WK7 series antibacterial peptide and application thereof in aspects of resisting bacteria and preventing and treating diabetic foot, pneumonia and septicemia

Abstract

The invention belongs to the technical field of polypeptides and biological medicines, and particularly relates to a WK7 series antibacterial peptide and application thereof in the aspects of resisting bacteria and preventing and treating diabetic foot, pneumonia and septicemia. According to the invention, a linear peptide WK7-line with an amino acid sequence of WKRWKRW is used as a minimum active unit, and modification is carried out to form a plurality of linear modified peptides; meanwhile, a cyclic peptide WK7-cyl is formed on the basis of the linear peptide WK7-line through cyclization, and the cyclic peptide WK7-cyl is further modified to form an annular modified peptide. The linear peptide WK7-line, the linear modified peptide, the cyclic peptide WK7-cyl and the cyclic modified peptide jointly form the WK7-series antibacterial peptide, and the WK7-series antibacterial peptide is artificially synthesized polypeptide, is small in molecular weight, easy to synthesize, free of cytotoxicity, almost free of hemolysis risk, high in sterilization speed and broad-spectrum antibacterial property and can be used for preparing the antibacterial peptide. The traditional Chinese medicine composition has a certain effect on prevention and treatment of diabetic foot ulcer, pneumonia and septicemia.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide and biomedical technology, specifically relating to a WK7 series antimicrobial peptide and its application in antibacterial, prevention and treatment of diabetic foot, pneumonia and sepsis. Background Technology

[0002] The discovery of antibiotics effectively reduced the incidence of infectious diseases. However, the widespread use of antibiotics in recent years has led to the emergence of multidrug-resistant bacteria, making existing antibiotics increasingly ineffective in controlling infectious diseases. Therefore, there is an urgent need to develop new antibacterial drugs.

[0003] Antimicrobial peptides, as a novel antimicrobial strategy, have shown broad application prospects in the fields of medicine and biotechnology. Antimicrobial peptides include both natural and synthetic polypeptides, which differ significantly in size, charge, hydrophobicity, and secondary structure characteristics. In addition to antimicrobial activity, antimicrobial peptides also possess antiviral, antifungal, anticancer, and immunomodulatory functions.

[0004] In recent years, the escalating multidrug resistance crisis has raised concerns about untreatable infections with rising morbidity and mortality rates. Of particular concern is diabetic foot ulcer (DFU), one of the most serious and costly complications of diabetes, affecting approximately 25% of diabetic patients worldwide. More than half of these cases become infected, making it the leading cause of lower limb amputations. In diabetic foot ulcers, over 90% of DFU infections involve biofilm formation, and the emergence of multidrug-resistant bacteria renders antibiotic treatment ineffective.

[0005] Antimicrobial peptides have shown great and irreplaceable potential in the treatment of diabetic foot ulcer infections. They offer new hope, particularly in addressing the two core challenges of biofilm infection and bacterial resistance. However, many current antimicrobial peptides are natural polypeptides with large molecular weights, making artificial synthesis difficult. Furthermore, some antimicrobial peptides pose risks of cytotoxicity or hemolysis, hindering their application in the prevention and treatment of diabetic foot ulcers. Summary of the Invention

[0006] The purpose of this invention is to provide a WK7 series antimicrobial peptide and its application in antibacterial treatment, prevention and treatment of diabetic foot, pneumonia and sepsis. The WK7 series antimicrobial peptide is a synthetic polypeptide with a small molecular weight, which is easy to synthesize artificially. It is non-cytotoxic and has almost no risk of hemolysis. It has broad-spectrum antibacterial activity, good repair and protection effects on diabetic foot ulcers, and certain effects on the prevention and treatment of sepsis and pneumonia.

[0007] This invention provides a WK7 series antimicrobial peptide, comprising at least one of the following: a) A linear peptide WK7-line with an amino acid sequence as shown in SEQ ID NO:1; b) A linear modified peptide obtained by modifying the linear peptide WK7-line described in a), wherein the modification includes at least one of b1) to b4): b1) N-terminal acetylation, b2) C-terminal amidation, b3) L- and D-type amino acid substitution, b4) N-terminal or C-terminal saturated fatty acid modification; c) A cyclic peptide WK7-cyl formed by cyclization of the linear peptide WK7-line described in a); d) A cyclic modified peptide obtained by modifying the cyclic peptide WK7-cyl described in c) with saturated fatty acids; e) Forming a polypeptide derivative based on any one of the antimicrobial peptides in a) to d).

[0008] Preferably, c) the cyclic peptide WK7-cyl is formed by cyclizing the N-terminus and C-terminus of the linear peptide WK7-line through an amide bond, or by introducing cysteine ​​residues at the N-terminus and C-terminus respectively to form a cyclic peptide through a disulfide bond.

[0009] Preferably, d) the position of the fatty acid chain modification is the first lysine residue starting from the N-terminus.

[0010] Preferably, the saturated fatty acid modification includes: linking saturated fatty acids and WK7 series antimicrobial peptides via amide bonds.

[0011] Preferably, the saturated fatty acid includes saturated fatty acids with an even number of carbon atoms.

[0012] This invention also provides the application of the WK7 series antimicrobial peptides described in the above technical solution in at least one of the following: A) Preparation of antibacterial products; B) Prepare medicines for the prevention and / or treatment of diabetic foot; C) Prepare drugs for the prevention and / or treatment of pneumonia; D) Prepare drugs for the prevention and / or treatment of sepsis.

[0013] Preferably, the bacteria include bacteria and / or fungi.

[0014] Preferably, the bacteria include at least one of Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Staphylococcus epidermidis, Staphylococcus hemolyticus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Streptococcus. The fungi include at least one of Candida albicans, Candida auris, Cryptococcus neoformans, and Candida albicans.

[0015] Preferably, the diabetic foot includes a diabetic foot ulcer.

[0016] The present invention also provides a drug and / or product, wherein the active ingredient comprises the WK7 series antimicrobial peptides described in the above technical solution.

[0017] Beneficial effects: This invention uses the linear peptide WK7-line with the amino acid sequence WKRWKRW as the smallest active unit, and performs modifications including N-terminal acetylation, C-terminal amidation, L- and D-type amino acid substitution, and N-terminal or C-terminal saturated fatty acid modification to form various linear modified peptides. Simultaneously, a cyclic peptide WK7-cyl is formed from the linear peptide WK7-line, and further modified to form a cyclic modified peptide. The linear peptide WK7-line, the linear modified peptide, the cyclic peptide WK7-cyl, and the cyclic modified peptide together constitute the WK7 series of antimicrobial peptides in this invention. These WK7 series antimicrobial peptides are synthetically produced polypeptides with small molecular weights, are easy to synthesize, are non-cytotoxic, have almost no risk of hemolysis, exhibit rapid bactericidal action, possess broad-spectrum antibacterial activity, show good repair effects on diabetic foot ulcers, and also have certain effects on the prevention and treatment of sepsis and pneumonia. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 This is a kinetic curve of the WK7 series antimicrobial peptides in Example 3; Figure 2 This is a TEM image of the treatment of Staphylococcus aureus with WK7 series antimicrobial peptides in Example 3; Figure 3 This is a graph showing the cytotoxicity and hemolytic activity results of the WK7 series antimicrobial peptides in Example 4; Figure 4 This is a diagram showing the results of WK7 series antimicrobial peptides promoting STZ-induced diabetic wound healing in Example 5. Figure 5 This is a diagram showing the results of WK7 series antimicrobial peptides clearing bacteria from STZ diabetic wounds in Example 5. "Indicates significant difference" P <0.05. "Indicates significant difference" P <0.01, "Indicates significant difference" P <0.001; Figure 6 This is a graph showing the results of the WK7 series antimicrobial peptides improving wound inflammation in Example 5; Figure 7 This is a graph showing the results of WK7 series antimicrobial peptides reducing MRSA-infected lung bacterial load in Example 6, where " "Indicates significant difference" P <0.0001; Figure 8 This is a diagram showing the results of the WK7 series antimicrobial peptides inhibiting the spread of MRSA-infected sepsis bacteria to target organs in Example 7, where " "Indicates significant difference" P <0.05. "Indicates significant difference" P <0.01, "Indicates significant difference" P <0.001, "Indicates significant difference" P <0.0001. Detailed Implementation

[0020] This invention provides a WK7 series antimicrobial peptide, comprising at least one of the following: a) A linear peptide WK7-line with an amino acid sequence as shown in SEQ ID NO:1; b) A linear modified peptide obtained by modifying the linear peptide WK7-line described in a), wherein the modification includes at least one of b1) to b4): b1) N-terminal acetylation, b2) C-terminal amidation, b3) L- and D-type amino acid substitution, b4) N-terminal or C-terminal saturated fatty acid modification; c) A cyclic peptide WK7-cyl formed by cyclization of the linear peptide WK7-line described in a); d) A cyclic modified peptide obtained by modifying the cyclic peptide WK7-cyl described in c) with saturated fatty acids; e) Forming a polypeptide derivative based on any one of the antimicrobial peptides in a) to d).

[0021] The amino acid sequence shown in SEQ ID NO:1 of this invention is WKRWKRW (tryptophan-lysine-arginine-tryptophan-lysine-arginine-tryptophan).

[0022] In one embodiment, c) the cyclic peptide WK7-cyl is formed by cyclizing the N-terminus and C-terminus of the linear peptide WK7-line through an amide bond, or by introducing cysteine ​​residues at the N-terminus and C-terminus respectively to form a cyclization through a disulfide bond, more preferably by cyclizing the N-terminus and C-terminus of the linear peptide WK7-line through an amide bond.

[0023] In one embodiment, d) the fatty acid chain modification is performed at the first lysine residue from the N-terminus. In another embodiment, the saturated fatty acid modification of this invention involves linking a saturated fatty acid and a WK7 series antimicrobial peptide via an amide bond, specifically, forming an amide bond through dehydration condensation between the carboxyl group of the saturated fatty acid and the amino group of the WK7 series antimicrobial peptide. In another embodiment, the saturated fatty acid includes saturated fatty acids with an even number of carbon atoms, including butyric acid, caprylic acid, capric acid, dodecanoic acid, myristic acid, palmitic acid, or stearic acid.

[0024] In one embodiment, the L-type and D-type amino acid substitution modification involves replacing some or all of the L-type amino acids in the linear peptide WK7-line with D-type amino acids.

[0025] In one embodiment, the polypeptide derivative may be a salt compound formed by an organic or inorganic acid based on any one of the antimicrobial peptides a) to d); or a compound containing ethers, esters, glycosides, or glycosides formed by the hydroxyl groups of any one of the antimicrobial peptides a) to d); or a compound containing disulfide bonds formed by the thiol groups of any one of the antimicrobial peptides a) to d); or a compound containing acylates, hydrocarbons, or glycosides formed by any one of the antimicrobial peptides a) to d) with cysteine ​​or cysteine-containing peptides; or a compound containing acylates, hydrocarbons, or glycosides formed by any one of the antimicrobial peptides a) to d); or a compound containing esters, amides, or glycosides formed by any one of the antimicrobial peptides a) to d); a complex, chelate, or compound formed by any one of the antimicrobial peptides a) to d) with metal ions; or a hydrate or solvent formed by any one of the antimicrobial peptides a) to d).

[0026] In this invention, the WK7 series antimicrobial peptides are artificially synthesized polypeptides with small molecular weights, making them easy to synthesize. This invention does not impose any particular limitation on the synthesis method of the WK7 series antimicrobial peptides; conventional polypeptide preparation steps in the art can be used, such as solid-phase synthesis, or synthesis can be outsourced to a third-party company.

[0027] In this invention, the WK7 series antimicrobial peptides possess broad-spectrum antimicrobial activity, exhibiting significant killing or inhibitory effects against a variety of common pathogenic microorganisms, including Gram-negative bacteria (such as Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, etc.), Gram-positive bacteria (such as Staphylococcus aureus, Streptococcus, hemolytic and epidermal Staphylococcus, etc.), and fungi (such as Candida albicans, Candida auris, Cryptococcus neoformans, and Candida albicans), and are equally effective against drug-resistant strains. Furthermore, the WK7 series antimicrobial peptides of this invention can rapidly kill bacteria and fungi, which is beneficial for timely control of the progression of bacterial and fungal-related diseases in the early stages of infection. Moreover, the WK7 series antimicrobial peptides exhibit low cytotoxicity to mammalian cells and almost no hemolytic activity against erythrocytes, indicating good biocompatibility and potential safety for clinical application. Furthermore, the WK7 series antimicrobial peptides can significantly promote the healing of diabetic foot ulcers, and the cyclic peptide WK7-cyl can effectively eliminate bacterial colonization in diabetic infected wounds, significantly promoting wound healing, showing important application potential in the treatment of refractory diabetic foot ulcers.

[0028] Based on the above advantages, the present invention also provides the application of the WK7 series antimicrobial peptides described in the above technical solution in at least one of the following: A) Preparation of antibacterial products; B) Prepare medicines for the prevention and / or treatment of diabetic foot; C) Prepare drugs for the prevention and / or treatment of pneumonia; D) Prepare drugs for the prevention and / or treatment of sepsis.

[0029] In one embodiment, the bacteria include bacteria and / or fungi; in another embodiment, the bacteria can be Gram-negative bacteria and / or Gram-positive bacteria; in yet another embodiment, the Gram-negative bacteria can be at least one of *Escherichia coli*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, and *Klebsiella pneumoniae*; the Gram-positive bacteria can be at least one of *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Staphylococcus hemolyticus*, and *Streptococcus*. In yet another embodiment, the fungi are at least one of *Candida albicans*, *Candida auris*, *Cryptococcus neoformans*, and *Candida albicans*. In one embodiment, the bacteria or fungi of the present invention can also be drug-resistant bacteria or fungi, such as drug-resistant Gram-negative or Gram-positive bacteria.

[0030] In one embodiment, the antibacterial effect is at least one of inhibiting bacterial growth, sterilization, disinfection, and antibacterial infection.

[0031] In one embodiment, the diabetic foot includes a diabetic foot ulcer. In another embodiment, the medication for treating diabetic foot includes a drug having at least one function of promoting the repair of diabetic foot ulcers, promoting the healing of diabetic foot ulcers, clearing bacteria from the diabetic foot ulcer site, and improving inflammation at the ulcer site.

[0032] As one implementation method, the prevention and / or treatment of pneumonia can reduce bacterial colonization in the lungs.

[0033] As one implementation, the prevention and / or treatment of pneumonia can be to inhibit the spread of bacteria from the blood to target organs; the target organs can be at least one of the spleen, liver, and lungs.

[0034] This invention also provides a drug and / or product, the active ingredient of which includes the WK7 series antimicrobial peptides described in the above-described technical solution. As one embodiment, the drug is a drug for the prevention and / or treatment of diabetic foot, or a drug for the prevention and / or treatment of pneumonia, or a drug for the prevention and / or treatment of sepsis. As one embodiment, the product is an antimicrobial product. As one embodiment, the antimicrobial product includes antimicrobial cosmetics. As one embodiment, when the antimicrobial product is an antimicrobial cosmetic, the excipients can be one or more of polyols, glycerin, lanolin, mineral oil, and vegetable oil. As one embodiment, the excipients of the drug can be pharmaceutically acceptable excipients, such as gels and / or liposomes.

[0035] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1 One type of WK7 series antimicrobial peptide is as follows: Linear peptides: 1) WK7-line: The amino acid sequence is WKRWKRW; 2) C4-WK7-line: obtained by modifying the N-terminus of WK7-line with butyric acid (C4); 3) C8-WK7-line: obtained by modifying the N-terminus of WK7-line with octanoic acid (C8); 4) C 10 -WK7-line: With decanoic acid (C 10 This is obtained by modifying the N-terminus of WK7-line; 5) C 12 -WK7-line: With dodecanoic acid (C 12 This is obtained by modifying the N-terminus of WK7-line; 6) C 14-WK7-line: With tetradecanoic acid (C 14 This is obtained by modifying the N-terminus of WK7-line; The method for preparing linear peptides includes the following steps: (1) Select 1.25g of Fmoc-Trp(Boc)-WangResin resin with a loading of 0.40mmol / g, and remove the Fmoc protecting group after swelling with DCM; (2) Weigh 0.974 g of Fmoc-Arg(Pbf)-OH, 0.78 g of PyBop (benzotriazol-1-yl-oxytripyrrolidinephosphide hexafluorophosphate), and 1.05 mL of DIEA. Dissolve them in 20 mL of DMF at 0 °C. After complete dissolution, add the solution to the reaction column and react at room temperature for 1 hour. The reaction progress is judged by a negative result of the ninhydrin detection method. After the coupling reaction is completed, the reaction solution is dried and washed with 20 mL of DMF. Add 25 mL of 20% hexahydropyridine DMF solution and react for 5 min. Wash once with DMF (N,N-dimethylformamide). After washing, add 25 mL of 20% hexahydropyridine DMF solution again and react for 10 min. Drain the solution and wash 3 times with DMF, 2 times with DCM, and 1 time with DMF.

[0037] (3) According to the polypeptide sequence, coupling is performed sequentially from the C-terminus to the N-terminus until Boc-Trp(Boc)-OH, where the side chain protecting groups of Trp, Arg, and Lys are Boc, Pbf, and Boc, respectively, and the second Lys at the N-terminus is Fmoc-Lys(Dde)-OH; all amino acids are protected with Fmoc at the α-amino position. (4) After the coupling reaction is complete, add 2% hydrazine hydrate / DMF solution and react for 30 min. The reaction progress is judged by a positive result using the ninhydrin detection method. After the reaction is complete, the reaction solution is dried and washed three times with 30 mL DMF. Weigh 0.216 g of fatty acid, 0.78 g of PyBop (benzotriazol-1-yl-oxytripyrrolidinephosphide hexafluorophosphate), and 1.05 mL of DIEA. Dissolve them in 20 mL DMF at 0 °C. After complete dissolution, add them to the reaction column and react at room temperature for 1 hour. The reaction progress is judged by a negative result using the ninhydrin detection method. After the coupling reaction is complete, the reaction solution is dried and washed three times with 20 mL DMF, three times with DCM (dichloromethane), and three times with methanol. After drying, the resin is weighed as 2.551 g. (5) The peptide was reacted with a linear peptide resin using a lysis buffer (TFA: triisopropylsilane: water = 95:2.5:2.5) to obtain approximately 0.825 g of peptide with all side chain protecting groups removed. (6) The crude linear peptide aqueous solution was filtered through a 0.45 μm filter membrane. The purified crude peptide was then purified using high performance liquid chromatography (HPLC): a DAC-HB50 dynamic axial compression column was used with mobile phase A being a 0.05% trifluoroacetic acid aqueous solution and mobile phase B being a 0.05% trifluoroacetic acid acetonitrile solution. Gradient elution and purification were performed, and the sample was detected using a UV detector. The peptide solution of the target peak was collected in segments. After HPLC purification, 100 mL of finished peptide liquid with a purity greater than 95% was obtained. After rotary evaporation and concentration, 55 mL of liquid was obtained. The liquid was pre-lyophilized and lyophilized to finally obtain 62 mg of refined peptide.

[0038] Cyclic peptides: 1) WK7-cyl: formed by the dehydration condensation of the first and last tryptophan of WK7-line (WKRWKRW) to form a peptide bond ring; 2) C4-WK7-cyl: Butyric acid (C4) reacts with WK7 (WK...) 1 RWK 2 RW)-cyl K 1 It is obtained by modifying the amino group; 3) C8-WK7-cyl: Caprylic acid (C8) reacts with WK7 (WK...) 1 RWK 2 RW)-cyl in K 1 It is obtained by modifying the amino group; 4) C 10 -WK7-cyl: decanoic acid (C 10 ) for WK7 (WK 1 RWK 2 RW)-cyl in K 1 It is obtained by modifying the amino group; 5) C 12 -WK7-cyl: In the form of dodecanoic acid (C 12 ) for WK7 (WK 1 RWK 2 RW)-cyl in K 1 It is obtained by modifying the amino group; 6) C 14 -WK7-cyl: In tetradecanoic acid (C 14 ) for WK7 (WK 1 RWK 2 RW)-cyl in K 1 It is obtained by modifying the amino group; 7)(d)WK7-cyl: Replace the L-type amino acid in WK7-cyl with the D-type amino acid.

[0039] The preparation method of cyclic peptides is as follows: (1) Select 2.85 g of Fmoc-Trp(Boc)-2-Chlorotrityl ChlorideResin resin with a loading of 0.35 mmol / g, and remove the Fmoc protecting group after swelling; (2) Weigh 1.94 g of Fmoc-Arg(Pbf)-OH, 1.56 g of PyBop (benzotriazol-1-yl-oxytripyrrolidinephosphide hexafluorophosphate), and 2.32 mL of DIEA. Dissolve them in 30 mL of DMF at 0 °C. After complete dissolution, add the solution to the reaction column and react at room temperature for 1 hour. The reaction progress is judged by a negative result of the ninhydrin detection method. After the coupling reaction is completed, the reaction solution is dried and washed with 30 mL of DMF. Then, 35 mL of 20% hexahydropyridine DMF solution is added and the reaction is carried out for 5 min. The solution is washed once with DMF (N,N-dimethylformamide). After washing, 35 mL of 20% hexahydropyridine DMF solution is added again and the reaction is carried out for 10 min. The solution is then dried and washed 3 times with DMF, 2 times with DCM, and 1 time with DMF.

[0040] (3) According to the polypeptide sequence, coupling is performed sequentially from the C-terminus to the N-terminus until the first amino acid at the N-terminus is Fmoc-Trp(Boc)-OH, where the side chain protecting groups of Trp, Lys, and Arg are Boc, Boc, and Pbf, respectively; all amino acids are protected with Fmoc at the α-amino position; the second amino acid at the N-terminus is Fmoc-Lys(Alloc)-OH; the coupling reaction of each amino acid is terminated after the ninhydrin test is negative. (4) After the coupling reaction is completed, the reaction solution is dried and washed three times with 15 mL DMF. 0.512 g of tetraphenylphosphine palladium is added and dissolved in 20 mL MDF and reacted for 30 min. The reaction progress is judged by the positive result of the ninhydrin test. After the reaction is completed, the solution is washed three times with 15 mg / mL sodium diethyldithiocarbamate DMF and three times with DMF. 0.422 g of fatty acid, 1.56 g of PyBop (benzotriazol-1-yl-oxytripyrrolylphosphide hexafluorophosphate), and 2.32 mL of DIEA are added and dissolved in 20 mL DMF at 0 °C. After complete dissolution, the solution is added to the reaction column and reacted at room temperature for 1 hour. The reaction progress is judged by the negative result of the ninhydrin test. After the coupling reaction was completed, Fmoc was removed, the reaction solution was dried, washed 6 times with DCM and 3 times with methanol, and then dried. The resin was weighed to be 4.43 g. The linear peptide resin was reacted with the lysis buffer (TFA: dichloromethane = 2:98) to obtain a mixed solution of linear peptide with all side chain protecting groups and TFA / DCM. (5) Adjust the pH of the linear peptide with side chain protecting group and TFA / DCM mixture to neutral, concentrate by rotary evaporation, dissolve it in 20mL DMF and adjust the pH to 7.5-8.0, add 0.624g PyBop and 0.41mL DIEA and carry out cyclization reaction. After the reaction is completed, add water and filter the filter cake of 2.051g after precipitation by mass spectrometry. (6) The above filter cake was reacted with the lysis buffer (TFA: triisopropylsilane: water = 95: 2.5: 2.5) to obtain 618 mg of cyclic polypeptide with all side chain protecting groups removed, which was then dissolved in an appropriate amount of pure water. (7) The crude linear peptide aqueous solution was filtered through a 0.45 μm filter membrane. The purified crude peptide was then purified using high performance liquid chromatography (HPLC): a DAC-HB50 dynamic axial compression column was used with mobile phase A being a 0.05% trifluoroacetic acid aqueous solution and mobile phase B being a 0.05% trifluoroacetic acid acetonitrile solution. Gradient elution and purification were performed, and the sample was detected using a UV detector. The peptide solution of the target peak was collected in segments. After HPLC purification, 40 mL of finished peptide liquid with a purity greater than 95% was obtained. After rotary evaporation and concentration, 18 mL of liquid was obtained. The liquid was pre-lyophilized and lyophilized to finally obtain 32.8 mg of refined peptide.

[0041] (d) Preparation method of WK7-cyl, the steps are as follows: The steps for preparing WK7-cyl are the same as those for preparing cyclic peptides, except that the L-amino acids in the target reaction of the cyclic peptide preparation method are replaced with D-amino acids.

[0042] Preparation method of CWKRWKRWC cyclic peptide (SS-WK7-cyl) with disulfide bonds at both ends: The preparation steps are the same as those for the cyclic peptides described above, except that step (3) is modified as follows: according to the polypeptide sequence, Cys(Trt) - Trp(Boc) - Lys(Boc) - Arg(Pbf) - Trp(Boc) - Lys(Boc) - Arg(Pbf) - Trp(Boc) - Cys(Trt), all amino acids are protected with Fmoc at the α-amino position; the coupling reaction of each amino acid is terminated after the ninhydrin test is negative; step (5) is modified as follows: the pH of the straight-chain peptide with the side chain protecting group and the TFA / DCM mixture is adjusted to neutral, concentrated by rotary evaporation, and then dissolved in 20 mL of 0.1 M ammonium phosphate buffer to adjust the pH to 7.5-8.0 for cyclization reaction. After the reaction is completed by mass spectrometry, water is added to precipitate and the filtered cake is 2.051 g.

[0043] Example 2 The antimicrobial activity of the WK7 series antimicrobial peptides in Example 1 against the standard strain was determined, and the results are as follows: The experiment used a two-fold serial dilution method. The specific experimental procedure is as follows: Prepare fresh bacterial culture, and use a UV spectrophotometer to detect the OD of the bacterial culture. 600 According to 1 OD 600 =1×10 9 CFU / mL, the above bacterial culture concentration was adjusted to 2×10⁻⁶ using 1640 medium containing 10% serum (FBS). 5 CFU / mL. Then, 100 μL of physiological saline was added to a sterile 96-well plate beforehand. The test sample was added to the first well, and the test samples were serially diluted twofold. Then, 100 μL of a 2×10⁻⁶ CFU / mL solution was added to each well. 5 The bacterial suspension of CFU / mL was mixed by pipetting and then incubated overnight (18h) at 37℃. Finally, the absorbance of the bacterial suspension at 600nm was detected by microplate reader. The minimum inhibitory concentration (MIC) was the average of the concentrations of the wells in which no bacterial growth was detected and the concentrations of the samples in adjacent wells. The results are shown in Tables 1-4.

[0044] Table 1. Minimum Inhibitory Concentrations (MICs) of WK7 Series Antimicrobial Peptides

[0045] Note: C n -WK7-line, C n The N-terminus of linear WK7 (WKRWKRW) was modified with saturated fatty acid chains, n=4,8,10,12,14; C n -WK7-cyl, C n WK7 (WK) with cyclotomic amides 1 RWK 2 In RW), the amino group of K1 is modified with a saturated fatty acid chain, n=4, 8, 10, 12, 14. (d)WK7-cyl: L-type amino acid is replaced with D-type amino acid. SS-WK7-cyl: CWKRWKRWC forms a ring with the first and last disulfide bonds.

[0046] EC: Escherichia coli ATCC 8739, SA: Staphylococcus aureus ATCC 6538, AB: Acinetobacter baumannii ATCC 19606, SEp: Staphylococcus epidermidis ATCC 12228, CA: Candida albicans BNCC 299343, PA: Pseudomonas aeruginosa ATCC 27853, KP: Klebsiella pneumoniae ATCC 700603, Strep: Streptococcus pneumoniae ATCC 49619, CAu: Candida auriculariae BNCC 357786, CF: Cryptococcus neoformans BNCC 225501.

[0047] The above-mentioned strains were purchased from the ATCC Culture Collection Center.

[0048] Table 1 shows that the WK7 series antimicrobial peptides have varying degrees of antimicrobial activity against Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Candida albicans. The MICs against Acinetobacter baumannii are 100–12.5 μg / mL, against Pseudomonas aeruginosa are 100–12.5 μg / mL, against Staphylococcus aureus are 50–0.78 μg / mL, against Escherichia coli are 100–6.25 μg / mL, against Candida albicans are 100–6.25 μg / mL, and against Pseudomonas aeruginosa are 100–12.5 μg / mL.

[0049] Table 2. MICs of WK7 series antimicrobial peptides against clinically derived Staphylococcus aureus

[0050] Table 2 shows that the MIC of WK7-cyl against clinically derived Staphylococcus aureus and MRSA is 6.25~0.78 μg / mL.

[0051] Table 3. MICs of WK7 series antimicrobial peptides against clinically derived bacteria

[0052] Table 3 shows that the WK7 series peptides have anti-Gram-negative bacterial activity.

[0053] Table 4. MICs of WK7 series antimicrobial peptides against clinically derived fungi

[0054] Table 4 shows that the WK7 series peptides have antifungal activity.

[0055] The clinical strains listed in Tables 2-4 were obtained from the First Affiliated Hospital of Kunming Medical University, Yunnan Province.

[0056] Example 3 The bactericidal curves and disruption of bacterial wall and membrane integrity of the WK7 series antimicrobial peptides against Staphylococcus aureus (ATCC 6538) are as follows: The bacteria were washed three times with physiological saline and then resuspended in RPMI 1640 medium containing 10% fetal bovine serum at a concentration of 2 × 10⁻⁶. 5CFU / mL. WK7 series antimicrobial peptides (1×MIC) were added to the bacterial suspension and incubated at 37°C for 0, 15, 30, 60, 120, 240, 360, 480, 720, and 1440 min, respectively. At each time point, 10 μL of the liquid was aspirated and diluted 100 times with physiological saline, with the resulting 100 μL diluted liquid spread onto agar plates. After incubation at 37°C for 24 h, viable bacterial colonies were measured, and the results are as follows: Figure 1 As shown, where Figure 1 The Control in this context refers to physiological saline.

[0057] Bacteria were treated with 1×MIC WK7 series antimicrobial peptides for 1 h, followed by centrifugation at 3000 rpm for 5 min. The supernatant was discarded, and 2.5% glutaraldehyde was slowly added for fixation at 4°C overnight. Then, 2.5% glutaraldehyde and 1% osmium tetroxide were slowly added to the precipitate and fixed at 4°C for 2 h. After dehydration, the precipitate was inoculated and stained, and observed under a transmission electron microscope (JEOL, JEM-1011, Japan). Figure 2 As shown, where Figure 2 The Control in this context refers to physiological saline.

[0058] Depend on Figure 1 The bactericidal curve results showed that the WK7 series antimicrobial peptides had a rapid bactericidal rate and disrupted the integrity of the Staphylococcus aureus cell wall and cell membrane. Figure 2 ).

[0059] Example 4 The steps for detecting the low cytotoxicity and hemolytic activity of WK7 series antimicrobial peptides are as follows: Cytotoxicity was determined using HaCaT and HEK293 cells from the cell bank of the Kunming Institute of Zoology, Chinese Academy of Sciences. HaCaT cells or HEK293 cells (1×10⁶) were used to determine cytotoxicity. 5 Cells were seeded into 96-well plates and cultured in DMEM medium containing 10% fetal bovine serum (FBS) and streptomycin (100 μg / mL) with penicillin (100 U / mL) in a 5% CO2 incubator. After 24 h of culture, different concentrations of WK7 series antimicrobial peptides (WK7-line, C8-WK7-line, WK7-cyl, (d)WK7-cyl, or C6-WK7-cyl) were added to the wells, and the cells were incubated for another 24 h under the same conditions. 10 μL of CCK8 was added to the cells, and after incubation for 2 h, cell viability was measured. The absorbance of the solution at 450 nm was measured using a microplate reader, and the results were analyzed based on the (OD) values. 实验组 -OD 对照组 ) / (OD 阳性对照组 -OD 对照组 Cell viability was calculated as (%) × 100%. The experiment was repeated three times. Results are as follows: Figure 3 As shown in A and B.

[0060] like Figure 3 As shown in Figures A and B, the WK7 series antimicrobial peptides did not exhibit significant cytotoxicity at concentrations ranging from 6.25 to 200 μg / mL.

[0061] In addition to cytotoxicity, the hemolytic activity of a drug is also an important indicator for its safety evaluation. Therefore, this embodiment studies the hemolytic activity of WK7 series antimicrobial peptides on mature human erythrocytes.

[0062] In the hemolysis assay, mature human erythrocytes were washed three times with physiological saline and then resuspended in physiological saline. 100 μL of the human erythrocyte suspension was incubated with 100 μL of different concentrations of WK7 series antimicrobial peptides (WK7-line, C8-WK7-line, WK7-cyl, (d)WK7-cyl, or C6-WK7-cyl) at 37°C for 30 min, followed by centrifugation (3500 rpm, 5 min). The supernatant was collected, and its absorbance was measured at 450 nm. The "zero hemolysis" value was determined using sterile physiological saline (negative control), while the 100% hemolysis value was determined using 1% (v / v) Triton X-100 (positive control). The absorbance was determined based on (OD). 实验组 -OD 对照组 ) / (OD 阳性对照组 -OD 对照组 The percentage of hemolysis is calculated as (%) × 100%. The result is as follows: Figure 3 As shown in C.

[0063] Depend on Figure 3 From C, it can be concluded that at concentrations of 3.125~200μg / mL, the WK7 series antimicrobial peptides have no significant toxicity to erythrocytes.

[0064] Example 5 The following steps were taken to determine the efficacy of WK7 series antimicrobial peptides in clearing wound bacteria and promoting wound healing in diabetic patients: Establishment of a full-thickness skin lesion model: First, diabetic mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (mice were induced with streptozotocin (intraperitoneal injection of streptozotocin, 150 mg / kg), while the control group received the same volume of 0.1 M citrate buffer. After the inducing agent injection, the mice were fed for 3 days, followed by a 6-hour fast. Blood glucose levels were measured using a glucometer. Mice with blood glucose levels between 15-28 mmol / L (3.9-6.1 mmol / L, normal fasting levels), accompanied by polydipsia, polyuria, and polyphagia, were considered diabetic mouse models and used for experiments. Successfully established diabetic models were monitored for blood glucose for one week, with stable blood glucose levels between 15-28 mmol / L, and were used for the subsequent wound healing model (0.1 mL / 20g) to ensure the mice remained conscious after surgery. Hair on the back was removed with electric clippers, and the skin was disinfected with 75% alcohol. A circular wound of approximately 8 mm in diameter was made on the back, with a depth of full-thickness skin defect extending to the fascia layer outside the muscle.

[0065] After establishing the full-thickness skin lesion model, the wound was cleaned of blood and dirt with sterile gauze and infected with Staphylococcus aureus (1×10⁻⁶). 8 Immediately after treatment, CFU / wound was photographed and recorded as wound day 0 post-traumatic period. WK7 series antimicrobial peptides or pexiganan samples were dissolved in PBS. Each wound was treated with carrier (20 μL, PBS), WK7 series antimicrobial peptides (20 μL, 20 μg / mL), or pexiganan (20 μL, 20 μg / mL) once daily from day 0 to day 9. Wound areas were monitored by taking digital photographs, and the wound area was obtained from the photographs using ImageJ. The relative wound healing rate (%) was calculated as follows: Relative Wound (%) = (A... t / A0) × 100% Calculate the healing rate at different time points (A0 represents the wound area on day 0, A t The data represents the wound area of ​​mice on days 3, 5, 7, and 9. Histopathological sections were examined to observe the level of inflammation, and Masson staining was performed to assess collagen production. Results are as follows: Figures 4-6 As shown.

[0066] Depend on Figures 4-6 As shown, treatment with WK7 series antimicrobial peptides promotes wound healing in diabetic mice. Figure 4 ), and can remove bacteria from wounds ( Figure 5 ), improve wound inflammation ( Figure 6 ).

[0067] Example 6 The determination of the effect of WK7 series antimicrobial peptides on reducing bacterial load in MRSA lung infections is performed as follows: Male C57BL / 6 mice (6 weeks old, 20g, purchased from Vital River) were randomly divided into four groups: NC treatment group, MRSA treatment group, Vancomycin treatment group, and peptide treatment group, with 5 mice in each group; NC processing group: No processing performed; MRSA treatment group: 20 μL of methicillin-resistant Staphylococcus aureus (MRSA) suspension (1×10⁻⁶) was added to the treatment group. 10 Male C57BL / 6 mice were injected intranasally with a solution of CFU / mL. Two hours after inoculation, the same volume of physiological saline was injected intranasally. Vancomycin treatment group: 20 μL of methicillin-resistant Staphylococcus aureus (MRSA) suspension (1×10⁻⁶) was added to the treatment group. 10 Male C57BL / 6 mice were injected intranasally with vancomycin (2 mg / kg) at a dose of CFU / mL. Two hours after inoculation, the mice were injected intranasally with vancomycin (2 mg / kg). Peptide treatment group: 20 μL of methicillin-resistant Staphylococcus aureus (MRSA) suspension (1×10⁻⁶) was added to the treatment group. 10 Male C57BL / 6 mice were injected intranasally with CFU / mL. Two hours after inoculation, WK7 series antimicrobial peptides (WK7-line, C8-WK7-line, WK7-cyl, (d)WK7-cyl or C8-WK7-cyl) were injected intranasally at 2 mg / kg. Mice in each treatment group were sacrificed 4 hours after MRSA inoculation, and lung tissue was collected. The tissue was homogenized on ice, serially diluted 100-fold with PBS, and then plated onto LB agar plates for bacterial counting. Results are as follows: Figure 7 As shown.

[0068] Depend on Figure 7 It can be concluded that the WK7 series antimicrobial peptides, like the positive control drug vancomycin, can reduce bacterial colonization in the lungs, thus indicating that they have a certain preventive or therapeutic effect on pneumonia.

[0069] Example 7 The inhibitory effect of WK7 series antimicrobial peptides on the spread of MRSA septicemia bacteria to target organs is as follows: Male C57BL / 6 mice were randomly divided into four groups: NC treatment group, MRSA treatment group, Vancomycin treatment group, and peptide treatment group, with five mice in each group. NC processing group: No processing performed; MRSA treatment group: 100 μL of methicillin-resistant Staphylococcus aureus suspension (1×10⁻⁶) 9 (CFU / mL) was injected intravenously into male C57BL / 6 mice, and 2 hours later, the same volume of physiological saline was injected intravenously. Vancomycin treatment group: 100 μL of Staphylococcus aureus MRSA-Z suspension (1×10⁻⁶) was added to the treatment group. 9 Male C57BL / 6 mice were intravenously injected with a concentration of CFU / mL, followed by an intravenous injection of vancomycin 2 mg / kg 2 h later. Polypeptide treatment group: 100 μL of Staphylococcus aureus MRSA-Z suspension (1×10⁻⁶) was added to the suspension. 9 Male C57BL / 6 mice were intravenously injected with CFU / mL. Two hours after inoculation, WK7 series antimicrobial peptides (WK7-line, C8-WK7-line, WK7-cyl, (d)WK7-cyl or C8-WK7-cyl) were intravenously injected at 2 mg / kg. Mice in each treatment group were sacrificed 4 hours after MRSA inoculation, and spleen, liver, lungs, and other organs were collected. These organs were homogenized on ice, serially diluted 100-fold with PBS, and the homogenates were plated onto LB agar plates for bacterial load determination. Results are as follows: Figure 8 As shown.

[0070] Depend on Figure 8 The results show that the WK7 series antimicrobial peptides, like the positive control drug vancomycin, can dose-dependently inhibit the spread of Staphylococcus aureus from the blood to target organs.

[0071] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A WK7 series of antibacterial peptides, characterized in that, comprises at least one of the following: a) a linear peptide WK7-line with an amino acid sequence as shown in SEQ ID NO: 1; b) a linear modified peptide obtained by modifying the linear peptide WK7-line in a), the modification comprising at least one of b1)~b4): b1) N-terminal acetylation, b2) C-terminal amidation, b3) L-form and D-form amino acid substitution, b4) N-terminal or C-terminal saturated fatty acid modification; c) a cyclic peptide WK7-cyl formed by cyclization of the linear peptide WK7-line in a); d) a cyclic modified peptide obtained by saturated fatty acid modification of the cyclic peptide WK7-cyl in c); e) a polypeptide derivative formed on the basis of any one of the antibacterial peptides in a)~d).

2. The WK7 series of antibacterial peptides according to claim 1, characterized in that, c) the cyclic peptide WK7-cyl is formed by cyclization of the N-terminal and C-terminal of the linear peptide WK7-line through an amide bond, or by introducing cysteine at the N-terminal and C-terminal respectively to form a disulfide bond.

3. The WK7 series of antibacterial peptides according to claim 1, characterized in that, d) the position comprising the fatty acid chain modification is the first lysine from the N-terminal.

4. The WK7 series of antibacterial peptides according to claim 1 or 3, characterized in that, The saturated fatty acid modification comprises connecting a saturated fatty acid and a WK7 series antibacterial peptide through an amide bond.

5. The WK7 series of antibacterial peptides according to claim 1 or 3, characterized in that, The saturated fatty acid comprises a saturated fatty acid with an even number of carbon atoms.

6. The WK7 series antibacterial peptide of any one of claims 1~5 for use in at least one of the following: A) preparation of an antibacterial product; B) preparation of a drug for preventing and / or treating diabetic foot; C) preparation of a drug for preventing and / or treating pneumonia; D) preparation of a drug for preventing and / or treating sepsis.

7. Use according to claim 6, characterized in that, The bacteria comprise bacteria and / or fungi.

8. Use according to claim 7, characterized in that, The bacteria include at least one of Escherichia coli (E. coli) Escherichia coli ), Staphylococcus aureus (S. aureus) Staphylococcus aureus ), Acinetobacter baumannii (A. baumannii) Acinetobacter baumannii ), Staphylococcus epidermidis (S. epidermidis) Staphylococcus epidermidis ), Staphylococcus hemolyticus (S. hemolyticus) Staphylococcus haemolyticus ), Pseudomonas aeruginosa (P. aeruginosa) Pseudomonas aeruginosa ), Klebsiella pneumoniae (K. pneumoniae) Klebsiella Pneumoniae ), and Streptococcus (S.) Streptococcus ). The fungus includes at least one of Candida albicans (C. albicans) Canidia Albicans ), Candida auris (C. auris) Candida auris ), Cryptococcus neoformans (C. neoformans) Cryptococcus neoformans ), and Candida albicans (C. albicans) Candida albicans ).

9. Use according to claim 6, characterized in that, The diabetic foot comprises diabetic foot ulcers.

10. A medicament and / or product, characterized in that, The active ingredient comprises the WK7 series antibacterial peptide of any one of claims 1~5.

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