Antimicrobial peptides

Abstract

Antimicrobial peptides (AMPs) exhibiting broad spectrum antimicrobial activity are described. Such peptides are useful in treating or preventing infections and other conditions, and are of special interest for treating antibiotic-resistant bacterial pathogens, viruses, fungi, and other pathogens, or for anti-cancer applications. Treatment or prevention of infection in humans or animals is described, such as for use in poultry and other livestock applications. Uses and methods are described for disinfecting or prevention of growth of microbes on a surface, a material, or in an environment, such as for environmental remediation.

Description

ANTIMICROBIAL PEPTIDESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. provisional patent application number 63 / 709,707 filed October 21, 2024, and entitled ANTIMICROBIAL PEPTIDES which is incorporated by reference herein in its entirety.BACKGROUND

[0002] The present disclosure relates generally to antimicrobial peptides for the treatment or mitigation of disease.

[0003] There is a need for peptides and pharmaceutical compositions thereof which are useful as therapies for microbial infections or as chemopreventative agents to slow or arrest the progression of microbial infections.

[0004] Use of antibiotics in livestock may have direct and indirect impact on medical use in addressing human disease. The ubiquitous use of antibiotics in all industries has contributed to the emergence of superbugs which have become resistant to the most common antibiotics. Some strains illustrate multi-drug resistance, which is a global concern. Although the search for new antibiotic approaches continues in earnest to address challenges in both human and animal health.

[0005] Consumers have concerns about the use of prophylactic antibiotics due to the potential environmental impact, increasing drug resistance, and the possible consumption of antibiotic-laced meat, egg, or dairy products. Restrictions on prophylactic antibiotic use in livestock that have been implemented to address these concerns, but have downstream consequences such as increased rates of animal infections, leading to productivity loss due to the increase disease burden. Sick animals that are then treated with antibiotics will continue to contribute to potential drug resistance. Poultry and swine raised in close quarters are particularly susceptible to the rapid spread of disease. Different approaches to reducing infections disease in livestock animals are under development, including investigation of new antibiotic approaches, and development of vaccines. While small molecule drugs have conventionally been used, antimicrobial peptide and polypeptide therapeutic approaches are also under consideration.

[0006] International Patent Publications WO 2020 / 118427 A1 (Birol et al.) and WO 2024 / 178484 A1 (Birol etal.) describe antimicrobial peptides.

[0007] It is desirable to find new antimicrobial approaches to reduce the onset and spread of disease in humans and animals.SUMMARY

[0008] Peptides and / or amino acid sequences with antimicrobial properties are described herein. A process for use as an antimicrobial peptide (AMP) sequence generation tool is described, for de novo AMP design.

[0009] A bioinformatics approach, starting with sequences exhibiting effect, and making strategic modifications thereto, has led to the discovery of antimicrobial peptides. In abioinformatics approach, sufficient similarity among sequences can be maintained so as to permit functional equivalency. Sequences similar to isolated sequences from which a consensus is derived are also described. Such similar sequences contain conserved amino acid substitutions and a limited number of non-conserved modifications.

[0010] It is an object of the present disclosure to provide antimicrobial peptides, which may obviate or mitigate at least one disadvantage of previous antimicrobial approaches.

[0011] There is described herein an antimicrobial peptide comprising an amino acid sequence according to any one of SEQ ID NO:258 (CCH33), SEQ ID NO: 1-257, and SEQ ID NO:259-361, or a variant thereof having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or having 100% amino acid sequence identity thereto.

[0012] Further, there is described herein a composition comprising the described antimicrobial peptide together with a suitable carrier, diluent, or excipient.

[0013] The composition comprising the described antimicrobial peptide may be used in treatment or prevention of a disease or condition, such as infectious disease or a tumour.

[0014] The composition comprising the described antimicrobial peptide may be used for application to a surface for disinfecting or prevention of growth of microbes. For example, such a use may be for disinfecting of a surface, a material, or an environment such as for use in environmental remediation. A method is described herein for cleaning or disinfecting of a surface, a material, or an environment comprising application of the described composition thereto.

[0015] A use for the antimicrobial peptide is provided, for treatment or prevention of a disease or condition in a subject in need thereof. Further, the use of the antimicrobial peptide for preparation of a medicament for treatment or prevention of a disease or condition in a subject in need thereof is also described herein. Additionally, a method of treating or preventing a disease or condition is described, comprising administering to a subject in need thereof an effective amount of the antimicrobial peptide or composition thereof. The disease may be, for example, an infectious disease or a tumour. The subject may be a human or an animal, such as a livestock animal, for example poultry, or a companion animal, such as a domestic animal or pet. The infectious agent may be a Gram-negative bacteria, Gram-positive bacteria, acid fast bacteria, bacteria resistant to other drugs, a virus, a fungi, or a parasite. For example, the bacteria may be selected from Escherichia coli, Salmonella enterica, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, Mycobacterium smegmatis, Methicillin-resistant Staphylococcus aureus (MRSA), Salmonella enteritidis, or Salmonella Heidelberg.

[0016] A lipid vesicle comprising the antimicrobial peptide is described. A nucleic acid molecule encoding the antimicrobial peptide is also provided, as is a vector comprising such a nucleic acid molecule.

[0017] A method of identifying a target molecule associated with an infectious agent is described, in which the target molecule targets, or is affected by, the antimicrobial peptide. The method comprises the step of screening a library of candidate target molecules associated with the infectious agent, for a molecule that targets or affects theantimicrobial peptide. A kit for conducting such a method for identifying a target molecule associated with an infectious agent is also described, in which the kit comprises the antimicrobial peptide described herein together with instructions.

[0018] Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures.BRIEF DESCRIPTION OF THE FIGURES

[0019] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0020] Figure 1 shows the carboxylate and amide C-terminus structures used for 110 peptide sequences.

[0021] Figure 2 provides a Venn diagram illustrating overlap in activity of AMP sequences in Example 1, for various microbes.

[0022] Figure 3 shows a phylogenetic tree of all 62 active AMPs identified by rAMPage. AMPs active in their C-terminal amide (square), carboxylate and amide (circle) or amide (triangle) forms are denoted on tip points and ring points.

[0023] Figure 4 shows the predicted secondary structure and antimicrobial activity against various microbes for active AMPs in relation to their length and helical content.

[0024] Figure 5 shows (Panel A) phylogenetic tree of AMPs with moderate to high activity against S. aureus, and (Panel B) sequence alignments of peptides active against S. aureus.

[0025] Figure 6 shows a phylogenetic tree of AMPs with high activity against (Panel A) E. coli ATCC 25922 (EC) and 317 (APEC) strains, and (Panel B) S. enterica LS101 (ASE) and 4931 (SE) strains. Sequence alignments of peptides active against EC / APEC (Panel C) and ASE / SE (Panel D) are shown.DETAILED DESCRIPTION

[0026] Peptides and / or amino acid sequences with antimicrobial properties are described herein. A bioinformatics approach, starting with sequences exhibiting effect, and making strategic modifications thereto, has led to the discovery of antimicrobial peptides. In a bioinformatics approach, sufficient similarity among sequences can be maintained so as to permit functional equivalency. Sequences similar to isolated sequences from which a consensus is derived are also described. Such similar sequences may contain conserved amino acid substitutions together with a limited number of non-conserved substitutions, such as modifications or deletions, but while still maintaining functionality.

[0027] These peptides and their pharmaceutical compositions and modifications thereof are also useful as therapies for microbial infections or as chemopreventative agents to slow or arrest the progression of microbial infections. Uses are also envisaged in agriculture and livestock applications, in addition to human health applications. Antimicrobial peptides may be used as disinfection or cleaning agents, on surfaces, or integrated into environments where environmental remediation may be desired.Modifications of peptides described herein may include but are not limited to incorporation of the peptides or their modifications in lipid vesicles for enhanced therapeutic delivery and the modulation of other ADMET properties (absorption, distribution, metabolism, excretion, toxicity) as well.

[0028] Chemical modifications of the peptides are described, which are known to individuals skilled in the art of peptide chemistry to be useful to enhance stability and otherwise make the peptides more drug-like and useful for the desired applications. Such modifications include peptide cyclization and the use of amino acids of opposite chirality; the so-called D- amino acids. Such modifications also include alternative backbone chemistries and side chains that retain function or specificity.

[0029] Also described is the application of the peptides, and modifications of the peptides obvious to those skilled in the art, to other microbial targets. Antimicrobial therapies useful and effective in one type of infection may be useful and effective in other diseases.

[0030] Also described are vector constructs incorporating the disclosed peptides and / or their amino acid sequences and coding nucleic acid sequences for the purposes of the production of antimicrobial peptides.

[0031] The peptides described herein, and the modifications thereof are also useful in combination with other antimicrobial agents for the treatment or prevention of disease, such as an infectious disease or a cancer.

[0032] Uses of the AMPs either alone or as part of a kit to isolate or identify target molecules, or molecules upon which an effect is observed, that may be associated with the infectious agent, are also described herein.

[0033] The peptides and / or amino acid sequences described herein have selective antimicrobial properties. Further aspects and advantages will become apparent from consideration of the ensuing description of various embodiments. A person skilled in the art will realize that other embodiments, combinations and variations are possible, and that the details described herein can be modified in a number of respects, all without departing from the overall concept. Thus, the following drawings, descriptions and examples are to be regarded as illustrative in nature and not restrictive.

[0034] The antimicrobial peptides (AMPs) described herein may possess activity to reduce morbidity or mortality caused by bacterial infections, such as by antibiotic resistant strains, and may be used to treat viral diseases, and / or cancer. AMPs have potential in therapeutic aspects of public health. High throughput methodology permits identification of a large number of AMPs with potential for therapeutic use, optionally in cocktail formats.

[0035] Table 1 provides a list of Antimicrobial Peptides (AMPs) referred to herein, as well as other sequences listed herein, in order of SEQ ID NO. Organism of origin may be reflected in the naming of the organism, such as Alytes multensis (A. multensis) being reflected in the name “AIMu”. Other origin organisms, such as R. Catesbeiana, may or may not be reflected in the naming convention.

[0036] Treatment or prevention of a disease or condition encompasses treatment before and after outward signs or symptoms of the disease or condition are present in the subject. For example, a subject exposed an infectious agent may or may not exhibit symptoms. Further, the prevention or prophylaxis of a disease or condition may encompass partial prevention, lessening of severity when onset occurs, decreasing likelihood of outward signs or symptoms, or preventing the spread of infection by keeping severity so low as to be undetectable or negligible. Treatment and prevention may involve modulating the immune system of the subject to the extent that the subject’s own defenses ward off the disease or condition, such as infection. An inflammatory or antiinflammatory effect of the peptides described herein may modulate the outward signs or symptoms of a disease or condition.

[0037] Anti-cancer activity, such as against solid tumours or liquid tumours, may be modulated by peptides as described herein. Indirect or direct attack on cancer cells by the peptides described herein through effects on the immune system by the peptides may alleviate cancerous cell growth.

[0038] An antimicrobial peptide comprising: an amino acid sequence according to any one of SEQ ID NO:1 to SEQ ID NO:361, such as SEQ ID NO:233, or a variant thereof, having at least 95% amino acid sequence identity thereto. The threshold of amino acid sequence identity for the variant may optionally be at least 96%, at least 97%, at least 98%, at least 99%, or may be 100% amino acid sequence identity to any one of SEQ ID NO:1 to SEQ ID NO:361.

[0039] The antimicrobial peptide may be modified, or may be a variant which comprises a modification that is a conservative amino acid substitution. Such amino acid sequences as are known in the art may include the following candidates, with the substitutable options shown in parentheses: Ala (Gly, Ser); Arg (Gly, Gin); Asn (Gin, His); Asp (Glu); Cys (Ser); Gin (Asn, Lys); Glu (Asp); Gly (Ala, Pro); His (Asn, Gin); lie (Leu, Vai); Leu (He, Vai); Lys (Arg, Gin); Met (Leu, He); Phe (Met, Leu, Tyr); Ser (Thr, Gly); Thr (Ser; Vai); Trp (Tyr); Tyr (Trp, Phe); and Vai (lie, Leu). Furthermore, ‘functional’ variants, mutations, insertions, or deletions encompass sequences in which the activity or function is substantially the same as that of the reference sequence from which the alteredsequence is derived. Activity or function may be tested according to such parameters as described herein, such as minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC). Further, it may be desirable to reduce the antigenicity of a peptide, for example by PEGylated, or the peptide may comprise a D-amino acid. The peptide may be cyclized.

[0040] A composition is described herein which comprises the antimicrobial peptide as described herein, together with a suitable excipient, such as a pharmaceutically acceptable carrier, excipient or diluent. The composition may be one that is suitable for use in treatment or prevention of a disease or condition, such as an infectious disease, or a cancer, such as may be attributable to a solid tumour or a liquid tumour.

[0041] The composition may be formulated for oral, injectable, rectal, topical, transdermal, nasal, or ocular delivery. Such compositions can thus be administered to subjects in need thereof through any acceptable route, such as topically (as by powders, ointments, or drops); oral tablets, capsules, gels or liquids; or rectal suppositories. Further modes of delivery include mucosally, sublingually, parenterally, intravaginally, intraperitoneally, bucally, ocularly, or intranasally. The composition may be formulated for application to a surface for prevention of growth of bacteria or other microbes, wherein the antimicrobial peptide would be formulated with a suitable carrier, excipient or diluent. Such a composition may be used in a method of cleaning or disinfecting of a surface, a material, or an environment, such as an environment in need of environmental remediation.

[0042] When formulated for oral use or administration in a liquid formulation, the excipients or ingredients may include but are not limited to those accepted in the art of pharmaceutical formulations, for example emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms may contain inert diluents such as water or other solvents, solubilizing agents, emulsifiers, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, or dimethylformamide. Further, a liquid formulation may comprise oils such as cottonseed, groundnut, corn, germ, olive, castor, and sesame oils; glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; and mixtures thereof. Besides inert diluents, such oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In livestock, such as poultry and in particular turkey or chicken farming, application in feed or liquid supplements may be considered.

[0043] The composition may be one that is lyophilized. The composition may comprise a suitable preservative.

[0044] The composition may be one that is distributed evenly in a diet intended for livestock, such as swine or poultry. Such a composition may be sprayed or mixed into a ground or powdered ingredient, and then mixed evenly into a coarser animal feed to ensure even distribution.

[0045] A composition for application to a surface is described herein, for use in the prevention of growth of bacteria, said composition comprising the antimicrobial peptide described herein and a suitable carrier or diluent, or combinations thereof.

[0046] A use of the antimicrobial peptide is provided herein, for treatment or prevention of a disease or condition in a subject in need thereof, such as an infectious disease. The disease or condition may also be a cancer, such as a solid tumour or a liquid tumour.

[0047] Further, a use is provided for preparation of a medicament for treatment or prevention of such a disease or condition in a subject in need thereof. A method of treating or preventing such a disease or condition is also described herein, which comprises administering to a subject in need thereof an effective amount of the peptide or the composition described herein.

[0048] The disease or condition may be one attributable to Gram-negative bacteria, or it may be a disease or condition attributable to Gram-positive bacteria. The disease or condition may be one that is attributable to acid fast bacteria, or one that is attributable to bacteria that has become resistant to other drugs. Such diseases or conditions may be ones attributable to E. coli, S. enterica, S. aureus, P. aeruginosa, S. pyogenes, M. smegmatis, MRSA, S. enterica serovar Enteritidis, S. enterica serovar Heidelberg, A. baumannii, K. pneumoniae, or E. faecalis bacteria, for example. The disease or condition may be one attributable to a virus, a fungi, or a parasite.

[0049] Further, the disease or condition may be a cancer, such as a solid tumour or a liquid tumour.

[0050] A lipid vesicle may be used to deliver the antimicrobial peptide described herein. A nucleic acid molecule encoding the antimicrobial peptide described is also envisioned. A vector comprising the nucleic acid molecule is also encompassed.

[0051] A method of identifying a target molecule associated with an infectious agent is described, wherein the target molecule targets the antimicrobial peptide described herein. Such a method involves the step of screening a library of candidate target molecules associated with the infectious agent, for a molecule that is affected by the antimicrobial peptide. The infectious agent may be Gram-negative bacteria, or may be Gram-positive bacteria. Further, the infections agent may be acid fast bacteria, bacteria that has become resistant to other drugs, a virus, a fungi, or a parasite. Exemplary infectious agents include but are not limited to E. coli, S. enterica, S. aureus, P. aeruginosa, S. pyogenes, M. smegmatis, MRSA, S. enterica serovar Enteritidis, S. enterica serovar Heidelberg, A. baumannii, K. pneumoniae, or E. faecalis bacteria. Further, a method of identifying a target molecule for modulating biological activity is described, wherein the target molecule targets a peptide as described herein. The method comprising the step of screening a library of candidate target molecules for a molecule that targets the peptide. Modulating of biological activity may comprise anti-tumour action, antiinflammatory action, or inflammatory action. In such methods of target identification, the screening of a library of candidate target molecules may comprise in silico screening.

[0052] A kit is encompassed herein for identifying a target molecule associated with an infectious agent. Such a kit comprises an antimicrobial peptide as described hereintogether with instructions for conducting the method described herein for identifying a target molecule associated with the infectious agent. Optionally, additional reagents may be provided with the kit. A kit for identifying a target molecule for modulating biological activity, is also described. Such a kit comprises a peptide, as described herein, together with instructions for conducting a screening method for molecules that bind to the peptide.

[0053] The term “putative” may be associated with the term “antimicrobial peptide”. The term makes no implication regarding antimicrobial action of the peptide but acknowledges difference between the establishment of antimicrobial effect versus use as an approved drug, given the years of downstream efforts after antimicrobial activity is established. Thus the term may be used to acknowledge the long term efforts required in establishing commercial viability and bringing such a product to market.

[0054] A naming strategy of peptides may be indicative of the parent peptide (organism base) and its AMP# name / numbering nomenclature. Organism base may be indicated by the first two letters of both parts of the organism's Latin name; AMP #: Next highest number based on total number of AMPs discovered in that organism. For example, an organism of origin such as Alytes multensis (A. multensis) being reflected in the name “AIMu”. Other origin organisms, such as R. Catesbeiana, may or may not be reflected in the naming convention used herein.

[0055] Mutants are listed following the AMP name where applicable following the format: original amino acid, position, desired amino acid. Modified peptides described herein follow the same naming conventions but include Dap, Dab, Orn or lower case letters to indicate D form (such as “k”) as the new amino acid substitutions. See below for codes and explanations. D-Lysine may be represented as lower case “k”, indicating D- instead of L-form. Dab is represented by “U”, indicative of diaminobutyric acid; Dap is represented by J, indicative of diaminopropionic acid; Hor is indicative of X**; which indicates homoarginine instead of L-Lysine; Orn is represented by O, indicative of ornithine; and lower case is indicative of the D-form of the amino acid at the stated position. The use of “X” in the peptide naming convention is not to be confused with the meaning of “any amino acid”.

[0056] If mutations are prepared, the indication of such a mutation may be reflected in a sequence name, for example a mutation A19K (Lysine (K) amino acid substitution for Alanine (A) at position 19) would be indicated as such. Naming with regard to truncations may be indicated by the peptide name followed by “tX_Y”, where X and Y are residue position numbers, meaning the residues from X to Y positions are present in the newly truncated sequence. For example “t15_23” would indicate a truncated version of the originally named peptide from positions 15 to 23. C-terminal amidation is indicated as “_a”, meaning “-CONH2”, for example “CCH1_a” indicates the peptide CCH1 is amidated.

[0057] Amidation of a peptide may enhance its activity or efficacy as a antimicrobial agent. When a peptide is amidated, its carboxyl group at the C-terminus is converted into an amide group which removes the negative charge on the carboxyl group and increases the overall peptide charge by +1. Charge may impact membrane permeability, and an increase in charge may improve electrostatic interactions with negatively chargedfeatures, such as on a bacterial surface and / or lipid bilayer, thereby impacting membranepenetrating ability. Amidation may permit a peptide to more readily disrupt and / or permeabilize a microbial cell membrane. Amidation may also enhance the stability of a peptide, making it more resistant to degradation.

[0058] Examples

[0059] The following Examples outline exemplary embodiments and / or studies conducted pertaining thereto. While the Examples are illustrative, they should not be viewed as limiting.

[0060] Example 1

[0061] Testing antimicrobial peptides in their C-terminal amide and carboxylate form against pathogens of importance in the poultry industry

[0062] Summary. With antibiotic resistance increasing worldwide, there is an urgent need for alternatives to existing antibiotics. The present work aims elucidate the impact of C-terminal amidation on the activity on a large dataset of antimicrobial peptides (AMPs). The rAMPage computational pipeline was previously used to identify 110 AMPs from amphibian RNA-sequencing datasets. Peptides were synthesized in both their C-terminal carboxylate and amide forms and evaluated for activity against pathogens important in the poultry industry and public health: avian pathogenic Escherichia coli 317 (APEC), Salmonella enterica serovar Enteritidis 4931 (SE), avian Salmonella enterica serovar Enteritidis LS101 (ASE), and Staphylococcus aureus 29213 (SA) plus a non-pathogenic Escherichia coli ATCC 25922 strains (EC). Of the 220 AMPs tested, 48-86 were classified as active (MIC ≤ 128 μg / mL) against the tested bacteria. Several peptides were successfully identified with high activity (MIC ≤ 4 μg / ml) and selectivity against EC (9 peptides), APEC (4 peptides), SE (3 peptides), and SA (13 peptides). Most of these displayed low cell toxicity against red blood and HEK293 cells. Additionally, the influence of C-terminus, length, charge, secondary structure, and hydrophobicity was investigated, using direct comparison and multiple linear regression. C-terminal amidation significantly increased activity of 36-58% peptides depending upon the bacterial type while longer chain lengths, a positive charge range of +1 to +5, and hydrophobic characteristics were all associated with increased antimicrobial activity. The present work provides actionable physicochemical information for incorporation into AMP discovery pipelines to augment the identification of AMPs as antibiotic alternatives from multiple genomic resources.

[0063] Introduction

[0064] Antimicrobial resistance (AMR) is an imminent global health crisis perpetuated by the overuse and misuse of antibiotics. Antibiotic use in animal husbandry is a known contributor to AMR, creating environmental reservoirs of resistant organisms and exacerbating AMR in the community. Prophylactic antibiotic administration was commonly implemented to increase food production and stave off infection in livestock and poultry; however, these practices exert a selective pressure towards bacteria with antibiotic resistance. Although government regulatory agencies have implemented policies torestrict antibiotic use in livestock production, insufficient global reporting and regulation, global food demands, and lack of antibiotic alternatives impede the ability to reach global milestones in antibiotic stewardship (Van Boeckel et al., 2015). Furthermore, the regulatory changes to antibiotic usage implemented in North America created unintended consequences in the farming industry, increasing mortality within the poultry industry and decreasing food production.

[0065] Poultry is one of the most consumed animal products worldwide. To maximize yield, flocks are often raised in close quarters at the expense of animal health, and given antibiotics to control disease and improve growth (Nhung et al., 2017). Overcrowded chicken coops allow flocks to be vulnerable to colibacillosis, salmonellosis, and staphylococcosis whose causative agents are, respectively, avian pathogenic Escherichia coll (APEC), Salmonella enterica serovar Enteritidis (SE), and Staphylococcal species such as Staphylococcus aureus (SA) (Nhung et al., 2017; Kabir 2010; Szafraniec 2022). These pathogens have economic and zoonotic importance, as they can decimate flocks and contaminate animal products, contributing to foodborne illness and the spread of antibiotic resistance genes (Nhung et al., 2017; Abd El-Ghany, 2021). While good husbandry practices are key in limiting disease, the development of new antimicrobials capable of both mitigating infection and restricting the development of resistance is critical to food security and animal health.

[0066] Antimicrobial peptides (AMPs) are an enticing option for preventing infection in poultry production. These peptides are endogenous to all classes of life and function as a primary defense against bacterial, fungal, and viral infections (Bahar et al., 2013; Shai 2002). AMPs are an extremely diverse group of antimicrobial molecules, typically ranging in length from 5 to 100 amino acids, with widespread differences in mechanism of action and physicochemical properties; however, the most common archetype is the cationic AMP. Cationic AMPs are characterized by a net positive charge at physiological pH, resulting from an abundance of cationic residues (Lys, Arg and His). In addition, cationic AMPs are often amphipathic and rich in non-polar amino acids (Seyfi et al., 2020; Hancock et al., 2006). Cationic AMPs can be further classified by secondary structure: alpha-helical, beta-sheet, extended, loop, or a combination of these conformations (Bahar et al., 2013; Seyfi et al., 2020).

[0067] AMPs are the first line of defense in the innate immune system. AMPs can either interact directly on the bacterial surface, causing membrane perturbation, or target other macromolecular features triggering cell death. Direct membrane action starts with an initial electrostatic interaction between the cationic AMPs and the negatively charged bacterial membrane components. The amphiphilic nature of the AMPs can then alter the curvature, thickness, and permeability of the membrane, ultimately leading to erosion through a detergent-like process or by perforation (Bahar et al., 2013; Shai 2002; Hancock et al., 2006; Huang et al., 2010). AMPs can also act through other mechanisms of action, either in tandem or as an alternative to membrane perturbation. These alternative targets include disrupting integral membrane protein function, inhibiting cell wall synthesis, disrupting DNA, RNA, and protein synthesis, and preventing enzymaticactivity (Bahar et al., 2013; Jenssen et al., 2006). AMPs are also powerful immune regulators that can signal, rally, and regulate the innate and adaptive immune response in addition to their direct antimicrobial effects. Their immunomodulatory abilities often occur at concentrations orders of magnitude lower than their minimum inhibitory concentrations (Lai et al., 2009). AMPs can also stimulate chemotaxis, modulate inflammation, neutralize pathogenic toxins, promote wound healing, and have anti-cancer properties (Lai et al., 2009; Mahlapuu et al., 2016). This multifactorial approach to pathogen clearance makes AMPs less likely to exert selective pressure for bacteria to exploit and develop resistance (Bahar et al. 2013, Hancock et al., 2006; Spohn etal., 2019).

[0068] Despite their acknowledged potential, several limitations prevent their translation from research labs to the clinic. AMP discovery using traditional wet lab techniques is time intensive. However, the emergence of new bioinformatic and machine learning tools for AMP discovery has revolutionized the ability to identify and characterize AMPs (Bahar et al., 2013; Helbing etal., 2019; Li C etal., 2022; Richter et al., 2022). Regardless of the number of AMPs identified, AMPs in their natural form may have poor pharmacodynamics in vivo (Baher et al., 2013). Their flexible structure and abundant cationic residues leave AMPs susceptible to proteases abundant in the gastrointestinal and circulatory systems. AMPs are also often rapidly cleared by the renal and hepatic systems (Lu et al., 2020; Zhang et al., 2021). Furthermore, some potent AMPs display toxicity against eukaryotic cell lines (Khabbaz et al., 2021). Current research has pivoted toward synthetically modified analogues of natural AMPs to address these challenges. Selective incorporation of unnatural amino acids as well as N-terminal and C-terminal modifications can enhance the proteolytic stability of AMPs (Molhoek et al., 2011; Wang 2012). C-terminal amidation is a potent AMP modification that can significantly enhance AMP activity and stability (Huan et al., 2020; Dennison et al., 2015; Strandberg et al., 2007). C-terminal amides are commonly found in amphibian AMPs. This modification is facilitated by peptidylglycine alpha-amidating monooxygenases (PAMs) which transform a C-terminal glycine residue to a primary amide after translation (Ladram et al., 2016; Wang 2020). This modification removes the negative charge on the carboxyl group and increases the overall AMP charge by +1³⁴. The increased charge enhances the electrostatic interactions with negatively charged bacterial membrane components. The amidated C terminus also facilitates stronger interactions with the lipid bilayer (Mura et al., 2016). Molecular dynamics simulations suggest that the C-terminal amide improves peptide membrane penetration when compared to their C-terminal carboxylate counterparts (Mura et al., 2016; Sforca et al., 2004; Kim et al., 2011). Moreover, C-terminal amides enhance AMP stability in the presence of exopeptidases, thereby extending their antimicrobial effects and improving their overall efficacy (Wang, 2012). Despite these advantages, systematic comparisons between the activities and properties of large sets of AMPs in both their C-terminal carboxylate and amide forms are lacking from the literature.

[0069] The impact of C-terminal amidation on 110 AMPs identified from amphibian RNA-seq datasets was assessed using the Rapid Antimicrobial Peptide Annotation and Gene Estimation (rAMPage) pipeline (Richter et al., 2022; Lin et al., 2022; Jackman et al.,2022). Each member of the set of AMPs was synthesized in both the C-terminal carboxylate and amide forms and subjected to antimicrobial susceptibility testing on a panel of Gram-negative and Gram-positive bacteria containing relevant pathogens in poultry production. The physicochemical properties of the compounds were also examined and linked to biological activity to provide a more comprehensive understanding of their structure-function relationships than available to date.

[0070] Materials and Methods

[0071] AMP identification and selection. The Rapid Antimicrobial Peptide Annotation and Gene Estimation (rAMPage) pipeline (Richter et al., 2022; Lin et al., 2022) is a homology-based pipeline that uses RNA-seq data to identify putative AMPs for downstream testing (Li et al., 2022). The rAMPage pipeline was applied to four amphibian RNA-seq datasets as input (Li et al., 2022; Jackman et al., 2022). Part of the rAMPage pipeline also includes AMPlify, an attentive deep learning model, capable of scoring putative peptide sequences fortheir probability of being active AMPs. From the putative AMPs identified using rAMPage, only peptides with AMPlify score >10, length < 30 amino acids, and charge > 1 were synthesized for in vitro validation of activity in this example. This resulted in 111 peptide sequences which were then synthesized in either or both their C-terminal carboxylate and C-terminal amide forms by GenScript (Piscataway, NJ, USA). The C-terminal carboxylate peptides are denoted as CCH-# and the C-terminal amide peptides as CCH-#a (where # = 1-111) (Li et al., 2022). One AMP, CCH-54, was not easily synthesized, but CCH-54a, was successfully synthesized but not included in the analysis.

[0072] Bacterial isolates. Antimicrobial susceptibility testing was performed on a panel of Gram-negative and positive bacteria containing relevant pathogens in poultry production. The panel contained Escherichia coli 25922 (EC), a non-pathogenic strain used as a drug susceptible control in antimicrobial susceptibility testing (Clinical & Laboratory Standards Institute (CLSI)), Salmonella enterica serovar Enteritidis 4931 (SE), a strain that contains three known antimicrobial resistance genes and is associated with food and water contamination, and Staphylococcus aureus 29213 (SA), a strain which contains three known resistance genes all sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA). Avian pathogenic E. coll 317 (APEC), a common poultry pathogen, and avian Salmonella enterica serovar Enteritidis LS101 (ASE), a flock isolate, were from the Vaccine and Infectious Disease Organization (VIDO; University of Saskatchewan, Saskatoon, Canada). Bacterial isolates were grown and stored as described previously (Richter et al., 2022).

[0073] Antimicrobial susceptibility and cell toxicity testing. The antimicrobial activity of all AMPs was measured by broth microdilution assays as describe previously (Richter et al., 2022) to determine their minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively), with some adaptations for testing cationic AMPs as described previously (Hancock et al., 2006). Peptides were considered active if the MIC of any of the bacteria tested was < 128 pg / mL and each experiment was performed independently three times. Cell toxicity testing (hemolysis assays and alamarBlue cell viability assays) ofthe AMPs proceeded as per protocols laid out in prior publications (Li etal., 2022; Richter et al., 2022). Briefly, the hemolysis assay was performed on porcine red blood cells (PRBCs) (Lampire Biological Laboratories, Pipersville, PA, USA) to study the ability of the AMPs to rupture eukaryotic membranes. PRBCs were selected over chicken red blood cells (CRBCs) for this assay due to the consistent availability of high-quality PRBCs. Preliminary tests indicated that the results from PRBCs and CRBCs were comparable within regular experimental variation of + / - one-fold change (data not shown). Hemolytic activity was denoted as the AMP concentration needed to lyse 50% of the PRBCs (HC50). The alamarBlue cell viability assay was used to measure cytotoxicity in human embryonic kidney cell line HEK293 (ATCC), measured as the AMP concentration needed to reduce cell viability by 50% (CC50). This assay was only performed on peptides with high antimicrobial activity and / or hemolytic activity. If these criteria were met, the cell viability assay was performed using both the C-terminal carboxylate and its amide counterpart. The antimicrobial activity of AMPs was classified using median MIC scores; “no” (MIC > 128 pg / mL), “low” (128 pg / mL > MIC >32 pg / mL), “moderate” (32 pg / mL > MIC > 4 pg / mL), or “high” (MIC < 4pg / mL) activities. Any peptide with low, moderate, or high activity (MIC of < 128 pg / mL) against any of the pathogens is denoted as a verified AMP. Hemolytic or cytotoxic activity was similarly classified as “no” (HC50 > 128 pg / mL), “moderate” (128 pg / mL > HC50 > 64 pg / mL), or “high” (HC50 < 64 pg / mL) activities.

[0074] Physicochemical property analysis. The 3D structures of AMPs were predicted with ColabFold (Mirdita et al., 2022). The ColabFold server generated five structures for each peptide with sub-models of AlphaFold (Jumper et al., 2021). The five predicted structures were relaxed using the Amber force field to limit stereochemical violations in the predictions. The five structures for each sequence were ranked by the per-residue estimate of AlphaFold’s confidence in its prediction (pLDDT score). The resulting PDB file of the Amber relaxed rank 1 model for each peptide was passed through STRIDE to categorize peptides based on their secondary structure (Frishman et al., 1995). The peptides were classified as: helical (>80% of the residues were assigned as participating in an a-, pi- or 310-helical structure); mainly helical (between 50-80% of the residues being helical); mainly extended (<50% of the residues being helical); extended peptides (only possessing turn secondary structure and / or coils, no helical or beta-strand containing residues); mixed (possessing both helical and beta strand motifs) or p-strand containing (only possessing beta-strand residues and no helical residues present).

[0075] To determine the hydrophobicity of the peptides, the Grand Average of Hydropathy (GRAVY) score was calculated using the sum of the hydropathy values for all amino acids (considering the hydrophilic and hydrophobic properties of the amino acid side chains) in the peptide divided by the total number of amino acids (Kyte et al., 1982; Stothard, 2000).

[0076] Statistical analyses. All XY-plots were created in R version 4.3.1 using ggplot2 (version 3.4.2) and all phylogenetic trees were created in R version 4.3.3 using ggtree (version 3.10.1) (Xu etal., 2022). Bubble plots were created using Prism (version 10.2.2). The nonparametric Mann-Whitney U (aka Mann-Whitney Wilcoxon) test was used toperform pairwise comparisons of the MIC and cell toxicity technical replicates for each AMP treatment using R version 3.6.3. A padj≤ 0.05 was deemed to be significant.

[0077] Multiple linear regression (MLR). Multiple linear regression (MLR) was performed using Python, incorporating functions from the SciPy (SciPy 1.0 Contributors, 2020), Scikit-learn (Pedregosa et al., 2011), and statsmodels (Seabold et al., 2010) packages. Analyses were performed individually for each pathogen, with its MIC being the dependent variable and length, charge, helicity, and hydrophobicity (GRAVY) being the independent variables, as well as other as required. AMPs measuring MIC > 128 pg / mL (no activity) were excluded from all analyses. A fabricated independent variable, amidation (0 and 1 for C-terminal carboxylated and amidated respectively), was added to combined carboxylated and amidated peptide regression. An intercept was also included in all regressions.

[0078] The transformation log2(MIC) was applied to maximize error normality. Individual independent variables (x) had different transformations (x, x-1, x2, x½x3, x⅓, 10x, log10x, ex, logex) systemically applied to maximize the goodness of fit compared to log2(MIC) for C-terminal carboxylate AMPs against EC. Only loge(charge) significantly improved the fit. These transformations were applied in all regressions.

[0079] Initially, models for C-terminal carboxylate, C-terminal amide, and all AMPs were fitted using every combination of parameters. For each combination, outliers were subsequently discounted one at a time, by removing the outlier with greatest Cook’s distance and refitting, until no factor had Cook’s distance three times greater than any other or more than ¼×number of data points, unless maximum Cook’s distance increased. The best model was primarily determined using the Akaike information criterion (AIC) but R2, adjusted R2, the Bayesian information criterion (BIC), and the requirement of outlier removal were also taken into consideration. Additionally, only p-values < 0.05 and R2> 0.4 were deemed significant, while variance inflation factors (VIF) < 10 and correlations < 0.8 were ideal. Model validity was confirmed by visually examining quantile-quantile (Q-Q), constant variance, and Cook’s distance plots.

[0080] The best models were subsequently cross validated by splitting the data into training (80%) and test (20%) sets and running the regression 100 times. Average root mean square error (RMSE) and average mean average error (MAE) indicated model suitability. Standard errors are reported for coefficients, and these cross-validations.

[0081] RESULTS

[0082] Heading. The rAMPage AMP discovery pipeline was applied to amphibia RNA-seq datasets, identified and selected 110 AMPs.

[0083] Chemical structure and measured activity against bacterial strains is shown in Figure 1 to Figure 3. Figure 1 shows the carboxylate and amide C-terminus structures used for all 110 peptide sequences. Figure 2 shows a Venn diagram of the overlap in activity of the active AMP sequences between E. coll 25922 (EC), avian pathogenic E. coll 317 (APEC), S. Enteritidis 4931 (SE) S. Enteritidis LS101 (ASE) and SA 29213.Figure 3 shows a phylogenetic tree of all 62 active AMPs identified by rAMPage. AMPsactive in their C-terminal amide (square), carboxylate and amide (circle) or amide (triangle) forms are denoted on tip points and ring points. Predicted secondary structure is highlighted by the colors in the external ring. Highly active AMPs (MIC < 4 pg / mL) in each strain tested are represented in the ring points.

[0084] These AMPs were synthesized in both their C-terminal carboxylate and C-terminal amide forms (Figure 1) to yield a set of 220 AMPs. This set was then subjected to in vitro antimicrobial susceptibility tests against a panel of bacterial pathogens, most are relevant to poultry production, and hemolysis testing against PRBCs. Of the 220 AMPs tested herein, 86, 75, 51, 34, and 48 of the AMPs, were classified as active (MIC ≤ 128 μg / mL) against EC, APEC, SE, ASE, and SA respectively (Figure 2). For a breakdown of activity per pathogen see Table 2, which shows Antimicrobial activity of the AMPs per pathogen. The total (carboxylated + amidated), carboxylated, and amidated numbers of peptides in each activity category per pathogen. AMP activity was determined using median MIC values scored as “No activity” (MIC > 128 pg / mL), “low” (128 pg / mL > MIC >32 pg / mL), “moderate” (32 pg / mL > MIC > 4 pg / mL), or “high” (MIC < 4 pg / mL).Table 2Antimicrobial Activity Against Select PathogensActivity EC APEC SE ASE SA High 1 0 1 0 5 Moderate 14 8 2 1 4 Low 16 16 IQ 7 4

[0085] There was overlap between the activities of AMPs against different pathogens, where 23 of the 94 active AMPs were active against all bacterial strains tested (Figure 2).Few active AMPs were selective for only one pathogen. Several peptides were successfully identified with high activity (MIC ≤ 4 μg / ml) and selectivity against EC (9), APEC (4), SE (3), and SA (13) (Figure 2 and Figure 3). While no highly active peptide was identified that was selective against ASE only, a number of moderately activepeptides were found (Table 2). APEC and ASE were less suspectable to peptide treatment compared to EC and SE.

[0086] Of the 110 AMP sequences, 62 sequences were particularly active. Of these, 32 were active AMPs regardless of the structure of the C-terminus (Figure 2 and Figure 3).Changing the structure from a C-terminal carboxylate to a C-terminal amide imparted AMP activity to 29 sequences, while just one sequence was exclusively active in its C-terminal carboxylate form. Almost all the high activity and moderate activity AMPs were active in both the C-terminal carboxylate and amide form (Figure 3).

[0087] Pairwise comparisons were performed of the antimicrobial activity of each active AMP in its C-terminal carboxylate and amide form, to detect any significant differences in MIC values (Table 3; p-value < 0.05).

[0088] Boxplots of individual pairwise comparisons (not shown) permitted evaluation of pairwise comparison of the antimicrobial activity of the carboxylated and amidated AMPs against Escherichia coli 25922 (EC) and avian pathogenic E. coli 317 (APEC), where AMPs with activity in at least one of the pairs of the comparison was observed. Further, pairwise comparison was also evaluated for antimicrobial activity of the carboxylated and amidated AMPs against Salmonella enterica serovar Enteritidis 4931 (SE) and S. Enteritidis LS101 (ASE); as well as against Staphylococcus aureus (SA). Pairwise comparison of the hemolytic (PRBC) or cytotoxic (HEK293) activity of the carboxylated and amidated AMPs, based on HC50 (defined as the AMP concentration needed to lyse 50% of the PRBCs), CC50 (defined as the concentration of AMP needed to reduce cell viability of the HEK293 cells by 50%) was also evaluated.

[0089] C-terminal amidation resulted in a significant increase in antimicrobial activity in all tested bacterial strains (Table 3). Of the 62 pairs of AMP sequences, 57% had a significant improvement in their antimicrobial activity in the C terminal amide form for both EC and APEC strains (Table 3). A significant increase in activity for SE and ASE was also observed in the C-terminal amide form of 44% and 36% of AMPs, respectively (Table 3). In SA, 37% of amidated AMPs increased their activity against the strain. 13% of AMPs had a significant increase in hemolytic activity in their C-terminal amide form. This mirrored cell viability testing which also resulted in 13% of AMPs significantly increasing their toxicity in HEK293 cells upon C-terminal amidation. Overall, these results suggest that the C-terminal amide form of certain AMPs increases their ability to interact with mammalian membranes resulting in cell damage; however, the concentration where the decrease in cell viability was observed far exceeded their respective MIC concentrations. There were rare cases when C-terminal amidated AMPs had decrease activity (CCH-63, -64, and -74 for EC and CCH-64 only for APEC).

[0090] Table 3 shows a pairwise comparison of antimicrobial activity, hemolysis and cell viability of the AMPs in their C-terminal carboxylate versus amide forms. Antimicrobial susceptibility testing was performed in E. coli 25922 (EC), avian pathogenic E. coli 317 (APEC), S. Enteritidis 4931 (SE), S. Enteritidis LS101 (ASE) and S. aureus 29213 (SA). Hemolysis assays were performed using porcine red blood cells (PRBC) and cell viability assays were performed using human embryonic kidney cells (HEK293). The number ofpairs with a significant change in activity due to the presence of the C-terminal amide (left) and the respective percentage out of the 62 AMP pairs tested that showed activity (MIC ≤ 128 μg / mL), and were therefore verified AMPs (right), is shown (p-value < 0.05). For the cell viability assay, only AMPs pairs with high antimicrobial activity and / or hemolytic activity were tested. Two percentage values were calculated; the upper is out of the total AMP pairs (62) and the lower is out of the number of AMPs tested in the cell viability assay (36).

[0091] AMP characterization by physicochemical properties. The lengths of the active AMPs ranged from 11 to 30 amino acids and charges for the active AMPs ranged from +1 to +14 for the natural peptides and correspondingly +2 to +15 for the amidated peptides. High and moderate antimicrobial activity was generally observed for longer peptides and in the +1 to +4 and +2 to +5 charge range for C-terminal carboxylate and amide AMPs respectively. Some low and low / moderate hemolysis was observed in PRBC after treatment with AMPs with a charge range of +2 to +5, regardless of their C-terminus. No increase in hemolysis was observed in the AMPs with a higher charge (> +5) due to amidation.

[0092] 3D structure prediction and peptide categorization were performed using ColabFold (Mirdita et al., 2022), and STRIDE (Frishman et al., 1995) on the 62 particularly active AMP sequences. The predicted 3D structure classification of the 62 AMP sequences in their C-terminal carboxylate form showed 60% of the sequences were alpha-helical and 31% were mainly helical. The relationship between predicted secondary structure and activity was then performed by plotting the antimicrobial activity of all active AMPs in relation to their percent helical content and length (Figure 4). Shorter AMPswere more likely to have an increase in AMP activity C-terminal amide form compared to longer AMP sequences.

[0093] Figure 4 shows the predicted secondary structure and antimicrobial activity against E. coli 25922 (EC), avian pathogenic E. coli 317 (APEC), S. Enteritidis 4931 (SE), S. Enteritidis LS101 (ASE) and S. aureus 29213 (SA) of all active AMPs in relation to their length (x-axis) and helical content (y-axis). The size of the circle correlates an AMP to the fold change in its activity when the C-terminal amide is present, where Increase in circle size indicates higher AMP activity. The color represents the predicted Colabfold secondary structure of the sequences.

[0094] Multiple linear regression (MLR) was used to quantify the relationship between log2(MIC) and physicochemical descriptors of AMPs (length, loge(charge), helicity, and hydrophobicity), as well as others as required, for each pathogen. The primary aim was to use quantitative statistical modeling to determine which physicochemical traits have significant influence on activity. Valid linear models were established for EC, APEC, and SA across different AMP subdivisions. However, no viable linear models were found for SE or ASE, likely due to these pathogens having too few active peptides for robust analysis.

[0095] In general, length, loge(charge), and hydrophobicity were negatively correlated to log2(MIC), with coefficients -0.09±0.04, -3.3±0.3, and -2.1±0.1 respectively, across the best valid models containing these terms. These coefficients indicated that longer, highly charged, and more hydrophobic AMPs were more potent.

[0096] For C-terminal carboxylate AMP models, the MLR analysis indicated that on average, length provided the most significant mean term contribution to potency (-3.2±0.3), with loge(charge) (-2.4±0.1) and hydrophobicity (-1.4±0.3) also showing relevance. Helicity was found to be insignificant across all pathogens, likely due to the highly correlated hydrophobicity better describing log2(MIC). These models had an adequate goodness of fit (R2= 0.59-0.85). In contrast, goodness of fit was poorer for C-terminal amide AMP models (R2= 0.42-0.63). Unlike previously, loge(charge) provided the most significant mean term contribution to amide AMP potency (-4.4±0.4), with the coefficients of length (-2.7±0.2) and hydrophobicity (-2.0±0.3) again showing relevance. For the models encompassing all AMPs, the fabricated “amidation” descriptor was usually insignificant, likely being accounted for more rigorously by the other descriptors. These models had characteristics similar to the two exclusive sets, with similar goodness of fits (R2= 0.47-0.62).

[0097] Additional models indicated which physicochemical peptide properties were related to a change in potency upon incorporation of the C-terminal amide. Model validity was confirmed by visually examining quantile-quantile (Q-Q), constant variance, and Cook’s distance plots.

[0098] Length was found to be by far the most significant descriptor, with coefficient 0.17±0.05 across the best valid models. Goodness of fit was more variable (R2= 0.18-0.79). This showed that shorter AMPs had a larger increase in potency when amidated.Table 4Characteristics of 111 AMPsTable 4...continuedCharacteristics of 111 AMPsCCH-4C VAAHVANGKRILGKILFLVSGLLGNLG ’5.7 27 3 C. B CCH-41 ILGKIFLVSG ’5.3 11 1 1.7 CCH-42 ILGKIL='L GGLL5 -f.3 15 1 1.4 CCH-43 ILGKILFLVGGLLS '4 3 14 1 1 9 CCH-44 ILGKI LVS ’4.7 13 1 1.3 C CH-45 GSLVKGIAAHVANGKRILGAILFLVSGLL ’4.7 23 3 1.1 CCH-45 G. AGRGCRRRGAQRGRRKR ’4.5 13 3 -2.7 CCH-47 S'A'FRKGCER YSS5LANGK=? '4 2 13 4 -1 3 CCH-48 IRG. A|L=LV3GLL LG ’3.4 17 1 1.3 C CH-46 GTGLSC EKRKAYNWRVTFCC Y ’3.4 21 3 -3.5 CCH-5C K LV L K T AD LKKK KK M AAF VANGK R ’3.4 25 3 -1.’ CCH-51 GA3RGRRKRGAQRG3RRRGACRGRRKR '3 3 2“ 14 -27 CCH-52 ILGKILFLVGGLLNh ’2.3 15 1 1.2 CCH-53 RF^KSKR ’2.3 7 4 -2.3 CCH-54 VD GKRILG IL=LF5GLLSFLG ’2.4 23 1 C.7 CCH-55 =LGK I5TI5=? LG 'C.9 13 2 C. S CCH-55 ILG=LI'-'VCKI ’C.3 11 1 2.3 a C CH-57 G = LDI KS AATSIVDLLLCKM. AACTHK 34.2 23 1 C.5 a C CH-58 = PAIICKV3KKLLKL'A'KLELEII 3C.3 23 3 C. B a CC4-5& S MLS VLK RC5 = 2&.4 11 2 C.3 a CCH-6C GLKKLESLRFGLKHT 25.5 15 3 -3.4 a CCH-61 F=AIICKVSKNC 24.1 12 2 C. B a C CH-62 E E E R FLF F I ARLAAKV FF S 11 CSVTKK C ’5.1 2B 2 C.4 a CCH- 3 FLPIIGKILSTVFGKKFR ETLKMELEII '5.9 33 2 C.4 a CCH-64 FLTFIARLAAKV = PSIICSVTKKC 53.4 24 4 1.1 a 0 CH-65 FLFFIARLAAKVFFSITCSVTKKC 55 24 4 C. S a CCH-65 FLFFTARLAAKV = P3IICSVTKKC 51.3 24 4 C. S s CCH-67 FLFFI RLAAK' / F 2S.9 13 2 1.5 a C CH-68 EEERFL=FIARLAAKVF=SITC5VTKKC ’5.3 2B 2 C.2 a CCH-66 EEERFL=TTARLAAKV = P3IIC5VTKKC ’5. B 2B 2 C.2 a CCH-7C II2GYRKRLISLIFSKGC ATK" ’3.5 22 4 C.1 s CCH-71 KFK4GK KLLKKKTTKL3RS3R ‘2.3 22 13 - 1.7 a CCH-72 FLPhVGSSGGY'.'RSKSSIAATMFIKRCF ’ 1.5 27 4 -3.2 a CCH-73 5RRSM5=RGAFF S= FF= E ’ 1.2 21 2 C. S a CCH-74 FLPFI AL LAAK YFF S 11 C SVTKK C 54.5 24 3 1.5 a CCH-7? =L = PLIT3 = L3K= 47.1 13 1 1.5 a C CH-75 =L==IARLAAKV=P5IIC5VTKKF 45.1 24 4 1.1 a C CH-77 GLFL 2TLK GAAK N 45. B 14 1 C.4 a C CH-78 GLFLDTLKGAAKDVAGKLLEGLKC KITRM 45.4 23 3 C.2 a CCH-7& FLPFI RLA.4KL 34.9 12 2 1.4 a CCH-oC GL = L2TLKG. AAK2,' / AGEIAGRSKM 34.4 24 1 C.1 a CCH-81 = L =' = I A R LA AKF = 33.3 13 2 1.4 a C CH-82 FLF Fl ARLAAKVFFSIICSVTKNVETLET 32. B 23 1 C. Ba CCH-83 =L = = IARL4> G= 22“ 13 2 1.2Table 4...continuedCharacteristics of 111 AMPsR. caf^iera.na CC4-51 SLI SIVSPLKG '4.3 11 1 C. S R. cafasrera.na CC4-52 TKRWL=HRTR "4.2 13 4 -2.’ R. ca? cieia.na C C 4-93 GA ETF KVWGK R GA ETF N RGKR S = '3.1 24 4 -1.5 R. cateiiera.na CC4-&4 G AETH RGK R3 = R S P R G '2.4 13 4 -1.3 R. catesiera.na CC4-95 COVR3KR "2.2 7 3 -1.5 R. cateiiera.na CC4-95 = LGGLIKIVPAMFC 12 14 1 1.3 R. caf^iera.na CC4-97 GAETFKVWGKRSFRGAET4NVRGKR M. S 25 5 -1.6 R. catecrera.na CC4-98 GAETFKV'A'GKRSFRSF ’ 1.4 15 3 -1.5 R. cateiiera.na CC4-95 GAETHKVWGHR5='R5='R ’ 1.1 17 4 -1.7 R. cateiiera.na CCF-’CC FLFFIARLAAKVF3IN"LF3N3KML 1’ 25 3 C. S R. caro.-i ra.na C C F - ’ C 1 GAETH KVWGK R3 =R S=GGA ETF NVWD KR "C. S 23 3 -1.5 R. cateiiera.na CCF-’C2 FLGGLIKIV=AMF'A' 'C.5 14 1 1.5 R. catescefa.ia CCF-'C3 SNIVIFC3LVLRAS5ELN5KRCKR "C.2 24 3 -3.3 R. Ctff?cr<?!:a.na CCF-’C4 GCYTVLRTGVCKLLIRVT 27.3 13 3 C.5 R. cateiiera.na CCF-’C5 VKAVLEKIGGHTK Y 22.1 14 2 -3.2 R. cateiiera.na CCF-’CC 3 R 33 RF'-TT Y LGC NC W3C W G3 -5.3 21 2 -3.4 R. cateiiera.na CCF-’C7 LMWWK43IKR "5.3 11 3 -3.3 R. cateiiera.na CCF-’C8 ILLA RFIR3R3GR '4.3 14 4 -3.’ R. catescefa.ia CCF-'CS GILTLKNTLSFSOKP "C.3 15 2 -3.’ R. catecrera.na CCF-’ 1C CKR = YTTYLGCNC,A'3C,A'G3 'C.7 13 2 -3.5 R. cateiiera.na CCF-’ 11 GLYGRVARRKFLL3ARNRK 'C.2 13 7 -1.0

[0099] To elucidate the physicochemical properties and sequence diversity of the 29 AMPs with an MIC < 32 pg / mL in SA (Table 2), a multiple sequence alignment was performed and a phylogenetic tree was generated.

[0100] Figure 5 shows (Panel A) phylogenetic tree of AMPs with moderate to high activity against SA. Antimicrobial activity is displayed (top) followed by its associated hemolysis and physicochemical properties (secondary structure, length, hydrophobicity and charge); and (Panel B) shows sequence alignments of peptides active against SA.

[0101] Aligned to the phylogenetic tree is a heatmap showing the MIC, HC50, predicted secondary structure, sequence length, hydrophobicity, and charge of each AMP (Figure 5, Panel A). All AMPs moderately active or greater against SA were classified as helical or mainly helical with lengths ranging from 12-30 amino acids in length and charge ranging from +2 to +5. The Grand Average of Hydropathy (GRAVY) score was used to generate a hydropathy index for the AMPs; hydrophilic peptides have a negative GRAVY score, and hydrophobic peptides have a positive GRAVY score (Kyte et al., 1982; Stothard, 2000). All AMPs with moderate or greater activity against SA activity had a positive a GRAVY score between 0 and +2.

[0102] AMPs which showed high activity in SA were aligned to identify conserved residues and create a consensus sequence (Figure 5, Panel B). There were 13 AMPs with high activity in SA. Four sequences, CCH-74 / CCH74a, CCH-66 / CCH-66a, CCH-76 / CCH-76a, and CCH-65 / CCH65a showed activity regardless of their C-terminus. One sequence, CCH 65 only had high activity (defined as < 4 pg / mL) in the C-terminal carboxylate form; however, CCH-65a retained a potent MIC of 6 pg / mL. Four AMPs, CCH-67a, CCH-81a, CCH-75a, and CCH-9a were only highly active in their C-terminal amide form. While CCH-81 retained moderate activity with an MIC of 8 pg / mL, CCH-67, CCH-75, and CCH-9 had low to no activity against SA, with MICs of 64 pg / mL, > 128 pg / mL and 128 pg / mL, respectively. Furthermore, there appears to be conserved FLPFIARLAAKVF sequence “CCH67” (SEQ ID NO: 316) within the highly active AMP sequences as shown by the generated consensus sequence. The shorter sequences appear to be more dependent on C-terminal amidation for activity, apart from CCH-81 / CCH-81a.

[0103] Figure 6 shows a phylogenetic tree of AMPs with high activity against (Panel A) EC / APEC and (Panel B) ASE / SE aligned to a heat map showing antimicrobial activity, hemolysis, and physicochemical attributes of the AMPs. Antimicrobial activity for each pathogen is displayed (top) followed by its associated hemolysis and physicochemical properties (secondary structure length hydrophobicity and charge, Sequence alignments of peptides active against EC / APEC (Panel C) and ASE / SE (Panel D).

[0104] A multiple sequence alignment was performed and generated phylogenetic trees for AMPs with moderate activity or greater in APEC and / or EC and ASE and / or SE (Figure 6, Panel A and Panel C). All AMPs moderately active or greater against EC and / or APEC were classified as helical or mainly helical with lengths ranging from 11 to 30 amino acids in length. All but one AMP had a charge ranging from +1 to +5 and all but two AMPs had positive a GRAVY score between 0 and +2. Interestingly, CCH-51a had a charge of +15 and a GRAVY score of -2.7 while CCH-107a had a GRAVY score of -0.8.

[0105] For APEC / EC, a total of nine AMPs were highly active. There were eight AMPs with high activity; CCH-33 / CCH-33a had high activity regardless of the C-terminus while CCH-67a, CCH-76a, CCH-66a, CCH-62a, CCH-65a, CCH-81 a, and CCH-45a were only highly active in their C-terminal amide form. All AMPs with only high activity in their C-terminal amide form had moderate activity in their C-terminal carboxylate form except for CCH-67, which had an MIC of > 128 pg / mL in APEC and 128 pg / mL in EC. The highly conserved FLPFIARLAAKVF sequence CCH67 (SEQ ID NO: 316) found in the high activity AMPs against SA is also found in the EC / APEC consensus sequence with the addition of a proline residue on the C-terminal end and three undefined residues between the alanine and arginine to give FLPFIAXXXRLAAKVFP (SEQ ID NO: 362). For AMPs moderately active or greater in SE and / or ASE, all were classified as helical or mainly helical with lengths ranging from 13-29 amino acids in length, charge ranging from +3 to +5 and a GRAVY score between 0 and +2. Only three AMPs were highly active against SE; CCH-33 / CCH-33a and CCH-45. CCH 33 / CCH-33a and CCH-45 / CCH-45a weremoderately or highly active against all bacteria tested. Interestingly, these two AMP sequences are highly similar and have a consensus sequence of XXXXKXIAAHVANGKRILGXILPVSGLLXXX (SEQ ID NO: 363) which is entirely shared between the two sequences. This consensus sequence does not share any commonality to the FLPFIAXXXRLAAKVFP (SEQ ID NO: 362) sequence found in the EC / APEC highly active AMPs or the FLPFIARLAAKVF (SEQ ID NO: 316) sequence found in SA highly active AMPs. In total, 12 AMP sequences showed high activity in at least one of the bacteria tested.

[0106] Table 5 lists AMPs in order of activity against five pathogens, from most active to least active in each column, as determined in heat maps (Figure 5).Table 5AMPs in Descending Order of Activity as Determined in Heat Maps E. coli ATCC APEC SE ATCC SE avian S. aureusCCH-33a CCH-33a CCH-33a CCH-33a CCH-66aCCH-45a CCH-45a CCH-45a CCH-81a CCH-65CCH-33 CCH-65a CCH-33 CCH-45a CCH-65aCCH-62a CCH-66a CCH-65a CCH-33 CCH-67aCCH-65a CCH-33 CCH-81a CCH-65a CCH-76aCCH-66a CCH-67a CCH-67a CCH-67a CCH-81aCCH-67a CCH-76a CCH-45 CCH-45 CCH-64CCH-76a CCH-81 a CCH-21a CCH-66a CCH-66CCH-81a CCH-64 CCH-66a CCH-65 CCH-74CCH-45 CCH-45 CCH-65 CCH-40a CCH-74aCCH-64 CCH-65 CCH-40a CCH-78a CCH-75aCCH-65 CCH-21a CCH-1a CCH-107a CCH-76CCH-21a CCH-40a CCH-51a CCH-9a CCH-9aCCH-40a CCH-66 CCH-76a CCH-5a CCH-64aCCH-66 CCH-76 CCH-66 CCH-79a CCH-20aCCH-76 CCH-78a CCH-78a CCH-21a CCH-79aCCH-78a CCH-64a CCH-64a CCH-51a CCH-81CCH-9a CCH-11a CCH-11a CCH-76a CCH-5aCCH-87a CCH-107a CCH-107a CCH-81 CCH-102aCCH-40 CCH-62a CCH-9a CCH-1a CCH-45aCCH-62 CCH-9a CCH-40 CCH-66 CCH-62CCH-74 CCH-40 CCH-5a CCH-11a CCH-87CCH-78 CCH-78 CCH-70a CCH-40 CCH-87aCCH-87 CCH-1a CCH-8a CCH-8a CCH-33CCH-1a CCH-5a CCH-81 CCH-20a CCH-62aCCH-5a CCH-20a CCH-79a CCH-78 CCH-100aCCH-8a CCH-75a CCH-64 CCH-37a CCH-14aCCH-11a CCH-70a CCH-62a CCH-14a CCH-83aCCH-20a CCH-87a CCH-20a CCH-16a CCH-8aCCH-64a CCH-87 CCH-76 CCH-51 CCH-33aCCH-75a CCH-8a CCH-78 CCH-83a CCH-37aCCH-5 CCH-62 CCH-62 CCH-31a CCH-5CCH-21 CCH-5 CCH-37a CCH-46a CCH-2aCCH-38 CCH-21 CCH-14a - CCH-36aCCH-81 CCH-81 CCH-34a CCH-67 CCH-37a CCH-37a CCH-38a CCH-6a CCH-38a CCH-51a CCH-16a CCH-21a CCH-51a CCH-79a CCH-69a CCH-16a CCH-57a CCH-14a CCH-68a CCH-20 CCH-70a CCH-34a CCH-57a CCH-30a CCH-79a CCH-111a CCH-51 CCH-107a CCH-107a CCH-38 CCH-86a CCH-11a CCH-20 CCH-38a CCH-83a CCH-22a CCH-51 CCH-16a CCH-107 CCH-38a CCH-14a CCH-69a CCH-31a CCH-43a CCH-16a CCH-68a CCH-36a CCH-68a CCH-69a CCH-57a CCH-67 CCH-82a CCH-74a CCH-20 CCH-46a CCH-9 CCH-89a CCH-51CCH-9 CCH-9CCH-6a CCH-6aCCH-34a CCH-82aCCH-68a CCH-86aCCH-82a CCH-83aCCH-86a CCH-11CCH-63 CCH-34CCH-83a CCH-68CCH-1 CCH-69CCH-8 CCH-107CCH-11 CCH-2aCCH-14 CCH-7aCCH-31 CCH-15aCCH-34 CCH-17aCCH-57 CCH-22aCCH-67 CCH-25aCCH-68 CCH-31aCCH-69 CCH-36aCCH-84 CCH-39aCCH-107 CCH-43aCCH-2a CCH-84aCCH-7aCCH-15aCCH-17aCCH-22aCCH-25aCCH-27aCCH-31aCCH-36aCCH-39aCCH-42aCCH-43aCCH-46aCCH-48aCCH-84aCCH-111a

[0107] DISCUSSION

[0108] The impact of C-terminal amidation was assessed on the activity on a large dataset of AMPs against a panel of bacteria important to poultry and public health. Multiple drug resistance has been characterized in all three pathogens tested (Abd El-Ghany, 2021; Furtula et al., 2010; Diarra et al., 2014; Waters et al., 2011; Kitai et al., 2005). Given that the EC isolate used herein (25922) is a non-pathogenic strain, while APEC is highly pathogenic and often associated with virulence and resistance genes (Furtula et al., 2010), it is understandable that EC was more susceptible to AMPs. In previous work, Furtula et al. (2010) isolated 69 E. coll strains from poultry litter in controlled feeding trials and commercial farms, where all isolates from commercial farms were resistant to at least seven antibiotics. Antibiotic resistance is also observed in Salmonella isolates. Greater than 43% of 193 Salmonella isolates from broiler chicken production facilities in British Columbia, Canada, were resistant to five antibiotics (Diarra et al., 2014). Additionally, 65% of the SE isolates were found to carry both the invasion gene ( / nva) and the Salmonella plasmid virulence gene (spvC). In US meat and poultry samples (n = 136), S. aureus contaminated 47% of the samples (41% of the chicken samples) and of those isolates, 52% were multi-drug resistant (26% of the chicken samples per Waters et al., 2011). Hence, the discovery of antibiotic alternatives for these agriculturally relevant pathogens is critical.

[0109] This example examines the influence the C-terminal amide modification on a large dataset of bioinformatically identified and experimentally verified AMPs. Previous studies focused on the effect of amidation on individual or a small number of peptides (Dennison etal., 2015; Mura etal., 2016; Shaley et al., 2002; Shahmiri etal., 2020), or on AMP database studies (Wang, 2020; Pirtskhalava M et al., 2021). Refinements of high throughput in silico AMP discovery methods continue (e.g., rAMPage), contributing to the understanding how predicted structure and physicochemical properties relate to AMP function and activity, so as to inform the decision-making process regarding which AMPs to test during in vitro validation.

[0110] The present dataset predominantly contained mainly helical or helical AMPs. While most AMPs with known structures are alpha-helical, the machine learning pipeline training dataset contained more helical AMPs than AMPs with alternative secondary structures, which could have resulted in a bias towards the identification of such AMPs from the present datasets. Additionally, there are limitations to using predicted secondary structure classifications. The software used cannot currently account for post-translational modifications, including C-terminal amides. This limited the ability to account for how C-terminal amidation might impact the secondary structure of the AMPs.

[0111] While the impact that C-terminal amides had on AMP structure is not fully elucidated herein, it is clear C-terminal amidation enhances AMP activity. It was found that C-terminal amidation increased the antimicrobial activity against all bacteria tested.Amidation significantly increased over half (APEC) and over a third (ASE and SA) of the activity of AMPs with little increase in toxicity. C-terminal amidation may be a promising route for development of therapeutics against avian pathogens. Studies of individual AMPs showed C-terminal amides stabilized AMP helix formation, improved cell membrane penetration, and increased charge (Mura et al., 2016; Shalev et al., 2002; Shahmiri et al., 2020; Dennison et al., 2011). While the mechanism of action is outside the scope of the examples herein, it is hypothesizes that C-terminal amides had a similar impact on the AMPs with improved activity. A slight increase in hemolysis and decrease in cell viability of AMPs in their C-terminal amide form was observed, as compared to their C-terminal carboxylate form. Other studies have shown that C-terminal amidation did not influence AMP selectivity for microbial over eukaryotic cells (Dennison et al., 2009; Pan et al., 2007).

[0112] The mechanisms of action of AMPs are dependent on both their structural and physicochemical properties (Torres et al., 2019). Initial cell membrane interaction is required for membrane-active AMPs as well as for AMPs with intracellular targets. Membrane-active AMPs are often amphipathic in nature, with a positive charge allowing for electrostatic interaction with the negatively charged microbial membrane and the hydrophobic part of the AMP contributing to membrane insertion (Bahar et al., 2013; Zasloff, 2002). Therefore, it was hypothesized that the hydrophobic and cationic properties of the tested peptides, strongly contribute to these interactions and activity of AMPs.

[0113] Loge(charge) was determined to be negatively correlated to log2(MIC) in MLR analysis indicating that as charge increases, potency increases. However, the logarithmic nature of the charge-potency relationship, determined to be most appropriate, indicates that variation in small charges is more significant than variation in larger charges, implying that while some charge is important for potency, benefits of further increasing charge are limited. Indeed, it was found that in general, positive charges +1 to +5 were associated with greater antimicrobial as well as hemolytic activity for all bacteria tested. AMPs with a positive charge > +5 had no / low antimicrobial and no hemolytic activity. C-terminal amidation increased activity but not cell toxicity at higher charge ranges. Chung et al. (2020) reported that approximately 50% of naturally occurring AMPs have a charge of +2 to +4 and in the Antimicrobial Peptide Database the average net charge is 3.47 (out of 3,705 peptides) (Wang et al., 2015). Therefore, the most active AMPs identified herein were consistent with the values previously described in the literature. Additionally, charge usually provided the most significant mean term contribution to potency in the MLR analysis of amide AMPs against pathogens and second most for carboxylate AMPs.

[0114] Hydrophobicity was associated with AMP activity in the present example, although to a lesser degree than charge. MLR analysis demonstrated a negative correlation between log2(MIC) and GRAVY score, suggesting that potency increases with hydrophobicity. Indeed, all AMPs with moderate to high antimicrobial activity had positive GRAVY scores between 0 and +2. This was interesting, as although an increase incharge and hydrophobicity was correlated to greater potency against pathogens, they also have an inverse correlation, both intuitively, and as observed in the MLR results. Hydrophobicity influences membrane permeability and cell targeting (Kustanovich et al., 2002; Zelezetsky et al., 2005). Increasing hydrophobicity of AMPs increases their lytic activity (Lee et al., 2006). In the present dataset, the average GRAVY score for AMPs with a HC50 value of < 128 pg / mL was 1.1 while AMPs without hemolytic activity had an average GRAVY score of 0.5.

[0115] In contrast to hydrophobicity, the best MLR models for carboxylate AMPs did not include a helicity term. This was likely due to the high correlation between calculated helicity and hydrophobicity, with log2(MIC) being predicted with greater statistical significance using hydrophobicity. This correlation is justified by the amino acids commonly believed to induce helicity, such as alanine, leucine, and valine, are being highly hydrophobic. To enhance confidence in whether helicity, hydrophobicity, or a combination of the descriptors statistically affect potency, further analysis would be required upon AMPs with more opposing helicity and hydrophobicity, such as those containing greater amounts of glutamic acid, and serine.

[0116] The length of an AMP may influence its activity, ability to penetrate a membrane, and other physicochemical characteristics. However, shorter AMP sequences are more desirable for therapeutic development as they reduce synthesis costs. A minimum of seven residues was cited as being necessary to form the amphipathic structures associated with membrane activity (Bahar et al., 2013). Therefore, attention is focused herein on peptides with a length of 7 to 30 residues. In general, MLR analysis demonstrated a negative correlation between log2(MIC) and length, suggesting that as length increases, potency increases. Indeed, it was observed that there was no activity in peptides below 10 residues in length for both the C-terminal carboxylate and amide AMPs. Additionally, length usually provided the most significant mean term contribution to potency in the MLR analysis of carboxylate AMPs against pathogens. Hemolytic activity was observed in AMPs that were >12 amino acids. AMP length is associated with cytotoxicity, with truncated peptides having significantly less toxicity than their original form (Park et al., 2007; Subbalakshmi et al., 1999). Moreover, relative leucine content is associated with peptide activity. In shorter peptides, and as the peptide length increased, the relative leucine content decreased33. In the present dataset, no significant relationship between leucine content and peptide activity was observed, with such MLR models performing poorly (data not shown).

[0117] Further models were produced to determine which AMPs most benefitted most from C terminal amidation, based upon their physicochemical properties. MLR analysis showed a positive correlation between length and change in log2(MIC) upon amidation. This indicated that shorter AMPs benefitted more from the presence of the C-terminal amide compared to longer sequences. While AMPs with a C-terminal amide also had increasing antimicrobial activity with increasing sequence length (AMPs > 22 amino acids), AMPs between 10-20 amino acids long had the largest improvement in MIC when amidated. Incorporating C-terminal amides into short AMP sequences could be used toimprove the activity of AMPs while decreasing production cost. In contrast, loge(charge) was usually found to be statistically insignificant in MLR analysis modelling change in log2(MIC) upon amidation. This was likely due to logarithmic nature of the charge-potency relationship causing changes in higher chances to affect potency less significantly than changes in higher lengths. These observations may suggest that shorter carboxylate AMPs are more vulnerable to debilitating degradation than longer carboxylate AMPs, reducing their potency, but such degradation is less significant for all protected amide AMPs. This would explain why length usually provided the most significant mean term contribution for carboxylate AMPs against pathogens while loge(charge) usually provided the most significant mean term contribution for amide AMPs.

[0118] Against all bacteria tested, the active AMPs demonstrated predominately alpha-helical predicted structure, hydrophobic properties, positive charge, and a minimum sequence length of 10 amino acids, with longer lengths associated with higher activity. The positively charged, often amphipathic alpha-helix is a common amphibian AMP motif associated with membrane disruption (Islam et al., 2023; Haney et al., 2009). This appears to be consistent with the distribution of the AMPs in the present dataset. Similar results were observed in Richter et al. (2022), where most validated AMPs from insect and amphibian datasets were predicted to be alpha-helical in nature. This is consistent with commonly observed amphibian AMP motifs and suggests that these AMPs may be associated with membrane disruption as a potential mechanism of action (Islam et al., 2023; Haney et al., 2009). The identification of conserved sequence motifs related to high activity are particularly intriguing and expand, yet challenge, the need for intact cysteines within the “Rana box” motif (Wang et al., 2015).

[0119] For the other peptides that were bioinformatically identified but did not show activity in the present data, it is possible that they are associated with other functions or mechanisms of action other than direct killing of the bacteria assessed in the present panel and the scope of their activity was not captured in the present analysis. While they were not identified as bioactive in the present tests, this does not exclude them from having antimicrobial activity on other bacteria or having alternative immunomodulatory functions.

[0120] AMPs illustrate potential to mitigate antibiotic use in public health and agricultural sectors. APEC, ASE, and SA are agriculturally relevant pathogens in the poultry industry that are responsible for large annual economic losses and pose public health risks. Here it is shown that a bioinformatic AMP discovery pipeline, rAMPage, could successfully identify AMPs from amphibian RNA-seq datasets and that some of them were active against these three critical poultry pathogens representing both Grampositive and Gram-negative bacteria. The present example provides a comprehensive analysis of a large dataset of AMPs in their natural and C-terminal amidated forms. Analysis of the physicochemical properties of these AMPs showed that generally, longer, more highly charged, and more hydrophobic AMPs are more potent, with the most active AMPs having a net positive charge of +1 to +5. Additionally, it was shown that amidation of the C-terminus significantly increases AMP activity against bacteria tested herein, withanalysis indicating that shorter AMPs benefit most from amidation. An increase in cell toxicity due to amidation was also observed, but to a lesser degree than the paired increase in antimicrobial activity. Amidation of the C-terminus of peptides may be a promising way to increase AMP activity in future therapeutic discovery studies. AMPs identified herein be used as antimicrobials in medicinal applications, or for agricultural applications such as the poultry industry, other for rearing of other.

[0121] Example 2

[0122] Antimicrobial Peptides - Toxicity and Activity

[0123] Select sequences described herein, such as in Example 1, are evaluated for eukaryotic toxicity and efficacy of antimicrobial activity against various microbes. The toxicity testing was conducted against Porcine RBC (HC50) and HEK293 (CC50). The testing with pRBC for toxicity was evaluated, and it was considered of whether both parameters of eukaryotic toxicity (also including CC50) also passed. Evaluation of minimum inhibitory concentration (MIC) for antimicrobial efficacy was deemed adequate in all peptides tested. Eukaryotic toxicity and antimicrobial efficacy (MIC) testing was conducted as previously described in Example 1, and for example also described in WO2024 / 178484 A1 (Birol et al.). In the tables below, the parameter of halfmaximal concentrations to cause hemolysis (HC50 or CC50) is provided, together with n of tests and median absolute deviation (MAD) as a measure of variability where relevant. The peptide naming and sequences shown correspond to the SEQ ID NOs provided in Table 1. For sequences without full MIC data, RNA-Seq data is used from generated skin samples.

[0124] Table 6 shows the results of toxicity testing for two parameters of eukaryotic toxicity: porcine RBC HC50 and HEK293 CC50.

[0125] Table 7 shows the results of antimicrobial activity testing against Escherichia coli ATCC 25922 and Avian Pathogenic Escherichia coli 317.

[0126] Table 8 shows the results of antimicrobial activity testing against Escherichia coli MCR-1 (BCCDC clinical isolate), Escherichia coli MCR-2 (BCCDC clinical isolate), and E. coli MCR / NDM (BCCDC clinical isolate).

[0127] Table 9 shows the results of antimicrobial activity testing against Salmonella enterica serovar Enteritidis ATCC 4931 and Salmonella enterica serovar Enteritidis LS101.

[0128] Table 10 shows the results of antimicrobial activity testing against Select Microbes, specifically: Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213.

[0129] Table 11 shows the overall results of the toxicity and efficacy testing from Table 6 to Table 10 above.

[0130] In this Example, all peptides tested showed efficacy, with the following peptides being of particular interest:

[0131] CCH1_a (SEQ ID NO:195) GIKDILKAGFGSLVKK_a;

[0132] CCH2_a (SEQ ID NO: 197) ILGAILPLVSGLRSHLG_a;

[0133] CCH5 (SEQ ID NO:202) ILGKIIPLVSVLLSHLG;

[0134] CCH5_a (SEQ ID NO:203) ILGKIIPLVSVLLSHLG_a;

[0135] CCH8 (SEQ ID NO:208) ILGKILPLVGGLLNNLC;

[0136] CCH8_a (SEQ ID NO:209) ILGKILPLVGGLLNNLC_a;

[0137] CCH9 (SEQ ID NO:210) ILGAILPLVSGLLRHLG;

[0138] CCH9_a (SEQ ID NO:211) ILGAILPLVSGLLRHLG_a;

[0139] CCH11_a (SEQ ID NO:215) IRGKIIPLVSGLLSHLG_a;

[0140] CCH20_a (SEQ ID NO:233) ILGKILPLVGVLLSNLG_a;

[0141] CCH21 (SEQ ID NO:234) KNIAAHVANGKRIIGALLSAATGLLSHLG;

[0142] CCH21_a (SEQ ID NO:235) KNIAAHVANGKRIIGALLSAATGLLSHLG_a;

[0143] CCH29 (SEQ ID NO:250) GLGSLVINIAAHVANGKR;

[0144] CCH29_a (SEQ ID NO:251) GLGSLVINIAAHVANGKR_a;

[0145] CCH32_a (SEQ ID NO:257) ILGKIVPLVGGLLNNLG_a;

[0146] CCH33 (SEQ ID NO:258) KNIAAHVANGKRILGKILPLVSGLLGNLG;

[0147] CCH33_a (SEQ ID NO:259) KNIAAHVANGKRILGKILPLVSGLLGNLG_a;

[0148] CCH37_a (SEQ ID NO:267) ILGKILPLVSGLLVNLG_a;

[0149] CCH38 (SEQ ID NO:268) IAAHVANGKRIIGALLSAATGLLSHLG;

[0150] CCH38_a (SEQ ID NO:269) IAAHVANGKRIIGALLSAATGLLSHLG_a;

[0151] CCH40 (SEQ ID NO:272) VAAHVANGKRILGKILPLVSGLLGNLG;

[0152] CCH40_a (SEQ ID NO:273) VAAHVANGKRILGKILPLVSGLLGNLG_a;

[0153] CCH44_a (SEQ ID NO:281) ILGKIIPLVS_a;

[0154] CCH45 (SEQ ID NO:282) GSLVKGIAAHVANGKRILGAILPLVSGLL;

[0155] CCH45_a (SEQ ID NO:283) GSLVKGIAAHVANGKRILGAILPLVSGLL_a;

[0156] CCH49 (SEQ ID NO:290) GTGLSCEKRKAYNWRVTFCCY;

[0157] CCH49_a (SEQ ID NO:291) GTGLSCEKRKAYNWRVTFCCY_a;

[0158] CCH54_a (SEQ ID NO:301) VDNGKRILGAILPLFSGLLSHLG_a; and

[0159] CCH56_a (SEQ ID NO:305) ILGFLIYVCKI_a.

[0160] In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the embodiments. However, it will be apparent to one skilled in the art that these specific details are not required.

[0161] The above-described embodiments are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

[0162] References

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Claims

CLAIMS1. An antimicrobial peptide consisting of the amino acid sequence as set forth in any one of the group consisting of SEQ ID NO:258 (CCH33), SEQ ID NO:1-257, and SEQ ID NO:259-361; or a variant thereof having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to any one of SEQ ID NO: 1-361.

2. The antimicrobial peptide of claim 1, wherein the amino acid sequence or variant thereof, has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or has 100% amino acid sequence identity with:(a) any one of the sequences selected from the group consisting of CCH1 to CCH56_a (SEQ ID NO: 194-305); CCH90 (SEQ ID NO:339), and CCH90_a (SEQ ID NO:340);(b) any one of the sequences selected from the group consisting of:CCH1_a (SEQ ID NO:195) GIKDILKAGFGSLVKK_a;CCH2_a (SEQ ID NO: 197) ILGAILPLVSGLRSHLG_a;CCH5 (SEQ ID NO:202) ILGKIIPLVSVLLSHLG;CCH5_a (SEQ ID NO:203) ILGKIIPLVSVLLSHLG_a;CCH8 (SEQ ID NO:208) ILGKILPLVGGLLNNLC;CCH8_a (SEQ ID NO:209) ILGKILPLVGGLLNNLC_a;CCH9 (SEQ ID NO:210) ILGAILPLVSGLLRHLG;CCH9_a (SEQ ID NO:211) ILGAILPLVSGLLRHLG_a;CCH11_a (SEQ ID NO:215) IRGKIIPLVSGLLSHLG_a;CCH20_a (SEQ ID NO:233) ILGKILPLVGVLLSNLG_a;CCH21 (SEQ ID NO:234) KNIAAHVANGKRIIGALLSAATGLLSHLG;CCH21_a (SEQ ID NO:235) KNIAAHVANGKRIIGALLSAATGLLSHLG_a;CCH29 (SEQ ID NO:250) GLGSLVINIAAHVANGKR;CCH29_a (SEQ ID NO:251) GLGSLVINIAAHVANGKR_a;CCH32_a (SEQ ID NO:257) ILGKIVPLVGGLLNNLG_a;CCH33 (SEQ ID NO:258) KNIAAHVANGKRILGKILPLVSGLLGNLG;CCH33_a (SEQ ID NO:259) KNIAAHVANGKRILGKILPLVSGLLGNLG_a;CCH37_a (SEQ ID NO:267) ILGKILPLVSGLLVNLG_a;CCH38 (SEQ ID NO:268) IAAHVANGKRIIGALLSAATGLLSHLG;CCH38_a (SEQ ID NO:269) IAAHVANGKRIIGALLSAATGLLSHLG_a;CCH40 (SEQ ID NO:272) VAAHVANGKRILGKILPLVSGLLGNLG;CCH40_a (SEQ ID NO:273) VAAHVANGKRILGKILPLVSGLLGNLG_a;CCH44_a (SEQ ID NO:281) ILGKIIPLVS_a;CCH45 (SEQ ID NO:282) GSLVKGIAAHVANGKRILGAILPLVSGLL;CCH45_a (SEQ ID NO:283) GSLVKGIAAHVANGKRILGAILPLVSGLL_a;CCH49 (SEQ ID NO:290) GTGLSCEKRKAYNWRVTFCCY;CCH49_a (SEQ ID NO:291) GTGLSCEKRKAYNWRVTFCCY_a;CCH54_a (SEQ ID NO:301) VDNGKRILGAILPLFSGLLSHLG_a; andCCH56_a (SEQ ID NO:305) ILGFLIYVCKI_a; or(c) CCH20_a (SEQ ID NO:233), CCH21 (SEQ ID NO:234), CCH33 (SEQ ID NO:258), CCH37_a (SEQ ID NO:267), CCH38 (SEQ ID NO:268), orCCH45 (SEQ ID NO:282).

3. The antimicrobial peptide of claim 1 or 2, wherein the variant comprises a modification that is a conservative amino acid substitution.

4. The antimicrobial peptide of claim 1, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:258, SEQ ID NO:1-257, and SEQ ID NO:259-361.

5. A composition comprising the antimicrobial peptide according to any one of claims 1 to 4, and a suitable diluent, carrier or excipient.

6. The composition of claim 5, for use in treatment or prevention of a disease or condition;wherein the disease or condition is infectious and / or attributable to Gram-negative bacteria, Gram-positive bacteria, acid fast bacteria, bacteria resistant to other drugs, a virus, a fungi, or a parasites; orwherein the disease or condition is cancer or a tumour, such as a solid tumour or a liquid tumour.

7. The composition of claim 5 or 6, formulated for oral, injectable, rectal, topical, transdermal, nasal, or ocular delivery.

8. A composition for application to a surface for disinfecting or prevention of growth of microbes, said composition comprising the antimicrobial peptide according to any one of claims 1 to 4 and a suitable carrier or diluent.

9. A method of cleaning or disinfecting of a surface, a material, or an environment comprising application of the composition of claim 8 thereto.

10. Use of the antimicrobial peptide as defined in any one of claims 1 to 4 or the composition of any one of claims 5 to 7 for treatment or prevention, or for preparation of a medicament for treatment or prevention, of a disease or condition in a subject in need thereof.

11. A method of treating or preventing a disease or condition comprising administering to a subject in need thereof an effective amount of the peptide according to any one of claims 1 to 4, or the composition according to any one of claims 5 to 7.

12. The use claim 10 or the method of claim 11, wherein the subject is a human or an animal, such as a livestock animal or a pet.

13. A lipid vesicle comprising the antimicrobial peptide of any one of claims 1 to 4.

14. A nucleic acid molecule encoding the antimicrobial peptide of any one of claims 1 to 5.

15. A vector comprising the nucleic acid molecule of claim 14.

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