Non-ribosomal polypeptide compounds, synthetic gene cluster BNP37 thereof, and use thereof

Abstract

Provided are non-ribosomal polypeptide compounds, a synthetic gene cluster BNP37 thereof, and the use thereof. The polypeptide compounds have the following structural sequence: X0-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11, which is a linear peptide or a cyclic peptide, wherein the cyclic peptide is a lactam cyclic peptide formed by linking any one of X1, X3 and X5 to X11, or a lactam cyclic peptide or a lactone cyclic peptide formed by linking X2 to X11. The compounds can target outer membrane lipid A of Gram-negative bacteria and teichoic acid of Gram-positive bacteria, thereby causing the efflux of important ions in a bacterium cell and resulting in bacterium death. The compounds have high in-vivo and in-vitro antibacterial activities against a variety of multidrug-resistant Gram-negative pathogens such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, Neisseria gonorrhoeae, etc., and a variety of multidrug-resistant Gram-positive pathogens such as Staphylococcus aureus and Enterococcus faecium, etc.

Description

A nonribosomal polypeptide compound, its synthetic gene cluster BNP37, and its applications Technical Field

[0001] This invention belongs to the field of microbial natural products, and particularly relates to a non-ribosomal polypeptide compound, its synthetic gene cluster BNP37, and its applications. Background Technology

[0002] The overuse of antibiotics has led to the faster emergence of drug-resistant bacteria and resistance genes, reducing the therapeutic potential of antibiotics in humans and animals. Bacterial resistance is becoming increasingly serious, with even multidrug-resistant bacteria emerging. Therefore, the need for the development of novel antibiotics is particularly urgent.

[0003] Among multidrug-resistant bacterial infections, Gram-negative bacterial infections are the most serious. Gram-negative strains account for as much as 70.1% of clinical infections, especially carbapenem-resistant Gram-negative bacteria (CROs), which pose a significant challenge to clinical anti-infective treatment. Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa are among the most common drug-resistant species. They are the most common, most concerning, and most clinically threatening drug-resistant bacteria in CROs. These species exhibit high resistance to many commonly used clinical antibiotics, and their detection rate is continuously increasing. Due to their unique double-membrane structure and complex resistance mechanisms such as multiple drug efflux pumps, Gram-negative bacteria have resulted in almost no innovative antibiotics being developed and marketed in the past 40 years. Polymyxins, introduced in 1947, were gradually withdrawn from clinical use due to their significant nephrotoxicity and neurotoxicity. However, with the emergence and prevalence of CRO infections, polymyxins have returned to clinical use due to their efficacy against almost all CROs, and are used as a last line of defense against Gram-negative bacterial infections. While providing good therapeutic effects, the current state of polymyxin resistance cannot be ignored. In addition to natural resistance, the emergence and widespread dissemination of the plasmid-mediated polymyxin resistance gene (mcr-1) in 2015 poses a significant threat to the efficacy of polymyxins. In conclusion, the global clinical application value of polymyxins remains irreplaceable, but they also face the challenges of increasing resistance and toxicity. Therefore, the development of novel antimicrobial compounds is particularly important to address the problem of bacterial resistance in pathogens. Summary of the Invention

[0004] Purpose of the invention: In order to solve the problems existing in the prior art, the present invention aims to provide a non-ribosomal polypeptide compound. The polypeptide compound synthesized by the present invention can effectively solve the problems of serious drug-resistant pathogens, especially drug-resistant Gram-negative bacteria, in clinical practice, the limited types of available antibacterial drugs, and the large toxic side effects.

[0005] This invention also provides a novel gene cluster for the synthesis of nonribosomal polypeptide compounds and its applications.

[0006] Technical solution: To achieve the above objectives, the present invention provides a polypeptide compound or a pharmaceutically acceptable salt thereof, wherein the structural sequence of the polypeptide compound is shown in formula (I), X0-X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 (I)

[0007] in,

[0008] X0 is selected from carboxylic acid compounds;

[0009] X1, X3, X5, and X8 are each independently selected from substituted or unsubstituted L-type or D-type basic amino acids;

[0010] X2 is selected from L-type or D-type amino acids with substituted or unsubstituted side chains containing amino or hydroxyl groups;

[0011] X4, X6, X7, X 10 Each amino acid is independently selected from substituted or unsubstituted L-type or D-type hydrophobic amino acids;

[0012] X9, X 11 Each amino acid is independently selected from substituted or unsubstituted L- or D-type amino acids;

[0013] The carboxyl group in X0 and the amino group in X1 form an amide bond;

[0014] The polypeptide compound is a linear peptide or a cyclic peptide, wherein the cyclic peptide is any one of X1, X3, and X5 and X. 11 The linked lactam cyclic peptide, or the cyclic peptide being X2 and X... 11 Linked lactam cyclic peptides or lactone cyclic peptides;

[0015] Each of the above substitutions is optionally replaced by one or more substituents R independently selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclic group, C(=O)NH2, NHC(=O)NH2 or COOH. a1 The 4-7 membered heterocyclic group comprises 1 to 3 heteroatoms independently selected from N, O or S.

[0016] Preferably, each substitution is optionally replaced by 1-3 substituents R independently selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclic group, C(=O)NH2, NHC(=O)NH2 or COOH. a1 The 4-7 membered heterocyclic group comprises 1 to 3 heteroatoms independently selected from N, O or S.

[0017] Wherein, X1, X3, X5, and X8 in formula (I) are each independently selected from the following substituted or unsubstituted basic amino acids of L-type or D-type: Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Orn (ornithine), Lys, Arg, His, D-Dab, D-Dap, D-Orn, D-Lys, D-Arg, or D-His;

[0018] X2 is selected from the following substituted or unsubstituted amino acids of L or D type with an amino or hydroxyl side chain: Dab, Dap, Orn, Lys, Arg, His, Tyr, Thr, allo-Thr (allo-threonine), Ser, D-Dab, D-Dap, D-Orn, D-Lys, D-Arg, D-His, D-Tyr, D-Thr, D-allo-Thr, or D-Ser;

[0019] X4, X6, X7, X 10 Each of the following substituted or unsubstituted hydrophobic amino acids, independently selected from L-type or D-type: Ala, Leu, Ile, Phe, Met, Trp, Pro, Val, D-Ala, D-Leu, D-Ile, D-Phe, D-Met, D-Trp, D-Pro, or D-Val;

[0020] X9, X 11Each of the following substituted or unsubstituted amino acids, independently selected from L- or D-type amino acids: Asp, Ala, Arg, Asn, Dab, Dap, Gln, Gly, His, Ile, Leu, Lys, Met, Orn, Phe, Ser, Thr, Trp, Tyr, Val, D-Asp, D-Ala, D-Arg, D-Asn, D-Dab, D-Dap, D-Gln, D-Gly, D-His, D-Ile, D-Leu, D-Ly s, D-Met, D-Orn, D-Phe, D-Ser, D-Thr, D-Trp, D-Tyr, or D-Val; wherein each of the above substitutions is optionally replaced by one or more substituents R independently selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclic group, C(=O)NH2, NHC(=O)NH2, or COOH. a1 The 4-7 membered heterocyclic group comprises 1 to 3 heteroatoms independently selected from N, O or S;

[0021] X0 is selected from fatty acids or aromatic carboxylic acids, wherein the fatty acid is a saturated fatty acid or an unsaturated fatty acid.

[0022] In formula (I), X1, X3, X5, and X8 are each independently selected from substituted or unsubstituted L-type or D-type amino acids: Dab, Dap, Orn, Lys, Arg, or His; X2 is selected from substituted or unsubstituted L-type or D-type amino acids: Thr or Ser; X4, X6, X7, X8 are selected from substituted or unsubstituted L-type or D-type amino acids: Thr or Ser; X4, X6, X7, X8 are selected from substituted or unsubstituted L-type or D-type amino acids: Thr or Ser; X8 is ... 10 Each amino acid is independently selected from substituted or unsubstituted L- or D-type amino acids: Leu, Phe, or Val; X9, X 11 Each of the following amino acids is independently selected from substituted or unsubstituted L- or D-type amino acids: Asp, Ala, Dab, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Tyr, Trp, or Val; wherein each of the above substitutions is optionally replaced by one or more substituents R independently selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclic group, C(=O)NH2, NHC(=O)NH2, or COOH. a1 The 4-7 membered heterocyclic group comprises 1 to 3 heteroatoms independently selected from N, O or S.

[0023] Preferably, in formula (I), X1 is a substituted or unsubstituted D-Dab, X2 is a substituted or unsubstituted Thr, X3 is a substituted or unsubstituted Dab, X4 is a substituted or unsubstituted Leu, X5 is a substituted or unsubstituted Dab, X6 is a substituted or unsubstituted D-Phe, X7 is a substituted or unsubstituted Leu, X8 is a substituted or unsubstituted Dab, and X9 is a substituted or unsubstituted D-Tyr or D-Dab. 10 For Val, whether or not it is replaced, X 11 The Asp can be substituted or unsubstituted; wherein each of the above substitutions is optionally replaced by one or more substituents R independently selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclic group, C(=O)NH2, NHC(=O)NH2 or COOH. a1 The 4-7 membered heterocyclic group comprises 1 to 3 heteroatoms independently selected from N, O or S.

[0024] Preferably, each of the above substitutions is optionally replaced by 1-3 C1-C6 alkyl groups.

[0025] Furthermore, in equation (I), X1 is D-Dab, X2 is Thr, X3 is Dab, X4 is Leu, X5 is Dab, X6 is D-Phe, X7 is Leu, X8 is Dab, X9 is D-Tyr or D-Dab, X 10 For Val, X 11 For Asp.

[0026] Wherein, X1 to X in the polypeptide compound represented by formula (I) 11 Amino acid sequences selected from any of the following groups;

[0027] X0 is selected from fatty acids or aromatic carboxylic acids, wherein the fatty acid is a saturated fatty acid or an unsaturated fatty acid.

[0028] In formula (I), X0 is selected from Myristic acid, Butyric acid, Decanoic acid, Lauric acid, Palmitic acid, Stearic acid, Sorbic acid, Neo-decanoic acid, 4-Methylnonanoic acid, Benzofuran-2-carboxylic acid, Indole-2-carboxylic acid, 2-Quinoxalinecarboxylic acid, 2-Biphenylcarboxylic acid, 9-Anthracenecarboxylic acid, 2-aminonicotinic acid, and 9-Fluorenone-4-carboxylic acid. 9-Fluorenone-2-carboxylic acid, 3-Biphenylcarboxylic acid, 4-piperidin-1-ylbenzoic acid, 4-Morpholinobenzoic Acid, 4-(4-Methyl-piperazin-1-yl)-benzoic acid, 3-(4-Methylpiperazin-1-yl)benzoic acid, 4”-(Pentyloxy)-1,1':4',1”-terphenyl-4-carboxylic acid (p-pentoxyterphenylcarboxylic acid), Undecanoic acid (undecanoic acid), Tridecylic acid (tetrate acid), Pentadecanoic acid (pentadecanoic acid), Heptadecanoic acid (heptadecanoic acid), 8-Phenyloctanoic acid (8-phenyloctanoic acid), 4-Cyanobenzoic acid (4-cyanobenzoic acid), 4-(4-Fluorophenyl)benzoic acid (4-fluorophenylbenzoic acid), 4-phenylcyclohexane-1-carboxylic acid (4-phenyl-cyclohexanecarboxylic acid), 4-cyclopropylbenzoic acid (4-cyclopropylbenzoic acid), 2,4-dichlorobenzoic acid (2,4-dichlorobenzoic acid), p-toluic acid (p-methylbenzoic acid), 4-chlorobenzoic acid (4-chlorobenzoic acid), 4-bromobenzoic acid (4-bromobenzoic acid), 4-fluorobenzoic acid (4-fluorobenzoic acid), 4-Phenylbenzoic acid 4-phenylbenzoic acid, 4′-chloro-[1,1′-biphenyl]-4-carboxylic acid, 4′-bromo-[1,1′-biphenyl]-4-carboxylic acid, or 4-(phenylethynyl)benzoic acid, 2-Ethylhexanoic acid, 10-Undecenoic acid, 2-hydroxynicotinic acid, or 3-hydroxytetradecanoic acid.

[0029] Preferably, X0 in formula (I) is selected from Myristic acid (tetradecanoic acid).

[0030] The polypeptide compound structure sequence represented by formula (I) is selected from X0-Dab-Thr-Dab-Leu-Dab-Phe-Leu-Dab-X9-Val-X. 11or X0-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-X9-Val-X 11 The peptide compound is a linear peptide or a cyclic peptide, wherein the cyclic peptide is any one of X1, X3, and X5, or a combination of D-Dab and X. 11 The linked lactam cyclic peptide, or the cyclic peptide being Thr and X 11 A cyclic lactone peptide formed by linkage.

[0031] Preferably, the polypeptide compound structure sequence shown in formula (I) is selected from X0-Dab-Thr-Dab-Leu-Dab-Phe-Leu-Dab-X9-Val-Asp, or X0-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-X9-Val-Asp, wherein the peptide compound is a linear peptide or a cyclic peptide, wherein the cyclic peptide is a lactam cyclic peptide formed by linking any one of the Dabs or D-Dabs in X1, X3, and X5 with Asp, or the cyclic peptide is a lactone cyclic peptide formed by linking Thr with Asp.

[0032] Further, the polypeptide compound structural sequence shown in formula (I) is selected from X0-Dab-Thr-Dab-Leu-Dab-Phe-Leu-Dab-X9-Val-Asp or X0-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-X9-Val-Asp, wherein X0 is selected from tetradecanoic acid, X9 is selected from L-type or D-type Tyr or L-type or D-type Dab, the peptide compound is a linear peptide or a cyclic peptide, the cyclic peptide is a lactam cyclic peptide formed by linking any one of X1, X3, X5 Dab or D-Dab with Asp, or the cyclic peptide is a lactone cyclic peptide formed by linking Thr with Asp.

[0033] The polypeptide compound is shown in formula (II):

[0034] Among them, R 1 R is the carbonyl compound obtained by removing the hydroxyl group from X0 when it forms an amide bond with X1 as described in claim 1. 2 for X9 is either D-Tyr or D-Dab, R 3 X is any amino acid side chain. 11The amino acid is either L-substituted or unsubstituted, wherein the substitution is optionally made by one or more substituents R independently selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclic group, C(=O)NH2, NHC(=O)NH2 or COOH. a1 The 4-7 membered heterocyclic group comprises 1 to 3 heteroatoms independently selected from N, O, or S; the peptide compound is a linear peptide or a cyclic peptide, wherein the cyclic peptide is any one of X1, X3, and X5 (Dab or D-Dab combined with X). 11 The linked lactam cyclic peptide, or the cyclic peptide being Thr and X2 of X2. 11 A cyclic lactone peptide formed by linkage.

[0035] Preferably, the substitution is optionally made by replacing 1-3 C1-C6 alkyl groups.

[0036] Preferably, the polypeptide compound represented by formula (II) has a linear polypeptide structure, and the R 1 It is a carbonyl compound obtained by removing the hydroxyl group when forming an amide bond with X1, selected from any one of the following: Butyric acid, Lauric acid, Palmitic acid, Stearic acid, Sorbic acid, 2-Ethylhexanoic acid, Neo-decanoic acid, 10-Undecenoic acid, Benzofuran-2-carboxylic acid, 4-Phenylbenzoic acid, 2-hydroxynicotinic acid, and 3-hydroxytetradecanoic acid.

[0037] The polypeptide compound is shown in formula (III):

[0038] The peptide compound is a linear peptide or a cyclic peptide. The cyclic peptide is a lactam cyclic peptide formed by linking any one of the Dabs or D-Dabs (X1, X3, X5) with Asp, or the cyclic peptide is a lactone cyclic peptide formed by linking Thr with Asp.

[0039] The polypeptide compound is shown in formula (IV):

[0040] Among them, R 1 It is the carbonyl compound obtained by removing the hydroxyl group when X0 and X1 form an amide bond.

[0041] Preferably, the polypeptide compound shown in formula (IV) is in the form of X2 and X 11 Formation of lactone ring, wherein R 1The following are selected from: Butyric acid, Decanoic acid, Lauric acid, Palmitic acid, Stearic acid, Sorbic acid, Neo-decanoic acid, 4-Methylnonanoic acid, Benzofuran-2-carboxylic acid, Indole-2-carboxylic acid, 2-Quinoxalinecarboxylic acid, 2-Biphenylcarboxylic acid, 9-Anthracenecarboxylic acid, 2-aminonicotinic acid, 9-Fluorenone-4-carboxylic acid, and 9-Fluorenone-2-carboxylic acid. 9-fluorenone-2-carboxylic acid, 3-Biphenylcarboxylic acid, 4-piperidin-1-ylbenzoic acid, 4-Morpholinobenzoic Acid, 4-(4-Methyl-piperazin-1-yl)-benzoic acid, 3-(4-Methylpiperazin-1-yl)benzoic acid, Benzoic acid, 4”-(Pentyloxy)-1,1':4',1”-terphenyl-4-carboxylic acid (p-pentoxyterphenylcarboxylic acid), Undecanoic acid (undecanoic acid), Tridecylic acid (tetrate acid), Pentadecanoic acid (pentadecanoic acid), Heptadecanoic acid (heptadecanoic acid), 8-Phenyloctanoic acid (8-phenyloctanoic acid), 4-Cyanobenzoic acid (4-cyanobenzoic acid), 4-(4-Fluorophenyl)benzoic acid (4-fluorophenylbenzoic acid), 4-phenylcyclohexane-1-carboxylic acid (4-phenyl-cyclohexanecarboxylic acid), 4-cyclopropylbenzoic acid (4-cyclopropylbenzoic acid), 2,4-dichlorobenzoic acid (2,4-dichlorobenzoic acid), p-toluic acid (p-methylbenzoic acid), 4-chlorobenzoic acid (4-chlorobenzoic acid), 4-bromobenzoic acid (4-bromobenzoic acid), 4-fluorobenzoic acid (4-fluorobenzoic acid), 4-Phenylbenzoic acid A carbonyl compound obtained by removing the hydroxyl group from any one of the following acids: 4-phenylbenzoic acid, 4′-chloro-[1,1'-biphenyl]-4-carboxylic acid, 4′-bromo-[1,1'-biphenyl]-4-carboxylic acid, and 4-(phenylethynyl)benzoic acid, when forming an amide bond with X1.

[0042] The polypeptide compound is shown in formula (V):

[0043] Among them, R 1 It is the carbonyl compound obtained by removing the hydroxyl group when X0 and X1 form an amide bond.

[0044] Preferably, the polypeptide compound of formula (V) is in the form of X2 and X... 11 Formation of lactone ring, wherein R 1The following are selected from: Decanoic acid, Lauric acid, Palmitic acid, 4-Methylnonanoic acid, Benzofuran-2-carboxylic acid, 9-Anthracenecarboxylic acid, 9-Fluorenone-4-carboxylic acid, 4-piperidin-1-ylbenzoic acid, 4”-(Pentyloxy)-1,1':4',1”-terphenyl-4-carboxylic acid, Tridecylic acid, Pentadecanoic acid, Heptadecanoic acid, 8-Phenyloctanoic acid, 4-Cyanobenzoic acid 4-Cyanobenzic acid, 4-(4-Fluorophenyl)benzoic acid, 4-phenylcyclohexane-1-carboxylic acid, 4-cyclopropylbenzoic acid, 2,4-dichlorobenzoic acid, p-toluic acid, 4′-chloro-[1,1'-biphenyl]-4-carboxylic acid, 4′-bromo-[1,1'-biphenyl]-4-carboxylic acid, 4-(phenylethynyl)benzoic acid, 4-chlorobenzoic acid, 4-bromobenzoic acid A carbonyl compound obtained by removing the hydroxyl group when either 4-bromobenzoic acid or 4-fluorobenzoic acid forms an amide bond with X1.

[0045] Furthermore, the amino acid sequence of the polypeptide compound is Myristic acid-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-D-Tyr-Val-X. 11 It exhibits a linear polypeptide structure, wherein X 11 Choose any one of Asp, Ala, Arg, Asn, Dab, Dap, Gln, Gly, His, Ile, Leu, Lys, Orn, Phe, Ser, Thr, Trp, Tyr, Val.

[0046] Furthermore, the polypeptide compound is based on the amino acid sequence Myristic acid-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-D-Tyr-Val-Asp, with X2 and X 11 Forming a lactone ring, X1, X3-X 11 The amino acids in the formula are replaced with alanine of the corresponding configuration.

[0047] Furthermore, the polypeptide compound is based on the amino acid sequence Myristic acid-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-D-Tyr-Val-Asp, with X2 and X 11 Forming a lactone ring, X4, X6, X7, X9-X 11 The amino acids in the formula are replaced with 2,4-diaminobutyric acid of the corresponding configuration.

[0048] Furthermore, the polypeptide compound is based on the amino acid sequence Myristic acid-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-D-Dab-Val-Asp, with X2 and X 11 Forming a lactone ring, with sites X1, X3, X5, X6, X7, X8, X9, X 10 X 11 The amino acids are replaced with different amino acids, and the specific sequences are shown below:

[0049] Preferably, the polypeptide compound is paenimycin, which forms a decacyclic lactone lipopeptide with the second amino acid as the cyclization site, as shown in formula (VI):

[0050] The use of the polypeptide compound or its pharmaceutically acceptable salt described in this invention in the preparation of antibacterial drugs.

[0051] The bacteria mentioned are any one of the following clinical pathogens: Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, Neisseria gonorrhoeae, Staphylococcus aureus, and Enterococcus faecalis.

[0052] The pharmaceutical composition of the antibacterial drug of the present invention comprises the aforementioned polypeptide compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0053] The pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhaler, ointment, suppository or patch.

[0054] The present invention relates to the use of the nonribosomal polypeptide biosynthesis gene cluster BNP37 in the synthesis of the aforementioned polypeptide compound or its pharmaceutically acceptable salt, wherein the GenBank accession number for the nucleotide sequence of the biosynthesis gene cluster BNP37 is: NZ_JAQAGY010000015.1

[0055] Furthermore, the polypeptide compound also includes derivatives obtained by chemical modification or alteration based on the side chain groups or sequence ends of the amino acids in the polypeptide compound, such as:

[0056] The hydroxyl groups of the polypeptide compound can form, but are not limited to, lactone compounds;

[0057] The hydroxyl groups of the polypeptide compound can form, but are not limited to, ethers, esters, glycosides, or glycosides.

[0058] The phenolic hydroxyl groups of the polypeptide compound can form, but are not limited to, ethers, esters, glycosides, or glycosides; the amino groups of the polypeptide compound can form, but are not limited to, lactams.

[0059] The amino groups of the polypeptide compounds may form, but are not limited to, acylates, hydrocarbons, glycosides, or glycosides.

[0060] The carboxyl group of the polypeptide compound can form, but is not limited to, esters and amides.

[0061] The polypeptide compound forms a salt compound with an organic acid or an inorganic acid;

[0062] The polypeptide compound forms a complex, chelate, or other compound with the metal ion.

[0063] Furthermore, all of the aforementioned polypeptide compounds were prepared using Fmoc protected solid-phase polypeptide synthesis and liquid-phase synthesis methods.

[0064] Furthermore, the use of all the aforementioned polypeptide compounds, their stereoisomers, and pharmaceutically acceptable salts in the preparation of antibacterial drugs.

[0065] The use of the polypeptide compound or its pharmaceutically acceptable salt or its stereoisomer described in this invention in the preparation of antibacterial drugs.

[0066] The bacteria are either Gram-negative or Gram-positive. Preferably, the Gram-negative bacteria are Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, or Neisseria gonorrhoeae. Preferably, the Gram-positive bacteria are Staphylococcus aureus or Enterococcus faecalis.

[0067] The pharmaceutical composition of the antibacterial drug of the present invention comprises the polypeptide compound or a pharmaceutically acceptable salt or stereoisomer thereof and a pharmaceutically acceptable carrier.

[0068] The pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhaler, ointment, suppository or patch.

[0069] On the other hand, the present invention provides a biosynthetic gene cluster BNP37 of a polypeptide compound, the nucleotide sequence of which is shown in SEQ ID NO.1 and the nucleotide sequence of which is registered in GenBank as NZ_JAQAGY010000015.1.

[0070] This invention employs a strategy of natural product structure prediction and directed chemical synthesis. Based on bioinformatics analysis, combined with genome sequencing, bioinformatics prediction, and sequence similarity network analysis, candidate biosynthetic functional gene cluster BNP37 was screened, with the gene sequence shown in GenBank accession number NZ_JAQAGY010000015.1. Through product structure analysis and design, chemical synthesis was guided by the candidate biosynthetic gene cluster to obtain a series of polypeptide compounds, the structural sequences of which are shown in formula (I) of the first aspect.

[0071] The compound of formula (VI) of the present invention (hereinafter referred to as paenimycin) has a special cyclic lipopeptide structure with a tetradecanoic acid linked to its nitrogen terminus. Its amino acid sequence is: D-type 2,4-diaminobutyric acid-threonine-2,4-diaminobutyric acid-leucine-2,4-diaminobutyric acid-D-type phenylalanine-leucine-2,4-diaminobutyric acid-D-type 2,4-diaminobutyric acid-valine-aspartic acid, wherein the hydroxyl group of the threonine side chain forms a lactone bond with the carboxyl group of aspartic acid, ultimately yielding an undecanoic acid compound containing a tetradecanoic acid fatty chain and a ten-membered macrocycle.

[0072] In exploring the mechanism of action of the synthesized compounds, this invention utilizes bacterial lysis experiments, membrane depolarization experiments, potassium ion release experiments, feeding experiments, scanning electron microscopy, and isothermal calorimetric titration.

[0073] This invention reveals that the compound paenimycin possesses a unique dual-target mechanism of action: against Gram-negative pathogens, paenimycin exerts its antibacterial effect by binding to the phosphate groups on both sides of the hexose sugar of lipid A in the cell wall and the hydroxyl group at the six-position of the side chain, thereby disrupting the cell membrane structure; against Gram-positive pathogens, paenimycin binds to teichoic acid in the bacterial cell wall and the phosphate groups in its long-chain repeating units, thereby disrupting the bacterial cell membrane, causing the efflux of important intracellular ions, leading to bacterial death.

[0074] The compound Paenimycin synthesized in this invention exhibits potent activity against resistant strains of common clinical antimicrobial drugs due to its unique dual-target mechanism of action, without inducing drug resistance under laboratory conditions. Currently, the only reported lipid A-binding antibiotics are from the polymyxin family. Paenimycin is effective against major clinical Gram-negative pathogens resistant to polymyxins, such as Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae. Simultaneously, it also shows good activity against naturally resistant polymyxin bacteria such as Neisseria gonorrhoeae and Neisseria meningitidis.

[0075] In this invention, amino acids represented by three-letter abbreviations generally refer to L-type amino acids. For example, Dab refers to L-2,4-diaminobutyric acid. However, in certain specific contexts, those skilled in the art will understand that the amino acids may also include both L-type and D-type structures. For example, in the statement "X1, X3, X5, X8 are each independently selected from the following basic amino acids of L-type or D-type: Dab (2,4-diaminobutyric acid), Dap, Orn, Lys, Arg, or His", Dab includes both L-Dab and D-Dab (also known as dDab) structures.

[0076] The terms “optional” or “optionally” mean that the event or condition subsequently described may or may not occur, including both the occurrence and non-occurrence of said event or condition. For example, “optionally” substituted with a halogen means that the ethyl group can be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). Those skilled in the art will understand that for any group containing one or more substituents, no substitution or substitution pattern that is spatially impossible and / or cannot be synthesized is introduced.

[0077] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0078] The polypeptide compounds obtained in this invention exhibit good antibacterial activity against various pathogenic bacteria. They not only show excellent antibacterial activity against a variety of Gram-positive and Gram-negative bacteria, but also retain strong antibacterial activity against a variety of drug-resistant bacteria. These include effectiveness against meropenem, third-generation cephalosporins, and polymyxin-resistant strains, activity against naturally resistant polymyxin strains, and activity against methicillin and vancomycin-resistant Gram-positive bacteria.

[0079] The polypeptide compounds of this invention are easy to prepare and inexpensive, with simple structures, ingenious designs, readily available and inexpensive raw materials, and safe and environmentally friendly synthesis processes, facilitating large-scale production. The polypeptide compounds of this invention possess a unique dual-target mechanism, targeting Gram-negative pathogens by binding to lipopolysaccharide components on the outer cell wall, K... d The value was 2.18 μM, while polymyxin E bound to K d The concentration was 2.00 μM. By knocking out the WaaC and WaaG genes, lipopolysaccharide (LPS)-deficient outer and inner nuclear strains were constructed, respectively. The compound retained its effective antibacterial activity in both strains, indicating its binding to lipid A in LPS. Further molecular dynamics simulations confirmed that the compound binds to the phosphate groups flanking the hexose group of lipid A and the hydroxyl group at the six-position of the side chain. Against Gram-positive pathogens, the compound binds to teichoic acid in the bacterial cell wall, and then to lipoteichoic acid (K+). d The value was 5.61 μM. Molecular dynamics simulations showed that the phosphate groups in the long-chain repeating units disrupted the bacterial cell membrane, causing the efflux of important intracellular ions and leading to bacterial death.

[0080] In vivo antibacterial activity tests in mice showed that the peptide compound exhibited dose-dependent efficacy against carbapenem- and third-generation cephalosporin-resistant Acinetobacter baumannii. Subcutaneous administration of 5 mg / kg reduced the bacterial load in the mouse thigh by 2.17 log10, and at a concentration of 20 mg / kg, it reduced it by 2.40 log10, comparable to the effect of polymyxin E. Meropenem, a carbapenem antibiotic, was ineffective. Against the newly emerging polymyxin-resistant Escherichia coli with resistance mediated by the mcr-1 plasmid, the peptide compound also showed dose-dependent efficacy. Subcutaneous administration of 5 mg / kg reduced the bacterial load in the mouse thigh by 2.47 log10, and at a concentration of 20 mg / kg, it reduced it by 3.42 log10, demonstrating significant efficacy. The efficacy of the peptide compound was superior to meropenem, while polymyxin E was ineffective. Against multidrug-resistant Klebsiella pneumoniae resistant to polymyxin, meropenem, and third-generation cephalosporins, the efficacy of the peptide compound showed a dose-dependent effect. Subcutaneous administration of 5 mg / kg reduced the bacterial load in the mouse thigh by 2.25 log10, and at a concentration of 20 mg / kg, it reduced it by 3.12 log10. Polymyxin and meropenem were ineffective. Against methicillin-resistant Staphylococcus aureus, the efficacy of the peptide compound showed a dose-dependent effect. Subcutaneous administration of 5 mg / kg reduced the bacterial load in the mouse thigh by 1.24 log10, and at a concentration of 20 mg / kg, it reduced it by 1.79 log10, comparable to vancomycin, while methicillin was ineffective. No acute toxicity was observed in in vivo animal experiments at all different concentrations. The peptide compound synthesized in this invention can be used to prepare antibacterial drugs.

[0081] Furthermore, this invention is the first to propose the non-ribosomal polypeptide natural product biosynthesis gene cluster BNP37. Based on the analysis of gene cluster BNP37, a series of synthetic polypeptide compounds were synthesized and designed, resulting in polypeptide compounds with potent and broad-spectrum activity against multidrug-resistant bacteria. Attached Figure Description

[0082] Figure 1 shows the process of mining the BNP37 biosynthetic gene cluster;

[0083] Figure 2 shows the BNP37 biosynthetic gene cluster and predicted products;

[0084] Figure 3 shows paeminycin 1 H-NMR (DMSO-d6) nuclear magnetic resonance spectrum;

[0085] Figure 4 shows paeminycin 13 C-NMR (DMSO-d6) nuclear magnetic resonance spectrum;

[0086] Figure 5 shows the COSY-NMR (DMSO-d6) nuclear magnetic resonance spectrum of paenimycin;

[0087] Figure 6 shows the HMBC-NMR (DMSO-d6) nuclear magnetic resonance spectrum of paenimycin;

[0088] Figure 7 shows the HSQC-NMR (DMSO-d6) nuclear magnetic resonance image of paenimycin;

[0089] Figure 8 shows the cytotoxicity of paenimycin on HepG2 cells;

[0090] Figure 9 shows the hemolytic activity of paenimycin;

[0091] Figure 10 shows the infection model of Acinetobacter baumannii ATCC BAA 1605 in the thigh muscle induced by paenimycin;

[0092] Figure 11 shows the thigh muscle infection model of Klebsiella pneumoniae BNCC 359393 induced by paenimycin;

[0093] Figure 12 shows the infection model of Escherichia coli MG1655-mcr-1 in thigh muscle by paenimycin;

[0094] Figure 13 shows the thigh muscle infection model of Staphylococcus aureus ATCC BAA44 induced by paenimycin;

[0095] Figure 14 shows the in vivo pharmacokinetic study of paenimycin;

[0096] Figure 15 shows the nephrotoxicity study of paenimycin;

[0097] Figure 16 shows the paenimycin cell membrane lysis experiment;

[0098] Figure 17 shows the depolarization experiment of the paenimycin membrane;

[0099] Figure 18 shows the change in K+ concentration after paenimycin treatment;

[0100] Figure 19 shows the bactericidal curve of paenimucin;

[0101] Figure 20 shows the bacterial cell morphology of Escherichia coli and Staphylococcus aureus after treatment with paenimycin under a scanning electron microscope.

[0102] Figure 21 shows the continuous passaging experiment of paenimycin;

[0103] Figure 22 shows the paenimycin feeding experiment;

[0104] Figure 23 shows the ITC assay for the binding of paenimycin and lipopolysaccharide.

[0105] Figure 24 shows the BC dye substitution experiment;

[0106] Figure 25 shows the growth curve determination of lipid A after feeding;

[0107] Figure 26 shows the molecular dynamics simulation of the binding of paenimyxin and lipid A;

[0108] Figure 27 shows the experiment on the accumulation of paenimycin cell wall precursors;

[0109] Figure 28 shows the paenimycin-bound cell wall teichoic acid. Detailed Implementation

[0110] The technical solution of the present invention will be further described below with reference to the accompanying drawings. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or according to the manufacturer's recommendations.

[0111] The amino acids in formula (I) of this invention are natural amino acids or non-natural amino acids, all of which are amino acids with known structures and can be purchased directly from the market or synthesized by methods in the prior art.

[0112] The sources of the non-natural amino acids used in the synthesis in the examples are as follows: Fmoc-Dab(Boc)-OH: Leyan, 1018956-25g; Fmoc-D-Dab(Boc)-OH: Leyan, 1066565-25g; Fmoc-Dap(Boc)-OH: Leyan, 1022952-10g; Fmoc-D-Dap(Boc)-OH: Leyan, 1136854-10g; Fmoc-Orn(Boc)-OH: Leyan, 1016499-25g Fmoc-D-Orn(Boc)-OH: Leyan, 1064968-10g; Fmoc-N-Me-Leu-OH: Leyan, 1015393-5g; Fmoc-N-Me-Val-OH: Leyan, 1044531-5g; Fmoc-allo-Thr-OH: Leyan, 1171620-5g; Fmoc-D-Phe-OH: Leyan, 1045032-25g; Fmoc-D-Tyr(tBu)-OH: Leyan, 1017786-25g.

[0113] Other non-natural amino acids used in synthesis are commercially available.

[0114] In the following examples, the abbreviations for commonly used reagents and methods are as follows:

[0115] SPE: Solid Phase Extraction

[0116] Fmoc: 9-fluorenylmethoxycarbonyl protecting group,

[0117] DCM: Dichloromethane

[0118] DMF: N,N-dimethylformamide

[0119] HBTU: O-benzotriazole-tetramethylurea hexafluorophosphate,

[0120] HOBt: 1-Hydroxybenzotriazole,

[0121] DIPEA: N,N-diisopropylethylamine

[0122] tBu: tert-butyl protecting group

[0123] Boc: tert-Butoxycarbonyl protecting group

[0124] TFA: Trifluoroacetic acid,

[0125] TIPS: Triisopropylsilane

[0126] Alloc: Allyloxycarbonyl protecting group

[0127] PyBOP: Benzotriazol-1-yl-oxotripyrrolidinyl-hexafluorophosphate

[0128] BzCl: Benzoyl chloride,

[0129] DIC: N,N'-Diisopropylcarbodiimide

[0130] FA: Formic acid

[0131] NMP: N-methylpyrrolidone,

[0132] DBU:1,8-diazabicyclo[5.4.0]undec-7-ene.

[0133] Example 1

[0134] Discovery of the BNP37 biosynthetic gene cluster

[0135] Figure 1 illustrates the process of mining BNP37 biosynthetic gene clusters. A total of 1024 bacterial genomes from the Paenibacillaceae family were collected from public databases and sequencing information of strains gathered within the laboratory. Using antiSMASH software, all genomic data were analyzed, yielding 17176 biosynthetic gene clusters (BGCs). Further screening was conducted to identify non-ribosomal polypeptide biosynthetic gene clusters. A Python script was written to determine the type of biosynthetic gene cluster, calculate the number of adenosine domains, and determine the presence of thioesterase domains as screening criteria, resulting in 776 candidate non-ribosomal polypeptide biosynthetic gene clusters. To exclude known natural product biosynthetic gene clusters and determine the similarity between candidate biosynthetic gene clusters, we used BIG-SCAPE to perform cluster analysis on all candidate biosynthetic gene clusters. Through screening, we obtained a single point that did not cluster with other biosynthetic gene clusters. We named this biosynthetic gene cluster BNP37, and its sequence is shown in the NCBI Genbank accession number: NZ_JAQAGY010000015.1. The functional annotations of the biosynthetic genes are shown in Table 1.

[0136] Table 1. Functional annotations of the biosynthetic gene cluster of BNP37.

[0137] Example 2

[0138] BNP37-guided synthesis of a series of polypeptide compounds: chemical synthesis of linear and cyclic peptides.

[0139] Using adenosine domain bioinformatics prediction and phylogenetic tree analysis, the structure and conformation of the polypeptide sequences encoded by the BNP37 biosynthetic gene cluster were analyzed and designed. The gene sequences were converted into structural sequences, and the obtained polypeptide sequences were synthesized using linear and cyclic polypeptide techniques. The polypeptide compounds in this invention can be directly synthesized using existing polypeptide synthesis methods from biotechnology companies, or synthesized according to the methods described below.

[0140] The specific synthesis method is as follows:

[0141] (1) Linear polypeptide synthesis:

[0142] Taking linear peptide synthesis as an example,

[0143] a) Loading of the first amino acid: Weigh 300 mg of 2-chlorotriphenylmethyl chlororesin (0.1 mmol synthetic equivalent) and load it into an empty SPE column equipped with a filter sieve. Add 8 mL of DCM and allow it to swell at room temperature for 30 minutes. Then, use a vacuum pump to remove the solvent. Weigh Fmoc-X... 11Amino acid (Fmoc-L-Asp(OtBu)-OH, 0.17g, 0.3g)

[0144] 2,4,6-trimethylpyridine (0.3 mL, 2 mmol) was dissolved in 8 mL of DCM, and then 2,4,6-trimethylpyridine (0.3 mL, 2 mmol) was added. After mixing and dissolving, the solution was added to an SPE column packed with resin and reacted at room temperature for 8-12 hours to prepare the resin loaded with the first amino acid.

[0145] b) Peptide chain elongation: Wash the resin three times each with 3 mL of DCM and DMF, then add 4 mL of a solution containing 20% ​​DCM.

[0146] The reaction of piperidine in DMF solution was repeated twice for 7.5 minutes to remove the Fmoc protecting group. Meanwhile, separately weigh F...

[0147] moc-X 10 Amino acids (Fmoc-L-Val-OH, 0.14 g, 0.3 mmol), HBTU (0.15 g, 0.3 mmol),

[0148] 0.3 mmol HOBt (0.055 g, 0.3 mmol) and DIPEA (0.071 mL, 0.3 mmol) were dissolved in 6 mL DMF. After deprotection, the resin was washed five times with 3 mL DMF, and the mixture of amino acids was poured in. The mixture was allowed to stand at room temperature for 1 hour. The coupling steps were repeated to sequentially condense and link X9-X8-X7-X6-

[0149] X5-X4-X3-X2-X1-X0, the amino acid raw materials are, in sequence: Fmoc-D-Tyr(tBu)-OH, Fmoc-L-Dab(Boc)-OH,

[0150] Fmoc-L-Leu-OH, Fmoc-D-Phe-OH, Fmoc-L-Dab(Boc)-OH, Fmoc-L-Leu-OH, Fmoc-

[0151] L-Dab(Boc)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-D-Dab(Boc)-OH, Myristic acid.

[0152] c) Final lysis: Prepare 5 mL of lysis solution (95% TFA, 2.5% TIPS, and 2.5% water), pour it into an SPE column containing the above linear peptide-resin mixture, and allow it to stand at room temperature for 2 hours. Collect the lysis solution, pour it into 45 mL of pre-cooled diethyl ether:n-hexane (1:1) mixture, and allow it to stand at -20°C for 1 hour to precipitate, yielding a white solid crude product.

[0153] All linear polypeptides were synthesized according to the above method based on the selection of different amino acid sequences and fatty acid chains.

[0154] (2) Synthesis of lactam cyclic peptides:

[0155] With X1 and X 11 Taking the synthesis of the formed lactam cyclic peptide as an example:

[0156] a) Linear polypeptide synthesis: The operation is the same as step (1) of this embodiment, except that the amino acid raw material at position X1 is replaced. The amino acid raw materials used are: Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Val-OH, Fmoc-D-Tyr(tBu)-OH, Fmoc-L-Dab(Boc)-OH, Fmoc-L-Leu-OH, Fmoc-D-Phe-OH, Fmoc-L-Dab(Boc)-OH, Fmoc-L-Leu-OH, Fmoc-L-Dab(Boc)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-D-Dab(Alloc)-OH, and Myristic acid, to obtain a linear polypeptide-resin mixture.

[0157] b) Removal of Alloc protecting groups from amino acid side chains: Weigh 0.25 mL of phenylsilane (1.5 mmol) and 0.078 g of tetra(triphenylphosphine)palladium (0.05 mmol), dissolve them in 6 mL of DCM, and pour the mixture into the linear peptide-resin mixture described above. React at room temperature for 3 hours. After the reaction is complete, wash thoroughly with 50 mL of DMF solution containing 10% sodium diethyldithiocarbamate.

[0158] c) Cyclation of peptide amide bonds: Add 5 mL of DCM solution containing 1% TFA to the SPE column, react at room temperature for 2 minutes, collect the cleaved linear peptide solution in a 50 mL centrifuge tube, add 40 mL of DCM solvent, and then add DIPEA (0.7 mL, 3 mmol) and PyBOP (0.56 g, 0.8 mmol) sequentially, mix well, and shake horizontally overnight. Finally, wash with an aqueous solution containing 5% formic acid, collect the lower DCM solution, and dry the solvent for final lysis.

[0159] d) Final lysis: Add 5 mL of the prepared lysis solution (95% TFA, 2.5% TIPS, and 2.5% water) to the dried peptide mixture, shake for 2 hours to remove all side-chain protecting groups. Then pour the lysis buffer into 45 mL of pre-cooled diethyl ether:n-hexane (1:1) mixture, and let it stand at -20°C for 1 hour to precipitate and obtain crude peptide.

[0160] Other sites such as X3, X5 and X 11The amide bond cyclization is formed by the same method as above, except that the amino acid raw material at the corresponding site is replaced with Fmoc-X(Alloc)-OH.

[0161] (3) Synthesis of lactone cyclic peptides

[0162] Taking the synthesis of paenimycin as an example:

[0163] a) Linear peptide synthesis: The seventh amino acid X7: leucine (Fmoc-L-Leu-OH) was used as the first amino acid loaded onto the resin. The operation was the same as step (1) in this embodiment. The second amino acid X2 was replaced with Fmoc-L-Thr-OH. X7-X6-X5-X4-X3-X2-X1-X0 were synthesized in sequence. The amino acid raw materials used were: Fmoc-L-Leu-OH, Fmoc-D-Phe-OH, Fmoc-L-Dab(Boc)-OH, Fmoc-L-Leu-OH, Fmoc-L-Dab(Boc)-OH, Fmoc-L-Thr-OH, Fmoc-D-Dab(Boc)-OH, and Myristic acid, to obtain a linear peptide-resin mixture.

[0164] b) Ester bond synthesis: Fmoc-Asp(OtBu)-OH (1.1 g, 2 mmol), DIPEA (0.95 mL, 4 mmol), BzCl (0.31 mL, 2 mmol), and 4-dimethylaminopyridine (0.016 g, 0.1 mmol) were added sequentially to 15 mL of DCM and mixed thoroughly. The linear peptide-resin mixture was then immediately added and the mixture was shaken at room temperature for 48 hours.

[0165] c) Amide cyclization: Continue loading the remaining amino acids according to step (1) of this embodiment, and sequentially link X 10 After removing the Fmoc protecting group at the X8 site with a 20% piperidine solution, the target polypeptide is cyclized using the PyBOP-DIPEA condensation system according to the polypeptide amide bond cyclization method in step (2) of this embodiment. Then, it is washed with an aqueous solution containing 5% formic acid, the lower layer DCM solution is collected, and the solvent is dried for the final cleavage of the polypeptide product.

[0166] d) Final lysis: Add 5 mL of the prepared lysis solution (95% TFA, 2.5% TIPS, and 2.5% water) to the dried peptide mixture, shake for 2 hours to remove all side-chain protecting groups. Then pour the lysis buffer into 45 mL of pre-cooled diethyl ether:n-hexane (1:1) mixture, and let it stand at -20°C for 1 hour to precipitate and obtain crude peptide.

[0167] (4) N-methylation synthesis

[0168] Taking the methylation of X6:D-phenylalanine (D-Phe) as an example, the amino acid site to be methylated (Fmoc-D-Phe-OH) is synthesized according to the linear peptide extension step in the above embodiment. Similarly, the Fmoc protecting group of the amino acid is removed with a 20% piperidine DMF solution. Weigh out o-nitrobenzenesulfonyl chloride (0.12 g, 0.4 mmol) and dissolve it in 2 mL of NMP. Add 2,4,6-trimethylpyridine (0.15 mL, 1 mmol) and mix well. Pour the mixture into the resin and react at room temperature for 15 minutes. Remove the reaction solution and repeat the above operation once. Wash the resin three times with 5 mL of NMP. Add DBU (0.1 mL, 0.5 mmol) to 1.5 mL of NMP, mix well, and add it into the resin. Let it stand for 3 minutes. Add dimethyl sulfate (0.09 mL, 0.5 mmol) to 1.5 mL of NMP, mix well, and pour it into the resin. Mix by pipetting and react at room temperature for 3 minutes. Remove the reaction solution and repeat the above operation once. Wash the resin three times with 5 mL of NMP. Add 2-mercaptoethanol (0.1 mL, 1 mmol) and DBU (0.1 mL, 0.5 mmol) to 2 mL of NMP. React at room temperature for 5 minutes. Remove the reaction solution and repeat the above operation twice. Except for Fmoc-N-Me-L-Leu-OH and Fmoc-N-Me-L-Val-OH, which were purchased directly from Leyan Reagent Company, the N-methylation of other amino acids can be completed according to the above steps. Subsequent peptide chain elongation, esterification, and cyclization are carried out by continuing the linear peptide synthesis, ester bond synthesis, and amide bond cyclization steps in the above examples.

[0169] Example 3

[0170] Purification of crude polypeptide compounds

[0171] The crude polypeptide compound prepared in Example 2 was purified by high performance liquid chromatography (HPLC). The HPLC conditions were as follows: C18 column (Shimadzu, ShimNet HE C18-AQ, 5 μm OBD, 19 × 250 mm column); mobile phase A was deionized water (containing 0.1% FA); mobile phase B was acetonitrile (containing 0.1% FA); flow rate was 3 mL / min; gradient of mobile phase B was 30%-65%; and UV detection wavelength was 220 nm. Elution peaks were collected at equal time intervals, and the components were detected using UPLC-MS. The UPLC-MS conditions were as follows: C18 column (Waters, C18-1.8μm, 2.1×100mm); mobile phase A was deionized water (containing 0.1% FA); mobile phase B was acetonitrile (containing 0.1% FA); flow rate was 0.6 mL / min; gradient of mobile phase B was 20%-90%; MS detection range was 200-1250; simultaneous scanning in positive and negative ion modes was used. The fraction with optimal purity was obtained and then freeze-dried to obtain a white, flocculent, pure polypeptide compound with a purity exceeding 95%.

[0172] Example 4

[0173] BNP37 series polypeptide compounds were synthesized using the methods described in Examples 2 and 3.

[0174] Minimum inhibitory concentration (MIC) determination: MIC determination was performed according to the protocol recommended by the Clinical and Laboratory Standards Institute (CLSI). All compounds were dissolved in sterile dimethyl sulfoxide (DMSO) (SCR, CN) to obtain a stock solution of 12.8 mg / mL. The stock solutions were serially diluted in 96-well plates using the 2-fold dilution method to obtain solutions with concentrations of 64 μg / mL–0.0625 μg / mL, 50 μL per well. Overnight cultures of each test strain (methicillin-resistant Staphylococcus aureus (MRSA, S. aureus ATCC BAA44) and carbapenem-resistant Acinetobacter baumannii (CRAB, A. baumannii ATCC BAA1605)) were diluted 5000-fold in fresh medium, and 50 μL of the dilution was added to each well. The lowest concentration at which no significant bacterial growth was observed after 16 hours of incubation at 37°C was recorded as the MIC value. All measurements were independently repeated three times.

[0175] High-resolution mass spectrometry (HPLC) detection: The synthesized BNP37 series peptide compounds were detected by HPLC using an ultra-high performance liquid chromatography-time-of-flight mass spectrometer (UPLC-QTOF). The LC conditions were as follows: CC18 column (Waters, C18-1.6 μm, 2.1 × 100 mm); mobile phase A was deionized water (containing 0.1% FA); mobile phase B was acetonitrile (containing 0.1% FA); the flow rate was 0.3 mL / min; and the gradient of mobile phase B was 30%–70%. MS detection used positive ion scanning, with real-time correction using leucine enkephalin solution (200 pg / μL, 10 μL / min); the detection range was 200–2000.

[0176] The following polypeptide compounds were obtained using the above preparation method:

[0177] 1. The polypeptide compound represented by formula (III)

[0178] Compounds of formula (III) can be linear (BNP37L) structures (Myristic acid-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-D-Tyr-Val-Asp) or X1-X 11 Lactam ring (BNP37C1), X2-X 11 Lactone ring (BNP37C2), X3-X 11 Lactam ring (BNP37C3), X5-X 11 The four cyclic peptide structures, including the lactam ring (BNP37C4), are shown in Table 2.

[0179] The polypeptide compounds represented by formula (III) in Table 2

[0180] 2. The polypeptide compound sequence shown in formula (II) is: R 1 -D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-D-Tyr-Val-Asp, exhibiting a linear polypeptide structure:

[0181] Among them, R 1 R represents the carbonyl compound obtained by removing the hydroxyl group from X0 in Table 3 when it forms an amide bond with X1. 2 for R 3 for

[0182] The polypeptide compounds represented by formula (II) in Table 3

[0183] 3. The polypeptide compound shown in formula (IV) has the following sequence: R 1 -D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-D-Tyr-Val-Asp, with X2 and X 11 lactone ring formation:

[0184] Among them, R 1 The carbonyl compound obtained by removing the hydroxyl group from X0 in Table 4 when it forms an amide bond with X1.

[0185] Table 4 shows the polypeptide compounds represented by formula (IV).

[0186] 3. The polypeptide compound shown in formula (V) has the following sequence: R 1 -D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-D-Dab-Val-Asp, where X2 and X 11 lactone ring formation:

[0187] Among them, R 1 The carbonyl compound obtained by removing the hydroxyl group from X0 in Table 5 when it forms an amide bond with X1.

[0188] Table 5 shows the polypeptide compounds represented by formula (V).

[0189] Example 5

[0190] X was performed based on the polypeptide compound Myristic acid-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-D-Tyr-Val-Asp from Example 4. 11 Amino acid scanning, X 11 By replacing Asp with different amino acids, the peptide compounds shown in Table 6-1 were obtained, and the corresponding high-resolution mass spectrometry information and activity information are shown in Table 6-2.

[0191] Table 6-1 BNP37L-X 11 amino acid structural sequence of polypeptide compounds

[0192] Table 6-2 BNP37L-X 11 High-resolution mass spectrometry of peptide compound structure and activity information

[0193] Example 6

[0194] Based on the BNP37C2 structure in Example 4: Myristic acid-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-D-Tyr-Val-Asp, an alanine scan was performed. All amino acids except X2 were replaced with alanine of the corresponding configuration (Ala), resulting in the polypeptide compounds shown in Table 7 below. The corresponding high-resolution mass spectrometry information and activity information are shown in Table 8.

[0195] Table 7. Scanned structural sequence of BNP37C2 alanine

[0196] Table 8 High-resolution mass spectrometry and activity information of BNP37C2 alanine.

[0197] Example 7

[0198] Based on the BNP37C2 structure in Example 4: Myristic acid-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-D-Tyr-Val-Asp, a 2,4-diaminobutyric acid (Dab) scan was performed. All amino acids except X2 were replaced with the corresponding Dab configurations, resulting in the polypeptide compounds shown in Table 9 below. The corresponding high-resolution mass spectrometry information and activity information are shown in Table 10.

[0199] Table 9. Scanned structural sequence of 2,4-diaminobutyric acid from BNP37C2

[0200] Table 10 High-resolution mass spectrometry and activity information of 2,4-diaminobutyric acid from BNP37C2

[0201] Example 8

[0202] Based on the BNP37C2-Dab9 structure in Example 7: Myristic acid-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-D-Dab-Val-Asp, for sites X1, X3, X5, X6, X7, X8, X9, X 10 X 11 Amino acid scanning was performed, and the amino acids were replaced with different amino acids to obtain the polypeptide compounds shown in Table 11 below. The corresponding high-resolution mass spectrometry information and activity information are shown in Table 12.

[0203] Table 11. Amino acid scanning sequence of each locus in BNP37C2-Dab9

[0204] Table 12 High-resolution mass spectrometry and activity information of amino acid structures at each point of BNP37C2-Dab9

[0205] Example 9

[0206] Using the methods described in Examples 2 and 3, paenimycin was synthesized with the sequence: Myristic acid-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-D-Dab-Val-Asp, and its structure is:

[0207] In this embodiment, the lidic acid moiety was synthesized as tetradecanoic acid, with the amino acid sequence: D-type 2,4-diaminobutyric acid-threonine-2,4-diaminobutyric acid-leucine-2,4-diaminobutyric acid-D-phenylalanine-leucine-2,4-diaminobutyric acid-D-tyrosine-valine-aspartic acid. The threonine side chain hydroxyl group forms a lactone bond with the α-carboxyl group of aspartic acid, ultimately yielding a decacyclic lactone undecyl lipopeptide containing a tetradecanoic acid fatty chain, named paenimycin, with the sequence: Myristic acid-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-D-Dab-Val-Asp. Paenimycin was dissolved in deuterated DMSO, and its structure was confirmed using a 600M NMR spectrometer, as shown in Figures 2-6.

[0208] Example 10

[0209] Bioactivity analysis of paenimycin

[0210] (1) In vitro detection of anti-multidrug-resistant bacteria activity

[0211] The antibacterial activity of paenimycin against common clinical bacterial pathogens was determined using the CLSI standard. The results are shown in Table 13 below, indicating that paenimycin has potent and broad-spectrum activity against multidrug-resistant bacteria.

[0212] Table 13 Antibacterial activity of paenimycin

[0213] (2) cytotoxicity detection of paenimycin

[0214] The cytotoxicity of paenimycin was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazole bromide (MTT) method. HepG2 cells were cultured in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. Cells were seeded into 96-well plates at a density of 5000 cells per well and cultured at 37°C and 5% CO2 for 24 hours. The medium was then removed, and 100 μL of fresh medium containing paenimycin at a final concentration of 64 μg / mL–0.25 μg / mL was added. Amphotericin B was used as a control. After incubation at 37°C and 5% CO2 for 48 hours, the medium was removed, and 110 μL of freshly prepared MTT solution (0.5 mg / mL) was added to each well. The mixture was incubated at 37°C for another 3 hours to form formazan crystals. Then, 100 μL of dissolving solution (40% DMF, 16% SDS, 2% acetic acid) was added to dissolve the precipitated formazan crystals. Finally, a microplate reader (Multiscan SkyHigh, Thermo Fisher) was used to analyze the OD... 570nm The absorbance of each well was measured. The effect of paenimycin on the IC50 of Hepg2 cells... 50 =34.84 μg / mL, far lower than the IC50 of amphotericin B. 50 =2.42 μg / mL. As shown in Figure 7, paenimycin exhibits low toxicity.

[0215] (3) Hemolytic activity test of paenimycin

[0216] Fresh, sterile, defiberized sheep blood was centrifuged, resuspended in PBS solution (pH 7.4), and diluted to 1×10⁻⁶. 9 Cells / mL. Paenimycin dilutions ranging from 100 μM to 0.38 μM were prepared and mixed with blood cell suspension to a final volume of 500 μL. 1% Triton X-100 and 1% DMSO were used as positive and negative controls, respectively. After incubation at 37°C for 3 hours, the cells were centrifuged at 3000 rpm for 20 minutes, and the supernatant was collected and transferred to 96-well plates. Hemolytic efficacy was measured using Multiscan SkyHigh absorbance at OD540 nm and calculated using Prism 9.0. All experiments were performed in triplicate (n=3). As shown in Figure 8, the results indicate that paenimycin at 100 μM has a low hemolytic effect. As shown in Figure 9, the HC50 value is greater than 100 μM, indicating low toxicity.

[0217] Example 11

[0218] In vivo antibacterial activity assessment of paenimycin

[0219] A mouse thigh muscle infection model with neutrophil deficiency was used.

[0220] Six-week-old, 23-27g female ICR mice free of specific pathogens were selected. Before the experiment, mice were divided into groups and allowed three days for environmental acclimatization. Cyclophosphamide (Sigma-Aldrich, CAS No.: 6055-19-2) was injected intraperitoneally at 150 mg / kg and 100 mg / kg, respectively, four days and one day before infection, to induce neutropenia in the mice. On the first day of infection, each mouse was injected intramuscularly with 50 μL of a suspension of Staphylococcus aureus ATCC BAA-44 / Escherichia coli MG1655-mcr-1 / Acinetobacter baumannii ATCC BAA-1605 / Klebsiella pneumoniae BNCC 353393 (approximately 1×10⁻⁶) in the thigh. 6 (cells). Two hours post-infection, mice were subcutaneously injected with different doses of paeminycin (1 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg) and their corresponding antibiotics meropenem, polymyxin E, vancomycin, and methicillin (20 mg / kg). Twenty-four hours post-infection, mice were euthanized, and the thigh muscle was aseptically excised, weighed, homogenized, and the bacterial load was calculated by colony-forming unit (CFU) count.

[0221] As shown in Figures 10-13, paenimycin showed a dose-dependent efficacy against carbapenem- and third-generation cephalosporin-resistant Acinetobacter baumannii. Subcutaneous administration of 5 mg / kg reduced the bacterial load in the mouse thigh by 2.17 log10, and at a concentration of 20 mg / kg, it reduced it by 2.40 log10, comparable to the effect of polymyxin E. Meropenem, a carbapenem antibiotic, was ineffective. Against the newly emerging polymyxin-resistant Escherichia coli with resistance mediated by the mgr-1 plasmid, paenimycin also showed a dose-dependent efficacy. Subcutaneous administration of 5 mg / kg reduced the bacterial load in the mouse thigh by 2.47 log10, and at a concentration of 20 mg / kg, it reduced it by 3.42 log10. Superior to meropenem, while ineffective against polymyxin E; against multidrug-resistant Klebsiella pneumoniae resistant to polymyxin, meropenem, and third-generation cephalosporins, paenimycin showed dose-dependent efficacy, with subcutaneous administration of 5 mg / kg reducing bacterial load in mouse thighs by 2.25 log10, and at a concentration of 20 mg / kg, reducing it by 3.12 log10, while polymyxin and meropenem were ineffective; against methicillin-resistant Staphylococcus aureus, paenimycin showed dose-dependent efficacy, with subcutaneous administration of 5 mg / kg reducing bacterial load in mouse thighs by 1.24 log10, and at a concentration of 20 mg / kg, reducing it by 1.79 log10, comparable to vancomycin, while methicillin was ineffective.

[0222] Paenimycin maintains good antibacterial activity in animals, suggesting its potential for development as a novel antibacterial drug.

[0223] Example 12

[0224] In vivo pharmacokinetic studies of Paenimycin

[0225] Pharmacokinetic studies were conducted using 6-8 week old male SD rats (200-300g). Paenimycin was administered intravenously (IV, 5 mg / kg) and subcutaneously (SC, 10 mg / kg). Blood samples were collected at the following time points after intravenous administration: before administration, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 24 hours, and 48 hours after administration; and before administration subcutaneously, 1 hour, 3 hours, 8 hours, 24 hours, 32 hours, 48 ​​hours, 56 hours, 72 hours, and 96 hours after administration. At each time point, 0.1 mL of blood was collected from each rat via jugular vein puncture. Samples were transferred to plastic microcentrifuge tubes containing EDTA-K2 and stored on ice. Blood samples were centrifuged at 4000g for 5 minutes at 4°C, and plasma was collected within 30 minutes. Plasma samples were stored at -80°C.

[0226] HPLC-MS / MS pharmacokinetic analysis: Paenimycin (1 mg / mL, in DMSO) was serially diluted with ACN:water (50:50) to establish a standard curve and quality control solution. 5 μL of peak-setting solution was added to 50 μL of drug-free plasma to establish standards and quality controls. 55 μL of standard, 55 μL of QC sample, and 55 μL of unknown sample (50 μL rat plasma with 5 μL blank solution) were added to 200 μL of dexamethasone (10 ng / mL internal standard) and acetonitrile, respectively, to precipitate proteins.

[0227] The extract was vortexed for 1 minute and centrifuged at 4000 rpm for 15 minutes. The supernatant was diluted 3-fold with water. 20 μL of the diluted supernatant was injected into an LC / MS / MS system for quantitative analysis. HPLC-MS / MS analysis was performed on a Sciex Applied Biosystems Triple Quad 5500+ or ​​6500+ mass spectrometer coupled to a Shimadzu Nexera series system controller CBM-40UPLC (ultra-high performance liquid chromatography) system to quantify each drug in plasma. Chromatography was performed using a Raptor biphenyl column (3x30 mm; particle size 2.7 μm) with a reversed-phase gradient method. The mobile phase consisted of 5% acetonitrile-0.1% formic acid-ultrapure water as the aqueous phase and 95% acetonitrile-0.1% formic acid-ultrapure water as the organic phase. Multiple reaction monitoring (MRM) was used for quantitative analysis of parent / daughter transition in positive electrospray ionization mode. The MRM variations of paenimycin (467.00 / 461.50) and dexamethasone (393.06 / 373.00) are as follows. Samples were accepted for analysis if the concentration of the quality control sample was within 20% of the nominal concentration. Data processing was performed using Analyst software (v1.7.3; Applied Biosystems Sciex). The results are shown in Figure 14. Paenimycin exhibited favorable kinetics, with a half-life of 20.2 hours, a maximum concentration (Cmax) of 5200 ng / mL, an area under the curve (AUC0-∞) of 115251 h*ng / mL, and a bioavailability of 102%. These in vitro and in vivo experiments collectively demonstrate that paenimycin has a broad therapeutic window.

[0228] Example 13

[0229] paenimycin in vivo nephrotoxicity evaluation

[0230] Six-week-old, 23-27g female ICR mice free of specific pathogens were selected. Mice were grouped and acclimatized for three days prior to the experiment. Paenimycin was prepared as a 0.9% saline solution containing 10% DMSO and 0.5% Tween 80. Positive controls and placebos were prepared using colistin or antibiotic-free solutions, respectively. The subcutaneous injection dose was 40 mg / kg of paenimycin once daily for 1 or 7 days. Serum samples were collected 24 hours after the last administration. The concentrations of toxicity-related biomarkers, including kidney injury molecule-1 (KIM-1), tissue inhibitor of metalloproteinases-1 (TIMP-1), neutrophil gelatinase-associated lipofuscin (NGAL), and bone growth factor (OPN), were then determined using a commercially available kit (Yunkron, China) according to the kit's protocol. Finally, all animals were euthanized, and kidney tissue was collected, fixed, dissected, and stained with H&E. As shown in Figure 15, after administration of paenimycin, the nephrotoxicity-related indicators in the body were not significantly different from those in the placebo group, while the nephrotoxicity indicators in the polymyxin E group increased significantly. The conclusion drawn from the slice scoring analysis was that paenimycin caused almost no kidney damage, which was the opposite of the polymyxin E group.

[0231] Example 14

[0232] Acute toxicity test of Paenimycin

[0233] Six-week-old, 23-27g female ICR mice free of specific pathogens were selected. Before the experiment, the mice were divided into groups and allowed to acclimatize to the environment for three days. Paenimycin was prepared as a 0.9% saline solution containing 10% DMSO and 0.5% Tween 80, and administered subcutaneously at doses of 100 mg / kg, 200 mg / kg, 300 mg / kg, and 400 mg / kg, respectively. As shown in Table 14, in the highest dose group, all mice survived 24 hours after administration, suggesting that paenimycin has a high safety profile.

[0234] Table 14 Acute toxicity test of paenimycin

[0235] Example 15

[0236] Study on the mechanism of action of paenimycin

[0237] (1) Bacterial lysis experiment

[0238] Single colonies of Staphylococcus aureus BNCC 186335 were picked and incubated overnight at 37°C and 220 rpm on a shaker. The bacteria were collected by centrifugation, resuspended in sterile PBS (pH 7.4), and diluted to OD200.600nm =0.35. Then, 900 μL of bacterial suspension was mixed with 100 μL of 17 μM SYTOX green nucleic acid dye (Thermo Fisher, USA), incubated at 37°C in the dark for 5 minutes, and transferred to 384-well flat-bottom black microplates (30 μL per well). The initial fluorescence intensity (excitation wavelength / emission wavelength = 488 / 523 nm) of each well was measured at 9-second intervals using a microplate reader (Infinite 200Pro, Tecan). After 5 minutes, the same volume of paenimycin solution with concentrations of 1, 2, 4, 8, and 10 × MIC was added to each well, with bee venom and DMSO used as positive and negative controls, respectively. Fluorescence intensity was continuously monitored for 25 minutes, and fluorescence intensity graphs were plotted using Prism 9.0. As shown in Figure 16, paenimycin at high concentrations causes an increase in fluorescence intensity, suggesting its bacterial lysis activity.

[0239] (2) Membrane depolarization test

[0240] Single colonies of Staphylococcus aureus BNCC 186335 were picked and incubated overnight at 37°C and 220 rpm on a shaker. The bacteria were collected by centrifugation, washed twice with 5 mM HEPES buffer, and resuspended in 5 mM HEPES buffer containing 20 mM glucose (OD200). 600nm =0.1). Take 1 mL of cell suspension, add 2 μL of 500 μM 3,3'-dipropylthiodinium dicyanocyanate [DiSC3(5)] dye (Macklin, CN), incubate at 37°C in the dark for 30 minutes, and transfer to 384-well flat-bottom black microplates (100 μL per well). Use a microplate reader to record the initial fluorescence intensity of each well at 9-second intervals (excitation wavelength / emission wavelength = 620 / 670 nm). After 5 minutes, add paenimycin solutions at concentrations of 1, 2, 4, 8, and 10 × MIC to each well, using Triton X-100 and DMSO as positive and negative controls, respectively, and continuously monitor the fluorescence intensity for 25 minutes. As shown in Figure 17, no increase in fluorescence intensity was detected in the paenimycin group at the highest test concentration, indicating that it does not cause cell membrane depolarization.

[0241] (3) Potassium ion release test

[0242] A single colony of Staphylococcus aureus BNCC 186335 was inoculated into 50 mL of LB medium and cultured overnight at 37°C and 220 rpm on a shaker. The bacteria were collected, washed twice with buffer (10 mM Tris-acetate, 100 mM NaCl, pH 7.4), and then resuspended in the same buffer to adjust the bacterial concentration to OD0.05. 600nm=1.0. Extracellular potassium ion concentration changes were measured using an Orion Dual Star pH / ISE meter at 5-second intervals. Paenimycin solutions at concentrations of 1, 2, 4, and 8 × MIC, and Gramicidin solution at a concentration of 8 × MIC (16 μg / mL), were added, and measurements were taken continuously for 8 minutes. As shown in Figure 18, after paenimycin treatment, the potassium ion concentration in the solution gradually increased, indicating that the bacteria began to excrete large amounts of potassium ions, and the efflux concentration was dose-dependent on the administered concentration.

[0243] (4) Sterilization curve test

[0244] Single colonies of Staphylococcus aureus BNCC 186335 and Escherichia coli ATCC 25922 were inoculated separately and cultured overnight in a shaker at 37°C and 220 rpm. The cultures were then diluted to a final bacterial concentration of 1 × 10⁻⁶. 6 CFU / mL. Paenimycin solution at 1, 4, and 8×MIC was added to the bacterial dilution; vancomycin at 8×MIC (8 μg / mL) (in Staphylococcus aureus BNCC 186335) or polymyxin E at 8×MIC (4 μg / mL) (in Escherichia coli ATCC 25922) was added. 1, 2, 4, 8, and 16 hours after the start of the experiment, 100 μL of bacterial suspension was taken, serially diluted, and plated onto LB agar plates. For Escherichia coli, the time intervals were increased to 0.25, 0.5, and 0.75 hours. The agar plates were incubated overnight at 37°C, and colonies were counted. As shown in Figure 19, paenimycin is a potent bactericide that exerts its bactericidal effect rapidly, killing pathogens within 4 hours.

[0245] (5) Scanning electron microscopy observation of the morphology of bacteria after paenimycin treatment

[0246] The bacterial samples of Staphylococcus aureus BNCC 186335 and Escherichia coli ATCC 259220h, 4h, and 8h were treated with 8×MIC in step (4), and the morphology of the bacteria at different treatment times was observed using scanning electron microscopy. The results are shown in Figure 20. Under the action of paeminycin, the cell membrane of Staphylococcus aureus ruptured and shrank after 1 hour, and completely shrank after 4 hours, losing its spherical structure; the cell membrane of Escherichia coli ruptured partially after 1 hour and 4 hours, and the bacterial morphology was maintained.

[0247] (6) Paenimycin resistance test

[0248] Single colonies of Staphylococcus aureus BNCC 186335 and Escherichia coli ATCC 25922 were inoculated separately and cultured in a shaker at 37°C and 220 rpm. The overnight cultures were then diluted 5000 times in the culture medium (final concentration 1×10⁻⁶). 6 The MIC values ​​of paenimycin, ciprofloxacin, and bacitracin against Staphylococcus aureus and paenimycin, ciprofloxacin, and tetracycline against Escherichia coli were determined using a assay (CFU / mL). For bacitracin, 50 μg / mL ZnCl2 was added to LB medium for measurement. The following day, bacterial cultures from the wells containing the sub-MIC concentrations of each antibiotic were diluted 500-fold in fresh medium, and the new MICs were tested using the same method described above. Subculture was performed for 28 consecutive days. As shown in Figure 21, unlike clinically used antibiotics, paenimycin did not induce resistance after continuous subculturing, suggesting that it is unlikely to induce drug-resistant bacteria in further clinical applications.

[0249] (7) Feeding experiment

[0250] Using *Staphylococcus aureus* BNCC 186335 and *Escherichia coli* ATCC 25922 as experimental materials, the effects of bacterial cell components on the antibacterial activity of paenimycin were studied. Cell components (peptidoglycan, total protein, genomic DNA, lipopolysaccharide, and lipoteichoic acid) from Gram-positive and Gram-negative bacteria were dissolved in water at a concentration of 5 mg / mL. Lipid A was dissolved in chloroform and premixed with 0.0156–1 mg / mL paenimycin solution for 15 minutes. The premixed solution was then air-dried. Each solution was added to a single well of a 96-well plate and diluted sequentially to a final concentration of 0.0078–0.5 mg / mL. The MIC values ​​were determined using the same method as for MIC determination. The fold change in MIC was calculated using the formula: Final MIC / Original MIC. As shown in Figure 22, paenimycin can bind to lipopolysaccharide, lipid A, and lipoteichoic acid.

[0251] (8) Target point determined by isothermal calorimetric titration

[0252] The binding of paenimycin to lipopolysaccharide (LPS) and lipoteichoic acid (LPA) was determined using the ITC method. The ITC experiment was performed using a PEAQ-ITC (Malvern, UK) instrument at 25°C. A 5 mM paenimycin or 1 mM polymyxin E solution was prepared using 5 mM HEPES buffer (pH 7.4), along with 100 μM LPS or LTA. The titration process involved an initial sample volume of 0.23 μL, with 2 μL injected every 80 seconds, and continuous stirring at 500 rpm. Data were analyzed using PEAQ-ITC software, and thermodynamic parameters [enthalpy (ΔH), entropy (ΔS), and equilibrium binding constant (Kd)] were calculated using a single-binding-site model. The results are shown in Figure 23. The Kd value for paenimycin binding to LPS was 2.18 μM, comparable to the Kd value of 2.00 μM for polymyxin E binding to LPS. In addition, paenimycin binds to lipoteichoic acid with a Kd value of 5.61 μM, while polymyxin E does not bind.

[0253] (10) BODIPY TR cadaverine (BC) dye substitution experiment

[0254] The affinity of Paenimycin for lipid A was determined based on its ability to displace BC from a lipid A-BC mixture. 20 μg / mL LPS was premixed with 20 μM BC in 5 mM HEPES buffer (pH 7.4) and incubated at 37°C for 30 min. The mixture was then transferred to 96-well flat-bottom black microplates (50 μL per well). 50 μL of Paenimycin solution dissolved in 5 mM HEPES was added, resulting in a final concentration of 0.0 μg / mL–10.0 μg / mL. Polymyxin E and kanamycin at the same concentrations (0.0 μg / mL–10.0 μg / mL) served as positive and negative controls, respectively. After incubation in the dark for 5 min, fluorescence intensity was measured using a microplate reader (Infinite 200Pro, Tecan) (excitation / emission wavelength = 580 / 620 nm). As shown in Figure 24, similar to polymyxin E, paenimycin can replace the BC dye bound to lipid A, which is different from kanamycin, suggesting that its binding site is lipid A.

[0255] (11) Lipid A extraction and feeding experiment

[0256] Lipid A and pEtN-modified lipid A were extracted from *Escherichia coli* MG1655 and *Escherichia coli* MG1655-mcr-1 according to the prescribed method. Briefly, the bacteria were chemically lysed using a mixture of chloroform, methanol, and water (Bligh-Dyer) solvent, and lipopolysaccharide was precipitated by centrifugation. Lipid A was then extracted from the particulate mixture using a combination of mild acid hydrolysis and solvent extraction. Lipid A was defined as the chloroform-soluble fraction of lipopolysaccharide after mild acid hydrolysis. After lyophilization, the crude lipid A was dissolved in 500 μL of chloroform and added to a feeding experiment mixture containing 4×MIC paeminycin and diluted *E. coli* MG1655 bacterial culture (concentration 1×10⁻⁶). 6 In a 96-well plate containing (CFU / mL) polymyxin E as a control, OD was measured using Multiscan SkyHigh at 37°C. 600nm After 16 hours, growth curves were plotted. As shown in Figure 25, unlike polymyxin E, paenimycin maintained its antibacterial activity after the addition of lipid A and pEtN-modified lipid A, suggesting that it can still bind to pEtN-modified lipid A.

[0257] (12) Molecular dynamics simulation

[0258] Lipid A and polymyxin E structures were extracted from PDB 1QFF and 8DEV, respectively. The 3D model of paeminycin was optimized using MOE. An initial complex model was obtained by placing the ligand close to the receptor, and ionic charge interactions between the receptor (lipid A or LTA) and the ligand (polymyxin E or paeminycin) were realized. The complex model was then submitted to Desmond (Desmond / Maestro non-commercial version 2022.1) for MD simulations. First, a 1,2-dipalmitoylphosphatidylcholine (DPPC) membrane model was automatically added, and then the orientation of the DPPC was modified so that the hydrophobic tail of the ligand was located at the center of the membrane plane. The complex was neutralized with Na or Cl ions, using a solution containing 0.15M NaCl. Solvation was performed using an orthogonal TIP3P water buffer box (default parameters in Desmond). The MD simulation consisted of a 5-step minimization with gradual constraint release, followed by a 200ns production run without constraints in the NPgT ensemble. The first step of the minimization was to confine the solute heavy atoms for 100 ps in the NVT ensemble at 10 K. The second step was to confine the membrane along the z-axis and protein atoms for 20 ps in the NPT ensemble at 100 K. The third step was to confine the membrane along the z-axis and protein atoms for 100 ps in the NPgT ensemble at 100 K. The fourth step was to heat the NPgT ensemble from 100 to 300 K for 150 ps. The fifth step was to remove all constraints in the NVT ensemble for 100 ps. The 200ns simulation was performed at 300 K with 1 bar in the NPgT ensemble, repeated 3 times, saving the ballistic coordinates every 100 ps. Geometric analysis was performed using the analyze_simulation.py script in Desmond. As shown in Figure 26, paenimycin binds to the phosphate groups on both sides of the hexose of lipid A and the hydroxyl group at the six-position of the side chain.

[0259] (13) Cell wall precursor accumulation experiment

[0260] The effect of paenimycin on cell wall biosynthesis was determined by measuring the accumulation of the cell wall precursor UDP-MurNAc-pentapeptide after antibiotic treatment. Single colonies of Staphylococcus aureus BNCC 186335 were inoculated into 50 mL of LB medium and cultured overnight at 37°C and 220 rpm on a shaker. The bacterial culture was then transferred to fresh medium and cultured until OD... 600nm =0.5. Then, chloramphenicol solution with a final concentration of 130 μg / mL was added, and the mixture was incubated at room temperature for 15 minutes. Paenimycin and vancomycin were added at a dose of 10×MIC, with DMSO as a negative control. After incubation for 1 hour, the bacteria were precipitated, and the bacterial resuspended in 30 μL of RO water and boiled for 15 minutes. After the solution cooled, it was centrifuged at 15000g for 5 minutes, and the supernatant was analyzed by UPLC-MS. As shown in Figure 27, unlike vancomycin, paenimycin did not lead to the accumulation of the cell wall precursor UDP-MurNAc-pentapeptide, suggesting that its mechanism of action is different from that of vancomycin.

[0261] (14) Extraction and conjugation of teichoic acid

[0262] Chitin was extracted from Staphylococcus aureus BNCC 186335. In short, crude peptidoglycan was extracted from Staphylococcus aureus, then completely washed with 4% SDS to remove lipoteichoic acid. The peptidoglycan mixture was then hydrolyzed with trichloroacetic acid to obtain water-soluble chitin. The lyophilized crude chitin was dissolved in water and diluted to 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL, respectively. These solutions were then mixed with 1 mg / mL paenimycin at w / w ratios of 0:1, 1:1, 2:1, 3:1, 4:1, and 5:1 and incubated at room temperature for 1 hour. The mixture was then centrifuged at 15,000 g for 5 minutes to precipitate the insoluble substances. The paenimycin content in the supernatant was detected by UPLC-MS, and the peak area change rate was calculated. As shown in Figure 28, paenimycin binds to chitin in a dose-dependent manner, unlike vancomycin.

[0263] In summary, the mechanism of action studies demonstrate that the BNP37 series compounds in this invention, represented by paenimycin, possess a unique and novel dual-target mechanism of action. They exhibit potent activity against both Gram-negative and Gram-positive drug-resistant bacteria, effectively addressing the challenge of treating complex drug-resistant bacterial infections. Against Gram-negative bacteria, paenimycin binds to the phosphate groups on both sides of the hexose group of lipid A in the cell wall and the hydroxyl group at the six-position of the side chain, disrupting the cell membrane structure and thus exerting its antibacterial effect. This mechanism differs from the action site of existing polymyxins, therefore exhibiting good in vitro and in vivo bactericidal effects against polymyxin-resistant strains, including acquired and naturally resistant strains. On the other hand, against Gram-positive bacteria, BNP37 binds to teichoic acid in the bacterial cell wall and the phosphate groups in its long-chain repeating units, disrupting the bacterial cell membrane and leading to bacterial death. Currently, no antibiotics exert their activity through this target.

Claims

1. A polypeptide compound or a pharmaceutically acceptable salt thereof, characterized in that, The structural sequence of the polypeptide compound is shown in formula (I), X0-X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 (I) in, X0 is selected from carboxylic acid compounds; X1, X3, X5, and X8 are each independently selected from substituted or unsubstituted L-type or D-type basic amino acids; X2 is selected from L-type or D-type amino acids with substituted or unsubstituted side chains containing amino or hydroxyl groups; X4, X6, X7, X 10 Each amino acid is independently selected from substituted or unsubstituted L-type or D-type hydrophobic amino acids; X9, X 11 Each amino acid is independently selected from substituted or unsubstituted L-type or D-type amino acids; The carboxyl group in X0 and the amino group in X1 form an amide bond; The polypeptide compound is a linear peptide or a cyclic peptide, wherein the cyclic peptide is any one of X1, X3, and X5 and X. 11 The linked lactam cyclic peptide, or the cyclic peptide being X2 and X... 11 Linked lactam cyclic peptides or lactone cyclic peptides; Each of the above substitutions is optionally replaced by one or more substituents R independently selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclic group, C(=O)NH2, NHC(=O)NH2 or COOH. a1 The 4-7 membered heterocyclic group comprises 1 to 3 heteroatoms independently selected from N, O or S.

2. The polypeptide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, In formula (I), X1, X3, X5, and X8 are each independently selected from the following substituted or unsubstituted basic amino acids of the L-type or D-type: Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Orn (ornithine), Lys, Arg, His, D-Dab, D-Dap, D-Orn, D-Lys, D-Arg, or D-His; X2 is selected from the following substituted or unsubstituted amino acids of L or D type with an amino or hydroxyl side chain: Dab, Dap, Orn, Lys, Arg, His, Tyr, Thr, allo-Thr (allo-threonine), Ser, D-Dab, D-Dap, D-Orn, D-Lys, D-Arg, D-His, D-Tyr, D-Thr, D-allo-Thr, or D-Ser; X4, X6, X7, X 10 Each of the following substituted or unsubstituted hydrophobic amino acids, independently selected from L-type or D-type: Ala, Leu, Ile, Phe, Met, Trp, Pro, Val, D-Ala, D-Leu, D-Ile, D-Phe, D-Met, D-Trp, D-Pro, or D-Val; X9, X 11 Each of the following substituted or unsubstituted amino acids, independently selected from L- or D-type amino acids: Asp, Ala, Arg, Asn, Dab, Dap, Gln, Gly, His, Ile, Leu, Lys, Met, Orn, Phe, Ser, Thr, Trp, Tyr, Val, D-Asp, D-Ala, D-Arg, D-Asn, D-Dab, D-Dap, D-Gln, D-Gly, D-His, D-Ile, D-Leu, D-Ly s, D-Met, D-Orn, D-Phe, D-Ser, D-Thr, D-Trp, D-Tyr, or D-Val; wherein each of the above substitutions is optionally replaced by one or more substituents R independently selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclic group, C(=O)NH2, NHC(=O)NH2, or COOH. a1 The 4-7 membered heterocyclic group comprises 1 to 3 heteroatoms independently selected from N, O or S; X0 is selected from fatty acids or aromatic carboxylic acids, wherein the fatty acid is a saturated fatty acid or an unsaturated fatty acid.

3. The polypeptide compound or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, In formula (I), X1, X3, X5, and X8 are each independently selected from substituted or unsubstituted L-type or D-type amino acids: Dab, Dap, Orn, Lys, Arg, or His; X2 is selected from substituted or unsubstituted L-type or D-type amino acids: Thr or Ser; X4, X6, X7, X8 are selected from substituted or unsubstituted L-type or D-type amino acids: Thr or Ser; 10 Each amino acid is independently selected from substituted or unsubstituted L- or D-type amino acids: Leu, Phe, or Val; X9, X 11 Each of the following amino acids is independently selected from substituted or unsubstituted L- or D-type amino acids: Asp, Ala, Dab, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Tyr, Trp, or Val; wherein each of the above substitutions is optionally replaced by one or more substituents R independently selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclic group, C(=O)NH2, NHC(=O)NH2, or COOH. a1 The 4-7 membered heterocyclic group comprises 1 to 3 heteroatoms independently selected from N, O or S.

4. The polypeptide compound or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that, In formula (I), X1 is substituted or unsubstituted D-Dab, X2 is substituted or unsubstituted Thr, X3 is substituted or unsubstituted Dab, X4 is substituted or unsubstituted Leu, X5 is substituted or unsubstituted Dab, X6 is substituted or unsubstituted D-Phe, X7 is substituted or unsubstituted Leu, X8 is substituted or unsubstituted Dab, X9 is substituted or unsubstituted D-Tyr or D-Dab, X... 10 For Val, whether or not it is replaced, X 11 The Asp can be substituted or unsubstituted; wherein each of the above substitutions is optionally replaced by one or more substituents R independently selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclic group, C(=O)NH2, NHC(=O)NH2 or COOH. a1 The 4-7 membered heterocyclic group comprises 1 to 3 heteroatoms independently selected from N, O or S.

5. The polypeptide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The polypeptide compound represented by formula (I) contains X1-X 11 Amino acid sequences selected from any of the following groups; X0 is selected from fatty acids or aromatic carboxylic acids, wherein the fatty acid is a saturated fatty acid or an unsaturated fatty acid.

6. The polypeptide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, In formula (I), X0 is selected from Myristic acid, Butyric acid, Decanoic acid, Lauric acid, Palmitic acid, Stearic acid, Sorbic acid, Neo-decanoic acid, 4-Methylnonanoic acid, Benzofuran-2-carboxylic acid, Indole-2-carboxylic acid, 2-Quinoxalinecarboxylic acid, 2-Biphenylcarboxylic acid, 9-Anthracenecarboxylic acid, 2-aminonicotinic acid, and 9-Fluorenone-4-carboxylic acid. 9-Fluorenone-2-carboxylic acid, 3-Biphenylcarboxylic acid, 4-piperidin-1-ylbenzoic acid, 4-Morpholinobenzoic Acid, 4-(4-Methyl-piperazin-1-yl)-benzoic acid, 3-(4-Methylpiperazin-1-yl)benzoic acid, 4”-(Pentyloxy)-1,1':4',1”-terphenyl-4-carboxylic acid (p-pentoxyterphenylcarboxylic acid), Undecanoic acid (undecanoic acid), Tridecylic acid (tetrate acid), Pentadecanoic acid (pentadecanoic acid), Heptadecanoic acid (heptadecanoic acid), 8-Phenyloctanoic acid (8-phenyloctanoic acid), 4-Cyanobenzoic acid (4-cyanobenzoic acid), 4-(4-Fluorophenyl)benzoic acid (4-fluorophenylbenzoic acid), 4-phenylcyclohexane-1-carboxylic acid (4-phenyl-cyclohexanecarboxylic acid), 4-cyclopropylbenzoic acid (4-cyclopropylbenzoic acid), 2,4-dichlorobenzoic acid (2,4-dichlorobenzoic acid), p-toluic acid (p-methylbenzoic acid), 4-chlorobenzoic acid (4-chlorobenzoic acid), 4-bromobenzoic acid (4-bromobenzoic acid), 4-fluorobenzoic acid (4-fluorobenzoic acid), 4-Phenylbenzoic acid 4-phenylbenzoic acid, 4′-chloro-[1,1′-biphenyl]-4-carboxylic acid, 4′-bromo-[1,1′-biphenyl]-4-carboxylic acid, or 4-(phenylethynyl)benzoic acid, 2-Ethylhexanoic acid, 10-Undecenoic acid, 2-hydroxynicotinic acid, or 3-hydroxytetradecanoic acid.

7. The polypeptide compound or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that, X0 in formula (I) is preferably selected from Myristic acid (tetradecanoic acid).

8. The polypeptide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The polypeptide compound structure sequence represented by formula (I) is selected from X0-Dab-Thr-Dab-Leu-Dab-Phe-Leu-Dab-X9-Val-X 11 or X0-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-X9-Val-X 11 The polypeptide compound is a linear peptide or a cyclic peptide, wherein the cyclic peptide is any one of X1, X3, and X5, or a combination of D-Dab and X. 11 The linked lactam cyclic peptide, or the cyclic peptide being Thr and X 11 A cyclic lactone peptide formed by linkage.

9. The polypeptide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The polypeptide compound structure sequence shown in formula (I) is selected from X0-Dab-Thr-Dab-Leu-Dab-Phe-Leu-Dab-X9-Val-Asp, or X0-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-X9-Val-Asp. The peptide compound is a linear peptide or a cyclic peptide. The cyclic peptide is a lactam cyclic peptide formed by linking any one of the Dabs or D-Dabs in X1, X3, and X5 with Asp, or the cyclic peptide is a lactone cyclic peptide formed by linking Thr with Asp.

10. The polypeptide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The polypeptide compound structure sequence shown in formula (I) is selected from X0-Dab-Thr-Dab-Leu-Dab-Phe-Leu-Dab-X9-Val-Asp or X0-D-Dab-Thr-Dab-Leu-Dab-D-Phe-Leu-Dab-X9-Val-Asp, wherein X0 is selected from tetradecanoic acid, X9 is selected from L-type or D-type Tyr or L-type or D-type Dab, the peptide compound is a linear peptide or a cyclic peptide, the cyclic peptide is a lactam cyclic peptide formed by linking any one of X1, X3, X5 Dab or D-Dab with Asp, or the cyclic peptide is a lactone cyclic peptide formed by linking Thr with Asp.

11. The polypeptide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The polypeptide compound is shown in formula (II): Among them, R 1 R is the carbonyl compound obtained by removing the hydroxyl group from X0 when it forms an amide bond with X1 as described in claim 1. 2 for X9 is either D-Tyr or D-Dab, R 3 X is any amino acid side chain. 11 The amino acid is either L-substituted or unsubstituted, wherein the substitution is optionally made by one or more substituents R independently selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclic group, C(=O)NH2, NHC(=O)NH2 or COOH. a1 The 4-7 membered heterocyclic group comprises 1 to 3 heteroatoms independently selected from N, O, or S; the peptide compound is a linear peptide or a cyclic peptide, wherein the cyclic peptide is any one of X1, X3, and X5 (Dab or D-Dab combined with X). 11 The linked lactam cyclic peptide, or the cyclic peptide being Thr and X2 of X2. 11 A cyclic lactone peptide formed by linkage.

12. The polypeptide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The polypeptide compound is shown in formula (III): The peptide compound is a linear peptide or a cyclic peptide. The cyclic peptide is a lactam cyclic peptide formed by linking any one of the Dabs or D-Dabs (X1, X3, X5) with Asp, or the cyclic peptide is a lactone cyclic peptide formed by linking Thr with Asp.

13. The polypeptide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The polypeptide compound is shown in formula (IV): Among them, R 1 It is the carbonyl compound obtained by removing the hydroxyl group from X0 when it forms an amide bond with X1 as described in claim 1.

14. The polypeptide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The polypeptide compound is shown in formula (V): Among them, R 1 It is the carbonyl compound obtained by removing the hydroxyl group from X0 when it forms an amide bond with X1 as described in claim 1.

15. The polypeptide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The polypeptide compound is shown in formula (VI):

16. Use of a polypeptide compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-15 in the preparation of an antibacterial drug.

17. The application according to claim 16, characterized in that, The bacteria are any one of the following clinical pathogens: Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, Neisseria gonorrhoeae, Staphylococcus aureus, and Enterococcus faecalis.

18. A pharmaceutical composition of an antibacterial drug, characterized in that, It comprises the polypeptide compound of any one of claims 1-15 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to claim 18, characterized in that, The pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhaler, ointment, suppository or patch.

20. The use of a nonribosomal polypeptide compound biosynthesis gene cluster BNP37 in the synthesis of any of the polypeptide compounds of claims 1-15 or pharmaceutically acceptable salts thereof, wherein the nucleotide sequence of the biosynthesis gene cluster BNP37 has the GenBank accession number: NZ_JAQAGY010000015.1.

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