Antimicrobial peptide compounds and methods of use

Abstract

Antimicrobial peptide compounds, pharmaceutical compositions, and methods of using them for inhibiting microbial growth are provided. The provided methods include inhibiting pan-drug-resistant Acinetobacter baumannii by administering the peptide compounds to a subject. A method for inhibiting pan-drug-resistant Acinetobacter baumannii using a synergistic combination of the peptide compound acetyl-as-aib-lrkl-aib-krll-amide (SEQ ID NO: 1) and polymyxin B is provided. Another method provides peptide compounds for inhibiting P. gingivalis, a keystone bacterium in periodontal disease, the sixth most common infectious disease worldwide.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 368,505, filed July 15, 2022, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates to antimicrobial peptide compounds, and in particular to their use in the treatment of microbial infections, including pan-drug resistant bacteria. [Background technology]

[0003] The emergence of resistance to multiple antimicrobial agents in pathogenic bacteria is a major public health problem. Standardized definitions of resistance have been established for pathogenic bacteria using documents and breakpoints from the Clinical and Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and the U.S. Food and Drug Administration (FDA). Multidrug resistance (MDR) is defined as acquired nonsusceptibility to at least one agent in three or more antibiotic categories, extensively drug resistant (XDR) is defined as nonsusceptibility to at least one agent in all but two or fewer antibiotic categories (i.e., the bacterial isolate remains susceptible to only one or two categories), and pandrug resistance (PDR) is defined as nonsusceptibility to all agents in all antibiotic categories.

[0004] One particularly problematic bacterium is Acinetobacter baumannii. Acinetobacter baumannii is a Gram-negative opportunistic pathogen that causes severe hospital-acquired infections in immunocompromised patients. A notable feature of this bacterium is its ability to rapidly develop antimicrobial resistance and survive for weeks on dry surfaces in healthcare facilities. As early as 2009, carbapenem-resistant A. baumannii was listed by the Infectious Diseases Society of America as one of six high-threat pathogens, also known as "ESKAPE" pathogens, urging global collaboration to address this threat. In particular, the emergence of chromosomally encoded and plasmid-mediated polymyxin resistance in A. baumannii breaches last-line defenses. The spread of A. baumannii infections is now considered a major and complex global health problem.

[0005] In addition to multidrug-resistant, extensively resistant, and pan-resistant bacteria, P. gingivalis is a keystone bacterium in periodontal disease (PD), the sixth most common infectious disease worldwide. In the United States, half of adults over the age of 30 and 70% of adults over the age of 65 suffer from some degree of P. gingivalis infection associated with gum disease. Older men and African Americans are at higher risk of developing periodontal disease. A study following 6,800 patients for up to 26 years found that gingivitis followed by more severe periodontal disease was associated with an increased risk of dementia, including Alzheimer's disease. Tooth loss, a frequent consequence of PD, also increases the risk of dementia. P. gingivalis products, including lipopolysaccharide (LPS), cysteine proteases known as gingipains, and 16S rRNA, have all been found in the brains of humans with AD. P. gingivalis can be found in the mouth and blood and is known to invade non-oral tissues, including the brain, where it can induce a pro-inflammatory state that is also associated with AD. Therefore, strategies to reduce the number and activity of P. gingivalis are already beginning to show positive effects on AD-related outcomes.

[0006] Therefore, there is an urgent and immediate need for new antibacterial agents that have activity against drug-resistant bacteria, including pan-drug-resistant bacteria, and P. gingivalis. The present disclosure provides such antibacterial agents. Summary of the Invention

[0007] In one aspect of the present invention, a series of peptide compounds having antibacterial activity are provided. The peptide compounds of the present invention have a structure selected from the following: (COG1410) Acetyl-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 1); Picolinyl-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 2); Picolinyl-AS-C-LRKL-aib-KRLL-C-amide (SEQ ID NO: 3); wherein a disulfide bond exists between the two cysteine residues; Acetyl-LLRK-aib-LKRL-aib-SA-CONH2 (SEQ ID NO: 4); Acetyl-llrk-Aib-lkkl-Aib-sa-amide (SEQ ID NO: 5), wherein all amino acid residues are D-amino acids; Acetyl-as-aib-lrkl-aib-krll-amide (SEQ ID NO: 6), wherein all amino acid residues are D-amino acids; Acetyl-LLRK-aib-LRKL-aib-SAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 7); Acetyl-LRVRCAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 8); Acetyl-LRVRLAS-aib-LKKL-aib-KRLL-amide (SEQ ID NO: 9); Acetyl-LRVRLAS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 10); Acetyl-llrk-aib-lkrl-aib-salrvrl-amide (SEQ ID NO: 11), wherein all amino acid residues are D-amino acids; Acetyl-LRVRLASHLRKLRKRLLAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 12); C8-as-aib-lrkl-aib-krll-amide (SEQ ID NO: 13); Acetyl-K(C8)-as-aib-lrkl-aib-krll-amide (SEQ ID NO: 14); Acetyl-K(picolinyl)-as-aib-lrkl-aib-krll-amide (SEQ ID NO: 15); Acetyl-LRVRLASHLRKLRKRLLR-amide (SEQ ID NO: 16); Acetyl-LRKLRKRLLLRKLRKRLL-amide (SEQ ID NO: 17); Acetyl-LRVRLASHLRKLRKRLLRDADDLQKRLAVY-amide (SEQ ID NO: 18); and Picolinyl-llrk-aib-lkrl-aib-salrvrl-amine (SEQ ID NO: 19), wherein all amino acid residues are D-amino acids; where aib is aminoisobutyric acid.

[0008] In another embodiment, a peptide compound of the invention, or a pharmaceutically acceptable salt or solvate thereof, is provided for use in a method of inhibiting microbial growth in a subject.

[0009] In another aspect, pharmaceutical compositions comprising peptide compounds of the invention are provided for use in methods of inhibiting microbial growth in a subject.

[0010] The pharmaceutical composition may comprise the peptide compound SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The pharmaceutical composition may comprise the peptide compound SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some cases, the pharmaceutical composition comprises the peptide compound SEQ ID NO: 1.

[0011] In other embodiments, the pharmaceutical composition comprises one or more other antibacterial compounds. The one or more antibacterial compounds may include polymyxin B.

[0012] In one aspect, a method for inhibiting microbial growth is provided, comprising contacting a microorganism with an effective amount of a peptide compound of the invention to inhibit microbial growth. In certain embodiments of this method, the peptide compound can comprise a peptide compound of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15. In another example, the peptide compound can comprise SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In some cases, the peptide compound comprises SEQ ID NO:1.

[0013] In a method comprising contacting a microorganism with an effective amount of a peptide compound of the present invention, the microorganism can include one or more of multidrug-resistant (MDR), extensively drug-resistant (XDR), or pan-drug-resistant (PDR) bacteria. In some examples, the microorganism includes Porphyromonas gingivalis. The microorganism can include a gram-negative bacterial pathogen or a gram-positive pathogen. The gram-negative bacterial pathogen can include one or a combination of Acinetobacter baumannii, P. gingivalis, or Escherichia coli, and the gram-positive pathogen can include one or a combination of Staphylococcus aureus or L. salivarius.

[0014] In some embodiments of the method, which includes contacting a microorganism with an effective amount of a peptide compound of the present invention, the peptide compound is contacted with the microorganism in combination with one or more other antibacterial compounds. The one or more antibacterial compounds can include polymyxin B. Microbial growth inhibited by the method can include microbial growth caused by pan-drug-resistant Acinetobacter baumannii or multidrug-resistant Staphylococcus aureus (S. aureus). The peptide can include SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the inhibited microorganism can include pan-drug-resistant Acinetobacter baumannii. In other examples, the peptide can include SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and the inhibited microorganism can include pan-drug-resistant Acinetobacter baumannii. In one example, the peptide comprises SEQ ID NO: 1 and the microorganisms inhibited include pan-drug resistant Acinetobacter baumannii.

[0015] In another embodiment of the present invention, there is provided a method for treating a subject having a microbial infection, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a peptide compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof, to inhibit microbial growth in the subject. In this method, the pharmaceutical composition may comprise one or more other antibacterial compounds. The one or more antibacterial compounds may include polymyxin B.

[0016] In a method of treating a subject having a microbial infection, the pharmaceutical composition can include the peptide compound SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In other examples, the pharmaceutical composition can include the peptide compound SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In one example, the pharmaceutical composition includes the peptide compound SEQ ID NO: 1.

[0017] In the method of treating a subject with a microbial infection, the microbial growth may be caused by bacteria containing one or more of multidrug resistance (MDR), extensively drug resistance (XDR), or pandrug resistance (PDR). In another example, the microbial growth is caused by an oral infection with bacteria including Porphoryomus gingivalis. The microbial growth may be caused by a gram-negative bacterial pathogen or a gram-positive pathogen. The pathogenic microorganism of the gram-negative bacterial pathogen may include one or a combination of Acinetobacter baumannii, P. gingivalis, or Escherichia coli, and the gram-positive pathogen may be Staphylococcus aureus or L. salivarius.

[0018] In one embodiment of the present invention, an antimicrobial composition is provided comprising a therapeutically effective amount of a peptide compound, or a pharmaceutically acceptable salt or solvate thereof, comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15; and a therapeutically effective amount of polymyxin B. In some cases, the antimicrobial composition can comprise SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In one aspect, the antimicrobial composition comprises SEQ ID NO:1.

[0019] In another embodiment, an antimicrobial composition is provided for use in a method of inhibiting microbial growth in a subject.

[0020] In another aspect, a pharmaceutical composition comprising the antimicrobial composition of the invention is provided for use in a method of inhibiting microbial growth in a subject.

[0021] Administration of the pharmaceutical compositions of the present invention to a subject can include single or multiple administrations of the pharmaceutical composition. Pharmaceutical compositions containing peptide compounds can be administered topically, enterally, systemically, or parenterally. In some cases, the pharmaceutical compositions can be administered in the form of mouthwash or toothpaste. When microbial growth is caused by oral infection with bacteria, including Porphoryomas gingivalis, the pharmaceutical compositions containing peptide compounds can be administered in the form of mouthwash or toothpaste.

[0022] The pharmaceutical composition may include one or more pharmaceutically acceptable excipients. The one or more excipients may include, but are not limited to, disintegrants, diluents, binders, solvents, cosolvents, lubricants, pH adjusters, buffers, preservatives, dispersants, suspending agents, ointment bases, emulsifiers, emollients, osmotic agents, surfactants, propellants, flavoring agents, sweeteners, or drug-release modifiers. The pharmaceutical composition may include a solvent, such as one or both of saline and glucose solution.

[0023] The pharmaceutical compositions of the present invention may be formulated as a lyophilized powder, or as a liquid suitable for administration by injection for sustained release of the peptide compound, or as a spray. In other embodiments, the pharmaceutical compositions of the present invention may be formulated as a mouthwash or toothpaste.

[0024] The features and advantages of the present invention will be more clearly understood from the following description taken in conjunction with the accompanying drawings, which are provided by way of illustration and not by way of limitation. The foregoing aspects and other features of the present disclosure are explained in the following description in connection with the accompanying illustrative figures (also referred to as "figures") of one or more embodiments. [Brief explanation of the drawings]

[0025] [Figure 1A] FIG. 1A is a graph showing the antibacterial activity of COG1410 (SEQ ID NO: 1), specifically the in vitro killing kinetics of COG1410 and polymyxin B (PMB) in PBS at 1×MIC and 5×MIC, respectively, against PDR-A. baumannii strain YQ4.

[0026] [Figure 1B] Figure 1B is a graph showing the antibacterial activity of COG1410 (SEQ ID NO: 1), specifically the bactericidal efficacy of COG1410 in PBS with or without 50% human plasma. CFU were counted after 2 hours of incubation at 37°C.

[0027] [Figure 1C] Figure 1C is a graph showing the antibacterial activity of COG1410 (SEQ ID NO: 1), specifically measuring the bactericidal efficacy of COG1410 under different conditions. Each experiment in Figures 1A-1C was performed in triplicate, and values represent the mean ± SD.

[0028] [Figure 2A]2A is a graph showing that COG1410 (SEQ ID NO: 1) exhibits biofilm inhibition and eradication activity against PDR-A. baumannii, specifically, preventing biofilm formation by COG1410. Results are expressed as biofilm mass measured using crystal violet staining (OD600).

[0029] [Figure 2B] Figure 2B is a graph showing that COG1410 (SEQ ID NO: 1) exhibits biofilm inhibition and eradication activity against PDR-A. baumannii, particularly eradication of established biofilms. Data are presented as the mean ± SD of eight replicates from three independent experiments. Statistical significance between each treatment and the control was analyzed by Student's t-test (unpaired), *p<0.5, ***p<0.001.

[0030] [Figure 3A] Figure 3A shows images demonstrating that COG1410 (SEQ ID NO: 1) treatment permeabilized the cell membrane of PDR-A. baumannii, as seen in SEM observations of A. baumannii YQ4 exposed to 1x MIC COG1410 or 1x MIC polymyxin B. Cells in PBS served as a negative control.

[0031] [Figure 3B] Figure 3B is an image showing that COG1410 (SEQ ID NO: 1) treatment permeabilized the cell membrane of PDR-A. baumannii, as seen by TEM observation of A. baumannii YQ4 exposed to 1x MIC COG1410 or 1x MIC polymyxin B. Cells in PBS served as a negative control.

[0032] [Figure 3C]Figure 3C is a graph showing that COG1410 (SEQ ID NO: 1) treatment permeabilized the cell membrane of PDR-A. baumannii, specifically the effect of 1xMIC COG1410 on ATP release (i.e., ATP leak) from A. baumannii YQ4. 1xMIC polymyxin B and 1xMIC tigecycline (TGC, 32 μg / ml) were used as positive and negative controls for ATP leakage, respectively. Untreated cells also served as a negative control.

[0033] [Figure 3D] Figure 3D is a graph showing that COG1410 (SEQ ID NO: 1) treatment permeabilized the cell membrane of PDR-A. baumannii, specifically showing measurements of ROS levels using a DCFH-DA probe in the presence or absence of COG1410 (16 μg / ml). Rosup is a positive control. Statistical significance between each treatment and the control was analyzed by one-way ANOVA with post-hoc testing for multiple comparisons; *p<0.05, **p<0.01.

[0034] [Figure 4] Figure 4 shows images demonstrating that FITC-COG1410 entered the cytoplasm of A. baumannii, E. faecium, K. pneumoniae, and S. aureus. Bacteria were treated with FITC-labeled COG1410, counterstained with FM4-64 dye, and observed by CLSM. Green fluorescence (i.e., hatched shapes) indicates FITC-COG1410 localization within the cells. Red fluorescence (i.e., open shapes) indicates the cell membrane. Scale bar: 4 μm.

[0035] [Figure 5A]Figure 5A is the first half of a graph showing the treatment of COG1410 (SEQ ID NO: 1) enriched genes involved in redox processes. Whole-transcriptome analysis of A. baumannii treated or not with COG1410 was performed by RNA-seq. Differentially expressed genes (DEGs) were analyzed using edgeR (v3.16.5). Gene Ontology (GO) enrichment analysis of DEGs was performed with clusterProfiler (v3.4.4). GO terms with an FDR ≤ 0.05 were considered significantly enriched by DEGs.

[0036] [Figure 5B] FIG. 5B is the second half of the graph depicted in FIG. 5A.

[0037] [Figure 6A] 6A is a graph showing that COG1410 (SEQ ID NO: 1) exhibits low hemolytic activity, which was determined by measuring the hemoglobin release at 414 nm from human red blood cells exposed to different concentrations of COG1410. PBS and Triton X-100 (0.1%) were used as negative and positive controls, respectively.

[0038] [Figure 6B] Figure 6B is a graph showing that COG1410 (SEQ ID NO: 1) exhibits moderate cytotoxicity. Specifically, the cytotoxicity of COG1410 was assessed by measuring the cell viability of normal human liver L02 cells treated with increasing concentrations of the peptide using a CCK8 assay. Experiments were performed in triplicate. Data shown are means ±SD.

[0039] [Figure 7A]7A is a graph showing that COG1410 (SEQ ID NO: 1) exhibited strong synergistic interaction with polymyxin B and that modification of the LPS did not alter the antibacterial activity of COG1410 against A. baumannii. MICs were determined in LB broth against the A. baumannii wild-type strain ATCC19606 and the corresponding LPS-deficient mutant strain harboring the pmrAP102R and pmrAP102RmiaAI221V mutations.

[0040] [Figure 7B] Figure 7B is a graph showing that COG1410 (SEQ ID NO: 1) exhibited strong synergistic interaction with polymyxin B; specifically, a combination of 2 μg / ml COG1410 and 1 μg / ml polymyxin B (i.e., 2 COG1410 + 1 PBM) was able to completely inhibit bacterial growth within 20 hours in LB broth. Growth curves were measured in duplicate, with eight wells per treatment in a 96-well plate each time. A representative curve is shown.

[0041] [Figure 7C] Figure 7C is a graph showing that COG1410 (SEQ ID NO: 1) exhibited strong synergistic interaction with polymyxin B; specifically, the combination of COG1410 and polymyxin B (i.e., 2 COG1410 + 1 PBM) significantly reduced CFU of A. baumannii YQ4 in PBS. Experiments were performed in triplicate. Data represent mean ± SD values.

[0042] Figure 8 is a graph showing the rescue of infected nematodes by combined treatment with COG1410 (SEQ ID NO: 1) and polymyxin B. Specifically, C. elegans worms were preinfected with A. baumannii YQ4 and then transferred to NGM plates supplemented with 16 μg / ml COG1410 or 2 μg / ml COG1410 and 1 μg / ml polymyxin B. Dead nematodes were counted daily for two weeks. Survival curves were analyzed using the Kaplan-Meier method, and statistical significance was determined using the log-rank test. ** , p<0.01. DETAILED DESCRIPTION OF THE INVENTION

[0043] To promote an understanding of the principles of the present disclosure, reference will be made to preferred embodiments and specific language will be used to describe the same. Nevertheless, it will be understood that no limitation on the scope of the disclosure is intended, and that variations and further modifications of the disclosure as exemplified herein are contemplated as would normally occur to one skilled in the art to which the disclosure pertains.

[0044] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0045] As used herein, the use of the terms "including," "comprising," or "having," and variations thereof, is meant to encompass the subsequently listed elements and equivalents thereof, as well as additional elements. As used herein, "and / or" refers to and includes all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0046] Furthermore, the indefinite articles "a" and "an" preceding an element or component of the invention are not intended to limit the number of occurrences of the element or component. Thus, "a" and "an" should be understood to include one or at least one, and the singular form of an element or component also includes the plural, unless the number is explicitly singular.

[0047] The recitation of ranges of values herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method for individually referencing each individual value falling within that range, and each individual value is incorporated herein as if it were individually recited herein. For example, if a concentration range is recited as 1% to 50%, it is intended herein to explicitly recite values such as 2% to 40%, 10% to 30%, 1% to 3%, etc. These are merely examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest and highest values recited should be considered to be expressly stated in this disclosure.

[0048] In the specification, drawings and claims, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0049] As used herein, the term "treatment" refers to administering a composition of the present invention to an individual suffering from a microbial infection, resulting in partial or complete relief of symptoms or preventing the symptoms of the microbial infection from worsening after treatment. Thus, treatment includes cure. As used herein, "efficacy" refers to the effect caused by treatment to alter, generally alter, alleviate, or improve the symptoms or characteristics of the microbial infection, or to cure the microbial infection. "Treatment" can also mean extending survival compared to the expected survival in the absence of treatment. In certain embodiments, "treatment" refers to both therapeutic and prophylactic or preventative measures. Treatment refers to inhibiting or reducing the increase in disease state or symptoms compared to the absence of treatment, and does not necessarily mean complete cessation of the microbial infection.

[0050] As used herein, the term "therapeutically effective amount" refers to the use or administration method of a composition of the present invention in an amount that achieves a desired therapeutic effect after administration.

[0051] As used herein, the term "subject" includes human and non-human (animal) patients. The term "non-human animals" includes vertebrates, e.g., mammals such as non-human primates, sheep, cows, dogs, cats, and rodents such as mice and rats.

[0052] The present inventors have discovered that a series of peptide compounds have excellent antibacterial effects. The peptide compounds are provided as antibacterial agents, and in some embodiments, as compositions for administration to a subject for the treatment of microbial infections. The peptide compounds of the present invention are provided below: (COG1410) Acetyl-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 1); Picolinyl-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 2); Picolinyl-AS-C-LRKL-aib-KRLL-C-amide (SEQ ID NO: 3); wherein a disulfide bond exists between the two cysteine residues; Acetyl-LLRK-aib-LKRL-aib-SA-CONH2 (SEQ ID NO: 4); Acetyl-llrk-Aib-lkkl-Aib-sa-amide (SEQ ID NO: 5), wherein all amino acid residues are D-amino acids; Acetyl-as-aib-lrkl-aib-krll-amide (SEQ ID NO: 6), wherein all amino acid residues are D-amino acids; Acetyl-LLRK-aib-LRKL-aib-SAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 7); Acetyl-LRVRCAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 8); Acetyl-LRVRLAS-aib-LKKL-aib-KRLL-amide (SEQ ID NO: 9); Acetyl-LRVRLAS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 10); Acetyl-llrk-aib-lkrl-aib-salrvrl-amide (SEQ ID NO: 11), wherein all amino acid residues are D-amino acids; Acetyl-LRVRLASHLRKLRKRLLAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 12); C8-as-aib-lrkl-aib-krll-amide (SEQ ID NO: 13); Acetyl-K(C8)-as-aib-lrkl-aib-krll-amide (SEQ ID NO: 14); Acetyl-K(picolinyl)-as-aib-lrkl-aib-krll-amide (SEQ ID NO: 15); Acetyl-LRVRLASHLRKLRKRLLR-amide (SEQ ID NO: 16); Acetyl-LRKLRKRLLLRKLRKRLL-amide (SEQ ID NO: 17); Acetyl-LRVRLASHLRKLRKRLLRDADDLQKRLAVY-amide (SEQ ID NO: 18); and Picolinyl-llrk-aib-lkrl-aib-salrvrl-amine (SEQ ID NO: 19), wherein all amino acid residues are D-amino acids.

[0053] The term "COG1410" is used interchangeably herein with the term "SEQ ID NO: 1."

[0054] In the above peptide compounds of the present invention, L, R, K, S, H, A, C, V, D, Q, and Y are one-letter abbreviations of amino acids, respectively representing leucine, arginine, lysine, serine, histidine, alanine, cysteine, valine, aspartic acid, glutamine, and tyrosine. Those skilled in the art will understand that in the peptide chain structure, the above amino acids are in the form of amino acid residues. In the present invention, the amino acids contained in the peptide chain structure refer to amino acid residues unless otherwise specified.

[0055] Unless otherwise specified, two adjacent amino acids are linked by an amide bond (-CO-NH-), also known as a peptide bond. For example, -AS- means that an alanine residue is attached to a serine residue, and the two are joined by an amide bond, also known as a peptide bond, in this case A and S and the amino acids.

[0056] In the above peptide compounds, L, R, K, S, H, A, C, V, D, Q, and Y may represent L-amino acids, unless the amino acid residue is otherwise specified as a D-amino acid.

[0057] In the peptide compounds of the present invention, "Aib" is aminoisobutyric acid.

[0058] In the peptide compound of SEQ ID NO: 3, there is a disulfide bond between the side chains of two cysteine residues (ie, -SS- or disulfide group or disulfide bridge).

[0059] In the peptide compounds of SEQ ID NOs: 1, 4-12, and 16-18, the amino-terminal amino acid is linked to acetyl (i.e., acetyl-CONH-). Exemplary schematic diagrams of the amino-terminal structures for peptide compounds beginning with acetyl-AS, i.e., SEQ ID NOs: 1 and 6, are shown below: [ka] Structure (I)

[0060] Structure (I) shows acetyl-alanine-serine with an acetyl group at the amino terminus.

[0061] For acetyl-LLRK, i.e., peptide compounds beginning with SEQ ID NOs: 4, 5, 7, and 11, the non-acetylated amino terminus is shown in structure (II) below: [ka] Structure (II)

[0062] The acetyl-LLRK structures of SEQ ID NOs: 4, 5, 7 and 11 are shown in structure (III) below: [ka] Structure (III).

[0063] For peptide compounds beginning with acetyl-LRVR, i.e., SEQ ID NOs: 8-10, 12, 16, and 18, the non-acetylated structure is shown in Structure (IV) below: [ka] Structure (IV)

[0064] The acetyl-LRVR structures of SEQ ID NOs: 8-10, 12, 16, and 18 are shown in structure (V) below: [ka] Structure (V)

[0065] For acetyl-LRK, i.e., the peptide compound beginning with SEQ ID NO: 17, the non-acetylated version is shown in structure (VI) below: [ka] Structure (VI)

[0066] The acetyl-LRK structure of SEQ ID NO: 17 is shown below in structure (VII): [ka] Structure (VII)

[0067] In the peptide compounds of SEQ ID NOS: 2-3 and 19, the amino terminal amino acid is linked to a picolinyl (i.e., picolinyl-CONH-). The amino termini of SEQ ID NOS: 2-3 begin with alanine-serine, while the amino terminus of SEQ ID NOS: 19 begins with leucine-leucine. The schematic structure shown below is for alanine-serine, but the same type of linkage to a picolinyl group is present in SEQ ID NOS: 19. The free amino group of SEQ ID NOS: 2-3 (i.e., without the picolinyl group) is shown below in structure (VIII): [ka] Structure (VIII)

[0068] The picolinyl-modified amino terminus (i.e., picolinyl-AS of SEQ ID NOs: 2-3) is shown in structure (IX) below: [ka] Structure (IX)

[0069] In the peptide compound SEQ ID NO: 13, the amino terminal amino acid is attached to "C8," which should be understood to mean an octanyl group of eight straight carbon atoms. The free amino terminus of SEQ ID NO: 13 is shown in the following structure (X): [ka] Structure (X)

[0070] The addition of a C8 group to the amino terminus of SEQ ID NO: 13 is shown in structure (XI) below: [ka] Structure (XI)

[0071] In the peptide compound SEQ ID NO: 14, the epsilon amino group of the amino terminal amino acid (lysine) is bonded to an eight linear carbon atom group (i.e., a C8 group) and the alpha amino group of the amino terminal amino acid (lysine) is bonded to an acetyl group, as shown in structure (XII) below: [ka] Structure (XII)

[0072] In the peptide compound SEQ ID NO: 15, the ε-amino group of the amino-terminal amino acid (lysine) is bonded to a picolinyl group and the α-amino group of the amino-terminal amino acid (lysine) is bonded to an acetyl group, as shown in structure (XIII): [ka] Structure (XIII)

[0073] Methods for preparing peptide compounds of the present invention

[0074] The following discussion illustrates the principles for obtaining the peptide compounds of the present invention and details some methods that can be used to prepare the peptide compounds of the present invention. However, this discussion is not intended to define or limit the scope of reactions or reaction sequences that can be used to prepare the peptide compounds of the present invention. The peptide compounds of the present invention can be prepared by the processes and techniques disclosed in the Examples section below, as well as by known organic synthesis techniques. The peptide compounds of the present invention can be synthesized using conventional solid-phase or solution-phase peptide synthesis and can be characterized by HPLC and mass spectrometry, as known to those skilled in the art.

[0075] Methods known to those skilled in the art can be identified in various references and published data. Detailed descriptions of reactants usable in the synthesis of compounds of the invention are available or can be found in, for example, "Practical Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York, 2011; S.R. Sandler et al., "Organic Functional Group Preparations", 2nd ed., Academic Press, New York, 1983; H.O. House, "Modern Synthetic Reactions", 2nd ed., W.A. Benjamin, Inc., Menlo Park, Calif., 1972; T.L. Gilchrist, "Heterocyclic Chemistry", 2nd ed., John Wiley & Sons, Inc., New York, 1992; J. March, "Advanced Organic Chemistry: Reactions, Mechanisms, Structures", vol. Chemistry: Reactions, Mechanisms and Structures, 4th ed., Wiley-Interscience, New York, 1992, and other suitable reference books. Other suitable reference books and monographs that provide detailed descriptions of the synthesis of reactants that can be used in the preparation of the compounds of the invention, or references to literature describing preparative methods, include, for example, Fuhrhop, J. and Penzlin G."Organic Synthesis: Concepts, Methods, Starting Materials", 2nd ed., revised and expanded (1994), John Wiley & Sons, ISBN: 3-527-29074-5; Hoffman, R.V. "Organic Chemistry, An Intermediate Text" (1996), Oxford University Press, ISBN: 0-19-509618-5; Larock, R.C. "Comprehensive Organic Transformations: A Guide to Functional Group Preparations", 2nd ed. (1999), Wiley-VCH, ISBN: 0-471-19031-4; Otera, J. (editor), "Modern Carbonyl Chemistry" "A Guide to the Chemistry of Functional Groups" (2000), Wiley-VCH, ISBN: 3-527-29871-1; Patai, S. "Guide to the Chemistry of Functional Groups" (1992), Interscience, ISBN: 0-471-93022-9; Quin, L.D. et al. "A Guide to Organophosphorous Chemistry" (2000), Wiley-Interscience, ISBN: 0-471-31824-8; Solomons, T.W.G. "Organic Chemistry," 7th Edition (2000), John Wiley & Sons, ISBN: 0-471-19095-0; Stowell, J.C."Intermediate Organic Chemistry," 2nd Edition (1993), Wiley-Interscience, ISBN: 0-471-57456-2; "Ullmann's Encyclopedia: Industrial Organic Chemicals: Starting Materials and Intermediates" (1999), John Wiley & Sons, ISBN: 3-527-29645-X-, Vol. 8, "Organic Reactions" (1942-2000), John Wiley & Sons, over 55 volumes in total; and "Chemistry of Functional Groups," John Wiley & Sons, 73 volumes in total.

[0076] In summary, the compounds used in the reactions described herein can be prepared according to organic synthesis techniques known to those skilled in the art from commercially available chemical reagents and / or compounds described in the chemical literature. "Commercially available chemical reagents" include Acros Organics (Pittsburgh, PA), Aldrich Chemical (Milwaukee, WI; including Sigma Chemical and Fluka), Apin Chemicals (Milton Park, UK), Avocado Research (Lancashire, UK), BDH (Toronto, Canada), Bionet (Cornwall, UK), Chemservice (West Chester, PA), Crescent Chemical Co. (Hauppauge, NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester, NY), Fisher Scientific Co. (Pittsburgh, PA), Fisons Chemicals (Leicestershire, UK), Frontier Scientific (Logan, UT), ICN Biomedicals Inc. (Costa Mesa, CA), Key Organics (Cornwall, UK), Lancaster Synthesis (Windham, NH), Maybridge Chemical Co. (Cornwall, UK), Parish Chemical Co. (Orem, UT), Pfaltz & Bauer, Inc. (Waterbury, CT), Polyorganix (Houston, TX), Pierce Chemical Co. (Rockford, IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland, OR), and Trans World Chemicals, Inc.(Rockville, MD) and Wako Chemicals USA, Inc. (Richmond, VA).

[0077] Specific reactants and similar reactants can be identified through the Index of Known Chemical Reagents compiled by the Chemical Abstracts Service of the American Chemical Society (available from most public or university libraries) or through online databases. Known but not commercially available chemical reagents can be prepared by custom chemical reagent synthesizers, many of which are standard chemical reagent providers (such as those listed above) that offer custom synthesis services.

[0078] For example, methods for forming -SS- between amino acids in SEQ ID NO: 3 of the cyclic peptide compounds of the present invention are known to those skilled in the art and can be found, for example, in Pohl, M. et al., J. Peptide Protein Res. 41(4):362-375. (1993); Tam, JP et al., J. ACS. 113(17):6657-6662. (1991); and in Example 1 of the present disclosure, which describes methods for preparing peptide compounds.

[0079] In some aspects of the present invention, the ApoE-based synthetic peptide acetyl-as-aib-LRKl-aib-krll-amide (SEQ ID NO: 1), designated "COG1410," is provided for use as an antibacterial agent. In one embodiment, the peptide COG1410 is provided for use in methods for inhibiting Gram-negative bacteria, including Enterobacter cloacae, Escherichia coli, Citrobacter freundii, and even the anaerobic bacterium Porphyromonas gingivalis, with MICs ranging from 16 to 64 μg / ml (see Examples 1 and 2 herein and Table 1 below). In another embodiment, the peptide compound COG1410 is provided for use in methods for inhibiting the pan-drug-resistant bacterium Acinetobacter baumannii YQ4. Surprisingly and unexpectedly, the experiments described in Examples 1-3 demonstrate that COG1410 can kill Acinetobacter baumannii YQ4 with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 16 μg / ml (11.3 μM). Additionally, COG1410 was found to inhibit 107 other clinically isolated A. baumannii strains with MICs ranging from 16 to 32 μg / ml.

[0080] In another aspect of the present invention, synthetic peptide compounds of the present invention are provided for use as antibacterial agents. The MICs of the peptide compounds against E. coli, L. salivarius, and P. gingivalis are shown in Table 1. Determination of the MIC values in Table 1 is described in Example 3. [Table 1]

[0081] COG1410 is located between residues 138 and 149 of the N-terminal domain of ApoE and contains amino acid residues with aminoisobutyric acid (Aib) substitutions at positions 140 and 145. COG1410 is a peptide variant based on residues 133 and 149 of ApoE, expanding the therapeutic window for treatment after TBI. It has demonstrated neuroprotective activity in several brain injury models, including intracerebral hemorrhage and focal cerebral ischemia. By reducing inflammation and apoptosis, COG1410 enhances retinal ganglion cell survival and alleviates early brain injury. Furthermore, COG1410 has been shown to have the ability to target the blood-brain barrier (BBB). COG1410 was fused with an Aβ-binding domain to form a multi-strategy peptide, which enhanced BBB targeting efficiency and ameliorated neuronal damage in a mouse model of Alzheimer's disease. Therefore, COG1410 is considered a promising therapeutic agent for diseases associated with neuronal injury.

[0082] In some embodiments of the present invention, COG1410 is provided for use as an antibacterial agent. Experimental data provided in Examples 1-3 and Figures 1-8 herein demonstrate the antibacterial effects of COG1410. Specifically, COG1410 exhibited broad-spectrum antibacterial activity and potent bactericidal activity, particularly against Acinetobacter baumannii strains, a pandrug-resistant bacterium. COG1410 exerted very rapid effects in vitro. For example, COG1410 at 1× MIC reduced A. baumannii CFU by 3 logs (1,000-fold) within 5 minutes, much faster than polymyxin B (PMB) (see Figure 1A) and comparable to the promising anti-A. baumannii antimicrobial peptide ZY4, a cathelicidin-derived peptide that killed A. baumannii within 30 minutes at 1× MIC

[24] . Figures 2A and 2B show that COG1410 can inhibit biofilm formation and eradicate mature biofilms in A. baumannii. A. baumannii is one of the major biofilm-producing bacteria, and its biofilm formation allows it to easily survive and spread in hospital environments. Thus, in one embodiment, COG1410 is provided for use as an antibacterial agent against A. baumannii.

[0083] One of the major challenges for the therapeutic application of antimicrobial peptides is their degradation or inactivation in plasma. The bactericidal efficacy of COG1410 was evaluated in plasma. The LC of COG1410 in PBS and 50% pooled plasma was 99.9 were 1.4 μM and 5.6 μM, respectively (see Figure 1B). 99.9This was better than SAAP-148, an LL-37-derived AMP, which showed activity of 1.6 μM and 12.8 μM against A. baumannii. Furthermore, stability studies have shown that COG1410 can be highly stable in plasma. Specifically, COG1410 was not significantly degraded within 2 hours in 100% human plasma. Even after 10 hours, over 80% of its activity was retained. Consistent with these results, COG1410 has been administered intravenously to a mouse model of traumatic brain injury (TBI). In that study, a single intravenous injection of COG1410 significantly improved vestibulomotor function and spatial learning and memory. Similar effects were also observed in a rat model of focal cerebral ischemia and a mouse model of traumatic optic nerve injury. Thus, in one embodiment, COG1410 is provided for administration to a subject as an antibacterial agent for systemic infection.

[0084] Most cationic AMPs, such as COG1410, directly target the cell membrane, causing pore formation and eventual cell lysis. As expected, the experiments described in Example 2 demonstrate that polymyxin B treatment leads to cell wall disintegration and release of cytoplasmic contents (see, e.g., Figures 3A-3D and 4). Unexpectedly, however, cells treated with COG1410 remained intact without pore formation. Cell death may have resulted from separation between the inner membrane and the cell wall. In the experiments described in Example 2, significant ATP leakage was observed in cells exposed to COG1410, indicating that the cell membrane had been disrupted or permeabilized. Consistently, fluorescent dye staining assays also confirmed this conclusion. Meanwhile, it was observed that COG1410 can cross the cell membrane and enter the cytoplasm of A. baumannii. RNA-seq analysis revealed that sub-MIC levels of COG1410 significantly affected genes in A. baumannii in minimal medium, among which genes were significantly enriched in oxidative and reductive biological processes (see Figure 5). The DCFH-DA probe also detected over 30% ROS after 20 minutes of COG1410 treatment. These data suggest that COG1410 can induce oxidative stress in A. baumannii. The experiments described in Example 2 suggest that COG1410 can nonspecifically bind to DNA. One theory previously described in the literature is that membrane disruption and DNA binding are two attacks on bacteria by the AMP NK18. However, although COG1410 was able to enter the cytoplasm of P. aeruginosa, S. aureus, and E. faecium, these bacteria were not susceptible to its antibacterial activity. Therefore, without wishing to be bound to any particular mechanism of action, DNA binding may not play an important role in the mechanism of antibacterial activity of COG1410.Again, without being limited to any one mechanism of action, the data taken together indicate that COG1410 may inhibit bacterial growth by mechanisms that include disruption of cell membrane integrity and / or induction of oxidative stress.

[0085] As a class of drugs, cationic AMPs can be particularly troublesome in terms of cytotoxicity. In the experiments described in Example 2, COG1410 at 128 μg / ml (8 times the MIC in LB) was observed to cause less than 5% hemolysis (FIG. 6A). The cytotoxic effect of COG1410 on normal human hepatic L02 cells was evaluated by the CCK8 assay described in Example 2. The EC 50 The concentration of LC-L100 in L02 cells was 58.9 μg / ml (Fig. 6B). 99.9 The cytotoxicity of COG1410 was 2 μg / ml and 8 μg / ml in PBS and 50% plasma, respectively. These results demonstrate an improved cytotoxicity profile of COG1410 compared to many existing antimicrobial peptides.

[0086] Another advantage of using COG1410 as an antibacterial agent is that a significant synergistic interaction was observed for the combination of COG1410 and polymyxin B. Specifically, the working concentrations were reduced to 2 μg / ml for COG1410 and 1 μg / ml for polymyxin B (Figures 7A-7C). Furthermore, the bactericidal effect of COG1410 was not dependent on electrostatic interactions with LPS, as modification of LPS did not inhibit COG1410 activity. This feature contrasts with previous observations for polymyxin B and LL-37, whose first step in attacking bacterial pathogens is binding to LPS. Generally, polymyxin B binds to the lipid A moiety of LPS in Gram-negative bacteria, resulting in Ca ions. 2+ and Mg 2+ It is recognized that LPS displaces cations such as α- and β-glucan, destabilizing LPS layers and membranes.

[0087] In another experiment, the graph in Figure 8 shows that a combination treatment of COG1410 and polymyxin B rescued infected worms. C. elegans were pre-infected with A. baumannii YQ4 and transferred to NGM plates supplemented with 16 μg / ml COG1410 or 2 μg / ml COG1410 and 1 μg / ml polymyxin B. Dead worms were counted daily for two weeks. The data in Figure 8 show the unexpected result that a combination treatment of COG1410 (2 μg / ml) plus polymyxin B (1 μg / ml) significantly rescued pre-infected worms and even extended their lifespan.

[0088] Combination drug therapy is frequently used in medical settings to treat microbial infections, especially in intensive care units. Therefore, the synergistic activity of polymyxin B and COG1410 is another advantage of using COG1410 in combination with polymyxin B to treat microbial infections.

[0089] The experimental results presented in Example 2 demonstrate the potent antibacterial potential of the ApoE mimetic peptide COG1410. The primary bactericidal mechanism of COG1410 was disruption of cell membrane integrity and induction of oxidative stress. COG1410 demonstrated potent bacterial killing, high stability in human plasma, and a low propensity for resistance development. The synergistic interaction between COG1410 and polymyxin B reduced the effective concentration of COG1410, avoiding the risk of eukaryotic cell toxicity. Given its simultaneous neuroprotective, anti-inflammatory, and antibacterial effects, COG1410, in one aspect of the present invention, is provided in a method for treating a subject with a microbial infection. The method includes administering to the subject a therapeutically effective amount of a composition containing the peptide compound COG1410 or a pharmaceutically acceptable salt or solvate thereof to inhibit microbial growth in the subject. The microbial growth may be the growth of pan-drug-resistant Acinetobacter baumannii, an infection considered a medical emergency.

[0090] In one aspect of the invention, a method for inhibiting microbial growth is provided, comprising contacting a microorganism with an effective amount of a peptide compound of the invention to inhibit microbial growth, wherein the peptide compound of the invention is selected from SEQ ID NOs: 1-19. In some embodiments, the peptide compound comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In other embodiments, the peptide compound comprises SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some cases, the peptide compound comprises SEQ ID NO: 1.

[0091] In this method, the microorganism can include one or more of multidrug-resistant (MDR), extensively drug-resistant (XDR), or pan-drug-resistant (PDR) bacteria. In some embodiments, the microorganism includes Porphoryomus gingivalis. The microorganism can include a gram-negative bacterial pathogen or a gram-positive pathogen. Gram-negative bacterial pathogens can include one or a combination of Acinetobacter baumannii, P. gingivalis, or Escherichia coli, and Gram-positive pathogens can include one or a combination of Staphylococcus aureus or L. salivarius.

[0092] In this method, the peptide compound can be contacted in combination with one or more other antimicrobial compounds. In one example, the one or more other antimicrobial compounds can include polymyxin B.

[0093] In some embodiments, the peptide compounds can be contacted in combination with polymyxin B to inhibit microorganisms, including pan-drug resistant Acinetobacter baumannii or multi-drug resistant Staphylococcus aureus.

[0094] In some embodiments, a peptide compound can be contacted in combination with polymyxin B to inhibit microorganisms including Acinetobacter baumannii, and the peptide compound comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In other examples, a peptide compound can be contacted in combination with polymyxin B to inhibit microorganisms including Acinetobacter baumannii. The peptide compound comprises SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In one example, a peptide compound can be contacted in combination with polymyxin B to inhibit microorganisms including Acinetobacter baumannii, and the peptide compound comprises SEQ ID NO: 1.

[0095] The peptide compounds of the present invention (SEQ ID NOS: 1-19) share similarity with the receptor binding domain of the holo-ApoE protein, and therefore can be expected to exhibit similar antibacterial activity.

[0096] Furthermore, peptide compounds SEQ ID NO:1 and SEQ ID NO:2 share similar structures and differ only in their amino termini. Specifically, SEQ ID NO:1 has an acetyl group at the amino terminus, while SEQ ID NO:2 has a picolinyl group at the amino terminus. As shown above and in Table 1, SEQ ID NO:1 has excellent antibacterial activity against Acinetobacter baumannii and is also effective against P. gingivalis. SEQ ID NO:2 is more effective against P. gingivalis than SEQ ID NO:1 and has also been shown to have antibacterial activity against E. coli and L. salivarius (see Table 1). Therefore, the antibacterial activity of SEQ ID NO:2 against pathogens such as Acinetobacter baumannii can be expected to be similar to that of SEQ ID NO:1. SEQ ID NO:3 has the same structure as SEQ ID NO:2, except that the Aib group is replaced with a cysteine that forms a disulfide bond. Similar to SEQ ID NO:2, SEQ ID NO:3 exhibits high antibacterial activity against P. gingivalis (see Table 1). Therefore, the antibacterial activity of SEQ ID NO:3 against pathogens such as Acinetobacter baumannii can be expected to be similar to that of SEQ ID NO:1.

[0097] The peptide compounds SEQ ID NOs: 13, 14, and 15 have similar structures to SEQ ID NOs: 1 and 2, differing only in their amino termini. Specifically, SEQ ID NO: 13 has a C8 group at the amino terminus, SEQ ID NO: 14 has an acetyl-K (C8) group at the amino terminus, and SEQ ID NO: 15 has an acetyl-K (picolinyl) group at the amino terminus. For the reasons described above, SEQ ID NOs: 13, 14, and 15 are expected to have antibacterial activity similar to that of SEQ ID NOs: 1 to 3.

[0098] The present invention includes a method for treating a subject having a microbial infection, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a peptide compound of the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, to inhibit microbial growth in the subject. The subject may be a mammal, a primate, or a human.

[0099] In various embodiments, the microbial growth can be caused by one or more bacteria that can be multi-drug resistant (MDR), extensively drug resistant (XDR), or pan-drug resistant (PDR).

[0100] In other embodiments, the microbial growth can be caused by an oral infection with bacteria, including Porphoryomas gingivalis.

[0101] Administration of a pharmaceutical composition containing a peptide compound to a subject can include single or multiple administrations of the pharmaceutical composition. The pharmaceutical composition containing the peptide compound can be administered topically, enterally, systemically, or parenterally. In some cases, the pharmaceutical composition containing the peptide compound can be administered in the form of a mouthwash or toothpaste. When the microbial growth is caused by an oral infection with bacteria, including Porphoryomas gingivalis, the pharmaceutical composition containing the peptide compound can be administered in the form of a mouthwash or toothpaste.

[0102] In some embodiments of the uses or methods described herein, the dose of the peptide compound of the invention generally depends on various factors, including the individual being treated or the severity of the microbial infection, the rate of administration, and the judgment of the prescribing physician. Generally, an effective daily dose per kg of body weight can range from about 0.01 to about 1.0 mg, e.g., from about 0.01 to about 1.0 mg, 0.01 to about 0.1 mg, 0.01 to about 0.09 mg, 0.01 to about 0.08 mg, 0.01 to about 0.07 mg, 0.01 to about 0.06 mg, 0.01 to about 0.05 mg, or 0.01 to about 0.04 mg / kg / day. In one embodiment, the dose is 0.051 mg / kg / day on the first day and 0.017 mg / kg / day on subsequent days. The exact dose may be varied from day to day to achieve the desired therapeutic efficacy.

[0103] In this treatment method, the bacteria to be inhibited may be a gram-negative or gram-positive pathogen. In the case of gram-negative bacteria, the bacteria may include, but are not limited to, one or a combination of Acinetobacter baumannii, P. gingivalis, and E. coli. In the case of gram-positive bacteria, the bacteria may include, but are not limited to, one or a combination of Staphylococcus aureus and L. salivarius.

[0104] In another aspect of the invention, pharmaceutical compositions comprising the peptide compounds of the present disclosure are administered in combination with one or more other antibacterial compounds. For example, the one or more antibacterial compounds can include polymyxin B.

[0105] In one example, a pharmaceutical composition comprising the peptide compound (COG1410) acetyl-as-aib-LRKl-aib-krll-amide (SEQ ID NO: 1) is administered in combination with polymyxin B to synergistically inhibit the pandrug-resistant bacterium Acinetobacter baumannii.

[0106] In another example, the pharmaceutical composition comprises the peptide compound SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and the pharmaceutical composition is administered in combination with polymyxin B to inhibit Acinetobacter baumannii.

[0107] In another example, a pharmaceutical composition comprising the peptide compound SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15 is administered in combination with polymyxin B to inhibit Acinetobacter baumannii.

[0108] In some embodiments, pharmaceutical compositions comprising a peptide compound are administered in combination with one or more antibacterial compounds to inhibit Acinetobacter baumannii or multidrug-resistant Staphylococcus aureus (S. aureus). The pharmaceutical compositions of the present invention comprise an antibacterial composition comprising: (i) a therapeutically effective amount of a peptide compound comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15; and (ii) a therapeutically effective amount of polymyxin B. In some embodiments, the peptide compound in the antibacterial composition comprises SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In other examples, the peptide compound in the antibacterial composition comprises SEQ ID NO:1.

[0109] In one embodiment, an antimicrobial composition comprising a peptide compound of the invention is provided for use in a method of inhibiting microbial growth in a subject. In another embodiment, a pharmaceutical composition comprising the antimicrobial composition is provided for use in a method of inhibiting microbial growth in a subject.

[0110] The peptide compounds of the present disclosure include pharmaceutically acceptable salts or solvates thereof for use in methods of inhibiting microbial growth in a subject.

[0111] Pharmaceutical compositions comprising the peptide compounds of the present disclosure, or pharmaceutically acceptable salts or solvates of the peptide compounds of the present disclosure, are provided.

[0112] In one embodiment, the pharmaceutical composition comprises the peptide compound SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In other embodiments, the pharmaceutical composition comprises the peptide compound SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In one example, the peptide compound included in the pharmaceutical composition comprises SEQ ID NO: 1.

[0113] Pharmaceutical compositions of the present disclosure that include a peptide compound can include one or more other antibacterial compounds. For example, the antibacterial compound can include polymyxin B.

[0114] In one embodiment, a pharmaceutical composition of the present disclosure is provided for use in a method of inhibiting microbial growth in a subject.

[0115] Administration of the pharmaceutical compositions of the present invention to a subject can include single or multiple administrations of the pharmaceutical composition. Pharmaceutical compositions containing peptide compounds can be administered topically, enterally, systemically, or parenterally. In some cases, the pharmaceutical compositions can be administered in the form of mouthwash or toothpaste. When microbial growth is caused by oral infection with bacteria, including Porphoryomas gingivalis, the pharmaceutical compositions containing peptide compounds can be administered in the form of mouthwash or toothpaste.

[0116] The pharmaceutical composition may include one or more pharmaceutically acceptable excipients. The one or more excipients may include, but are not limited to, disintegrants, diluents, binders, solvents, cosolvents, lubricants, pH adjusters, buffers, preservatives, dispersants, suspending agents, ointment bases, emulsifiers, emollients, osmotic agents, surfactants, propellants, flavoring agents, sweeteners, or drug-release modifiers. The pharmaceutical composition may include a solvent, such as one or both of saline and glucose solution.

[0117] The pharmaceutical compositions of the present invention may be formulated as a lyophilized powder, or as a liquid suitable for administration by injection for sustained release of the peptide compound, or as a spray. In other embodiments, the pharmaceutical compositions of the present invention may be formulated as a mouthwash or toothpaste.

[0118] The present invention will now be described in more detail by way of examples, which should not be construed as limiting the disclosure. [Example]

[0119] Example 1. Peptide synthesis, purification and identification COG1410 was synthesized by conventional solid-phase peptide synthesis to 95% purity and characterized by HPLC and mass spectrometry at Polypeptide Labs (San Diego, CA). COG1410 is acetyl-AS-Aib-LRKLAib-KRLL-amide (SEQ ID NO: 1), derived from ApoE residues 138-149 with Aib (aminoisobutyric acid) substitutions at positions 140 and 145. In all experiments, the peptide was dissolved in sterile saline immediately before use.

[0120] Peptide compounds SEQ ID NOs: 2-19 were synthesized in a similar manner as described above for the peptide compound SEQ ID NO: 1. Specifically, the peptide compounds were synthesized to 95% purity using conventional solid-phase peptide synthesis and qualified by HPLC and mass spectrometry at Polypeptide Labs (San Diego, CA).

[0121] More specifically, solid-phase synthesis of peptide compounds represented by SEQ ID NOS: 1-19 was performed using a condensation reaction well known to those skilled in the art as follows: The solid-phase peptide resin was added one by one, and the condensation reaction yielded the correct peptide sequence. The amino acid condensation was performed using the solid-phase polypeptide synthesis (SPPS) method, starting from the N-terminus. The amino acid side chains were protected, and their amino groups were protected with Fmoc. A typical SPPS method involves alternating cycles of N-terminal deprotection and condensation reactions, requiring resin washing between each step. After condensation of all amino acids was completed, picolinic acid was condensed into the sequence to yield the full-length polypeptide bound to the support resin. Each condensation reaction was monitored by appropriate in-process control tests (ninhydrin test, TNBS test, and / or analytical HPLC test).

[0122] The distinguishing feature of adding an acetyl, octanyl, or picolinyl group is that each involves adding a carboxyl group to a free amino group to form an amide bond between them. This amide, when used to join two amino acids together, is usually called a peptide bond. Thus, the amide bond is the result of a dehydration condensation reaction, which covalently bonds the carboxyl group to the amino group with the removal of a water molecule.

[0123] Cleavage of the polypeptide from the support resin is carried out under temperature-controlled conditions using trifluoroacetic acid (TFA), triisopropylsilane (TIS), 3,6-dioxin-1,8-octanedithiol (DODT), and water. After cleavage is complete, the mixture is filtered, the resin is washed, and the collected filtrate is centrifuged. The collected peptide in the filtrate is precipitated with methyl tert-butyl ether (MTBE) and filtered. The resulting crude peptide solution is dried and packaged.

[0124] The crude peptide was subjected to pre-purification and recycling purification using a trifluoroacetic acid, acetonitrile, and water buffer system. According to the purity results obtained by analytical HPLC, the co-eluting polypeptide fractions obtained from the purification were collected, analyzed, and pooled. This pool was then subjected to further chromatography to exchange the counterion salt.

[0125] Final salt exchange (FSE) and elution were performed using preparative HPLC, converting the purified peptide to the acetate salt using a buffer system of ammonium acetate, acetonitrile (ACN), and water. This was followed by a steep gradient wash with acetic acid, ACN, and water buffer to elute the peptide. HPLC analysis was used to determine the purity and pooling of the salt-exchanged components. Pooled subbatches that met the target yield purity criteria were lyophilized. The salt-exchanged purified peptide was lyophilized using a tray lyophilizer to remove water and residual organic solvents, yielding the final peptide drug substance.

[0126] Example 2. COG1410 has antibacterial activity against PDR-A. baumannii. Materials and Methods Strains and cultures The study included Bacillus subtilis, vancomycin-resistant Enterococcus faecalis, E. faecium, Mycobacterium tuberculosis, M. smegmatis, Enterobacter cloacae, Escherichia coli, Citrobacter freundii, Porphyromonas gingivalis, Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, and others. A panel of Gram-positive and Gram-negative bacterial strains was evaluated, including P. pneumoniae, Pseudomonas aeruginosa, and pan-drug-resistant (PDR) Acinetobacter baumannii YQ4. Strains were maintained in 10% glycerol at -80°C. Bacteria were streaked onto fresh plates before each experiment. Most were grown at 37°C in LB broth containing 10 g / L NaCl, except for Mycobacteria in 7H9 broth and Enterococcus in BHI broth. Porphyromonas gingivalis was grown in an anaerobic chamber at 37°C in BHI broth supplemented with 1 μg / ml vitamin K1, 5 μg / ml hemin, and 5 mg / ml L-cysteine hydrochloride. The minimum inhibitory concentrations (MICs) of antimicrobial agents were determined using microdilutions in the corresponding broths.

[0127] Pan-drug-resistant A. baumannii YQ4 was collected from a clinical laboratory, and the complete genome sequence was deposited in GenBank under accession number CP053033.

[0128] Antibacterial activity of COG1410

[0129] For the dynamic time-kill assay, overnight cultures of A. baumannii YQ4 were grown at initial OD 600 The culture was transferred to fresh broth with an OD of 0.01 and grown to logarithmic phase. 1 ml of the culture was harvested, washed twice with 1× phosphate-buffered saline (PBS, pH 7.4) containing 8 mM NaH2PO4, 2 mM KH2PO4, 2.6 mM KCl, and 136 mM NaCl, and finally incubated at OD 600 is 0.5 (approximately 1×10 8 The cells were suspended in 1 ml of PBS at 16 μg / ml (1×MIC) or 80 μg / ml (5×MIC) of COG1410, respectively, and incubated at 37°C without shaking. 100 μl aliquots were taken at 0, 5, 10, and 30 minutes, respectively, serially diluted with PBS, and plated on LB agar. CFU were counted after 18 hours of incubation at 37°C. Polymyxin B at 16 μg / ml (1×MIC) or 80 μg / ml (5×MIC) was used as a positive control. Three independent experiments were performed.

[0130] The antibacterial activity of COG1410 in plasma was performed as previously described

[22] . Briefly, logarithmic phase cultures of A. baumannii YQ4 were exposed to different concentrations of COG1410 in PBS or PBS supplemented with 50% (v / v) pooled human plasma. After incubation with shaking at 200 rpm at 37°C for 2 hours, CFU were counted on LB agar. LC 99.9 indicates the lowest peptide concentration that killed >99.9% of the bacteria (i.e., reduced by 1000-fold). Experiments were performed independently three times.

[0131] Biofilm inhibition and eradication assays

[0132] Static biofilm inhibition was performed as previously described

[23] . Briefly, logarithmic phase cultures of A. baumannii YQ4 were prepared as described above. OD 600 is 0.01 (approximately 1 x 106 Each cell of a 96-well PVC plate was inoculated with 200 μl of culture (CFU / ml) and exposed to various COG1410 solutions at final concentrations ranging from 0.5 to 128 μg / ml. Each concentration was measured in eight wells. After 48 hours of incubation at 37°C, planktonic bacteria were removed by washing three times with sterile water, followed by fixation with methanol for 15 minutes and staining with 0.1% crystal violet (CV) for 15 minutes. Excess CV was washed away, and bound CV was eluted from the biofilm in 150 μl of 95% ethanol and quantified spectrophotometrically at 600 nm using a Biotek Synergy H1 plate reader. Experiments were performed in triplicate.

[0133] Biofilm eradication assays were performed as previously described

[24] . Logarithmic-phase A. baumannii YQ4 cultures were cultured in fresh LB broth at OD . 600 The biofilm was diluted to a concentration of 0.01 and 200 μl was dispensed per well into a 96-well PVC plate. The plate was incubated at 37°C for 48 hours and washed three times with PBS. Serial dilutions of COG1410 (0.5–128 μg / ml) were prepared using the same medium, and 200 μl was dispensed into each well. Each concentration was measured in eight wells. LB without COG1410 was used as an untreated control. After an additional 24 hours of incubation, the remaining biofilm was quantified as described above. Experiments were performed in triplicate.

[0134] Stability of COG1410 in human plasma

[0135] To determine stability in human plasma, COG1410 was dissolved in 1 ml of 100% human plasma at a final concentration of 10 mg / ml and incubated at 37°C without shaking. 100 μl aliquots were taken after 0, 1, 2, 4, 6, 8, and 10 hours. A logarithmic-phase culture of A. baumannii YQ4 was prepared as described above. 200 μl of the culture was mixed with 6 mL of 0.8% soft agar to create a two-layer plate. After air-drying for 30 minutes, four 6 mm paper disks were placed on top, and 6 μl aliquots of COG1410 at different time points were dropped onto the paper disks. After 18 hours of incubation at 37°C, the zone of inhibition was recorded with a digital camera, and the inhibition diameter was measured using Image J. Three independent experiments were performed.

[0136] Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM)

[0137] Log-phase cultures of A. baumannii YQ4 were prepared as described above, harvested, and washed once with PBS. The pellets were suspended in PBS supplemented with 1x COG1410 (16 μg / ml) or 1x polymyxin B (16 μg / ml) and incubated at 37°C for 30 minutes. Untreated cultures were used as positive controls to observe intact cells. The pellets were then fixed with 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate and stored overnight at 4°C. After three washes with PBS, the cells were postfixed in 1% osmium chloride for 1 hour at 4°C. After extensive washing in distilled water for 1 hour, the bacteria were dehydrated in increasing concentrations of ethanol. Finally, these samples were transferred to a Thermo Fisher Quattro SEM for image collection after tert-butanol lyophilization and sputter coating.

[0138] Bacteria were treated with COG1410 or polymyxin B as described above. Samples were then fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2), postfixed in 1% buffered osmium tetroxide, dehydrated through gradient ethanol, embedded in epoxy resin, and polymerized at 60°C for 2 days. Ultrathin sections (50–70 nm) of the samples were prepared, placed on copper grids, stained with uranyl acetate and lead citrate, and transferred to a Thermo Fisher Talos L120C TEM for image collection.

[0139] Confocal laser scanning microscope

[0140] Cell membrane damage was determined using the Live / Dead BacLight bacterial viability kit (Invitrogen L7012) as previously described

[25] . Log-phase cultures of A. baumannii YQ4 were washed twice and incubated at a final OD 600 The cells were suspended in 0.1% PBS. The suspension was incubated with 1x MIC COG1410 (16 μg / ml) at 37°C for 30 minutes. 5x MIC polymyxin B (40 μg / ml) and untreated cells served as positive and negative controls, respectively. The cells were then stained with 7.5 μM SYTO-9 and 30 μM propidium iodide (PI) for 15 minutes in the dark. Five μl of the culture was spotted onto a clean slide coated with a thin layer of 1% agarose. Fluorescence was observed with an Olympus FV3000 confocal laser scanning microscope. For SYTO-9, excitation and emission were 483 nm and 503 nm, respectively. For PI, excitation and emission were 493 nm and 636 nm, respectively.

[0141] To investigate the localization of COG1410 in bacteria, logarithmic phase cultures of A. baumannii YQ4, K. pneumonia ATCC 2146, S. aureus ATCC 29213, and E. faecium 11-47 were prepared, washed, and assayed at a final OD 600The strains were suspended in 0.1% PBS. These cultures were exposed to FITC-labeled COG1410 (8 μg / ml) at room temperature for 30 minutes, followed by costaining with 1.6 μM membrane dye FM4-64. For each strain, 5 μl of culture was spotted onto a clean slide coated with a thin layer of 1% agarose. Fluorescence was observed with an Olympus FV3000 confocal laser scanning microscope. For FITC, excitation and emission were 488 nm and 525 nm, respectively. For FM4-64, excitation and emission were 558 nm and 734 nm, respectively.

[0142] ATP leakage assay

[0143] ATP leakage assays were performed using Beyotime's Enhanced ATP Assay Kit (S0027) according to the manufacturer's instructions. Briefly, logarithmic-phase cultures of A. baumannii YQ4 were prepared as described above, washed, and suspended in PBS. The suspensions were exposed to 16 μg / ml COG1410, 16 μg / ml polymyxin B, and 32 μg / ml tigecycline, respectively, and incubated at 37°C for 30 minutes. The supernatants were collected and used to measure ATP levels. 100 μl of the supernatant was mixed with 100 μl of working solution, and chemiluminescence was measured using a Biotek Synergy H1 plate reader. An untreated sample served as a negative control. Experiments were performed in triplicate.

[0144] DNA binding assay

[0145] Gel retardation experiments were performed as previously described

[26] . Briefly, 300 ng of plasmid pUC18 was mixed with various concentrations of COG1410 in 30 μl of buffer (10 mM Tris-HCl, 1 mM EDTA buffer, pH 8.0) and incubated at room temperature for 30 minutes. The reaction mixture was mixed with 1x native loading buffer and subjected to 1.5% agarose gel electrophoresis. DNA migration was detected by GelRed dye fluorescence.

[0146] RNA-seq analysis

[0147] To determine the effect of COG1410 on gene transcription, A. baumannii YQ4 was cultured in 50 ml of M9 medium supplemented with 20% glucose as the sole carbon source and treated with or without 0.25×MIC of COG1410 (4 μg / ml). Experiments were performed in triplicate. When the OD600 reached 0.8, cells were collected by centrifugation at 4°C and frozen in liquid nitrogen. Samples were shipped on dry ice to Guangdong Magigene Biotechnology Co., Ltd. (Guangzhou, China). Total RNA was extracted and purified using the TransZol Up Plus RNA Kit and EasyPure RNA Purification Kit, and rRNA was removed using the Ribo-Zero rRNA Removal Kit according to the manufacturer's instructions. Total libraries for Illumina sequencing were prepared using the NEB Next® Ultra™ Directional RNA Library Prep Kit. After cluster generation, the libraries were sequenced on an Illumina Novaseq6000 platform to generate 150-bp paired-end reads. The raw data were filtered with fastp to remove rRNA sequences

[27] . Differentially expressed genes were identified using the edgeR program. Genes with an FDR of 0.05 and a log2 (fold change) of >1 were considered candidate genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes was performed using Cluster Profiler. The raw data have been deposited in the SRA database under the accession number PRJNA833738.

[0148] ROS detection

[0149] Intracellular ROS levels were measured using a reactive oxygen species assay kit (S0033S) from Beyotime Biotechnology according to the manufacturer's instructions. Log-phase cultures of A. baumannii YQ4 were harvested, washed with PBS, and diluted 10-fold to approximately 10 7 The CFU / mL was calculated. 1 μl of DCFH-DA (10 μM) was added to 1 ml of cell culture and incubated at 37°C for 20 minutes. The fluorescent probe was then completely removed by three PBS washes and resuspended in PBS, after which COG1410 (16 μg / ml) or water was added. Rosup (50 μg / ml) was used as the kit's positive control. The culture was incubated at 37°C for 30 minutes. Fluorescence intensity was measured on a plate reader at 488 nm excitation and 525 nm emission. Experiments were performed in duplicate.

[0150] Evaluation of resistance induction by serial passage

[0151] To evaluate the drug resistance barrier of COG1410, A. baumannii YQ4 was grown in LB broth at 37°C with constant shaking at 150 rpm and exposed to sub-MIC levels of COG1410 or polymyxin B. 20 μl of culture was transferred to 2 ml of fresh medium daily. The initial concentration of the test compound was set at 1 / 32 × MIC and doubled every 10 passages. 1 ml of bacterial culture was stored in 10% sterile glycerol at -80°C every 5 passages. MIC values of the harvested cultures and the original strains were determined by microdilution.

[0152] Hemolysis and cytotoxicity assays

[0153] The human red blood cell (RBC) hemolytic activity of COG1410 was measured according to a previously described protocol with minor modifications

[28] . Anticoagulated (citrated) whole blood was pelleted by centrifugation at 700 g for 8 minutes, washed three times with PBS, and suspended at 0.5% (vol / vol) in PBS. 75 μl of RBC suspension was transferred to each well of a V-bottom 96-well plate, and an equal volume of COG1410 was diluted two-fold with PBS. The highest concentration was 512 μg / ml (363 μM). PBS and Triton X-100 (0.1%) were used as negative and positive controls, respectively. The plate was incubated at 37°C for 1 hour and then centrifuged at 1,000 rpm for 5 minutes at 4°C. A 60 μl aliquot of the supernatant from each well was quickly transferred to a new flat-bottom 96-well plate. OD 414 The absorbance at 100°C was measured using a microplate reader (BioTek, synergy H1). The hemolysis rate was then normalized to the averaged negative (0%) and positive (100%) controls. Three independent experiments were performed.

[0154] The cytotoxicity of COG1410 against normal liver cells L02 was evaluated using Cell Counting Kit 8 (Solarbio, CA1210) according to the manufacturer's instructions. Briefly, 100 μl of human liver L02 cells were plated in RPMI-1640 medium containing 20% FBS at 4 × 10 cells per well in a 96-well plate. 3 Cells were seeded at 1000 kJ / well and incubated at 37°C under a 5% CO2 atmosphere for 24 hours. Cells not exposed to the peptide served as a negative control. L02 cells were then incubated with various concentrations of COG1410 for an additional 24 hours. 10 μl of CCK8 solution was added to each well. After 2 hours of incubation, the optical absorbance at 450 nm was measured using a BioTek synergy H1 plate reader. Cell activity was expressed as a percentage of the mean absorbance of the peptide-exposed cells divided by the result of incubation with the control. Experiments were performed in triplicate.

[0155] Synergistic effect with antibacterial agents

[0156] The checkerboard method was performed to determine the fractional inhibitory concentration (FIC) index between COG1410 and other antimicrobial agents. Two-fold serial dilutions of the antimicrobial agents and COG1410 were prepared and mixed in a 96-well plate. Logarithmic phase cultures of A. baumannii YQ4 were cultured at initial OD . 600 0.01 was added to each well. The plates were incubated at 37°C for 20 hours, and the FIC values were calculated based on the formula: FIC = MIC (COG1410 combination) / MIC (COG1410 alone) + MIC (antimicrobial combination) / MIC (antimicrobial alone). Experiments were performed twice independently.

[0157] Nematode killing assay

[0158] This assay used the wild-type Caenorhabditis elegans strain Bristol N2. The relative experimental procedures followed a previously described protocol

[29] . C. elegans were grown in nematode growth medium (NGM) at 20°C with a lawn of E. coli OP50 as a food source. For synchronization, C. elegans eggs were harvested and hatched to the L1 stage in M9 medium at 20°C, then transferred to a lawn of E. coli and grown to the L4 stage. For the in vivo killing assay, synchronized L4 nematodes were harvested from several NGM plates and cultured in 20% LB, 1 × 10 A. baumannii YQ4 medium. 9Log-phase cells were transferred to 15 ml of M9 medium containing 10 μM FeCl3. The infection model was established at 20°C for 24 hours. E. coli OP50 was used as a negative control. Pre-infected nematodes were washed twice with M9 medium and distributed at 30 nematodes per 60 mm NGM agar plate, supplemented with different concentrations of COG1410 and / or polymyxin B, as well as 2 mM 5-fluoro-2'-deoxyuridine-floxuridine (FUDR). Uninfected nematodes were used as a positive control for worm lifespan. Live and dead nematodes were counted and recorded every 24 hours under a stereomicroscope for 16 days. C. elegans survival curves were analyzed using the Kaplan-Meier method using GraphPad Prism 9. In vivo killing assays were performed in triplicate.

[0159] result

[0160] COG1410 has broad-spectrum antibacterial activity

[0161] COG1410 is a synthetic cationic peptide with a simple α-helical structure, consisting of 12 amino acids, including four positively charged amino acids, five nonpolar amino acids, and one polar amino acid, as well as two unnatural amino acids, Aib, acetyl-as-aib-LRKl-aib-krll-amide (SEQ ID NO: 1). The antibacterial activity of COG1410 was determined by measuring minimum inhibitory concentration (MIC) values in a panel of Gram-positive and Gram-negative bacterial strains. For Gram-positive strains, COG1410 inhibited the growth of Bacillus subtilis, vancomycin-resistant Enterococcus faecalis and E. faecium, Mycobacterium tuberculosis, and M. smegmatis with MICs ranging from 1 to 32 μg / ml. Regarding Gram-negative bacteria, COG1410 exhibited antibacterial activity against Enterobacter cloacae, Escherichia coli, Citrobacter freundii, and the anaerobic bacterium Porphyromonas gingivalis, with MICs ranging from 16 to 64 μg / ml. In contrast, COG1410 was inactive against Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, and Pseudomonas aeruginosa. Notably, COG1410 was able to kill pan-drug-resistant (PDR) Acinetobacter baumannii YQ4 with an MIC and minimum bactericidal concentration (MBC) of 16 μg / ml (11.3 μM). Testing of 107 other clinically collected A. baumannii strains revealed that the MICs of COG1410 ranged from 16 to 32 μg / ml.

[0162] COG1410 exhibits potent and rapid bactericidal activity against PDR A. baumannii in vitro

[0163] The in vitro time-kill kinetics of COG1410 against PDR A. baumannii YQ4 was analyzed. COG1410 at 1× MIC reduced CFU by nearly 3 logs (i.e., 1000-fold) within 5 minutes in 20 μM phosphate-buffered saline (PBS) (Figure 1A). When the concentration of AMP was increased to 5× MIC, the inoculum (1×10 8 CFU / ml were completely eliminated within 5 minutes. Within 30 minutes, COG1410 at 1× MIC reduced CFU by 6 logs and killed the bacteria. In comparison, polymyxin B at 1× and 5× MIC reduced CFU by 2 logs and 4 logs, respectively, after 30 minutes (Figure 1A). These data indicated that COG1410 acts more rapidly than polymyxin B.

[0164] To determine the efficacy of COG1410 in pooled human plasma, its LC 99.9 The LC values (99.9% killing within 2 hours of incubation) were measured in PBS with or without 50% human plasma. As shown in Figure 1B, 2 μg / ml (1.4 μM) COG1410 achieved 99.9% killing in PBS. Consistently, the antibacterial activity of this AMP was reduced in the presence of human plasma, resulting in a LC 99.9 The maximum concentration was 8 μg / ml (5.6 μM).

[0165] To address the activity of COG1410 under other conditions, the bactericidal efficacy of COG1410 was measured in PBS, LB broth, and saline, respectively. While nearly eliminated in PBS, COG1410 at 1× MIC only killed 78.6% and 78.3% of A. baumannii in LB broth (250 mM NaCl) and saline (225 mM NaCl), respectively, after 2 hours of incubation (Figure 1C). These data suggested that high salt concentrations may interfere with the bactericidal effect of COG1410.

[0166] COG1410 remains stable in human plasma

[0167] To measure the stability of COG1410 in human plasma, 10 mg / ml of COG1410 was incubated with 100% human plasma at 37°C. Samples were collected at different time points and dropped onto paper discs. Antibacterial activity was assessed by measuring the diameter of the inhibition zone. Interestingly, the inhibition diameter of COG1410 did not change significantly within the first 2 hours (data not shown). At 4 hours, the activity decreased by 6.8%. After 10 hours of incubation, 20% of the activity was lost. These data suggested that COG1410 is highly stable in human plasma.

[0168] COG1410 exhibits biofilm inhibition and eradication activity

[0169] Bacterial biofilm formation is often associated with chronic wound infections and drug resistance. To examine the efficacy of COG1410 against biofilms, the biofilm mass formed by A. baumannii YQ4 was measured by crystal violet staining. COG1410 inhibited biofilm formation in a dose-dependent manner (Figure 2A). COG1410 at 0.5×MIC significantly reduced biofilm formation.

[0170] COG1410 at 1× MIC reduced biofilm mass by 55% (46%) compared with untreated controls.

[0171] In the case of preformed biofilms, COG1410 at 1× the MIC dispersed approximately 46% of the mature biofilms. When the AMP concentration was increased to 4× the MIC, more than 88% of the biofilms were eradicated (Figure 2B). Collectively, these data indicate that COG1410 can be used as an antibiofilm agent.

[0172] COG1410 treatment increases the permeability of the bacterial cell membrane

[0173] To evaluate the mechanism of action of COG1410, cell morphology was first examined by scanning electron microscopy (SEM). As shown in Figure 3A, untreated A. baumannii cells resembled peanuts with small spike-like patterns on their surfaces. In contrast, polymyxin B treatment resulted in cell lysis and release of intracellular contents. In contrast, COG1410-treated cells were largely intact, and little cell debris was observed. No pores were detected on the cell surface. These data indicated that COG1410 may not cause pore formation or cell lysis.

[0174] To determine the integrity of the bacterial cell membrane, the same batch of treated cells was observed under a transmission electron microscope (TEM). Untreated cells had intact cell membranes. Exposure to polymyxin B gradually induced cell wall disintegration in most cells (Figure 3B). However, COG1410-treated cells appeared shrunken and smaller compared to untreated cells. The inner membrane also appeared to be detached from the cell wall.

[0175] To verify COG1410-induced cell membrane permeabilization, we stained COG1410-treated A. baumannii YQ4 cells with two fluorescent nucleic acid dyes: SYTO-9 and propidium iodide (PI). The former stains both live and dead cells, exhibiting green fluorescence (shown as hatched shapes in Figure 4), whereas the latter enters only non-viable cells, emitting red fluorescence (shown as open shapes in Figure 4). As expected, untreated cells exhibited green fluorescence, indicating that all cells were viable. Both COG1410- and polymyxin B-treated cells exhibited red fluorescence, confirming cell death (data not shown). Notably, these treated dead cells stained with SYTO-9 exhibited no green fluorescence, which may be due to competition with PI. Therefore, this staining assay confirmed that COG1410 permeabilized the bacterial membrane.

[0176] Membrane disruption can be further characterized by measuring the leakage of intracellular components from bacterial cells. To address the mechanism of action of COG1410, we measured the extracellular ATP concentration in cells exposed to COG1410 at 1× MIC using the Enhanced ATP Assay Kit (Beyotime). Tigecycline, which binds to bacterial 30S ribosomes and blocks transfer RNA entry, was used as a negative control. As expected, tigecycline-treated cells had similar ATP leakage levels to untreated cells. Cells exposed to polymyxin B released more ATP than untreated cells. Notably, COG1410 treatment caused ATP leakage, which was significantly greater than that of the untreated control and tigecycline-treated groups (Figure 3C). In summary, the cationic AMP COG1410 disrupted bacterial cell membranes.

[0177] COG1410 localizes to the cytoplasm

[0178] In addition to direct membrane disruption, some sub-MIC AMPs may target the cytoplasm. To address whether COG1410 directly binds to bacterial membranes or enters the cytoplasm, a small number of bacterial pathogens were treated with 8 μg / ml COG1410 for 30 minutes and then costained with a red fluorescent membrane dye. COG1410 entered the cytoplasm of not only A. baumannii and E. faecium but also K. pneumoniae and S. aureus (Figure 4). COG1410 was effective against the former two strains but did not kill the latter two. These results suggest that COG1410 may bind more readily to the cell membrane of A. baumannii than to other bacteria, or that COG1410 specifically inhibits cytoplasmic targets in A. baumannii.

[0179] COG1410 binds nonspecifically to DNA

[0180] To investigate whether cationic AMP binds to DNA, we performed gel retardation experiments. The electrophoretic mobility of the plasmid pUC18 was measured after incubation with different concentrations of COG1410. In the absence of AMP, the plasmid migrated normally in the gel. In the presence of 1× MIC of COG1410, some DNA remained in the loading well, and only a small portion of the DNA migrated in the gel. With increasing concentrations of COG1410, DNA mobility was completely retarded (data not shown). In summary, COG1410 was able to nonspecifically bind to DNA in a concentration-dependent manner.

[0181] Sub-MIC treatment with COG1410 induces the expression of genes involved in redox processes

[0182] To further identify putative intracellular targets of COG1410, we used RNA-seq to compare the transcriptomes of A. baumannii YQ4 in the presence or absence of COG1410 at 0.25×MIC (4 μg / ml). To reduce interference from rich media, M9 minimal medium was selected for bacterial culture preparation. Analysis of RNA-seq data identified 92 significantly differentially expressed genes (DEGs) with at least a two-fold change. Compared to untreated controls, the transcription levels of 55 and 37 genes increased and decreased, respectively, in the presence of AMP. The 92 DEGs were classified into 12 categories at the GO level 2, including catalytic activity, cellular anatomical entity, metabolic process, and response to stimuli (data not shown). Interestingly, genes involved in redox processes were enriched (Figure 5). This raised the question of whether treatment with COG1410 increases reactive oxygen species. To address this concern, we measured intracellular ROS levels using a DCFH-DA probe. As shown in Figure 3D, COG1410 treatment significantly increased ROS production, indicating that ROS may be another killing mechanism of COG1410.

[0183] COG1410 is highly resistant to induced resistance

[0184] Because bacterial populations constantly seek to develop resistance to survive, the drug resistance barrier is a crucial parameter for new antibacterial compounds. In particular, A. baumannii naturally possesses the ability to absorb exogenous DNA and acquire resistance to various antibiotics. To evaluate the resistance rate of A. baumannii to COG1410, PDR strain YQ4 was serially passaged in the presence of sub-MIC concentrations of COG1410. Polymyxin B was included as an antibiotic control. The MIC value of polymyxin B increased 64-fold after 55 passages (data not shown). In contrast, the MIC of COG1410 increased only 4-fold in YQ4 after 55 passages. These data indicated that A. baumannii cannot easily neutralize the toxicity of COG1410 through genetic mutations.

[0185] COG1410 exhibits low hemolytic activity and moderate eukaryotic cell toxicity

[0186] The hemolytic potential of COG1410 was measured by exposing human red blood cells to various concentrations of COG1410 and measuring hemoglobin release. Controls consisted of 100% hemoglobin release after treatment with 0.1% Triton X-100 and 0% release after treatment with PBS. COG1410 at 128 μg / ml (8×MIC) caused less than 5% hemolysis (Figure 6A). The minimum concentration at which half of the red blood cells were lysed (EC 50 ) was 441 μg / ml. Therefore, the selectivity index (SI) of PDR A. baumannii YQ4 was 50 / MIC) was 27.5.

[0187] The cytotoxic effect of COG1410 on normal human hepatic L02 cells was evaluated by CCK8 assay. As shown in Figure 6B, 16 μg / ml was 7% toxic to L02 cells. The half-maximal dose (EC 50 The toxic concentration was 58.9 μg / ml.

[0188] The bactericidal activity of COG1410 differed from that of polymyxin B

[0189] To address whether the antibacterial effect of COG1410 depends on negatively charged lipopolysaccharide (LPS) on the cell surface, we investigated the effects of A. baumannii ATCC 19606 and two LPS-modified strains, pmrA and pmrB. P102R and pmrA P102R miaA I221V

[34] . The pmrA mutation results in derepression of PmrC, which encodes lipid A phosphoethanolamine transferase

[35] . Lipid A modification of LPS was found not to alter the antibacterial efficacy of COG1410. This is in contrast to polymyxin B, the last resort for MDR Gram-negative bacteria, whose bactericidal effect relies on electrostatic interactions with LPS (Figure 7A). These data suggest that COG1410 may have a different antibacterial mechanism from polymyxin B and that there is no cross-resistance between them.

[0190] COG1410 exhibits strong synergistic effects with polymyxin B

[0191] To investigate whether COG1410 has synergistic interactions with conventional antibiotics, we measured synergistic effects using COG1410 in combination with several antibiotics frequently used against A. baumannii in clinical settings. As shown in Figures 7A-7C, COG1410 exhibited synergistic activity with polymyxin B, ceftazidime, and tetracycline, with FIC values of 0.13, 0.31, and 0.31, respectively. Surprisingly, only 2 μg / ml COG1410 plus 1 μg / ml polymyxin B was able to completely inhibit the growth of PDR strains (Figure 7B). Time-kill kinetics indicated that this combination exhibited bactericidal activity similar to that of 16 μg / ml COG1410 alone (Figure 7C).

[0192] Combination therapy of COG1410 and polymyxin B can rescue C. elegans infected with A. baumannii

[0193] To address in vivo efficacy, we developed a bacterial infection model using C. elegans. L4 worms were preinfected in M9 buffer and transferred to NGM agar plates with or without COG1410. To enhance the virulence of A. baumannii, 10 μM FeCl3 was added, as previously reported

[36] . Figure 8 shows the rescue of infected worms by combined treatment with COG1410 and polymyxin B. C. elegans worms were preinfected with A. baumannii YQ4 and transferred to NGM plates supplemented with 16 μg / ml COG1410 or 2 μg / ml COG1410 and 1 μg / ml polymyxin B. Dead worms were counted daily for two weeks. Survival curves were analyzed using the Kaplan-Meier method, and statistical significance was determined using the log-rank test. ** , p<0.01. Compared with the untreated group, 16 μg / ml of COG1410 reduced the mortality of infected worms, but the change was not statistically significant. Unexpectedly, the combination treatment of COG1410 (2 μg / ml) + polymyxin B (1 μg / ml) significantly rescued pre-infected worms and even extended their lifespan.

[0194] Example 3. Antibacterial activity of peptide compounds against bacterial strains including P. gingivalis Minimum inhibitory concentrations (MICs) were measured for the peptide compounds listed in Table 1 above. Briefly, P. gingivalis was grown in anaerobic BHI broth (Brain Heart Infusion Broth containing 0.5 mg / ml L-cysteine, 0.5 mg / ml arginine, 5 μg / ml hemin, and 1.0 μg / ml vitamin K) for 24–96 hours at 37°C under anaerobic conditions to mid-logarithmic phase according to Dominy et al. (2019). L. salivarius was grown in BHI broth under 5% CO2 conditions at 37°C for 24–48 hours. MICs were measured using the microtiter plate method outlined by Jansen (ibid.). Compounds and bacteria are added to wells of a microtiter plate and grown anaerobically (P. gingivalis) or with CO2 (L. salivarius) for 24-72 hours. Growth is measured using light scattering at 592 nm on a plate reader, and absorbance is plotted against μg / ml of drug in each well. The MIC is the μg / ml of each compound at which absorbance does not increase significantly above background (medium alone) by the end of the time course.

[0195] Those skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present disclosure is representative of exemplary embodiments and is not intended to limit the scope of the disclosure. Modifications and other uses will occur to those skilled in the art which are encompassed within the spirit of the disclosure as defined by the scope of the claims.

[0196] No admission is made that any reference, including non-patent or patent literature, cited herein constitutes prior art. Unless otherwise expressly stated, it will be understood that reference to any document herein is not an admission that any of such documents forms part of the common general knowledge in the art in the United States or any other country. Any discussion of a reference states what its author asserts, and applicants reserve the right to challenge the accuracy and pertinence of the documents cited herein. All references cited herein are incorporated by reference in their entirety unless expressly stated otherwise. In the event of a conflict between definitions and / or explanations found in a cited reference, the present disclosure controls. References

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Claims

1. 1. A method for inhibiting microbial growth, comprising contacting a microorganism with an effective amount of a peptide compound to inhibit said microbial growth, said peptide compound comprising: (COG1410) Acetyl-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 1); Picolinyl-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 2); Picolinyl-AS-C-LRKL-aib-KRLL-C-amide (SEQ ID NO: 3); where a disulfide bond exists between the two cysteine residues; Acetyl-LLRK-aib-LKRL-aib-SA-CONH2 (SEQ ID NO: 4); Acetyl-llrk-Aib-lkkl-Aib-sa-amide (SEQ ID NO: 5), wherein all amino acid residues are D-amino acids; acetyl-as-aib-lrkl-aib-krll-amide (SEQ ID NO: 6), wherein all amino acid residues are D-amino acids; Acetyl-LLRK-aib-LRKL-aib-SAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 7); Acetyl-LRVRCAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 8); Acetyl-LRVRLAS-aib-LKKL-aib-KRLL-amide (SEQ ID NO: 9); Acetyl-LRVRLAS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 10); Acetyl-llrk-aib-lkrl-aib-salrvrl-amide (SEQ ID NO:11), wherein all amino acid residues are D-amino acids; Acetyl-LRVRLASHLRKLRKRLLAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 12); C8-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 13); Acetyl-K(C8)-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 14); Acetyl-K(picolinyl)-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 15); Acetyl-LRVRLASHLRKLRKRLLR-amide (SEQ ID NO: 16); Acetyl-LRKLRKRLLLRKLRKRLL-amide (SEQ ID NO: 17); Acetyl-LRVRLASHLRKLRKRLLRDADDLQKRLAVY-amide (SEQ ID NO: 18); or Picolinyl-llrk-aib-lkrl-aib-salrvrl-amine (SEQ ID NO: 19), wherein all said amino acid residues are D-amino acids; wherein aib is aminoisobutyric acid. A method comprising:

2. 2. The method of claim 1, wherein the peptide compound comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:

15.

3. 2. The method of claim 1, wherein the peptide compound comprises SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:

3.

4. The method of claim 1 , wherein the peptide compound comprises SEQ ID NO:

1.

5. 10. The method of claim 1, wherein the microorganism comprises a bacterium that is one or more of multidrug resistant (MDR), extensively drug resistant (XDR), or pan-drug resistant (PDR).

6. 10. The method of claim 1, wherein the microorganism comprises Porphoryomas gingivalis.

7. The method of claim 1 , wherein the microorganism comprises a gram-negative bacterial pathogen.

8. 8. The method of claim 7, wherein the microorganism comprises one or a combination of Acinetobacter baumannii, P. gingivalis, or E. coli.

9. The method of claim 1 , wherein the microorganism comprises a gram-positive pathogen.

10. 10. The method of claim 9, wherein the microorganism comprises one or a combination of S. aureus or L. salivarius.

11. The method of claim 1 , wherein the peptide compound is contacted in combination with one or more antimicrobial compounds.

12. 12. The method of claim 11, wherein the one or more antimicrobial compounds comprises polymyxin B.

13. 13. The method of claim 12, wherein the microorganism comprises pan-drug resistant Acinetobacter baumannii or multi-drug resistant S. aureus.

14. 13. The method of claim 12, wherein the peptide comprises SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and the bacterium comprises pan-drug resistant Acinetobacter baumannii.

15. 13. The method of claim 12, wherein the peptide comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, and the bacterium comprises pan-drug resistant Acinetobacter baumannii.

16. 1. A method of treating a subject having a microbial infection, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a peptide compound, or a pharmaceutically acceptable salt or solvate thereof, to inhibit microbial growth in the subject, wherein the peptide compound is (COG1410) Acetyl-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 1); Picolinyl-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 2); Picolinyl-AS-C-LRKL-aib-KRLL-C-amide (SEQ ID NO: 3); where a disulfide bond exists between the two cysteine residues; Acetyl-LLRK-aib-LKRL-aib-SA-CONH2 (SEQ ID NO: 4); Acetyl-llrk-Aib-lkkl-Aib-sa-amide (SEQ ID NO: 5), wherein all amino acid residues are D-amino acids; acetyl-as-aib-lrkl-aib-krll-amide (SEQ ID NO: 6), wherein all amino acid residues are D-amino acids; Acetyl-LLRK-aib-LRKL-aib-SAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 7); Acetyl-LRVRCAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 8); Acetyl-LRVRLAS-aib-LKKL-aib-KRLL-amide (SEQ ID NO: 9); Acetyl-LRVRLAS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 10); Acetyl-llrk-aib-lkrl-aib-salrvrl-amide (SEQ ID NO:11), wherein all amino acid residues are D-amino acids; Acetyl-LRVRLASHLRKLRKRLLAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 12); C8-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 13); Acetyl-K(C8)-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 14); Acetyl-K(picolinyl)-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 15); Acetyl-LRVRLASHLRKLRKRLLR-amide (SEQ ID NO: 16); Acetyl-LRKLRKRLLLRKLRKRLL-amide (SEQ ID NO: 17); Acetyl-LRVRLASHLRKLRKRLLRDADDLQKRLAVY-amide (SEQ ID NO: 18); or Picolinyl-llrk-aib-lkrl-aib-salrvrl-amine (SEQ ID NO: 19), wherein all said amino acid residues are D-amino acids; wherein aib is aminoisobutyric acid A method comprising:

17. 17. The method of claim 16, wherein the microbial growth is caused by bacteria comprising one or more of multidrug resistant (MDR), extensively drug resistant (XDR), or pan-drug resistant (PDR).

18. 17. The method of claim 16, wherein the microbial growth is caused by an oral infection with bacteria, including Porphoryomas gingivalis.

19. 17. The method of claim 16, wherein the microbial growth is caused by a gram-negative bacterial pathogen.

20. 20. The method of claim 19, wherein the bacterial pathogen comprises one or a combination of Acinetobacter baumannii, P. gingivalis, or E. coli.

21. 17. The method of claim 16, wherein the microbial growth is caused by a gram-positive pathogen.

22. 22. The method of claim 21, wherein the Gram-positive pathogen comprises one or a combination of S. aureus or L. salivarius.

23. 17. The method of claim 16, wherein the administration is in combination with one or more antibacterial compounds.

24. 24. The method of claim 23, wherein the one or more antimicrobial compounds comprises polymyxin B.

25. 25. The method of claim 24, wherein the microbial growth is caused by bacteria including pan-drug resistant Acinetobacter baumannii or multi-drug resistant S. aureus.

26. 25. The method of claim 24, wherein the peptide comprises SEQ ID NO: 1, SEQ ID NO: 2, or (SEQ ID NO: 3) and the microbial growth is caused by bacteria comprising pan-drug resistant Acinetobacter baumannii.

27. 25. The method of claim 24, wherein the peptide comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, and the microbial growth is caused by bacteria comprising Acinetobacter baumannii.

28. 25. The method of claim 24, wherein the peptide comprises SEQ ID NO: 1 and the microbial growth is caused by bacteria comprising Acinetobacter baumannii.

29. 17. The method of claim 16, wherein said administering comprises a single administration or multiple administrations of said composition comprising said peptide compound.

30. 17. The method of claim 16, wherein the composition comprising the peptide compound is administered topically, enterally, systemically, or parenterally.

31. 17. The method of claim 16, wherein the microbial growth is caused by an oral infection with bacteria, including Porphoryomas gingivalis, and the composition comprising the peptide compound is administered in a mouthwash or toothpaste formulation.

32. 17. The method of claim 16, wherein the subject is a mammal, a primate, or a human.

33. 1. An antimicrobial composition comprising: a therapeutically effective amount of a peptide compound comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, or a pharmaceutically acceptable salt or solvate thereof; a therapeutically effective amount of polymyxin B; 1. An antimicrobial composition comprising:

34. 34. The antimicrobial composition of claim 33, wherein the peptide compound comprises SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:

3.

35. 35. The antimicrobial composition of claim 34, wherein the peptide compound comprises SEQ ID NO:

1.

36. 36. The antimicrobial composition of any one of claims 33 to 35 for use in a method of inhibiting microbial growth in a subject.

37. 36. A pharmaceutical composition comprising the antimicrobial composition of any one of claims 33 to 35 for use in a method of inhibiting microbial growth in a subject.

38. A peptide compound having a structure selected from: (COG1410) Acetyl-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 1); Picolinyl-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 2); Picolinyl-AS-C-LRKL-aib-KRLL-C-amide (SEQ ID NO: 3); where a disulfide bond exists between the two cysteines; Acetyl-LLRK(Aib)LKRL(Aib)SA-CONH2 (SEQ ID NO: 4); Acetyl-llrk(Aib)lkkl(Aib)sa-amide (SEQ ID NO: 5), (lowercase letters represent D amino acids); acetyl-as-aib-lrkl-aib-krll-amide (SEQ ID NO: 6), wherein all amino acid residues are D-amino acids; Acetyl-LLRK-Aib-LRKL-Aib-SAS-Aib-LRKL-Aib-KRLL-CONH2 (SEQ ID NO: 7); Acetyl-LRVRCAS(Aib)LRKL(Aib)KRLL-CONH2 (SEQ ID NO: 8); Acetyl-LRVRLAS(Aib)LKKL(Aib)KRLL-amide (SEQ ID NO: 9); Acetyl-LRVRLAS(Aib)LRKL(Aib)KRLL-amide (SEQ ID NO: 10); Acetyl-llrk(Aib)lkrl(Aib)salrvrl-amide (SEQ ID NO: 11), (lowercase letters represent D amino acids); and Acetyl-LRVRLASHLRKLRKRLLAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 12); C8-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 13); Acetyl-K(C8)-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 14); Acetyl-K(picolinyl)-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 15); Acetyl-LRVRLASHLRKLRKRLLR-amide (SEQ ID NO: 16); Acetyl-LRKLRKRLLLRKLRKRLL-amide (SEQ ID NO: 17); Acetyl-LRVRLASHLRKLRKRLLRDADDLQKRLAVY-amide (SEQ ID NO: 18); and Picolinyl-llrk-aib-lkrl-aib-salrvrl-amine (SEQ ID NO: 19), wherein all amino acid residues are D-amino acids; where aib is aminoisobutyric acid.

39. 40. The peptide compound of claim 38, or a pharmaceutically acceptable salt or solvate thereof, for use in a method of inhibiting microbial growth in a subject.

40. A pharmaceutical composition comprising the peptide compound of claim 39.

41. 41. The pharmaceutical composition of claim 40, wherein the peptide compound comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:

15.

42. 41. The pharmaceutical composition of claim 40, wherein the peptide compound comprises SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:

3.

43. 41. The pharmaceutical composition of claim 40, wherein the peptide compound comprises SEQ ID NO:

1.

44. 44. A pharmaceutical composition according to any one of claims 40 to 43, comprising one or more antibacterial compounds.

45. 45. The pharmaceutical composition of claim 44, wherein the one or more antibacterial compounds comprises polymyxin B.

46. 46. The pharmaceutical composition of any one of claims 40 to 45 for use in a method for inhibiting microbial growth in a subject.

47. 47. The pharmaceutical composition of claim 37 or 46, further comprising one or more pharmaceutically acceptable excipients.

48. 48. The pharmaceutical composition of claim 47, wherein the pharmaceutically acceptable excipient comprises one or more of a filler, disintegrant, diluent, binder, solvent, co-solvent, lubricant, pH adjuster, buffer, preservative, dispersant, suspending agent, ointment base, emulsifier, emollient, penetrant, surfactant, propellant, flavoring agent, sweetening agent, or drug release modifier.

49. 48. The pharmaceutical composition of claim 47, wherein the solvent comprises one or more of saline or glucose solution.

50. 48. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is formulated as a lyophilized powder, or as a liquid suitable for administration by injection for sustained release of the peptide compound, or as a spray.

51. 48. The pharmaceutical composition of claim 47, formulated as a mouthwash or toothpaste.

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