RU2804774C2 - Bacteriophage-derived antimicrobial polypeptides and their use against gram-negative bacteria - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to bacteriophage-derived antimicrobial polypeptides, and can be used in medicine for the treatment of infections caused by Gram-negative bacteria. The following is proposed: a pharmaceutical composition for growth inhibition, population reduction or destruction of at least one type of gram-negative bacteria, containing an effective amount of the isolated Chp peptide with the following SEQ ID NO: 2, or an active fragment thereof, in which the alpha helical domain is retained, or a modified Chp peptide, the amino acid sequence of which is at least 80% identical to the following SEQ ID NO: 2 and in which the alpha helical domain is retained.

EFFECT: invention provides obtaining antimicrobial agents having a broad spectrum activity against gram-negative pathogens and the ability to persist in the presence of serum.

18 cl, 3 dwg, 13 tbl, 7 ex

Description

LINK TO RELATED APPLICATIONS

[001] This application relies on its filing date and claims benefit from U.S. Provisional Patent Application No. 62/650,235, filed March 29, 2018, the disclosure of which is incorporated herein by reference in its entirety.

LIST OF SEQUENCES

[002] This application contains a Sequence Listing that was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy in question, created on March 28, 2019, is named 0341_0002-PCT_SL.txt and is 28097 bytes in size.

TECHNICAL FIELD

[003] The present invention relates to the field of antimicrobial agents and, in particular, to phage-derived antimicrobial peptides amurins that infect gram-negative bacteria, and the use of these peptides to kill gram-negative bacteria and combat bacterial infection and contamination.

BACKGROUND OF THE ART

[004] Gram-negative bacteria, particularly members of the genus Pseudomonas and the emerging multidrug-resistant pathogen Acinetobacter baumannii, are an important cause of serious and potentially life-threatening invasive infections. Pseudomonas infection is a major concern in burn wounds, chronic wounds, chronic obstructive pulmonary disease (COPD), cystic fibrosis, bacterial growth on implanted biomaterial surfaces and hospital surfaces, and in water supplies, where it poses multiple threats to vulnerable patients.

[005] Once P. aeruginosa has become established in a patient, the infection can be particularly difficult to treat. The P. aeruginosa genome encodes multiple resistance genes, including efflux pumps that mediate multidrug resistance and enzymes that confer resistance to beta-lactam and aminoglycoside antibiotics, making therapy particularly challenging for this Gram-negative pathogen due to the lack of new antimicrobial drugs. Contributing to this problem is the ability of P. aeruginosa to grow in biofilm, which may enhance its ability to cause infections by protecting the bacteria from host immune defenses and chemotherapy.

[006] The incidence of drug-resistant strains of Pseudomonas aeruginosa is increasing in health care settings. In an observational study of health care-associated bloodstream infections (BSIs) in community hospitals, P. aeruginosa was one of the four most common multidrug-resistant pathogens (MDR), contributing 18% to overall hospital mortality. In addition, MDR-P flashes. aeruginosa is supported by numerous documents. Poor outcomes are associated with MDR strains of P. aeruginosa, which often require treatment with drugs of last resort such as colistin.

[007] Other drug-resistant bacteria considered a significant threat by the World Health Organization (WHO) and the Centers for Disease Control (CDC) include the following Gram-negative bacteria: Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacteriaceae (including Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae), Salmonella spp., Neisseria gonorrhoeae and Shigella spp. (Tillotson G. 2018. A crucial list of pathogenes. Lancet Infect Dis 18:234-236).

[008] To meet the need for new antimicrobial agents with new mechanisms, researchers are studying various drugs and biologics. One such class of antimicrobial agents includes lysines. Lysines are cell wall peptidoglycan hydrolases that act as "molecular scissors" to disrupt the peptidoglycan network structure responsible for maintaining cell shape and resisting internal osmotic pressure. The destruction of peptidoglycan leads to osmotic lysis. However, some lysines were ineffective against Gram-negative bacteria, at least in part due to the presence of an outer membrane (OM), which is absent in Gram-positive bacteria and which limits access to the underlying peptidoglycan. Modified lysines (“artilisins”) have also been developed. These agents, which contain lysines fused to special α-helical domains with polycationic, amphipathic and hydrophobic properties, are able to move through the NM. However, some artilisins exhibit low activity in vivo. The reason for this may be components of human serum and, in particular, physiological salt and divalent cations. These components compete for lipopolysaccharide binding sites and may interfere with α-helical lysine translocation domains, thereby limiting activity in the blood and limiting the effectiveness of some lysines and artilisins for the treatment of invasive infections. Similar lack of activity in the blood has been reported for a variety of different outer membrane-permeating and membrane-destabilizing antimicrobial peptides.

[009] In addition to klysins and artilisins, other phage-encoded host lysis systems have been discovered, including “amurines” (Chamakura KR et al., 2017. Mutational analysis of the MS2 lysis protein L. Microbiology 163:961-969). The term amurin describes a limited set of non-muralytic (not “wall-killing”, i.e. not based on hydrolysis of cell wall peptidoglycans) lysis activities from both ssDNA and ssRNA phages (Microviridae and Leviviridae, respectively). For example, the amur E protein of phage ϕX174 (family Microviridae, genus Microvirus) is a 91-amino acid membrane protein that causes lysis by inhibiting the bacterial translocase MraY, an important membrane-bound enzyme that catalyzes the formation of the murein precursor lipid I (Zheng Y et al. ., 2009. Purification and functional characterization of phiX174 lysis protein E. Biochemistry 48:4999-5006). In addition, the capsid protein A2 of phage Qβ (family Leviviridae, genus Allolevivirus) is a structural protein (and amurine) of 420 amino acids that causes lysis by interfering with MurA activity and disrupting the regulation of peptidoglycan biosynthesis (Gorzelnik KV et al., 2016. Proc Natl Acad Sci USA 113:11519–11524). Other non-limiting examples include amurin LysM from phage M, which is a specific inhibitor of E. coli lipid II flippase MurJ, and amurin protein L from phage MS2 (family Levivirdae, genus Levivirus), which is a 75 amino acid integral membrane protein and causes lysis in a manner requiring the activity of the host chaperone DnaJ (Chamakura KR et al., 2017. J Bacterid 199). The putative structure of the L-like amurine domains has been determined and contains an internal dipeptide leucylserine immediately preceded by a region of 10–17 hydrophobic residues. These amurins are integral membrane proteins and have not yet been purified or used as lysines. In addition, their targets are located in the cytoplasm. They have not been studied as lytic agents. Some amurines have been described in detail, for example in published PCT application No. WO 2001/009382, but at best they are the basis for drug development and have not been developed into antibacterial drugs.

[0010] Although recent publications have described lysines/artilisins and other host lysis systems (eg, amurines) that can be used against Gram-negative bacteria with varying degrees of effectiveness in vivo, there remains a need for additional antibacterial compounds that target Multidrug-resistant P. aeruginosa and other Gram-negative bacteria for the treatment of invasive infections, and especially antibacterial compounds that are highly soluble, remain active in vivo in the presence of serum, and/or lack hemolytic activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1A shows three-dimensional models predicted by I-Tasser of the structures of the Chlamydia phage peptide (Chp) family members Chp1, Chp 2, Chp4, Chp5, Spp6, Spp7, Esp1, Esp2, and Osp1. The human innate immune effector peptide LL-37 is included for comparison. Alpha-helical structures are clearly visible, and the top end is typically the N-terminus.

[0012] Figure 1B shows the consensus secondary structure prediction of Chp2 (SEQ ID NO: 2) using JPRED4. Alpha helices are indicated by thick shaded bands.

[0013] Figure 1C shows the consensus secondary structure prediction of Chp4 (SEQ ID NO: 4) using JPRED4. Alpha helices are indicated by thick shaded bands.

[0014] Figure 2A shows a rooted (UPGMA) phylogenetic tree of some members of the Chp family resulting from the ClustalW alignment.

[0015] Figure 2B shows an unrooted (neighbor-joining clustering) phylogenetic tree of some Chp family members resulting from a ClustalW alignment.

[0016] Figure 3 is a series of micrographs showing microscopic analysis (2000x magnification) of Pseudomonas aeruginosa strain 1292 treated for 15 minutes with Chp2 (10 μg/ml) or buffer control ("untreated"), in 100% human serum. Samples were stained using the Live/Dead Cell Viability Kit (ThermoFisher) and examined by both differential interference contrast (DIC) and fluorescence microscopy. The microphotographs show the absence of dead bacteria in the untreated series and a decrease in the number of live bacteria in the treated series.

[0017] This application describes a new class of phage-derived lytic agents that are derived, for example, from Microviridae genomic sequences and are distinct from other similar agents, including known lysines/artilisins and amurines. The phage-derived lytic agents described herein are referred to as chlamydial phage peptides (Chp), also called "amurine peptides" (a functional term not implying sequence similarity to amurines). This application describes 40 Chp peptides that have been identified to constitute a family of specific bacteriolytic proteins. Several of the Chp peptides described herein show significant sequence similarity to each other, but are distinct from other known peptides present in sequence databases. Despite the unique sequences of the Chp peptides, they are all predicted to form alpha-helical structures similar to those of some previously described antimicrobial peptides (AMPs) of the vertebrate innate immune systems (E.F. Haney et al., 2017, in Hansen PR (ed.) , Antimicrobial Peptides: Methods and Protocols, Methods in Molecular Biology, volume 1548), but do not have sequence similarity with such AMPs. Consistent with the antibacterial function of the Chp class, this application describes potent broad-spectrum bactericidal activity against Gram-negative pathogens for several different purified Chp peptides. Unlike previously described Microviridae amurines, which have cytoplasmic targets in the biosynthetic machinery of the cell wall that may be limitedly accessible to externally introduced proteins, the Chp peptides described in this application can be used, in purified forms, to exert bactericidal activity “from the outside.” ", i.e., by acting externally on the cell wall. The Chp peptides identified herein represent a new class of antimicrobial agents with broad spectrum activity against Gram-negative pathogens and the ability to persist in the presence of serum.

[0018] In one aspect of the present invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of (i) an isolated Chp peptide having an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 1 - 4, 6 - 26 and 54 - 66, or active fragments thereof, or (ii) a modified Chp peptide, the sequence of which is at least 80%, for example at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, at least 99% identical to at least one of SEQ ID NOs: 1 - 4, 6 - 26 and 54 - 66, wherein the modified Chp peptide inhibits growth, reduces the population and/or destroys at least one species of gram-negative bacteria, optionally in the presence of human serum. In some embodiments, the at least one species of gram-negative bacteria includes Pseudomonas aeruginosa.

[0019] In another embodiment described herein, the pharmaceutical composition contains a pharmaceutically acceptable carrier and an effective amount of an isolated Chp peptide selected from the group consisting of Chp1, Chp 2, Chp3, Chp4, Chp6, Chp7, Chp8, Chp9, Chp10, Chp11, Chp12, CPAR39, Gkh1, Gkh2, Unp1, Ecp1, Tma1, Ecp2, Osp1, Unp2, Unp3, Gkh3, Unp5, Unp6, Spi1, Spi2, Eср3, Ecp4, Lvp1, Lvp2, ALCES1, AVQ206, AVQ244, CDL907, AGT915, HH3930, Fen7875 and SBR77, or active fragments thereof.

[0020] In some embodiments, the Chp peptide is Chp2, Chp4, Chp6, Ecp1, or Ecp2.

[0021] In various embodiments of the present invention, the pharmaceutical composition contains a pharmaceutically acceptable carrier and an effective amount of (i) an isolated Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65 and SEQ ID NO: 66, or active fragments thereof.

[0022] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of (i) an isolated Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6, SEQ ID NO: 16; SEQ ID NO: 18; and SEQ ID NO: 54, or active fragments thereof.

[0023] In some embodiments, the Chp peptide provided herein, or active fragments thereof, contains at least one unnatural modification relative to any of the amino acid sequences of SEQ ID NOs: 1 - 4, 6 - 26, and 54 - 66, and in some embodiments, said non-naturally occurring modification is selected from the group consisting of modification by substitution, such as an amino acid substitution; modification by N-terminal acetylation and modification by C-terminal amidation. In some embodiments, the modified Chp peptide comprises at least one amino acid substitution, insertion, or deletion relative to any of the amino acid sequences of SEQ ID NOs: 1 to 4, 6 to 26, and 54 to 66, wherein the modified Chp peptide inhibits growth, reduces population, and/or or kills at least one species of gram-negative bacteria, optionally in the presence of human serum. In some embodiments, the at least one species of gram-negative bacteria includes Pseudomonas aeruginosa. In some embodiments, the at least one amino acid substitution is a conservative amino acid substitution. In some embodiments, the modified Chp peptide containing at least one amino acid substitution relative to any of the amino acid sequences of SEQ ID NO: 1 - 4, 6 - 26, and 54 - 66 is a cationic peptide containing at least one alpha-helical domain .

[0024] The pharmaceutical composition in some embodiments may be a solution, suspension, emulsion, inhalable powder, aerosol, or spray. In some embodiments, the pharmaceutical composition may also contain one or more antibiotics suitable for treating gram-negative bacteria. Optionally, the Chp1 peptide is excluded, so that the pharmaceutical composition does not contain Chp1.

[0025] In some embodiments, this application provides a vector containing a nucleic acid that encodes (i) a Chp peptide having an amino acid sequence selected from the group consisting of the sequences SEQ ID NOs: 1 - 4, 6 - 26, and 54 - 66, or active fragments thereof, or (ii) a Chp peptide, the sequence of which is at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, or at least 99% identical to at least one of SEQ ID NOs: 1 - 4, 6 - 26 and 54 - 66, wherein the modified Chp peptide inhibits growth, reduces population and/or or kills at least one species of gram-negative bacteria, optionally in the presence of human serum. In some embodiments, the at least one species of gram-negative bacteria includes Pseudomonas aeruginosa.

[0026] Also provided herein are recombinant expression vectors containing a nucleic acid encoding (i) a Chp peptide containing an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 1 - 4, 6 - 26 and 54 - 66, or active fragments thereof, or (ii) a modified Chp peptide, the sequence of which is at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95 %, at least 98% or at least 99% identical to at least one of SEQ ID NOs: 1 - 4, 6 - 26 and 54 - 66, wherein the modified Chp peptide inhibits growth, reduces population and/or kills at least one species of gram-negative bacteria, optionally in the presence of human serum. In some embodiments, the at least one species of gram-negative bacteria includes Pseudomonas aeruginosa. In some embodiments, the nucleic acid is operably linked to a heterologous promoter. In some embodiments, the nucleic acid encodes a Chp peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 62; SEQ ID NO: 63 and SEQ ID NO: 66, or active fragments thereof, and in some embodiments, the nucleic acid encodes a Chp peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 16; SEQ ID NO: 18 and SEQ ID NO: 54, or active fragments thereof.

[0027] Additional embodiments described herein include an isolated host cell containing the vectors mentioned above. In some embodiments, the nucleic acid sequence is a cDNA sequence.

[0028] In another further aspect, the present invention provides an isolated purified nucleic acid encoding a Chp peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-26 and 54-66, or active fragments thereof. In some embodiments, the nucleic acid encodes a Chp peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4, 6 to 26, and 54 to 66, or active fragments thereof. In an alternative embodiment, the isolated purified DNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27 - 53 and 68 - 80, and in some embodiments, the isolated purified DNA comprises a nucleotide sequence selected from the group consisting of SEQ sequences ID NOs: 27 - 30, 32 - 53 and 68 - 79. Optionally, the nucleic acid is cDNA. In some embodiments, the nucleotide sequence comprises at least one non-naturally occurring modification, such as a mutation (eg, substitution, insertion, or deletion), or a nucleic acid sequence encoding an N-terminal modification or a C-terminal modification.

[0029] In other aspects of the present invention, various methods/applications are provided. One such use is a method of inhibiting the growth, population reduction and/or destruction of at least one species of gram-negative bacteria, the method comprising contacting the bacteria with a composition containing an effective amount of (i) a Chp peptide containing an amino acid sequence selected from the group, consisting of the sequences SEQ ID NO: 1 - 4, 6 - 26 and 54 - 66, or active fragments thereof, or (ii) a modified Chp peptide that is at least 80% consistent, for example, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, or at least 99% identical to it, wherein the modified Chp peptide inhibits said growth, reduces said population and/or destroys said at least one species of gram-negative bacteria. In some embodiments, the Chp peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 62; SEQ ID NO: 63 and SEQ ID NO: 66, or active fragments thereof, and in some embodiments, the Chp peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 16; SEQ ID NO: 18 and SEQ ID NO: 54, or active fragments thereof.

[0030] Also provided herein is a method of inhibiting the growth, population reduction and/or destruction of at least one species of gram-negative bacteria, the method comprising contacting the bacteria with a composition containing an effective amount of a Chp peptide selected from the group consisting of Chp1, Chp 2, Chp3, Chp4, Chp6, Chp7, Chp8, Chp9, Chp10, Chp11, Chp12, CPAR39, Gkh1, Gkh2, Unp1, Ecp1, Tma1, Ecp2, Osp1, Unp2, Unp3, Gkh3, Unp5, Unp6, Spi1, Spi2 , Ecp3, Ecp4, Lvp1, Lvp2, ALCES1, AVQ206, AVQ244, CDL907, AGT915, HH3930, Fen7875 and SBR77, or active fragments thereof, wherein said Chp peptide or active fragments thereof have the ability to inhibit growth, reduce population and/or destroy at least one species of gram-negative bacteria.

[0031] In some embodiments, the at least one species of gram-negative bacteria is Pseudomonas aeruginosa, and in some embodiments, the method further includes killing at least one other species of gram-negative bacteria in addition to Pseudomonas aeruginosa.

[0032] Also provided herein is a method of treating a bacterial infection caused by gram-negative bacteria, comprising administering a pharmaceutical composition described herein to a subject who has been diagnosed with, is at risk of developing, or is exhibiting symptoms of a bacterial infection.

[0033] In any of the preceding methods/applications, said gram-negative bacteria may be at least one gram-negative bacterium selected from the group consisting of Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Salmonella spp., Neisseria gonorrhoeae, and Shigella spp. In some embodiments, the gram-negative bacterium is Pseudomonas aeruginosa.

[0034] Also provided herein is a method of treating or preventing a topical or systemic pathogenic bacterial infection caused by gram-negative bacteria, comprising administering a pharmaceutical composition described herein to a subject in need of such treatment or prevention.

[0035] Additionally, this application provides a method of preventing or treating a bacterial infection, comprising co-administering to a subject who has been diagnosed with, is at risk of developing, or exhibiting symptoms of a bacterial infection, a combination of a first amount of a pharmaceutical composition described herein and a second amount of an antibiotic, suitable for treating a gram-negative bacterial infection, wherein said first and said second amounts are collectively effective to prevent or treat a gram-negative bacterial infection.

[0036] In some embodiments, an antibiotic suitable for treating a gram-negative bacterial infection is selected from one or more of the following: ceftazidime, cefepime, cefoperazone, ceftobiprole, ciprofloxacin, levofloxacin, aminoglycosides, imipenem, meropenem, doripenem, gentamicin, tobramycin, amikacin, piperacillin, ticarcillin, penicillin, rifampicin, polymyxin B and colistin. In some embodiments, the antibiotic is selected from one or more of amikacin, azithromycin, aztreonam, ciprofloxacin, colistin, fosfomycin, gentamicin, imipenem, piperacillin, rifampicin, and tobramycin.

[0037] In yet another embodiment, there is provided a method of increasing the effectiveness of an antibiotic suitable for treating a Gram-negative bacterial infection, comprising co-administrating the antibiotic in combination with a pharmaceutical composition described herein, wherein administration of such combination is more effective in inhibiting the growth, reducing the population or eradicating Gram-negative bacteria. bacteria than administering either the antibiotic or the pharmaceutical composition separately.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0038] As used herein, the following terms and their cognates shall have the following meanings unless the context clearly indicates otherwise.

[0039] "Carrier" refers to the solvent, excipient, excipient, dispersion medium, solubilizing agent, coating, preservative, isotonic and absorption retardant agent, surfactant, propellant, diluent, vehicle and the like with which the active compound. Such carriers may be sterile liquids such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions and oils, including petroleum, animal, vegetable or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil and similar oils.

[0040] "Pharmaceutically acceptable carrier" refers to any and all solvents, excipients, excipients, dispersion media, solubilizing agents, coatings, preservatives, isotonic and absorption retarding agents, surfactants, propellants, diluents, media and the like agents that are physiologically compatible. The carrier(s) must be "acceptable" in the sense that it is not harmful to the subject to be treated in amounts typically used in medicinal products. Pharmaceutically acceptable carriers are compatible with the other ingredients of the composition without rendering the composition unsuitable for its intended purpose. Moreover, pharmaceutically acceptable carriers are suitable for use in the subjects provided herein without undue undesirable side effects (such as toxicity, irritation and allergic response). Side effects are "unreasonable" when their risk outweighs the benefit provided by the composition. Non-limiting examples of pharmaceutically acceptable carriers or excipients include any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water and emulsions such as oil/water emulsions and microemulsions. Suitable pharmaceutical carriers are described, for example, in Remington's Pharmaceutical Sciences, E.W. Martin, 18th edition. The pharmaceutically acceptable carrier may be a carrier that does not exist in nature.

[0041] "Bactericidal" or "bactericidal activity" refers to the ability to cause bacterial death or the ability to kill bacteria to a reduction in number of at least 3 orders of magnitude (99.9%) or greater reduction from the original bacterial population over a period 18-24 hours.

[0042] “Bacteriostatic” or “bacteriostatic activity” refers to the ability to inhibit bacterial growth, including inhibition of growing bacterial cells, thereby causing a reduction in their number of 2 orders of magnitude (99%) or greater, and up to a reduction of almost 3 order of magnitude of the original bacterial population over a period of 18-24 hours.

[0043] "Antibacterial" refers to both bacteriostatic and bactericidal agents.

[0044] "Antibiotic" refers to a compound that has properties that have a negative effect on bacteria, such as the ability to cause their death or reduce their growth. An antibiotic may have a negative effect on gram-positive bacteria, gram-negative bacteria, or both types of bacteria. As an example, an antibiotic may affect cell wall peptidoglycan biosynthesis, cell membrane integrity, or DNA or protein synthesis in bacteria. Non-limiting examples of antibiotics active against gram-negative bacteria include cephalosporins such as ceftriaxone-cefotaxime, ceftazidime, cefepime, cefoperazone and ceftobiprole; fluoroquinolones such as ciprofloxacin and levofloxacin; aminoglycosides such as gentamicin, tobramycin and amikacin; piperacillin, ticarcillin, imipenem, meropenem, doripenem, broad-spectrum penicillins with or without beta-lactamase inhibitors, rifampicin, polymyxin B and colistin.

[0045] “Drug-resistant” generally refers to bacteria that are resistant to the antibacterial activity of the drug. In some cases, "drug-resistant" may specifically refer to antibiotic resistance. In some cases, a bacterium that is typically sensitive to a particular antibiotic can develop resistance to that antibiotic, causing it to become a drug-resistant microbe or strain. A "multidrug-resistant" ("MDR") pathogen is a pathogen that has developed resistance to at least two classes of antimicrobial drugs, each used as monotherapy. For example, some strains of S. aureus have been found to be resistant to multiple antibiotics, including methicillin and/or vancomycin (Antibiotic Resistant Threats in the United States, 2013, US Department of Health and Human Services, Centers for Disease Control and Prevention). One skilled in the art can readily determine whether a bacterium is drug resistant by using routine laboratory techniques that can determine the sensitivity or resistance of a bacterium to a drug or antibiotic.

[0046] "Effective amount" refers to an amount that, when used or administered at an appropriate frequency or dosage schedule, is sufficient to prevent, reduce, inhibit or eliminate bacterial growth or bacterial load or to prevent, reduce or reduce the severity of onset, severity, duration, or progression of the disorder being treated (eg, growth of a gram-negative bacterial pathogen or infection caused by it), preventing progression of the disorder being treated, to cause regression of the disorder being treated, or enhancing or improving prophylactic(s) or the therapeutic effect(s) of another therapy, such as antibiotic or bacteriostatic therapy.

[0047] "Co-administer" refers to the administration of two agents, such as a Chp peptide and an antibiotic or any other antibacterial agent, in a sequential manner, as well as the administration of these agents in a substantially simultaneous manner, for example, in a single mixture/composition or in doses, which are given separately but are nonetheless administered substantially simultaneously to a subject, for example, at different times in the same day or 24 hour period. Such co-administration of Chp peptides with one or more additional antibacterial agents can be administered as a continuous treatment over days, weeks or months. In addition, depending on the application, coadministration need not be continuous or uniform in duration. For example, if the use is as a topical antibacterial agent to treat, for example, a bacterial ulcer or an infected diabetic ulcer, the Chp peptide can only be administered initially within 24 hours of administration of the additional antibiotic, and then the use of the additional antibiotic can be continued without further administration of the Chp peptide.

[0048] "Subject" refers to a mammal, plant, lower animal, single-celled organism, or cell culture. For example, the term “subject” is intended to include organisms, such as prokaryotes and eukaryotes, that are affected by or susceptible to bacterial infection, such as gram-positive or gram-negative bacterial infections. Examples of subjects include mammals, for example, humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats and transgenic non-human animals. In some embodiments, the subject is a human, such as a human, suffering from, at risk of suffering from, or susceptible to infection with gram-negative bacteria, which infection may be systemic, topical, or otherwise concentrated or limited to a particular organ or tissue.

[0049] "Polypeptide" is used interchangeably with the term "peptide" herein and refers to a polymer composed of amino acid residues and typically containing at least about 30 amino acid residues. The term includes not only polypeptides in isolated form, but also their active fragments and derivatives. Also included within the scope of the term “polypeptide” are fusion proteins or fusion polypeptides containing the Chp peptide described herein and retaining, for example, lytic function. Depending on the context, the polypeptide may be a naturally occurring polypeptide or a recombinant, engineered or synthetically produced polypeptide. A particular Chp peptide, for example, can be prepared or separated from the native protein by enzymatic or chemical digestion, or can be prepared using conventional peptide synthesis techniques (eg, solid phase synthesis) or molecular biology techniques (such as those described in Sambrook, J. et al. ., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (1989)), or can, following a certain strategy, be shortened or fragmented to obtain active fragments that retain, for example, lytic activity against the same or at least one target bacterium of common origin.

[0050] "Fusion polypeptide" refers to an expression product resulting from the joining of two or more nucleic acid fragments, resulting in a fusion expression product typically containing two or more domains or fragments that typically have different properties or sets of functionalities. characteristics. More specifically, the term "fusion polypeptide" may also refer to a polypeptide or peptide containing two or more heterologous polypeptides or peptides linked by a covalent bond, either directly or through an amino acid or peptide linker. The polypeptides forming a fusion polypeptide are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The term "fusion polypeptide" may be used interchangeably with the term "fusion protein." The non-limiting phrase "polypeptide comprising" a structure includes molecules larger than the structure, such as fusion polypeptides.

[0051] “Heterologous” refers to sequences of nucleotides, peptides, or polypeptides that are not naturally contiguous. For example, in the context of the present invention, the term "heterologous" can be used to describe a combination or fusion of two or more peptides and/or polypeptides, wherein the fusion peptide or polypeptide is not typically found in nature, such as, for example, a Chp peptide or an active fragment thereof and a cationic and/or polycationic peptide, amphipathic peptide, sushi peptide (Ding et al. Cell Mol Life Sci., 65(7-8): 1202-19 (2008)), defensin peptide (Ganz, T. Nature Reviews Immunology 3, 710-720 (2003)), a hydrophobic peptide and/or an antimicrobial peptide that may have increased lytic activity. The scope of this definition includes two or more Chp peptides or active fragments thereof. They can be used to produce a fusion polypeptide with lytic activity.

[0052] "Active fragment" refers to a portion of a polypeptide that retains one or more functions or biological activities of the isolated polypeptide from which the fragment was derived, for example, bactericidal activity against one or more gram-negative bacteria.

[0053] "Amphipathic peptide" refers to a peptide containing both hydrophilic and hydrophobic functional groups. In some embodiments, the secondary structure may position hydrophobic and hydrophilic amino acid residues on opposite sides (eg, on the inside and on the outside when the peptide is in a solvent, such as water) of the amphipathic peptide. These peptides, in some embodiments, may adopt a helical secondary structure, such as an alpha helical secondary structure.

[0054] "Cationic peptide" refers to a peptide containing a high percentage of positively charged amino acid residues. In some embodiments, the pKa value of the cationic peptide is 8.0 or greater. Also included within the scope of the term “cationic peptide” in the context of the present invention are polycationic peptides, which are synthetically produced peptides predominantly consisting of positively charged amino acid residues such as lysine (Lys) and/or arginine (Arg) residues. Amino acid residues that are not positively charged may be neutrally charged amino acid residues, negatively charged amino acid residues and/or hydrophobic amino acid residues.

[0055] "Hydrophobic group" refers to a chemical group, such as a side chain of an amino acid, that has little or no affinity for water molecules but has a greater affinity for oil molecules. Hydrophobic substances tend to have low or no solubility in water or aqueous phases and are generally non-polar, but tend to be more soluble in oil phases. Examples of hydrophobic amino acids include glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met) and tryptophan (Trp).

[0056] "Enhancement" refers to the degree of activity of an agent, such as antimicrobial activity, that is greater than it would otherwise be. The scope of the term “enhancement” includes additive as well as synergistic (superadditive) effects.

[0057] "Synergistic" or "superadditive" refers to the beneficial effect produced by two agents in combination that is greater than the sum of the effects of the two agents acting independently. In some embodiments, the synergistic or superadditive effect is significantly, i.e., statistically significantly greater than the sum of the effects of the two agents acting independently. One or both active ingredients can be used at a subthreshold level, i.e., a level at which the active substance, when used alone, causes no or very limited effect. This effect can be measured using an assay such as a checkerboard assay. boards" described in this application.

[0058] “Treatment” refers to any process, act, application, therapy, or the like in which a subject, such as a person, receives medical care for the purpose of treating a disorder, eliminating a pathogen, or improving the condition of the subject, directly or indirectly. Treatment also refers to reducing the incidence, alleviating symptoms, preventing relapse, preventing relapse, preventing occurrence, reducing the risk of occurrence, improving symptoms, improving prognosis, or a combination of these effects. The term "treatment" may further include reducing the population, growth rate or virulence of bacteria in a subject, thereby controlling or reducing bacterial infection in the subject or bacterial contamination of an organ, tissue or environment. Thus, a “treatment” that reduces the incidence, for example, may be effective in inhibiting the growth of at least one gram-negative bacterium in a particular habitat, whether the subject or the environment. On the other hand, "treating" an already established infection refers to inhibition of growth, population reduction, destruction, including elimination, of the gram-negative bacteria responsible for the infection or contamination.

[0059] "Prevention" refers to preventing the occurrence, recurrence, spread, onset or development of a disorder, such as a bacterial infection. It is not intended that the present invention be limited to completely preventing or preventing the development of infection. In some embodiments, the onset is delayed or the severity of a subsequently acquired disease is reduced or the likelihood of acquiring a disease is reduced, and the above is an example of prevention.

[0060] "Acquired diseases" refer to diseases manifested by clinical or subclinical symptoms, such as the detection of fever, sepsis or bacteremia, as well as diseases that can be detected by the growth of a bacterial pathogen (for example, in culture), when symptoms associated with such pathology has not yet manifested itself.

[0061] The term “derivative” in the context of a peptide or polypeptide, or active fragments thereof, is intended to include, for example, a polypeptide modified such that it contains one or more chemical molecules other than an amino acid that is not substantially has an adverse effect and does not neutralize lytic activity. The chemical molecule may be linked to the peptide covalently, for example, through an amino-terminal amino acid residue, a carboxy-terminal amino acid residue, or an internal amino acid residue. Such modifications may be natural or non-natural. In some embodiments, the non-naturally occurring modification may include the addition of a protecting or capping group on the reactive group, the addition of a detectable label such as an antibody and/or fluorescent label, the addition or modification by glycosylation, or the addition of a bulking group such as PEG (PEGylation), and other changes known to those skilled in the art. In some embodiments, the non-naturally occurring modification may be a capping modification, such as N-terminal acetylations and C-terminal amidations. Examples of protecting groups that can be added to Chp peptides include, but are not limited to, t-Boc and Fmoc. Widely used fluorescent tagged proteins, such as but not limited to green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and mCherry, are compact proteins that which can be covalently or non-covalently linked to the Chp peptide or fused to the Chp peptide without interfering with the normal functions of cellular proteins. In some embodiments, a polynucleotide encoding a fluorescent protein can be inserted upstream or downstream of a Chp polynucleotide sequence. The result is a fusion protein (e.g., a Chp::GFP peptide) that does not interfere with cellular function or the function of the Chp peptide, to to which it is attached. Conjugation of polyethylene glycol (PEG) to proteins has been used as a method to extend the circulating half-life of many pharmaceutical proteins. Thus, in the context of Chp peptide derivatives, the term “derivative” includes Chp peptides chemically modified by the covalent attachment of one or more PEG molecules. PEGylated Chp peptides are expected to exhibit an extended half-life in the circulation compared to non-PEGylated Chp peptides while maintaining biological and therapeutic activity.

[0062] “Percent amino acid sequence identity” refers to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence, such as a particular Chp peptide sequence, after sequence alignment and introduction of gaps as necessary to achieve the maximum percentage sequence identity , and not considering any conservative substitutions as part of sequence identity. Alignments to determine percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available software such as BLAST or commercially available software such as from DNASTAR. Two or more polypeptide sequences may be any percentage identical from 0 to 100%, or any integer value in between. As used herein, two polypeptides are "substantially identical" if at least 80% of the amino acid residues (e.g., at least about 85%, at least about 90%, at least about 92.5%, at least about 95%, at least about 98%, or at least about 99%) are identical. The term "percentage (%) amino acid sequence identity" described in this application is also used in relation to Chp peptides. Thus, the scope of the term "substantially identical" will include mutated, truncated, fused or otherwise modified in sequence variants of the isolated polypeptides and Chp peptides described herein and their active fragments, as well as polypeptides with significant sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, or at least 99% identical, as measured, for example, by one or more of the methods mentioned above) relative to a reference (wild type or another intact) polypeptide.

[0063] As used herein, two amino acid sequences are "substantially homologous" if at least about 80% of the amino acid residues (e.g., at least about 85%, at least about 90%, at least about 92.5%, at least about 95%, at least about 98%, or at least about 99%) are identical or represent conservative replacements. The sequences of the polypeptides of the present invention are substantially homologous if one or more, for example, up to 10%, up to 15%, or up to 20% of the amino acids of a polypeptide, such as the Chp peptides described herein, are replaced by a similar amino acid or a conservative amino acid substitution, and wherein the resulting peptides have at least one activity (eg, antibacterial action) and/or bacterial specificity of a reference polypeptide, such as the Chp peptides described herein.

[0064] As used herein, a “conservative amino acid substitution” is one in which an amino acid residue is replaced by an amino acid residue containing a side chain of similar charge. Families of amino acid residues containing side chains with similar charges have been identified in the field. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine , tryptophan, histidine).

[0065] "Inhalation composition" refers to pharmaceutical compositions as described herein that are formulated for direct delivery to the respiratory tract during or in conjunction with conventional breathing or assisted ventilation (e.g., by intratracheobronchial, pulmonary, and/or nasal administration), including , but not limited to: atomized, spray, dry powder and/or aerosol formulations.

[0066] “Biofilm” refers to bacteria that adhere to surfaces and aggregate to form a water-containing polymer matrix, which may be composed of bacterial and/or host-derived components. A biofilm is an aggregate of microorganisms in which cells adhere to each other on a biotic or abiotic surface. These adherent cells are often embedded in a matrix consisting of, but not limited to, extracellular polymeric substance (EPS). EPS biofilm, also called mucus (although not everything described as mucus is biofilm) or plaque, is a polymeric conglomerate typically composed of extracellular DNA, proteins, and polysaccharides.

[0067] "Suitable" in the context of an antibiotic suitable for use against certain bacteria refers to an antibiotic that has been found to be effective against those bacteria even if resistance subsequently develops.

[0068] "Outer membrane" or "OM" refers to a feature of gram-negative bacteria. The outer membrane consists of a lipid bilayer with an inner lining of phospholipids and an outer amphiphilic lining consisting mostly of lipopolysaccharide (LPS). LPS contains three main parts: a hexaacylated glucosamine-based phospholipid called lipid A, a polysaccharide core, and a protruding outer polysaccharide chain called the O-antigen. NM is a non-liquid continuous medium stabilized by three main interactions, including: i) avid binding of LPS molecules to each other, especially if cations are present to neutralize phosphate groups; ii) close packing of highly saturated acyl chains; and iii) hydrophobic folding of the lipid A molecule. The resulting structure is a barrier to both hydrophobic and hydrophilic molecules. Beneath the NM, the peptidoglycan forms a thin layer that is very sensitive to hydrolytic degradation: unlike the peptidoglycan of Gram-positive bacteria, which is 30 - 100 nanometers (nm) thick and consists of up to 40 layers, the peptidoglycan of Gram-negative bacteria is only 2 - 3 nm thick and consists only from 1 - 3 layers.

Microviridae phages

[0069] Members of the Microviridae family of phages may be of particular interest as potential sources of anti-infective agents for a number of reasons. As described herein, it has been discovered that a large subpopulation of these phages, including phages of the chlamydial genus Microvirus (family Microvirus, subfamily Gokushovirinae), do not contain the conserved amurin sequence, but instead encode small, uncharacterized cationic peptides that appear to form the basis a hitherto uncharacterized lytic system. In addition, bacteriophages of the Microviridae family infect medically important organisms, including members of the Enterobacteriaceae, Pseudomonadaceae, and Chlamydiaceae families (Doore SM et al. 2016. Virology 491:45-55.). They also lack amurins and instead, as described herein, encode unique uncharacterized antimicrobial-like peptides (termed amurin peptides) that have not previously been identified or have no defined function. We conclude that if putative antimicrobial-like peptides act in a manner similar to previously described antimicrobial peptides (AMPs), they are predicted to allow “external lysis” in a manner not possible for amurins and their cytoplasmic targets.

[0070] Based on a bioinformatic analysis of all annotated Microviridae genomic sequences in GenBank (with an emphasis on phages that lack amurins), 40 novel and syntenic open reading frames were identified. They encode small cationic peptides with a predicted alpha-helical structure similar to AMP (but with amino acid sequences dissimilar to AMP) from the innate immune systems of various vertebrates. These peptides, collectively termed “Chp peptides” or “amurine peptides,” are primarily found in chlamydiae of the genus Microvirus and, to a lesser extent, in other related members of the subfamily Gokushovirinae. See, for example, Tables 1 and 2 below. Chp peptides from a number of Microviridae phages may exhibit 30 to 100% identity to each other and may have little or no homology to other peptides in the protein sequence database. See for example table 3 below. Based on the prediction that Chp peptides have AMP-like activities, 39 different members of the family (Chp2 and Chp3 were identical amino acid sequences) were synthesized for testing in various aspartate aminotransferase (ACT) assays. Based on minimum inhibitory concentration (MIC) values of 0.25 - 4 μg/mL in the presence of human serum, several Chp peptides demonstrated superior activity in serum compared with a panel of up to 17 known AMPs tested (including innate immune effectors and their derivatives). Moreover, have demonstrated activity against a range of Gram-negative pathogens, including several on the World Health Organization (WHO) and Centers for Disease Control (CDC) priority list, including P. aeruginosa, E coli, E, cloacae, K. pneumoniae, A. baumannii, and S. typhimurium.

[0071] For at least two of the effective Chp peptides, Chp2 and Chp4 have demonstrated the ability to synergize in vitro with a range of up to 11 antibiotics against P. aeruginosa, including antibiotics used in the clinical treatment of Gram-negative infections. In addition, both Chp2 and Chp4 showed strong anti-biofilm activities in the MBA assay format (Minimum Biofilm Interruptive Concentration, MBA = 0.25 μg/ml) and bactericidal activity in the time-kill assay format at concentrations up to 1 μg/ml or lower. See example 5 below.

[0072] Overall, these findings are consistent with both the role of Chp family members in host cell lysis (in the context of the bacteriophage life cycle) and the use of purified Chp peptides or their derivatives as broad-spectrum antimicrobial agents to target Gram-negative organisms. pathogens. One major drawback of previously described AMPs as a treatment for invasive infections concerns red blood cell toxicity and generalized membranolytic activity (i.e., hemolysis) (Oddo A. et al., 2017. Hemolytic Activity of Antimicrobial Peptides. Methods Mol Biol 1548: 427-435). Typically, this can be examined in vitro using a standardized human red blood cell lysis detection assay. Many of the Chp peptides described herein do not exhibit hemolytic activity against human red blood cells, in contrast to several AMPs that have been reported in the literature (as well as Triton X-100) to have hemolytic activity. In some embodiments, the Chp peptides described herein may exhibit only minimal or no hemolytic activity against human red blood cells compared to AMPs. Another disadvantage of AMP described in the literature concerns the loss of activity in the presence of the extracellular matrix of human blood and at physiological salt concentrations (Mohanram N. et al., 2016. Salt-resistant short antimicrobial peptides. Biopolymers 106:345-356); indeed, this effect of known AMPs can be seen below in Table 6. The results provided herein demonstrate that some Chp peptides are active in the presence of either human serum or plasma and/or are active in growth media such as Mueller-Hinton and medium based on casamino acids containing physiological concentrations of salts. Without being bound by any theory, it is believed that the differences observed in the activities of Chp peptides and AMP peptides (in the literature) can be explained by differences in the sources of the two types of agents, with Chp peptides being derived from phage and AMPs being largely derived from innate effectors. vertebrate immune systems. The high activity of Chp peptides, the activity of Chp peptides in the extracellular matrix of blood and/or the lack of hemolytic activity make them suitable for use in the treatment of invasive diseases. For example, in some embodiments, Chp peptides may be active in nanomolar amounts.

[0073] In summary, while pathogen-targeted lysine-based drugs have the potential to serve as targeted therapies for the treatment of severe single-microbial infections caused by known MDR pathogens, there is still an unmet medical need for agents to combat severe and life-threatening infections, e.g. , polymicrobial resistant gram-negative infections (for example, some intra-abdominal infections, as well as infections of severe burns, surgical and other wounds). The Chp peptides described herein help meet this need as they have been shown to exhibit potent activity against all major ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter) commonly associated with MDR and are expected to be active against a variety of Gram-negative bacteria. The Chp peptides described herein can be active at high nanomolar concentrations comparable to those of active lysines. The Chp peptides described herein may also be responsible for highly effective, rapid, bacteriolytic effects, biofilm elimination, synergy with conventional antibiotics, and synergy with each other, such as synergy between two or more Chp peptides.

[0074] Although the Chp peptides described herein do not necessarily need to be modified by the addition of antimicrobial peptides, in some embodiments, the Chp peptides described herein can be included in the fusion protein. For example, the fusion protein may comprise the Chp peptide described herein and a lysine, such as lysine active against Gram-negative bacteria. In some embodiments, a Chp peptide can be added to the N-terminus or C-terminus of a lysine with or without a linker sequence. It is expected that fusion polypeptides containing more than one bacteriolytic fragment may contribute positively to the bacteriolytic activity of the parent lysine and/or the parent Chp peptide.

Polypeptides.

[0075] This application demonstrates and explains that the Chp peptides described in this section, including wild-type Chp peptides, modified Chp peptides, and derivatives or active fragments thereof, can be used in the pharmaceutical compositions and methods described herein.

[0076] In some embodiments, the Chp peptide is selected from at least one of: Chp1 (SEQ ID NO: 1), Chp2 (SEQ ID NO: 2), CPAR39 (SEQ ID NO: 3), Chp3 (SEQ ID NO: 54); Chp4 (SEQ ID NO: 4), Chp6 (SEQ ID NO: 6), Chp7 (SEQ ID NO: 7), Chp8 (SEQ ID NO: 8), Chp 9 (SEQ ID NO: 9), Chp 10 (SEQ ID NO: 10), Chp 11 (SEQ ID NO: 11), Chp12 (SEQ ID NO: 12), Gkh1 (SEQ ID NO: 13), Gkh2 (SEQ ID NO: 14), Unp1 (SEQ ID NO: 15 ), Ecp1 (SEQ ID NO: 16), Tma1 (SEQ ID NO: 17), Ecp2 (SEQ ID NO: 18), Osp1 (SEQ ID NO: 19), Unp2 (SEQ ID NO: 20), Unp3 (SEQ ID NO: 21), Gkh3 (SEQ ID NO: 22), Unp5 (SEQ ID NO: 23), Unp6 (SEQ ID NO: 24), Spi1 (SEQ ID NO: 25), Spi2 (SEQ ID NO: 26) , Ecp3 (SEQ ID NO: 55), Ecp4 (SEQ ID NO: 56); Lvp1 (SEQ ID NO: 57), Lvp2 (SEQ ID NO: 58), ALCES1 (SEQ ID NO: 59), AVQ206 (SEQ ID NO: 60), AVQ244 (SEQ ID NO: 61), CDL907 (SEQ ID NO : 62), AGT915 (SEQ ID NO: 63), HH3930 (SEQ ID NO: 64), Fen7875 (SEQ ID NO: 65), SBR77 (SEQ ID NO: 66) and Bdp1 (SEQ ID NO: 67), or their active fragments with lytic activity.

[0077] The Chp peptide may be a modified Chp peptide or an active fragment thereof. In some embodiments, the Chp peptide or active fragment thereof comprises at least one modification relative to at least one of SEQ ID NOs: 1 - 4, 6 - 26, and 54 - 66, such as at least one substitution, insertion, or deletion amino acids. In some embodiments, the modified Chp peptide comprises a polypeptide sequence that is at least 80% identical, e.g., at least 85%, e.g., at least 90%, e.g., at least 92.5%, e.g. at least 95%, for example at least 98% or, for example, at least 99% of the amino acid sequence of at least one Chp peptide selected from the group consisting of the sequences SEQ ID NO: 1 - 4, 6 - 26 and 54 - 66, or an active fragment thereof, wherein the modified Chp peptide inhibits the growth, reduces the population of and/or destroys at least one species of gram-negative bacteria, such as P. aeruginosa, and optionally at least one additional species of gram-negative bacteria, described in this application, optionally in the presence of human serum.

[0078] In some embodiments, the Chp peptide is selected from (i) at least one of Chp1 (SEQ ID NO: 1), Chp2 (SEQ ID NO: 2), CPAR39 (SEQ ID NO: 3), Chp3 (SEQ ID NO: 54); Chp4 (SEQ ID NO: 4), Chp6 (SEQ ID NO: 6), Chp7 (SEQ ID NO: 7), Chp8 (SEQ ID NO: 8), Chp10 (SEQ ID NO: 10), Chp11 (SEQ ID NO : 11), Ecp1 (SEQ ID NO: 16), Ecp2 (SEQ ID NO: 18), Esp3 (SEQ ID NO: 55), Ecp4 (SEQ ID NO: 56), Osp1 (SEQ ID NO: 19), Unp2 (SEQ ID NO: 20), Gkh3 (SEQ ID NO: 22), Unp5 (SEQ ID NO: 23), Unp6 (SEQ ID NO: 24), Spi1 (SEQ ID NO: 25), Lvp1 (SEQ ID NO: 57), ALCES1 (SEQ ID NO: 59), AVQ206 (SEQ ID NO: 60), CDL907 (SEQ ID NO: 62), AGT915 (SEQ ID NO: 63) and SBR77 (SEQ ID NO: 66), or their active fragments, or (ii) a modified Chp peptide that is at least 80% consistent, e.g., at least 85%, at least 90%, at least 92.5%, at least 95 %, at least 98%, or at least 99% identical to at least one of SEQ ID NO: 1 - 4, 6 - 8, 10, 11, 16, 18, 19, 21-25, 54 -57, 59, 60, 62, 63 and 66, wherein the modified Chp peptide inhibits the growth, depopulation and/or eradication of Pseudomonas aeruginosa, and at least one additional species of gram-negative bacteria, optionally in the presence of human serum.

[0079] In some embodiments, the Chp peptide is selected from (i) at least one of Chp2 (SEQ ID NO: 2), Chp3 (SEQ ID NO: 54), Chp4 (SEQ ID NO: 4), Chp6 (SEQ ID NO: 6), Ecp1 (SEQ ID NO: 16) and Ecp2 (SEQ ID NO: 18), or active fragments thereof, or (ii) a modified Chp peptide that is at least 80% consistent, e.g. 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, or at least 99% identical to at least one of the SEQ ID sequences NO: 2, 4, 6, 16, and 18, wherein the modified Chp peptide inhibits the growth, reduces the population of and/or kills at least one species of gram-negative bacteria, such as Pseudomonas aeruginosa, and at least one additional species of gram-negative bacteria , optionally in the presence of human serum.

[0080] In some embodiments, the Chp peptide is selected from (i) at least one Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 4; and SEQ ID NO: 6, or active fragments thereof, or (ii) a modified Chp peptide, the sequence of which is at least 92.5% identical to at least one of the sequences of SEQ ID NOs: 2, 4 and 6, wherein the modified peptide Chp inhibits the growth, reduces the population of and/or kills at least one species of gram-negative bacteria, such as Pseudomonas aeruginosa, and at least one additional species of gram-negative bacteria, optionally in the presence of human serum.

[0081] In some embodiment, the Chp peptide of the present disclosure is a derivative of one of the reference Chp peptides that has been chemically modified. Chemical modification includes, but is not limited to, the addition of chemical groups, the creation of new bonds, and the removal of chemical groups. Chemical modifications can occur anywhere on the Chp peptide, including amino acid side chains and the amino or carboxy termini. For example, in some embodiments, the Chp peptide contains an N-terminal acetylation modification. In some embodiments, the Chp peptide or active fragment thereof comprises a modification by C-terminal amidation. Such modifications may be present at more than one site in the Chp peptide.

[0082] Moreover, one or more side groups or terminal groups of the Chp peptide or active fragment thereof can be protected using protecting groups known to one of ordinary skill in the art.

[0083] In some embodiments, Chp peptides, or active fragments thereof, are conjugated to a life-enhancing moiety. In some embodiments, the life-enhancing group is polyethylene glycol. Polyethylene glycol ("PEG") has been used to produce therapeutic polypeptides with extended lifespan (Zalipsky, S., Bioconjugate Chemistry, 6:150-165 (1995); Mehvar, R., J. Pharm, Pharmaceut. Sci., 3:125 -136 (2000), which is incorporated herein by reference in its entirety). The PEG backbone (CH2CH2-0-)n, where n represents the number of repeating monomers, is flexible and amphiphilic. When attached to another chemical molecule, such as a Chp peptide or an active fragment thereof, the PEG polymer chains can protect such polypeptides from the immune response and other clearance mechanisms. As a result, PEGylation may lead to improved efficacy and safety by optimizing pharmacokinetics, increasing bioavailability and reducing immunogenicity and the amount and/or frequency of dosing.

Polynucleotides.

Chp peptides and their active fragments.

[0084] In one aspect of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid molecule encoding a Chp peptide or active fragments thereof having lytic activity. As used herein, the term "lytic activity" includes the ability of a Chp peptide to kill bacteria, reduce bacterial populations, or inhibit bacterial growth, for example, by permeating the outer membrane of gram-negative bacteria (eg, P. aeruginosa) in the presence or absence of human serum. Also included within the scope of the term "lytic activity" is the ability to remove or reduce biofilm and/or the ability to reduce the minimum inhibitory concentration (MIC) of an antibiotic in the presence and/or absence of human serum.

[0085] In some embodiments, the nucleic acid molecule encodes a Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65 and SEQ ID NO: 66 or active fragments thereof.

[0086] In some embodiments, the nucleic acid molecule encodes a Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65 and SEQ ID NO: 66 or active fragments thereof.

[0087] In some embodiments, the nucleic acid molecule encodes a Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 62; SEQ ID NO: 63 and SEQ ID NO: 66 or active fragments thereof, and in some embodiments, the nucleic acid encodes a Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6, SEQ ID NO: 16; SEQ ID NO: 18; and SEQ ID NO: 54 or active fragments thereof.

[0088] In some embodiments, the Chp peptides described herein and active fragments thereof are capable of permeating the outer membrane of gram-negative bacteria. Without being limited by theory, after permeating the outer membrane, Chp peptides or active fragments thereof can destroy peptidoglycan, a major structural component of the bacterial cell wall, resulting in cell lysis. In some embodiments, the Chp peptides or active fragments thereof described herein contain positively charged (and amphipathic) N- and/or C-terminal α-helical domains that promote binding to the anionic outer membrane of gram-negative bacteria to effect translocation into the peptidoglycan located directly below it.

[0089] The ability of a Chp peptide or an active fragment thereof to penetrate the outer membrane of gram-negative bacteria can be assessed using any method known in the art, such as that described in WO 2017/049233, which is incorporated herein by reference in its entirety. For example, the Chp peptide or an active fragment thereof can be incubated with gram-negative bacteria and a hydrophobic compound. Most Gram-negative bacteria are highly resistant to hydrophobic compounds due to the presence of an outer membrane and therefore do not allow the uptake of hydrophobic agents such as 1-N-phenylnaphthylamine (NPN), crystal violet or 8-anilino-1-naphthalenesulfonic acid (ANS). NPN, for example, fluoresces strongly under hydrophobic conditions and weakly under aqueous conditions. Accordingly, NPN fluorescence can be used as a measure of outer membrane permeability.

[0090] In particular, the ability of the Chp peptide or its active fragment to penetrate the outer wall can be assessed by incubating, for example, NPN with gram-negative bacteria, for example, P. aeruginosa strain PA01, in the presence of the Chp peptide or active fragment thereof, the activity of which is to be tested . The higher induction of fluorescence compared to the fluorescence emitted in the absence of Chp peptide (negative control) indicates penetration through the outer membrane. In addition, fluorescence induction can be compared with that of known permeabilizing agents such as EDTA (ethylenediaminetetraacetate), or an antibiotic such as the antibiotic of last resort used to treat P. aeruginosa, i.e., polymyxin B (PMB), to evaluate level of permeabilization of the outer membrane.

[0091] In some embodiments, the Chp peptides described herein, or active fragments thereof, exhibit lytic activity in the presence and/or absence of human serum. Suitable methods for assessing the activity of a Chp peptide or active fragment thereof in human serum are known in the art and are described in the Examples. Briefly, the MIC value (i.e., the minimum concentration of peptide sufficient to inhibit bacterial growth by at least 80% relative to control) can be determined for the Chp peptide or its active fragment and compared, for example, with a compound that is inactive in serum human, for example, phage T4 lysozyme or artilisin GN126. Phage T4 lysozyme is available for purchase, for example, from Sigma-Aidrich, Inc. GN126 corresponds to Art-175, which is described in the literature and is prepared by coupling the AMP SMAP-29 with the GN lysine KZ144. See Briers et al. 2014, Antimicrob, Agents Chemother. 58:3774-3784, which is incorporated herein by reference in its entirety.

[0092] In particular, it is possible to determine the MIC values for the Chp peptide or its active fragment against, for example, laboratory strain PA01 of P. aeruginosa, for example, in Mueller-Hinton medium, Mueller-Hinton medium supplemented with human serum, casamino acid (CAC), described in this application, which contains physiological concentrations of salt, and AS, supplemented with human serum. The use of PA01 allows testing in the presence of elevated serum concentrations because, unlike most clinical isolates, PA01 is insensitive to the antibacterial activity of the extracellular matrix of human blood.

[0093] In some embodiments, the Chp peptides described herein, or active fragments thereof, are capable of reducing biofilm. Methods for estimating the minimum biofilm disruptive concentration (MBIC) of a Chp peptide or its active fragment can be determined using a variant of the MIC broth microdilution method with modifications (see Ceri et al. 1999. J. Clin Microbial. 37:1771-1776, which is incorporated herein by reference in its entirety, and Schuch et al., 2017, Antimicrob.Agents Chemother.61, pages 1-18, which is incorporated herein by reference in its entirety). In this method, fresh colonies of, for example, a P. aeruginosa strain such as ATCC 17647 are suspended in a medium, for example, phosphate buffer solution (PBS), diluted, for example, 1:100 in TSBg (tryptic soy broth supplemented with 0.2 % glucose), add, for example, in 0.15 ml aliquots to a Calgary biofilm growth device (96-well plate with a lid containing 96 polycarbonate tabs; Innovotech Inc.) and incubate, for example, 24 hours at 37°C. The biofilms are then washed and treated, for example, with a series of 2-fold dilutions of lysine in TSBg, for example at 37°C for 24 hours. After treatment, the wells are washed, air dried, for example, at 37°C and stained, for example, with 0.05% crystal violet for 10 minutes. After staining, the dye is removed from the biofilms, for example, in 33% acetic acid and the optical density is determined 600 nm (OD600), for example, extracted crystal violet. The MPBC of each sample represents the minimum concentration of Chp peptide required to remove at least 95% of the biofilm biomass, as assessed by determining the amount of crystal violet.

[0094] In some embodiments, the Chp peptides described herein, or active fragments thereof, reduce the minimum inhibitory concentration (MIC) of an antibiotic in the presence and/or absence of human serum. Any known method for assessing BMD can be used. In some embodiments, a checkerboard assay is used to determine the effect of the Chp peptide or active fragment thereof on the concentration of the antibiotic. The checkerboard assay is based on a modification of the CLSI method for determining broth microdilution MICs (see Clinical and Laboratory Standards Institute (CLSI), CLSI. 2015. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, 10th edition Clinical and Laboratory Standards Institute, Wayne, Pennsylvania, which is incorporated herein by reference in its entirety, and Ceri et al. 1999 J Clin Microbiol 37: 1771-1776, which is also incorporated herein by reference in its entirety).

[0095] Checkerboards are constructed by first preparing columns, for example in a 96-well polypropylene microtiter plate, in which each well contains the same amount of antibiotic diluted 2-fold along the horizontal axis. In a separate plate, comparable rows are prepared in which each well contains the same amount of Chp peptide or its active fragment, diluted, for example, 2 times along the vertical axis. Dilutions of the Chp peptide or active fragment thereof and the antibiotic are then combined such that each column contains a constant amount of the antibiotic and twice the dilution of the Chp peptide, with each row containing a constant amount of the Chp peptide and twice the dilution of the antibiotic. Therefore, each well contains a unique combination of Chp peptide and antibiotic. Bacteria are added to drug combinations at concentrations of 1×10 ⁵ CFU/ml in AS, for example, with or without human serum. The MIC of each drug, alone and in combination, is then recorded, for example, after 16 hours at 37°C in ambient atmosphere. The sum of fractional inhibitory concentrations (ΣFIC) is calculated for each drug and the minimum ΣFIC value (ΣFICmin) is used to determine the effect of the Chp peptide/antibiotic combination.

[0096] In some embodiments, the Chp peptides described herein, or active fragments thereof, exhibit low toxicity to red blood cells. Any technique known in the art can be used to evaluate the possible hemolytic activity of the Chp peptides of the present invention or active fragments thereof.

[0097] In some embodiments, the isolated polynucleotides described herein comprise a nucleic acid molecule that encodes a modified Chp peptide, e.g., a Chp peptide containing one or more insertions, deletions, and/or amino acid substitutions relative to a reference Chp peptide. Such reference peptides Chp contain any of the sequences of SEQ ID NO. 1 - 4, 6 - 26, and 54 - 66. In some embodiments, the sequence of the modified Chp peptide is at least 80%, at least 85%, at least 90%, at least 95%, at least is at least 98% or at least 99% identical to the sequence of a reference Chp polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 - 4, 6 - 26 and 54 - 66.

[0098] Modified Chp peptides according to the present disclosure are typically designed to retain an α-helical domain, the presence or absence of which can be readily determined using various software such as Jpred4 (compio.dundee.ac.uk/jpred) and Helical Wheel (hael.net/helical.htm).

[0099] In some embodiments, the α-helical domain spans most of the molecule. See, for example, Chp1 and Chp4 in Figure 1. In some embodiments, the α-helical domain is interrupted (see, for example, Chp2 in Figure 1) and in some embodiments, the α-helical domain is shortened (see, for example, Chp6 and Osp1 in figure 1). The α-helical domain of Chp peptides as described herein ranges in size from about 3 to 32 amino acids, more typically from about 10 to 25 amino acid residues.

[00100] Modified Chp peptides according to the present description generally retain one or more functional or biological activities of the reference Chp peptide. In some embodiments, the modification improves the antibacterial activity of the Chp peptide. Typically, the modified Chp peptide has improved antibacterial activity in vitro (e.g., in buffer and/or medium) compared to the reference Chp peptide. In other embodiments, the modified Chp peptide has improved antibacterial activity in vitro vivo (eg, in an animal model of infection). In some embodiments, the modification improves the antibacterial activity of the Chp peptide in the absence and/or presence of human serum.

[00101] In some embodiments, the Chp peptides described herein, or variants or active fragments thereof, are capable of inhibiting the growth of, or reducing the population of, or killing P. aeruginosa and optionally at least one other species of gram-negative bacteria in the absence or presence of, or how in the absence and presence of human serum.

[00102] In some embodiments, the nucleic acid molecules described herein encode the active fragment of Chp peptides or modified Chp peptides described herein. The term "active fragment" refers to the portion of the full-length Chp peptide that retains one or more of the biological activities of the reference peptide. Thus, the active fragment of the Chp peptide or modified Chp peptide herein inhibits the growth of, or reduces the population of, or destroys P. aeruginosa and optionally at least one species of gram-negative bacteria described in this application, in the absence or presence, or both in the absence , and in the presence of human serum. Typically, the α-helical domain is retained in active fragments. In some embodiments, the active moiety is a cationic peptide that retains the α-helical domain.

Vectors and host cells.

[00103] In another aspect of the present invention, there is provided a vector containing an isolated polynucleotide containing a nucleic acid molecule encoding any of the Chp peptides or active fragments thereof described herein, or a sequence complementary to the sequence of the isolated polynucleotides of the present invention. In some embodiments, the vector is a plasmid or cosmid. In other embodiments, the vector is a viral vector, and additional DNA fragments can be ligated into the viral vector. In some embodiments, the vector can autonomously replicate in a host cell into which it is introduced. In some embodiments, the vector may integrate into the genome of a host cell upon entry into the host cell and thereby replicate along with the host genome.

[00104] In some embodiments, certain vectors, referred to herein as “recombinant expression vectors” or “expression vectors,” can direct the expression of genes to which they are operably linked. A polynucleotide sequence is "operably linked" when it is placed in a functional relationship with another nucleotide sequence. For example, a promoter or DNA regulatory sequence is said to be “operably linked” to a DNA sequence that encodes an RNA and/or protein if the two sequences are operably linked or positioned such that the promoter or DNA regulatory sequence influences the level of expression of the coding or structural DNA sequence . Functionally related DNA sequences are usually, but not necessarily, in contact.

[00105] In some embodiments, the present invention provides a vector containing a nucleic acid molecule that encodes a Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65 and SEQ ID NO: 66 or active fragments thereof.

[00106] In some embodiments, the vector contains a nucleic acid molecule that encodes a Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65 and SEQ ID NO: 66 or active fragments thereof.

[00107] In some embodiments, the vector contains a nucleic acid molecule that encodes a Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 62; SEQ ID NO: 63 and SEQ ID NO: 66 or active fragments thereof, and in some embodiments, the vector contains a nucleic acid molecule that encodes a Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6, SEQ ID NO: 16; SEQ ID NO: 18 and SEQ ID NO: 54 or active fragments thereof.

[00108] In general, any system or vector suitable for maintaining, propagating or expressing a polypeptide in a host can be used to express the Chp peptides described herein or active fragments thereof. A suitable DNA sequence/polynucleotide sequence can be incorporated into an expression system using any of various well known and conventional techniques, such as, for example, those described in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual (3rd ed.) , volumes 1 - 3, Cold Spring Harbor Laboratory (2001). In addition, tags can also be added to Chp peptides or their active fragments to provide convenient isolation methods, such as c-myc, biotin, poly-His, etc. Kits for such expression systems are available for purchase.

[00109] A wide variety of host/expression vector combinations can be used to express polynucleotide sequences encoding the Chp peptides described herein, or active fragments thereof. A large number of suitable vectors are known to those skilled in the art and are commercially available. Examples of suitable vectors are provided, for example, in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual (3rd ed.), volumes 1 - 3, Cold Spring Harbor Laboratory (2001). Such vectors include, but are not limited to, chromosomal, episomal, and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses , papovaviruses such as SV40, vaccinia viruses, adenoviruses, fowlpox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as vectors derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.

[00110] Moreover, vectors can provide constitutive or inducible expression of Chp peptides or active fragments thereof as described herein. Suitable vectors include, but are not limited to, derivatives of SV40 and known bacterial plasmids, for example, E. coli colEI, pCRI, pBR322, pMB9 plasmids and their derivatives, such as RP4, pBAD24 and pBAD-TOPO; Phage DNA, for example, many derivatives of phage A, for example, NM989, and other phage DNA, for example, M13 DNA and single-stranded filamentous phage DNA; yeast plasmids such as plasmid 2D or derivatives thereof; vectors suitable for eukaryotic cells, such as vectors suitable for insect or mammalian cells; vectors derived from combinations of plasmids and phage DNA, such as plasmids that have been modified to use phage DNA or other expression control sequences; and similar vectors. Many of the vectors mentioned above are available for purchase from suppliers such as New England Biolabs Inc., Addgene, Takara Bio Inc., ThermoFisher Scientific Inc., etc.

[00111] In addition, vectors may contain various regulatory elements (including a promoter, ribosome binding site, terminator, enhancer, various cis-acting elements to control the level of expression), and the vector is designed depending on the host cell. These polynucleotide sequence expression vectors encoding Chp peptides or active fragments thereof as described herein can use any of a wide variety of expression control sequences (sequences that control the expression of a polynucleotide sequence operably linked thereto). Suitable control sequences include, but are not limited to: early or late promoters of SV40, CMV, vaccinia virus, polyoma virus or adenovirus, lac system, trp system, TAC system, TRC system, LTR system, phage A operator and promoter core regions, control sequences fd coat protein regions, promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, acid phosphatase promoters (e.g. Pho5), yeast mating factor promoters, E promoter, coli for expression in bacteria and other promoter sequences known to control prokaryotic gene expression or eukaryotic cells or their viruses, and various combinations of the above sequences. Typically, polynucleotide sequences encoding Chp peptides or active fragments thereof are operably linked to a heterologous promoter or regulatory element.

[00112] In another aspect of the present invention, a host cell is provided containing any of the vectors described herein, including expression vectors containing polynucleotide sequences encoding Chp peptides or active fragments thereof as described herein. A wide variety of host cells are suitable for expression of the polypeptides of the present invention. Non-limiting examples of host cells suitable for expressing the polypeptides of the present invention include well-known eukaryotic and prokaryotic hosts such as E. coli, Pseudomonas, Bacillus, Streptomyces strains, fungi such as yeast, and animal cells such as CHO cells, RI.I, B-W and L-M, African green monkey kidney cells (eg COS 1, COS 7, BSCI, BSC40 and BMT10), insect cells (eg Sf9), and human and plant cells in culture. Although the expression host can be any known expression host cell, in a typical embodiment the expression host is one of the E. coli strains. These strains include, but are not limited to, commercially available E coli strains such as Top10 (ThermoFisher Scientific, Inc.), DH5a (Thermo Fisher Scientific, Inc.), XLI-Blue (Agilent Technologies, Inc.), SCSIIO (Agilent Technologies, Inc.), JM109 (Promega, Inc.), LMG194 (ATCC), and BL21 (Thermo Fisher Scientific, Inc.)

[00113] There are several advantages to using E. coli as a host system, including: rapid growth kinetics, with a doubling time of approximately 20 minutes under optimal environmental conditions (Sezonov et al., J, Bacterial. 189 8746-8749 (2007 )), ease of obtaining high-density cultures, simple and rapid transformation of exogenous DNA, etc. Details regarding protein expression in E. coli, including plasmid selection as well as strain selection, are discussed in detail in Rosano, G. and Ceccarelli, E., Front Microbial., 5: 172 (2014).

[00114] Efficient expression of the Chp peptides of the present invention or active fragments thereof depends on various factors, such as optimal expression signals (at both transcriptional and translational levels), proper protein folding, and cell growth patterns. With respect to methods for constructing a vector and methods for transducing a host cell with a constructed recombinant vector, conventional methods known in the art can be used. While it should be apparent that not all sequence expression control vectors and hosts will function equally well in expressing polynucleotide sequences encoding Chp peptides or active fragments thereof as described herein, one skilled in the art will be able to select suitable sequence expression control vectors and hosts without undue experimentation in order to achieve the desired expression without deviating from the scope of the present invention.

[00115] Chp peptides or active fragments thereof as described herein can be isolated and purified from recombinant cell cultures by well known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion exchange or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. High performance liquid chromatography can also be used to purify the Chp peptide.

[00116] Alternatively, the vector system used to produce Chp peptides or active fragments according to the present disclosure may be a cell-free expression system. A variety of cell-free expression systems are commercially available, including but not limited to systems available from Promega, LifeTechnologies, Clonetech, etc.

[00117] As stated above, when the need arises for protein production and purification, a number of alternatives exist. Examples of suitable methods and strategies to be considered in protein production and purification are proposed in WO 2017/049233, which is incorporated herein by reference in its entirety, and further proposed in Structural Genomics Consortium et al., Nat. Methods., 5(2): 135-146 (2008).

Pharmaceutical compositions.

[00118] The compositions herein may be formulated as solutions, suspensions, emulsions, tablets, pills, pellets, capsules, liquid capsules, powders, sustained release formulations, suppositories, tampon emulsions, aerosols, sprays, suspensions, lozenges , troches, lozenges, injectables, chewing gums, ointments, lubricants, time-release patches, liquid-impregnated wipes, and combinations thereof.

[00119] Administration of the compositions herein or pharmaceutically acceptable forms thereof may be topical, that is, the pharmaceutical composition may be applied directly to the site where its action is desired (eg, directly to a wound), or systemic. In turn, systemic administration can be enteral or oral, i.e., the composition can be administered through the digestive tract, parenteral, i.e., the composition can be administered by routes other than the digestive tract, for example, by injection or inhalation. Thus, the Chp peptides of the present description and compositions containing them can be administered to a subject orally, parenterally, by inhalation, topically, rectally, nasally, buccally, through an implanted reservoir, or by any other known method. Chp peptides according to the present description, or active fragments thereof, can also be administered via sustained release dosage forms.

[00120] For oral administration, the Chp peptides of the present disclosure or active fragments thereof can be formulated into solid or liquid preparations, for example, tablets, capsules, powders, solutions, suspensions and dispersions. The composition may include excipients such as, for example, lactose, sucrose, corn starch, gelatin, potato starch, alginic acid and/or magnesium stearate.

[00121] To prepare solid compositions such as tablets and pills, the Chp peptide of the present disclosure or an active fragment thereof can be mixed with a pharmaceutical excipient to form a solid composition that has not yet been formulated into a dosage form. If desired, the tablets can be sugar-coated or enteric-coated using standard techniques. Tablets or pills may be coated or otherwise mixed to form a dosage form providing the benefit of a sustained release. For example, a tablet or pill may contain an internal dosage component and an external dosage component, the latter being a coating over the former. These two components can be separated by an enteric layer, which serves to prevent degradation in the stomach and allows the internal component to pass intact into the duodenum or slow its release. A variety of materials can be used for such enteric layers or coatings, such materials include a variety of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol and cellulose acetate.

[00122] Compositions for topical use according to the present disclosure may further comprise a pharmaceutically or physiologically acceptable carrier, such as a dermatologically or ear-acceptable carrier. Such carriers, in the case of dermatologically acceptable carriers, may be compatible with skin, nails, mucous membranes, tissues and/or hair, and may include any commonly used dermatological carrier that satisfies these requirements. In the case of media suitable for insertion into the ear, said media may be compatible with all parts of the ear. Such media can be easily selected by one of ordinary skill in the art. Carriers for topical administration of the compositions herein include, but are not limited to: mineral oil, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene and/or polyoxypropylene compounds, emulsion wax, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water. When formulating skin ointments, the active ingredients herein may be formulated in a formulation, for example, containing an oleaginous hydrocarbon base, an anhydrous absorbent base, a water-in-oil absorbent base, a water-washable oil-in-water base, and/or a water-soluble base. When formulating compositions for administration to the ear, the active components according to the present description may be formulated, for example, containing an aqueous polymer suspension, including carriers such as dextrans, polyethylene glycols, polyvinylpyrrolidone, polysaccharide gels, gellan gums such as Gelrit®, cellulosic polymers such as hydroxypropyl methylcellulose, and carboxy-containing polymers such as acrylic acid polymers or copolymers, as well as other polymeric emollients. Compositions for topical use according to the present description may be in any form suitable for topical use, including aqueous, hydroalcoholic or oily solutions; dispersive lotions or serums; aqueous, anhydrous or oily gels; emulsions obtained by dispersing a fatty phase in an aqueous phase (O/W, or oil in water) or vice versa (W/O, or water in oil); microemulsions or, alternatively, microcapsules, microparticles or liposome dispersions of the ionic and/or non-ionic type; creams; lotions; gels; foams (which may use a pressurized container, a suitable applicator, an emulsifier and an inert propellant); essences; milk; suspensions and patches. Compositions for topical use herein may also contain adjuvants such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, excipients, photoprotective filters, odor absorbents and colorants. In an additional aspect, the topical compositions described herein can be administered in combination with devices such as transdermal patches, dressings, pads, wraps, matrices, and bandages that are capable of adhering to or otherwise being associated with the skin or other tissue of a subject. , which are capable of delivering a therapeutically effective amount of one or more Chp peptides or active fragments thereof described in this application.

[00123] In one embodiment, the topical compositions described herein further comprise one or more components used to treat topical burns. Such components may include, but are not limited to: propylene glycol hydrogel; a combination of glycol, cellulose derivative and water-soluble aluminum salt; antiseptic; antibiotic and corticosteroid. Humectants such as solid or liquid wax esters can also be added; absorption stimulants such as hydrophilic clays or starches; thickening agents and skin protecting agents. Formulations for topical use may be in the form of rinses, such as mouthwash. See for example WO2004/004650.

[00124] Compositions according to the present description can also be administered by injection of a therapeutic agent containing a suitable amount of Chp peptide or active fragment thereof and a carrier. For example, the Chp peptide or an active fragment thereof can be administered intramuscularly, intrathecally, subdermally, subcutaneously or intravenously to treat infections with gram-negative bacteria, such as those caused by P. aeruginosa. The carrier may consist of distilled water, saline, albumin, serum, or any combination thereof. In addition, pharmaceutical compositions for parenteral injection may contain pharmaceutically acceptable aqueous or non-aqueous solutions of the Chp peptides described herein, or active fragments thereof, in addition to one or more of the following: pH buffer solutions, adjuvants (e.g., preservatives, wetting agents , emulsifying agents and dispersing agents), liposomal formulations, nanoparticles, dispersions, suspensions or emulsions, and sterile powders for dissolution in sterile injectable solutions or dispersions immediately before use.

[00125] In cases where the chosen route of administration is parenteral injection, an isotonic formulation may be used. Typically, isotonicity auxiliaries may include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate-buffered saline are preferred. Stabilizers may include gelatin and albumin. A vasoconstrictor agent may be added to the formulation. Pharmaceutical preparations suitable for this type of application may be offered as sterile and pyrogen-free.

[00126] The diluent may further contain one or more other excipients, such as ethanol, propylene glycol, oil, or a pharmaceutically acceptable emulsifier or surfactant.

[00127] In another embodiment, the compositions according to the present description are compositions for inhalation. Compositions for inhalation according to the present description may further contain a pharmaceutically acceptable carrier. In one embodiment, the Chp peptides described herein or active fragments thereof may be formulated as a dry powder for inhalation. In specific embodiments, an inhalation solution containing Chp peptides or active fragments thereof may further be formulated with a propellant for aerosol delivery. In some embodiments, the solutions may be sprayable.

[00128] A surfactant can be added to an inhalable pharmaceutical composition according to the present description to reduce the surface and interfacial tension between the drugs and the propellant. If the drugs, propellant and excipient are in the form of a suspension, a surfactant may or may not be used. If a solution is to be made from the drugs, propellant and excipient, a surfactant may or may not be used, depending, for example, on the solubility of the particular drug and excipient. The surfactant may be any suitable non-toxic compound that does not react with the drug and that reduces the surface tension between the drug, the excipient and the propellant and/or acts as a lubricant for the valve.

[00129] Examples of suitable surfactants include, but are not limited to: oleic acid; sorbitan trioleate; cetylpyridinium chloride; soy lecithin; polyoxyethylene (20) sorbitan monolaurate; polyoxyethylene(10) stearyl ether; polyoxyethylene (2) oleyl ether; polyoxypropylene-polyoxyethylene-ethylenediamine block copolymers; polyoxyethylene (20) sorbitan monostearate; polyoxyethylene (20) sorbitan monooleate; polyoxypropylene-polyoxyethylene block copolymers; castor oil ethoxylate and combinations thereof.

[00130] Examples of suitable propellants include, but are not limited to, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide.

[00131] Examples of suitable excipients for use in inhalation compositions include, but are not limited to: lactose, starch, propylene glycol diesters of medium chain fatty acids; triglyceride esters of medium chain fatty acids, short chain or long chain, or any combination thereof; perfluorodimethylcyclobutane; perfluorocyclobutane; polyethylene glycol; menthol; lauroglycol; diethylene glycol monoethyl ether; polyglycolyzed glycerides of medium chain fatty acids; alcohols; Eucalyptus oil; short-chain fatty acids and their combinations.

[00132] Some embodiments provide nasal administration of the compositions described herein. Nasal applications include direct use, such as nasal sprays, nasal drops, nasal ointments, nasal rinses, nasal injections, nasal tampons, bronchial sprays and inhalation formulations, as well as indirect use, such as lozenges and mouth or throat rinses, or through the use of ointments applied to the nostrils or face, and any combination of these and similar methods of application.

[00133] In another embodiment, the pharmaceutical compositions according to the present description contain a complementary agent, including one or more antimicrobial agents and/or one or more conventional antibiotics. In order to speed up the treatment of an infection or enhance the antibacterial effect, a therapeutic agent containing the Chp peptide according to the present description or an active fragment thereof may further contain at least one complementary agent, which can also enhance the bactericidal activity of the peptide. The complementary agent may be one or more antibiotics used to treat infection with gram-negative bacteria. In one embodiment, the complementary agent is an antibiotic or antimicrobial agent used to treat infections caused by P. aeruginosa.

[00134] The compositions herein may be presented in unit dosage form and may be prepared by any methods well known in the art. The amount of active ingredients that can be combined with the carrier material to form a unit dosage form will vary, for example, depending on the host being treated, the duration of exposure of the recipient to infectious bacteria, the body size and weight of the subject, and the particular route of administration. The amount of active ingredients that can be combined with the carrier material to form a unit dosage form, for example, may be that amount of each compound that produces a therapeutic effect. In some embodiments, of one hundred percent, the total amount may range from about 1 percent to about ninety-nine percent of the active ingredients, such as from about 5 percent to about 70 percent, or from about 10 percent to about 30 percent.

Dosage and administration.

[00135] The doses administered depend on many factors, including the activity of the infection being treated; the age, health and general physical condition of the subject being treated; the activity of a particular Chp peptide or its active fragment; the nature and activity of the antibiotic, if any, with which the Chp peptide or active fragment thereof is combined according to the present description; and the overall effect of this combination. In some embodiments, effective amounts of Chp peptide or active fragment thereof to be administered may range from about 1 to 50 mg/kg (or 1 to 50 μg/ml). In some embodiments, the Chp peptide or active fragment thereof can be administered 1 to 4 times daily for a period ranging from 1 to 14 days. The antibiotic, if also used, will be administered in standard dosing schedules or in smaller quantities based on the presence of any synergism. However, all such dosages and dosage regimens (either of the Chp peptide or its active fragment, or of any antibiotic that will be administered in combination with it) are subject to optimization. Optimal dosages can be determined by conducting in vitro and in vivo efficacy trials that are within the scope of the art but subject to the present invention.

[00136] It is believed that the Chp peptides described herein, or active fragments thereof, may have a rapid bactericidal effect and, when used in amounts below the MIC, may have a bacteriostatic effect. It is further contemplated that the Chp peptides described herein, or active fragments thereof, may be active against a range of antibiotic-resistant bacteria and may not be associated with the development of resistance. Based on the present disclosure, in a clinical setting, the Chp peptides or active fragments thereof of the present invention may represent an effective alternative (or complement) to the treatment of infections arising from drug- and multidrug-resistant bacteria, alone or together with antibiotics (including antibiotics to which resistance has developed). It is believed that existing resistance mechanisms of Gram-negative bacteria do not affect the sensitivity to lytic activity of the Chp peptides of the present invention or active fragments thereof.

[00137] In some embodiments, exposure time to the Chp peptides described herein or active fragments thereof may influence the required concentration of active peptide units per ml. Carriers that are classified as "extended" or "sustained" release carriers (such as, for example, some nasal sprays or lozenges) may contain or provide a lower concentration of peptide units per ml, but over a longer period of time, whereas the carrier "short" or "quick" release (such as, for example, a gargle) may contain or provide a high concentration of peptide units (µg) per ml, but over a shorter period of time. There are circumstances in which a higher unit/mL dosage or a lower unit/mL dosage may be necessary.

[00138] For Chp peptides or active fragments thereof herein, a therapeutically effective dose can first be assessed either in cell culture assays or in animal models, typically mice, rabbits, dogs or pigs. An animal model can also be used to achieve the desired concentration range and route of administration. The information obtained can then be used to determine effective doses as well as routes of administration in humans. Dosage and administration may be further adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Additional factors that may be considered include the severity of the disease state; age, body weight and gender of the patient; diet; desired duration of treatment; method of administration; time and frequency of administration; drug combinations; allergic reactions; tolerability/response to therapy and the decision of the treating physician.

[00139] The treatment regimen may include daily administration (e.g., once, twice, three times, etc. per day), every other day administration (e.g., once, twice, three times, etc. every other day ), twice a week, once a week, once every two weeks, once a month, etc. In one embodiment of the present invention, the treatment can be administered as a continuous infusion. Single doses can be administered multiple times. The intervals may also be irregular, depending on the clinical symptoms being monitored. Alternatively, the unit dose may be administered as a sustained release formulation, in which case less frequent administration may be used. Dosage and frequency may vary depending on the patient. One skilled in the art will appreciate that such guidelines will be adjusted for localized administration, e.g., intranasal, inhalation, rectal, etc., or for systemic administration, e.g., oral, rectal (eg, enema), intramuscular (i.v.). m), intraperitoneal (i/p), intravenous (i.v.), subcutaneous (s.c.), transurethal, etc.

Ways.

[00140] Chp peptides and active fragments thereof according to the present description can be used in vivo, for example, to treat bacterial infections caused by gram-negative bacteria, such as P. aeruginosa, in a subject, as well as in vitro, for example, to reduce the level of bacterial contamination, for example, on the surface of, for example, a medical device.

[00141] For example, in some embodiments, the Chp peptides or active fragments thereof of the present invention can be used to prevent, control, disrupt, and treat bacterial biofilms formed by gram-negative bacteria. Biofilm formation occurs when microbial cells adhere to each other and are surrounded by a matrix of extracellular polymeric substance (EPS) on the surface. Microbial growth in such a protected environment, which is enriched in biomacromolecules (eg, polysaccharides, nucleic acids and proteins) and nutrients, allows for improved signaling between microbes and increased virulence. Biofilm can form in any supporting environment, including living and nonliving surfaces such as mucus plugs from a cystic fibrosis (CF) lung, contaminated catheters, contact lenses, etc. (Sharma et al. Biologicals, 42(1):1-7 (2014), which is incorporated herein by reference in its entirety). Thus, in one embodiment, Chp peptides or active fragments thereof as described herein can be used to prevent, control, interrupt, and treat bacterial infections caused by gram-negative bacteria when the bacteria are protected by a bacterial biofilm.

[00142] In one aspect of the present invention, there is provided a method of treating a bacterial infection caused by one or more additional gram-negative bacteria described herein, comprising administering to a subject diagnosed with, at risk of developing, or exhibiting symptoms of a bacterial infection, a pharmaceutical composition described herein application.

[00143] The terms “infection” and “bacterial infection” are intended to include respiratory tract infections (RTIs), such as respiratory tract infections in patients with cystic fibrosis (MB), lower respiratory tract infections, such as acute attacks of chronic bronchitis (ACB) , acute sinusitis, community-acquired pneumonia (CAP), hospital-acquired pneumonia (AP) and nosocomial respiratory tract infections; sexually transmitted diseases such as gonococcal cervicitis and gonococcal urethritis; urinary tract infections; acute inflammation of the middle ear; sepsis, including neonatal sepsis and catheter-associated sepsis; and osteomyelitis. Infections caused by drug-resistant and multidrug-resistant bacteria are also included.

[00144] Non-limiting examples of infections caused by gram-negative bacteria such as P. aeruginosa include: A) nosocomial infections: 1. respiratory tract infections especially in patients with cystic fibrosis and in mechanically ventilated patients; 2. bacteremia and sepsis; 3. wound infections, especially in patients with burns; 4. urinary tract infections; 5. postoperative infections on invasive devices; 6. endocarditis as a result of intravenous administration of contaminated drug solutions; 7. infections in patients with acquired immunodeficiency syndrome, cancer chemotherapy, steroid therapy, hematologic malignancies, organ transplantation, renal replacement therapy, and other conditions with severe neutropenia; C) community-acquired infections: 1. community-acquired respiratory tract infections; 2. meningitis; 3. folliculitis and infections of the external auditory canal caused by contaminated water; 4. malignant otitis externa in the elderly and diabetics; 5. osteomyelitis of the calcaneus in children; 6. eye infections, usually associated with a contaminated contact lens; 7. skin infections such as nail infections in people whose hands frequently come into contact with water; 8. gastrointestinal tract infections; and 9. infections of the musculoskeletal system.

[00145] The one or more species of gram-negative bacteria according to the methods of the present invention may include any of the species of gram-negative bacteria described in this application. Typically, the additional gram-negative bacterial species are selected from one or more of the following: Acinetobacter baumannii, Acinetobacter haemolyticus, Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, Bacteroides species such as Bacteroides fragilis, Bacteroides theataioatamicron, Bacteroides distasonis, Bacteroides ovatus, Bacteroides vuigatus, Bartonella Quintana, Bordetella pertussis , Brucella species such as Brucella melitensis, Burkhoideria species such as Burkhoideria cepacia, Burkhoideria pseudomallei and Burkhoideria mallei, Fusobacterium, Prevotella corporis, Prevotella intermedia, Prevotella endodontalis, Porphyromonas asaccharolytica, Campylobacter jejuni, Campylobacter fetus, Campylobacter coli, Chlamydia species , such such as Chlamydia pneumoniae and Chlamydia trachomatis, Citrobacter freundii, Citrobacter koseri, Coxiella burnetii, Edwarsiella species such as Edwarsiella tarda, Eikenella corrodens, Enterobacter species such as Enterobacter cloacae, Enterobacter aerogenes and Enterobacter agglomerans, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Haemophilus ducreyi, Helicobacter pylori, Kingella kingae, Klebsiella species such as Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella rhinoscleromatis and Klebsiella ozaenae, Legionella penumophila, Moraxella species such as Moraxella catarrhalis, Morganella species such as Morganella morganii, Neisseria gonorrhoeae, Neisseria mening itidis , P. aeruginosa, Pasteurella multocida, Plesiomonas shigelloides, Proteus mirabilis, Proteus vulgaris, Proteus penneri, Proteus myxofaciens, Providencia species such as Providencia stuartii, Providencia rettgeri, Providencia alcalifaciens, Pseudomonas fluorescens, Salmonella typhi, Salmonella typhimur ium, Salmonella paratyphi, Serratia species such as Serratia marcescens, Shigella species such as Shigella flexneri, Shigella boydii, Shigella sonnei and Shigella dysenteriae, Stenotrophomonas maltophilia, Streptobacillus moniliformis, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Yersinia enteroc olitica, Yersinia pestis, Yersinia pseudotuberculosis, Chlamydia pneumoniae, Chlamydia trachomatis, Ricketsia prowazekii, Coxiella burnetii, Ehrlichia chafeensis and/or Bartonella hensenae.

[00146] More commonly, at least one other species of gram-negative bacteria is selected from one or more of the following: Acinetobacter baumannii, Bordetella pertussis, Burkhoideria cepacia, Burkhoideria pseudomallei, Burkhoideria mallei, Campylobacter jejuni, Campylobacter coli, Enterobacter cloacae, Enterobacter aerogenes, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Haemophilus ducreyi, Helicobacter pylori, Klebsiella pneumoniae, Legionella penumophila, Moraxella catarrhalis, Morganella morganii, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Proteus mirabilis, Proteus vulgaris, Salmonella typhi, Serratia marcescens, Shi gella flexneri, Shigella boydii , Shigella sonnei, Shigella dysenteriae, Stenotrophomonas maltophilia, Vibrio cholerae and/or Chlamydia pneumoniae.

[00147] More often, at least one other species of gram-negative bacteria is selected from one or more of the following: Salmonella typhimurium, Salmonella typhi, Shigella spp., Escherichia coli, Acinetobacter baumanii, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitides, Serratia spp., Proteus mirabilis , Morganella morganii, Providencia spp., Edwardsiella spp., Yersinia spp., Haemophilus influenza, Bartonella quintana, Brucella spp., Bordetella pertussis, Burkhoideria spp., Moraxella spp., Francisella tularensis, Legionella pneumophila, Coxiella burnetii, Bacteroides spp., Enterobacter spp., and/or Chlamydia spp. .

[00148] Even more commonly, at least one other species of gram-negative bacteria is selected from one or more of Klebsiella species, Enterobacter species, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Yersinia pestis, and/or Franciscella tulerensis.

[00149] In some embodiments, infection with gram-negative bacteria results in a localized infection, such as a topical bacterial infection, such as a skin wound. In other embodiments, the bacterial infection is a systemic pathogenic bacterial infection. Common gram-negative pathogens and associated infections are listed in Table A of this specification. These are intended to be examples of bacterial infections that can be treated, reduced or prevented by the Chp peptides and active fragments thereof of the present invention, and are not intended to be limiting.

[00150] In some embodiments, the Chp peptides and active fragments thereof as described herein are used to treat a subject at risk of contracting a gram-negative bacterial infection. Subjects at risk of acquiring a gram-negative bacterial infection include, for example, patients with cystic fibrosis, patients with neutropenia, patients with necrotizing enterocolitis, burn patients, patients with wound infections and, more generally, patients in hospital settings, particularly surgical patients and patients being treated with an implantable medical device such as a catheter, such as a central venous catheter, a Hickman device, or cardiac electrophysiological devices, such as pacemakers and implantable defibrillators. Other patient groups at risk for infection with gram-negative bacteria include, but are not limited to, patients with implanted prostheses, such as total joint replacements (eg, total knee or total hip replacement).

[00151] In another aspect of the present invention, there is provided a method of preventing or treating a bacterial infection, comprising co-administering to a subject diagnosed with, at risk of developing, or exhibiting symptoms of a bacterial infection, a combination of a first effective amount of a composition comprising an effective amount of a Chp peptide or an active fragment thereof, described herein, and a second effective amount of an antibiotic suitable for treating a gram-negative bacterial infection.

[00152] Chp peptides and active fragments thereof as described herein may be co-administered with standard of care antibiotics or antibiotics of last resort, alone or in various combinations as is within the skill of the art. Common antibiotics used against P. aeruginosa are described in Table B. Antibiotics for other gram-negative bacteria, such as Klebsiella spp., Enterobacter spp., Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Yersinia pestis, and Franciscella tulerensis, are similar to those suggested in table B for P. aeruginosa.

[00153] In more specific embodiments, the antibiotic is selected from one or more of the following: ceftazidime, cefepime, cefoperazone, ceftobiprole, ciprofloxacin, levofloxacin, aminoglycosides, imipenem, meropenem, doripenem, gentamicin, tobramycin, amikacin, piperacillin, ticarcillin, penicillin, rifampicin , polymyxin B and colistin.

[00154] Combining Chp peptides or active fragments thereof as described herein with antibiotics provides an effective antibacterial treatment regimen. In some embodiments, coadministration of Chp peptides or active fragments thereof as described herein with one or more antibiotics can be accomplished at lower doses and amounts of either Chp peptides or active fragments thereof or the antibiotic or both and/or at lower frequency and/or or duration of treatment with improved bactericidal and bacteriostatic activity, a reduced risk of antibiotic resistance, and a reduced risk of harmful neurological or renal side effects (such as those associated with colistin or polymyxin B). Previous studies have shown that the total cumulative dose of colistin is associated with kidney damage, suggesting that reducing the dosage or shortening the duration of treatment using combination therapy with Chp peptides or their active fragments may reduce the incidence of nephrotoxicity (Spapen et al. Ann Intensive Care, 1: 14 (2011), which is incorporated herein by reference in its entirety). As used herein, the term "reduced dose" refers to the dose of one active ingredient in combination compared to monotherapy with the same active ingredient. In some embodiments, the dosage of the Chp peptides or active fragments thereof or the antibiotic in combination may be suboptimal or even subthreshold compared to the corresponding monotherapy.

[00155] In some embodiments, the present invention provides a method of increasing the antibiotic activity of one or more antibiotics against gram-negative bacteria compared to the activity of said antibiotics used alone by administering to a subject Chp peptides or active fragments thereof described herein together with the antibiotic of interest . This combination is effective against bacteria and allows one to overcome antibiotic resistance and/or use the antibiotic at lower doses, reducing unwanted side effects such as the nephrotoxic and neurotoxic effects of polymyxin B.

[00156] Chp peptides or active fragments thereof, optionally in combination with antibiotics according to the present description, can be further combined with additional agents that permeabilize the outer membrane of gram-negative bacteria, including, but not limited to: metal chelates, such as, for example, EDTA, Tris, lactic acid, lactoferrin, polymyxins, citric acid (Vaara M. Microbial Rev, 56(3):395-441 (1992), which is incorporated herein by reference in its entirety).

[00157] In another further aspect of the present invention, there is provided a method of inhibiting the growth of, or reducing the population of, or killing at least one species of gram-negative bacteria, the method comprising contacting the bacteria with a composition containing an effective amount of a Chp peptide or an active fragment thereof described in this application, wherein said Chp peptide or an active fragment thereof inhibits the growth of, or reduces the population of, or destroys at least one species of gram-negative bacteria.

[00158] In some embodiments, inhibiting the growth, or reducing the population, or killing at least one species of gram-negative bacteria includes contacting the bacteria with Chp peptides or active moieties described herein, wherein said bacteria are present on the surface of, for example, medical devices, floors, stairs, walls and countertops in hospitals and other healthcare facilities or public buildings, and on equipment surfaces in operating rooms, emergency rooms, wards, clinics and sanitary facilities, and similar areas.

[00159] Examples of medical devices that can be protected using Chp peptides or active fragments thereof described herein include, but are not limited to, tubing and other medical devices that come into contact with the surface of the human body, such as urinary catheters, mucus collection catheters , Suction Catheters, Umbilical Catheters, Contact Lenses, Intrauterine Devices, Intravaginal and Intestinal Devices, Endotracheal Tubes, Bronchoscopes, Dentures and Orthodontic Devices, Surgical Devices, Dental Devices, Tubes, Dental Water Lines, Tissues, Paper, Test Strips ( such as paper test strips or plastic test strips), adhesives (such as hydrogel adhesives, hot-melt adhesives, or solvent-based adhesives), bandages, tissue dressings or healing devices and occlusive patches, and any other products in contact with the surface of the human body that used in the field of medicine. These devices may include electrodes, ectoprostheses, fixation tapes, compression bandages and various types of monitoring devices. Medical devices may also include any device that can be placed at an insertion or implantation site, such as skin near the insertion or implantation site, and which may contain at least one surface that is susceptible to colonization by gram-negative bacteria.

Examples

Materials and methods

[00160] Bacterial strains and growth conditions. Much of the research described in this application was conducted using the carbapenam-resistant P. aeruginosa clinical isolate CFS-1292, obtained from human blood at the Hospital for Special Surgery in New York (provided by Dr. Lars Westblade, Professor in the Department of Pathological Anatomy and Laboratory Medicine) , but commercially available antibiotic-resistant isolates can also be used. All other isolates were obtained from either the American Type Culture Collection ("ATCC"), the d'Herelle Collection ("HER"), the BEI Resources Repository ("HM"), or the Hospital for Special Surgery in New York ("HSS"). Isolates were cultured and examined in any of the following media: lysogeny broth (LB; Sigma-Aldrich), casamino acid (CA) media (5 g/L casamino acid, Ameresco/VWR; 5.2 mM K ₂ HPO ₄ , Sigma-Aldrich ; 1 mM MgSO ₄ , Sigma-Aldrich), AS supplemented with 100 mM NaCl, or AS supplemented with 2.5% human serum (type AB, male, pooled; Sigma-Aldrich). All antibiotics and protein reagents (e.g., T4 lysozyme) were obtained from Sigma-Aldrich unless otherwise noted.

[00161] Bioinformatics research. All proteins were identified in annotated entries in the GenBank database for all genomes of Microviridae and Leviviridae. The accession number for each Chp group peptide is listed below in Tables 1 and 2. Blastp analysis was performed using the UniProt server available at uniprot.org/blast/. Protein secondary structure predictions were performed using JPRED4, available at www.compbio.dundee.ac.uk/jpred/index, and I-Tasser, available at www.zhanglab.ccmb.med.umich.edu/I-TASSER/ . Phylogenetic analysis was performed using the multiple sequence alignment program ClustalW, available at www.genome.jp/tools-bin/clustalw. Predicted molecular weights and isoelectric points were determined using the ExPASy resource portal available at web.expasy.org/compute_pi/.

[00162] Determination of minimum inhibitory concentrations (MICs). MIC values were determined using a modification of the standard recommended broth microdilution method described by the Clinical and Laboratory Standards Institute (CLSI) (2015. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, 10th edition. Clinical and Laboratory Standards Institute, Wayne , Pennsylvania). The modification was based on replacing the Mueller-Hinton medium, in some cases, with either KAK media (with or without NaCl) or KAK supplemented with 2.5% human serum. As used herein, the MIC is the minimum concentration of peptide sufficient to inhibit at least 80% of bacterial growth compared to control.

[00163] Determination of minimum biofilm disrupting concentrations (MBICs). MPBC values were determined using a variant of the broth microdilution method for MIC determination with modifications (Ceri N et al. 1999. J Clin Microbiol 37:1771-1776; and Schuch R et al. 2017. Antimicrob Agents Chemother 61). Fresh colonies of P. aeruginosa strain ATCC 17647 were suspended in PBS (0.5 McFarland units), diluted 1:100 in LB with 0.2% glucose, and added as 0.15 ml aliquots to each well of a 96-well Calgary device for biofilm growth (Innovotech) and incubated for 24 hours at 37 C to form biofilms on the polycarbonate protrusions. Biofilms were washed and treated with a series of 2-fold dilutions of each peptide in TSBg at 37°C for 16 hours. After treatment, the wells were washed, air dried at 37°C, stained with 0.05% crystal violet for 10 minutes, and the dye was removed in 33% acetic acid. The OD600 of the extracted crystal violet was determined. The MPBC value of each sample was determined as the minimum drug concentration required to remove >95% of biofilm biomass, as assessed by determining the amount of crystal violet (compared to untreated controls). Phage T4 lysozyme was used as a negative control and showed no activity against biofilm.

[00164] Checkerboard analysis. The checkerboard test is based on a modification of the CLSI method for determining MIC by broth microdilution (CLSI 2015; and Moody J. 2010. Synergy testing: broth microdilution checkerboard and broth macrodilution methods, pp. 5.18.1-5.18.23. In Garcia LS (ed), Clinical Microbiology Procedures Handbook, volume 2). Checkerboards were constructed by first preparing columns in a 96-well polypropylene microtiter plate in which each well contained an equal amount of antibiotic diluted 2-fold along the horizontal axis. In a separate plate, comparable rows were prepared in which each well contained the same amount of peptide, diluted, for example, 2-fold along the vertical axis. The peptide and antibiotic dilutions were then combined so that each column contained a constant amount of antibiotic and doubling dilutions of Gram-negative bacterial lysine, with each row containing a constant amount of Gram-negative bacterial lysine and doubling dilutions of antibiotic. Therefore, each well contained a unique combination of peptide and antibiotic. Bacteria were added to drug combinations at a concentration of 1×10 ⁵ CFU/ml in CAC with 2.5% human serum. The MIC of each agent, alone and in combination, was then recorded after 16 hours at 37°C in ambient atmosphere. The sum of fractional inhibitory concentration indices (FICI) was calculated for each drug, and the minimum FICI value was used to determine synergy. FICI was calculated as follows: FICI=FIC A+FIC B, where FIC A is the MIC of each antibiotic in combination/MIC of each antibiotic alone, and FIC B is the MIC of each Gram-negative bacterial lysine in combination/MIC of each Gram-negative bacterial lysine alone . Combinations were considered synergistic if the FICI was ≤0.5, highly additive if the FICI was >0.5 to <1, additive if the FICI was 1-<2, and antagonistic if the FICI was >2. A checkerboard assay was performed using P. aeruginosa strain CFS-1292 in KAK/HuS with combinations of either Chp2 or Chp4 against a range of 11 different antibiotics, including amikacin, azithromycin, aztreonam, ciprofloxacin, colistin, fosfomycin, gentamicin, imipenem , piperacillin, rifampicin and tobramycin. FICI values <0.5 were observed for most combinations, indicating the ability of Chp2 and Chp4 to act synergistically with a wide range of antibiotics (see Table 8 below). These findings suggest that Chp peptides may exhibit potent antibacterial activity in the presence of antibiotics.

[00165] Analysis of hemolytic activity of lysine of gram-negative bacteria. Hemolytic activity was measured as the amount of hemoglobin released by lysis of human red blood cells (Lv Y et al. 2014. PLoS One 9:e86364). Briefly, 3 ml of fresh human blood cells (hRBCs) obtained from pooled blood of healthy donors (BioreclamationlVT), in a polycarbonate tube containing heparin, were centrifuged at 1000xg for 5 min at 4 C. The resulting red blood cells were washed three times with phosphate-buffered saline. solution (PBS, pH 7.2) and resuspended in 30 ml PBS. A volume of 50 μl of erythrocyte solution was incubated with 50 μl of each lysine of gram-negative bacteria (in PBS) in a range of 2-fold dilutions (from 128 μg/ml to 0.25 μg/ml) for 1 hour at 37 C. Intact erythrocytes were pelleted by centrifugation at 1000xg for 5 min at 4°C, and the supernatant was transferred to a new 96-well plate. Hemoglobin release was monitored by measuring absorbance using optical density (OD) at 570 nm. The minimum hemolytic concentration was defined as the lowest concentration of peptide resulting in visible lysis (corresponding to the minimum concentration resulting in an OD value ≥5% of that of the untreated control sample). Additional controls were used, including hRBC in PBS, treated as described above with either 0.1% Triton X-100 or each of a number of antimicrobial peptides with known hemolytic activity, including RR12, RR12 polar and RR12 hydrophobic (Mohanram N. et al ., 2016. Biopolymers 106:345-356), and with little or no hemolytic activity, including RI18 (Lyu Y. et al., 2016. Sci Rep 6:27258) and RR22.

[00166] Analysis of lysine activity of gram-negative bacteria by time of death. An overnight culture of P. aeruginosa strain CFS-1292 was diluted 1:00 into fresh KAK medium with 2.5% human serum (KAK/HuS) and grown for 2.5 hours at 37°C with shaking. Exponential growth phase bacteria were then diluted 1:100 in KAK/HuS and peptide was added at a final concentration of either 1 or 10 μg/ml. Control cultures without the addition of peptide (i.e., with buffer as a control) were included in the analysis. Cultures were incubated at 37°C with aeration, and samples were collected at 1 h, 3 h, and 24 h for quantitative plating on KAC agar plates.

[00167] Microscopy. Aliquots of P. aeruginosa strain CFS-1292 grown for 2.5 hours in LB were washed with PBS and resuspended in either PBS or 100% human serum and treated for 15 minutes at room temperature with and without Chp2 peptide. at a final concentration of 10 μg/ml. Subsets of samples were stained using the Live/Dead Cell Viability Kit (ThermoFisher) following the manufacturer's protocol and examined by differential interference contrast (DIC) microscopy and fluorescence microscopy. Example 1: Identification of Chp peptides.

[00168] With knowledge of a certain poorly characterized bacteriophage (Chlamydiamicroviridae) that specifically infects and kills gram-negative chlamydia bacteria, the published genomes of these organisms were examined, initially with the goal of identifying new lysines, although neither lysine-like sequences nor any sequences were observed, similar to previously described amurines. Chlamydiae do not use peptidoglycans (a known target of lysines) in their structures as extensively as other bacteria, but instead chlamydiae tend to only use peptidoglycans during division. Consequently, the question arose as to what was the target of the chlamydia phage. It was hypothesized that the mechanism by which chlamydial phage invades a target may be different from previously known mechanisms and that its target may be different, and focused on lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria and an obstacle to the entry of lysines. through the outer membrane.

[00169] Published Chlamydiamicrovirus genomes were examined to identify syntenic loci, i.e., similar genes at the same genomic position in a group of genetically related phages that would suggest similar function. Small, highly cationic peptides were discovered that had a very similar molecular charge profile to previously identified antimicrobial peptides (AMPs). Although chlamydial phage protein sequences were not observed to be similar to those of AMPs, lysines, or known amurin proteins (such as protein A2, protein E, and other proteins), the overall positive charge was a characteristic feature. Using the bioinformatics techniques described above (JPRED and iTASSAR), structure predictions were made that revealed the presence of alpha helices, a hallmark of many AMPs. All of the findings—alpha helices, net charge, conservation among chlamydia, and phage-related Gram-negative bacterial genomes—suggested that these proteins may represent a family of previously uncharacterized phage lytic polypeptides and that they may mediate a previously undescribed phage lytic mechanism. The fact that they were predicted to be small and soluble (based on their charge profile) also meant that once synthesized they were likely to be immediately suitable for testing simply by adding them to cultures of sensitive bacteria.

[00170] Based on the description above, 12 conserved sequences within syntenic loci were selected from the Microviridae genomes in the GenBank database, and in particular from the Chlamydiamicrovirus genomes (as well as several other viruses described below). These 12 conserved sequences were annotated only as hypothetical, uncharacterized, or nonstructural proteins and encoded small (presumably) cationic proteins with predicted alpha-helical structures. These 12 sequences are described in Table 1. One of the peptides from Table 1, Chp5, was synthesized so that its molecular charge differs from that of Chp4 by replacing arginines and lysines, which are positively charged, with negatively charged amino acid residues. Chp5 was predicted to be inactive. Although these peptides do not exhibit sequence similarity with other lytic or antimicrobial proteins, they are predicted to adopt alpha helical structures (e.g., see Figure 1), similar to subfamilies of the large AMP family of antibacterial agents. It has been proposed that Chp peptides have a host lysis function for the phages from which they are derived.

[00171] Based on the factors described above, additional examination of the genomes of other phages (related to Chlamydiamicrovirus, from the same family Microviridae) that infect Gram-negative bacteria, as well as other undescribed sources that had the same synteny and charge profile, yielded 14 additional peptides , listed in Table 2. Together, all 39 peptides (except Chp5) form a related family of novel phage-derived lytic agents. All are derived from Microviridae sources.

[00172] Thus, a complete list of all members of the Chp family (including some features of each peptide) is presented in Table 1 and Table 2. This group includes the peptides Chp1 - 4 and 6 -12 and CPAR39, which are derived from 11 different Chlamydiamicroviruses and have been described in table 1; peptides Chp2 and Chp3 are two identical peptides from two different phages. As stated above, Chp5 is a modified derivative of Chp4, obtained by replacing all positively charged amino acids, including arginines and lysines, with negatively charged amino acids, including glutamine and glutamic acid. An additional 27 Chp family members were identified by homology to Chlamydiamicrovirus proteins and are described in Table 2 (“Additional Chp family members”). An additional 27 members of the Chp family are derived not from Chlamydiamicrovirus sources, but from putative sources, Microviridae phages.

[00173]

[00174]

[00175] Additional information regarding protein sequence homologies of several Chp family members is presented in Table 3. Chp1, Bdp1, Lvp1, and Lvp2 are the only Chp family members with predicted activity in the GenBank annotation. Chp1 (GenBank sequence NP_044319.1) is annotated as a DNA binding protein, although no data are provided to support this fact and the annotation is inconsistent with a proposed role in host lysis. Overall, the Chp proteins are 39 to 100% identical to each other and are not homologous to other peptides in the protein sequence database. Rooted and unrooted phylogenetic trees showing some members of the Chp family are presented in Figures 2A and 2B, respectively.

[00176]

Example 2. Synthesis of Chp peptides.

[00177] All Chp peptides were synthesized at GenScript, New Jersey, USA, with capping [N-terminal acetylation (Ac) and C-terminal amidation (NH ₂ )] on a piecemeal basis. GenScript assessed the purity of each peptide using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). GenScript also performed a solubility test for all peptides and determined net peptide content (NPC%) using a Vario MICRO organic elemental analyzer. With the exception of Chp5, Lvp1, and Lvp2, all peptides were water soluble and were suspended at either 5 mg/mL or 10 mg/mL. Chp5 and Lvp1 were suspended in DMSO at a concentration of 10 mg/ml; Lvp2 was suspended in DMSO at a concentration of 2 mg/ml. Control peptides RI18, RP-1, WLBU2, BAC3, GN-2 amp, GN-3 amp, GN-4 amp, GN-6 amp, and Bac8c were also synthesized in GenScript as described above. All additional peptides were commercially available products purchased from either GenScript or Anaspec. Example 3: Activity of Chp peptides: minimum inhibitory concentration (MIC) against Gram-negative bacteria.

[00178] Thirty-nine of these Chp peptides (excluding Chp3, whose peptide sequence was identical to Chp2) were synthesized and tested in antimicrobial susceptibility testing (ACT) formats. First, MIC values were determined against the carbapenam-resistant clinical isolate P aeruginosa CFS-1292 in KAC medium supplemented with 2.5% human serum (Table 4). Several peptides including Chp1, Chp2, Chp4, Chp6, CPAR39 (with dithiothreitol (DTT)), Chp7, Chp8, Chp10, Chp11, Ecp1, Ecp2, Osp1, Spi1, Gkh3, Unp2, Unp5, Unp6, Ecp3, Ecp4, Lvp1 , ALCES1, AVQ206, CDL907, AGT915 and SBR77 showed the best MIC values in the range of 0.25 - 4 μg/ml. The peptides Chp5, CPAR39 (without DTT), Gkh1, Unp1, Spi2 and Bdp1 were only weakly active and exhibited MIC values of ≥32 μg/ml. CPAR39 is unique in this group because it contains internal cysteine residues and requires the presence of 0.5 mM DTT for its activity. Chp5 was designed as a derivative of Chp4 in which all positively charged residues were replaced by negatively charged residues; based on studies of cationic AMPs, it was predicted that cationic residues are essential for antibacterial activity, and replacing the cationic residues with anionic residues would disrupt the activity. Accordingly, Chp5 (MIC >64 μg/ml) is an inactive variant of Chp4 (MIC = 0.5 μg/ml). Both CPAR39 (without DTT) and Chp5 were used as negative controls.

[00179] Additional MIC testing was performed using the peptides Cpr1, Chp2, Chp4, CPAR39 (without DTT), Chp6, Esp1 and Esp2 against a number of gram-negative organisms, including Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae and Acinetobacter baumannii, which included some major ESKAPE pathogens (Table 5). Testing was performed in BAC (containing physiological concentrations of salt) which was not supplemented with 2.5% human serum due to the varying sensitivity of the target organisms to the presence of human serum. The best MIC values of 1 - 4 μg/ml were observed against all strains tested for Chp2, Chp4, Chp6, Ecp1 and Ecp2, indicating broad spectrum activity for the Chp peptides of the present invention in the context of physiological salt concentrations. Chp2 and Esp1 were further tested against Salmonella typhimurium and demonstrated an MIC of 2 μg/ml.

[00180] MIC values for both Chp2 and Chp4 were also determined and compared with those for a number of AMPs from the literature (including innate immune effectors and their derivatives) against P. aeruginosa laboratory strain PA01 in Mueller-Hinton medium supplemented with either 50% human plasma or or 50% human serum (Table 6). Here, the use of PA01 (laboratory isolate) allows testing in the presence of elevated concentrations of serum or plasma; PA01, unlike most clinical isolates, is insensitive to the antibacterial activity of the extracellular matrix of human blood. In Table 6, the MIC values for Chp2 and Chp4 were 2 μg/ml; in comparison, only RI18 and protegrin had similar activity (MIC = 1-4 μg/ml), and 18 additional peptides tested were either inactive or weakly active.

Example 4. Activity of Chp peptides: destruction of biofilm of gram-negative bacteria.

[00181] In order to evaluate anti-biofilm activity, the MBC values for peptides Chp2 and Chp4 were determined against mature biofilms formed by P. aeruginosa strain ATCC 17647 in tryptic soybean culture medium supplemented with 2% glucose. MPBA values of 0.25 μg/ml were observed for both Chp2 and Chp4 (Table 7), consistent with the ability to effectively disrupt mature biofilms. In comparison, the observed activity of RI18, a highly active AMP (15), was significantly lower at 4 μg/ml, and the observed activity of lysozyme T4, a weakly active lysine, was >64 μg/ml.

Example 5. Combination of Chp peptides and antibiotics.

[00182] To evaluate the synergy between either Chp2 or Chp4 and a panel of 11 antibiotics, each combination of Chp2 with 11 antibiotics and Chp4 with 11 antibiotics was tested in a standard checkerboard assay format using P. aeruginosa strain CFS-1292 in KAK medium supplemented with 2.5% human serum. In the checkerboard analysis, fractional inhibitory concentration index (FICI) values were calculated. FICI values ≤0.5 correspond to synergism, values >0.5 - 1 correspond to highly additive activity, values 1 - 2 correspond to additive activity and values >2 are considered antagonistic. Table 8 below shows that for both Chp2 and Chp4 the values were consistent with either synergistic (i.e., ≤0.5) or highly additive (i.e., >0.5 - 1) interactions between the Chp peptide and the antibiotic .

Example 6. Evaluation of hemolytic activity of Chp peptides.

[00183] Antimicrobial peptides suitable for use in the treatment of invasive infections should exhibit low toxicity to red blood cells (Oddo A. et al., 2017. Methods Mol Biol 1548:427-435). To investigate possible hemolytic activity, a conventional technique (described above in the Materials and Methods section) for measuring the ability of AMPs to lyse red blood cells, based on the determination of minimum hemolytic concentrations (MHCs) for human red blood cells, was used. No hemolysis was observed for 33 of the 37 Chp peptides tested, with MGC values >128 μg/mL (Table 9). The Triton X100 control was tested at an initial concentration of 2%, and MHA was observed at 0.007%. In comparison, four AMPs with known hemolytic activity, including RI18, R12, RR12p, and RR12h, had MHA values in the range of 4–128 μg/mL. Triton X-100, a membrane-lying detergent commonly used as a positive control in hemolytic assays, was hemolytic over a concentration range of 2% to 0.007%. These findings suggest that Chp peptides do not exhibit the in vitro toxicity (i.e., hemolytic activity) typically observed with AMPs. This property is expected to also be present in the remaining Chp peptides from Tables 1 and 2, based not only on percent sequence identity, 3D structure similarity, and charge profile, but also on the prediction that, as lytic agents, the peptides of the present invention are most likely to will be very highly specific to the membrane of gram-negative cells.

Example 7 Duration of lytic activity against Gram-negative bacteria.

[00184] The activity of Chp2 and Chp4 against P aeruginosa strain CFS-1292 was tested in a time-kill activity assay format using KAK with 2.5% human serum as described in Materials and Methods. Assessment of bacterial viability 1, 3 and 24 hours after treatment with concentrations of 1 μg/ml and 10 μg/ml of either Chp2 or Chp4 showed a decrease by several orders of magnitude, corresponding to effective bactericidal activity in all cases (Table 10). Table 10 shows the orders of magnitude (log) reduction in colony-forming units (compared to untreated controls) as determined using the time-of-kill activity assay format for P. aeruginosa strain CFS-1292 following treatment with CAK supplemented with 2.5% human serum .

[00185] In addition, stability was assessed to detect the fold change in MIC after incubation of the peptides prepared as described above in Example 2. Stability was assessed after incubation in 100% human serum at 37°C after 10 minutes, 1 hour and 2 hours. The results are presented below in Table 11.

Table 11 shows that all of Chp1, Chp2, CPAR39, Chp4, Chp5, Chp6, Chp7, Chp8, Chp9, Chp10, Chp11, Chp12, Gkh1, Gkh2, Gkh3, Ecp1, Ecp2, Ecp3, Ecp4, Osp1, Unp1, Unp2, Unp3, Unp5, Unp6, Spi1, Spi2, Bdp1, Lvp1, Lvp2, ALCES1, AVQ206, AVQ244, CDL907, AGT915, HH3930, Fen7875 and SBR77 were quite stable at 10 minutes, 1 hour and 2 hours.

--->

LIST OF SEQUENCES

<110> CONTRAFECT CORPORATION

<120> ANTI-MICROBIAL POLYPEPTIDES OBTAINED FROM BACTERIOPHAGES AND THEIR

APPLICATION AGAINST GRAM NEGATIVE BACTERIA

<130> 0341.0002-PCT

<140>

<141>

<150> 62/650.235

<151> 2018-03-29

<160> 80

<170> PatentIn version 3.5

<210> 1

<211> 36

<212> PRT

<213> Chlamydia virus Chp1

<400> 1

Met Val Arg Arg Arg Arg Leu Arg Arg Arg Ile Ser Arg Arg Ile Phe

1 5 10 15

Arg Arg Thr Val Ala Arg Val Gly Arg Arg Arg Arg Ser Phe Arg Gly

20 25 30

Gly Ile Arg Phe

35

<210> 2

<211> 44

<212> PRT

<213> Chlamydia phage 2

<400> 2

Met Arg Leu Lys Met Ala Arg Arg Arg Tyr Arg Leu Pro Arg Arg Arg

1 5 10 15

Ser Arg Arg Leu Phe Ser Arg Thr Ala Leu Arg Met His Pro Arg Asn

20 25 30

Arg Leu Arg Arg Ile Met Arg Gly Gly Ile Arg Phe

35 40

<210> 3

<211> 35

<212> PRT

<213> Chlamydia virus CPAR39

<400> 3

Met Cys Lys Lys Val Cys Lys Lys Cys Pro Lys Lys Gly Pro Lys Asn

1 5 10 15

Ala Pro Lys Ile Gly Ala Phe Tyr Glu Arg Lys Thr Pro Arg Leu Lys

20 25 30

Gln Ser Thr

35

<210> 4

<211> 39

<212> PRT

<213> Chlamydia phage 4

<400> 4

Met Ala Arg Arg Tyr Arg Leu Ser Arg Arg Arg Ser Arg Arg Leu Phe

1 5 10 15

Ser Arg Thr Ala Leu Arg Met His Arg Arg Asn Arg Leu Arg Arg Ile

20 25 30

Met Arg Gly Gly Ile Arg Phe

35

<210> 5

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 5

Met Ala Glu Gln Tyr Glu Leu Ser Gln Glu Gln Ser Glu Gln Leu Phe

1 5 10 15

Ser Glu Thr Ala Leu Gln Met His Glu Gln Asn Glu Leu Gln Glu Ile

20 25 30

Met Gln Gly Gly Ile Glu Phe

35

<210> 6

<211> 40

<212> PRT

<213> Guinea pig chlamydia phage

<400> 6

Met Ala Arg Arg Arg Tyr Arg Leu Pro Arg Arg Arg Ser Arg Arg Leu

1 5 10 15

Phe Ser Arg Thr Ala Leu Arg Met His Pro Arg Asn Arg Leu Arg Arg

20 25 30

Ile Met Arg Gly Gly Ile Arg Phe

35 40

<210> 7

<211> 38

<212> PRT

<213> Chlamydia trachomatis

<400> 7

Met Lys Arg Arg Lys Met Thr Arg Lys Gly Ser Lys Arg Leu Phe Thr

1 5 10 15

Ala Thr Ala Asp Lys Thr Lys Ser Ile Asn Thr Ala Pro Pro Pro Met

20 25 30

Arg Gly Gly Ile Arg Leu

35

<210> 8

<211> 39

<212> PRT

<213> Chlamydia trachomatis

<400> 8

Met Ser Lys Lys Arg Ser Arg Met Ser Arg Arg Arg Ser Lys Lys Leu

1 5 10 15

Phe Ser Lys Thr Ala Leu Arg Thr Lys Ser Val Asn Thr Arg Pro Pro

20 25 30

Met Arg Gly Gly Phe Arg Phe

35

<210> 9

<211> 40

<212> PRT

<213> Chlamydia trachomatis

<400> 9

Met Ser Leu Arg Arg His Lys Leu Ser Arg Lys Ala Ser Lys Arg Ile

1 5 10 15

Phe Arg Lys Gly Ala Ser Arg Thr Lys Thr Leu Asn Thr Arg Ala Thr

20 25 30

Pro Met Arg Gly Gly Phe Arg Ile

35 40

<210> 10

<211> 38

<212> PRT

<213> Chlamydia trachomatis

<400> 10

Met Lys Arg Arg Lys Leu Ser Lys Lys Lys Ser Arg Lys Ile Phe Thr

1 5 10 15

Arg Gly Ala Val Asn Val Lys Lys Arg Asn Leu Arg Ala Arg Pro Met

20 25 30

Arg Gly Gly Phe Arg Ile

35

<210> 11

<211> 37

<212> PRT

<213> Chlamydia trachomatis

<400> 11

Met Ala Lys Lys Met Thr Lys Gly Lys Asp Arg Gln Val Phe Arg Lys

1 5 10 15

Thr Ala Asp Arg Thr Lys Lys Leu Asn Val Arg Pro Leu Leu Tyr Arg

20 25 30

Gly Gly Ile Arg Leu

35

<210> 12

<211> 39

<212> PRT

<213> Chlamydia trachomatis

<400> 12

Met Ala Gly Lys Lys Met Val Ser Lys Gly Lys Asp Arg Gln Ile Phe

1 5 10 15

Arg Lys Thr Ala Asp Arg Thr Lys Lys Met Asn Val Arg Pro Leu Leu

20 25 30

Tyr Arg Gly Gly Ile Arg Leu

35

<210> 13

<211> 41

<212> PRT

<213> Marine gokushovirus

<400> 13

Met Arg Arg Pro Arg Lys Met Asn Tyr Lys Lys Ser Lys Arg Met Phe

1 5 10 15

Ser Arg Thr Ala Ala Arg Thr His Arg Lys Asn Ser Leu Arg Gly Ser

20 25 30

Arg Pro Met Arg Gly Gly Ile Arg Leu

35 40

<210> 14

<211> 34

<212> PRT

<213> Gokushovirinae Fen672_31

<400> 14

Met Ser Lys Lys Ala Ser Arg Lys Ser Phe Thr Lys Gly Ala Val Lys

1 5 10 15

Val His Lys Lys Asn Val Pro Thr Arg Val Pro Met Arg Gly Gly Ile

20 25 30

Arg Leu

<210> 15

<211> 35

<212> PRT

<213> Unknown

<220>

<223> description of the unknown:

Microviridae phage sequence

<400> 15

Met Lys Met Arg Lys Arg Thr Asp Lys Arg Val Phe Thr Arg Thr Ala

1 5 10 15

Ala Lys Ser Lys Lys Val Asn Ile Ala Pro Lys Ile Phe Arg Gly Gly

20 25 30

Ile Arg Leu

35

<210> 16

<211> 39

<212> PRT

<213> Escherichia coli

<400> 16

Met Ala Arg Ser Arg Arg Arg Met Ser Lys Arg Ser Ser Arg Arg Ser

1 5 10 15

Phe Arg Lys Tyr Ala Lys Thr His Lys Arg Asn Phe Lys Ala Arg Ser

20 25 30

Met Arg Gly Gly Ile Arg Leu

35

<210> 17

<211> 47

<212> PRT

<213> Cognatishimia maritima

<400> 17

Met Glu Ser Pro Asn Ser Arg Ser Gln Leu Gly Ile Thr Leu Tyr Leu

1 5 10 15

Leu Ser Thr Ile Phe Pro Asp Ala Cys Phe Arg Tyr Arg Arg Glu Leu

20 25 30

Pro Tyr Pro Leu Val Ile Trp Gly Val Ala Thr Leu Cys Leu Gln

35 40 45

<210> 18

<211> 39

<212> PRT

<213> Escherichia coli

<400> 18

Met Ala Arg Ser Arg Arg Arg Met Ser Lys Arg Ser Ser Arg Arg Ser

1 5 10 15

Phe Arg Lys Tyr Ala Lys Ser His Lys Lys Asn Phe Lys Ala Arg Ser

20 25 30

Met Arg Gly Gly Ile Arg Leu

35

<210> 19

<211> 37

<212> PRT

<213> Oscillibacter species

<400> 19

Met Arg Lys Arg Met Ser Lys Arg Val Asp Lys Lys Val Phe Arg Arg

1 5 10 15

Thr Ala Ala Ser Ala Lys Lys Ile Asn Ile Asp Pro Lys Ile Tyr Arg

20 25 30

Gly Gly Ile Arg Leu

35

<210> 20

<211> 37

<212> PRT

<213> Unknown

<220>

<223> description of the unknown:

Microviridae phage sequence

<400> 20

Met Arg Arg Arg Arg Leu Ser Arg Arg Thr Ser Arg Arg Phe Phe Arg

1 5 10 15

Lys Gly Leu Lys Val Arg Arg Arg Asn Leu Arg Ala Arg Pro Met Arg

20 25 30

Gly Gly Phe Arg Ile

35

<210> 21

<211> 39

<212> PRT

<213> Unknown

<220>

<223> description of the unknown:

Microviridae phage sequence

<400> 21

Met Ala Arg Arg Lys Lys Met Lys Gly Lys Arg Asp Lys Arg Val Phe

1 5 10 15

Lys Gln Thr Ala Asn Lys Thr Lys Ala Ile Asn Ile Ser Pro Lys Asn

20 25 30

Met Arg Gly Gly Thr Arg Leu

35

<210> 22

<211> 53

<212> PRT

<213> Marine gokushovirus

<400> 22

Met Leu Thr Val Trp Ser Asp Thr Pro Thr Ile Lys Arg Arg Lys Asp

1 5 10 15

Met Tyr Arg Lys Arg Met Ser Arg Lys Lys Ser Lys Lys Val Phe Ala

20 25 30

Lys Thr Ala Met Lys Val Asn Lys Arg Asn His Val Lys Pro Met Arg

35 40 45

Gly Gly Tyr Arg Ile

50

<210> 23

<211> 39

<212> PRT

<213> Marine gokushovirus

<400> 23

Met Met Lys Tyr Arg Lys Lys Met Ser Ala Lys Ser Ser Arg Lys Gln

1 5 10 15

Phe Thr Lys Gly Ala Met Lys Val Lys Gly Lys Asn Phe Thr Lys Pro

20 25 30

Met Arg Gly Gly Ile Arg Leu

35

<210> 24

<211> 38

<212> PRT

<213> Marine gokushovirus

<400> 24

Met Arg Arg Tyr Asn Val Asn Lys Gly Lys Ser Ala Lys Lys Phe Arg

1 5 10 15

Lys Gln Val Ser Lys Thr Lys Val Ala Asn Leu Arg Ser Asn Pro Met

20 25 30

Arg Gly Gly Trp Arg Leu

35

<210> 25

<211> 28

<212> PRT

<213> Spiroplasma virus

<400> 25

Met Ala Tyr Arg Gly Phe Lys Thr Ser Arg Val Val Lys His Arg Val

1 5 10 15

Arg Arg Arg Trp Phe Asn His Arg Arg Arg Tyr Arg

20 25

<210> 26

<211> 38

<212> PRT

<213> Spiroplasma virus

<400> 26

Met Arg Arg Lys Val Lys Asn Thr Lys Arg His Gln Trp Arg Leu Thr

1 5 10 15

His Ser Ala Arg Ser Ile Lys Arg Ala Asn Ile Met Pro Ser Asn Pro

20 25 30

Arg Gly Gly Arg Arg Phe

35

<210> 27

<211> 111

<212> DNA

<213> Chlamydia virus Chp1

<400> 27

atggttcgta gaagacgttt gagaagaaga ataagtagaa gaatttttag aagaacagta 60

gctagagttg gtagaaggcg aaggtctttt cgtggtggta ttagatttta a 111

<210> 28

<211> 135

<212> DNA

<213> Chlamydia phage 2

<400> 28

atgaggttaa aaatggcacg aagaagatac agacttccgc gacgtagaag tcgaagactt 60

ttttcaagaa ctgcattgag gatgcatcca agaaataggc ttcgaagaat tatgcgtggc 120

ggcattaggt tctag 135

<210> 29

<211> 108

<212> DNA

<213> Chlamydia virus CPAR39

<400> 29

ttgtgcaaaa aagtgtgcaa aaaatgccca aaaaaagggc caaaaaatgc ccccaaaatc 60

ggagcatttt acgagaaaa aacacctaga cttaaacagt ctacttga 108

<210> 30

<211> 120

<212> DNA

<213> Chlamydia phage 4

<400> 30

atggcacgaa gatacagact ttcgcgacgc agaagtcgac gacttttttc aagaactgca 60

ttaagaatgc atcgaagaaa tagacttcga agaattatgc gtggcggcat taggttttag 120

<210> 31

<211> 120

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 31

atggcggaac agtatgaact gagccaggaa cagagcgaac agctgtttag cgaaaccgcg 60

ctgcagatgc atgaacagaa cgaactgcag gaaattatgc agggcggcat tgaattttaa 120

<210> 32

<211> 123

<212> DNA

<213> Guinea pig chlamydia phage

<400> 32

atggcacgaa gaagatacag acttccgcga cgtagaagtc gaagactttt ttcaagaact 60

gcattaagga tgcatccaag aaataggctt cgaagaatta tgcgtggcgg cattaggttc 120

tag 123

<210> 33

<211> 117

<212> DNA

<213> Chlamydia trachomatis

<400> 33

atgaaacgta gaaaaatgac aagaaaaggt tctaagcgtc tttttactgc aactgctgat 60

aaaactaaat ctatcaatac tgccccgccg ccaatgcgtg gcggtatccg gttgtaa 117

<210> 34

<211> 120

<212> DNA

<213> Chlamydia trachomatis

<400> 34

atgtctaaaa agcgttctcg catgtctcgc cgccgttcta agaagttgtt ctcgaaaacg 60

gctctccgca cgaagagtgt caacacccgt ccgcctatgc gcggagggtt ccggttctga 120

<210> 35

<211> 123

<212> DNA

<213> Chlamydia trachomatis

<400> 35

atgtctcttc gtcgtcataa gctttctcgt aaggcgtcta agcgtatttt tcgtaaaggt 60

gcatcacgca cgaagacttt gaatactcgt gctacgccta tgcgcggcgg tttccgtatt 120

taa 123

<210> 36

<211> 117

<212> DNA

<213> Chlamydia trachomatis

<400> 36

atgaaacgtc gtaaactgtc caaaaagaaa tctcgcaaga ttttcactcg cggtgctgta 60

aatgtgaaaa agcgtaacct tcgcgctcgc ccaatgcgcg gcggtttccg gatctaa 117

<210> 37

<211> 114

<212> DNA

<213> Chlamydia trachomatis

<400> 37

atggctaaaa aaatgactaa aggcaaggat cgtcaggttt ttcgtaaaac cgctgatcgt 60

actaagaaac tcaatgttag accgttgtta tatcgaggag gtatcagatt atga 114

<210> 38

<211> 120

<212> DNA

<213> Chlamydia trachomatis

<400> 38

atggcaggaa aaaaaatggt atcaaaagga aaagatagac agattttccg aaaaactgct 60

gatcgcacta aaaaaatgaa tgtgcgcccg ctattatatc gtggaggtat tagattatga 120

<210> 39

<211> 126

<212> DNA

<213> Marine gokushovirus

<400> 39

atgagaagac caagaaaaat gaactataaa aaatcaaaaa gaatgttttc acgcacagca 60

gcgagaacac acagaaaaaa ctctctaaga ggtagccgac ctatgagagg cggaatacgt 120

cttaa 126

<210> 40

<211> 105

<212> DNA

<213> Gokushovirinae Fen672_31

<400> 40

atgtcgaaga aggcgtcgag gaagagtttt actaagggtg ccgttaaggt tcataagaaa 60

aatgttccta ctcgtgttcc tatgcgtggc ggtattaggc tttag 105

<210> 41

<211> 108

<212> DNA

<213> Unknown

<220>

<223> description of the unknown:

Microviridae phage sequence

<400> 41

atgaaaatgc gtaagcggac ggacaagcga gtgtttaccc gcaccgctgc taagtccaag 60

aaagtgaaca ttgccccgaa aatttttaga ggaggtatcc gtctgtga 108

<210> 42

<211> 120

<212> DNA

<213> Escherichia coli

<400> 42

atggctcgtt ctcgccgtcg tatgtccaag cgttcttccc gtcgttcgtt ccgtaagtac 60

gcaaagacgc ataaacgtaa ctttaaagcc cgctctatgc gtggtggaat tcgtctttga 120

<210> 43

<211> 144

<212> DNA

<213> Cognatishimia maritima

<400> 43

atggaaagcc cgaacagccg cagccagctg ggcattaccc tgtatctgct gagcaccatt 60

tttccggatg cgtgctttcg ctatcgccgc gaactgccgt atccgctggt gatttggggc 120

gtggcgaccc tgtgcctgca gtaa 144

<210> 44

<211> 120

<212> DNA

<213> Escherichia coli

<400> 44

atggctcgtt cccgtagacg tatgtctaag cgttcttccc gccgttcgtt ccgcaagtat 60

gcgaagtcgc ataagaagaa ctttaaagcc cgctcaatgc gtggcggtat ccgtttataa 120

<210> 45

<211> 114

<212> DNA

<213> Oscillibacter species

<400> 45

atgagaaagc gaatgtctaa gcgtgttgac aagaaggtgt tccgtcgtac tgccgcatct 60

gccaagaaga ttaacattga ccccaagatt taccgtggag gtattcgcct atga 114

<210> 46

<211> 114

<212> DNA

<213> Unknown

<220>

<223> description of the unknown:

Microviridae phage sequence

<400> 46

atgagacgtc gtcgtctatc ccgcagaact tcccgccgtt ttttccgtaa aggacttaag 60

gttcgccgtc gtaacctccg cgcgagaccc atgagaggcg gattcagaat ttga 114

<210> 47

<211> 120

<212> DNA

<213> Unknown

<220>

<223> description of the unknown:

Microviridae phage sequence

<400> 47

atggcacgac gcaagaagat gaaaggcaag cgggataaac gggtgtttaa gcagacagcc 60

aacaaaacca aggctatcaa catcagccca aaaaacatga gagggggtac gagactgtga 120

<210> 48

<211> 162

<212> DNA

<213> Marine gokushovirus

<400> 48

atgttaactg tgtggagtga cacccctacc ataaaaagga gaaaagacat gtatagaaag 60

agaatgtcaa gaaagaaaag taaaaaggtt tttgcaaaaa ccgcaatgaa agtaaataaa 120

agaaaccacg ttaaacctat gcgtggtgga tatagaatat aa 162

<210> 49

<211> 120

<212> DNA

<213> Marine gokushovirus

<400> 49

atgatgaagt acagaaaaaa aatgagcgct aaaagtagcc gaaagcaatt tacaaaaggc 60

gccatgaaag tgaagggtaa aaacttcaca aaaccaatgc gcggaggcat ccgtctatag 120

<210> 50

<211> 117

<212> DNA

<213> Marine gokushovirus

<400> 50

atgcgacgtt acaatgtaaa taaaggtaaa tctgctaaga agtttcgaaa gcaggtaagt 60

aagacgaagg ttgcaaacct acgttctaat ccaatgcgag gtggttggag actctaa 117

<210> 51

<211> 87

<212> DNA

<213> Spiroplasma virus

<400> 51

atggcttatc gtggttttaa aacgagtcgt gttgtaaaac atagagtacg tagaagatgg 60

tttaatcata gaagacgtta tagatag 87

<210> 52

<211> 117

<212> DNA

<213> Spiroplasma virus

<400> 52

gtgagacgca aggttaagaa cacaaagcgt catcagtgga ggttgactca ttctgcacgt 60

tcaattaaac gtgctaatat aatgccgtca aatcctcgtg gtggacgtcg tttttag 117

<210> 53

<211> 135

<212> DNA

<213> Chlamydia phage 3

<400> 53

atgaggttaa aaatggcacg aagaagatac agacttccgc gacgtagaag tcgaagactt 60

ttttcaagaa ctgcattaag gatgcatcca agaaataggc ttcgaagaat tatgcgtggc 120

ggcattaggt tctag 135

<210> 54

<211> 44

<212> PRT

<213> Chlamydia phage 3

<400> 54

Met Arg Leu Lys Met Ala Arg Arg Arg Tyr Arg Leu Pro Arg Arg Arg

1 5 10 15

Ser Arg Arg Leu Phe Ser Arg Thr Ala Leu Arg Met His Pro Arg Asn

20 25 30

Arg Leu Arg Arg Ile Met Arg Gly Gly Ile Arg Phe

35 40

<210> 55

<211> 39

<212> PRT

<213> Escherichia species

<400> 55

Met Ala Arg Ser Arg Arg Arg Met Ser Lys Arg Ser Ser Arg Arg Ser

1 5 10 15

Phe Arg Lys Tyr Ala Lys Thr His Lys Lys Asn Phe Lys Ala Arg Ser

20 25 30

Met Arg Gly Gly Ile Arg Leu

35

<210> 56

<211> 39

<212> PRT

<213> Escherichia species

<400> 56

Met Ala Arg Ser Arg Arg Arg Met Ser Lys Arg Ser Ser Arg Arg Ser

1 5 10 15

Phe Arg Lys Tyr Ala Lys Ser His Lys Lys Asn Phe Lys Ala Arg Ser

20 25 30

Met Arg Gly Gly Ile Arg Leu

35

<210> 57

<211> 55

<212> PRT

<213> phage PP7 Pseudomonas

<400> 57

Met Ser Ser Thr Leu Cys Arg Trp Ala Val Lys Ala Leu Arg Cys Thr

1 5 10 15

Arg Val Tyr Lys Glu Phe Ile Trp Lys Pro Leu Val Ala Leu Ser Tyr

20 25 30

Val Thr Leu Tyr Leu Leu Ser Ser Val Phe Leu Ser Gln Leu Ser Tyr

35 40 45

Pro Ile Gly Ser Trp Ala Val

50 55

<210> 58

<211> 35

<212> PRT

<213> Phage AP205 Acinetobacter

<400> 58

Met Lys Lys Arg Thr Lys Ala Leu Leu Pro Tyr Ala Val Phe Ile Ile

1 5 10 15

Leu Ser Phe Gln Leu Thr Leu Leu Thr Ala Leu Phe Met Tyr Tyr His

20 25 30

Tyr Thr Phe

35

<210> 59

<211> 38

<212> PRT

<213> moose feces-associated Microvirus MP12 5423

<400> 59

Met Ala Lys Lys Ile Arg Asn Lys Ala Arg Asp Arg Arg Ile Phe Thr

1 5 10 15

Arg Thr Ala Ser Arg Met His Lys Ala Asn Arg Thr Pro Arg Phe Met

20 25 30

Arg Gly Gly Ile Arg Leu

35

<210> 60

<211> 38

<212> PRT

<213> Unknown

<220>

<223> description of the unknown:

sequence of environmental Gokushovirinae specimens

<400> 60

Met Arg Arg Lys Lys Met Ser Arg Gly Lys Ser Lys Lys Leu Phe Arg

1 5 10 15

Arg Thr Ala Lys Arg Val His Arg Lys Asn Leu Arg Ala Arg Pro Met

20 25 30

Arg Gly Gly Ile Arg Met

35

<210> 61

<211> 39

<212> PRT

<213> Unknown

<220>

<223> description of the unknown:

sequence of environmental Gokushovirinae specimens

<400> 61

Met Ala Lys Arg His Lys Ile Pro Gln Arg Ala Ser Gln His Ser Phe

1 5 10 15

Thr Arg His Ala Gln Lys Val His Pro Lys Asn Val Pro Arg Leu Pro

20 25 30

Met Arg Gly Gly Ile Arg Leu

35

<210> 62

<211> 37

<212> PRT

<213> Unknown

<220>

<223> description of the unknown:

sequence of an uncultured bacterium

<400> 62

Met Arg Lys Lys Met His Lys Ser Leu Asp Lys Arg Val Phe Asn Arg

1 5 10 15

Thr Ala Lys Lys Ser Lys Lys Ile Asn Val Asn Pro Val Val Tyr Arg

20 25 30

Gly Gly Ile Arg Leu

35

<210> 63

<211> 38

<212> PRT

<213> Marine gokushovirus

<400> 63

Met Arg Arg Tyr Asn Val Asn Lys Gly Lys Ser Ala Lys Lys Phe Arg

1 5 10 15

Lys Gln Val Ser Lys Thr Lys Val Ala Asn Leu Arg Ser Asn Pro Met

20 25 30

Arg Gly Gly Trp Arg Leu

35

<210> 64

<211> 41

<212> PRT

<213> Unknown

<220>

<223> description of the unknown:

sequence HH01 Richelia intracellularis

<400> 64

Met Arg Pro Val Lys Arg Ser Arg Val Asn Lys Ala Arg Ser Ala Gly

1 5 10 15

Lys Phe Arg Lys Gln Val Gly Lys Thr Lys Met Ala Asn Leu Arg Ser

20 25 30

Asn Pro Met Arg Gly Gly Trp Arg Leu

35 40

<210> 65

<211> 41

<212> PRT

<213> Gokushovirinae Fen7875_21

<400> 65

Met Lys Pro Leu Lys Arg Lys Pro Val Gln Lys Ala Arg Ser Ala Ala

1 5 10 15

Lys Phe Arg Arg Asn Val Ser Thr Val Lys Ala Ala Asn Met Ala Val

20 25 30

Lys Pro Met Arg Gly Gly Trp Arg Phe

35 40

<210> 66

<211> 44

<212> PRT

<213> mycobacterial phage BabyRay

<400> 66

Met Thr Lys Arg Asp Ile Glu Tyr Arg Lys Ala Leu Gly Leu Asn Pro

1 5 10 15

Ser Glu Pro Leu Pro Lys Ile Val Gly Ala Val Thr Arg His Gly Ala

20 25 30

Thr Leu Lys Arg Pro Arg Val Thr Ala Leu Ala Arg

35 40

<210> 67

<211> 38

<212> PRT

<213> Unknown

<220>

<223> description of the unknown:

Bdellovibrio phage phiMH2K sequence

<400> 67

Met Lys Arg Lys Pro Met Ser Arg Lys Ala Ser Gln Lys Thr Phe Lys

1 5 10 15

Lys Asn Thr Gly Val Gln Arg Met Asn His Leu Asn Pro Arg Ala Met

20 25 30

Arg Gly Gly Ile Arg Leu

35

<210> 68

<211> 120

<212> DNA

<213> Escherichia species

<400> 68

atggctcgtt ctcgtcgtcg tatgtctaaa cgttcttctc gtcgttcttt tcgtaaatat 60

gctaaaactc ataaaaaaaa ttttaaagct cgttctatgc gtggaggaat tcgtttataa 120

<210> 69

<211> 117

<212> DNA

<213> Escherichia species

<400> 69

atggcgcgca gccgccgccg catgagcaaa cgcagcagcc gccgcagctt tcgcaaatat 60

gcgaaaagcc ataaaaaaaa ctttaaagcg cgcagcatgc gcggcggcat tcgcctg 117

<210> 70

<211> 168

<212> DNA

<213> phage PP7 Pseudomonas

<400> 70

ttgtcgtcaa ccttgtgccg ctgggccgtt aaggccctgc ggtgtacccg tgtgtataag 60

gagtttatat ggaaaccctt agtagcgctc agttacgtga cgttgtatct tctgagctcg 120

gtcttcctgt cccaactcag ctaccccatc gggagctggg cggtgtag 168

<210> 71

<211> 108

<212> DNA

<213> Phage AP205 Acinetobacter

<400> 71

atgaagaaaa ggacaaaagc cttgcttccc tatgcggttt tcatcatact cagctttcaa 60

ctaacattgt tgactgcctt gtttatgtat taccattata ccttttag 108

<210> 72

<211> 117

<212> DNA

<213> moose feces-associated Microvirus MP12 5423

<400> 72

atggcaaaga aaattagaaa caaagcacgt gataacgta tcttcacaag aacagcttca 60

cgcatgcaca aggcaaaccg cacaccaaga tttatgagag gcggtattag gttatga 117

<210> 73

<211> 117

<212> DNA

<213> Unknown

<220>

<223> description of the unknown:

sequence of environmental Gokushovirinae specimens

<400> 73

atgcgtcgta aaaaaatgtc acgcggtaaa tcaaaaaaac tctttcgccg aacagcaaaa 60

cgcgttcatc gaaaaaacct acgagctcgc ccaatgcgtg gcggcatacg catgtag 117

<210> 74

<211> 120

<212> DNA

<213> Unknown

<220>

<223> description of the unknown:

sequence of environmental Gokushovirinae specimens

<400> 74

atggcgaagc gacacaaaat cccgcaacgc gcgtcacaac attccttcac gcgccatgcg 60

caaaaggtcc accctaagaa cgttccccgc ctgccaatgc gaggcggtat ccgtctctaa 120

<210> 75

<211> 114

<212> DNA

<213> Unknown

<220>

<223> description of the unknown:

sequence of an uncultured bacterium

<400> 75

atgcgtaaaa aaatgcacaa atcattagac aagcgagtgt ttaaccgcac tgcaaaaaaa 60

tcaaaaaaaa taaatgttaa tcctgtagtt tatcgtggag gtattagatt atga 114

<210> 76

<211> 117

<212> DNA

<213> Marine gokushovirus

<400> 76

atgcgacgtt acaatgtaaa taaaggtaaa tctgctaaga agtttcgaaa gcaggtaagt 60

aagacgaagg ttgcaaacct acgttctaat ccaatgcgag gtggttggag actctaa 117

<210> 77

<211> 126

<212> DNA

<213> Unknown

<220>

<223> description of the unknown:

sequence HH01 Richelia intracellularis

<400> 77

atgcgtccag ttaaaagatc aagagtaaat aaggcccgat ctgcaggcaa gtttcgtaag 60

caggtcggta aaacaaagat ggcaaatctg cgtagtaatc cgatgcgcgg cggatggcgg 120

ctgtga 126

<210> 78

<211> 126

<212> DNA

<213> Gokushovirinae Fen7875_21

<400> 78

atgaagccat tgaagcgtaa gccggttcag aaggcgcggt cagcagccaa gttccgtcga 60

aatgtgtcta ccgttaaggc tgccaatatg gcggtgaagc cgatgcgcgg cggttggcgg 120

ttctga 126

<210> 79

<211> 135

<212> DNA

<213> mycobacterial phage BabyRay

<400> 79

atgaccaaga gagacatcga gtaccggaaa gctttggggc tcaacccatc tgagccgctc 60

ccgaagattg tgggtgccgt cacccgccac ggggccactc tgaaacgccc acgggtcacc 120

gcactggcccgatag 135

<210> 80

<211> 117

<212> DNA

<213> Unknown

<220>

<223> description of the unknown:

Bdellovibrio phage phiMH2K sequence

<400> 80

atgaaaagaa aaccaatgag ccgcaaggcc tctcaaaaaa ccttcaaaaa gaacacaggc 60

gttcaacgca tgaaccatct caacccacgc gccatgcgtg gtggcattag actataa 117

<---

Claims (22)

1. A pharmaceutical composition for inhibiting the growth, reducing the population or destroying at least one type of gram-negative bacteria, containing: an effective amount of: (i) an isolated Chp peptide having the amino acid sequence of SEQ ID NO: 2, or an active fragment thereof, in which the α-helical domain is retained; or (ii) a modified Chp peptide, the amino acid sequence of which is at least 80% identical to the amino acid sequence of SEQ ID NO: 2, and in which the α-helical domain is retained, wherein said Chp peptide, active fragment or Chp peptide having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, inhibits the growth of, reduces the population of or eliminates at least one species of gram-negative bacteria; And pharmaceutically acceptable carrier. 2. The pharmaceutical composition according to claim 1, characterized in that the Chp peptide further contains at least one non-naturally occurring modification with respect to the amino acid sequence SEQ ID NO: 2 or active fragments thereof. 3. The pharmaceutical composition according to claim 2, characterized in that said non-naturally occurring modification is selected from the group consisting of modifications by substitution, modifications by N-terminal acetylation, and modifications by C-terminal amidation. 4. Pharmaceutical composition according to any one of paragraphs. 1 - 3, characterized in that the amino acid sequence is selected from SEQ ID NO: 2 or active fragments thereof. 5. The pharmaceutical composition according to claim 1, characterized in that the amino acid sequence is SEQ ID NO: 2. 6. The pharmaceutical composition according to claim 1, which is a solution, suspension, emulsion, inhalation powder, aerosol or spray. 7. The pharmaceutical composition according to claim 1, further containing one or more antibiotics suitable for the treatment of gram-negative bacteria. 8. The pharmaceutical composition according to claim 1, characterized in that said amino acid sequence is SEQ ID NO: 4. 9. A method of preventing or treating a bacterial infection caused by at least one species of gram-negative bacteria, comprising administering to a subject diagnosed with, at risk of developing, or exhibiting symptoms of a bacterial infection, an effective amount of a pharmaceutical composition according to any one of claims. 18. 10. The method according to claim 9, characterized in that the gram-negative bacteria are selected from the group consisting of Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Salmonella, Neisseria gonorrhoeae and Shigella . 11. The method according to claim 9, characterized in that at least one species of gram-negative bacteria is Pseudomonas aeruginosa . 12. The method according to claim 9, characterized in that the bacterial infection is a topical or systemic bacterial infection. 13. The method of claim 9, further comprising administering to said subject an antibiotic suitable for treating an infection caused by a gram-negative bacterium. 14. The method according to claim 13, characterized in that the antibiotic is selected from one or more of the following: azithromycin, aztreonam, fosfomycin, ceftazidime, cefepime, cefoperazone, ceftobiprole, ciprofloxacin, levofloxacin, aminoglycosides, imipenem, meropenem, doripenem, gentamicin, tobramycin , amikacin, piperacillin, ticarcillin, penicillin, rifampicin, polymyxin B and colistin. 15. The method according to claim 14, characterized in that the antibiotic is selected from one or more of the following: amikacin, azithromycin, aztreonam, ciprofloxacin, colistin, fosfomycin, gentamicin, imipenem, piperacillin, rifampicin and tobramycin. 16. The method according to claim 13, characterized in that the introduction of the pharmaceutical composition according to claims. 1 - 8 are more effective in inhibiting the growth, reducing populations, or killing Gram-negative bacteria than administering the antibiotic alone. 17. A method of preventing or treating a bacterial infection caused by at least one species of gram-negative bacteria, comprising co-administering to a subject diagnosed with, at risk of developing, or exhibiting symptoms of a bacterial infection, a combination of an effective amount of a pharmaceutical composition according to any one of claims. 1 to 8 and an effective amount of antibiotic suitable for treating infection caused by a gram-negative bacterium, wherein said first and second amounts are collectively effective to prevent or treat said bacterial infection. 18. The method according to claim 17, characterized in that the antibiotic is selected from one or more of the following: azithromycin, aztreonam, fosfomycin, ceftazidime, cefepime, cefoperazone, ceftobiprole, ciprofloxacin, levofloxacin, aminoglycosides, imipenem, meropenem, doripenem, gentamicin, tobramycin , amikacin, piperacillin, ticarcillin, penicillin, rifampicin, polymyxin B and colistin. 19. The method according to claim 17 or 18, characterized in that the antibiotic is selected from one or more of the following: amikacin, azithromycin, aztreonam, ciprofloxacin, colistin, fosfomycin, gentamicin, imipenem, piperacillin, rifampicin and tobramycin.