Antimicrobial peptide compounds and methods of use

Abstract

The present invention provides antimicrobial peptide compounds, pharmaceutical compositions and methods of their use for inhibiting the growth of microorganisms. The present invention also relates to a method for inhibiting the total drug resistance of Acinetobacter baumannii, and a method for inhibiting the total drug resistance of Acinetobacter baumannii by administering the peptide compound to a subject. Provided is a method for the inhibition of fully 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. The invention also provides a method for the inhibition of fully drug resistant Acinetobacter baumannii. In other methods, a peptide compound is provided for the inhibition of Porphyromonas gingivalis (P.gingivalis), which is the key bacterium of the most six most common infectious periodontal disease in the world.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 368,505, filed on July 15, 2022, the entire contents of which are incorporated herein by reference. Field of the Invention

[0003] The present invention relates to antimicrobial peptide compounds, and in particular to their use in treating microbial infections including those with pan-drug resistant bacteria.

[0004] background

[0005] The emergence of resistance to multiple antimicrobial agents in pathogenic bacteria has become a major public health concern. Using documents and breakpoints from the Clinical Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and the U.S. Food and Drug Administration (FDA), standardized definitions of the degree of resistance were created for pathogenic bacteria. Multidrug resistance (MDR) is defined as acquired insusceptibility to at least one agent in three or more antimicrobial classes, extensive resistance (XDR) is defined as insusceptibility to at least one agent in all but two or fewer antimicrobial classes (i.e., the bacterial isolate is sensitive to only one or two classes), and pandrug resistance (PDR) is defined as insusceptibility to all agents in all antimicrobial classes.

[0006] One particularly problematic bacterium is Acinetobacter baumannii. A. baumannii is a Gram-negative opportunistic pathogen that can cause serious nosocomial infections in immunocompromised patients. A notable feature of this bacterium is its ability to rapidly develop resistance to antimicrobial agents and to survive for weeks on dry surfaces in healthcare facilities. As early as 2009, carbapenem-resistant A. baumannii was listed as one of six high-threat pathogens (also known as “ESKAPE” pathogens) by the Infectious Diseases Society of America, calling for global collaboration to address this threat. In particular, the emergence of chromosomally encoded and plasmid-mediated polymyxin resistance in A. baumannii has breached the last means of defense. Currently, the prevalence of A. baumannii infections is considered a major and complex problem for global health.

[0007] In addition to multi-resistant, pan-resistant and pan-resistant bacteria, Porphyromonas gingivalis (P. gingivalis) is the key bacterium in periodontal disease (PD), the sixth most common infectious disease worldwide. In the United States, half of adults over 30 years old and 70% of adults over 65 years old have some degree of P. gingivalis infection associated with gum disease. Older men and African Americans are at greater risk for periodontal disease. In a study of 6,800 patients followed for up to 26 years, more severe periodontal disease after gingivitis was associated with an increased risk of dementia, including Alzheimer's dementia. Tooth loss is a common consequence of PD and also increases the risk of dementia. P. gingivalis products have been found in human AD brains, including lipopolysaccharide (LPS), cysteine ​​proteases called gingipasins, and 16S rRNA. Because P. gingivalis is present in the oral cavity, blood, and is known to invade non-oral tissues including the brain, where it can induce a proinflammatory state that has also been linked to AD, strategies to reduce the abundance and activity of P. gingivalis have begun to show positive effects on AD-related outcomes.

[0008] Therefore, there is an urgent and pressing need for new antimicrobial agents that have activity against drug-resistant bacteria (including pan-resistant bacteria) and against Porphyromonas gingivalis. The present disclosure provides such antimicrobial agents.

[0009] Overview

[0010] In one aspect of the present invention, a series of peptide compounds with antimicrobial activity are provided. The peptide compounds of the present invention have a structure selected from the following:

[0011] (COG1410) acetyl-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 1);

[0012] Picolinyl-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 2);

[0013] Picolinyl-AS-C-LRKL-aib-KRLL-C-amide (SEQ ID NO: 3); wherein there is a disulfide bond between the two cysteine ​​residues;

[0014] Acetyl-LLRK-aib-LKRL-aib-SA-CONH2 (SEQ ID NO: 4);

[0015] Acetyl-llrk-Aib-lkkl-Aib-sa-amide (SEQ ID NO:5), wherein all amino acid residues are D-amino acids;

[0016] Acetyl-as-aib-lrkl-aib-krll-amide (SEQ ID NO:6), wherein all amino acid residues are D-amino acids;

[0017] Acetyl-LLRK-aib-LRKL-aib-SAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 7);

[0018] Acetyl-LRVRCAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 8);

[0019] Acetyl-LRVRLAS-aib-LKKL-aib-KRLL-amide (SEQ ID NO: 9);

[0020] Acetyl-LRVRLAS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 10);

[0021] Acetyl-llrk-aib-lkrl-aib-salrvrl-amide (SEQ ID NO: 11), wherein all amino acid residues are D-amino acids;

[0022] Acetyl-LRVRLASHLRKLRKRLLAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 12);

[0023] C8-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 13);

[0024] Acetyl-K(C8)-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 14);

[0025] Acetyl-K(picolinyl)-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 15);

[0026] Acetyl-LRVRLASHLRKLRKRLLR-amide (SEQ ID NO: 16);

[0027] Acetyl-LRKLRKRLLLRKLRKRLL-amide (SEQ ID NO: 17);

[0028] Acetyl-LRVRLASHLRKLRKRLLRDADDLQKRLAVY-amide (SEQ ID NO: 18); and

[0029] Picolinyl-llrk-aib-lkrl-aib-salrvrl-amine (SEQ ID NO: 19), wherein all amino acid residues are D-amino acids,

[0030] Among them, aib is aminoisobutyric acid.

[0031] In another embodiment, provided is a peptide compound of the present invention or a pharmaceutically acceptable salt or solvate thereof for use in a method of inhibiting the growth of a microorganism in a subject.

[0032] In other aspects, provided are pharmaceutical compositions comprising a peptide compound of the invention for use in a method of inhibiting microbial growth in a subject.

[0033] 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.

[0034] In other embodiments, the pharmaceutical composition comprises one or more additional antimicrobial compounds. The one or more antimicrobial compounds may include polymyxin B.

[0035] In one aspect, a method for inhibiting the growth of a microorganism is provided, comprising: contacting the microorganism with an effective amount of a peptide compound of the present invention to inhibit the growth of the microorganism. In certain embodiments of the method, the peptide compound may comprise 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 case, the peptide compound may 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.

[0036] In a method comprising contacting a microorganism with an effective amount of a peptide compound of the present invention, the microorganism may include one or more bacteria of multidrug resistance (MDR), pan-drug resistance (XDR) or full drug resistance (PDR). In some cases, the microorganism includes Porphyromonas gingivalis. The microorganism may include a gram-negative bacterial pathogen or a gram-positive pathogen. The gram-negative bacterial pathogen may include one or a combination of Acinetobacter baumannii, Porphyromonas gingivalis or Escherichia coli (E.coli); the gram-positive pathogen may include one or a combination of Staphylococcus aureus (S.aureus) or Lactobacillus salivarius (L.salivarius).

[0037] In some embodiments of the method comprising contacting a microorganism with an effective amount of a peptide compound of the present invention, the peptide compound and one or more other antimicrobial compounds are contacted with the microorganism. The one or more antimicrobial compounds may include polymyxin B. The microbial growth inhibited in the method may include microbial growth caused by fully resistant Acinetobacter baumannii or multi-resistant Staphylococcus aureus. The peptide may include SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and the inhibited microorganism may include fully resistant Acinetobacter baumannii. In other cases, the peptide includes 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 includes fully resistant Acinetobacter baumannii. In one case, the peptide includes SEQ ID NO: 1, and the inhibited microorganism includes fully resistant Acinetobacter baumannii.

[0038] In another embodiment of the present invention, a method for treating a subject suffering from a microbial infection is provided, 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 the method, the pharmaceutical composition may comprise one or more other antimicrobial compounds. The one or more antimicrobial compounds may comprise polymyxin B.

[0039] In a method for treating a subject with a microbial infection, 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. In other cases, the pharmaceutical composition may comprise the peptide compound SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In one example, the pharmaceutical composition comprises the peptide compound SEQ ID NO: 1.

[0040] In the method for treating a subject suffering from a microbial infection, the microbial growth can be caused by bacteria comprising one or more of multidrug resistance (MDR), pan-drug resistance (XDR) or full drug resistance (PDR). In other cases, the microbial growth is caused by an oral infection of a bacterium comprising Porphyromonas gingivalis. The microbial growth can be caused by a gram-negative bacterial pathogen or a gram-positive pathogen. The gram-negative bacterial pathogen can comprise one or a combination of Acinetobacter baumannii, Porphyromonas gingivalis or Escherichia coli, and the gram-positive pathogen can be Staphylococcus aureus or Lactobacillus salivarius.

[0041] 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 may 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.

[0042] In another embodiment, the antimicrobial composition is provided for use in a method of inhibiting the growth of a microorganism in a subject.

[0043] In other aspects, provided are pharmaceutical compositions comprising the antimicrobial compositions of the invention for use in a method of inhibiting the growth of a microorganism in a subject.

[0044] The administration of the pharmaceutical composition of the present invention to a subject may include a single administration or multiple administrations of the pharmaceutical composition. The pharmaceutical composition comprising the peptide compound may be administered topically, enterally, systemically or parenterally. In some cases, the pharmaceutical composition may be administered in a formulation of a mouthwash or toothpaste. In some cases where the microbial growth is caused by an oral infection of a bacterium comprising Porphyromonas gingivalis, the pharmaceutical composition comprising the peptide compound may be administered in a formulation of a mouthwash or toothpaste.

[0045] The pharmaceutical composition may include one or more pharmaceutically acceptable excipients. One or more excipients may include but are not limited to disintegrants, diluents, adhesives, solvents, co-solvents, lubricants, pH regulators, buffers, preservatives, dispersants, suspending agents, ointment bases, emulsifiers, softeners, permeants, surfactants, propellants, flavoring agents, sweeteners or drug release regulators. The pharmaceutical composition may include a solvent, such as one or both of physiological saline and glucose solution.

[0046] The pharmaceutical composition of the present invention may be formulated into a lyophilized powder or a liquid suitable for administration by injection to cause a slow release of the peptide compound, or formulated into a spray. In other embodiments, the pharmaceutical composition of the present invention may be formulated into a mouthwash or toothpaste.

[0047] BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The features and advantages of the present invention will be more clearly understood through the following description in conjunction with the accompanying drawings. The accompanying drawings are for illustration only and are not limiting. The above aspects and other features of the present disclosure are explained in the following description in conjunction with the attached example drawings (also referred to as "drawings") associated with one or more embodiments.

[0049] Figure 1A is a graph illustrating the antimicrobial activity of COG1410 (SEQ ID NO: 1), specifically the in vitro killing kinetics of COG1410 and polymyxin B (PMB) against PDR-Acinetobacter baumannii YQ4 strain at 1x MIC and 5x MIC in PBS, respectively.

[0050] Figure 1B is a graph illustrating the antimicrobial 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 incubation at 37°C for 2 hours.

[0051] Figure 1C is a graph illustrating the antimicrobial activity of COG1410 (SEQ ID NO: 1), specifically determining the bactericidal efficacy of COG1410 under different conditions. Figures 1A-1C Each experiment was performed in triplicate and values ​​are expressed as mean ± SD.

[0052] Figure 2A : is a graph illustrating that COG1410 (SEQ ID NO: 1) exhibits biofilm inhibition and elimination activity against PDR-Acinetobacter baumannii, specifically, COG1410 prevents biofilm formation. The results are expressed as crystal violet staining (OD 600 ) measured biofilm mass.

[0053] Figure 2B 1 is a graph illustrating that COG1410 (SEQ ID NO: 1) exhibits biofilm inhibition and removal activity against PDR-Acinetobacter baumannii, specifically the removal of established biofilms. Data are presented as mean ± SD of 8 replicates from 3 independent experiments. Statistical significance between each treatment and control was analyzed by Student's t-test (unpaired), *p < 0.5, ***p < 0.001.

[0054] Figure 3A are images illustrating that COG1410 (SEQ ID NO: 1) treatment permeabilizes the cell membrane of PDR-A. baumannii as seen by SEM observation of A. baumannii YQ4 exposed to 1x MIC COG1410 or 1x MIC polymyxin B. Cells in PBS served as negative controls.

[0055] Figure 3B are images illustrating that COG1410 (SEQ ID NO: 1) treatment permeabilizes the cell membrane of PDR-A. baumannii as observed by TEM of A. baumannii YQ4 exposed to 1x MIC COG1410 or 1x MIC polymyxin B. Cells in PBS served as negative controls.

[0056] Figure 3C 1 is a diagram illustrating the permeabilization of PDR-Acinetobacter baumannii cell membranes by COG1410 (SEQ ID NO: 1), specifically the effect of 1x MIC COG1410 on ATP release (i.e., ATP leakage) in Acinetobacter baumannii YQ4. 1x MIC polymyxin B and 1x MIC tigecycline (TGC, 32 μg / ml) were used as positive and negative controls for ATP leakage, respectively. Untreated cells were also negative controls.

[0057] Figure 3D1 is a diagram illustrating that COG1410 (SEQ ID NO: 1) treated the permeabilized cell membrane of PDR-Acinetobacter baumannii, specifically showing the measurement of ROS levels by the DCFH-DA probe in the presence or absence of COG1410 (16 μg / ml). Rosup is a positive control. The statistical significance between each treatment and control was analyzed by one-way ANOVA method, and multiple comparisons were performed by post hoc test, *p<0.05, **p<0.01.

[0058] Figure 4 Shown are images illustrating that FITC-COG1410 has entered the cytoplasm of Acinetobacter baumannii, Enterococcus faecium, Klebsiella pneumoniae, and Staphylococcus aureus. Bacteria were treated with FITC-labeled COG1410, counterstained with FM4-64 dye, and observed by CLSM. Green fluorescence (i.e., shaded shapes) indicates the localization of FITC-COG1410 in the cell. Red fluorescence (i.e., open shapes) indicates the cytoplasmic membrane. Scale bar: 4 μm.

[0059] Figure 5A Figure 1 is the first half of a graph showing the enrichment of genes involved in redox processes by treatment with COG1410 (SEQ ID NO: 1). Whole transcriptome analysis of A. baumannii treated or untreated 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 using clusterProfiler (v3.4.4). GO terms with FDR ≤ 0.05 were considered to be significantly enriched by DEGs.

[0060] Figure 5B yes Figure 5A The second half of the diagram is described.

[0061] Fig. 6A is a graph illustrating that COG1410 (SEQ ID NO: 1) exhibits low hemolytic activity, specifically determined by measuring the release of hemoglobin at 414 nm from human erythrocytes exposed to different concentrations of COG1410. PBS and Triton X-100 (0.1%) were used as negative and positive controls, respectively.

[0062] Figure 6Bis a graph illustrating 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 the CCK8 assay. The experiment was performed in triplicate. The data shown are means ± SD.

[0063] Fig. 7A is a graph showing that COG1410 (SEQ ID NO: 1) exhibits a strong synergistic interaction with polymyxin B, specifically that modification of LPS did not alter the antimicrobial activity of COG1410 against Acinetobacter baumannii. MICs were determined in LB liquid medium against the wild-type strain ATCC19606 of Acinetobacter baumannii and the corresponding LPS-deficient mutants with pmrAP102R and pmrAP102RmiaAI221V mutations.

[0064] Figure 7B 1 is a graph showing that COG1410 (SEQ ID NO: 1) exhibits a strong synergistic interaction with polymyxin B, specifically a combination of 2 μg / ml COG1410 and 1 μg / ml polymyxin B (i.e., 2COG1410+1PBM) can completely inhibit bacterial growth in LB liquid medium within 20 h. Growth curves were measured in duplicate, with 8 wells per treatment set in a 96-well plate each time. Representative results are shown.

[0065] Figure 7C A graph showing that COG1410 (SEQ ID NO: 1) exhibits a strong synergistic interaction with polymyxin B, specifically the combination of COG1410 and polymyxin B (i.e. 2COG1410+1PBM) significantly reduced the CFU of Acinetobacter baumannii YQ4 in PBS. The experiment was performed in triplicate. Data are expressed as mean ± SD.

[0066] Figure 8 is a diagram illustrating that the combination therapy of COG1410 (SEQ ID NO: 1) and polymyxin B rescued infected nematodes. Specifically, C. elegans was pre-infected with Acinetobacter 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 nematodes were counted every day for 2 weeks. Survival curves were analyzed using the Kaplan-Meier method, and statistical significance was analyzed using the log-rank test. **, p < 0.01. DETAILED DESCRIPTION OF THE INVENTION

[0068] In order to promote an understanding of the principles of the present disclosure, reference will now be made to the preferred embodiments, and specific language will be used to describe the same. However, it will be appreciated that it is not intended to limit the scope of the present disclosure, and such changes and further modifications to the present disclosure as described herein would be considered generally by those skilled in the art to which the present disclosure relates.

[0069] Unless defined otherwise, 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.

[0070] The terms "including," "comprising," or "having," and variations thereof, used herein, are intended to encompass the elements listed thereafter and their equivalents as well as additional elements. As used herein, "and / or" refers to and encompasses all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreting an alternative ("or").

[0071] In addition, the indefinite articles "a" and "an" in front of the elements or components of the present invention are not intended to limit the number of times the elements or components appear. Therefore, "a" and "an" should be understood to include one or at least one, and unless the number is clearly singular, the singular form of the element or component also includes the plural form.

[0072] Unless otherwise specified herein, references to ranges of values ​​herein are intended merely as a shorthand method of individually referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is specified as 1% to 50%, it is intended that values ​​of 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly recited in this specification. These are merely examples of specific intent, and all possible combinations of values ​​between and including the lowest value and the highest value recited should be considered to be expressly stated in this disclosure.

[0073] For the purposes of this specification, drawings and claims, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0074] As used herein, the term "treatment" refers to the administration of the composition of the present invention to an individual suffering from a microbial infection, which results in partial or complete relief of symptoms, or prevents an increase in symptoms of microbial infection after treatment / treatment. Therefore, treatment / treatment includes cure. As used herein, "efficacy" represents the effect caused by treatment / treatment, which changes, generally changes, alleviates or improves the symptoms or characteristics of microbial infection, or cures microbial infection. "Treatment / treatment" may also mean prolonged survival compared to the expected survival without treatment / treatment. "Treatment / treatment" refers to both therapeutic treatment / treatment and preventive or preventive measures in certain embodiments. Treatment / treatment refers to suppressing or reducing the increase in pathology or symptoms compared to the absence of treatment / treatment, and does not necessarily mean the complete cessation of microbial infection.

[0075] As used herein, the term "therapeutically effective amount" refers to a use or method of administering an amount of a composition of the present invention that will achieve the desired therapeutic effect upon administration.

[0076] As used herein, the term "subject" includes human patients and non-human (animal) patients. The term "non-human animal" includes vertebrates, such as mammals (such as non-human primates, sheep, cows, dogs, cats, and rodents (such as mice and rats)).

[0077] The present inventors have found that a series of peptide compounds have excellent antimicrobial effects. The peptide compounds are provided as antimicrobial agents, and in some embodiments are provided in the form of compositions for use in administering to a subject to treat microbial infection. The peptide compounds of the present invention are provided as follows:

[0078] (COG1410) acetyl-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 1);

[0079] Picolinyl-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 2);

[0080] Picolinyl-AS-C-LRKL-aib-KRLL-C-amide (SEQ ID NO: 3); wherein there is a disulfide bond between the two cysteine ​​residues;

[0081] Acetyl-LLRK-aib-LKRL-aib-SA-CONH2 (SEQ ID NO: 4);

[0082] Acetyl-llrk-Aib-lkkl-Aib-sa-amide (SEQ ID NO:5), wherein all amino acid residues are D-amino acids;

[0083] Acetyl-as-aib-lrkl-aib-krll-amide (SEQ ID NO:6), wherein all amino acid residues are D-amino acids;

[0084] Acetyl-LLRK-aib-LRKL-aib-SAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 7);

[0085] Acetyl-LRVRCAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 8);

[0086] Acetyl-LRVRLAS-aib-LKKL-aib-KRLL-amide (SEQ ID NO: 9);

[0087] Acetyl-LRVRLAS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 10);

[0088] Acetyl-llrk-aib-lkrl-aib-salrvrl-amide (SEQ ID NO: 11), wherein all amino acid residues are D-amino acids;

[0089] Acetyl-LRVRLASHLRKLRKRLLAS-aib-LRKL-aib-KRLL-CONH2 (SEQ ID NO: 12);

[0090] C8-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 13);

[0091] Acetyl-K(C8)-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 14);

[0092] Acetyl-K(picolinyl)-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 15);

[0093] Acetyl-LRVRLASHLRKLRKRLLR-amide (SEQ ID NO: 16);

[0094] Acetyl-LRKLRKRLLLRKLRKRLL-amide (SEQ ID NO: 17);

[0095] Acetyl-LRVRLASHLRKLRKRLLRDADDLQKRLAVY-amide (SEQ ID NO: 18); and

[0096] Picolinyl-llrk-aib-lkrl-aib-salrvrl-amine (SEQ ID NO: 19), wherein all amino acid residues are D-amino acids.

[0097] As used herein, the term "COG1410" is used interchangeably with the term "SEQ ID NO: 1".

[0098] In the above-mentioned peptide compounds of the present invention, L, R, K, S, H, A, C, V, D, Q and Y are single-letter abbreviations of amino acids, representing leucine, arginine, lysine, serine, histidine, alanine, cysteine, valine, aspartic acid, glutamine and tyrosine, respectively. It is understood by those skilled in the art that in the peptide chain structure, the above-mentioned amino acids exist in the form of amino acid residues. In the present invention, unless otherwise specified, the amino acids included in the peptide chain structure refer to amino acid residues.

[0099] Unless otherwise specified, two adjacent amino acids are linked by an amide bond (-CO-NH-), also known as a peptide bond. For example, -AS- indicates that an alanine residue is linked to a serine residue, and the two are linked by an amide bond, in which case, since A and S are amino acids, the amide bond is also known as a peptide bond.

[0100] In the above-mentioned peptide compounds, unless otherwise specified that the amino acid residue is a D-amino acid, L, R, K, S, H, A, C, V, D, Q and Y may represent an L-amino acid.

[0101] In the peptide compounds of the present invention, "Aib" is aminoisobutyric acid.

[0102] In the peptide compound SEQ ID NO: 3, there is a disulfide bond (ie, -SS- or a disulfide group or a disulfide bridge) between the side chains of the two cysteine ​​residues.

[0103] In the peptide compounds SEQ ID NOs: 1, 4-12 and 16-18, the amino terminal amino acid is bonded to an acetyl group (i.e., acetyl-CONH-). For the peptide compounds beginning with acetyl-AS, i.e., SEQ ID NOs: 1 and 6, an exemplary schematic diagram of the amino terminal structure is shown below:

[0104]

[0105] Structure (I) shows acetyl-alanine-serine, which has an acetyl group at the amino terminus.

[0106] For peptide compounds beginning with acetyl-LLRK, i.e., SEQ ID NOs: 4, 5, 7, and 11, the non-acetylated amino terminus is shown in the following structure (II):

[0107]

[0108] The acetyl-LLRK structures of SEQ ID NOs: 4, 5, 7 and 11 are shown in the following structure (III):

[0109]

[0110] 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 the following structure (IV):

[0111]

[0112] The acetyl-LRVR structures of SEQ ID NOs: 8-10, 12, 16 and 18 are shown in the following structure (V):

[0113]

[0114] For the peptide compound beginning with acetyl-LRK, i.e., SEQ ID NO: 17, the non-acetylated version is shown in the following structure (VI):

[0115]

[0116] The structure of acetyl-LRK of SEQ ID NO: 17 is shown in the following structure (VII):

[0117]

[0118] In the peptide compounds SEQ ID NO: 2-3 and 19, the amino terminal amino acid is bonded to a picolinyl group (i.e., picolinyl-CONH-). The amino terminal of SEQ ID NO: 2-3 begins with alanine-serine, and the amino terminal of SEQ ID NO: 19 begins with leucine-leucine. The structural schematic diagram shown below is for alanine-serine, but the same type of bond as the picolinyl group is present in SEQ ID NO: 19. The free amino group of SEQ ID NO: 2-3 (i.e., without a picolinyl group) is shown in the following structure (VIII):

[0119]

[0120] The amino terminus modified with picolinyl (i.e., picolinyl-AS of SEQ ID NO: 2-3) is shown in the following structure (IX):

[0121]

[0122] In the peptide compound SEQ ID NO: 13, the amino terminal amino acid is bonded to "C8", where C8 can be understood as an octanyl group of eight straight-chain carbon atoms. The free amino terminal of SEQ ID NO: 13 is shown in the following structure (X):

[0123]

[0124] The addition of a C8 group to the amino terminus of SEQ ID NO: 13 is shown in the following structure (XI):

[0125]

[0126] In the peptide compound SEQ ID NO: 14, the epsilon amino group of the amino terminal amino acid (lysine) is bonded to eight straight-chain carbon atom groups (i.e., C8 groups), and the alpha amino group of the amino terminal amino acid (lysine) is bonded to an acetyl group, as shown in the following structure (XII):

[0127]

[0128] In the peptide compound SEQ ID NO: 15, the epsilon amino group of the amino terminal amino acid (lysine) is bonded to the picolinyl group, and the alpha amino group of the amino terminal amino acid (lysine) is bonded to the acetyl group, as shown in the following structure (XIII):

[0129]

[0130] Preparation method of the peptide compound of the present invention

[0131] The following discussion provides principles for obtaining the peptide compounds of the present invention and gives details of some available methods for preparing 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 steps and techniques disclosed in the Examples section below and known organic synthesis techniques. As known to those skilled in the art, the peptide compounds of the present invention can be synthesized using conventional solid phase or liquid phase peptide synthesis methods and identified by HPLC and mass spectrometry.

[0132] Methods known to those of ordinary skill in the art can be confirmed by various reference books and published data. A detailed description of the reactants available in the synthesis of the compounds of the present invention is available or provided in the references described in the literature and appropriate reference books, including, for example, "Practical Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York 2011; and SR Sandler et al., "Organic Functional Group Preparations", Second Edition, Academic Press, New York, 1983; HO House, "Modern Synthetic Reactions", Second Edition, WA Benjamin, Inc. Menlo Park, Calif. 1972; TL Gilchrist, "Heterocyclic Chemistry", Second Edition, John Wiley & Sons, New York, 1992; J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structures", Fourth Edition, Wiley.-Interscience, New York, 1992. Other suitable reference books and monographs that provide detailed descriptions of the synthesis of reactants that can be used to prepare the compounds of the present invention or provide references to literature describing preparation methods include, for example, Fuhrhop, J. and Penzlin G. "Organic Synthesis: Concepts, Methods, Starting Materials"; Second Edition, Revised and Expanded Edition (1994) John Wiley & Sons ISBN: 3-527-29074-5; Hoffman, RV "Organic Chemistry, An Intermediate Text" (1996) Oxford University Press, ISBN 0-19-509618-5; Larock, RC "Comprehensive Organic Transformations: A Guide to Functional Group Preparations" Second Edition (1999) Wiley-VCH, ISBN: 0-471-19031-4; Otera, J.(Editor) “Modern Carbonyl Chemistry” (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 Organophosphorus 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-, Volume 8 “Organic Reaction” (1942 - 2000) John Wiley & Sons, over 55 volumes in total; and “Chemistry of Functional Groups” John Wiley & Sons, 73 volumes in total.

[0133] In general, the compounds used in the reactions described herein can be prepared from commercially available chemical reagents and / or compounds described in the chemical literature according to organic synthesis techniques known to those skilled in the art. "Commercial chemicals" can be obtained from standard commercial sources, including Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee WI, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park, UK), Avocado Research (Lancashire, UK), BDH Inc. (Toronto, Canada), Bionet (Cornwall, UK), Chemservice Inc. (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. Ltd. (Cornwall, UK), Parish Chemical Co. (Orem UT), Pfaltz & Bauer, Inc. (Waterbury CN), Polyorganix (Houston TX), Pierce Chemical Co. (Rockford IL), Riedel de Haen AG (Hanover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland OR), Trans World Chemicals, Inc. (Rockville MD), and Wako Chemicals USA, Inc. (Richmond VA).

[0134] Specific reactants and similar reactants can be identified by indexing known chemical reagents prepared by the Chemical Abstracts Service of the American Chemical Society or by online databases available from most public or university libraries. Chemical reagents known in the catalog but not commercially available can be prepared by custom chemical reagent synthesizers, many of which are standard chemical reagent suppliers (such as the above-mentioned suppliers) that provide custom synthesis services.

[0135] For example, the manner in which the cyclic peptide compound SEQ ID NO: 3 of the present invention forms -SS- between amino acids is known to those skilled in the art, and can be found in, for example, 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, a method for preparing the peptide compound is described.

[0136] In some aspects of the invention, an ApoE-based synthetic peptide acetyl-AS-aib-LRKL-aib-KRLL-amide (SEQ ID NO: 1) named "COG1410" is provided, which is used as an antimicrobial agent. In one embodiment, the peptide COG1410 is provided for a method of inhibiting Gram-negative bacteria, including Enterobacter cloacae, Escherichia coli, Citrobacter freundii, and even the anaerobic Porphyromonas gingivalis, with an MIC range of 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 a method of inhibiting the fully resistant bacterium Acinetobacter baumannii YQ4. It is noteworthy and unexpected that the experiments described in Examples 1-3 show that COG1410 is able to kill Acinetobacter baumannii YQ4 with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 16 μg / ml (11.3 μM). In addition, COG1410 was found to inhibit 107 other clinically collected Acinetobacter baumannii strains with a MIC range of 16 to 32 μg / ml.

[0137] In other aspects of the present invention, there is provided a synthetic peptide compound of the present invention for use as an antimicrobial agent. The MICs of the peptide compound against Escherichia coli, Lactobacillus salivarius and Porphyromonas gingivalis are shown in Table 1. Example 3 describes the determination of the MIC values ​​in Table 1.

[0138] Table 1.

[0139]

[0140] "Aib" in Table 1 is aminoisobutyric acid

[0141] COG1410 contains amino acid residues 138-149 located in the N-terminal domain of ApoE, with aminoisobutyric acid (Aib) substitutions at positions 140 and 145. COG1410 is a modification of a peptide based on ApoE residues 133-149 to extend the therapeutic window after TBI treatment and has demonstrated neuroprotective activity in multiple 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 damage. In addition, COG1410 has been shown to have the ability to target the blood-brain barrier (BBB). COG1410 has been fused to the Aβ binding region to form a multi-strategy peptide that improves BBB targeting efficiency and improves neural damage in a mouse Alzheimer's disease model. Therefore, COG1410 has been considered a promising therapeutic agent for diseases related to neural injury.

[0142] In some embodiments of the present invention, COG1410 is provided for use as an antimicrobial agent. The experimental data provided herein in Examples 1-3 and Figures 1-8 illustrate the antibacterial efficacy of COG1410. Specifically, COG1410 exhibits broad-spectrum antibacterial and potent bactericidal activity, especially against fully resistant strains of Acinetobacter baumannii. COG1410 works very quickly in vitro. For example, 1x MIC COG1410 reduced the CFU of Acinetobacter baumannii by 3 logs (1000 times) in 5 minutes, which is much faster than polymyxin B (PMB) (see Figure 1A ), and was comparable to the promising antimicrobial peptide ZY4 against Acinetobacter baumannii, a cathepsin-derived peptide that killed Acinetobacter baumannii within 30 min at 1x MIC

[24] . Figure 2A and 2B COG1410 was shown to inhibit the formation of biofilms in Acinetobacter baumannii and remove mature biofilms. Acinetobacter baumannii is one of the main bacteria that produce biofilms, and due to the formation of its biofilms, it is easy to survive and spread in hospital environments. Therefore, in one embodiment, COG1410 is provided, which is used as an antimicrobial agent for Acinetobacter baumannii.

[0143] A major challenge for therapeutic applications of antimicrobial peptides is degradation or inactivation in plasma. The bactericidal efficacy of COG1410 was evaluated in plasma. The LC values ​​of COG1410 in PBS and 50% pooled plasma were 99.9 1.4 μM and 5.6 μM respectively (see Figure 1BThis is superior to the LL-37-derived AMP, SAAP-148, which targets the corresponding LC against Acinetobacter baumannii. 99.9 1.6 μM and 12.8 μM, respectively. In addition, stability studies have shown that COG1410 can be very stable in plasma. Specifically, COG1410 did not significantly degrade in 100% human plasma within 2 hours. Even after 10 hours, more than 80% of the activity was retained. Consistent with these results, COG1410 has been administered by intravenous injection in a murine model of traumatic brain injury (TBI). In this study, a single intravenous injection of COG1410 significantly improved vestibular motor function and spatial learning and memory. Similar effects were observed in a rat model of focal cerebral ischemia and a mouse model of traumatic optic nerve injury. Therefore, in one embodiment, COG1410 is provided for administration to a subject as an antimicrobial agent to respond to systemic infection.

[0144] Most cationic AMPs (such as COG1410) directly target the cell membrane, leading to pore formation and eventual cell lysis. As expected, the experiments described in Example 2 showed that treatment with polymyxin B resulted in collapse of the cell wall and release of cytoplasmic contents (see e.g. Figures 3A-3D and Figure 4). However, unexpectedly, cells treated with COG1410 remained intact without the formation of pores. Cell death may be due to separation between the inner membrane and the cell wall. Significant ATP leakage of cells exposed to COG1410 was observed in the experiments described in Example 2, indicating that the cell membrane was damaged or permeabilized. Consistently, fluorescent dye staining assays also confirmed this conclusion. On the other hand, it was observed that COG1410 can pass through the cell membrane and enter the cytoplasm of Acinetobacter baumannii. RNA-seq analysis revealed that sub-MIC levels of COG1410 significantly affected the genes of Acinetobacter baumannii in minimal culture medium, with genes in oxidative and reductive biological processes significantly enriched (see Figure 5). The DCFH-DA probe also detected 30% more ROS after 20 minutes of COG1410 treatment. These data indicate that COG1410 can induce oxidative stress in Acinetobacter baumannii. The experiments described in Example 2 show that COG1410 can bind to DNA non-specifically. One theory that has been proposed in the literature is that membrane disruption and DNA binding are the two-hit-hit of the AMP, NK18, against bacteria. However, COG1410 is able to enter the cytoplasm of Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecium, but these bacteria are not susceptible to antimicrobial activity. Therefore, and without wishing to be limited to any specific mechanism of action, DNA binding may not play a key role in the mechanism of antimicrobial activity of COG1410. Again, without being limited to any one mechanism of action, the data combined suggest that COG1410 may inhibit bacterial growth through mechanisms that include one or both of disruption of cell membrane integrity and induction of oxidative stress.

[0145] As a class of drugs, cationic AMPs can be particularly troublesome in terms of cytotoxicity. In the experiments described in Example 2, it was observed that 128 μg / ml (8x MIC in LB) of COG1410 caused less than 5% hemolysis ( Fig. 6A ). As described in Example 2, the cytotoxic effect of COG1410 on normal human liver L02 cells was evaluated by CCK8 assay. 50 58.9 μg / ml ( Figure 6B ). In addition, the LC in PBS and 50% plasma 99.9 These results suggest that COG1410 has an improved cytotoxic profile relative to many existing antimicrobial peptides.

[0146] Another advantage of using COG1410 as an antimicrobial agent is that a significant synergistic interaction was observed with the combination of COG1410 and polymyxin B. Specifically, the working concentration of COG1410 was reduced to 2 μg / ml and that of polymyxin B was reduced to 1 μg / ml ( Figures 7A-7C ). In addition, it is noteworthy that the bactericidal effect of COG1410 does not depend on electrostatic interactions with LPS, as modifications of LPS do not inhibit the activity of COG1410. This feature is different from what has been previously observed for polymyxin B and LL-37, in which the initial step in their attack on bacterial pathogens is binding to LPS. It is generally believed that polymyxin B binds to the lipid A portion of LPS in Gram-negative bacteria, replacing the Ca 2+ and Mg 2+ This can destabilize the LPS layer and membrane.

[0147] In another experiment, Figure 8 The graph in illustrates that the combination therapy of COG1410 and polymyxin B rescues infected nematodes. C. elegans was 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 nematodes were counted daily for 2 weeks. Figure 8 The data in showed the unexpected result that the combination therapy of COG1410 (2 μg / ml) plus polymyxin B (1 μg / ml) significantly rescued the pre-infected nematodes and even extended their lifespan.

[0148] Combination drug therapy is frequently used in medical practice to treat microbial infections, particularly in intensive care units. Therefore, the synergistic activity between polymyxin B and COG1410 is another advantage afforded to the use of COG1410 in combination with polymyxin B for the treatment of microbial infections.

[0149] The experimental results provided in Example 2 show the potent antibacterial ability of the ApoE mimetic peptide COG1410. The main bactericidal mechanism of COG1410 is to destroy cell membrane integrity and induce oxidative stress. COG1410 exhibits strong bacterial killing, high stability in human plasma and a low tendency to develop drug resistance. The synergistic interaction between COG1410 and polymyxin B reduces the working concentration of COG1410 and avoids the risk of its eukaryotic cell toxicity. Considering that it has neuroprotective, anti-inflammatory and antibacterial effects at the same time, in one aspect of the present invention, COG1410 is provided for use in a method for treating a subject with a microbial infection. The method comprises administering to the subject a therapeutically effective amount of a composition comprising 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 fully resistant Acinetobacter baumannii, an infection that has been considered a healthcare crisis.

[0150] In one aspect of the present invention, a method for inhibiting the growth of a microorganism is provided, comprising: contacting the microorganism with an effective amount of a peptide compound of the present invention to inhibit the growth of the microorganism, wherein the peptide compound of the present invention is selected from SEQ ID NO: 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.

[0151] In the method, the microorganism may include one or more bacteria of multi-drug resistance (MDR), pan-drug resistance (XDR) or full drug resistance (PDR). In some embodiments, the microorganism includes Porphyromonas gingivalis. The microorganism may include a Gram-negative bacterial pathogen or a Gram-positive pathogen. The Gram-negative bacterial pathogen may include one or a combination of Acinetobacter baumannii, Porphyromonas gingivalis or Escherichia coli; the Gram-positive pathogen may include one or a combination of Staphylococcus aureus or Lactobacillus salivarius.

[0152] In the 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.

[0153] In some embodiments, the peptide compound can be contacted in combination with polymyxin B to inhibit microorganisms including pan-resistant Acinetobacter baumannii or multi-resistant Staphylococcus aureus.

[0154] In some embodiments, the peptide compound can be contacted in combination with polymyxin B to inhibit microorganisms comprising Acinetobacter baumannii, 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. In other cases, the peptide compound can be contacted in combination with polymyxin B to inhibit microorganisms comprising Acinetobacter baumannii, wherein the peptide compound comprises: SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In one case, the peptide compound can be contacted in combination with polymyxin B to inhibit microorganisms comprising Acinetobacter baumannii, wherein the peptide compound comprises SEQ ID NO: 1.

[0155] The peptide compounds SEQ ID NO: 1-19 of the present invention have similarities with the receptor binding region of the holo-ApoE protein. Therefore, it can be expected that all the peptide compounds of the present invention exhibit similar antimicrobial activities.

[0156] In addition, peptide compounds SEQ ID NO:1 and 2 have similar structures and differ only at the amino terminus. Specifically, SEQ ID NO:1 has an acetyl group at the amino terminus, and SEQ ID NO:2 has a picolinyl group at the amino terminus. As described above and as shown in Table 1, SEQ ID NO:1 has excellent antimicrobial activity against Acinetobacter baumannii, and is also effective against Porphyromonas gingivalis. SEQ ID NO:2 has been shown to be more effective than SEQ ID NO:1 against Porphyromonas gingivalis, and also has antimicrobial activity against Escherichia coli and Lactobacillus salivarius (see Table 1). Therefore, it can be expected that the antimicrobial activity of SEQ ID NO:2 against pathogens such as Acinetobacter baumannii is 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 by a cysteine ​​that forms a disulfide bond. Like SEQ ID NO:2, SEQ ID NO:3 exhibits high antimicrobial activity against Porphyromonas gingivalis (see Table 1). Therefore, it can be expected that the antimicrobial activity of SEQ ID NO: 3 against pathogens such as Acinetobacter baumannii is also similar to that of SEQ ID NO: 1.

[0157] The peptide compounds SEQ ID NOs: 13, 14 and 15 have structures similar to those of SEQ ID NOs: 1 and 2, and differ only at the amino terminus. 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 above reasons, it can be expected that SEQ ID NOs: 13, 14 and 15 have antimicrobial activity similar to that of SEQ ID NOs: 1-3.

[0158] The present invention includes a method for treating a subject suffering from 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.

[0159] In various embodiments, the microbial growth may be caused by one or more bacteria that may be multi-drug resistant (MDR), extensively drug resistant (XDR), or pan-drug resistant (PDR).

[0160] In other embodiments, the microbial growth may result from an oral infection with bacteria including Porphyromonas gingivalis.

[0161] The administration of the pharmaceutical composition comprising the peptide compound to the subject may include a single administration or multiple administrations of the pharmaceutical composition. The pharmaceutical composition comprising the peptide compound may be administered topically, enterally, systemically or parenterally. In some cases, the pharmaceutical composition comprising the peptide compound may be administered in a formulation of a mouthwash or toothpaste. In some cases where the microbial growth is caused by an oral infection of bacteria comprising Porphyromonas gingivalis, the pharmaceutical composition comprising the peptide compound may be administered in a formulation of a mouthwash or toothpaste.

[0162] In some embodiments of the purposes or methods described herein, the dosage of the peptide compounds of the present invention generally depends on various factors, including the severity of the individual or microbial infection being treated, the rate of administration, and the judgment of the prescribing physician. In general, the effective daily dose per kg body weight can range from about 0.01 to about 1.0 mg, for example, from about 0.01 to about 1.0, 0.01 to about 0.1, 0.01 to about 0.09, 0.01 to about 0.08, 0.01 to about 0.07, 0.01 to about 0.06, 0.01 to about 0.05 or 0.01 to about 0.04 mg / kg / day. In one embodiment, the dosage is 0.051 mg / kg / day on the first day and 0.017 mg / kg / day on the following days. The exact dosage can change on each day of administration to achieve the desired therapeutic efficacy.

[0163] In the treatment method, the inhibited bacteria can be gram-negative or gram-positive pathogens. In the case of gram-negative bacteria, the bacteria can include but are not limited to one or a combination of Acinetobacter baumannii, Porphyromonas gingivalis and Escherichia coli. In the case of gram-positive bacteria, the bacteria can include but are not limited to one or a combination of Staphylococcus aureus and Lactobacillus salivarius.

[0164] In other aspects of the invention, pharmaceutical compositions comprising the peptide compounds of the present disclosure are administered in combination with one or more other antimicrobial compounds. For example, the one or more antimicrobial compounds may include polymyxin B.

[0165] In one instance, 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 pan-resistant bacterium Acinetobacter baumannii.

[0166] In other cases, 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.

[0167] In another instance, 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.

[0168] In some embodiments, a pharmaceutical composition comprising a peptide compound is administered in combination with one or more antimicrobial compounds to inhibit Acinetobacter baumannii or multidrug-resistant Staphylococcus aureus. The pharmaceutical composition of the present invention comprises an antimicrobial 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 antimicrobial composition comprises SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In other cases, the peptide compound in the antimicrobial composition comprises SEQ ID NO: 1.

[0169] 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.

[0170] The peptide compounds of the present disclosure, including pharmaceutically acceptable salts or solvates thereof, are used in a method of inhibiting the growth of a microorganism in a subject.

[0171] Provided are pharmaceutical compositions comprising a peptide compound of the present disclosure, or a pharmaceutically acceptable salt or solvate of a peptide compound of the present disclosure.

[0172] In one embodiment, the pharmaceutical composition includes a 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 includes a peptide compound: SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In one case, the peptide compound included in the pharmaceutical composition comprises SEQ ID NO: 1.

[0173] The pharmaceutical composition comprising the peptide compound of the present disclosure may include one or more other antimicrobial compounds. For example, the antimicrobial compound may include polymyxin B.

[0174] In one embodiment, a pharmaceutical composition of the present disclosure is provided for use in a method of inhibiting the growth of a microorganism in a subject.

[0175] The administration of the pharmaceutical composition of the present invention to a subject may include a single administration or multiple administrations of the pharmaceutical composition. The pharmaceutical composition comprising the peptide compound may be administered topically, enterally, systemically or parenterally. In some cases, the pharmaceutical composition may be administered in a formulation of a mouthwash or toothpaste. In some cases where the microbial growth is caused by an oral infection of a bacterium comprising Porphyromonas gingivalis, the pharmaceutical composition comprising the peptide compound may be administered in a formulation of a mouthwash or toothpaste.

[0176] The pharmaceutical composition may include one or more pharmaceutically acceptable excipients. One or more excipients may include but are not limited to disintegrants, diluents, binders, solvents, co-solvents, lubricants, pH regulators, buffers, preservatives, dispersants, suspending agents, ointment bases, emulsifiers, softeners, permeants, surfactants, propellants, flavoring agents, sweeteners or drug release regulators. The pharmaceutical composition may include a solvent, such as one or both of physiological saline and glucose solution.

[0177] The pharmaceutical composition of the present invention may be formulated into a lyophilized powder or a liquid suitable for administration by injection to cause a slow release of the peptide compound, or formulated into a spray. In other embodiments, the pharmaceutical composition of the present invention may be formulated into a mouthwash or toothpaste.

[0178] The present invention will be described in more detail below by way of examples, but these examples are not intended to limit the present disclosure. Example

[0179] Example 1. Peptide synthesis, purification and identification

[0180] COG1410 was synthesized using conventional solid phase peptide synthesis methods with a purity of 95% and qualified by HPLC and mass spectrometry at Polypeptide Labs (San Diego, CA). COG1410 is acetyl-AS-Aib-LRKLAib-KRLL-amide (SEQ ID NO: 1), which is derived from apoE residues 138-149 with Aib (aminoisobutyric acid) substitutions at positions 140 and 145. For all experiments, the peptide was used immediately after dissolution in sterile saline.

[0181] Peptide compounds SEQ ID NO: 2-19 were synthesized similarly to the above-mentioned peptide compound SEQ ID NO: 1. Specifically, the peptide compounds were synthesized using conventional solid phase peptide synthesis methods with a purity of 95%, and were qualified by HPLC and mass spectrometry at Polypeptide Labs (San Diego, CA).

[0182] More specifically, the solid phase synthesis of the peptide compounds SEQ ID NO: 1-19 is performed as follows using condensation reactions well known to those of ordinary skill in the art. Solid phase peptide resins are added one by one, and the condensation reaction results in the correct peptide sequence. The amino acid condensation uses a polypeptide solid phase synthesis (SPPS) process, the condensation starts from the N-terminus, and the amino acid side chains are protected, and their amino groups are protected by Fmoc. The general SPPS process is a repeated cycle of alternating N-terminal deprotection and condensation reactions, and the resin needs to be cleaned between each step. After all amino acid condensations are completed, picolinic acid is condensed into the sequence to obtain a full-length polypeptide bound to a supporting resin. Each condensation reaction is monitored by an applicable in-process control test (ninhydrin test, TNBS test and / or analytical HPLC test).

[0183] The salient feature of the addition of an acetyl, octanoyl or picolinyl group is that each involves the addition of a carboxyl group to a free amino group to form an amide bond between them. When such an amide is used to link two amino acids together, it is often referred to as a peptide bond. Thus, the amide bond is the result of a dehydration condensation reaction that covalently links the carboxyl group to the amino group with the elimination of a water molecule.

[0184] The peptides were cleaved from the support resin using trifluoroacetic acid (TFA), triisopropylsilane (TIS), 3,6-dioxin-1,8-octanedithiol (DODT) and water under temperature controlled conditions. After cleavage, the mixture was filtered, the resin was washed, and the collected filtrate was centrifuged. The collected peptides in the filtrate were precipitated with methyl tert-butyl ether (MTBE) and filtered. The resulting crude peptide solution was dried and packaged.

[0185] The crude peptide was subjected to initial purification and cyclic purification using a trifluoroacetic acid, acetonitrile and water buffer system. Based on the purity results obtained by the analytical HPLC method, the co-eluted polypeptide fractions obtained from the purification were collected, analyzed and pooled. The pool was then subjected to further chromatography to change the counterion salt.

[0186] Use preparative HPLC to carry out final salt exchange (FSE) and elution, and the purified peptide is converted into acetate using the buffer system of ammonium acetate, acetonitrile (ACN) and water.Then use acetic acid, ACN and aqueous buffer to carry out large gradient (steep gradient) washing to elute peptide.The analytical method of HPLC is used to determine the purity and collection of salt exchange components.The sub-batch (sub-batch) collected that will meet the target harvest purity standard will be freeze-dried.With tray freeze dryer, the peptide freeze-dried through salt exchange and purification, to remove moisture and residual organic solvent, obtain final peptide bulk drug substance.

[0187] Example 2. COG1410 has antibacterial activity against PDR-Acinetobacter baumannii.

[0188] Materials and methods

[0189] Strains and cultures

[0190] A panel of Gram-positive and Gram-negative strains including Bacillus subtilis, vancomycin-resistant Enterococcus faecalis, Enterococcus faecium, Mycobacterium tuberculosis, M. smegmatis, Enterobacter cloacae, Escherichia coli, Citrobacter freundii, Porphyromonas gingivalis, Streptococcus pneumoniae, methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, Pseudomonas aeruginosa, and pan-drug-resistant (PDR) Acinetobacter baumannii YQ4 were evaluated in this study. Strain stocks were stored in 10% glycerol at -80°C. Bacteria were streaked onto fresh plates before each experiment. Most of them were cultured in LB liquid medium containing 10 g / L NaCl at 37°C, except that Mycobacteria were cultured in 7H9 liquid medium and Enterococcus were cultured in BHI liquid medium. Porphyromonas gingivalis was cultured in BHI liquid medium (supplemented with 1 μg / ml vitamin k1, 5 μg / ml hemin and 5 mg / ml L-cysteine ​​hydrochloride) in an anaerobic chamber at 37°C. The minimum inhibitory concentration (MIC) of the antimicrobial agent was determined using the microdilution method in the corresponding liquid medium.

[0191] The pan-resistant A. baumannii YQ4 was collected from a clinical laboratory, and the complete genome sequence was deposited in GenBank with the accession number CP053033.

[0192] Antibacterial activity of COG1410

[0193] For the dynamic time-kill assay, an overnight culture of Acinetobacter baumannii YQ4 was transferred to an initial OD 600 0.01 in fresh liquid medium and cultured to the logarithmic phase. Collect 1 ml of the culture, wash twice with 1x phosphate buffered saline (PBS, pH 7.4) (containing 8 mM NaH2PO4, 2 mM KH2PO4, 2.6 mM KCl, 136 mM NaCl), and finally suspend in 1 ml PBS. OD 600 is 0.5, which is approximately equivalent to 1×10 8CFU / ml. The suspension was supplemented with 16 μg / ml (1x MIC) COG1410 or 80 μg / ml (5x MIC) COG1410, respectively, and incubated at 37°C without shaking. 100 μl aliquots were taken at 0, 5, 10 and 30 minutes, serially diluted in PBS and plated on LB agar. CFU were counted after incubation for 18 hours at 37°C. Polymyxin B at 16 μg / ml (1x MIC) or 80 μg / ml (5x MIC) was used as a positive control. Three independent experiments were performed.

[0194] 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 dissolved in PBS or in PBS supplemented with 50% (v / v) pooled human plasma. After incubation at 37°C with shaking at 200 rpm for 2 h, CFU were counted on LB agar. LC 99.9 The lowest peptide concentration that killed ≥99.9% of the bacteria (i.e., a 1000-fold reduction) was indicated. The experiment was performed three times independently.

[0195] Biofilm inhibition and clearance assays

[0196] Static biofilm inhibition was performed as previously described

[23] . Briefly, logarithmic phase cultures of Acinetobacter baumannii YQ4 were prepared as described above. 200 μl of culture was plated at OD 600 0.01 (about 1×10 6 CFU / ml) were inoculated in each well (cell) of a 96-well PVC plate and exposed to different COG1410 solutions with a final concentration range of 0.5 to 128 μg / ml. The concentration of each was determined in 8 wells. After incubation at 37°C for 48 hours, planktonic bacteria were removed by washing 3 times with sterile water, then fixed in methanol for 15 minutes and stained 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 microplate reader. The experiment was performed in triplicate.

[0197] Biofilm clearance assays were performed as described previously

[24] . A logarithmic phase culture of A. baumannii YQ4 was diluted into fresh LB broth to an OD of 600The concentration of COG1410 was 0.01 and 200 μl was dispensed into a 96-well PVC plate per well. The plates were incubated at 37°C for 48 hours and washed 3 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. The concentration of each was determined in 8 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. The experiment was performed in triplicate.

[0198] Stability of COG1410 in human plasma

[0199] To determine stability in human plasma, COG1410 was dissolved in 1 ml of 100% human plasma to a final concentration of 10 mg / mL and incubated at 37°C without shaking. 100 μl aliquots were taken at 0, 1, 2, 4, 6, 8, and 10 hours. Logarithmic phase cultures of Acinetobacter baumannii YQ4 were prepared as described above. 200 μl of culture was mixed with 6 mL of 0.8% soft agar to make two-layer plates. 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 on the paper disks. After incubation at 37°C for 18 hours, the inhibition zone was recorded with a digital camera and the inhibition diameter was measured by image J. Three independent experiments were performed.

[0200] Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)

[0201] Logarithmic phase cultures of Acinetobacter baumannii YQ4 were prepared as described above, collected 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 for observing intact cells. The pellets were then fixed with 0.1 M sodium dimethyl caesium containing 4% paraformaldehyde and 2.5% glutaraldehyde and stored overnight at 4°C. After washing three times with PBS, the cells were post-fixed in 1% osmium at 4°C for 1 hour. After a thorough wash in distilled water for 1 hour, the bacteria were dehydrated in increasing concentrations of ethanol. Finally, the samples were transferred to a Thermo fisher Quattro SEM for image acquisition after freeze drying and sputter coating in tert-butyl alcohol.

[0202] Bacteria were treated with COG1410 or polymyxin B as described above. Samples were then fixed in 0.2 M sodium cacodylate buffer (pH 7.2) containing 4% paraformaldehyde and 2.5% glutaraldehyde, postfixed in 1% buffered osmium tetroxide, dehydrated in graded ethanol, embedded in epoxy resin, and polymerized at 60°C for 2 days. Ultrathin sections (50-70 nm) of the samples were made and placed on copper grids, stained with uranyl acetate and lead citrate, and transferred to a Thermo fisher Talos L120CTEM for image acquisition.

[0203] Confocal laser scanning microscopy

[0204] Cytoplasmic membrane damage was determined using the Live / Dead BacLight Bacterial Viability Kit (Invitrogen L7012) as described previously

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

[0205] To explore the localization of COG1410 in bacteria, logarithmic phase cultures of Acinetobacter baumannii YQ4, Klebsiella pneumoniae ATCC2146, Staphylococcus aureus ATCC29213, and Enterococcus faecium 11-47 were prepared, washed, and suspended in PBS, with a final OD 600 The culture was exposed to FITC-labeled COG1410 (8 μg / ml) for 30 minutes at room temperature and then co-stained with 1.6 μM membrane dye FM4-64. For each strain, 5 μl of culture was spotted on a clean slide coated with a thin layer of 1% agarose. Fluorescence was observed using an Olympus FV3000 confocal laser scanning microscope. For FITC, the excitation and emission lights were 488 nm and 525 nm, respectively. For FM4-64, the excitation and emission lights were 558 nm and 734 nm, respectively.

[0206] ATP leak assay

[0207] According to the manufacturer's manual, ATP leakage assay was performed using Beyotime's enhanced ATP assay kit (S0027). In brief, logarithmic cultures of Acinetobacter baumannii YQ4 were prepared as described above, washed and suspended in PBS. The suspension was 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. Supernatants were collected and used for the measurement of ATP levels. 100 μl of supernatant was mixed with 100 μl of working solution and chemiluminescence was measured by Biotek Synergy H1 microplate reader. Untreated samples were set as negative controls. The experiment was performed in triplicate.

[0208] DNA binding assay

[0209] Gel retardation experiments were performed as previously described

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

[0210] RNA-seq analysis

[0211] To determine the effect of COG1410 on gene transcription, Acinetobacter baumannii YQ4 was cultured in 50 ml M9 medium supplemented with 20% glucose as the sole carbon source and treated with or without 0.25x MIC of COG1410 (4 μg / ml). The experiment was performed in triplicate. When OD600 reached 0.8, the cells were collected by a 4°C centrifuge and frozen in liquid nitrogen. The samples were shipped with 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 according to the manufacturer's instructions, and rRNA was removed using the Ribo-Zero rRNA removal kit. NEB Ultra TMThe Directional RNA library preparation kit generated complete libraries for Illumina sequencing. After cluster generation, the libraries were sequenced on the Illumina Novaseq6000 platform and paired-end reads of 150 bp were generated. The raw data were filtered by fastp and rRNA sequences were removed

[27] . The edgeR program was used to identify differentially expressed genes. Genes with an FDR of 0.05 and log2 (fold change) > 1 were selected as candidate genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes was achieved using cluster Profiler. The raw data have been deposited in the SRA database with the accession number PRJNA833738.

[0212] ROS detection

[0213] Intracellular ROS levels were determined by the Reactive Oxygen Species Assay Kit (S0033S) from Beyotime Biotechnology according to the manufacturer’s instructions. Logarithmic phase cultures of Acinetobacter baumannii YQ4 were collected and washed in PBS and then diluted 10-fold to approximately 10 7 CFU / mL. 1μl DCFH-DA (10μM) was added to 1ml cell culture and incubated at 37°C for 20 minutes. The fluorescent probe was then completely removed by washing 3 times with PBS and resuspended in PBS, followed by the addition of COG1410 (16μg / ml) or water. Rosup (50μg / ml) is the positive control in the kit. The culture was incubated at 37°C for 30 minutes. The fluorescence intensity was measured by a microplate reader at 488nm excitation light and 525nm emission light. The experiment was performed in duplicate.

[0214] Evaluation of induction of drug resistance by serial passaging

[0215] To evaluate the resistance barrier of COG1410, Acinetobacter baumannii YQ4 was cultured in LB liquid medium, continuously shaken at 150 rpm at 37°C, and exposed to sub-MIC levels of COG1410 or polymyxin B. 20 μl of culture was transferred to 2 mL of fresh medium every day. The initial concentration of the tested compound was set to 1 / 32x MIC, and the concentration was doubled every 10 passages. 1 ml of bacterial culture was stored in 10% sterile glycerol at -80°C and stored every 5 passages. The MIC values ​​of the collected cultures and the original strains were measured by microdilution method.

[0216] Hemolysis assay and cytotoxicity assay

[0217] The human red blood cell (RBC) hemolytic activity of COG1410 was measured according to a previously described protocol with slight modifications

[28] . Anticoagulated (citrated) whole blood was pelleted by centrifugation at 700 g for 8 min, washed three times with PBS, and suspended in PBS at 0.5% (vol / vol). 75 μl of the RBC suspension was transferred to each well of a V-bottom 96-well plate and diluted 2-fold in PBS to prepare an equal volume of COG1410. 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 plates were incubated at 37°C for 1 h and then centrifuged at 1,000 rpm for 5 min at 4°C. A 60 μl aliquot of the supernatant from each well was quickly transferred to a new flat-bottom 96-well plate. The OD was measured using a microplate reader (BioTek, synergy H1). 414 The absorbance at 40°C was measured. The percentage of hemolysis was then normalized to the mean negative (0%) and positive (100%) controls. Three independent experiments were performed.

[0218] The cytotoxicity of COG1410 to normal liver L02 cells was evaluated by Cell Counting Kit 8 (Solarbio, CA1210) according to the manufacturer's manual. Briefly, 100 μl of human liver L02 cells were seeded in a 96-well plate, with 4 × 10 cells per well. 3 The cells were placed in RPMI-1640 medium containing 20% ​​FBS and incubated at 37°C and 5% CO2 for 24 hours. Cells not exposed to the peptide were used as negative controls. L02 cells were then incubated with different concentrations of COG1410 for another 24 hours. 10 μl of CCK8 solution was added to each well. After 2 hours of incubation, the absorbance at 450 nm was measured using a BioTek synergy H1 microplate reader. Cell activity was expressed as the average absorbance divided by the percentage of cells exposed to the peptide relative to the results of incubation with the control. The experiment was performed in triplicate.

[0219] Synergistic effect with antimicrobial agents

[0220] The checkerboard method was performed to determine the fractional inhibitory concentration (FIC) index between COG1410 and other antimicrobial agents. Two-fold serial dilutions of antimicrobial agents and COG1410 were performed in a 96-well plate and mixed. A logarithmic phase culture of Acinetobacter baumannii YQ4 was added to each well, and the initial OD 600The plate was incubated at 37°C for 20 hours and the FIC value was calculated according to the following equation: FIC = MIC (COG1410 combination) / MIC (COG1410 alone) + MIC (antimicrobial combination) / MIC (antimicrobial alone). The experiment was performed twice independently.

[0221] Nematode killing assay

[0222] This assay used the wild-type strain of C. elegans, Bristol N2. Experimental procedures were performed according to previously described protocols

[29] . C. elegans was grown at 20°C on nematode growth medium (NGM) using Escherichia coli OP50 lawns as food source. For synchronization, C. elegans eggs were collected and hatched in M9 medium at 20°C to the L1 stage and then transferred to E. coli lawns to grow to the L4 stage. For in vivo killing assays, synchronized L4 worms were collected from several NGM plates and transferred to medium containing 20% ​​LB, 1 × 10 9 baumannii YQ4 and 10μM FeCl3 in 15mlM9 medium. The infection model was established at 20°C for 24h. Escherichia coli OP50 was used as a negative control. The pre-infected nematodes were washed twice with M9 medium, and 30 nematodes were distributed to each 60mm NGM agar plate, which was supplemented with different concentrations of COG1410 and / or polymyxin B, and 2mM 5-fluoro-2'-deoxyuridine-fluorouridine (FUDR). Uninfected nematodes were used as a positive control for worm lifespan. Live and dead or lost nematodes were counted every 24 hours and recorded by stereomicroscope for 16 days. The survival curve of Caenorhabditis elegans was analyzed by Kaplan-Meier using GraphPad Prism 9. The in vivo killing assay was performed in triplicate.

[0223] result

[0224] COG1410 has broad-spectrum antimicrobial activity

[0225] COG1410 is a synthetic cationic peptide with a simple alpha helix, which consists of 12 amino acids: acetyl-AS-Aib-LRKL-Aib-KRLL-amide (SEQ ID NO: 1), which includes 4 positively charged amino acids, 5 non-polar amino acids and 1 polar amino acid and two unnatural amino acids Aib. The antimicrobial activity of COG1410 was determined by measuring the minimum inhibitory concentration (MIC) values ​​of a group of Gram-positive and Gram-negative strains. In the case of Gram-positive, COG1410 inhibited the growth of Bacillus subtilis, vancomycin-resistant Enterococcus faecalis and Enterococcus faecium, Mycobacterium tuberculosis and Mycobacterium smegmatis, with a MIC range of 1 to 32 μg / ml. As for Gram-negative bacteria, COG1410 showed antimicrobial activity against Enterobacter cloacae, Escherichia coli, Citrobacter freundii, and even the anaerobic bacterium Porphyromonas gingivalis, with a MIC of 16 to 64 μg / ml. In contrast, COG1410 was inactive against S. pneumoniae, methicillin-sensitive S. aureus, methicillin-resistant S. aureus (MRSA), K. pneumoniae, and P. aeruginosa. Notably, COG1410 killed pan-drug resistant (PDR) Acinetobacter baumannii YQ4 with a MIC and minimum bactericidal concentration (MBC) of 16 μg / ml (11.3 μM). 107 other clinically collected strains of Acinetobacter baumannii were tested, and the MIC of COG1410 ranged from 16 to 32 μg / ml.

[0226] COG1410 shows potent and rapid bactericidal efficacy against PDR Acinetobacter baumannii in vitro

[0227] The in vitro time-kill kinetics of COG1410 against PDR Acinetobacter baumannii YQ4 were analyzed. In 20 μM phosphate-buffered saline (PBS), 1x MIC COG1410 reduced CFU by almost 3 logs (i.e., 1000-fold) within 5 minutes ( Figure 1A When the AMP concentration was increased to 5x MIC, the inoculum (1×10 8 CFU / ml) was completely eliminated within 5 minutes. Within 30 minutes, 1xMIC COG1410 killed bacteria with a 6-log reduction in CFU. In contrast, 1x and 5x MIC polymyxin B reduced CFU by 2 and 4 logs, respectively, after 30 minutes ( Figure 1A ). These data show that COG1410 is faster acting than polymyxin B.

[0228] To determine the efficacy of COG1410 in pooled human plasma, its LC was measured in PBS with or without 50% human plasma. 99.9 Value (kills 99.9% within 2 hours of incubation). Figure 1B As shown, 2 μg / ml (1.4 μM) COG1410 achieved a 99.9% killing rate in PBS. Consistently, in the presence of human plasma, the antimicrobial activity of this AMP was reduced, and LC 99.9 Up to 8μg / mL (5.6μM).

[0229] To investigate the activity of COG1410 under other conditions, the bactericidal efficacy of COG1410 in PBS, LB liquid medium, and saline was determined. Compared to near elimination in PBS, 1x MIC COG1410 killed only 78.6% and 78.3% of A. baumannii in LB liquid medium (250 mM NaCl) and saline (225 mM NaCl), respectively, after 2 hours of incubation. Figure 1C ). These data suggest that high salt may interfere with the bactericidal effect of COG1410.

[0230] COG1410 maintains stability in human plasma

[0231] To determine the stability of COG1410 in human plasma, 10 mg / ml COG1410 was incubated with 100% human plasma at 37°C. Samples were taken at different time points and dropped onto paper disks. The antimicrobial activity was evaluated by measuring the diameter size 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 was reduced by 6.8%. After 10 hours of incubation, 20% of the activity was lost. These data indicate that COG1410 is very stable in human plasma.

[0232] COG1410 exhibits biofilm inhibition and clearance activities

[0233] Bacterial biofilm formation is often associated with chronic wound infection and drug resistance. To investigate the anti-biofilm efficacy of COG1410, the mass of biofilm formed by Acinetobacter baumannii YQ4 was measured by crystal violet staining. COG1410 inhibited biofilm formation in a dose-dependent manner ( Figure 2A ). COG1410 at 0.5x MIC significantly reduced biofilm formation.

[0234] 1x MIC COG1410 reduced biofilm mass by 55% (?46%?) compared to untreated controls.

[0235] For preformed biofilms, 1x MIC of COG1410 dissipated about 46% of mature biofilms. If the AMP concentration was increased to 4x MIC, more than 88% of the biofilms were cleared ( Figure 2B). In conclusion, the data show that COG1410 can be used as an anti-biofilm agent.

[0236] COG1410 treatment increases bacterial cell membrane permeability

[0237] To evaluate the mechanism of action of COG1410, the cell morphology was first examined by scanning electron microscopy (SEM). Figure 3A As shown, untreated A. baumannii cells look like peanuts with small spike-like patterns on their surface. As a control, polymyxin B treatment resulted in cell lysis and release of cell contents. In contrast, most of the COG1410-treated cells were intact, with little cell debris observed. No pores were detected on the cell surface. These data suggest that COG1410 may not cause pore formation or cell lysis.

[0238] To determine the integrity of the bacterial cell membrane, treated cells from the same batch were observed by transmission electron microscopy (TEM). Untreated cells had an intact cytoplasmic membrane. Exposure to polymyxin B gradually led to the collapse of the cell wall of most cells ( Figure 3B However, cells treated with COG1410 became wrinkled and smaller than untreated cells. In addition, the inner membrane appeared to be separated from the cell wall.

[0239] In order to verify the permeabilization effect of COG1410 on the plasma membrane, it was decided to stain the COG1410-treated Acinetobacter baumannii YQ4 cells with two fluorescent nucleic acid dyes, SYTO-9 and propidium iodide (PI). The former can stain both live and dead cells and show green fluorescence (in Figure 4 ), but the latter only enter non-viable cells and emit red fluorescence (in Figure 4 ). As expected, untreated cells showed green fluorescence, indicating that they were all alive. Both COG1410 and polymyxin B treated cells showed red fluorescence, indicating that the cells were dead (data not shown). Of note, these treated dead cells stained with SYTO-9 had no green fluorescence, which may be due to competition with PI. Therefore, this staining assay confirmed that COG1410 permeabilized the bacterial membrane.

[0240] Membrane disruption can be further characterized by measuring the leakage of intracellular components of bacterial cells. To investigate the mechanism of action of COG1410, extracellular ATP concentrations of cells exposed to 1x MIC COG1410 were measured by an enhanced ATP assay kit (Beyotime). Tigecycline, which binds to bacterial 30S ribosomes and blocks the entry of transfer RNA, was used as a negative control. As expected, cells treated with tigecycline showed similar levels of ATP leakage to untreated cells. Cells exposed to polymyxin B released more ATP compared to untreated cells. Notably, COG1410 treatment resulted in ATP leakage, which was significantly higher than that of the untreated control and tigecycline-treated groups ( Figure 3C ). In conclusion, the cationic AMP COG1410 disrupts the bacterial cell membrane.

[0241] COG1410 is located in the cytoplasm

[0242] In addition to direct membrane disruption, some sub-MIC AMPs may have cytoplasmic targets. To investigate whether COG1410 directly binds to bacterial membranes or enters the cytosol, several bacterial pathogens were treated with 8 μg / ml COG1410 for 30 min and then co-stained with a red fluorescent membrane dye. COG1410 entered the cytoplasm of not only Acinetobacter baumannii and Enterococcus faecium, but also Klebsiella pneumoniae and Staphylococcus aureus ( Figure 4 ). COG1410 was active against the first two strains, but did not kill the latter two strains. These results suggest that COG1410 may bind to the cell membrane of Acinetobacter baumannii more easily than other bacteria, or that COG1410 specifically inhibits cytoplasmic targets in Acinetobacter baumannii.

[0243] COG1410 binds to DNA nonspecifically

[0244] In order to investigate whether the cationic AMP binds to DNA, a gel retardation experiment was performed. The electrophoretic mobility of plasmid pUC18 was measured after incubation with different concentrations of COG1410. In the absence of AMP, the plasmid would normally migrate into the gel. In the presence of 1x MIC COG1410, some DNA remained in the loading wells and only part of the DNA migrated into the gel. When the COG1410 concentration was high, the DNA mobility was completely blocked (data not shown). In summary, COG1410 can bind to DNA non-specifically in a concentration-dependent manner.

[0245] Sub-MIC COG1410 treatment induces the expression of genes involved in redox processes

[0246] To further identify putative intercellular targets of COG1410, RNA-seq was used to compare the transcriptome of A. baumannii YQ4 in the presence or absence of 0.25x MIC of COG1410 (4 μg / ml). To reduce interference from rich culture media, M9 basal medium was selected to prepare bacterial cultures. RNA-seq data analysis identified 92 significantly differentially expressed genes (DEGs) that changed by at least 2-fold. Compared with the untreated control, the transcript levels of 55 and 37 genes were increased and decreased in the presence of AMP, respectively. The 92 DEGs were classified into 12 categories at the GO 2 level, such as catalytic activity, cellular anatomical entities, metabolic processes, and responses to stimuli (data not shown). Interestingly, genes involved in redox processes were enriched (Figure 5). This raises the question: whether treatment with COG1410 increases reactive oxygen species. To address this question, intracellular ROS levels were measured using the DCFH-DA probe. As Figure 3D As shown, COG1410 treatment significantly increased the production of ROS, suggesting that ROS may be another killing mechanism of COG1410.

[0247] COG1410 is highly resistant to induced drug resistance (refractory)

[0248] For new antimicrobial compounds, its resistance barrier is a key parameter because bacterial populations always try to develop resistance in order to survive. In particular, Acinetobacter baumannii is naturally able to adsorb exogenous DNA to obtain resistance to different types of antibiotics. In order to evaluate the resistance development rate of Acinetobacter baumannii for COG1410, PDR strain YQ4 was continuously 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 by 64 times after 55 passages (data not shown). In contrast, the MIC of COG1410 increased by only 4 times in YQ4 after 55 passages. These data show that Acinetobacter baumannii may not be easy to invalidate the toxicity of COG1410 by genetic mutation.

[0249] COG1410 shows low hemolytic activity and moderate eukaryotic cytotoxicity

[0250] The hemolytic potential of COG1410 was determined by exposing human erythrocytes to different concentrations of COG1410 and measuring the release of hemoglobin. Controls consisted of 100% release of hemoglobin after treatment with 0.1% Triton X-100 and 0% release after treatment with PBS. 128 μg / ml (8x MIC) of COG1410 resulted in less than 5% hemolysis ( Fig. 6A The lowest concentration that lyses half of the red blood cells (EC 50) was 441 μg / ml. Therefore, the selectivity index SI (EC 50 / MIC) is 27.5.

[0251] The cytotoxic effect of COG1410 on normal human liver L02 cells was evaluated by CCK8 assay. Figure 6B As shown, at 16 μg / ml, the toxicity to L02 cells was 7%. The half-maximal (EC 50 )The toxic concentration is 58.9μg / ml.

[0252] The bactericidal effect of COG1410 is different from that of polymyxin B

[0253] To investigate whether the antimicrobial effect of COG1410 depends on negatively charged lipopolysaccharide (LPS) on the cell surface, we compared the antimicrobial activity of Acinetobacter baumannii ATCC19606 and two LPS-modified strains (pmrA). P102R and pmrA P102R a I221V The susceptibility of COG1410 to pmrA was determined by the presence of pmrA, which is a phosphoethanolamine transferase that encodes lipid A.

[34] Mutations in pmrA lead to derepression of PmrC, which encodes lipid A phosphoethanolamine transferase.

[35] Modification of lipidA by LPS did not alter the antimicrobial efficacy of COG1410. This is in contrast to polymyxin B, which is a last resort for MDR Gram-negative bacteria and whose bactericidal efficacy depends on electrostatic interactions with LPS ( Fig. 7A ). These data suggest that COG1410 may have a different antibacterial mechanism from polymyxin B and there is no cross-resistance between them.

[0254] COG1410 shows strong synergy with polymyxin B

[0255] To investigate whether COG1410 has synergistic interactions with conventional antibiotics, COG1410 was used in combination with several commonly used anti-Acinetobacter baumannii antibiotics in medical practice to measure synergistic effects. Figures 7A-7C As shown, COG1410 showed synergistic effects with polymyxin B, ceftazidime, and tetracycline, with FIC values ​​of 0.13, 0.31, and 0.31, respectively. Unexpectedly, 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 showed that this combination exhibited similar bactericidal activity to 16 μg / ml COG1410 alone ( Figure 7C ).

[0256] Combination therapy of COG1410 and polymyxin B rescues the nematode worm infected with Acinetobacter baumannii

[0257] To investigate the efficacy in vivo, a bacterial infection model was developed using C. elegans. L4 nematodes were pre-infected in M9 buffer and transferred to NGM agar plates supplemented with or without COG1410. To increase the pathogenicity of A. baumannii, 10 μM FeCl3 was added as described previously

[36] . Figure 8 is a graph illustrating that the combination therapy of COG1410 and polymyxin B rescued infected nematodes. Caenorhabditis elegans was pre-infected with Acinetobacter baumannii YQ4 and transferred to NGM plates supplemented with 16μg / ml COG1410 or 2μg / ml COG1410 and 1μg / ml polymyxin B. Dead nematodes were counted every day for 2 weeks. Survival curves were analyzed using the Kaplan-Meier method, and statistical significance was analyzed using the log-rank test. **, p<0.01. Compared with the untreated group, 16μg / ml COG1410 alleviated the mortality of infected nematodes, but the change was not statistically significant. Unexpectedly, the combination therapy of COG1410 (2μg / ml) plus polymyxin B (1μg / ml) significantly rescued pre-infected nematodes and even extended their lifespan.

[0258] Example 3. Antibacterial activity of peptide compounds against bacterial strains including Porphyromonas gingivalis.

[0259] The minimum inhibitory concentrations (MICs) of the measured peptide compounds are shown in Table 1 above. Briefly, according to Dominy et al. (2019), Porphyromonas 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) at 37°C under anaerobic conditions for 24 to 96 hours to mid-log-phase. Lactobacillus salivarius was grown in BHI broth at 37°C under 5% CO2 for 24 to 48 hours. The microtiter plate method outlined by Jansen (supra) was used to measure MICs. Compounds and bacteria were added to the wells of a microtiter plate and grown for 24 to 72 hours under anaerobic conditions (Porphyromonas gingivalis) or in CO2 (Lactobacillus salivarius). Growth was measured using light scattering at 592 nm on a microplate reader and the absorbance in each well was plotted against μg / ml drug. The μg / ml of each compound where the absorbance did not increase significantly over background (medium alone) before the end of the time course was the MIC.

[0260] Those skilled in the art will readily appreciate that the present disclosure is well adapted to achieve the stated objectives and obtain the results and advantages mentioned and inherent therein. The present disclosure is representative of embodiments, which are exemplary and not intended to limit the scope of the present disclosure. Those skilled in the art will envision variations and other uses which are within the spirit of the present disclosure as defined by the scope of the claims.

[0261] It is not recognized that any reference (including any non-patent or patent document cited in this specification) constitutes prior art. It is understood that, unless otherwise stated, any document cited herein does not constitute an admission that any of these documents constitute part of the common knowledge in this field in the United States or any other country. Any discussion of the references states the claims of their authors, and the applicant reserves the right to question the accuracy and relevance of any document cited herein. Unless otherwise expressly stated, all references cited herein are fully incorporated by reference. If there is any difference between any definition and / or description in the cited references, the present disclosure shall prevail.

[0262] References

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Claims

1. A method for inhibiting the growth of microorganisms, the method comprising: The microorganism is contacted with an effective amount of a peptide compound to inhibit the growth of the microorganism, wherein the peptide compound comprises: -(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 there is a disulfide bond 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 amino acid residues are D-amino acids, Among them, aib is aminoisobutyric acid.

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

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

1.

5. The method according to claim 1, wherein the microorganisms comprise one or more types of bacteria among multi-drug resistance (MDR), extensive drug resistance (XDR) or pan-drug resistance (PDR).

6. The method of claim 1, wherein the microorganism comprises Porphoryomasgingivalis.

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

8. The method according to claim 7, wherein the microorganism comprises one or a combination of Acinetobacter baumannii, Porphyromonas gingivalis or Escherichia coli.

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

10. The method according to claim 9, wherein the microorganism comprises one or a combination of Staphylococcus aureus (S. aureus) or Lactobacillus salivarius (L. salivarius).

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

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

13. The method according to claim 12, wherein the microorganisms comprise pan-resistant Acinetobacter baumannii or multi-drug resistant Staphylococcus aureus.

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

15. 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 wherein the bacteria comprises pan-resistant Acinetobacter baumannii.

16. A method for treating a subject suffering from 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 comprises: -(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 there is a disulfide bond 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 amino acid residues are D-amino acids, Among them, aib is aminoisobutyric acid.

17. The method of claim 16, wherein the microbial growth is caused by bacteria of one or more classes including multi-drug resistance (MDR), extensive drug resistance (XDR) or pan-drug resistance (PDR).

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

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

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

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

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

23. The method of claim 16, wherein said administering is in combination with one or more antimicrobial compounds.

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

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

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

27. 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 wherein the microbial growth is caused by bacteria comprising Acinetobacter baumannii.

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

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

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

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

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

33. An antimicrobial composition 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.

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

1.

36. An antimicrobial composition according to any one of claims 33 to 35 for use in a method of inhibiting the growth of microorganisms in a subject.

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

38. A peptide compound having a structure selected from the group consisting of: -(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 there is a disulfide bond 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) (lower case 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, Among them, aib is aminoisobutyric acid.

39. The peptide compound according to claim 38, or a pharmaceutically acceptable salt or solvate thereof, for use in a method of inhibiting the growth of a microorganism in a subject.

40. A pharmaceutical composition comprising the peptide compound according to claim 39.

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

1.

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

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

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

47. A pharmaceutical composition according to claim 37 or 46, further comprising one or more pharmaceutically acceptable excipients.

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

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

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

51. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is formulated as a mouthwash or toothpaste.

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