Non-ribosome polypeptide compound as well as synthetic gene cluster BNP37 and application thereof

Abstract

The invention provides a non-ribosome polypeptide compound as well as a synthetic gene cluster BNP37 and application thereof, the structural sequence of the polypeptide compound is X0-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11, the polypeptide compound is a linear peptide or a cyclic peptide, and the cyclic peptide is a lactam cyclic peptide formed by connecting any one of X1, X3 and X5 with X11, or a lactam cyclic peptide or a lactone cyclic peptide formed by connecting X2 with X11. The compound disclosed by the invention can cause discharge of important ions in bacterial cells by targeting Gram-negative bacterium outer membrane lipid A and Gram-positive bacterium teichoic acid, so that bacterial death is caused. The compound disclosed by the invention has strong in-vivo and in-vitro antibacterial activity on a plurality of multi-drug-resistant gram-negative pathogenic bacteria such as escherichia coli, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, enterobacter cloacae and neisseria gonorrhoeae and a plurality of multi-drug-resistant gram-positive pathogenic bacteria such as staphylococcus aureus and enterococcus faecium.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial natural products, and particularly relates to a non-ribosomal polypeptide compound, a synthetic gene cluster BNP37 thereof and application thereof. BACKGROUND

[0002] The overuse of antibiotics has led to the faster emergence of drug-resistant bacteria and drug-resistant genes, reducing the therapeutic potential of antibiotics for humans and animals, and the problem of bacterial drug resistance is becoming increasingly serious, and even multiple drug-resistant bacteria have emerged. Therefore, the demand for the development of new antibiotics is particularly urgent.

[0003] Among multiple drug-resistant bacterial infections, gram-negative bacterial infections are the most serious. In clinical infections, gram-negative bacterial strains account for as high as 70.1%, especially multiple drug-resistant bacteria such as carbapenem-resistant gram-negative bacteria (CRO) pose a great challenge to clinical anti-infection treatment. Among them, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa are the most common drug-resistant bacteria, which are the most common and most concerned among CROs, and are also the most serious drug-resistant bacteria threatening the clinic. These bacteria have high drug resistance to many clinically commonly used antibacterial drugs, and the detection rate is showing a continuous upward trend. Due to the unique double-layer cell membrane structure of gram-negative bacteria and the complex drug resistance mechanism of various drug efflux pumps, the development of new antibiotics against this type of bacteria has almost no innovative drugs on the market in the past 40 years. Polymyxin drugs were marketed and applied in the clinic in 1947, and due to their obvious nephrotoxicity and neurotoxicity, they were once gradually withdrawn from clinical use. However, with the emergence and prevalence of CRO infections, polymyxin drugs have returned to the clinic because they are effective against almost all CROs and are used as the last line of defense against gram-negative bacterial infections. However, while bringing good therapeutic effects, the drug resistance of polymyxin drugs cannot be ignored. In addition to natural drug resistance, the emergence and widespread transmission of the plasmid-mediated polymyxin resistance (mcr-1) gene in 2015 pose a major threat to the efficacy of polymyxins. In summary, the clinical application value of polymyxin drugs worldwide cannot be replaced, but at the same time, they are facing the problems of increasing drug resistance and toxicity. Therefore, the development of new antibacterial compounds is particularly important to cope with the problem of drug resistance of pathogenic bacteria. SUMMARY

[0004] The present application aims to provide a non-ribosomal polypeptide compound, which can effectively solve the problems of serious drug-resistant pathogens, especially gram-negative bacterial drug-resistant pathogens, few available antibacterial drugs, and large toxic and side effects in the prior art.

[0005] The present application also provides a synthetic gene cluster of a new non-ribosomal polypeptide compound and application thereof.

[0006] Technical solution: In order to achieve the above-mentioned purpose, the polypeptide compound or its pharmaceutically acceptable salt according to the present application, the structure sequence of the polypeptide compound is shown as formula (I),

[0007] X0-X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11

[0008] (I)

[0009] Among them,

[0010] X0 is selected from carboxylic acid compounds;

[0011] X1, X3, X5, X8 are each independently selected from substituted or non-substituted L-type or D-type basic amino acids;

[0012] X2 is selected from substituted or non-substituted L-type or D-type amino acids with side chain containing amino or containing hydroxyl;

[0013] X4, X6, X7, X 10 are each independently selected from substituted or non-substituted L-type or D-type hydrophobic amino acids;

[0014] X9, X 11 are each independently selected from substituted or non-substituted L-type or D-type amino acids;

[0015] The carboxyl in X0 and the amino in X1 form an amide bond;

[0016] The polypeptide compound is a linear peptide or a cyclic peptide, the cyclic peptide is a lactam cyclic peptide formed by any one of X1, X3, X5 and X 11 connected, or the cyclic peptide is a lactam cyclic peptide or a lactone cyclic peptide formed by X2 and X 11 connected;

[0017] Each substitution is optionally substituted by one or more substituents R 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 heterocyclyl, C(=O)NH2, NHC(=O)NH2 or COOH, a1 The 4-7 membered heterocyclyl contains 1-3 heteroatoms independently selected from N, O or S.

[0018] As preferred, each of the substituents is optionally substituted with 1-3 substituents R independently selected from halogen, CN, =0, C1-C6alkyl, OH, 0(C1-C6alkyl), NH2, NH(C1-C6alkyl), N(C1-C6alkyl)2, C3-C6cycloalkyl, 4-7 membered heterocyclyl, C(=0)NH2, NHC(=0)NH2, or COOH a1 substituted, the 4-7 membered heterocyclyl comprises 1-3 heteroatoms independently selected from N, O, or S.

[0019] wherein X1, X3, X5, X8in the formula (I) are each independently selected from the following substituted or unsubstituted basic amino acids in L-form or D-form: 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;

[0020] X2is selected from the following substituted or unsubstituted amino acid with side chain containing amino group or containing hydroxyl group in L-form or D-form: Dab, Dap, Orn, Lys, Arg, His, Tyr, Thr, allo-Thr (allothreonine), Ser, D-Dab, D-Dap, D-Orn, D-Lys, D-Arg, D-His, D-Tyr, D-Thr, D-allo-Thr, or D-Ser;

[0021] X4, X6, X7, X 10 are each independently selected from the following substituted or unsubstituted hydrophobic amino acids in L-form or D-form: 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;

[0022] X9, X 11each independently selected from the following substituted or non-substituted amino acids in L or D form: Asp, Ala, Arg, Asn, Dab, Dap, Gin, Gly, His, lie, 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-Lys, D-Met, D-Orn, D-Phe, D-Ser, D-Thr, D-Trp, D-Tyr, or D-Val; wherein each of the above is optionally substituted with one or more substituents R independently selected from halogen, CN, =0, C1-C6 alkyl, OH, 0(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclyl, C(=0)NH2, NHC(=0)NH2, or COOH a1 substituted, the 4-7 membered heterocyclyl comprises 1-3 heteroatoms independently selected from N, O, or S;

[0023] X0 is selected from a fatty acid, which is a saturated fatty acid or an unsaturated fatty acid, or an aromatic carboxylic acid.

[0024] wherein X1, X3, X5, X8 in the formula (I) are each independently selected from the following substituted or non-substituted amino acids in L or D form: Dab, Dap, Orn, Lys, Arg, or His; X2 is selected from the following substituted or non-substituted amino acids in L or D form: Thr or Ser; X4, X6, X7, X 10 each independently selected from the following substituted or non-substituted amino acids in L or D form: Leu, Phe, or Val; X9, X 11 each independently selected from the following substituted or non-substituted amino acids in L or D form: Asp, Ala, Dab, Gly, His, lie, Leu, Lys, Met, Phe, Ser, Thr, Tyr, Trp, or Val; wherein each of the above is optionally substituted with one or more substituents R independently selected from halogen, CN, =0, C1-C6 alkyl, OH, 0(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclyl, C(=0)NH2, NHC(=0)NH2, or COOH a1 substituted, the 4-7 membered heterocyclyl comprises 1-3 heteroatoms independently selected from N, O, or S.

[0025] As preferred, X1in the formula (I) is substituted or unsubstituted D-Dab, X2is substituted or unsubstituted Thr, X3is substituted or unsubstituted Dab, X4is substituted or unsubstituted Leu, X5is substituted or unsubstituted Dab, X6is substituted or unsubstituted D-Phe, X7is substituted or unsubstituted Leu, X8is substituted or unsubstituted Dab, X9is substituted or unsubstituted D-Tyr or D-Dab, X 10 is substituted or unsubstituted Val, X 11 is substituted or unsubstituted Asp; wherein each of the above substitutions is optionally substituted with one or more substituents R independently selected from halogen, CN, =O, C1-C6alkyl, OH, O(C1-C6alkyl), NH2, NH(C1-C6alkyl), N(C1-C6alkyl)2, C3-C6cycloalkyl, 4-7 membered heterocyclyl, C(=O)NH2, NHC(=O)NH2or COOH. a1 The 4-7 membered heterocyclyl comprises 1-3 heteroatoms independently selected from N, O or S.

[0026] As preferred, each of the above substitutions is optionally substituted with 1-3 C1-C6alkyl.

[0027] Further, X1in the formula (I) is D-Dab, X2is Thr, X3is Dab, X4is Leu, X5is Dab, X6is D-Phe, X7is Leu, X8is Dab, X9is D-Tyr or D-Dab, X 10 is Val, X 11 is Asp.

[0028] wherein X1-X 11 are selected from any one of the following groups of amino acid sequences;

[0029]

[0030]

[0031]

[0032]

[0033]

[0034] X0is selected from a fatty acid or an aromatic carboxylic acid, the fatty acid being a saturated fatty acid or an unsaturated fatty acid.

[0035] wherein X0in formula (I) is selected from Myristic acid (tetradecanoic acid), Butyric acid (n-Butyric acid), Decanoic acid (capric acid), Lauric acid (dodecanoic acid), Palmitic acid (hexadecanoic acid), Stearic acid (octadecanoic acid), Sorbic acid (hexan-2-enoic acid), Neo-decanoic acid (2-ethyldecanoic acid), 4-Methylnonanoic acid (4-methyl-nonanoic acid), Benzofuran-2-carboxylic acid (2,3-benzofuran carboxylic acid), Indole-2-carboxylic acid (1H-Indole-2-carboxylic acid), 2-Quinoxalinecarboxylic acid (2-benzopyrrole carboxylic acid), 2-Biphenylcarboxylic acid (2-biphenylcarboxylic acid), 9-Anthracenecarboxylic acid (9-anthracene carboxylic acid), 2-aminonicotinic acid (2-aminonicotinic acid), 9-Fluorenone-4-carboxylic acid (9-fluorenone-4-carboxylic acid), 9-Fluorenone-2-carboxylic acid (9-fluorenone-2-carboxylic acid), 3-Biphenylcarboxylic acid (3-biphenylcarboxylic acid), 4-piperidin-1-ylbenzoic acid (4-piperidin-1-ylbenzoic acid), 4-Morpholinobenzoic Acid (4-morpholinobenzoic acid), 4-(4-Methyl-piperazin-1-yl)-benzoic acid (4-(4-methyl-piperazin-1-yl)-benzoic acid), 3-(4-Methylpiperazin-1-yl)benzoic acid (3-(4-methylpiperazin-1-yl)benzoic acid), Benzoic acid (benzoic acid), 4”-(Pentyloxy)-1,1':4',1”-terphenyl-4-carboxylic acid (p-pentoxyterphenylcarboxylic acid), Undecanoic acid (undecanoic acid), Tridecylic acid (tetrazolic 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-carboxylicacid (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.

[0036] Preferably, X0 in formula (I) is selected from Myristic acid (tetradecanoic acid).

[0037] 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.

[0038] 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.

[0039] 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.

[0040] The polypeptide compound is shown in formula (II):

[0041]

[0042] 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.

[0043] Preferably, the substitution is optionally made by replacing 1-3 C1-C6 alkyl groups.

[0044] 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.

[0045] The polypeptide compound is shown in formula (III):

[0046]

[0047] 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.

[0048] The polypeptide compound is shown in formula (IV):

[0049]

[0050] Among them, R 1 It is the carbonyl compound obtained by removing the hydroxyl group when X0 and X1 form an amide bond.

[0051] 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 (tetrazolic 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-carboxylicacid (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.

[0052] The polypeptide compound is shown in formula (V):

[0053]

[0054] Among them, R 1 It is the carbonyl compound obtained by removing the hydroxyl group when X0 and X1 form an amide bond.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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:

[0060]

[0061]

[0062] 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):

[0063]

[0064] The use of the polypeptide compound or its pharmaceutically acceptable salt described in this invention in the preparation of antibacterial drugs.

[0065] 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.

[0066] 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.

[0067] The pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhaler, ointment, suppository or patch.

[0068] 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

[0069] 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:

[0070] The hydroxyl groups of the polypeptide compound can form, but are not limited to, lactone compounds;

[0071] The hydroxyl groups of the polypeptide compound can form, but are not limited to, ethers, esters, glycosides, or glycosides.

[0072] 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.

[0073] The amino groups of the polypeptide compounds may form, but are not limited to, acylates, hydrocarbons, glycosides, or glycosides.

[0074] The carboxyl group of the polypeptide compound can form, but is not limited to, esters and amides.

[0075] The polypeptide compound forms a salt compound with an organic acid or an inorganic acid;

[0076] The polypeptide compound forms a complex, chelate, or other compound with the metal ion.

[0077] Furthermore, all of the aforementioned polypeptide compounds were prepared using Fmoc protected solid-phase polypeptide synthesis and liquid-phase synthesis methods.

[0078] Furthermore, the use of all the aforementioned polypeptide compounds, their stereoisomers, and pharmaceutically acceptable salts in the preparation of antibacterial drugs.

[0079] The use of the polypeptide compound or its pharmaceutically acceptable salt or its stereoisomer described in this invention in the preparation of antibacterial drugs.

[0080] 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.

[0081] 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.

[0082] The pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhaler, ointment, suppository or patch.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] The compound Paenimycin synthesized in this invention exhibits potent activity against resistant strains of common clinical antibacterial 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 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.

[0089] 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.

[0090] 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.

[0091] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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

[0096] Figure 1 This refers to the process of mining the BNP37 biosynthetic gene cluster.

[0097] Figure 2 The BNP37 biosynthetic gene cluster and its predicted products;

[0098] Figure 3 for paenimycin 1 H-NMR (DMSO-d6) nuclear magnetic resonance spectrum;

[0099] Figure 4 for paenimycin 13 C-NMR (DMSO-d6) nuclear magnetic resonance spectrum;

[0100] Figure 5 The image shows the COSY-NMR (DMSO-d6) nuclear magnetic resonance spectrum of paenimycin.

[0101] Figure 6 The image shows the HMBC-NMR (DMSO-d6) nuclear magnetic resonance spectrum of paenimycin.

[0102] Figure 7 The image shows the HSQC-NMR (DMSO-d6) nuclear magnetic resonance image of paenimycin.

[0103] Figure 8 Paenimycin is cytotoxic to HepG2 cells;

[0104] Figure 9 The hemolytic activity of paenimycin;

[0105] Figure 10 A paenimycin-induced infection model of Acinetobacter baumannii ATCC BAA 1605 in the thigh muscle;

[0106] Figure 11 A paenimycin-induced infection model of Klebsiella pneumoniae BNCC 359393 in the thigh muscle;

[0107] Figure 12 This model demonstrates the effect of paenimycin on the thigh muscle infection of Escherichia coli MG1655-mcr-1;

[0108] Figure 13 This model of paenimycin-induced infection of the thigh muscle of Staphylococcus aureus ATCC BAA44;

[0109] Figure 14 For in vivo pharmacokinetic studies of paenimycin;

[0110] Figure 15 For the study of paenimycin nephrotoxicity;

[0111] Figure 16 For the paenimycin cell membrane lysis experiment;

[0112] Figure 17 For the depolarization experiment of the paenimycin film;

[0113] Figure 18 The change in K+ concentration after paenimycin treatment;

[0114] Figure 19 The bactericidal curve of paenimucin;

[0115] Figure 20 Scanning electron microscopy observation of bacterial cell morphology after paenimycin treatment of Escherichia coli and Staphylococcus aureus;

[0116] Figure 21 For continuous passaging experiments of paenimycin;

[0117] Figure 22 For the paenimycin feeding experiment;

[0118] Figure 23 For the ITC assay to determine the binding of paenimycin and lipopolysaccharide;

[0119] Figure 24 The experiment was conducted using BC dye as a substitute.

[0120] Figure 25 For the determination of lipid A growth curve;

[0121] Figure 26 Molecular dynamics simulations were performed to demonstrate the binding of paenimyxin to lipid A;

[0122] Figure 27 Experiments to accumulate paenimycin cell wall precursors;

[0123] Figure 28 It is paenimycin-bound cell wall teichoic acid. Detailed Implementation

[0124] 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.

[0125] 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.

[0126] 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.

[0127] Other non-natural amino acids used in synthesis are commercially available.

[0128] In the following examples, the abbreviations for commonly used reagents and methods are as follows:

[0129] SPE: Solid Phase Extraction

[0130] Fmoc: 9-fluorenylmethoxycarbonyl protecting group,

[0131] DCM: Dichloromethane

[0132] DMF: N,N-dimethylformamide

[0133] HBTU: O-benzotriazole-tetramethylurea hexafluorophosphate,

[0134] HOBt: 1-Hydroxybenzotriazole,

[0135] DIPEA: N,N-diisopropylethylamine

[0136] tBu: tert-butyl protecting group

[0137] Boc: tert-Butoxycarbonyl protecting group

[0138] TFA: Trifluoroacetic acid,

[0139] TIPS: Triisopropylsilane

[0140] Alloc: Allyloxycarbonyl protecting group

[0141] PyBOP: Benzotriazol-1-yl-oxotripyrrolidinyl-hexafluorophosphate

[0142] BzCl: Benzoyl chloride,

[0143] DIC: N,N'-Diisopropylcarbodiimide

[0144] FA: Formic acid

[0145] NMP: N-methylpyrrolidone,

[0146] DBU:1,8-diazabicyclo[5.4.0]undec-7-ene.

[0147] Example 1

[0148] Discovery of the BNP37 biosynthetic gene cluster

[0149] like Figure 1 The diagram illustrates the process of discovering 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 of these BGCs for encoding non-ribosomal polypeptide biosynthetic genes was conducted. A Python script was written to identify 776 candidate non-ribosomal polypeptide biosynthetic gene clusters, based on criteria including the type of biosynthetic gene cluster, the number of adenosine domains, and the presence of thioesterase domains. 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.

[0150] Table 1. Functional annotations of the biosynthetic gene cluster of BNP37.

[0151]

[0152]

[0153] Example 2

[0154] BNP37-guided synthesis of a series of polypeptide compounds: chemical synthesis of linear and cyclic peptides.

[0155] 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.

[0156] The specific synthesis method is as follows:

[0157] (1) Linear polypeptide synthesis:

[0158] Taking linear peptide synthesis as an example,

[0159] 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 LDM and allow it to swell at room temperature for 30 minutes. Then, use a vacuum pump to remove the solvent. Weigh Fmoc-X... 11 Amino acid (Fmoc-L-Asp(OtBu)-OH, 0.17g, 0.3g)

[0160] 2,4,6-trimethylpyridine (0.3 mL, 2 mmol) was dissolved in 8 mL of LCM, 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.

[0161] 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.

[0162] The reaction of piperidine in DMF solution was repeated twice for 7.5 minutes to remove the Fmoc protecting group. Meanwhile, separately weigh F...

[0163] moc-X 10 Amino acids (Fmoc-L-Val-OH, 0.14 g, 0.3 mmol), HBTU (0.15 g, 0.3 mmol),

[0164] 0.3 mmol HOBt (0.055 g, 0.3 mmol) and DIPEA (0.071 mL, 0.3 mmol) were mixed and dissolved in 6 mL of DMF. After deprotection, the resin was washed five times with 3 mL of DMF, and the mixture was poured into the above-mentioned amino acid solution. The mixture was allowed to stand at room temperature for 1 hour. The above coupling steps were repeated to sequentially condense and link X9-X8-X7-X6-

[0165] X5-X4-X3-X2-X1-X0, the amino acid raw materials are, in sequence: Fmoc-D-Tyr(tBu)-OH, Fmoc-L-Dab(Boc)-OH,

[0166] Fmoc-L-Leu-OH, Fmoc-D-Phe-OH, Fmoc-L-Dab(Boc)-OH, Fmoc-L-Leu-OH, Fmoc-

[0167] L-Dab(Boc)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-D-Dab(Boc)-OH, Myristic acid.

[0168] 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.

[0169] All linear polypeptides were synthesized according to the above method based on the selection of different amino acid sequences and fatty acid chains.

[0170] (2) Synthesis of lactam cyclic peptides:

[0171] With X1 and X 11 Taking the synthesis of the formed lactam cyclic peptide as an example:

[0172] 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.

[0173] 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.

[0174] c) Peptide amide bond cyclization: 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.

[0175] 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.

[0176] Other sites such as X3, X5 and X 11 The 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.

[0177] (3) Synthesis of lactone cyclic peptides

[0178] Taking the synthesis of paenimycin as an example:

[0179] 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.

[0180] 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 LCM and mixed thoroughly. The linear peptide-resin mixture was then immediately added and the mixture was shaken at room temperature for 48 hours.

[0181] 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.

[0182] 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.

[0183] (4) N-methylation synthesis

[0184] 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.

[0185] Example 3

[0186] Purification of crude polypeptide compounds

[0187] 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; and simultaneous scanning was performed in positive and negative ion modes. The fraction with optimal purity was obtained and then freeze-dried to obtain a white, flocculent, pure polypeptide compound with a purity exceeding 95%.

[0188] Example 4

[0189] BNP37 series polypeptide compounds were synthesized using the methods described in Examples 2 and 3.

[0190] 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.

[0191] 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); 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.

[0192] The following polypeptide compounds were obtained using the above preparation method:

[0193] 1. The polypeptide compound represented by formula (III)

[0194] 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-X11 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.

[0195]

[0196] The polypeptide compounds represented by formula (III) in Table 2

[0197]

[0198] 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:

[0199]

[0200] 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

[0201] The polypeptide compounds represented by formula (II) in Table 3

[0202]

[0203]

[0204] 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:

[0205]

[0206] 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.

[0207] Table 4 shows the polypeptide compounds represented by formula (IV).

[0208]

[0209]

[0210]

[0211] 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:

[0212]

[0213] 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.

[0214] Table 5 shows the polypeptide compounds represented by formula (V).

[0215]

[0216]

[0217] Example 5

[0218] 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.

[0219] Table 6-1 BNP37L-X 11 amino acid structural sequence of polypeptide compounds

[0220]

[0221] Table 6-2BNP37L-X 11 High-resolution mass spectrometry of peptide compound structure and activity information

[0222]

[0223] Example 6

[0224] 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.

[0225] Table 7. Scanned structural sequence of BNP37C2 alanine

[0226]

[0227]

[0228] Table 8 High-resolution mass spectrometry and activity information of BNP37C2 alanine.

[0229]

[0230] Example 7

[0231] 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.

[0232] Table 9. Scanned structural sequence of 2,4-diaminobutyric acid from BNP37C2.

[0233]

[0234]

[0235] Table 10 High-resolution mass spectrometry and activity information of 2,4-diaminobutyric acid from BNP37C2

[0236]

[0237] Example 8

[0238] 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 X11 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.

[0239] Table 11. Amino acid scanning sequence of each locus from BNP37C2 to Dab9

[0240]

[0241]

[0242]

[0243]

[0244] Table 12 High-resolution mass spectrometry and activity information of amino acid structures at each point of BNP37C2-Dab9

[0245]

[0246]

[0247]

[0248] Example 9

[0249] 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:

[0250]

[0251] In this embodiment, the Lipid moiety is 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 analyzed using a 600M NMR instrument. Figures 2-6 As shown, the paeminycin structure is confirmed.

[0252] Example 10

[0253] Bioactivity analysis of paenimycin

[0254] (1) In vitro detection of anti-multidrug-resistant bacteria activity

[0255] 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.

[0256] Table 13 Antibacterial activity of paeminycin

[0257]

[0258]

[0259]

[0260]

[0261]

[0262] (2) cytotoxicity detection of paenimycin

[0263] 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 64 μg / mL - 0.25 μg / mL paenimycin was added to each well. 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. For example...Figure 7 As shown, paenimycin has low toxicity.

[0264] (3) Hemolytic activity test of paenimycin

[0265] 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 the 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 MultiscanSkyHigh absorbance at OD540 nm and calculated using Prism 9.0. All experiments were performed in triplicate (n=3). Figure 8 As shown, the results indicate that paenimycin has a low hemolytic effect at 100 μM, such as... Figure 9 As shown, the HC50 value is greater than 100 μM, indicating low toxicity.

[0266] Example 11

[0267] In vivo antibacterial activity assessment of paenimycin

[0268] A mouse thigh muscle infection model with neutrophil deficiency was used.

[0269] Six-week-old, 23-27g female ICR mice without specific pathogens were selected. Before the experiment, the 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 four days and one day before infection, respectively, 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 ATCCBAA-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.

[0270] like Figures 10-13 As shown, paenimycin showed 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 mcr-1 plasmid-mediated resistance of polymyxin-resistant Escherichia coli, paenimycin 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, showing superior efficacy compared to meropenem. Peenimycin was ineffective against polymyxin E and other drugs, but not against polymyxin-, meropenem-, and third-generation cephalosporin-resistant Klebsiella pneumoniae. Against these multidrug-resistant strains, paenimycin showed dose-dependent efficacy, reducing bacterial load in mouse thighs by 2.25 log10 after subcutaneous administration of 5 mg / kg, and by 3.12 log10 at a concentration of 20 mg / kg. Polymyxin and meropenem were ineffective. Against methicillin-resistant Staphylococcus aureus (MRSA), paenimycin also showed dose-dependent efficacy, reducing bacterial load in mouse thighs by 1.24 log10 after subcutaneous administration of 5 mg / kg, and by 1.79 log10 at a concentration of 20 mg / kg, comparable to vancomycin, while methicillin was ineffective.

[0271] Paenimycin maintains good antibacterial activity in animals, suggesting its potential for development as a novel antibacterial drug.

[0272] Example 12

[0273] In vivo pharmacokinetic studies of Paenimycin

[0274] 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.

[0275] 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.

[0276] 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 electrospray positive ionization mode. The MRM changes for 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). Results are as follows... Figure 14As shown, paenimycin exhibits 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 possesses a broad therapeutic window.

[0277] Example 13

[0278] paenimycin in vivo nephrotoxicity evaluation

[0279] 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. Figure 15 As shown, 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.

[0280] Example 14

[0281] Acute toxicity test of Paenimycin

[0282] 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.

[0283] Table 14 Acute toxicity tests of paenimycin

[0284] Mode of administration Dose of administration Number of mice Time Survival rate Subcutaneous injection 100 mg / kg 5 24 hours 100％ Subcutaneous injection 200 mg / kg 5 24 hours 100％ Subcutaneous injection 300 mg / kg 5 24 hours 100％ Subcutaneous injection 400 mg / kg 5 24 hours 100％

[0285] Example 15

[0286] Study on the mechanism of action of paenimycin

[0287] (1) Bacterial lysis experiment

[0288] 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 paeminycin 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. (See attached image.) Figure 16 As shown, paenimycin at high concentrations causes an increase in fluorescence intensity, suggesting that it has a bacterial lysis effect.

[0289] (2) Membrane depolarization test

[0290] 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-bottomed 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 solution 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. Figure 17 As shown, 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.

[0291] (3) Potassium ion release test

[0292] 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. Figure 18 As shown, after treatment with paenimycin, the concentration of potassium ions in the solution gradually increased, indicating that the bacteria began to excrete a large amount of potassium ions, and the concentration of ion excretion was dose-dependent on the concentration of the drug administered.

[0293] (4) Sterilization curve test

[0294] 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. 6CFU / mL. Add 1, 4, and 8×MIC of paeminycin solution to the bacterial dilution; 8×MIC (8 μg / mL) of vancomycin (from Staphylococcus aureus BNCC 186335) or 8×MIC (4 μg / mL) of polymyxin E (from Escherichia coli ATCC 25922) to the dilution. At 1, 2, 4, 8, and 16 hours after the start of the experiment, take 100 μL of bacterial suspension, serially dilute it, and plate it onto LB agar plates. For Escherichia coli, increase the time intervals by 0.25, 0.5, and 0.75 hours. Incubate the agar plates overnight at 37°C and then count the colonies. Figure 19 As shown, paenimycin is a potent bactericide that works quickly to kill pathogens within 4 hours.

[0295] (5) Scanning electron microscopy observation of the morphology of bacteria after paenimycin treatment

[0296] Bacterial samples of Staphylococcus aureus BNCC 186335 and Escherichia coli ATCC259220h, 4h, and 8h treated with 8×MIC in step (4) were selected, and the morphology of the bacteria treated at different time points was observed using scanning electron microscopy. The results are as follows: Figure 20 As shown, under the action of paenimycin, 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.

[0297] (6) Paenimycin resistance test

[0298] 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 the method described above (CFU / mL). For bacitracin, 50 μg / mL ZnCl2 was added to LB medium for measurement. The following day, bacterial cultures from wells containing sub-MIC concentrations of each antibiotic were diluted 500-fold in fresh medium, and the new MICs were tested according to the above method. Subculture was performed for 28 consecutive days. Results... Figure 21 It can be seen that, unlike antibiotics used in clinical practice, paenimycin does not produce drug resistance after continuous passage, suggesting that it is unlikely to produce drug-resistant bacteria when further used in clinical practice.

[0299] (7) Feeding experiment

[0300] Using *Staphylococcus aureus* BNCC 186335 and *Escherichia coli* ATCC 25922 as experimental materials, the effect of bacterial cell components on the antibacterial activity of paeminycin was studied. Cell components (peptidoglycan, total protein, genomic DNA, lipopolysaccharide, and lipoteichoic acid) of 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 the 0.0156–1 mg / mL paeminycin 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 serially diluted 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. Figure 22 As shown, paenimycin can bind to lipopolysaccharide, lipid A, and lipoteichoic acid.

[0301] (8) Target point determined by isothermal calorimetric titration

[0302] The binding of paenimycin to lipopolysaccharide and lipoteichoic acid was determined using the ITC method. The ITC experiment was performed using a PEAQ-ITC (Malvern, UK) instrument at 25°C. 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. Results are shown below. Figure 23 The Kd value of paenimycin binding to lipopolysaccharide is 2.18 μM, which is comparable to the Kd value of 2.00 μM for polymyxin E binding to lipopolysaccharide. Furthermore, paenimycin binds to lipoteichoic acid with a Kd value of 5.61 μM, while polymyxin E does not bind.

[0303] (10) BODIPYTR cadaverine (BC) dye substitution experiment

[0304] 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). Results are as follows. Figure 24 Similar to polymyxin E, paenimycin can replace the BC dye bound to lipid A, unlike kanamycin, suggesting that its binding site is lipid A.

[0305] (11) Lipid A extraction and feeding experiment

[0306] 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 Plot a growth curve after 16 hours. Figure 25 As shown, unlike polymyxin E, paeminycin retains 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.

[0307] (12) Molecular dynamics simulation

[0308] 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. Figure 26 As shown, 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.

[0309] (13) Cell wall precursor accumulation experiment

[0310] 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. 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. The bacterial culture was then transferred to fresh medium and cultured until OD... 600nm=0.5. Then add chloramphenicol solution to a final concentration of 130 μg / mL, incubate at room temperature for 15 minutes, and add paeminycin and vancomycin at a dose of 10×MIC respectively, with DMSO as a negative control. After incubation for 1 hour, precipitate the bacteria, resuspend the bacterial body in 30 μL RO water, and boil for 15 minutes. After the solution cools, centrifuge at 15000g for 5 minutes, and take the supernatant for UPLC-MS analysis. Figure 27 As shown, unlike vancomycin, paenimycin does 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.

[0311] (14) Extraction and conjugation of teichoic acid

[0312] 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 concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL, respectively. These 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 insoluble substances. The paenimycin content in the supernatant was detected by UPLC-MS, and the peak area change rate was calculated. Figure 28 As shown, paenimycin binds to paenichoic acid in a dose-dependent manner, unlike vancomycin.

[0313] 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- 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 it is a substitute or not, 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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