Derivative peptide based on frog-derived antibacterial peptide CPF-C1 and preparation method and application thereof

Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to a derived peptide based on frog-derived antibacterial peptide CPF-C1 as well as a preparation method and application of the derived peptide. The general formula of the derivative peptide is as follows: Trp1-Phe2-X3-X4-Leu5-Leu6-X7-X8-Ala9-Leu10-Arg11-Leu12-Trp13-X14-X15-Val16-Leu17-CONH2, X represents lysine, the lysine at the third site, the lysine at the fourth site, the lysine at the seventh site, the lysine at the eighth site, the lysine at the fourteenth site and the lysine at the fifteenth site can be independently selected from alpha-lysine or epsilon-lysine, and the amino acid at the sixth site to the amino acid at the eleventh site can be independently selected from L-type amino acid or D-type amino acid. The derived peptide obtained by the invention has broad-spectrum antibacterial activity, the antibacterial activity on gram-negative bacteria, especially klebsiella pneumoniae, salmonella typhimurium and pseudomonas aeruginosa is obviously higher than that of a parent peptide CPF-C1, and the derived peptide shows relatively low hemolytic activity and relatively good stability; the compound shows a wide application prospect in preparation of clinical antibacterial drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and specifically relates to a derivative peptide based on frog-derived antimicrobial peptide CPF-C1, and a preparation method and application thereof. Background Art

[0002] The irrational use of antibiotics in the early stages has accelerated the occurrence and spread of bacterial resistance. Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii can cause fatal infections in hospitals, and the above pathogens have developed a high degree of resistance to most commonly used antibiotics.

[0003] Biofilm is one of the main reasons why bacteria develop resistance to antibiotics. According to statistics, about 60%-78% of chronic wound infections are closely related to the formation of bacterial biofilms. Implantable medical devices are also very likely to induce bacterial biofilm infections, which usually show adverse characteristics of recurrence and long-term treatment. Unfortunately, no effective drugs for the treatment of bacterial biofilm infections have been developed clinically.

[0004] Antimicrobial peptides (AMPs) are produced by the natural immune defense system of organisms. They are peptide compounds that have resistance to a variety of pathogens such as bacteria, fungi and viruses. Studies have shown that most antimicrobial peptides mainly destroy the integrity of bacterial cell membranes, causing the leakage of cell contents and leading to bacterial death. This unique bactericidal mechanism is generally not easy to cause microbial resistance. In addition, some antimicrobial peptides also have intracellular targets and can bind to bacterial DNA and RNA to inhibit the synthesis of certain proteins. This multi-pathway action mode makes it one of the most promising candidate drugs for the treatment of bacterial infections, especially multi-drug resistant bacterial infections. However, antimicrobial peptides from natural sources also have some defects, such as weak antibacterial activity, poor enzymatic stability and high toxicity.

[0005] CPF-C1 (GFGSLLGKALRLGANVL-NH2) is an antimicrobial peptide isolated from the skin secretions stimulated by norepinephrine of tetraploid frogs. It shows moderate antimicrobial activity against standard strains of common clinical bacteria and multidrug-resistant bacteria, and exhibits systemic toxicity at high concentrations, which limits its application in vivo. To develop it into an antimicrobial candidate compound, its antimicrobial activity and selectivity must be improved. Summary of the invention

[0006] The present invention aims at the shortcomings of frog-derived antimicrobial peptide CPF-C1 and provides a derivative peptide with significantly improved antimicrobial activity, lower hemolytic activity and better stability.

[0007] Another object of the present invention is to provide a method for preparing the derivatized peptide.

[0008] Another object of the present invention is to provide the use of the derivative peptide in the preparation of drugs for preventing or treating diseases related to drug-resistant bacterial infection and biofilm infection.

[0009] The frog-derived antimicrobial peptide CPF-C1 derivative peptide according to a specific embodiment of the present invention is based on the wild sequence of the frog-derived antimicrobial peptide CPF-C1, in which the 1st and 13th amino acids in the wild sequence are replaced by tryptophan, and the 3rd, 4th, 7th, 14th and 15th amino acids are replaced by lysine.

[0010] The amino acid sequence of the CPF-C1 derived peptide is shown in SEQ ID NO.11:

[0011] Trp 1 -Phe 2 -X 3 -X 4 -Leu 5 -Leu 6 -X 7 -X 8 -Ala 9 -Leu 10 -Arg 11 -Leu 12 -Trp 13 -X 14 -X 15 -Val 16 -Leu 17 -CONH2, wherein X represents lysine.

[0012] Preferably, the 3rd, 4th, 7th, 8th, 14th and 15th lysines of the CPF-C1 derived peptide sequence are independently selected from α-lysine or ε-lysine, α-lysine forms a peptide bond, and ε-lysine forms an isopeptide bond, as shown in formula (I):

[0013]

[0014] According to a specific embodiment of the CPF-C1 derived peptide of the present invention, the amino acids at positions 6 to 11 in the amino acid sequence are independently selected from the corresponding L-type or D-type amino acids in the sequence.

[0015] According to a specific embodiment of the present invention, the CPF-C1 derived peptide is any one of the following compounds:

[0016] The derived peptide CPF-IP1, whose amino acid sequence is shown in SEQ ID NO.1:

[0017] Trp 1 -Phe 2 -ε-Lys 3 -α-Lys4 -Leu 5 -Leu 6 -α-Lys 7 -α-Lys 8 -Ala 9 -Leu 10 -Arg 11 -Leu 12 -Trp 13 -α-Lys 14 -α-Lys 15 -Val 16 -Leu 17 -CONH2;

[0018] The derived peptide CPF-IP2, whose amino acid sequence is shown in SEQ ID NO.2:

[0019] Trp 1 -Phe 2 -α-Lys 3 -ε-Lys 4 -Leu 5 -Leu 6 -α-Lys 7 -α-Lys 8 -Ala 9 -Leu 10 -Arg 11 -Leu 12 -Trp 13 -α-Lys 14 -α-Lys 15 -Val 16 -Leu 17 -CONH2;

[0020] The derived peptide CPF-IP3, whose amino acid sequence is shown in SEQ ID NO.3:

[0021] Trp 1 -Phe 2 -α-Lys 3 -α-Lys 4 -Leu 5 -Leu 6 -ε-Lys 7 -α-Lys 8 -Ala 9 -Leu 10 -Arg 11 -Leu 12 -Trp 13 -α-Lys 14 -α-Lys 15 -Val16 -Leu 17 -CONH2;

[0022] The derived peptide CPF-IP4, whose amino acid sequence is shown in SEQ ID NO.4:

[0023] Trp 1 -Phe 2 -α-Lys 3 -α-Lys 4 -Leu 5 -Leu 6 -α-Lys 7 -ε-Lys 8 -Ala 9 -Leu 10 -Arg 11 -Leu 12 -Trp 13 -α-Lys 14 -α-Lys 15 -Val 16 -Leu 17 -CONH2;

[0024] The derived peptide CPF-IP5, whose amino acid sequence is shown in SEQ ID NO.5:

[0025] Trp 1 -Phe 2 -α-Lys 3 -α-Lys 4 -Leu 5 -Leu 6 -α-Lys 7 -α-Lys 8 -Ala 9 -Leu 10 -Arg 11 -Leu 12 -Trp 13 -ε-Lys 14 -α-Lys 15 -Val 16 -Leu 17 -CONH2;

[0026] The derived peptide CPF-IP6, whose amino acid sequence is shown in SEQ ID NO.6:

[0027] Trp 1 -Phe 2 -α-Lys 3 -α-Lys 4 -Leu 5 -Leu6 -α-Lys 7 -α-Lys 8 -Ala 9 -Leu 10 -Arg 11 -Leu 12 -Trp 13 -α-Lys 14 -ε-Lys 15 -Val 16 -Leu 17 -CONH2;

[0028] The amino acid sequence of the derivative peptide IP2-D1 is shown in SEQ ID NO.7:

[0029] Trp 1 -Phe 2 -α-Lys 3 -ε-Lys 4 -Leu 5 -Leu 6 -α-Lys 7 -α-Lys 8 -Ala 9 - Leu 10 - Arg 11 -Leu 12 -Trp 13 -α-Lys 14 -α-Lys 15 -Val 16 -Leu 17 -CONH2

[0030] The amino acid sequence of the derivative peptide IP2-D2 is shown in SEQ ID NO.8:

[0031] Trp 1 -Phe 2 -α-Lys 3 -ε-Lys 4 -Leu 5 -Leu 6 -α-Lys 7 -α-Lys 8 - Land 9 - Leu 10 - Arg 11 -Leu 12 -Trp 13 -α-Lys 14 -α-Lys 15 -Val 16 -Leu 17 -CONH2;

[0032] The derivative peptide IP2-D3, whose amino acid sequence is shown in SEQ ID NO.9:

[0033] Trp 1 -Phe 2 -α-Lys 3 -ε-Lys 4 -Leu 5 - Leu 6 - α-Lys 7 -α-Lys 8 -Ala 9 - Leu 10 - Arg 11 -Leu 12 -Trp 13 -α-Lys 14 -α-Lys 15 -Val 16 -Leu 17 -CONH2;

[0034] The derivative peptide IP2-D4, whose amino acid sequence is shown in SEQ ID NO.10:

[0035] Trp 1 -Phe 2 -α-Lys 3 -ε-Lys 4 -Leu 5 - Leu 6 - α-Lys 7 - α-Lys 8 - Land 9 - Leu 10 - Arg 11 -Leu 12 -Trp 13 -α-Lys 14 -α-Lys 15 -Val 16 -Leu 17 -CONH2;

[0036] SEQ ID NO.1-10 Italic underlined letters Indicates D-amino acid.

[0037] According to the present invention, a method for preparing the derivatized peptide is provided, comprising the following steps:

[0038] Step 1: Using the Fmoc solid phase synthesis method, the first amino acid at the C-terminus is connected to the MBHA resin, and amino acids are connected in sequence from the C-terminus to the N-terminus until the peptide chain is completed. The amino acid used when connecting α-Lys is L-Fmoc-Lys(Boc)-OH, and the amino acid used when connecting ε-Lys is L-Boc-Lys(Fmoc)-OH;

[0039] Step 2: Prepare a cutting solution according to the volume ratio of TFA: Tis: H2O 95: 2.5: 2.5, cut the resin in step 1, and remove the remaining protective groups of the peptide chain. Collect the cutting solution containing the crude polypeptide peptide chain;

[0040] Step 3: Purify the crude peptide obtained in step 2 by desalting using RP-HPLC to obtain a derivatized peptide with a purity of >99%, and identify the isolated product by mass spectrometry to confirm the m / z value of the protonated molecular ion peak.

[0041] The present invention provides the use of the above-mentioned derivative peptide in the preparation of a drug for preventing or treating a drug-resistant bacterial infection disease, specifically comprising:

[0042] The CPF-C1 derivative peptide of the present invention can be used as the sole effective ingredient or one of the effective ingredients in treating Gram-negative bacteria and / or Gram-positive bacteria resistant bacterial infections, and has therapeutic and preventive effects on the diseases.

[0043] The CPF-C1 derivative peptide of the present invention has a therapeutic and preventive effect on bacterial biofilm infection-related diseases, and can be used as the only effective ingredient or one of the effective ingredients to be directly or indirectly applied to bacterial biofilm infection diseases.

[0044] The specific meanings of the abbreviations used in the present invention are as follows:

[0045] Trp is tryptophan, Phe is phenylalanine, Lys is lysine, Leu is leucine, Ala is alanine, Arg is arginine, Val is valine, DCM is dichloromethane, DMF is N,N-dimethylformamide, HOBT is 1-hydroxybenzotriazole, HBTU is benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, DIEA is N,N-diisopropylethylamine, TFA is trifluoroacetic acid, Fmoc is fluorenylmethyloxycarbonyl, Boc is tert-butyloxycarbonyl, and Tis is triisopropylsilane.

[0046] Beneficial effects of the present invention:

[0047] The present invention designs and synthesizes a series of derivative peptides based on the wild sequence of frog-derived antimicrobial peptide CPF-C1, the general formula of which is:

[0048] Trp 1 -Phe 2 -X 3 -X 4 -Leu 5 -Leu 6 -X 7 -X 8 -Ala 9 -Leu 10 -Arg 11-Leu 12 -Trp 13 -X 14 -X 15 -Val 16 -Leu 17 -CONH2, wherein X represents lysine. Preferably, the lysine at positions 3, 4, 7, 8, 14 and 15 in the sequence can be independently selected as α-lysine or ε-lysine, and the amino acids at positions 6 to 11 can be independently selected as L-type or D-type amino acids.

[0049] The CPF-C1 derivative peptide designed and synthesized by the present invention was comprehensively evaluated for its antibacterial activity, toxicity, stability and other efficacy indicators, including the minimum inhibitory concentration (MIC) detection of standard strains with a high clinical isolation rate, and the study of its anti-biofilm activity and anti-persistent bacteria ability as well as the therapeutic effect on the mouse model infected with drug-resistant bacteria. In addition, the present invention also evaluated the serum stability, salt stability and hemolytic activity of the derivative peptide.

[0050] The results showed that the antibacterial activity of the derived peptides obtained by using ε-Lys to form isopeptide bonds based on the introduction of tryptophan was significantly improved, and the hemolytic activity was lower after the isopeptide bonds were formed at sites 3, 4, 7, and 8, which increased the safety. 平均最小抑菌浓度 =5.8-11.6 μg / mL) was significantly better than the parent peptide CPF-C1 (GM 平均最小抑菌浓度 =52.9μg / mL), among which the average minimum inhibitory concentrations of CPF-IP2, CPF-IP5, CPF-IP6, IP2-D1 and IP2-D4 were 6.2, 7.1, 8.4, 7.6 and 5.8μg / mL, respectively, which was increased by about 10 times. Further studies have shown that the antibacterial spectrum of the derivative peptides has been expanded, and the MICs of Pseudomonas aeruginosa, Salmonella typhimurium and Klebsiella pneumoniae have been reduced from 32-128μg / mL to 4-8μg / mL. Toxicity studies have shown that the hemolysis rates of CPF-IP2, CPF-IP3, CPF-IP4 and IP2-D4 are less than 10% at the highest detection concentration (128μg / mL), indicating that they have good safety.

[0051] The present invention screened out the derivative peptide IP2-D4, which had the highest therapeutic index (TI=44.14) and showed good salt solution stability and serum stability (T 1 / 2 =2.932h). Compared with the parent peptide CPF-C1, IP2-D4 has stronger antibacterial activity against drug-resistant bacteria and biofilms and a broader antibacterial spectrum, which verifies the feasibility of the design and modification strategy in this study and has the potential for further promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0053] Figure 1 This is the mass spectrometry characterization diagram of the derived peptides CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, and CPF-IP6 in Example 1.

[0054] Figure 2 The mass spectrometry characterization diagram of the derived peptides IP2-D1, IP2-D2, IP2-D3 and IP2-D4 in Example 1.

[0055] Figure 3 This is a graph of the minimum inhibitory concentrations of the test substances CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, CPF-IP6, IP2-D1, IP2-D2, IP2-D3 and IP2-D4 against the tested strains in Example 2.

[0056] Figure 4 This is a graph showing the hemolytic activity of the test substances CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, CPF-IP6 and IP2-D4 in Example 3 on mouse red blood cells.

[0057] Figure 5 It is the change multiple of the minimum inhibitory concentration of the test substances CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, CPF-IP6 and IP2-D4 in Example 4 in various salt environments.

[0058] Figure 6 This is a half-life graph of the test substances CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, CPF-IP6, IP2-D3 and IP2-D4 in Example 5 after incubation with mouse serum at different time points.

[0059] Figure 7 It is the inhibition rate of the test substance IP2-D4 on bacterial biofilm formation in Example 6.

[0060] Figure 8 This is the antibacterial activity of the test substance IP2-D4 in Example 7 against bacterial biofilm persisters.

[0061] Fig. 9The bactericidal abilities of 1×MIC, 2×MIC, 4×MIC IP2-D4 and 4×MIC antibiotics in Example 8 against bacteria.

[0062] Fig.10 The induction resistance of the test substance IP2-D4 and the control antibiotic in Example 9 on bacteria.

[0063] Fig.11 The results are as follows: 3 days after treatment in Example 10, the skin resistant bacterial infection of mice in the normal group, the model group, the experimental group IP2-D4 (2.5 mg / kg, 5 mg / kg, 10 mg / kg), the experimental group ceftazidime (5 mg / kg) and the experimental group polymyxin B (5 mg / kg). DETAILED DESCRIPTION

[0064] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0065] Materials and Methods

[0066] The experimental conditions and methods described in the following examples are conventional conditions and methods unless otherwise specified, and the reagents and instruments described are all commercially available.

[0067] GraphPad Prism 8.0 software was used for data statistics and analysis. Data were expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA and Tukey test were used for significant difference analysis ( *** P < 0.001, ** P<0.01, * P<0.05).

[0068] Example 1 Preparation of Derivatized Peptides

[0069] Step 1: Synthesis of derivatized peptides

[0070] The Fmoc solid phase synthesis method was used to synthesize from the carboxyl end to the amino end. The specific steps are as follows:

[0071] (1) Activation of MBHA resin: Weigh the resin, add appropriate amount of DCM and swell on a shaker for 30 min. After draining, add DMF to wash three times, each time for 3 min.

[0072] (2) Indole test: Add ninhydrin: pyridine: phenol = 1:2:1 indole test reagent into a test tube, dip a small amount of resin into it, and boil it in boiling water for 3 minutes. If the indole test result is transparent and colorless, it indicates that the resin is normal;

[0073] (3) Resin deprotection: Add DMF solution containing 3% redistilled piperidine to remove the protecting group, remove the residual reagent, add DMF to wash, each time for 3 minutes, repeat 4 times, and remove the residual reagent;

[0074] (4) Indole test: Add ninhydrin: pyridine: phenol = 1:2:1 indole test reagent to a test tube, take a small amount of resin into the test tube, and boil it in boiling water for 3 minutes. If the indole test result is blue-purple, it proves that the protecting group has been removed;

[0075] (5) Amino acid condensation reaction: weigh 3 times the amount of amino acid, 3 times the amount of HOBT, 3 times the amount of HBTU, and 6 times the amount of DIEA in a beaker, dissolve in a small amount of DMF, immediately add to the resin, stir for 1 hour, drain the solvent, wash with DMF for 3 minutes, and repeat 3 times;

[0076] (6) Indene test: If the indene test result is transparent and colorless, it indicates that the condensation is successful;

[0077] (7) Repeat steps (3), (4), (5), and (6) in the order of the amino acid sequence in the compound until all the amino acids in the compound to be synthesized are condensed.

[0078] For example: when preparing the derivative peptide of SEQ ID NO.1, first weigh 1 times the amount of MBHA resin and add it to the synthesizer, add 10-15mL DCM at the same time, shake for 30 minutes to fully wet the resin, then drain the solvent and wash the resin with DMF 3 times, each time for 3 minutes; pick a small amount of resin from the synthesizer, put it into the pre-prepared indene test solution, heat it in a boiling water bath for 3 minutes, observe whether the resin is transparent, if it is transparent, it means that the resin is normal and you can continue to operate; add 10-15mL of DMF solution containing 3% redistilled piperidine to the synthesizer to remove the protecting group of the resin. After the reaction is completed, wash the resin with DMF 4 times, each time for 3 minutes, and perform indene test again. If the resin is blue-purple, it means that the protecting group is successfully removed and the condensation reaction can be carried out; weigh 3 times the amount of Fmoc-Leu-OH and 3 times the amount of HOBT, 3 times the amount of HBTU, and 6 times the excess of DIEA are added and dissolved with a small amount of DMF. The solution is immediately added to the resin, shaken on a shaker for 1 hour, and then the solvent is drained off. After washing with DMF, an indene test is performed. If the resin is transparent, it means that the amino acid is successfully connected and the deprotection reaction can be carried out. The deprotection reaction is carried out according to steps (3) and (4). After success, the condensation reaction (5) and (6) are repeated according to the CN end sequence of the amino acids of the compound. Note that when connecting α-Lys, L-Fmoc-Lys(Boc)-OH is used, and when connecting ε-Lys, L-Boc-Lys(Fmoc)-OH is used. Through these steps, a complete SEQ ID NO.1 derivative peptide can be synthesized to ensure the accurate connection of each amino acid and the effective removal of the protecting group.

[0079] The structural formula of the peptide derived from SEQ ID NO.1 is as follows:

[0080]

[0081] The same method was used to prepare the derivative peptide of SEQ ID NO.2-10, the structural formula of which is as follows:

[0082]

[0083]

[0084] Step 2: Cutting the polypeptide chain:

[0085] After all amino acids are connected, deprotection is performed, and the resin is washed with DMF for 3 minutes, repeated twice; washed with methanol for 3 minutes; washed with DCM for 3 minutes; and finally washed with methanol for 3 minutes, repeated twice. The solvent is pumped dry with an oil pump until the resin is in powder form. Prepare a cutting solution of TFA: Tis: H2O = 95: 2.5: 2.5 (V / V / V), add it to the resin for cutting for 3 hours, and collect the cutting solution. Use a rotary evaporator to spin off the solvent, add pre-cooled ice ether to precipitate the derivative peptide, and add deionized water for extraction after completion. Collect the aqueous phase and divide it into 50 mL beakers, freeze it in a -80°C refrigerator overnight for freeze-drying, and obtain the crude peptide.

[0086] Step 3: Preparation and purification of derivatized peptides:

[0087] (1) Weigh about 40 mg of crude peptide and dissolve it in deionized water to prepare a peptide solution. After it is completely dissolved, use a 0.45 μm filter to remove insoluble substances in the peptide solution, and add 0.1% TFA to the elution solvent (acetonitrile and deionized water);

[0088] (2) HPLC using C18 reverse preparative column with 100% acetonitrile flushing until the spectrum is stable, setting the flow gradient, and injecting after initial concentration equilibrium;

[0089] (3) After injection, detect the absorption peak at 220 nm, collect the eluate corresponding to the main peak, mark it, seal it with plastic wrap, pierce the vent hole, freeze it in a -80°C refrigerator overnight, and then freeze-dry it.

[0090] (4) After freeze-drying, a small amount of the compound was dissolved and eluted with 5%-95% acetonitrile / deionized water on a C18 reverse analytical column for 30 min. The peak area of ​​the chromatogram at 220 nm was integrated to calculate the retention time and purity of the compound.

[0091] (5) Use mass spectrometry to characterize and identify the isolated product and confirm the m / z value of the protonated molecular ion peak. Figure 1 , 2 shown.

[0092] Table 1 Physicochemical properties of the compounds synthesized in Example 1

[0093]

[0094] Example 2 In vitro antibacterial activity detection

[0095] Gram-negative bacteria Escherichia coli ATCC 25922, Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 14028 and Klebsiella pneumoniae ATCC 700603, and Gram-positive bacteria Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 23857, methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591 and Staphylococcus epidermidis ATCC12228 were selected to detect the minimum inhibitory concentration (MIC) of the derivative peptide obtained in Example 1 against sensitive bacteria to evaluate the in vitro antibacterial activity of the compound.

[0096] (1) Add 3 mL of broth culture medium to a sterile test tube, inoculate a monoclonal colony, and place it in a shaking incubator at 37°C and 180 rpm for 5-7 hours until the logarithmic growth phase. Compare the turbidity by McFarland turbidimetry and dilute with MH to obtain a concentration of 1.0×10 6 CFU / mL of bacterial solution.

[0097] (2) Weigh the derived peptide obtained in Example 1 and prepare a 2048 μg / mL stock solution with ultrapure water. Duplicate the solution with MH medium to obtain a series of drug solutions with a concentration gradient of 128, 64, 32, 16, 8 and 4 μg / mL.

[0098] (3) Add different concentrations of the drug solution obtained in step (2) and an equal volume of bacterial solution to a sterile 96-well plate, set up 3 replicate wells for each concentration, place in an incubator, and culture overnight at 37°C for 16-18 hours.

[0099] (4) Observe the bacterial turbidity. The minimum drug concentration in the clear well is the minimum inhibitory concentration of the compound. The results are shown in the table below.

[0100] Table 2 Minimum inhibitory concentration (MIC) of the derived peptides synthesized in Example 1 against sensitive bacteria

[0101]

[0102] Table 3 Average minimum inhibitory concentration (GM) of the synthesized derivative peptides in Example 1

[0103]

[0104]

[0105] Note: GM - = the sum of the minimum inhibitory concentrations of the compounds against the tested Gram-negative bacteria / the number of tested Gram-negative bacteria; GM + = the sum of the minimum inhibitory concentrations of the compounds against the tested Gram-positive bacteria / the number of tested Gram-positive bacteria; GM ALL= Total minimum inhibitory concentration of the compound against the tested strains / number of tested bacteria. When MIC>128μg / mL, calculate as 256μg / mL.

[0106] Figure 3 This is a graph of the minimum inhibitory concentrations of the test substances CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, CPF-IP6, IP2-D1, IP2-D2, IP2-D3 and IP2-D4 against the tested strains in Example 2.

[0107] Figure 3 A is the minimum inhibitory concentration of the test substance against the tested Gram-negative bacterial strains;

[0108] Figure 3 B is the minimum inhibitory concentration of the test substance against the tested Gram-positive bacterial strains.

[0109] From the above Table 2, Table 3 and Figure 3 It can be seen that the minimum inhibitory concentration of the test substance against bacteria found that the above derivative peptides have good antibacterial activity against both Gram-positive and Gram-negative bacteria. Especially for Gram-negative bacteria, compared with the parent peptide CPF-C1, the minimum inhibitory concentrations of the derivative peptides CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, CPF-IP6, IP2-D1, and IP2-D4 against Pseudomonas aeruginosa, Salmonella typhimurium, and Klebsiella pneumoniae were all 4-8μg / mL, and the antibacterial activity was significantly improved.

[0110] Example 3 Hemolytic Activity Experiment

[0111] The derived peptides CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, CPF-IP6 and IP2-D4 obtained in Example 1 were selected as test substances. Fresh red blood cells from Kunming mice were selected to detect the lysis activity of the derived peptides on red blood cells as an indicator for evaluating the in vitro toxicity of the compound.

[0112] (1) Prepare heparin sodium into a solution with a concentration of 2 mg / mL using PBS, and add 200 μL per tube into a centrifuge tube. Take blood from the eyeball and put it into a centrifuge tube, centrifuge at 800 g for 10 min. Discard the supernatant, wash the lower layer of red blood cells with PBS three times, and dilute with PBS to obtain an 8% red blood cell suspension.

[0113] (2) The compound was prepared into a stock solution with a concentration of 2048 μg / mL using PBS, and then two-fold dilutions were used to obtain drug solutions with concentrations of 128, 64, 32, 16, and 8 μg / mL.

[0114] (3) Add samples in groups in a 90-well plate: add an equal volume of red blood cell suspension and PBS buffer to the negative control group; add an equal volume of red blood cell suspension and test substance solutions of different concentrations to the experimental group; and add an equal volume of red blood cell suspension and 1% TritonX-100 to the positive control group.

[0115] (4) Place the plate in a 37°C incubator and incubate for 1 hour. Centrifuge the plate and transfer the supernatant to another clean 96-well plate. Detect the absorbance of the supernatant at 490 nm.

[0116] (5) Calculate the hemolysis rate of the compound according to the following formula.

[0117] Hemolysis rate (%) = (OD 490 Experimental group-OD 490 Negative control group) / (OD 490 Positive control group-OD 490 Negative control group)×100%.

[0118] The in vitro toxicity and selectivity of the derived peptides were evaluated by detecting the hemolytic activity of the test substances. Figure 4 As shown, the derivative peptides all showed low hemolytic activity within the antibacterial concentration range (4-8 μg / mL), especially the derivative peptides CPF-IP2, CPF-IP3, CPF-IP4 and IP2-D4, whose hemolytic rates were still less than 10% at the highest concentration tested (128 μg / mL), showing good safety.

[0119] Table 4 Therapeutic index values ​​(TI) of the compounds synthesized in Example 1

[0120]

[0121] Note: MHC 20 It represents the lowest concentration of the compound that causes 20% hemolysis rate, TI=lowest concentration of the compound that causes 20% hemolysis rate / average minimum inhibitory concentration of the compound.

[0122] From the results, it can be seen that the therapeutic index (TI) of the derived peptides obtained is higher than that of the parent peptide CPF-C1, especially CPF-IP2, CPF-IP3, CPF-IP4 and IP2-D4, the therapeutic index of which increases by 5-9 times. Among them, the derived peptide IP2-D4 with the highest therapeutic index is 44.14, indicating that the selectivity of the derived peptides is significantly improved compared with the parent peptide.

[0123] Example 4 Salt stability experiment

[0124] 150mM NaCl, 4.5mM KCl, 2mM CaCl2, 6μM NH4Cl, 8μM ZnCl2, 1mM MgCl2, and 4μMFeCl3 were used to detect the stability of the test subjects CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, CPF-IP6, and IP2-D4 in a physiological environment.

[0125] (1) The bacteria were cultured in nutrient broth medium in a 37°C constant temperature shaker until the logarithmic growth phase, and then diluted to 1.5×10 6 CFU / mL of bacterial suspension.

[0126] (2) The compound was diluted twice with MH medium to a final concentration of 4-128 μg / mL. Deionized water was used to prepare a salt solution with a final concentration of 150 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 6 μM NH4Cl, 8 μM ZnCl2, 1 mM MgCl2, and 4 μM FeCl3.

[0127] (3) Add equal volumes of bacterial solution, saline solution, and test substance of corresponding concentration to a 96-well plate and incubate at 37°C overnight. Record the minimum drug concentration in the clear wells and calculate the multiple of the minimum inhibitory concentration.

[0128] The experimental results are as follows Figure 5 As shown, except for the reduced antibacterial activity of the tested derivative peptides against Pseudomonas aeruginosa in CaCl2 salt solution, the antibacterial activity against methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae in NaCl, KCl, NH4Cl, ZnCl2, MgCl2 and FeCl3 salt solutions remained almost unchanged, and the MIC varied between 1-2 times, indicating that the derivative peptides have good salt stability.

[0129] Example 5 Serum stability test

[0130] The derived peptides CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, CPF-IP6, IP2-D1, IP2-D2 and IP2-D4 obtained in Example 1 were used as test substances, and the experimental steps were as follows:

[0131] (1) Blood was collected from anesthetized Kunming mice and centrifuged at 8000 rpm / min for 30 min. The upper serum was collected and set aside.

[0132] (2) Weigh an appropriate amount of the test substance and prepare a stock solution with a concentration of 10 mM. Mix the test substance with mouse serum at a ratio of 1:4 (V:V) and incubate at 37°C. Take 70 μL of the sample at different time points (0 h, 1 h, 2 h, 3 h, 4 h, 5 h), immediately add an equal volume of glacial acetonitrile, vortex and let stand on ice for 10 min. Centrifuge the sample at 13,000 g for 15 min and collect the supernatant.

[0133] (3) RP-HPLC was performed with a gradient elution of 5%-95% acetonitrile / deionized water for 30 min. The absorption peak at 220 nm was detected and the peak area of ​​the test substance was recorded and compared with the peak area of ​​the test substance at 0 h. The half-life of the test substance at different time points was calculated.

[0134] Table 5 Half-life of the derived peptides of Example 1

[0135]

[0136] The experimental results are as follows Figure 6 As shown in Table 5, except for CPF-IP2, the half-lives of the other test substances after incubation with mouse serum were >0.5h, especially CPF-IP6 and IP2-D4, whose half-lives were 3.304h and 2.932h, respectively, showing good in vitro stability.

[0137] Example 6 Bacterial biofilm inhibition experiment

[0138] The derivative peptide obtained in Example 1 and CPF-C1 were used as test substances, and the experimental steps were as follows:

[0139] (1) The bacteria were cultured in nutrient broth medium in a 37°C constant temperature shaker until the logarithmic growth phase, and then diluted to 1.0×10 6 CFU / mL of bacterial suspension.

[0140] (2) Dilute the test substance twice with TSBG medium to a concentration gradient of 64, 32, 16, 8 and 4 μg / mL, and use polymyxin B as an antibiotic control. Add samples in groups in a 96-well plate:

[0141] Experimental group: equal volumes of peptide drugs and bacterial suspensions with different concentrations.

[0142] Positive control group: equal volume of TSBG medium and bacterial suspension,

[0143] Negative control group: double volume of TSBG medium.

[0144] The cells were placed in a 37°C constant temperature incubator and cultured for 24 h to form a biofilm.

[0145] (3) The 96-well plate was removed, the floating bacteria on the surface were removed and gently washed with PBS buffer, fixed with anhydrous methanol for 15 min, dried, and then 0.1% crystal violet stain was added to stain the biofilm for 15 min. The crystal violet stain was removed, and the plate was washed with deionized water for 2-3 times. Finally, 95% ethanol was added and left for 15 min, and the absorbance at 595 nm was detected.

[0146] Biofilm formation inhibition rate (%) = [1-(OD 595 - Negative control group OD 595 ) / (OD of positive control group 595 - Negative control group OD 595 )]×100%.

[0147] Figure 7 The inhibition rate of the test substances CPF-C1, IP2-D4 and polymyxin B on the biofilm formation of drug-resistant bacteria in Example 6;

[0148] Figure 7 A is the biofilm inhibition activity of CPF-C1 and IP2-D4 against drug-resistant Pseudomonas aeruginosa NO.86;

[0149] Figure 7 B is the biofilm inhibition activity of CPF-C1 and IP2-D4 against drug-resistant Pseudomonas aeruginosa NO.157;

[0150] Figure 7 C is the biofilm inhibition activity of CPF-C1 and IP2-D4 against drug-resistant Acinetobacter baumannii NO.91152;

[0151] Figure 7 D is the biofilm inhibition activity of CPF-C1 and IP2-D4 against drug-resistant Acinetobacter baumannii NO.9336.

[0152] The experimental results are as follows Figure 7 As shown, IP2-D4 has an inhibitory effect on the biofilm formation of drug-resistant Pseudomonas aeruginosa NO.86, NO.157 and drug-resistant Acinetobacter baumannii NO.91152, NO.9336. It can inhibit 50% of the biofilm formation of Acinetobacter baumannii at 2-4 μg / mL and 50% of the biofilm formation of Pseudomonas aeruginosa at 4-8 μg / mL, indicating that the derived peptide IP2-D4 can inhibit bacterial biofilm formation at low concentrations and has potent anti-biofilm activity.

[0153] The derived peptides CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, and CPF-IP6 have inhibitory effects on the biofilms of drug-resistant Pseudomonas aeruginosa and drug-resistant Acinetobacter baumannii, can inhibit bacterial biofilm formation at low concentrations, and have strong anti-biofilm activity.

[0154] Example 7 In vitro anti-biofilm persister activity experiment

[0155] Resistant strains of Pseudomonas aeruginosa and Acinetobacter baumannii were selected to test the antibacterial activity of IP2-D4 against bacteria persisting in bacterial biofilms.

[0156] (1) Dilute the bacteria in the logarithmic growth phase to 1×10 8 CFU / mL, 200 μL per well was plated in a 96-well plate and incubated at 37 °C for 24 h to form a biofilm.

[0157] (2) The biofilms were washed twice with PBS to remove planktonic bacteria, and then exposed to high-concentration antibiotics diluted in 200 μL TSBG medium and cultured at 37°C for 24 h.

[0158] (3) Wash twice with PBS to remove planktonic bacteria, transfer attached bacteria to 100 μL PBS by ultrasonication for 5 min, and then expose the bacteria to 1×MIC, 2×MIC, and 4×MIC of IP2-D4. PBS was used as a control group. After incubation at 37°C for 0.5 h, 1 h, 2 h, 4 h, and 6 h, the plates were spread to determine the number of live bacteria.

[0159] Figure 8 The antibacterial activity of the test substance IP2-D4 against bacteria persisting in bacterial biofilm in Example 7;

[0160] Figure 8 A is the antibacterial activity of IP2-D4 against drug-resistant Pseudomonas aeruginosa NO.86 biofilm persisters;

[0161] Figure 8 B is the antibacterial activity of IP2-D4 against drug-resistant Pseudomonas aeruginosa NO.157 biofilm persisters;

[0162] Figure 8 C is the antibacterial activity of IP2-D4 against drug-resistant Acinetobacter baumannii NO.91152 biofilm persisters;

[0163] Figure 8 D is the antibacterial activity of IP2-D4 against drug-resistant Pseudomonas aeruginosa NO.9336 biofilm persisters.

[0164] From the results, it can be seen that after the bacterial biofilm was treated with high concentrations of antibiotics, a large number of biofilm bacteria survived, and high concentrations of antibiotics could not kill the biofilm persisters. IP2-D4 has a significant bactericidal ability against persisters. 2×MIC and 4×MIC IP2-D4 killed the persisters formed by resistant Acinetobacter baumannii NO.91152 and resistant Pseudomonas aeruginosa NO.157 within 1h and 0.5h, respectively, and could kill the persisters formed by resistant Acinetobacter baumannii NO.9336 and resistant Pseudomonas aeruginosa NO.86 within 2h and 1h, indicating that the derivative peptide IP2-D4 has a fast and significant bactericidal effect on the persisters induced by traditional antibiotics.

[0165] The derived peptides CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, and CPF-IP6 can also kill the persisters formed by drug-resistant Acinetobacter baumannii NO.9336 and drug-resistant Pseudomonas aeruginosa NO.86 in a short period of time, indicating that the derived peptides have a fast and significant killing effect on the persisters induced by traditional antibiotics.

[0166] Example 8 In vitro bactericidal kinetics

[0167] Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606, Klebsiella pneumoniae ATCC700603 and MRSA ATCC 33591 were selected to detect the rapid bactericidal ability of IP2-D4 against bacteria.

[0168] (1) Bacteria in logarithmic growth phase were diluted to 1.0×10 6 CFU / mL. Dilute the test substance IP2-D4 to a final concentration of 1×MIC, 2×MIC, and 4×MIC using MH medium. Dilute the antibiotic to a final concentration of 4×MIC.

[0169] (2) Mix equal volumes of IP2-D4 / antibiotics and bacterial solution, and culture in a constant temperature incubator at 37°C. Take out appropriate amounts of bacterial solution at different time intervals (0 min, 10 min, 15 min, 30 min, 1 h, 2 h, 3 h, 6 h, and 24 h), dilute them appropriately with MH medium, and evenly apply them on MH solid medium.

[0170] (3) Place the MH solid plates in a 37°C constant temperature incubator for overnight culture and record the number of colonies grown on each agar plate.

[0171] Fig. 9 The bactericidal ability of 1×MIC, 2×MIC, 4×MIC IP2-D4 and 4×MIC antibiotics in Example 8 against bacteria;

[0172] Fig. 9A is the bactericidal ability of IP2-D4 against Pseudomonas aeruginosa ATCC 27853;

[0173] Fig. 9 B is the bactericidal ability of IP2-D4 against Acinetobacter baumannii ATCC 19606;

[0174] Fig. 9 C is the bactericidal ability of IP2-D4 against Klebsiella pneumoniae ATCC 700603;

[0175] Fig. 9 D is the bactericidal ability of IP2-D4 against MRSA ATCC 33591;

[0176] As can be seen from the figure, IP2-D4 has a very fast killing time for Gram-negative bacteria. At a concentration of 4×MIC, it can kill Pseudomonas aeruginosa ATCC 27853 and Acinetobacter baumannii ATCC 19606 within 5-10 minutes. In contrast, the killing time for Gram-positive bacteria is slower. At a concentration of 4×MIC, it can kill methicillin-resistant Staphylococcus aureus ATCC 33591 within 2 hours. The results show that at the same concentration, IP2-D4 kills bacteria much faster than antibiotics.

[0177] Similarly, the derivative peptides CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, and CPF-IP6 killed bacteria faster than antibiotics.

[0178] Example 9 Induced drug resistance experiment

[0179] Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606, Klebsiella pneumoniae ATCC 700603 and MRSA ATCC 33591 were selected to detect the ability of the derived peptides and multiple control antibiotics to induce resistance to bacteria.

[0180] (1) Dilute the logarithmic phase bacteria to 1.0×10 6 CFU / ml of bacterial suspension.

[0181] (2) IP2-D4 was double-diluted to 128, 64, 32, 16, 8, and 4 μg / mL using MH liquid culture medium, and antibiotics were diluted to drug concentrations of 4, 2, 1, 0.5, and 0.125 μg / mL, and the minimum inhibitory concentrations of IP2-D4 and antibiotics were detected.

[0182] (3) Take the sub-inhibitory concentration of bacterial solution and add it to 3 mL of nutrient broth medium to culture until the logarithmic growth phase of bacteria, and dilute it to 1.0 × 10 6CFU / ml. Test the minimum inhibitory concentration of each drug group again. Repeat this process for 20 consecutive days and record the results.

[0183] Fig.10 is the ability of the test substance IP2-D4 and the control antibiotic in inducing drug resistance on bacteria in Example 9;

[0184] Fig.10 A is the induction resistance of IP2-D4, polymyxin B, ceftazidime and ciprofloxacin to Pseudomonas aeruginosa ATCC 27853;

[0185] Fig.10 B is the inducible resistance of IP2-D4, polymyxin B, ceftazidime and imipenem to Acinetobacter baumannii ATCC 19606;

[0186] Fig.10 C is the induction resistance of IP2-D4, polymyxin B, ceftazidime and imipenem to Klebsiella pneumoniae ATCC 700603;

[0187] Fig.10 D is the ability of IP2-D4 to induce resistance to vancomycin and tigecycline against MRSA ATCC 33591;

[0188] As can be seen from the figure, after 20 consecutive days of induction, the minimum inhibitory concentration of IP2-D4 against Pseudomonas aeruginosa ATCC 27853 and Acinetobacter baumannii ATCC 19606 remained unchanged, while the minimum inhibitory concentrations against Klebsiella pneumoniae ATCC 700603 and MRSA ATCC 33591 increased by 4 times and 2 times, respectively, indicating that IP2-D4 is not easy to induce bacterial resistance. In contrast, antibiotics are easy to induce bacterial resistance. The minimum inhibitory concentration of ceftazidime against Pseudomonas aeruginosa increased by 64 times; the minimum inhibitory concentration of tigecycline against MRSA increased by 128 times. The above results show that IP2-D4 is not easy to induce bacterial resistance.

[0189] Similarly, the derived peptides CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, and CPF-IP6 are less likely to induce bacterial resistance.

[0190] Example 10 Mouse skin wound infection experiment

[0191] Fifty-six female BALB / c mice (8 weeks old, weighing about 18 g, purchased from the Animal Experiment Center of Lanzhou University) were randomly divided into 7 groups, with 8 mice in each group, namely the normal group, the model group, the peptide treatment group (derivative peptide, 0.25 mg / kg, 1 mg / kg, 4 mg / kg), the polymyxin B experimental group (1 mg / kg), and the ceftazidime experimental group (1 mg / kg).

[0192] Establishment of mouse skin wound infection model: After BALB / c mice were anesthetized, they were fixed on the operating table. The hair on the back of the mice was shaved and the mouse skin was cleaned with 75% alcohol. Two full-thickness circular wounds with a diameter of 5 mm were created on the back of the mice using a punch, and the wound sites were covered with medical transparent dressings. A microsyringe was used to penetrate the dressing, and 20 μL of sterile saline was injected into the wound sites of mice in the normal group, and 20 μL of resistant Pseudomonas aeruginosa NO.157 (5×10 6 CFU / mL) to induce mouse skin infection. 1h, 24h and 48h after bacterial infection, 50μL PBS was injected into the wound site of mice in the normal group and model group, and different doses of IP2-D4, ceftazidime and polymyxin B were injected into the wound site of mice in the treatment group. 72h after infection, 4 mice in each group were euthanized, and the skin tissue of the wound site was homogenized with PBS to detect the bacterial load. The wound sites of the remaining mice were photographed on 0 days, 3 days, 7 days and 10 days.

[0193] Fig.11 The bacterial infection of mouse skin wounds in the normal group, model group, and experimental group of IP2-D4 (0.25 mg / kg, 1 mg / kg, 4 mg / kg), ceftazidime (1 mg / kg), and polymyxin B (1 mg / kg) after 3 days of treatment in Example 10;

[0194] Fig.11 A is the load of drug-resistant Pseudomonas aeruginosa NO.157 in the skin wounds of mice in the normal group, model group and experimental group after 3 days of treatment. From the results, the bacterial load in the model group was 1.51×10 7 CFU / tissue, after treatment with IP2-D4 at 0.25 mg / kg, 1 mg / kg, and 4 mg / kg, the bacterial count in wound tissue was reduced to 4.47 × 10 5 CFU / tissue, 2.09×10 5 CFU / tissue and 1.67×10 5 CFU / tissue, significantly reduced the bacterial load in the wound skin of mice compared with the control group (P<0.001). The therapeutic effect of 1 mg / kg ceftazidime was comparable to that of the 1 mg / kg IP2-D4 group.

[0195] Fig.11 B is the load of drug-resistant Pseudomonas aeruginosa NO.157 in the wound dressings of mice in the normal group, model group and experimental group after 3 days of treatment. Similar to the above results, the bacterial load in the dressings of the model group was 1.41×10 7 CFU / dressing. After treatment with IP2-D4 at 0.25 mg / kg, 1 mg / kg, and 4 mg / kg, the amount of bacteria in the dressings was reduced to 1.55 × 10 5 CFU / dressing, 2.29×10 5 CFU / dressing and 4.57×10 4 CFU / dressing, significantly reduced the bacterial load in mouse wound dressings compared with the control group and inhibited the growth of bacterial biofilm on the dressings.

[0196] Fig.11 C shows the degree of wound healing in the normal group, model group and experimental group after 3 days of treatment. As can be seen from the figure, the wound in the model group was severely inflamed and had pus exudation. After treatment with 0.25mg / kg, 1mg / kg and 4mg / kg doses of IP2-D4, the wound scabs formed on the 7th day and the wound was basically healed on the 10th day. The effect is comparable to that of ceftazidime and polymyxin B, indicating that IP2-D4 has the effect of promoting wound healing.

[0197] Fig.11 D is the content of inflammatory factors in the wounds of mice in the normal group, model group and experimental group after 3 days of treatment. The secretion of tumor necrosis factor (TNF-α) and interleukin 1β (IL-1β). The content of IL-1β and TNF-α in the wounds of mice in the model group was significantly increased compared with that in the normal group. After IP2-D4 treatment, the content of these two inflammatory factors in the wounds was significantly reduced, indicating that IP2-D4 has anti-inflammatory activity.

[0198] In vivo activity experiments showed that IP2-D4 can effectively reduce the bacterial load in mouse skin wounds and dressings, and has the ability to promote wound healing and anti-inflammatory. Similarly, the derived peptides CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, and CPF-IP6 can also effectively reduce the bacterial load in mouse skin wounds and dressings, and have the ability to promote wound healing and anti-inflammatory.

[0199] In summary, the antibacterial activity of the derivative peptides CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, CPF-IP6, IP2-D1, IP2-D2, IP2-D3 and IP2-D4 was significantly improved compared with the parent peptide, and they showed good stability in a variety of physiological salt environments. In addition, the derivative peptide IP2-D4 had a strong inhibitory effect on bacterial biofilms and persisters, with a fast bactericidal rate and was not easy to induce bacterial resistance. Further studies have found that IP2-D4 showed a good therapeutic effect on mouse skin wound infection and had anti-inflammatory activity.

[0200] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A peptide derived from frog-derived antimicrobial peptide CPF-C1, characterized in that: The derivative peptide is based on the wild sequence of frog-derived antimicrobial peptide CPF-C1, in which the 1st and 13th amino acids in the wild sequence are replaced by tryptophan, and the 3rd, 4th, 7th, 14th and 15th amino acids are replaced by lysine.

2. The frog-derived antimicrobial peptide CPF-C1 derivative peptide according to claim 1, characterized in that: Positions 3, 4, 7, 8, 14 and 15 are each independently selected from α-lysine or ε-lysine.

3. The frog-derived antimicrobial peptide CPF-C1 derivative peptide according to claim 1, characterized in that: The amino acids at positions 6 to 11 of the derived peptide sequence are independently selected from L-type amino acids or D-type amino acids.

4. The frog-derived antimicrobial peptide CPF-C1 derivative peptide according to claim 1, characterized in that: The derived polypeptide is any one of the following sequences a1)-a10): a1): a derivative polypeptide with an amino acid sequence as shown in SEQ ID No. 1; a2): a derivative polypeptide with an amino acid sequence as shown in SEQ ID No. 2; a3): a derivative polypeptide with an amino acid sequence as shown in SEQ ID No. 3; a4): a derivative polypeptide with an amino acid sequence as shown in SEQ ID No.4; a5): a derivative polypeptide with an amino acid sequence as shown in SEQ ID No.5; a6): a derivative polypeptide with an amino acid sequence as shown in SEQ ID No.6; a7): a derivative polypeptide with an amino acid sequence as shown in SEQ ID No.7; a8): a derivative polypeptide with an amino acid sequence as shown in SEQ ID No.8; a9): a derivative polypeptide with an amino acid sequence as shown in SEQ ID No.9; a10): A derivative polypeptide having an amino acid sequence as shown in SEQ ID No.

10.

5. The method for preparing the frog-derived antimicrobial peptide CPF-C1 derivative peptide according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: The first amino acid at the C-terminus is connected to the MBHA resin by using the Fmoc solid phase synthesis method, and the amino acids are connected in sequence from the C-terminus to the N-terminus to synthesize the polypeptide resin corresponding to the derivative peptide; after the connection is completed, a cutting solution is prepared to remove the resin and the protective group for purification.

6. Use of the frog-derived antimicrobial peptide CPF-C1 derivative peptide according to any one of claims 1 to 4 in the preparation of an antimicrobial drug or composition for treating bacterial infection.

7. Use of the frog-derived antimicrobial peptide CPF-C1 derivative peptide according to any one of claims 1 to 4 in the preparation of an antibacterial biofilm drug or composition.

8. An antibacterial drug for treating bacterial infection, characterized in that: The drug uses the frog-derived antimicrobial peptide CPF-C1 derivative peptide according to any one of claims 1 to 4 as an active ingredient.

9. An antibacterial biofilm drug, characterized in that: The drug uses the frog-derived antimicrobial peptide CPF-C1 derivative peptide according to any one of claims 1 to 4 as an active ingredient.

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