Antimicrobial polypeptides and their use in medicine

Abstract

The application belongs to the field of polypeptide drugs, and particularly relates to an antibacterial polypeptide and application thereof in pharmacy. The application takes Rink amide MBHA amino resin as a solid phase carrier, and modifies and transforms according to a template polypeptide raniseptin PL:Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2 amino acid sequence, wherein amino acid residues 19G and 26N are replaced by R8 and S5, to obtain a target stapled peptide PL-16. The stapled peptide PL-16 can significantly improve the antibacterial activity on Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis and Staphylococcus epidermidis relative to the template polypeptide PL-0.

Description

Technical Field

[0001] This invention belongs to the field of peptide drugs, specifically relating to an antimicrobial peptide that can improve the activity against drug-resistant bacteria and enhance conformational stability based on a template peptide, and its application in pharmaceutical manufacturing. Background Technology

[0002] In recent years, the prolonged and widespread overuse of antibiotics has led to widespread bacterial resistance, posing a serious threat to public health. Infections caused by antibiotic-resistant bacteria result in approximately 700,000 deaths globally each year, and this number is estimated to exceed 10 million annually by 2050. However, antibiotic development has lagged significantly, and effective treatments for infections caused by drug-resistant bacteria are lacking, thus necessitating the urgent development of novel antibacterial drugs.

[0003] Enterococci are Gram-positive cocci, usually arranged in pairs or short chains, and catalase-negative (a key difference from staphylococci). Enterococci are typical opportunistic pathogens, usually causing disease when the host's defense mechanisms are impaired or the normal flora is imbalanced. Diseases caused by enterococcal infection mainly include: (1) Urinary tract infection: accounting for 15-20% of hospital-acquired urinary tract infections, mostly related to indwelling catheters. (2) Abdominal and pelvic infections: often as part of mixed infections, seen in abdominal abscesses, peritonitis, biliary tract infections, pelvic inflammatory disease, etc., mostly due to translocation of intestinal flora. (3) Bacteremia and infective endocarditis: are common causes of hospital-acquired bacteremia. Enterococcus faecalis is the third leading cause of infective endocarditis, often affecting natural valves, with a subacute course. (4) Medical device-related infections: infections of implants such as central venous catheters, urinary catheters, artificial joints, and pacemakers. (5) Others: Postoperative wound infection of the abdomen / pelvis, neonatal sepsis / meningitis, and in rare cases, osteomyelitis and endophthalmitis. There are two main pathogenic species of enterococci: Enterococcus faecalis and Enterococcus faecium. More than 90% of enterococcal infections in clinical practice are caused by these two species. Enterococcus faecalis is more common, accounting for about 80-90% of clinical isolates, and has traditionally been more virulent. Enterococcus faecium accounts for about 10-15%, but its drug resistance problem is more prominent, which is the main challenge of nosocomial infections. The drug resistance of enterococci is mainly manifested as: (1) Inherent drug resistance: Naturally insensitive or resistant to many commonly used antibiotics such as cephalosporins, compound sulfamethoxazole, and clindamycin. (2) Acquired drug resistance: By producing modifying enzymes, even high doses of gentamicin and streptomycin cannot produce synergistic bactericidal effects with cell wall activators (such as ampicillin), which seriously affects the treatment of severe infections such as endocarditis. Resistance to ampicillin and penicillin is achieved through the production of low-affinity penicillin-binding proteins or β-lactamases. Vancomycin resistance: Vancomycin-resistant enterococci are a major global public health threat. Although the proportion of vancomycin-resistant enterococci among *Enterococcus faecalis* is generally lower than that among *Enterococcus faecium*, once they occur, treatment options are extremely limited.

[0004] Antimicrobial peptides (AMPs) are a class of host defense peptides with unique mechanisms of action, such as disrupting cell membrane structure and inhibiting the synthesis of intracellular biomolecules. Due to their unique mechanisms of action, they act on multiple targets without involving specific protein binding, are less likely to induce drug resistance, and can combat bacterial infections unresponsive to traditional antibiotics. Therefore, AMPs have the potential to be a new generation of antibiotics for treating drug-resistant bacterial infections. There are numerous research reports on antimicrobial peptides. For example, patent CN116217669A discloses a staple peptide that can enhance broad-spectrum antimicrobial activity, its preparation method, and its applications. This staple peptide exhibits very significant broad-spectrum antimicrobial activity, including activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Patent CN117756905A discloses a staple peptide and its pharmaceutical uses; the staple peptide prepared by this invention has inhibitory activity against Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. Patent CN116655766A discloses a staple peptide, its preparation method, and its applications. This staple peptide can significantly improve the inhibitory activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. While the staple peptide in the aforementioned study can enhance the antibacterial activity against some drug-resistant bacteria, there are no reports on its activity against enterococcal resistance. Solving the problem of enterococcal drug resistance remains an urgent issue.

[0005] Michael Conlon's team isolated raniseptin PL, a multifunctional host defense peptide, from the skin secretions of the banana tree frog. Raniseptin PL contains 27 residues and has an α-helix structure, effectively inhibiting common ESKAPE pathogens such as Staphylococcus aureus, Klebsiella pneumoniae, and the Gram-positive anaerobic spore-forming bacterium Clostridium difficile. However, as a linear antimicrobial peptide, raniseptin PL inherently possesses several drawbacks, such as being easily enzymatically hydrolyzed and having limited antimicrobial activity. These limitations severely hinder its development into a novel antibiotic. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing an antimicrobial peptide, specifically a staple peptide. Compared to template peptides, this staple peptide exhibits enhanced activity against drug-resistant bacteria and improved conformational stability, resulting in superior overall performance and making it more suitable for drug development.

[0007] Another object of the present invention is to provide pharmaceutical use of the said antimicrobial polypeptide.

[0008] To achieve the first objective mentioned above, the technical solution adopted by the present invention is as follows: An antimicrobial polypeptide, wherein the antimicrobial polypeptide is a staple peptide, and the staple peptide is: PL-1: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 6V and 10G are replaced by S5. PL-2: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 9I and 13V are replaced by S5; PL-4: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 12A and 16F are replaced by S5; PL-5: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 13V and 17A are replaced by S5; PL-6: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 16F and 20V are replaced by S5; PL-8: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 19G and 23N are replaced by S5; PL-9: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 20V and 24Y are replaced by S5; PL-10: The peptide chain template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 6V and 13V are replaced by R8 and S5. PL-11: The peptide chain template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 9I and 16F are replaced by R8 and S5. PL-13: The peptide chain template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 13V and 20V are replaced by R8 and S5. PL-14: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 16F and 23N are replaced by R8 and S5. PL-16: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 19G and 26N are replaced by R8 and S5.

[0009] Preferably, in the above-mentioned antimicrobial polypeptides, the stapler peptide is: PL-2: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 9I and 13V are replaced by S5; PL-4: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 12A and 16F are replaced by S5; PL-5: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 13V and 17A are replaced by S5; PL-6: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 16F and 20V are replaced by S5; PL-8: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 19G and 23N are replaced by S5; PL-9: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 20V and 24Y are replaced by S5; PL-10: The peptide chain template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 6V and 13V are replaced by R8 and S5. PL-11: The peptide chain template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 9I and 16F are replaced by R8 and S5. PL-13: The peptide chain template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 13V and 20V are replaced by R8 and S5. PL-14: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 16F and 23N are replaced by R8 and S5. PL-16: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 19G and 26N are replaced by R8 and S5.

[0010] More preferably, in the above-mentioned antimicrobial polypeptides, the stapler peptide is: PL-2: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 9I and 13V are replaced by S5; PL-4: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 12A and 16F are replaced by S5; PL-6: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 16F and 20V are replaced by S5; PL-8: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 19G and 23N are replaced by S5; PL-9: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 20V and 24Y are replaced by S5; PL-10: The peptide chain template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 6V and 13V are replaced by R8 and S5. PL-11: The peptide chain template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 9I and 16F are replaced by R8 and S5. PL-13: The peptide chain template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 13V and 20V are replaced by R8 and S5. PL-14: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 16F and 23N are replaced by R8 and S5. PL-16: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 19G and 26N are replaced by R8 and S5.

[0011] To achieve the second objective mentioned above, the technical solution adopted by the present invention is as follows: The above-mentioned antimicrobial peptides are used in the preparation of drugs against Staphylococcus aureus.

[0012] The above-mentioned antimicrobial peptides are used in the preparation of drugs against Enterococcus faecalis.

[0013] The above-mentioned antimicrobial peptides are used in the preparation of drugs against Enterococcus faecalis.

[0014] The above-mentioned antimicrobial peptides are used in the preparation of drugs against Staphylococcus epidermidis.

[0015] In this invention, the abbreviations are explained as follows: Fmoc: fluorenemethyloxycarbonyl; DCE: 1,2-dichloroethane; Oxyme: ethyl 2-oxime cyanoacetate; DCM: dichloromethane; DMF: N,N-dimethylformamide; DIC: N,N-diisopropylcarbodiimide; S5: 2-amino-2-methyl-9-heptenoic acid; R8: 2-amino-2-methyl-9-decenoic acid; TFA: trifluoroacetic acid; EDT: 1,2-ethylenedithiol; GrubbsⅠ: phenylmethylenebis(tricyclohexylphosphine)ruthenium dichloride; MS: mass spectrometry; HR-Q-TOF-MS: high-resolution matrix-assisted laser desorption / ionization time-of-flight mass spectrometry; S. aureus: Staphylococcus aureus; E. faecium: Enterococcus faecium; E. faecalis: Enterococcus faecalis; S. epidermidis: Staphylococcus epidermidis.

[0016] The advantages of this invention are: 1. This invention uses Rink amide MBHA amino resin as a solid-phase support and modifies the template peptide raniseptinPL: Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2 amino acid sequence. While retaining the key amino acid residues, the original amino acids at positions i, i+4 and i, i+7 are replaced with S5 and R8, respectively, to obtain the target stapling peptide.

[0017] 2. The 16 staple peptides obtained in this invention exhibit significantly improved antibacterial properties compared to the template peptide. Among them, PL-2, PL-4, PL-6, PL-8, PL-9, PL-10, PL-11, PL-13, PL-14, and PL-16 showed good in vitro antibacterial properties against Staphylococcus aureus ATCC25923; PL-1, PL-2, PL-4, PL-5, PL-6, PL-8, PL-9, PL-10, PL-11, PL-13, PL-14, and PL-16 showed good in vitro antibacterial properties against Staphylococcus aureus ATCC43300; and PL-2, PL-4, PL-6, PL-8, PL-11, and PL-13... PL-14 and PL-16 showed good in vitro antibacterial activity against Enterococcus faecalis ATCC19434; PL-13 and PL-14 showed good in vitro antibacterial activity against Enterococcus faecalis ATCC29212; PL-2, PL-4, PL-6, PL-8, PL-9, PL-10, PL-11, PL-13, PL-14, and PL-16 showed good in vitro antibacterial activity against Enterococcus faecalis ATCC51299; and PL-2, PL-4, PL-6, PL-8, PL-10, PL-11, PL-13, PL-14, and PL-16 showed good in vitro antibacterial activity against Staphylococcus epidermidis ATCC12228. Among them, the antibacterial activity of binding peptides PL-2, PL-4, PL-9, and PL-11 against Staphylococcus aureus ATCC25923 was 3 times higher than that of template peptide PL-0; PL-6, PL-8, PL-13, PL-14, and PL-16 against Staphylococcus aureus ATCC25923 was 7 times higher than that of template peptide PL-0; and PL-10 against Staphylococcus aureus ATCC25923 was 1 time higher than that of template peptide PL-0. PL-14 against Staphylococcus aureus ATCC43300 was 31 times higher than that of template peptide PL-0; PL-6, PL-13, and PL-16 against Staphylococcus aureus ATCC43300 was 15 times higher than that of template peptide PL-0; and PL-2, PL-4, PL-8, and PL-11 against Staphylococcus aureus ATCC43300 was 7 times higher than that of template peptide PL-0. PL-5 and PL-9 showed a 3-fold increase in antibacterial activity against Staphylococcus aureus ATCC43300 compared to the template peptide PL-0, while PL-1 showed a 1-fold increase in antibacterial activity against Staphylococcus aureus ATCC43300 compared to the template peptide PL-0.PL-9 and PL-10 showed a 1-fold increase in antibacterial activity against Enterococcus faecalis ATCC19434 compared to the template peptide PL-0; PL-2, PL-4, PL-6, PL-11, and PL-16 showed a 3-fold increase in antibacterial activity against Enterococcus faecalis ATCC19434 compared to the template peptide PL-0; PL-13 showed a 7-fold increase in antibacterial activity against Enterococcus faecalis ATCC19434 compared to the template peptide PL-0; PL-14 showed a 15-fold increase in antibacterial activity against Enterococcus faecalis ATCC19434 compared to the template peptide PL-0; and PL-8 showed a 31-fold increase in antibacterial activity against Enterococcus faecalis ATCC29212 compared to the template peptide PL-0. PL-9 and PL-11 showed a 1-fold increase in antibacterial activity against Enterococcus faecalis ATCC51299 compared to the template peptide PL-0. PL-2, PL-4, PL-6, PL-8, PL-10, and PL-16 showed a 3-fold increase in antibacterial activity against Enterococcus faecalis ATCC51299 compared to the template peptide PL-0. PL-13 and PL-14 showed a 7-fold increase in antibacterial activity against Enterococcus faecalis ATCC51299 compared to the template peptide PL-0. PL-8 showed a 15-fold increase in antibacterial activity against Enterococcus faecalis ATCC51299 compared to the template peptide PL-0. PL-10 and PL-16 showed a 1-fold increase in antibacterial activity against Staphylococcus epidermidis ATCC12228 compared to the template peptide PL-0. PL-2, PL-4, PL-6, PL-11, and PL-13 showed a 3-fold increase in antibacterial activity against Staphylococcus epidermidis ATCC12228 compared to the template peptide PL-0. PL-14 showed a 7-fold increase in antibacterial activity against Staphylococcus epidermidis ATCC12228 compared to the template peptide PL-0. This invention successfully prepared a modified staple peptide based on PL-0. In vitro experiments demonstrated that the synthesized staple peptide can significantly inhibit the growth and reproduction of pathogenic bacteria, showing promise for development into a novel antibacterial drug.

[0018] 3. The present invention prepared a modified staple peptide based on PL-0. Through circular dichroism spectroscopy analysis, except for PL-6, the helicity of the other staple peptides was greater than that of the linear peptide PL-0, indicating that the staple locking after the staple peptide strategy modification played a certain role in the reinforcement of the peptide chain and improved the conformational stability of the peptide. Attached Figure Description

[0019] Figure 1 The diagram shows the amino acid sequence of PL-0 and its characterization spectrum, where A is the amino acid sequence of PL-0, B is the HPLC chromatogram of PL-0, and C is the mass spectrum of PL-0.

[0020] Figure 2 The diagram shows the amino acid sequence of PL-1 and its characterization spectrum, where A is the amino acid sequence of PL-1, B is the HPLC chromatogram of PL-1, and C is the mass spectrum of PL-1.

[0021] Figure 3 The diagram shows the amino acid sequence of PL-2 and its characterization spectrum. In the diagram, A is the amino acid sequence of PL-2, B is the HPLC chromatogram of PL-2, and C is the mass spectrum of PL-2.

[0022] Figure 4 The diagram shows the amino acid sequence of PL-3 and its characterization spectrum. In the diagram, A is the amino acid sequence of PL-3, B is the HPLC chromatogram of PL-3, and C is the mass spectrum of PL-3.

[0023] Figure 5 The diagram shows the amino acid sequence of PL-4 and its characterization spectrum, where A is the amino acid sequence of PL-4, B is the HPLC chromatogram of PL-4, and C is the mass spectrum of PL-4.

[0024] Figure 6 The diagram shows the amino acid sequence of PL-5 and its characterization spectrum, where A is the amino acid sequence of PL-5, B is the HPLC chromatogram of PL-5, and C is the mass spectrum of PL-5.

[0025] Figure 7 The diagram shows the amino acid sequence of PL-6 and its characterization spectrum. In the diagram, A is the amino acid sequence of PL-6, B is the HPLC chromatogram of PL-6, and C is the mass spectrum of PL-6.

[0026] Figure 8 The diagram shows the amino acid sequence of PL-7 and its characterization spectrum. In the diagram, A is the amino acid sequence of PL-7, B is the HPLC chromatogram of PL-7, and C is the mass spectrum of PL-7.

[0027] Figure 9 The diagram shows the amino acid sequence of PL-8 and its characterization spectrum, where A is the amino acid sequence of PL-8, B is the HPLC chromatogram of PL-8, and C is the mass spectrum of PL-8.

[0028] Figure 10 The diagram shows the amino acid sequence of PL-9 and its characterization spectrum. In the diagram, A is the amino acid sequence of PL-9, B is the HPLC chromatogram of PL-9, and C is the mass spectrum of PL-9.

[0029] Figure 11 The diagram shows the amino acid sequence of PL-10 and its characterization spectrum. In the diagram, A is the amino acid sequence of PL-10, B is the HPLC chromatogram of PL-10, and C is the mass spectrum of PL-10.

[0030] Figure 12 The diagram shows the amino acid sequence of PL-11 and its characterization spectrum. In the diagram, A is the amino acid sequence of PL-11, B is the HPLC chromatogram of PL-11, and C is the mass spectrum of PL-11.

[0031] Figure 13 The diagram shows the amino acid sequence of PL-12 and its characterization spectrum. In the diagram, A is the amino acid sequence of PL-12, B is the HPLC chromatogram of PL-12, and C is the mass spectrum of PL-12.

[0032] Figure 14 The diagram shows the amino acid sequence of PL-13 and its characterization spectrum. In the diagram, A is the amino acid sequence of PL-13, B is the HPLC chromatogram of PL-13, and C is the mass spectrum of PL-13.

[0033] Figure 15 The diagram shows the amino acid sequence of PL-14 and its characterization spectrum. In the diagram, A is the amino acid sequence of PL-14, B is the HPLC chromatogram of PL-14, and C is the mass spectrum of PL-14.

[0034] Figure 16 The diagram shows the amino acid sequence of PL-15 and its characterization spectrum. In the diagram, A is the amino acid sequence of PL-15, B is the HPLC chromatogram of PL-15, and C is the mass spectrum of PL-15.

[0035] Figure 17 The diagram shows the amino acid sequence of PL-16 and its characterization spectrum. In the diagram, A is the amino acid sequence of PL-16, B is the HPLC chromatogram of PL-16, and C is the mass spectrum of PL-16. Detailed Implementation

[0036] The present application will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present application. However, these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. That is, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0037] This invention designs and synthesizes 16 staple peptides based on the amino acid sequence of the template polypeptide raniseptin PL:Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2. A schematic diagram, HPLC chromatogram, and mass spectrum of the template polypeptide PL-0 are shown below. Figure 1 .

[0038] The experimental materials involved in the embodiments of this invention were sourced as follows: Fmoc-amino acids and RinkamideMBHA amino resin were purchased from Nankai Synthetic Co., Ltd.; Fmoc-amino acids, N,N-dimethylformamide, N,N-diisopropylcarbodiimide, and ethyl 2-oxime cyanoacetate were purchased from Jier Biochemical (Shanghai) Co., Ltd.; trifluoroacetic acid, acetonitrile (chromatographic grade), benzyl sulfide, 1,2-ethanedithiol, anhydrous diethyl ether, dichloromethane, 1,2-dichloroethane, piperidine, and phenol were all analytical grade and purchased from Shanghai Titan Technology Co., Ltd.

[0039] Example 1: Preparation of PL-0-based staple peptide 1. General Synthesis Process All stapled peptides (PL-1 to PL-16) were prepared using a solid-phase peptide synthesis method based on the Fmoc (fluorenemethyloxycarbonyl) protection strategy. This method used Rink amide MBHA resin as the solid-phase support at a loading capacity of 0.30 mmol / g. Synthesis was carried out in solid-phase synthesis reaction tubes, and the entire process included resin swelling, Fmoc protecting group removal, amino acid condensation, N-terminal acetylation, olefin metathesis cyclization, and final peptide cleavage.

[0040] (1) Resin pretreatment and activation: Take 400 mg of amino resin and soak it in dichloromethane solvent for 30 minutes to allow it to swell fully. Then, treat it twice (5 minutes each time) with 7 mL of 20% piperidine N,N-dimethylformamide solution at 35 °C to remove the Fmoc protecting groups on the resin surface. Finally, wash the resin three times each with N,N-dimethylformamide, dichloromethane and N,N-dimethylformamide in sequence.

[0041] (2) Amino acid condensation: Following the sequence of the target polypeptide PL-0 (Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2), amino acids were sequentially linked from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus). For common amino acids, 1 mmol of Fmoc-protected amino acid, 142 mg of ethyl 2-oxime cyanoacetate, and 200 μL of N,N-diisopropylcarbodiimide were dissolved in 7 mL of N,N-dimethylformamide, activated at 37°C for 15 minutes, and then added to a reaction tube for coupling with resin. The reaction was carried out at 60°C for 20 minutes. For non-natural amino acids S5 or R8, the amount used was 0.2 mmol, and the condensing agent was 43 mg of ethyl 2-oxime cyanoacetate and 60 μL of N,N-diisopropylcarbodiimide. After activation in the same solvent, the reaction was carried out at 60°C for 3 hours or at 37°C overnight to ensure complete coupling. After each amino acid is attached, the Fmoc protecting group must be removed with a 20% piperidine N,N-dimethylformamide solution, followed by washing.

[0042] (3) N-terminal acetylation: After sequence synthesis, the Fmoc protecting group of the terminal amino acid was removed using a 20% piperidine N,N-dimethylformamide solution. Then, 10 mL of acetylation reagent (V diisopropylethylamine: V acetic anhydride: V N,N-dimethylformamide = 1:1:8) was added, and the reaction was carried out at 37°C for 5 minutes to acetylate the N-terminus of the peptide. After the reaction, the resin was dried and washed.

[0043] (4) Olefin metathesis reaction (cyclization): The resin was rinsed three times with 1,2-dichloroethane. A solution of 56 mg of phenylmethylene bis(tricyclohexylphosphine) ruthenium dichloride dissolved in 6 mL of 1,2-dichloroethane was added, and the reaction was carried out at room temperature for 8 hours. This caused a ring-closing metathesis reaction between the introduced non-natural amino acid side chain olefins, forming a full-carbon scaffold, thereby stabilizing the α-helical conformation of the peptide. The resin was thoroughly washed after the reaction was completed.

[0044] (5) Peptide cleavage and purification: The resin was placed in a 50 mL centrifuge tube, and 20 mL of cleavage reagent K (trifluoroacetic acid: water: 1,2-ethylenedithiol: benzyl sulfide: phenol = 82.5: 5: 2.5: 5: 5, V / V / V / V / V) was added. The mixture was shaken at 37°C for 3 hours. After the reaction was complete, the cleavage solution was collected, dried under nitrogen, and concentrated. The crude peptide was then precipitated with pre-cooled ice-cold ether. After centrifugation (3500 r / min, 3 min), the supernatant was discarded, and the precipitate was air-dried to obtain the crude target peptide. The crude peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and its structure was identified by mass spectrometry.

[0045] 2. Specific synthesis of each binding peptide The general synthetic procedure described above applies to all staple peptides from PL-1 to PL-16. The difference between each staple peptide lies in the substitution of a specific position in its template sequence by a non-natural amino acid S5 or a combination of S5 and R8, as shown in Table 1 below. These substitutions are the basis for the olefin metathesis reaction to proceed and form the specific "staple" structure.

[0046] Table 1. Specific amino acid substitution sites for each binding peptide Note: S5 and R8 are specific non-natural amino acids that are introduced for subsequent olefin metathesis reactions.

[0047] 2. Purification of staple peptide samples The crude peptide was dissolved in a mixed solvent of acetonitrile and water, and purified by reversed-phase preparative RP-HPLC to obtain the purified staple peptide product. The separation conditions were as follows: Instrument: Shimadzu LC-20A reversed-phase high-performance liquid chromatograph; Column: UltimateXB-C18, 21.2×250mm, 5μm; Mobile phase: Mobile phase A is an acetonitrile solution of 0.1% trifluoroacetic acid by volume, and mobile phase B is an aqueous solution of 0.1% trifluoroacetic acid by volume; Procedure and parameters: Elute with 90%B for 3 min, then elute with 90%B~50%B for 40 min; flow rate is 8 mL / min, injection volume is 3 mL, and detection wavelengths are 214 nm and 254 nm.

[0048] Each peptide is purified individually.

[0049] During the gradient elution process, when the volume fraction of mobile phase A increased to approximately 60%, compound PL-0 was eluted to obtain PL-0, with a separation rate of 9.5%.

[0050] During gradient elution, when the volume fraction of mobile phase A increased to approximately 81%, compound PL-1 was eluted to obtain PL-1, with a separation rate of 22.6%.

[0051] During gradient elution, when the volume fraction of mobile phase A increased to approximately 65%, compound PL-2 was eluted to obtain PL-2, with a separation rate of 31.3%.

[0052] During the gradient elution process, when the volume fraction of mobile phase A increased to approximately 60%, compound PL-3 was eluted to obtain PL-3, with a separation rate of 11.0%.

[0053] During the gradient elution process, when the volume fraction of mobile phase A increased to approximately 69%, compound PL-4 was eluted to obtain PL-4, with a separation rate of 30.6%.

[0054] During the gradient elution process, when the volume fraction of mobile phase A increased to approximately 74%, compound PL-5 was eluted to obtain PL-5, with a separation rate of 24.7%.

[0055] During the gradient elution process, when the volume fraction of mobile phase A increased to approximately 65%, compound PL-6 was eluted to obtain PL-6, with a separation rate of 25.5%.

[0056] During gradient elution, when the volume fraction of mobile phase A increased to approximately 83%, compound PL-7 was eluted to obtain PL-7, with a separation rate of 30.5%.

[0057] During gradient elution, when the volume fraction of mobile phase A increased to approximately 72%, compound PL-8 was eluted to obtain PL-8, with a separation rate of 18.5%.

[0058] During gradient elution, when the volume fraction of mobile phase A increased to approximately 68%, compound PL-9 was eluted to obtain PL-9, with a separation rate of 10.4%.

[0059] During gradient elution, when the volume fraction of mobile phase A increased to approximately 72%, compound PL-10 was eluted to obtain PL-10, with a separation rate of 23.6%.

[0060] During gradient elution, when the volume fraction of mobile phase A increased to approximately 68%, compound PL-11 was eluted to obtain PL-11, with a separation rate of 33.4%.

[0061] During gradient elution, when the volume fraction of mobile phase A increased to approximately 75%, compound PL-12 was eluted to obtain PL-12, with a separation rate of 27.4%.

[0062] During gradient elution, when the volume fraction of mobile phase A increased to approximately 67%, compound PL-13 was eluted to obtain PL-13, with a separation rate of 22.9%.

[0063] During gradient elution, when the volume fraction of mobile phase A increased to approximately 75%, compound PL-14 was eluted to obtain PL-14, with a separation rate of 27.3%.

[0064] During gradient elution, when the volume fraction of mobile phase A increased to approximately 90%, compound PL-15 was eluted to obtain PL-15, with a separation rate of 5.4%.

[0065] During gradient elution, when the volume fraction of mobile phase A increased to approximately 65%, compound PL-16 was eluted to obtain PL-16, with a separation rate of 8.5%.

[0066] Identification and structural analysis of the product in Example 2 The product obtained in step 2 of Example 1 was identified by reversed-phase HPLC. Analytical column: Welch C18; mobile phase A was an acetonitrile solution of 0.1% trifluoroacetic acid (v / v), and mobile phase B was an aqueous solution of 0.1% trifluoroacetic acid (v / v). Gradient elution was used (0–2 min, mobile phase B: 90%; 3–25 min, mobile phase B: 90%–10%); flow rate: 1.0 mL / min. -1 The detection wavelengths were 214 nm and 254 nm, and the injection volume was 24 μl. The peak elution time was consistent with that of the crude product, and the purity of the staple peptide prepared by this method was >95%. The HPLC chromatograms of PL-1-PL-16 staple peptides are shown below. Figures 2-17 .

[0067] Structural analysis was performed using HR-Q-TOF-MS, and the mass spectrometry analysis results of the obtained L-1-PL-16 binding peptide are shown below. Figures 2-17 The structure of the staple peptide obtained after analysis is shown in Table 2.

[0068] Table 2. Sequences of the template peptide and the modified binding peptide used in this invention. The amino acid sequences of the template peptides and the obtained staple peptides involved in Tables 1 and 2 of this invention are shown in SEQ ID NO: 1-17.

[0069] Example 3: Experiment on the inhibition of bacteria by the staple peptide of the present invention In vitro antimicrobial resistance assay: Solid LB medium was prepared, autoclaved, plated, and liquid LB medium was prepared and stored at 4°C. Bacterial suspension was spread onto solid LB medium and incubated overnight at 37°C. Single colonies were added to 3 mL of liquid LB medium and incubated at 37°C, 220 rpm for 6 h in a shaker to allow the bacteria to reach the logarithmic growth phase. 1 mL of bacterial suspension was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and PBS was added. The bacterial concentration was adjusted to 2 × 10⁶ CFU / mL based on the OD value. Different concentrations of antimicrobial peptides and bacterial suspensions were added to 96-well plates and incubated at 37°C for 8 h. Detection was performed using a microplate reader at 595 nm. The assay was repeated three times, and the MIC values ​​were statistically analyzed. The results are shown in Table 3.

[0070] Table 3. Antibacterial experimental results of the peptides in this invention. Table 3 shows that the 16 binding peptides obtained in this invention have significantly improved antibacterial properties compared to the template peptides. Among them, PL-2, PL-4, PL-6, PL-8, PL-9, PL-10, PL-11, PL-13, PL-14, and PL-16 showed good in vitro antibacterial properties against Staphylococcus aureus ATCC25923; PL-1, PL-2, PL-4, PL-5, PL-6, PL-8, PL-9, PL-10, PL-11, PL-13, PL-14, and PL-16 showed good in vitro antibacterial properties against Staphylococcus aureus ATCC43300; and PL-2, PL-4, PL-5, PL-6, PL-8, PL-9, PL-10, PL-11, PL-13, PL-14, and PL-16 showed good in vitro antibacterial properties against Staphylococcus aureus ATCC43300. 6. PL-8, PL-11, PL-13, PL-14, and PL-16 showed good in vitro antibacterial activity against Enterococcus faecalis ATCC19434; PL-13 and PL-14 showed good in vitro antibacterial activity against Enterococcus faecalis ATCC29212; PL-2, PL-4, PL-6, PL-8, PL-9, PL-10, PL-11, PL-13, PL-14, and PL-16 showed good in vitro antibacterial activity against Enterococcus faecalis ATCC51299; and PL-2, PL-4, PL-6, PL-8, PL-10, PL-11, PL-13, PL-14, and PL-16 showed good in vitro antibacterial activity against Staphylococcus epidermidis ATCC12228. Among them, the antibacterial activity of binding peptides PL-2, PL-4, PL-9, and PL-11 against Staphylococcus aureus ATCC25923 was 3 times higher than that of template peptide PL-0; PL-6, PL-8, PL-13, PL-14, and PL-16 against Staphylococcus aureus ATCC25923 was 7 times higher than that of template peptide PL-0; and PL-10 against Staphylococcus aureus ATCC25923 was 1 time higher than that of template peptide PL-0. PL-14 against Staphylococcus aureus ATCC43300 was 31 times higher than that of template peptide PL-0; PL-6, PL-13, and PL-16 against Staphylococcus aureus ATCC43300 was 15 times higher than that of template peptide PL-0; and PL-2, PL-4, PL-8, and PL-11 against Staphylococcus aureus ATCC43300 was 7 times higher than that of template peptide PL-0. PL-5 and PL-9 showed a 3-fold increase in antibacterial activity against Staphylococcus aureus ATCC43300 compared to the template peptide PL-0, while PL-1 showed a 1-fold increase in antibacterial activity against Staphylococcus aureus ATCC43300 compared to the template peptide PL-0.PL-9 and PL-10 showed a 1-fold increase in antibacterial activity against Enterococcus faecalis ATCC19434 compared to the template peptide PL-0; PL-2, PL-4, PL-6, PL-11, and PL-16 showed a 3-fold increase in antibacterial activity against Enterococcus faecalis ATCC19434 compared to the template peptide PL-0; PL-13 showed a 7-fold increase in antibacterial activity against Enterococcus faecalis ATCC19434 compared to the template peptide PL-0; PL-14 showed a 15-fold increase in antibacterial activity against Enterococcus faecalis ATCC19434 compared to the template peptide PL-0; and PL-8 showed a 31-fold increase in antibacterial activity against Enterococcus faecalis ATCC29212 compared to the template peptide PL-0. PL-9 and PL-11 showed a 1-fold increase in antibacterial activity against Enterococcus faecalis ATCC51299 compared to the template peptide PL-0. PL-2, PL-4, PL-6, PL-8, PL-10, and PL-16 showed a 3-fold increase in antibacterial activity against Enterococcus faecalis ATCC51299 compared to the template peptide PL-0. PL-13 and PL-14 showed a 7-fold increase in antibacterial activity against Enterococcus faecalis ATCC51299 compared to the template peptide PL-0. PL-8 showed a 15-fold increase in antibacterial activity against Enterococcus faecalis ATCC51299 compared to the template peptide PL-0. PL-10 and PL-16 showed a 1-fold increase in antibacterial activity against Staphylococcus epidermidis ATCC12228 compared to the template peptide PL-0. PL-2, PL-4, PL-6, PL-11, and PL-13 showed a 3-fold increase in antibacterial activity against Staphylococcus epidermidis ATCC12228 compared to the template peptide PL-0. PL-14 showed a 7-fold increase in antibacterial activity against Staphylococcus epidermidis ATCC12228 compared to the template peptide PL-0.

[0071] The above embodiments demonstrate that the present invention successfully prepared a modified staple peptide based on PL-0. In vitro experiments proved that the synthesized staple peptide can significantly inhibit the growth and reproduction of pathogenic bacteria, and has the potential to be developed into a novel antibacterial drug.

[0072] Example 4: Experimental Study of the Circular Dichroism Chromatography Method for the Bound Peptide of the Present Invention Linear peptide PL-0 and stapled peptides PL-1 through PL-16 were accurately weighed at 1 mg and dissolved separately in water and trifluoroethanol (V / V = 1:1) to a final concentration of 50 mM. The concentration was measured at room temperature using circular dichroism JASCO. J Characterization was performed using -1500 and 1 mm quartz cuvettes. The following experimental parameters were measured: wavelength, 190-260 nm; velocity, 20 nm / min. -1Bandwidth, 1 nm. For the α-helical structure, there is a positive band near 192 nm and two negative bands at 222 nm and 208 nm. Based on the ellipticity of the peptide spectrum at 222 nm and the number of amino acids in the peptide sequence, the helicity of each peptide is calculated using the equation helicity (%) = [θ]222 / (-39500(1-2.57 / n)) ×100.

[0073] The results are shown in Table 4.

[0074] Table 4 Helicity of Peptides Table 4 shows that the staple peptide of the present invention can improve the α-helix degree of the template peptide. The above examples demonstrate that the modified staple peptide based on PL-0 prepared by the present invention, through circular dichroism spectroscopy analysis, shows that, except for PL-6, the helix degree of the other staple peptides is greater than that of the linear peptide PL-0, indicating that the staple locking modified by the staple peptide strategy plays a certain role in reinforcing the peptide chain and improving the conformational stability of the peptide.

Claims

1. An antimicrobial polypeptide, characterized in that, The antibacterial polypeptide is a stapler peptide, and the stapler peptide is: PL-16: The peptide template is Ac-GVFDTVKKIGKAVGKFALGVAKNYLNS-NH2, in which amino acid residues 19G and 26N are replaced by R8 and S5.

2. The use of the antimicrobial polypeptide according to claim 1 in the preparation of an anti-Staphylococcus aureus drug.

3. The use of the antimicrobial polypeptide according to claim 1 in the preparation of anti-Enterococcus faecalis drugs.

4. The use of the antimicrobial polypeptide according to claim 1 in the preparation of an anti-Enterococcus faecium drug.

5. The use of the antimicrobial polypeptide according to claim 1 in the preparation of an anti-Staphylococcus epidermidis drug.

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