“heteroaralkyl” when used without the“substituted” modifier refers to the monovalent group -alkanediyl-heteroaryl, in which the terms alkanediyl and heteroaryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are pyridinylmethyl or 2-quinolinylethyl. When the term heteroaralkyl is used with the“substituted” modifier one or more hydrogen atom from the alkanediyl and/or the heteroaryl group has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(0)CH₃, -NHCH3, -NHCH2CH3, -N(CH₃)2, -C(0)NH₂, -C(0)NHCH₃, -C(0)N(CH₃)₂, -OC(0)CH₃, -NHC(0)CH₃, -S(0)₂OH, or -S(0)₂NH₂. Non-limiting examples of substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-l-yl.

[0068] The term“heteroaryl” when used without the“substituted” modifier refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more aromatic ring structures wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. As used herein, the term heteroaryl does not preclude the presence of one or more alkyl or aryl groups (carbon number limitation permitting) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term N-heteroaryl refers to a heteroaryl group with a nitrogen atom as the point of attachment. A“heteroarene” refers to the class of compounds having the formula H-R, wherein R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes. Similarly, the term“heteroaroyl” refers to the class of compounds having the formula: -C(0)-R, wherein R is heteroaryl as this term is defined above. When these terms are used with the“substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH₂·, -N0₂, -C0₂H, -C0₂CH₃, -CN, -SH, -OCH₃, -OCH₂CH₃, -C(0)CH₃, -NHCH₃, -NHCH₂CH₃, -N(CH₃)₂, -C(0)NH₂, -C(0)NHCH₃, -C(0)N(CH₃)₂, -OC(0)CH₃, -NHC(0)CH₃, -S(0)₂OH, or -S(0)₂NH₂.

[0069] The term“heterocycloalkyl” when used without the“substituted” modifier refers to a monovalent non-aromatic group with a carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more non-aromatic ring structures wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heterocycloalkyl group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the ring or ring system. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term“A-heterocycloalkyl” refers to a heterocycloalkyl group with a nitrogen atom as the point of attachment. A-pyrrolidinyl is an example of such a group. When these terms are used with the“substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(0)CH₃, -NHCH3, -NHCH2CH3, -N(CH₃)2, -C(0)NH₂, -C(0)NHCH₃, -C(0)N(CH₃)2, -OC(0)CH3, -NHC(0)CH₃, -S(0)₂OH, or -S(0)₂NH₂.

[0070] The term“acyl” when used without the“substituted” modifier refers to the group -C(0)R, in which R is a hydrogen, alkyl, cycloalkyl, or aryl as those terms are defined above. The groups, -CHO, -C(0)CH3 (acetyl, Ac), -C(0)CH2CH3, -C(0)CH(CH3)2, -C(0)CH(CH2)2, -C(0)CeH5, and -C(0)C6H4CH3 are non-limiting examples of acyl groups. A“thioacyl” is defined in an analogous manner, except that the oxygen atom of the group -C(0)R has been replaced with a sulfur atom, -C(S)R. The term“aldehyde” corresponds to an alkyl group, as defined above, attached to a -CHO group. When any of these terms are used with the“substituted” modifier one or more hydrogen atom (including a hydrogen atom directly attached to the carbon atom of the carbonyl or thiocarbonyl group, if any) has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(0)CH₃, -NHCH3, -NHCH2CH3, -N(CH₃)₂, -C(0)NH₂, -C(0)NHCH₃, -C(0)N(CH₃)₂, -OC(0)CH₃, -NHC(0)CH₃, -S(0)₂OH, or -S(0)₂NH₂. The groups, -C(0)CH2CF3, -CO2H (carboxyl), -CO2CH₃ (methylcarboxyl), -CO2CH2CH3, -C(0)NH2 (carbamoyl), and -CON(CH3)2, are non-limiting examples of substituted acyl groups.

[0071] The term“alkoxy” when used without the“substituted” modifier refers to the group -OR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: -OCH3 (methoxy), -OCH2CH3 (ethoxy), -OCH2CH2CH3, -OCH(CH3)2 (isopropoxy), -OC(CH3)3 (tert- butoxy), -OCH(CH2)2, -O-cyclopentyl, and -O-cyclohexyl. The terms “cycloalkoxy”, “alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”, “heteroaryloxy”,“heterocycloalkoxy”, and“acyloxy”, when used without the“substituted” modifier, refers to groups, defined as -OR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term“alkylthio” and “acylthio” when used without the“substituted” modifier refers to the group -SR, in which R is an alkyl and acyl, respectively. The term“alcohol” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a hydroxy group. The term“ether” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with an alkoxy group. When any of these terms is used with the“substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH₂, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(0)CH₃, -NHCH3, -NHCH2CH3, -N(CH₃)2, -C(0)NH₂, -C(0)NHCH₃, -C(0)N(CH₃)2, -OC(0)CH3, -NHC(0)CH₃, -S(0)₂OH, or -S(0)₂NH₂.

[0072] The term“alkylamino” when used without the“substituted” modifier refers to the group -NHR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: -NHCH₃ and -NHCH2CH3. The term“dialkylamino” when used without the “substituted” modifier refers to the group -NRR', in which R and R' can be the same or different alkyl groups, or R and R' can be taken together to represent an alkanediyl. Non- limiting examples of dialkylamino groups include: -NCCHifi and -N(CH3)(CH2CH3). The terms “cycloalkylamino”, “alkenylamino”, “alkynylamino”, “arylamino”, “aralkylamino”, “heteroarylamino”,“heterocycloalkylamino”,“alkoxyamino”, and“alkylsulfonylamino” when used without the“substituted” modifier, refers to groups, defined as -NHR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. A non-limiting example of an arylamino group is -NHC6H5. The term“amido” (acylamino), when used without the“substituted” modifier, refers to the group -NHR, in which R is acyl, as that term is defined above. A non-limiting example of an amido group is -NHC(0)CH3. The term“alkylimino” when used without the“substituted” modifier refers to the divalent group =NR, in which R is an alkyl, as that term is defined above. When any of these terms is used with the“substituted” modifier one or more hydrogen atom attached to a carbon atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(0)CH₃, -NHCH3, -NHCH2CH3, -N(CH₃)2, -C(0)NH₂, -C(0)NHCH₃, -C(0)N(CH₃)2, -OC(0)CH₃, -NHC(0)CH₃, -S(0)₂OH, or -S(0)₂NH₂. The groups -NHC(0)OCH₃ and -NHC(0)NHCH₃ are non- limiting examples of substituted amido groups.

[0073] The terms “alkylsulfonyl” and “alkylsulfmyl” when used without the “substituted” modifier refers to the groups -S(0)₂R and -S(0)R, respectively, in which R is an alkyl, as that term is defined above. The terms“cycloalkylsulfonyl”,“alkenylsulfonyl”, “alkynylsulfonyl”, “arylsulfonyl”, “aralkylsulfonyl”, “heteroarylsulfonyl”, and “heterocycloalkylsulfonyl” are defined in an analogous manner. When any of these terms is used with the“substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH₂, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(0)CH₃, -NHCH3, -NHCH2CH3, -N(CH₃)2, -C(0)NH₂, -C(0)NHCH₃, -C(0)N(CH₃)2, -OC(0)CH₃, -NHC(0)CH₃, -S(0)₂OH, or -S(0)₂NH₂.

[0074] The term "fusion protein" as used herein refers to a recombinant protein of two or more proteins. Fusion proteins can be produced, for example, by a nucleic acid sequence encoding one protein is joined to the nucleic acid encoding another protein such that they constitute a single open-reading frame that can be translated in the cells into a single polypeptide harboring all the intended proteins. The order of arrangement of the proteins can vary. Fusion proteins can include an epitope tag or a half-life extender. Epitope tags include biotin, FLAG tag, c-myc, hemaglutinin, His6, digoxigenin, FITC, Cy3, Cy5, green fluorescent protein, V5 epitope tags, GST, b-galactosidase, AU1, AU5, and avidin. Half-life extenders include Fc domain and serum albumin.

II. Antimicrobial Peptides

[0075] Embodiments of the disclosure concern isolated peptide or polypeptides that have antimicrobial activity as well as methods of their use. These include peptides or expression vectors encoding the peptides disclosed herein as well as that structurally similar compounds (i.e., small molecules) that may be formulated to mimic the key portions of peptide. In certain embodiments, the peptide or polypeptide has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity or similarity with any one of SEQ ID NOs: 1-7968. In particular aspects, the peptide comprises an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity or similarity with SEQ ID NO: 7, 106, 130, 135, 227, 249, 323, 473, 506, 537, 646, 1136, 1341, 1611, 2101, 2744, 6519, or 6624.

[0076] In general, the peptides of the present disclosure may be 50 residues or less. The overall length may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 residues. Ranges of peptide length of 10-50 residues, 15-50 residues, 20-25 residues 21-25, residues, 20- 30 residues, 30-40 residues, and 35-45 residues, and 25-35 residues are contemplated. The present disclosure may utilize L-configuration amino acids, D-configuration amino acids, or a mixture thereof.

[0077] In some embodiments, the peptide is a variant comprising at least one amino acid substitution, deletion, or insertion relative to the amino acid sequence of any one of SEQ ID NOs: 1-7968. Variants can be synthetic, recombinant, or chemically modified polypeptides isolated or generated using methods well known in the art. Variants can include conservative or non-conservative amino acid changes, as described below. Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Variants can also include insertions, deletions or substitutions of amino acids, including insertions and substitutions of amino acids and other molecules that do not normally occur in the peptide sequence that is the basis of the variant, for example but not limited to insertion of ornithine which do not normally occur in human proteins. The term conservative substitution, when describing a polypeptide, refers to a change in the amino acid composition of the polypeptide that does not substantially alter the polypeptide's activity. For example, a conservative substitution refers to substituting an amino acid residue for a different amino acid residue that has similar chemical properties. Conservative amino acid substitutions include replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

[0078] Conservative amino acid substitutions result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Thus, a conservative substitution of a particular amino acid sequence refers to substitution of those amino acids that are not critical for polypeptide activity or substitution of amino acids with other amino acids having similar properties ( e.g acidic, basic, positively or negatively charged, polar or non-polar) such that the substitution of even critical amino acids does not reduce the activity of the peptide. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company (1984), incorporated by reference in its entirety.) In some embodiments, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids can also be considered conservative substitutions if the change does not reduce the activity of the peptide. Insertions or deletions are typically in the range of about 1 to 5 amino acids. The choice of conservative amino acids may be selected based on the location of the amino acid to be substituted in the peptide, for example if the amino acid is on the exterior of the peptide and expose to solvents, or on the interior and not exposed to solvents.

[0079] In alternative embodiments, one can select the amino acid which will substitute an existing amino acid based on the location of the existing amino acid, i.e. its exposure to solvents (i.e. if the amino acid is exposed to solvents or is present on the outer surface of the peptide or polypeptide as compared to internally localized amino acids not exposed to solvents). Selection of such conservative amino acid substitutions are well known in the art, for example as disclosed in Dordo et al, J. Mol Biol, 1999, 217, 721-739 and Taylor et al, J. Theor. Biol. 119(1986);205-218 and S. French and B. Robson, J. Mol. Evol. 19(1983)171. Accordingly, one can select conservative amino acid substitutions suitable for amino acids on the exterior of a protein or peptide (i.e. amino acids exposed to a solvent), for example, but not limited to, the following substitutions can be used: substitution of Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with T or A, R with K, G with N or A, K with R, A with S, K or P.

[0080] In alternative embodiments, one can also select conservative amino acid substitutions encompassed suitable for amino acids on the interior of a protein or peptide, for example one can use suitable conservative substitutions for amino acids is on the interior of a protein or peptide (i.e. the amino acids are not exposed to a solvent), for example but not limited to, one can use the following conservative substitutions: where Y is substituted with F, T with A or S, I with L or V, W with Y, M with L, N with D, G with A, T with A or S, D with N, I with L or V, F with Y or L, S with A or T and A with S, G, T or V. In some embodiments, non conservative amino acid substitutions are also encompassed within the term of variants.

[0081] In some aspects, the peptides or polypeptides disclosed herein are derivatives of the SEQ ID NOs: 1-7968. The term“derivative” as used herein refers to peptides which have been chemically modified, for example but not limited to by techniques such as ubiquitination, labeling, pegylation (i.e., derivatization with polyethylene glycol), lipidation, glycosylation, or addition of other molecules. A molecule is also a“derivative” of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties can improve the molecule's solubility, absorption, biological half-life, etc. The moieties can alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., MackPubl, Easton, PA (1990), incorporated herein, by reference, in its entirety.

[0082] The term "functional" when used in conjunction with“derivative” or“variant” refers to a polypeptide of the present disclosure which possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of the entity or molecule it is a functional derivative or functional variant thereof. The term functional derivative is intended to include the fragments, analogues or chemical derivatives of a molecule.

[0083] In some aspects, amino acid substitutions can be made in a polypeptide at one or more positions wherein the substitution is for an amino acid having a similar hydrophilicity. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Thus such conservative substitution can be made in a polypeptide and will likely only have minor effects on their activity. As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine ( 0.5); histidine -0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). These values can be used as a guide and thus substitution of amino acids whose hydrophilicity values are within ±2 are preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. Thus, any of the peptides or polypeptides described herein may be modified by the substitution of an amino acid, for different, but homologous amino acid with a similar hydrophilicity value. Amino acids with hydrophilicities within +/- 1.0, or +/- 0.5 points are considered homologous. [0084] The antimicrobial peptides or polypeptides may comprise co-translational and post- translational (C-terminal peptide cleavage) modifications, such as, for example, disulfide- bond formation, glycosylation, acetylation, phosphorylation, and proteolytic cleavage ( e.g cleavage by furins or metalloproteases) to the extent that such modifications do not affect the antimicrobial properties of the isolated peptides.

[0085] In some aspects, the antimicrobial peptide (or polypeptide) comprises non- naturally occurring amino acids. The antimicrobial peptides can comprise a combination of naturally occurring and non-naturally occurring amino acids, or may comprise only non- naturally occurring amino acids. The non-naturally occurring amino acids can include synthetic non-native amino acids, substituted amino acids, or one or more D-amino acids into the peptides (or other components of the composition, with exception for protease recognition sequences) is desirable in certain situations. D-amino acid- containing peptides exhibit increased stability in vitro or in vivo compared to L-amino acid- containing forms. Thus, the construction of peptides incorporating D-amino acids can be particularly useful when greater in vivo or intracellular stability is desired or required. More specifically, D- peptides are resistant to endogenous peptidases and proteases, thereby providing better oral trans-epithelial and transdermal delivery of linked drugs and conjugates, improved bioavailability of membrane -permanent complexes, and prolonged intravascular and interstitial lifetimes when such properties are desirable. The use of D- isomer peptides can also enhance transdermal and oral trans-epithelial delivery of linked drugs and other cargo molecules. Additionally, D- peptides cannot be processed efficiently for major histocompatibility complex class II- restricted presentation to T helper cells, and are therefore less likely to induce humoral immune responses in the whole organism. Peptide conjugates can therefore be constructed using, for example, D-isomer forms of cell penetrating peptide sequences, L-isomer forms of cleavage sites, and D-isomer forms of therapeutic peptides.

[0086] In addition to the 20“standard” L-amino acids, D-amino acids or non-standard, modified or unusual amino acids which are well-defined in the art are also contemplated for use in the present disclosure including phosphorylated amino acids (Ser, Thr, Tyr), glycosylated amino acids (Ser, Thr, Asn), //-amino acids, GABA, and cw-amino acids.

[0087] There are a vast number of“non-standard” amino acids. Two of these can be specified by the genetic code, but are rather rare in proteins. Selenocysteine is incorporated into some proteins at a UGA codon, which is normally a stop codon. Pyrrolysine is used by some methanogenic archaea in enzymes that they use to produce methane. It is coded for with the codon UAG. Examples of non-standard amino acids that are not found in proteins include lanthionine, 2-aminoisobutyric acid, dehydroalanine and the neurotransmitter gamma- aminobutyric acid. Non-standard amino acids often occur as intermediates in the metabolic pathways for standard amino acids - for example ornithine and citrulline occur in the urea cycle, part of amino acid catabolism. Non-standard amino acids are usually formed through modifications to standard amino acids. For example, homocysteine is formed through the transsulfuration pathway or by the demethylation of methionine via the intermediate metabolite S-adenosyl methionine, while hydroxyproline is made by a posttranslational modification of proline.

[0088] The amino acids of the antimicrobial peptides of the present disclosure may also be modified. For example, amino groups may be acylated, alkylated, or arylated. Benzyl groups may be halogenated, nitrosylated, alkylated, sulfonated or acylated.

[0089] Carboxy terminal modifications include acylation with carboxylic acids: formic, acetic, propionic, fatty acids (myristic, palmitic, stearic), succinic, benzoic, carbobenzoxy (Cbz); and biotinylation. Amino terminal modifications include: (i) acylation with carboxylic acids: formic, acetic, propionic, fatty acids (myristic, palmitic, stearic, etc) succinic, benzoic, carbobenzoxy (Cbz); (ii) biotinylation; (iii) attachment of dyes such as fluorescein (FITC, FAM, etc.), 7-hydroxy-4-methylcoumarin-3-acetic acid, 7- hydroxycoumarin-3-acetic acid, 7-methoxycoumarin-3-acetic acid and other coumarins; rhodamines (5-carboxyrhodamine 110 or 6G, 5(6)-TAMRA, ROX); /V-|4-(4- dimethylamino)phenylazo]benzoic acid (Dabcyl), 2,4-dinitrobenzene (Dnp), 5- dimethylaminonaphthalene-l -sulfonic acid (Dansyl) and other dyes; and (iv) polyethyleneglycol. These include, for example, include //-alanine (//-Ala) and other cw-amino acids such as 3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; a-aminoisobutyric acid (Aib); e-aminohexanoic acid (Aha); ri-aminovaleric acid (Ava); X-methylglycine or sarcosine (MeGly); ornithine (Om); citrulbne (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); X-methylisoleucine (Melle); phenylglycine (Phg); norleucine (Nle); 4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F)); 3- fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); l,2,3,4-tetrahydroisoquinoline-3-carboxybc acid (Tic); homoarginine (hArg); X-acetyl lysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,4-diaminobutyric acid (Dab); p- aminophenylalanine (Phe(pNH2)); N-methyl valine (MeVal); homocysteine (hCys), homophenylalanine (hPhe) and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro), iV-methylated amino acids and peptoids (TV-substituted glycines).

[0090] The polypeptide may be capped at its N and C termini with an acyl (abbreviated “Ac”) -and an amido (abbreviated“Am”) group, respectively, for example acetyl (CH3CO-) at the N terminus and amido (-NH2) at the C terminus. A broad range of N-terminal capping functions, preferably in a linkage to the terminal amino group, is contemplated, for example:

• formyl;

• alkanoyl, having from 1 to 10 carbon atoms, such as acetyl, propionyl, or butyryl;

• cycloalkanoyl, having from 1 to 10 carbon atoms;

• alkenoyl, having from 1 to 10 carbon atoms, such as hex-3-enoyl;

• alkynoyl, having from 1 to 10 carbon atoms, such as hex-5-ynoyl;

• aroyl, such as benzoyl or l-naphthoyl;

• heteroaroyl, such as 3-pyrroyl or 4-quinoloyl;

• alkylsulfonyl, such as methanesulfonyl;

• arylsulfonyl, such as benzenesulfonyl or sulfanilyl;

• heteroarylsulfonyl, such as pyridine-4-sulfonyl;

• substituted alkanoyl, having from 1 to 10 carbon atoms, such as 4-aminobutyryl;

• substituted alkenoyl, having from 1 to 10 carbon atoms, such as 6-hydroxy -hex- 3-enoyl;

• substituted alkynoyl, having from 1 to 10 carbon atoms, such as 3 -hydroxy -hex- 5-ynoyl;

• substituted aroyl, such as 4-chlorobenzoyl or 8-hydroxy-naphth-2-oyl;

• substituted heteroaroyl, such as 2,4-dioxo-l,2,3,4-tetrahydro-3-methyl- quinazolin-6-oyl;

• substituted alkylsulfonyl, such as 2-aminoethanesulfonyl;

• substituted arylsulfonyl, such as 5-dimethylamino-l-naphthalenesulfonyl;

• substituted heteroarylsulfonyl, such as l-methoxy-6-isoquinolinesulfonyl;

• carbamoyl (^~C(0)NH2) or thiocarbamoyl (^~C(S)NH2);

• substituted carbamoyl (-C(O)NR'R") or substituted thiocarbamoyl (-C(S)NR'R") wherein R' and R" are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl, or substituted heteroaryl;

• substituted carbamoyl (-C(O)NR'R'') and substituted thiocarbamoyl

(-C(S)NR'R'') wherein R' and R" are each independently hydrogen, alkanoyl, alkenoyl, alkynoyl, aroyl, heteroaroyl, substituted alkanoyl, substituted alkenoyl, substituted alkynoyl, substituted aroyl, or substituted heteroaroyl, all as above defined.

[0091] The C-terminal capping function can either be in an amide or ester bond with the terminal carboxyl. Capping functions that provide for an amide bond are designated as NR' R² wherein R¹ and R² may be independently drawn from the following group:

• hydrogen;

• alkyl, preferably having from 1 to 10 carbon atoms, such as methyl, ethyl, or isopropyl;

• cycloalkyl, preferably having from 1 to 10 carbon atoms, such as cyclopropyl, cyclobutyl, or cyclopentyl;

• alkenyl, preferably having from 1 to 10 carbon atoms, such as prop-2-enyl;

• alkynyl, preferably having from 1 to 10 carbon atoms, such as prop-2-ynyl;

• substituted alkyl having from 1 to 10 carbon atoms, such as hydroxy alkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl, haloalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkanoylalkyl, carboxyalkyl, carbamoylalkyl;

• substituted alkenyl having from 1 to 10 carbon atoms, such as hydroxy alkenyl, alkoxyalkenyl, mercaptoalkenyl, alkylthioalkenyl, haloalkenyl, cyanoalkenyl, aminoalkenyl, alkylaminoalkenyl, dialkylaminoalkenyl, alkanoylalkenyl, carboxyalkenyl, carbamoylalkenyl;

• substituted alkynyl having from 1 to 10 carbon atoms, such as hydroxy alkynyl, alkoxyalkynyl, mercaptoalkynyl, alkylthioalkynyl, halogenoalkynyl, cyanoalkynyl, aminoalkynyl, alkylaminoalkynyl, dialkylaminoalkynyl, alkanoylalkynyl, carboxyalkynyl, carbamoylalkynyl;

• aroylalkyl having up to 10 carbon atoms, such as phenacyl or 2-benzoylethyl;

• aryl, such as phenyl or 1 -naphthyl;

• heteroaryl, such as 4-quinolyl; • alkanoyl having from 1 to 10 carbon atoms, such as acetyl or butyryl;

• aroyl, such as benzoyl;

• heteroaroyl, such as 3-quinoloyl;

• OR' or NR'R" where R' and R" are independently hydrogen, alkyl, aryl, heteroaryl, acyl including aroyl, sulfonyl, sulfmyl, or SO2-R'" or SO-R'" where R'" is substituted or unsubstituted alkyl, aryl, heteroaryl, alkenyl, or alkynyl.

[0092] Alternatively, Ri and R2 may be taken together to form a heterocycloalkyl group or a substituted version thereof containing from 3 to 10 ring members including heteroatoms.

[0093] Capping functions that provide for an ester bond are designated as OR, wherein R may form an alkoxy; aryloxy; heteroaryloxy; aralkoxy; heteroaralkoxy; substituted alkoxy; substituted aryloxy; substituted heteroaryloxy; substituted aralkoxy; or substituted heteroaralkoxy group.

[0094] Either the AMerminal or the C-terminal capping function, or both, may be of such structure that the capped molecule functions as a prodrug (a pharmacologically inactive derivative of the parent drug molecule) that undergoes spontaneous or enzymatic transformation within the body in order to release the active drug and that has improved delivery properties over the parent drug molecule (Bundgaard H, Ed: Design of Prodrugs, Elsevier, Amsterdam, 1985).

[0095] Judicious choice of capping groups allows the addition of other activities on the peptide. For example, the presence of a sulfhydryl group linked to the N- or C-terminal cap will permit conjugation of the derivatized peptide to other molecules.

[0096] In yet a further aspect, the peptides or variants or derivatives thereof can be "retro-inverso peptides." A "retro-inverso peptide" refers to a peptide with a reversal of the direction of the peptide bond on at least one position, i.e., a reversal of the amino- and carboxy- termini with respect to the side chain of the amino acid. Thus, a retro-inverso analogue has reversed termini and reversed direction of peptide bonds while approximately maintaining the topology of the side chains as in the native peptide sequence. The retro-inverso peptide can contain L-amino acids or D-amino acids, or a mixture of L-amino acids and D-amino acids, up to all of the amino acids being the D-isomer. Partial retro-inverso peptide analogues are polypeptides in which only part of the sequence is reversed and replaced with enantiomeric amino acid residues. Since the retro- inverted portion of such an analogue has reversed amino and carboxyl termini, the amino acid residues flanking the retro-inverted portion are replaced by side-chain-analogous a-substituted geminal-diaminomethanes and malonates, respectively. Retro-inverso forms of cell penetrating peptides have been found to work as efficiently in translocating across a membrane as the natural forms. Synthesis of retro-inverso peptide analogues are described in Bonelli, F. et al, Int J Pept Protein Res. 24(6):553-6 (1984); Verdini, A and Viscomi, G. C, J. Chem. Soc. Perkin Trans. 1 :697-70l (1985); and U.S. Patent No. 6,261,569, which are incorporated herein in their entirety by reference. Processes for the solid-phase synthesis of partial retro-inverso peptide analogues have been described (EP 97994-B) which is also incorporated herein in its entirety by reference.

[0097] Embodiments of the present disclosure also include longer polypeptides built from repeating units of an antimicrobial polypeptide. A polypeptide multimer may comprise different combinations of polypeptide. Such multimeric polypeptides can be made by chemical synthesis or by recombinant DNA techniques as discussed herein. When produced by chemical synthesis, the oligomers preferably have from 2-5 repeats of a core polypeptide sequence, and the total number of amino acids in the multimer should not exceed about 160 residues, preferably not more than 100 residues (or their equivalents, when including linkers or spacers).

A. Peptidomimetics

[0098] In addition to the peptides disclosed herein, the present disclosure also contemplates that structurally similar compounds may be formulated to mimic the key portions of peptide or polypeptides of the present disclosure. Such compounds, which may be termed peptidomimetics, may be used in the same manner as the peptides of the present disclosure and, hence, also are functional equivalents.

[0099] A peptidomimetic agent may be an unnatural peptide or a non-peptide agent that recreates the stereospatial properties of the binding elements of the antimicrobial peptide such that it has the binding activity and biological activity of the unmodified peptide.

[00100] In some aspects, the present disclosure also includes compounds that retain partial peptide characteristics. For example, any proteolytically unstable bond within a peptide of the present disclosure could be selectively replaced by a non-peptidic element such as an isostere (N-methylation; D-amino acid) or a reduced peptide bond while the rest of the molecule retains its peptidic nature. It is contemplated that one, two, three, four, or five peptide bonds have been reduced in the antimicrobial peptides described herein. These reduced peptide bonds result in the conversion of an amide into a amine.

[00101] Additionally contemplated are azapeptide analogs wherein the a-carbon atom is replaced with an isoelectronic nitrogen atom. Within the azapeptide, the side chains remain unchanged but the hydrogen atom on the a-carbon atom is missing. It is contemplated that that the entire peptide may be constructed of azapeptide linkages or only one, two, three, four, five, six, seven, eight, nine, ten, fifteen, or all a-carbon atoms are replaced with azapeptide linkages.

[00102] In yet another aspect, the antimicrobial polypeptides described herein may be chemically prepared as a comparable peptoid. The peptoids may be prepared using a glycine backbone in which the respective side chain of the peptide has been attached to the nitrogen atom rather than the a-carbon atom. The conversion of a peptide sequence to a peptoid is taught by at least Tan, et al, Bioorg Med. Chem., 16(1 l):5853-586l, 2008. In some aspects, the conversion of the peptides into isomerically similar peptoids results in the production of compound which is more resistant to the activity of proteases or peptidases. It is contemplated that one or more side chain from one or more amino acid has been converted into the corresponding peptoid molecule. In some embodiments, the number of amino acid residues converted into their peptoid counterpart is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or all of the amino acid residues.

[00103] Peptidomimetic compounds, either agonists, substrates or inhibitors, have been described for a number of bioactive peptides/polypeptides such as opioid peptides, VIP, thrombin, HIV protease, etc. Methods for designing and preparing peptidomimetic compounds are known in the art (Hruby, VJ, Biopolymers 33: 1073-1082 (1993); Wiley, RA et al., Med. Res. Rev. 73:327-384 (1993); Moore et al., Adv. in Pharmacol 33:91-141 (1995); Giannis et al, Adv. in Drug Res. 29: 1-78 (1997). Certain mimetics that mimic secondary structure are described in Johnson et al. , In: Biotechnology and Pharmacy, Pezzuto et al. , Chapman and Hall (Eds.), NY, 1993. These methods are used to make peptidomimetics that possess at least the binding capacity and specificity of the desired antimicrobial polypeptide and also possess the biological activity. Knowledge of peptide chemistry and general organic chemistry available to those skilled in the art are sufficient, in view of the present disclosure, for designing and synthesizing such compounds. [00104] For example, such peptidomimetics may be identified by inspection of the three-dimensional structure of a peptide or polypeptide of the present disclosure either free or bound in complex with a ligand. Alternatively, the structure of a polypeptide of the present bound to its ligand can be gained by the techniques of nuclear magnetic resonance spectroscopy. Greater knowledge of the stereochemistry of the interaction of the peptide with its ligand or receptor will permit the rational design of such peptidomimetic agents. The structure of a peptide or polypeptide of the invention in the absence of ligand could also provide a scaffold for the design of mimetic molecules.

B. Stabilized Peptides

[00105] A particular modification is in the context of peptides as therapeutics is the so-called“Stapled Peptide” technology of Aileron Therapeutics. The general approach for “stapling” a peptide is that two key residues within the peptide are modified by attachment of linkers through the amino acid side chains. Once synthesized, the linkers are connected through a catalyst, thereby creating a bridge that physically constrains the peptide into its native a- helical shape. In addition to helping retain the native structure needed to interact with a target molecule, this conformation also provides stability against peptidases as well as promotes cell permeating properties.

[00106] More particularly, the term“peptide stapling” may encompasses the joining of two double bond-containing sidechains, two triple bond-containing sidechains, or one double bond-containing and one triple bond-containing side chain, which may be present in a polypeptide chain, using any number of reaction conditions and/or catalysts to facilitate such a reaction, to provide a singly“stapled” polypeptide. In a specific embodiment, the introduction of a staple entails a modification of standard peptide synthesis, with a-methyl, a-alkenyl amino acids being introduced at two positions along the peptide chain, separated by either three or six intervening residues (i + 4 or / + 7). These spacings place the stapling amino acids on the same face of the a-helix, straddling either one ( i + 4) or two ( i + 7) helical turns. The fully elongated, resin-bound peptide can be exposed to a ruthenium catalyst that promotes cross-linking of the alkenyl chains through olefin metathesis, thereby forming an all-hydrocarbon macrocyclic cross-link. U.S. Patents 7,192,713 and 7,183,059, and U.S. Patent Publications 2005/02506890 and 2006/0008848, describing this technology, are hereby incorporated by reference. See also Schafmeister et al, Journal of the American Chemical Society, 122(24): p. 5891-5892 (2000); Walensky et al, Science 305: 1466-1470 (2004). Additionally, the term“peptide stitching” refers to multiple and tandem“stapling” events in a single peptide chain to provide a“stitched” (multiply stapled) polypeptide, each of which is incorporated herein by reference. See WO 2008/121767 for a specific example of stitched peptide technology.

C. PEGylation

[00107] The antimicrobial peptides may be conjugated with heterologous polypeptide segments or polymers, such as polyethylene glycol. The peptides may be linked to PEG to increase the hydrodynamic radius and hence increase the serum persistence. The peptides may be conjugated to any targeting agent, such as a ligand having the ability to specifically and stably bind to an external receptor (U.S. Patent Publication No. 2009/0304666). [00108] In certain aspects, methods and compositions of the embodiments relate to

PEGylation of disclosed polypeptides. PEGylation is the process of covalent attachment of poly(ethylene glycol) polymer chains to another molecule, normally a drug or therapeutic protein. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target macromolecule. The covalent attachment of PEG to a drug or therapeutic protein can “mask” the agent from the host's immune system (reduced immunogenicity and antigenicity) or increase the hydrodynamic size (size in solution) of the agent, which prolongs its circulatory time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins.

[00109] The first step of the PEGylation is the suitable functionalization of the PEG polymer at one or both terminals. PEGs that are activated at each terminus with the same reactive moiety are known as“homobifunctional,” whereas if the functional groups present are different, then the PEG derivative is referred as“heterobifunctional” or“heterofunctional.” The chemically active or activated derivatives of the PEG polymer are prepared to attach the PEG to the desired molecule. [00110] The choice of the suitable functional group for the PEG derivative is based on the type of available reactive group on the molecule that will be coupled to the PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. The /V-terminal amino group and the C-terminal carboxylic acid can also be used. [00111] The techniques used to form first generation PEG derivatives are generally reacting the PEG polymer with a group that is reactive with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates, and carbonates. In the second generation PEGylation chemistry more efficient functional groups, such as aldehyde, esters, amides, etc., are made available for conjugation.

[00112] As applications of PEGylation have become more and more advanced and sophisticated, there has been an increase in need for heterobifunctional PEGs for conjugation. These heterobifunctional PEGs are very useful in linking two entities, where a hydrophilic, flexible, and biocompatible spacer is needed. Preferred end groups for heterobifunctional PEGs are maleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids, and NHS esters.

[00113] The most common modification agents, or linkers, are based on methoxy PEG (mPEG) molecules. Their activity depends on adding a protein-modifying group to the alcohol end. In some instances polyethylene glycol (PEG diol) is used as the precursor molecule. The diol is subsequently modified at both ends in order to make a hetero- or homo-dimeric PEG- linked molecule.

[00114] Proteins are generally PEGylated at nucleophilic sites, such as unprotonated thiols (cysteinyl residues) or amino groups. Examples of cysteinyl-specific modification reagents include PEG maleimide, PEG iodoacetate, PEG thiols, and PEG vinylsulfone. All four are strongly cysteinyl-specific under mild conditions and neutral to slightly alkaline pH but each has some drawbacks. The thioether formed with the maleimides can be somewhat unstable under alkaline conditions so there may be some limitation to formulation options with this linker. The carbamothioate linkage formed with iodo PEGs is more stable, but free iodine can modify tyrosine residues under some conditions. PEG thiols form disulfide bonds with protein thiols, but this linkage can also be unstable under alkaline conditions. PEG- vinylsulfone reactivity is relatively slow compared to maleimide and iodo PEG; however, the thioether linkage formed is quite stable. Its slower reaction rate also can make the PEG- vinylsulfone reaction easier to control.

[00115] Site-specific PEGylation at native cysteinyl residues is seldom carried out, since these residues are usually in the form of disulfide bonds or are required for biological activity. On the other hand, site-directed mutagenesis can be used to incorporate cysteinyl PEGylation sites for thiol-specific linkers. The cysteine mutation must be designed such that it is accessible to the PEGylation reagent and is still biologically active after PEGylation.

[00116] Amine-specific modification agents include PEG NHS ester, PEG tresylate, PEG aldehyde, PEG isothiocyanate, and several others. All react under mild conditions and are very specific for amino groups. The PEG NHS ester is probably one of the more reactive agents; however, its high reactivity can make the PEGylation reaction difficult to control on a large scale. PEG aldehyde forms an imine with the amino group, which is then reduced to a secondary amine with sodium cyanoborohydride. Unlike sodium borohydride, sodium cyanoborohydride will not reduce disulfide bonds. However, this chemical is highly toxic and must be handled cautiously, particularly at lower pH where it becomes volatile.

[00117] Due to the multiple lysine residues on most proteins, site-specific PEGylation can be a challenge. Fortunately, because these reagents react with unprotonated amino groups, it is possible to direct the PEGylation to lower-pK amino groups by performing the reaction at a lower pH. Generally the pK of the alpha-amino group is 1-2 pH units lower than the epsilon- amino group of lysine residues. By PEGylating the molecule at pH 7 or below, high selectivity for the A-terminus frequently can be attained. However, this is only feasible if the A- terminal portion of the protein is not required for biological activity. Still, the pharmacokinetic benefits from PEGylation frequently outweigh a significant loss of in vitro bioactivity, resulting in a product with much greater in vivo bioactivity regardless of PEGylation chemistry.

[00118] There are several parameters to consider when developing a PEGylation procedure. Fortunately, there are usually no more than four or five key parameters. The “design of experiments” approach to optimization of PEGylation conditions can be very useful. For thiol-specific PEGylation reactions, parameters to consider include: protein concentration, PEG-to-protein ratio (on a molar basis), temperature, pH, reaction time, and in some instances, the exclusion of oxygen. (Oxygen can contribute to intermolecular disulfide formation by the protein, which will reduce the yield of the PEGylated product.) The same factors should be considered (with the exception of oxygen) for amine-specific modification except that pH may be even more critical, particularly when targeting the N-terminal amino group.

[00119] For both amine- and thiol-specific modifications, the reaction conditions may affect the stability of the protein. This may limit the temperature, protein concentration, and pH. In addition, the reactivity of the PEG linker should be known before starting the PEGylation reaction. For example, if the PEGylation agent is only 70 percent active, the amount of PEG used should ensure that only active PEG molecules are counted in the protein- to-PEG reaction stoichiometry.

D. Fusion Proteins

[00120] Certain embodiments of the present disclosure concern fusion proteins of the antimicrobial peptides or polypeptides. These molecules may have the peptides of the embodiments linked at the N- or C-terminus to a heterologous domain. For example, fusions may also employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Fusion proteins can comprise a half-life extender. Another useful fusion includes the addition of a protein affinity tag, such as a serum albumin affinity tag or six histidine residues, or an immunologically active domain, such as an antibody epitope, preferably cleavable, to facilitate purification of the fusion protein. Non-limiting affinity tags include polyhistidine, chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S -transferase (GST).

[00121] In a particular embodiment, the peptides or polypeptides of the embodiments may be linked to a peptide that increases the in vivo half-life, such as an XTEN polypeptide (Schellenberger et al, 2009), IgG Fc domain, albumin, or albumin binding peptide.

[00122] Methods of generating fusion proteins are well known to those of skill in the art. Such proteins can be produced, for example, by de novo synthesis of the complete fusion protein, or by attachment of the DNA sequence encoding the heterologous domain, followed by expression of the intact fusion protein.

[00123] Production of fusion proteins that recover the functional activities of the parent proteins may be facilitated by connecting genes with a bridging DNA segment encoding a peptide linker that is spliced between the polypeptides connected in tandem. The linker would be of sufficient length to allow proper folding of the resulting fusion protein.

1. Linkers

[00124] In certain embodiments, the antimicrobial peptides or polypeptides of the embodiments may be chemically conjugated using bifunctional cross-linking reagents or fused at the protein level with peptide linkers. Bifunctional cross-linking reagents have been extensively used for a variety of purposes, including preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies. Suitable peptide linkers may also be used to link the peptide or polypeptide of the embodiments, such as Gly-Ser linkers.

[00125] Homobifunctional reagents that carry two identical functional groups proved to be highly efficient in inducing cross-linking between identical and different macromolecules or subunits of a macromolecule, and linking of polypeptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By taking advantage of the differential reactivities of the two different functional groups, cross-linking can be controlled both selectively and sequentially. The bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g., amino-, sulfhydryl- , guanidine-, indole-, carboxyl-specific groups. Of these, reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis, and the mild reaction conditions under which they can be applied. A majority of heterobifunctional cross-linking reagents contain a primary amine-reactive group and a thiol- reactive group.

[00126] In another example, heterobifunctional cross-linking reagents and methods of using the cross-linking reagents are described (U.S. Pat. No. 5,889,155, specifically incorporated herein by reference in its entirety). The cross-linking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing coupling, in one example, of aldehydes to free thiols. The cross-linking reagent can be modified to cross link various functional groups.

[00127] Additionally, any other linking/ coupling agents and/or mechanisms known to those of skill in the art may be used to combine polypeptides of the embodiments, such as, for example, antibody-antigen interaction, avidin biotin linkages, amide linkages, ester linkages, thioester linkages, ether linkages, thioether linkages, phosphoester linkages, phosphoramide linkages, anhydride linkages, disulfide linkages, ionic and hydrophobic interactions, bispecific antibodies and antibody fragments, or combinations thereof.

[00128] It is preferred that a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo. These linkers are thus one group of linking agents.

[00129] In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP, and 2-iminothiolane (Wawrzynczak and Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.

[00130] Once chemically conjugated, the peptide generally will be purified to separate the conjugate from unconjugated agents and from other contaminants. A large number of purification techniques are available for use in providing conjugates of a sufficient degree of purity to render them clinically useful.

[00131] Purification methods based upon size separation, such as gel filtration, gel permeation, or high performance liquid chromatography, will generally be of most use. Other chromatographic techniques, such as Blue-Sepharose separation, may also be used. Conventional methods to purify the fusion proteins from inclusion bodies may be useful, such as using weak detergents, such as sodium N-lauroyl-sarcosine (SLS).

2. Cell Penetrating and Membrane Translocation Peptides

[00132] Furthermore, in certain aspects, the present disclosure contemplates fusing or conjugating a cell-penetrating domain (also called a cell delivery domain, or cell transduction domain) to an antimicrobial peptide. Such domains are well known in the art and are generally characterized as short amphipathic or cationic peptides and peptide derivatives, often containing multiple lysine and arginine resides (Fischer, 2007). Of particular interest are the TAT sequence from HIVl (Y GRKKRRQRRR; SEQ ID NO:7969), and poly-D-Arg and poly- D-Lys sequences (e.g., dextrorotary residues, eight residues in length). As used herein the terms “cell penetrating peptide” and “membrane translocation domain” are used interchangeably and refer to segments of polypeptide sequence that allow a polypeptide to cross the cell membrane (e.g., the plasma membrane in the case a eukaryotic cell).

[00133] Examples of CPP segments include, but are not limited to, segments derived from HIV Tat (e.g, GRKKRRQRRRPP Q (SEQ ID NO: 7970)), herpes virus VP22, the Drosophila Antennapedia homeobox gene product, protegrin I, Penetratin (RQIKIWF QNRRMKWKK (SEQ ID NO:797l)) or melittin

(GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO:7972)). In certain aspects, the CPP comprises the Tl (TKIESLKEHG (SEQ ID NO:7973)), T2 (TQIENLKEKG (SEQ ID NO:7974)), 26 (AALEALAEALEALAEALEALAEAAAA (SEQ ID NO:7975)) or INF7 (GLFEAIEGFIENGWEGMIEGWY GCG (SEQ ID NO:7976) CPP sequence.

[00134] Peptides may be modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the peptide in vivo are contemplated. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. These agents can be added either chemically during the synthesis of the peptide, or by recombinant DNA technology by methods familiar in the art. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues. In addition, nanoparticles could be used for the packaging and delivery of the peptide.

E. Peptide Delivery

[00135] A nucleic acid encoding a peptide of the present disclosure may be made by any technique known to one of ordinary skill in the art. Non-limiting examples of a synthetic nucleic acid, particularly a synthetic oligonucleotide, include a nucleic acid made by in vitro chemical synthesis using phosphotriester, phosphite or phosphoramidite chemistry and solid phase techniques such as described in EP 266,032, or via deoxynucleoside H-phosphonate intermediates as described in U.S. Patent Serial No. 5,705,629. A non-limiting example of enzymatically produced nucleic acids includes one produced by enzymes in amplification reactions such as PCR™ (see for example, U.S. Patent 4,683,202 and U.S. Patent 4,682,195), or the synthesis of oligonucleotides described in U.S. Patent No. 5,645,897. A non-limiting example of a biologically produced nucleic acid includes recombinant nucleic acid production in living cells, such as recombinant DNA vector production in bacteria (see for example, Sambrook et al. 1989).

[00136] The nucleic acid(s), regardless of the length of the sequence itself, may be combined with other nucleic acid sequences, including but not limited to, promoters, enhancers, polyadenylation signals, restriction enzyme sites, multiple cloning sites, coding segments, and the like, to create one or more nucleic acid construct(s). The overall length may vary considerably between nucleic acid constructs. Thus, a nucleic acid segment of almost any length may be employed, with the total length preferably being limited by the ease of preparation or use in the intended recombinant nucleic acid protocol.

III. Methods of Use

[00137] The present disclosure also provides methods of using the antimicrobial peptides and antimicrobial compositions of the present disclosure to prevent, inhibit or terminate the growth of at least one microbe which may include, for example, bacteria, archaea, fungi, algae, protozoa, multicellular parasites, and viruses.

[00138] In one embodiment, the compositions of the present disclosure provide antimicrobial effect to a target microbial organism and can be used to treat a disease or infection associated with the target microbial organism. An antimicrobial effect includes inhibiting the growth or killing of the target microbial organisms, or interfering with any biological functions of the target microbial organisms. In general, the compositions of the present disclosure can be used to treat a disease or infection at any place in a host, e.g., at any tissue including surfaces of any tissue or implant. In one embodiment, the compositions are used to specifically kill or inhibit bacterial target microbial organisms in body fluid (e.g., blood, sputum).

[00139] In some embodiments, compositions of the present disclosure are effective against bacteria including Gram-positive and Gram-negative cocci, Gram-positive and Gram negative straight, curved and helical/vibroid and branched rods, sheathed bacteria, sulfur- oxidizing bacteria, sulfur or sulfate-reducing bacteria, spirochetes, actinomycetes and related genera, myxobacteria, my coplasmas, rickettsias and chlamydias, cyanobacteria, archea, fungi, parasites, viruses and algae. For example, the target microbial organisms of the present disclosure include, without limitation, Escherichia coli, Candida, Salmonella, Staphylococcus, and Pseudomonas , especially Campylobacter jejuni, Candida albicans, Candida krusei, Chlamydia trachomatis, Clostridium difficile, Cryptococcus neoformans, Haempohilus influenzae, Helicobacter pylori, Moraxella catarrhalis, Neisseria gonorrhoeae, Pseudomonas aeroginosa, Salmonella typhimurium, Shigella disenteriae, Staphylococcus aureus, and Streptococcus pneumoniae. In addition, the microbial peptide composition may be used to treat chronic skin ulcers, infected acute wounds or bum wounds, infected skin eczema, impetigo, atopic dermatitis, acne, external otitis, vaginal infections, seborrhoic dermatitis, oral infections, paradontitis, conjunctivitis or pneumonia. [00140] In particular embodiments, the compositions of the present disclosure are effective against gram-negative bacteria. Gram-positive and Gram-negative cocci include, but are not limited to, Aerococcus, Enterococcus, Halococcus, Leuconostoc, Micrococcus, Mobiluncus, Moraxella catarrhalis, Neisseria (Including N. gonorrheae and /V. meningitidis), Pediococcus, Peptostreptococcus, Staphylococcus species (Including S. aureus, methicillin- resistant S. aureus, coagulase-negative S. aureus, and S. saprophyticus), Streptococcus species (Including S. pyogenes, S. agalactiae, S. bovis, S. pneumoniae, S. mutans, S. sanguis, S. equi, S. equinus, S. thermophilus , S. morbillorum, S. hansenii, S. pleomorphus, and S. parvulus), and Veillonella.

[00141] The Gram-positive and Gram-negative straight, curved, helical/vibrioid and branched rods include, but are not limited to, Acetobacter, Acinetobacter, Actinobacillus equuli, Aeromonas, Agrobacterium, Alcaligenes, Aquaspirillum, Arcanobacterium haemolyticum, Bacillus species (Including B. cereus and B. anthracis), Bacteroides species (Including B. fragilis), Bartonella, Bordetella species (including B. pertussis), Brochothrix, Brucella, Burkholderia cepacia, Calymmatobacterium granulomatis, Campylobacter species (Including C. jejuni), Capnocytophaga, Caulobacter, Chromobacterium violaceum, Citrobacter, Clostridium species (Including C. perfringens, C. tetani and C. difficile), Comamonas, Curtobacterium, Edwardsiella, Eikenella, Enterobacter, Erwinia, Erysipelothrix, Escherichia species (Including E. coli), Flavobacterium species (Including E. meninosepticum), Francisella species (Including E. tularensis), Fusobacterium (Including E. nucleatum), Gardnerella species (Including G. vaginalis), Gluconobacter , Haemophilus species (Including H. influenzae and H. ducreyi), Hafnia, Helicobacter (Including H. pylori), Herpetosiphon, Klebsiella species (Including K. pneumoniae), Kluyvera, Lactobacillus, Legionella species (Including E. pneumophila), Leptotrichia, Listeria species (Including E. monocytogenes), Microbacterium, Morganella, Nitrobacter, Nitrosomonas, Pasteurella species (Including P. multocida), Pectinatus, Porphyromonas gingivalis, Proteus species (Including E. mirabilis), Providencia, Pseudomonas species (Including E. aeruginosa, P. mallei, P. pseudomallei and E. solanacearum) , Rahnella, Renibacterium salmoninarum, Salmonella, Serratia, Shigella, Spirillum, Streptobacillus species (Including S. moniliformis), Vibrio species (Including V. cholerae and V. vulnificus), Wolinella, Xanthobacter, Xenorhabdus, Yersinia species (Including Y. pestis and Y. enter ocoliticd), Zanthomonas and Zymomonas. [00142] The clinical diseases or infections caused by Gram-positive and/or Gram negative bacteria, treatable with the present disclosure include abscesses, bacteremia, contamination of peritoneal dialysis fluid, endocarditis, pneumonia, meningitis, osteomyelitis, cellulitis, pharyngitis, otitis media, sinusitis, scarlet fever, arthritis, urinary tract infection, laryngotracheitis, erysipeloid, gas gangrene, tetanus, typhoid fever, acute gastroenteritis, bronchitis, epiglottitis, plague, sepsis, chancroid, wound and bum infection, cholera, glanders, periodontitis, genital infections, empyema, granuloma inguinale, Legionnaire's disease, paratyphoid, bacillary dysentary, brucellosis, diphtheria, pertussis, botulism, toxic shock syndrome, mastitis, rheumatic fever, cystic fibrosis, eye infections, plaque, and dental caries. Other uses include swine erysipelas, peritonitis, abortion, encephalitis, anthrax, nocardiosis, pericarditis, mycetoma, peptic ulcer, melioidosis, HaverhiU fever, tularemia, Moko disease, galls ( e.g crown, cane and leaf), hairy root, bacterial rot, bacterial blight, bacterial brown spot, bacterial wilt, bacterial fin rot, dropsy, columnaris disease, pasteurellosis, furunculosis, enteric redmouth disease, vibriosis offish, and fouling of medical devices.

[00143] Another embodiments of the present disclosure relates to administering an antimicrobial peptide provided herein in combination with an antibiotic. Antibiotics suitable for co-administration with the antimicrobial peptides disclosed herein include substances, produced synthetically or naturally, which can inhibit the growth of or kill microbial organisms. Such antibiotics include, without any limitation, b-lactam antibiotics (e.g., ampicillin, aziocillin, aztreonam, carbenicillin, cefoperazone, ceftriaxone, cephaloridine, cephalothin, cloxacillin, moxalactam, penicillin, piperacillin, and ticarcillin), amoxicillin, bacitracin, chloramphenicol, clindamycin, capreomycin, colistimethate, ciprofloxacin, doxycycline, erythromycin, fusidic acid, fosfomycin, fusidate sodium, gramicidin, gentamycin, lincomycin, minocycline, macrolides, monobactams, nalidixic acid, novobiocin, ofloxcin, rifamycins, tetracyclines, vancomycin, tobramycin, and trimethoprim.

[00144] Another aspect of the present disclosure relates to a composition comprising an antimicrobial peptide and an agent which can enhance, maintain, or facilitate the function or activity of the peptide. In one embodiment, the chemical is a protease inhibitor. The peptide is exposed to a protease-present environment where the presence of the protease may reduce the antimicrobial activity of the peptide via, for example, enzymatic degradation. The combination of a protease inhibitor and the peptide stabilizes the peptide from the protease degradation and thus enhances the activity of the antimicrobial peptide. The protease-present environment includes, for example, body fluid ( e.g ., urine, blood, serum, salvia, sputum, and mucosal fluid). The protease includes, for example, neutrophil elastase, proteinase-3, cycteine protease, metalloprotease, serine-protease, or other proteases derived from bacteria and/or hosts. The protease inhibitor includes, for example, BMF, EDTA, PMSF, benzamidine, and/or recombinant a-l antitrypsin (rAAT).

A. Disinfectant Compositions

[00145] The antimicrobial peptides of the present disclosure are useful in a variety of environments including industrial, clinical, the household, and personal care. The peptide compositions of the present disclosure for industrial, pharmaceutical, household and personal care use may comprise at least one active ingredient, of which the peptide of the present disclosure is an active ingredient acting alone, additively, or synergistically against the target microbe.

[00146] Accordingly, the antimicrobial compositions of the present disclosure may be used to form contact-killing coatings or layers on a variety of substrates including personal care products (e.g., toothbrushes, contact lens cases and dental equipment), healthcare products, household products, food preparation surfaces and packaging, and laboratory and scientific equipment. Further, other substrates include medical devices such as catheters, urological devices, blood collection and transfer devices, tracheotomy devices, intraocular lenses, wound dressings, sutures, surgical staples, membranes, shunts, gloves, tissue patches, prosthetic devices (e.g., heart valves) and wound drainage tubes. Still further, other substrates include textile products such as cacpets and fabrics, paints and joint cement. A further use is as an antimicrobial soil fumigant.

B. Pharmaceutical Compositions

[00147] The antimicrobial peptides of the present disclosure may be delivered in a pharmaceutically acceptable composition. The antimicrobial peptide(s) and any suitable carrier may be prepared for delivery in forms including solution, microemulsion, suspension or aerosol.

[00148] The antimicrobial peptides of the invention may be incorporated into a polymer, such as, for example, a polysaccharide, a glycol polymer, a polyester, a polyurethane, a polyacrylate, a polyacrylonitrile, a polyamide, a polyolefin, a polystyrene, a vinyl polymer, a polypropylene, silk, a biopolymer, and mixtures thereof. [00149] In the antimicrobial compositions of the present disclosure, the peptides are typically present in an amount of about 0.000001 to about 99%. In other embodiments, the peptides are present in an amount of about 0.001 to about 50%. In other embodiments, the peptides are present in an amount of about 0.01 to about 25%.

[00150] In the antimicrobial compositions of the present disclosure, the carrier, or mixture of carriers, is typically present in an amount of about 1 to about 99% by weight of the composition. In other embodiments, the carrier, or mixture of carriers, is typically present in an amount of about 50 to about 99% by weight of said composition. In other embodiments, the carrier, or mixture of carriers, is typically present in an amount of 75 to about 99% by weight of said composition.

[00151] Where clinical applications are contemplated, it may be necessary to prepare pharmaceutical compositions comprising proteins, antibodies, and drugs in a form appropriate for the intended application. Generally, pharmaceutical compositions may comprise an effective amount of one or more of the polypeptides of the embodiments or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases“pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one polypeptide of the embodiments isolated by the method disclosed herein, or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference. Moreover, for animal ( e.g human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.

[00152] As used herein,“pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[00153] Certain embodiments of the present disclosure may comprise different types of carriers depending on whether it is to be administered in solid, liquid, or aerosol form, and whether it needs to be sterile for the route of administration, such as injection. The compositions can be administered intravenously, intrathecally, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, mucosally, orally, topically, locally, by inhalation ( e.g ., inhalation of a nebulized formulation), by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, via a lavage, in lipid compositions (e.g., liposomes), or by other methods or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference).

[00154] The modified polypeptides may be formulated into a composition in a free base, neutral, or salt form. Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine, or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as formulated for parenteral administrations, such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations, such as drug release capsules and the like.

[00155] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an antimicrobial peptide as an active ingredient. A compound may also be administered as a bolus, electuary, or paste.

[00156] Further in accordance with certain aspects of the present disclosure, the composition suitable for administration may be provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent, or carrier is detrimental to the recipient or to the therapeutic effectiveness of a composition contained therein, its use in administrable composition for use in practicing the methods is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives, such as various antibacterial and antifungal agents, including but not limited to parabens ( e.g methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[00157] In accordance with certain aspects of the present disclosure, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption, and the like. Such procedures are routine for those skilled in the art.

[00158] In a specific embodiment of the present disclosure, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner, such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in a composition include buffers, amino acids, such as glycine and lysine, carbohydrates or lyoprotectants, such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[00159] In some aspects, a pharmaceutical formulation comprises one or more surfactants. Surfactants used in accordance with the disclosed methods include ionic and non ionic surfactants. Representative non-ionic surfactants include polysorbates such as TWEEN®-20 and TWEEN-80® surfactants (ICI Americas Inc. of Bridgewater, N.J.); poloxamers (e.g., poloxamer 188); TRITON® surfactants (Sigma of St. Louis, Mo.); sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palnidopropyl-, or(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; MONAQUAT™ surfactants (Mona Industries Inc. of Paterson, N.J.); polyethyl glycol; polypropyl glycol; block copolymers of ethylene and propylene glycol such as PLURONIC® surfactants (BASF of Mt. Olive, N.J.); oligo (ethylene oxide) alkyl ethers; alkyl (thio) glucosides, alkyl maltosides; and phospholipids. For example, the surfactant can be present in a formulation in an amount from about 0.01% to about 0.5% (weight of surfactant relative to total weight of other solid components of the formulation;“w/w”), from about 0.03% to about 0.5% (w/w), from about 0.05% to about 0.5% (w/w), or from about 0.1% to about 0.5% (w/w). However, in further aspects, a pharmaceutical formulation of the embodiments is essentially free of non-ionic surfactants or essentially free of all surfactants.

[00160] With respect to the therapeutic methods of the present disclosire, it is not intended that the administration of the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof be limited to a particular mode of administration, dosage, or frequency of dosing; the present disclosure contemplates all modes of administration, including intramuscular, intravenous, intraperitoneal, intravesicular, intraarticular, intralesional, subcutaneous, or any other route sufficient to provide a dose adequate to treat the inflammation- related disorder. The therapeutic may be administered to the patient in a single dose or in multiple doses. When multiple doses are administered, the doses may be separated from one another by, for example, one hour, three hours, six hours, eight hours, one day, two days, one week, two weeks, or one month. For example, the therapeutic may be administered for, e.g., 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more weeks. It is to be understood that, for any particular subject, specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. For example, the dosage of the therapeutic can be increased if the lower dose does not provide sufficient therapeutic activity.

[00161] The term“unit dose” or“dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the effect desired. The actual dosage amount of a composition of the present embodiments administered to a patient or subject can be determined by physical and physiological factors, such as body weight, the age, health, and sex of the subject, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic interventions, idiopathy of the patient, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance. For example, a dose may also comprise from about 1 pg/kg/body weight to about 1000 mg/kg/body weight (this such range includes intervening doses) or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 pg/kg/body weight to about 100 mg/kg/body weight, about 5 pg/kg/body weight to about 500 mg/kg/body weight, etc., can be administered. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. In some embodiments, the dosage of antigen-specific T cell infusion may comprise about 100 million to about 30 billion cells, such as 10, 15, or 20 billion cells.

[00162] While the attending physician ultimately will decide the appropriate amount and dosage regimen, therapeutically effective amounts of the one or more polypeptides as disclosed herein or a mutant, variant, analog or derivative thereof may be provided at a dose of 0.0001, 0.01, 0.01 0.1, 1, 5, 10, 25, 50, 100, 500, or 1,000 mg/kg or g/kg. A typical dosage, for example is about 0.01 to about 100 mg/kg of peptide. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test bioassays or systems.

[00163] Dosages for a particular patient or subject can be determined by one of ordinary skill in the art using conventional considerations, ( e.g . by means of an appropriate, conventional pharmacological protocol). A physician may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. The dose administered to a patient is sufficient to effect a beneficial therapeutic response in the patient over time, or, e.g., to reduce symptoms, or other appropriate activity, depending on the application. The dose is determined by the efficacy of the particular formulation, and the activity, stability or serum half- life of the one or more polypeptides as disclosed herein or a mutant, variant, analog or derivative thereof and the condition of the patient, as well as the body weight or surface area of the patient to be treated. IV. Examples

[00164] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 - Gram-Negative Bacteria Peptide-Screening Platform

[00165] The Surface Localized Antimicrobial displaY (SLAY), a high-throughput screening platform was used to rapidly identify lead antimicrobial peptides to combat multi drug resistant Gram-negative bacteria (see, e.g., International (PCT) Patent Publication No. WO 2016/176573, which is incorporated herein by reference). SLAY drives bacteria to express and self-test peptides of any size, structure, or sequence complexity for antimicrobial activity through a physiologically and therapeutically meaningful interface and provides readout of the interactions via high-throughput DNA sequencing. Using SLAY -800,000 20-mer peptides were quickly screened for antimicrobial activity and identified 7,968 new fully synthetic sequences covering an broad range of peptide physicochemical space. Selected peptides with properties far removed from CAMPs showed activity against multi-drug resistant bacteria, new potential mechanisms of action, and low eukaryotic toxicity. SLAY offers a different approach to peptide discovery and aims to advance the understanding of antimicrobial peptide chemistry that can serve to supplement our antibiotic arsenal, generate new antibiotic scaffolds, and expand the knowledge of potential antimicrobial targets to combat the spread of antibiotic- resistant bacteria.

Development of Surface Localized Antimicrobial display (SLAY).

[00166] During infection treatment, drugs first interact with a bacterium at its cell surface and then migrate to their target. To recapitulate this scenario during screening, SLAY localizes peptides on the Gram-negative bacterial cell surface as part of a fusion protein consisting of: (1) a murein lipoprotein (lpp) signal sequence that directs proteins for export from the cytoplasm and is subsequently cleaved, (2) five transmembrane domains (residues 46- 159) of the OmpA membrane protein for outer membrane localization (Georgiou et al, 1996), (3) a flexible tether that allows spatial freedom (Li et al, 2011), and (4) a C-terminal peptide the tether was engineered to extend up to 180 A from its fusion to OmpA, enabling the C- terminal peptide flexibility to interact with the growth environment, the outer membrane, and periplasmic components. With the fluid nature of periplasmic space ranging anywhere from 106 to 253 A, peptides have the potential to penetrate as far as the cytoplasmic membrane(Graham et al, 1991) (FIG. 1A).

[00167] Cecropin Pl is a well-studied CAMP that acts by binding and disrupting the structure of the bacterial outer membrane (Gazit et al, 1995). As a test case, cecropin Pl was cloned as the C-terminal peptide and the construct was expressed in wild-type E. coli K-12 strain W3110. A tandem influenza hemagglutinin peptide (2xHA) was cloned as a C-terminal peptide control. Expression was induced with increasing concentrations of IPTG and monitored optical density as an initial measure of cell growth and viability. The cultures expressing the control 2xHA peptide grew similarly at all IPTG concentrations (FIG. IB). The cultures expressing cecropin Pl showed an induction-dependent decrease in optical density (FIG. IB). Colony-forming units (CFUs) for cecropin Pl cultures were measured and found a correlative decrease in viable cells following induction (FIG. 1C). Cytosolic expression of cecropin Pl alone did not affect W3110 growth or viability (FIG. 3). The length of the flexible tether strongly influenced cecropin Pl -dependent growth effects. In addition to the full-length tether (2X), cecropin Pl with a half-length tether (IX) and no tether (OX) was also cloned. Induction of each construct at 0.1 mM IPTG showed that cecropin Pl displayed with the full 2X tether length had the strongest activity (FIG 4).

Displayed Peptides Mimic Native Interactions

[00168] To further demonstrate bacterium-relevant physiological interactions recapitulated through this approach, the 2X tether cecropin Pl construct were introduced in E. coli strain WDl0l(Trent et al, 2001). WD101 is a derivate of strain W3110 and carries a mutation that decreases its overall surface charge through the addition of amine-containing residues to lipopolysaccharide (LPS) and makes it resistant to CAMPs like cecropin Pl. Consistent with the ability the engineered system to recapitulate natural interactions, WD101 was more resistant to antimicrobial activity of surface expressed cecropin Pl compared to the parent CAMP sensitive strain W3110 (FIG. ID). Furthermore, deletion of the eptA gene, which is required for the LPS modification conferring CAMP resistance, sensitized WD101 to surface displayed cecropin Pl (Herrera et ctl, 2010). The action of peptides displayed by the platform is also sensitive to relevant environmental conditions. CAMP activity is decreased by the addition of magnesium ions that fortify bacterial cell surfaces and are also sensitive to trypsin degradation due the large numbers of arginine and lysine residues they contain. Addition of up to 2 mM magnesium to the growth medium greatly reduced the antimicrobial action of surface-displayed cecropin Pl, and addition of trypsin to the culture medium greatly lessens cecropin Pl -induced growth effects (FIG. 5).

SLAY Allows Functional Display of Cyclic Peptides in a Broad Range of Gram-Negative Bacteria.

[00169] In addition to cecropin Pl, antimicrobial peptides dermaseptin, protegrin 1, and defensin HNP-l showed strong antimicrobial activity against W3110 in the system (FIG. IE, 6). Defensin HNP-l and protegrin 1 were particularly interesting since they require disulfide bonds for activity. Defensin HNP-l was reconstructed without disulfides and demonstrated that its activity was dramatically reduced, in agreement with biochemical studies (Varkey and Nagaraj, 2005). This indicates that the system supports the formation of cyclic, disulfide bond-dependent antimicrobial peptides.

[00170] To ensure the application of the system in a wide range of Gram-negative bacteria engineered expression and replication of ubiquitous OmpA surface localization on a broad RSF1010 origin-based plasmid. Without any change to the system it was demonstrated that the system was transferable and functional in a broad range of Gram-negative bacteria including ESKAPE pathogens like Acinetobacter baumannii and Pseudomonas aeruginosa (FIG. IF)

Demonstration of SLAY Batch Screening with Defined Peptide Library.

[00171] While the full-length tether (2X) is required for the spatial freedom of a peptide to interact with its host bacterium, it is short enough to prevent an antimicrobial peptide from exerting an effect on neighbor cells in culture. W3110 surface expressing cecropin Pl, which shows potent CAMP activity, was co-cultured with W3110 containing only the empty plasmid pMMB67EH. When co-cultured in a 1 :1 ratio and induced, cecropin Pl only affects the viability of cells expressing it (FIG. 1G). Thus, multiple peptides can be assayed for activity in a single tube. [00172] The screening workflow for SLAY is shown in FIG. 2. Peptides are cloned into our surface display system and transformed into a Gram-negative strain of interest. Peptide surface expression is then induced by IPTG. Bacteria expressing bactericidal or bacteriostatic peptides will decrease in abundance during the induction period. One PCR reaction generates Illumina next-generation sequencing samples for sequencing from plasmid libraries pre- and post-induction. In silico translation and comparison identifies each peptide in the library and its abundance pre- and post-induction to identify potential antimicrobial hits.

[00173] To validate SLAY, a small library of three antimicrobial peptides and two control peptides (TABLE 1) were transformed into E. coli then pooled, induced, and harvested at 0 (input), 2, 3, and 4 hours. Following next-gen library construction and sequencing, reads were normalized to the input counts (FIG. 8). Log2 fold values, reported in TABLE 1, indicate the degree to which the peptides were removed from the population. Control peptides 2xHA and defensin HNP-l cysteine mutant showed a near neutral log2 fold change over the time course examined. Meanwhile, the remaining antimicrobial peptides show a log2 fold change of -1 or lower indicating they were removed from the population over the time course. From these data, protegrin 1, cecropin Pl, and defensin HNP-l were determined to have effective antimicrobial activity against the E. coli strain with protegrin 1 exhibiting the strongest activity. Indeed, minimal bactericidal concentration (MBC) assays using synthesized peptides showed correlative bactericidal activity with log2 fold values, with MBCs of <0.125 mM, 1 mM and 8 pM measured for protegrin 1, cecropin Pl and defensin HNP-l, respectively (TABLE 1).

SLAY Identifies New Antimicrobial Sequences from a Massive Random Pool.

[00174] The breadth of peptide chemical space with antimicrobial and potential therapeutic value is likely much larger than current screening approaches allow to be assessed. To test this hypothesis, SLAY was applied to screen a massive and unbiased peptide library of fully random sequences for antimicrobial activity. A library of approximately 800,000 peptides encoding random 20-mer sequences was constructed in E. coli W3110. This library was sequenced and then a sequence logo based on all peptides in the library was generated (FIG. 10). A sequence logo based on a computationally generated random 20-mer peptide library was also generated (FIG. 10). The sequence logo generated in both analysis was nearly identical indicating our -800,000 peptide library did indeed contain a random assortment of 20-mer sequences. [00175] Library samples were collected pre-induction and post four-hour induction with O. lmM IPTG in duplicate. Sequencing read counts are listed in TABLE 2. Peptides were taken through two triage stages to identify hits with a high likelihood of true activity. Peptides were first sorted by their log2 reduction values. Peptides with a significant decrease of at least log2 fold -1 were considered to be depleted from the input library and to have potential antimicrobial activity. Next, peptides that had less than 50 reads in each replicate were removed. While somewhat arbitrary, samples with fewer reads will be more affected by machine errors than those with larger read counts so removing these decreases the overall noise in the analysis. As anticipated from screening a random library the vast majority (98.3%) of these 20-mer sequences showed no depletion following induction indicating these peptides had no antimicrobial activity (FIG. 9A). However, due to the massive throughput of SLAY, the 1.7% of the peptide library that did show depletion and potential antimicrobial activity representing 7,968 new peptides. This single screen nearly doubled the number of unique antimicrobial peptides reported in six publicly available databases (Hammami el al, 2009; Novkovic et al, 2012; Piotto et al, 2012; Waghu et al, 2016; Wang et al, 2016; Zhao et al, 20l3b).

SLAY Reveals Untapped Chemical Diversity of Antimicrobial Peptides.

[00176] Natural antimicrobial peptides are dominated by cationic and amphipathic composition. To begin to explore the range of hits identified by SLAY in the context of currently known antimicrobial chemistry, each active and inactive peptide from the screen were plotted by their charge and hydrophobicity. On average, the chemical composition of the library was centered near neutral charge and neutral hydrophobicity (FIG. 9B). Remarkably, no bias was observed in these parameters between inactive and active sequences with the bulk of both peptide populations centered near neutral charge and neutral hydrophobicity (FIG. 9B). Active sequences did not show a propensity towards any specific charge or hydrophobic character. This lack of selection is in sharp contrast to the bulk of naturally occurring antimicrobial peptides in current databases, which are dominated by positive charge and hydrophobic character (FIG. 9C). Comparing amino acid frequency further highlights these observations (FIG. 9D). When examining the library little enrichment of any specific amino acid was observed in active vs. inactive sequences. Meanwhile positively charged lysine was found at a much higher frequency in known antimicrobial peptides compared to active sequences from this screen. Hydrophobic residues including alanine, isoleucine, leucine, valine, were also more frequent in known antimicrobial peptides compared to the active sequences from this screen. This initial analysis indicates that SLAY explores antimicrobial peptide chemical space well beyond what is currently known.

[00177] To further explore the composition of active sequences identified in this screen and identify possible sequence patterns, a clustering analysis based on amino acid side chain properties was performed to identify subclasses of peptide sequences that may be present in the hits. To facilitate this analysis, the amino acid sequence were first simplified such that all the amino acids except glycine, proline, and cysteine were grouped into the broad categories of hydrophobic, polar uncharged, positively charged, and negatively charged amino acids. Glycine, proline, and cysteine were left as their own groups due to their unique biochemical properties and effects on peptide structure and functionality (Ageitos et al, 2016; Krauson el ah, 2015). This simplification of peptide sequences was used due to the diversity of the hits, which hindered clustering of non-simplified peptide sequences. All sequences that were shorter than 10 amino acids were also excluded. The pairwise Levenshtein edit distances between all remaining simplified sequences was then calculated. The edit distances were generally found to be high, even for simplified sequences (min = 2, median = 13, max = 21). The hierarchical clustering were performed and sub-divided the peptides into 79 subgroups, applying an arbitrary distance cutoff of less than 17 among all sequences within one cluster (FIG. 11). To confirm that the cutoff for the subgroupings was reasonable, a multiple sequence alignment on the simplified sequences was performed on each subgroup using Clustal W. This analysis further supports that our screen identified a diverse set of antimicrobial hits and that SLAY offers a path for exploring previously unrecognized and intractable regions of antimicrobial chemical space. For example, the group sizes ranged from 4 to 523 peptides with a median of 52 peptides. Some groups were observed to have a simplified consensus sequence with an apparent hydrophobic domain in addition to variable domains. From the variance in cluster sizes and in the simplified consensus sequences, outlined in Supplemental Dataset 1, it is evident that the peptides discovered in this screen are extremely diverse and represent a vast potential for research into new antimicrobial peptides. These results further support that active antimicrobial sequences exist in a much wider range of peptide chemical space than previously recognized that extends far beyond what has evolved in nature.

SLAY Hits are Active in Synthetic Form. [00178] To validate the hits, 22 peptides were selected based on chemical composition, predicted aqueous solubility (Pepcalc.com, Innovagen), and clustering diversity for chemical synthesis and antimicrobial activity testing. This included two cationic peptides, Pl and P2, that were selected to show SLAY can identify antimicrobial sequences reminiscent of naturally occurring CAMPs. In contrast, the remaining peptides (P3-P18) were selected for opposing characteristics with low hydrophobicity and neutral to negative charge. These sequences were chosen to test if SLAY could identify peptide chemistry not typically associated with antimicrobial activity. One control peptide (Cl) that had a neutral log2 fold reduction in the screen was used. These peptide sequences that were synthesized and tested for antimicrobial activity are listed in TABLE 3.

[00179] Antimicrobial activity were tested against the host strain used in the screen ( E . coli W3110) and three multi-drug resistant strains: Acinetobacter baumannii (Ab 5075), Pseudomonas aeruginosa (PA14), and E. coli conferring New-Delhi metallo-beta-lactamase (NDM) resistance. Antimicrobial peptide activity is highly sensitive to medium conditions (Friedrich et al, 1999; Giacometti et al, 2000; Schwab et al, 1999). Minimal inhibitory concentration (MIC) assays by standard methods using Mueller-Hinton medium were first performed. Cationic peptides, Pl and P2 showed robust activity, like that of our standard CAMP cecropin Pl (TABLE 4). Peptides P3-P23 did not show activity in this standard medium. Antimicrobial activity were next assayed using a simple and defined Tris based medium. Since the bacteria did not grow robustly in this medium the minimal bactericidal concentration (MBC) of each peptide was assayed. In this medium, cationic peptides Pl and P2 had potent antibacterial activity, with minimal bactericidal concentration (MBC) values of less than 2 mM for Pl for all bacteria tested. Peptides P3-P18 had activity against the strain W3110 except for P3 and P5. Both peptides P3 and P5 contained cysteine residues suggesting possible cyclic formation is needed for activity. P5 contains two cysteine residues within its sequence, while P3 contains four. P5 was then synthesized as a cyclic peptide with a disulfide bond and retested its activity. This cyclic analog of P5 exhibited much higher antimicrobial activity, with MBC changing from >128 pM to <2-8 pM. Similarly, a cyclic configuration of P3 with disulfides C2-C19 and C8-C17 was tested and its antimicrobial activity increased from MBC of 128 pM to <2-4 pM. This further reiterates that SLAY can screen and select for cyclic peptides. Peptides P19-P22 as well as the control peptide Cl did not show activity in any medium tested (TABLE 3). Thus, 18 of 22 (-80%) sequences identified by SLAY as active showed antimicrobial activity in at least one medium indicating a high true-positive rate. [00180] Cationic-hydrophobic peptide Pl showed universal activity, which is commonly associated with non-specific CAMP activity. Interestingly, P2, which is cationic but non-hydrophobic, showed a larger range of activity. Furthermore, many of the atypical, non- cationic, non-hydrophobic peptides (P3-P18) showed varying ranges in activity across the four Gram-negative bacteria tested. For example, P6, P8, P13, and P16 showed antimicrobial action against some strains while having no activity (>128 mM) against others. This suggests many of the peptides may act through a more targeted mechanism.

SLAY Hits Present with New Potential Mechanisms of Peptide Action.

[00181] Traditional antimicrobial peptides are considered to act non-specifically through membrane disruption with extremely rapid killing. In addition to the new chemical landscape SLAY provides access to, without wishing to be bound by any theory, it is believed that peptides identified by SLAY might also act through new mechanisms of peptide action. Defining the target(s) and mechanism(s) of antibiotic action is challenging and is still debated for many clinically used antibiotics(Dwyer e/a/., 2015; Miller et al, 2016; Trimble et al. , 2016; Zipperer et al. , 2016). To begin to explore the mechanism of action of peptides identified by SLAY their pore-forming activity and killing kinetics were compared to the traditional CAMP cecropin PL Peptide-dependent membrane damage is commonly assayed with propidium iodide (PI), which penetrates cells with compromised membranes to stain nucleic acids (Belloc et al, 1994; Darzynkiewicz et al, 1997). The effect of peptides on E. coli was probed by incubating peptide-treated cells with PI followed by flow cytometry analysis to determine peptide-induced membrane damage as previously described(Zhang et al. , 2016) (FIG. 12A). As a known pore-forming peptide, treatment of E. coli with cecropin Pl resulted in 33.7% of the population staining Pi-positive indicating membrane damage. Cationic peptides Pl and P2 identified by SLAY exhibited even stronger membrane damage compared to cecropin Pl, with 85.3% and 71.6% Pi-positive cells respectively. Remarkably, peptides P3-P18 identified in the screen that contained atypically antimicrobial amino acid compositions compared to known CAMPs, did not cause cell fluorescence over 4%, with majority under 1%. This indicates that peptides P3-P18 identified through SLAY do not damage bacterial membranes, suggesting these peptides kill bacteria via alternative mechanism(s) of action.

[00182] The mechanism of SLAY peptides was further probed with time-dependent killing assays. While membrane-targeting CAMPs kill rapidly, antimicrobials targeting specific cellular processes tend to elicit their effect over a long period of time(Yang et al. , 2006). The top five non-cationic peptides, P3 cyclic, P4, P5 cyclic, P6, and P7 were selected for testing. All peptides were assayed at 4X MBC. In the time-kill assay, cecropin Pl killed >99.9% of bacteria in less than 30 minutes (FIG. 12B). In contrast, the selected peptides acted over a much longer time period. Peptides P3, P4, and P5 acted over 12 hours, while P6 and P7 acted over 18 hours. Combined with their non-pore forming action, these results suggest that peptides identified through SLAY may represent non-pore forming and diverse mechanism of action.

[00183] Hemolysis is a known off-target effect of CAMPs, with peptides such as protegrin-l showing marked hemolysis at therapeutically relevant concentrations(Edwards el ah, 2016). The hemolytic activities of the peptides against human red blood cells were determined as an indication of their toxicity towards mammalian cells. The hemolytic activities of all peptides are summarized in FIGURE 12C. PBS was used as a negative control and 1% triton was used as a positive control for 100% lysis. None of the peptides P3-P18 identified in the screen exhibited notable hemolytic activity, with all well under 20% hemolysis.

5

Example 2 - Materials and Methods

[00184] Bacterial strains, plasmids, growth conditions, and antibiotics. Strains were grown at 37°C in Luria Bertani (LB) broth/agar. The antibiotics carbenicilbn 75 pg/mL or 150 pg/mL were added for selection as needed for E. coli, A. baumannii, or P. aeruginosa, respectively.

[00185] Growth curves. Strains were grown overnight at 37°C. The following day cultures were inoculated and grown to log phase. The cultures were then back diluted to OD 600nm 0.01. IPTG was added to the cultures where appropriate. Data points were collected every 20 mins over a 6 hour period using a SpectraMax Plus384 absorbance microplate reader with SOFTmax Pro v6.2.2 software.

[00186] Mixed-culture assay. Strains were grown overnight at 37°C. The following day cultures were inoculated and grown to log phase. The cultures were then back diluted to OD 600nm 0.01 in 5 mLs of LB containing 75 pg/mL carbenicillin. Mixed cultures were at 1 : 1 ratio and total OD 0.01. 1 mM IPTG was added to the cultures. Cultures were serial diluted and spotted on plates containing carbenicillin 75 pg/mL with 80 pg/mL X-gal at 0 and 3 hours.

[00187] Peptide library construction. The surface display system was constructed on the broad host plasmid pMMB67EH. Random 20-mer peptides were cloned into the Kpnl and Sall sites using primers with homology to the tether sequence and a 60-base random nucleotide segment on the reverse primer. The library was then transformed into C2987 competent cells (NEB) in batch and plated. Roughly 800,000 colonies were plated and pooled. Cells were harvested and aliquoted into glycerol stocks. Plasmid DNA was isolated from the library and re-transformed into the E. coli W3110 strain at 3 to 5 times coverage. Colonies were collected and frozen.

[00188] Library screen and build. An aliquot of the frozen library was thawed and added to lOml of LB supplemented with carbenicillin 75 pg/mL for growth, shaking at 37°C for about 1 hour. The culture was then back diluted into 5 mL LB with carbenicillin 75 pg/mL to OD 600nm 0.01 supplemented with 0.1 mM IPTG. The remaining culture was collected as the“Input” sample. Induced cultures were allowed to grow, shaking at 37°C. Cells were harvested after 2, 3, and 4 hours for the defined peptide library and harvest after 4 hours for the random peptide library. Plasmids were isolated from each culture using the Zyppy Plasmid Miniprep kit from Zymo Research Corp. (Irvine, CA). Samples were collected in duplicate. Plasmid concentrations were measured using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). Primers with homologous regions to the plasmid were used to amplify and attach adaptors for sequencing. Briefly, 10 ng plasmid DNA and 1 pl of 10 mM primer mix [2 pL of forward primer and 2 pL of reverse primer diluted with 16 pL dFEO] were added to a 2* master mix of Phusion high-fidelity polymerase (NEB) in a total volume of 50 pL. Four reactions per sample were run for a total of 12 cycles. The reactions were pooled and cleaned using Zymo DNA Clean and Concentrator (Irvine, CA). The complete libraries were further gel purified and extracted using the Zymoclean Gel DNA Recovery kit (Irvine, CA). The defined peptide library was sequenced using Illumina Mi-seq. The random peptide library was sequenced using Illumina Hi-seq supplemented with Phi-X. DNA was sequenced at The University of Texas Genomic Sequencing and Analysis Facility.

[00189] Read trimming and counting. Flexbar (Dodt et al. , 2012) was used to trim the reads of excess sequence. To do this, a known sequence was searched for as part of the peptide display sequence, “CACCGGCAGCCGGTATC,” (SEQ ID N0:8004) and then retained 73 nucleotides downstream of this sequence. This allowed retention of the test peptide sequence, as well as a preceding‘GG’ motif and ending stop codon for each read. Next, ustacks (from the stacks computational pipeline (Catchen et al. , 2013; Catchen et al. , 2011)) was used to consolidate reads that originated from a particular nucleotide sequence. This allowed collection of reads for each tested peptide as well as reads with <2 nucleotide mismatches. The nucleotide sequences for each nucleotide sequence was translated into an amino acid sequence with a custom python script using the Biopython (Cock et al. , 2009) library, and then summed the total reads for each unique peptide. To reduce false positives, only peptides that started with the expected‘GG’ motif were retained and were present in both input libraries.

[00190] Differential abundance analysis. A file containing each peptide sequence and the total number of reads in each library was then used as input for the DESeq2(Love et al. , 2014) R/Bioconductor(Huber et al. , 2015) package. DESeq2 is a commonly used R/Bioconductor package for count based differential testing with next generation sequencing data. A standard DESeq2 workflow, which includes read count normalization, peptide dispersion estimates, and Wald tests for significance of differential abundance was used. For each peptide, the total abundance (normalized reads) in the induced (IPTG) libraries were compared to the abundance in the input libraries, resulting in a log2 FoldChange (log2FC) and p-value for each peptide. P-values were adjusted (padj) for multiple testing using Benjamini- Hochberg correction as part of the standard DESeq2 workflow detailed in the package manual (available here: www.bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.pdf).

Peptides with lfcMLE < or = -1 were considered active (7,968 peptides). Peptides with lfcMLE > -1 were considered inactive. Peptides were considered removed if the initial reads in either replicate of less than 50. Generally, peptides that were selected for further experimental validation had a log2FC < -1 and padj < .05.

[00191] Calculating Peptide Properties. After calculating the change in abundance for each peptide, we computed expected properties for each peptide sequence using a custom R script. After removing the preceding‘GG’ motif and trailing stop codon, the“Peptides” R package (cran.r-project.org/package=Peptides) was used to calculate charge and hydrophobicity for the remaining 20 amino acids. Specific methods used to calculate charge (Nelson and Cox, 2004) and hydrophobicity (Kyte and Doolittle, 1982) are detailed in the Peptides package documentation. The calculated properties (hydrophobicity and charge) of the screened peptides were compared with those of 8685 known antimicrobial peptides from available online databases (Hammami et al, 2009; Novkovic et al, 2012; Piotto et al, 2012; Waghu et al, 2016; Wang et al, 2016; Zhao et al, 20l3b).

[00192] Logo Plots. Logo plots were generated using R package“RWeblogo” (Omar Wagih (2014). RWebLogo: plotting custom sequence logos. R package version 1.0.3. CRAN.R-project.org/package=RWebLogo), a programmatic interface to make sequence logos (Crooks et al, 2004; Schneider and Stephens, 1990). Briefly, sequence logos were generated from either the entire set of possible killing peptides (7,968 sequences), 10,000 randomly sampled sequences of the total library, or an amino acid translation of 10,000 randomly generated nucleotide sequences of a repeated“NNB” motif. All logos are plotted in units of probability.

[00193] Amino acid frequency. Individual amino acid frequencies were determined for each peptide (simply # of amino acids/length of peptide). Then average frequencies were calculated per group, which is what is graphed. The error bars represent the SEM (standard error of the mean) and the asterisks correspond to Bonferroni adjusted p-values (*, **, and *** denote p-value <0.05, <0.01, and <0.001 respectively) derived from Tukey's range test performed in conjunction with an ANOVA. [00194] Clustering Analysis. The clustering analysis was conducted on the 7,968 peptides with at least a -1 log2 fold depletion from the antimicrobial peptide screen. These peptides were then screened for those that were 10 amino acids or longer and continued with the clustering the resulting 5,733 peptides that fit into this group. This was done to limit inaccurate clustering that could result from biochemically unrepresentative edit distances due to large differences in peptide length. With these 5,733 peptides, their amino acid sequence were then simplified such that all the amino acids except glycine, proline, and cysteine were grouped into the broad categories of hydrophobic (Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp), polar uncharged (Ser, Thr, Asn, Gln), positively charged (Arg, His, Lys), and negatively charged (Asp, Glu) amino acids. Glycine, proline, and cysteine were left as their own groups do to their unique biochemical properties and how they can affect peptide structure and functionality. This simplification of peptide sequences was used due to the incredible diversity of the hits which hindered clustering of non-simplified peptide sequences. A Levenshtein distance for every pairing of peptides in this list of 5,733 simplified peptide sequences were then acquired. These distances were then used as the edit distance inputs for a complete-linkage hierarchal clustering analysis utilizing R’s hclust command. The resulting clustering dendrogram was then arbitrarily sub-divided into 79 subgroups representing different groups of similar peptides identified in this analysis. To check to see if the cutoff for subgroups was reasonable and whether any patterns could be identified in the groupings multiple sequence alignments for each group’s simplified sequence was generated using the R package msa. The multiple sequence alignment used was Clustal W with default settings. The consensus sequence for a group is made up of amino acids with presence in at least 50% of the sequences for a given position.

[00195] Antimicrobial activity. The antibacterial activities of the peptides against four Gram-negative strains were measured as previously described (Mah, 2014; Qaiyumi, 2007). Briefly, strains were grown overnight on an LB agar plate at 37°C. A small amount of bacteria was scraped from the plate and added to LB and grown to log phase. Cells were collected and washed twice in 20 mM Tris (pH 7.4), 50 mM NaCl. Cells were diluted to 1 x 10⁶ CFU/mL and 50 pL were added to each well in a polypropylene 96-well plate (Coming Inc., Lowell, MA, USA). Peptides with >90% purity were synthesized by Genscript (GenScript USA Inc., NJ). Synthesized peptides were diluted into 0.2% BSA, 0.01% acetic acid solution to 256 pM and serial diluted for a total volume of 100 pL of each dilution. Then, 50 pL of each peptide solution was added to 50 pL of cells. Plates were parafilmed and incubated at 37°C overnight. After 20 hours, each well was spotted onto LB agar to assess cell viability. MBCs were determined where cells had a 3-log reduction in growth. MIC assays were performed as reported previously (Wiegand el al, 2008). Briefly, strains were grown overnight on an LB agar plate at 37°C. A small amount of bacteria was scraped from the plate and added to Mueller- hinton growth media and grown to log phase. Cells were diluted to 1 x 10⁶ CFU/mL and 50ul were added to each well in a polypropylene 96-well plate. Synthesized peptides were diluted into 0.2% BSA, 0.01% acetic acid solution to 64 mM and serial diluted for a total volume of 100 pL of each dilution. Then, 50 pL of each peptide solution was added to 50 pL of cells. Plates were parafilmed and incubated at 37 °C overnight. The MIC was determined by OD600nm where cell density was 0.

[00196] Peptide-induced membrane permeability. Bacterial cell membrane damage and pore formation induced by the peptides was examined by detection of propidium iodide (PI) influx(Zhang el al, 2016). The bacteria were cultured at 37 °C to mid-log phase and then diluted to OD600 0.1 in 10 mM Tris (pH 7.4), 25 mM NaCl. Synthesized peptides, at a concentration of 25 pM, were added to a 500 pL bacterial suspension and incubated for 30 min. Bacteria were collected and resuspended in buffer. PI solution was added to a final concentration of 2 pg/mL. The fluorescence signal in treated cells was determined by flow cytometry (BD Accuri) and further analyzed with FlowJo (Treestar, USA).

[00197] Hemolytic activity of the peptides. Hemolytic assays were performed as described previously(Zhao el al. , 20l3a). Briefly, 50 pM solutions of synthesized peptides were prepared by mixing the peptides by inversion in 10 mM PBS at pH 7.4 for a total volume of 0.5 mL. A human red blood cell solution was made by washing 0.4 mL of the red blood cells twice with 7 mL of PBS by centrifugation at 2500 rpm for 10 minutes. The precipitates were then resuspended in 4 mL of PBS. Hemolytic activity of the peptides was measured by first mixing by inversion the 0.5 mL peptide solutions with 0.4 mL of the human red blood cell solution. The mixtures were placed in a 37°C water bath for 1 h. A negative control of 0.5 mL PBS plus 0.4 mL human red blood cell solution and a positive control of 1% (w/v) Triton X- 100 plus 0.4 mL of human red blood cell solution were also incubated in the water bath. After one hour, the samples were centrifuged at 2500 rpm for 10 minutes. The absorbance of the supernatant was measured at 540 nm. The percent hemolysis was calculated using the following equation. % hemolysis= (absorbance_Sampie - absorbancenegative) / (absorbancepositive) x 100 [00198] Time-kill analysis assay. Kinetics assays were set up identically to the MBC assay with the following exceptions. A total volume of 200 pL was added to 96-well plates in triplicate. At time points of 30 min, 1, 3, 6, 9, 12, 18, and 24 hours, 15 pL of sample was removed from each well. Aliquots were serial diluted and plated to assess viability. * * *

[00199] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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

Claims

WHAT IS CLAIMED IS:

1. An isolated peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1- 7968.

2. The peptide of claim 1, wherein the peptide essentially consists of an amino acid sequence of any one of SEQ ID NOs: 1-7968.

3. The peptide of claim 1, wherein the peptide consists of an amino acid sequence of any one of SEQ ID NOs: 1-7968.

4. The peptide of any one of claims 2-3, wherein the peptide further comprises no more than 5 additional amino acids on either end of the peptide.

5. The peptide of any one of claims 2-3, wherein the peptide further comprises no more than 10 additional amino acids on either end of the peptide.

6. The peptide of claim 1, wherein the peptide exhibits antimicrobial activity.

7. The peptide of claim 6, wherein the antimicrobial activity is further defined as antimicrobial activity against a gram-negative bacterial strain.

8. The peptide of claim 6, wherein the antimicrobial activity is further defined as antimicrobial activity against a multi-drug resistant bacterial strain.

9. The peptide of claim 1, wherein the peptide is less than 50 amino acids in length.

10. The peptide of claim 1, wherein the peptide is 15 to 25 amino acids in length.

11. The peptide of claim 1, wherein the peptide is 19 to 21 amino acids in length.

12. The peptide of claim 1, wherein the peptide is 20 amino acids in length.

13. The peptide of claim 1, wherein the peptide is further defined as a linear peptide.

14. The peptide of claim 1, wherein the peptide is further defined as a cyclic peptide.

15. The peptide of claim 14, wherein the cyclic peptide comprises one or more disulfide bonds.

16. The peptide of claim 1, further comprising a cell penetrating peptide (CPP).

17. The peptide according to any one of claims 1-16, wherein the N terminus or the C terminus has been chemically modified.

18. The peptide of claim 17, wherein the chemical modification results in the peptide having a reduced susceptibility of enzymatic cleavage.

19. The peptide of claim 17, wherein the N terminus has been chemically modified.

20. The peptide of claim 17, wherein the C terminus has been chemically modified.

21. The peptide according to any one of claims 17-20, wherein both the N terminus and the

C terminus have been chemically modified.

22. A polypeptide multimer comprising at least two peptides according to any one of claims

1 21

23. The multimer of claim 22, wherein a first peptide of the at least two peptides is essentially identical to a second peptide.

24. The multimer of claim 22, wherein a first peptide of the at least two peptides is not identical to a second peptide.

25. A pharmaceutical composition comprising a peptide of any one of claims 1-21 and a pharmaceutically acceptable carrier.

26. The composition of claim 25, wherein the pharmaceutical composition is formulated for oral, intravenous, intraarticular, parenteral, enteral, topical, subcutaneous, intramuscular, buccal, sublingual, rectal, intravaginal, intrapenile, intraocular, epidural, intracranial, or inhalational administration.

27. A polynucleotide comprising a nucleic acid sequence encoding the peptide of any one of claims 1-21.

28. A method of treating a microbial infection in a subject comprising administering to the subject an effective amount of a peptide of any one of claims 1-21.

29. The method of claim 28, wherein the microbial infection was caused by a bacterium, a fungus, a virus, or a protozoan.

30. The method of claim 28, wherein the microbial infection was caused by a bacterium.

31. The method for claim 30, wherein the bacterium is a gram-negative bacterium.

32. The method of claim 31, wherein the gram-negative bacterium is Acinetobacter baumannii or Pseudomonas aeruginosa.

33. The method of claim 30, wherein the bacterium is a multi-drug resistant bacterium.

34. The method of claim 28, wherein the subject is a human.

35. The method of claim 28, wherein the subject has shown resistance to one or more antibiotics.

36. The method of claim 28, wherein the peptide is administered orally, enterically, topically, intravenously, intraperitoneally, intramuscularly, endoscopically, percutaneously, subcutaneously, regionally, or by direct injection.

37. The method of claim 36, wherein the orally administered peptide is a capsule or tablet.

38. The method of claim 37, wherein the capsule or tablet is enterically-coated.

39. The method of claim 28, further comprising administering at least a second therapeutic agent.

40. The method of claim 39, wherein the second therapeutic agent is an antibiotic or a protease inhibitor.

41. The method of 40, wherein the antibiotic is a b-lactam antibiotic, amoxicillin, bacitracin, chloramphenicol, clindamycin, capreomycin, colistimethate, ciprofloxacin, doxy cy cline, erythromycin, fusidic acid, fosfomycin, fusidate sodium, gramicidin, gentamycin, lincomycin, minocycline, macrolides, monobactams, nalidixic acid, novobiocin, ofloxcin, rifamycins, tetracyclines, vancomycin, tobramycin, and/or trimethoprim.

42. A method of disinfecting a surface comprising applying to said surface an effective amount of a composition comprising at least one peptide according to any one of claims 1-16.

43. An antimicrobial disinfecting solution comprising a peptide according to any one of claims 1-16 and an acceptable carrier.