Derivative peptide based on frog-derived antibacterial peptide cpf-c1, and preparation method and application thereof

Abstract

The application belongs to the technical field of biochemistry, and particularly relates to a derivative peptide based on frog-derived antibacterial peptide CPF-C1 and a preparation method and application thereof. 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, the 3rd, 4th, 7th, 8th, 14th and 15th lysines can be independently selected as alpha-lysine or epsilon-lysine, and the 6th to 11th amino acids can be independently selected as L-type or D-type amino acids. The derivative peptide obtained by the application has broad-spectrum antibacterial activity, and the antibacterial activity of the derivative peptide on gram-negative bacteria, particularly Klebsiella pneumoniae, Salmonella typhimurium and Pseudomonas aeruginosa, is obviously stronger than that of the parent peptide CPF-C1. Moreover, the derivative peptide shows lower hemolytic activity and better stability, and shows a wide application prospect in the preparation of clinical antibacterial drugs.

Description

TECHNICAL FIELD

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

[0002] The early irrational use of antibiotics has accelerated the occurrence and spread of bacterial drug resistance. Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii can cause fatal infections in hospitals, and the above-mentioned pathogenic bacteria have developed high resistance to most commonly used antimicrobial drugs.

[0003] Biofilm is one of the main reasons for bacterial resistance to antibiotics. According to statistics, about 60%-78% of chronic wound infections are closely related to the formation of bacterial biofilm, and implanted medical devices are also prone to bacterial biofilm infections. Such infections often show adverse characteristics of repeated attacks and long-term non-healing. Unfortunately, there is no effective drug for treating bacterial biofilm infections in the clinic at present.

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

[0005] CPF-C1 (GFGSLLGKALRLGANVL-NH2) is an antimicrobial peptide isolated from the skin secretions of tetraploid frogs stimulated by norepinephrine, which shows moderate antimicrobial activity against standard strains and multi-drug resistant bacteria of clinically common bacteria, and presents systemic toxicity at high concentrations, which limits its application in organisms. To develop an antimicrobial candidate compound, it is necessary to improve its antimicrobial activity and selectivity. SUMMARY

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

[0007] Still another object of the present application is to provide a preparation method of the derivative peptide.

[0008] Still another object of the present application is to provide use of the derived peptide in preparation of a medicament for preventing or treating drug-resistant bacterial infection and biofilm infection related diseases.

[0009] The frog-derived antibacterial peptide CPF-C1 derived peptide according to the embodiment of the present application is based on the wild sequence of frog-derived antibacterial peptide CPF-C1, wherein the 1st and 13th amino acids are replaced by tryptophan, and the 3rd, 4th, 7th, 14th and 15th amino acids are all 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 in the sequence of the CPF-C1 derived peptide are each independently selected from α-lysine or ε-lysine, the α-lysine forms a peptide bond, and the ε-lysine forms an isopeptide bond, as shown in formula (I):

[0013]

[0014] The CPF-C1 derived peptide according to the embodiment of the present application is that the 6th to 11th amino acids in the amino acid sequence are each independently selected from the corresponding L-type or D-type amino acid in the sequence.

[0015] The CPF-C1 derived peptide according to the embodiment of the present application is any one of the following compounds:

[0016] The derived peptide CPF-IP1 has an amino acid sequence 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 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 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 derivative 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 derivative 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 derivative 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 - a-Lys 7 - a-Lys 8 - Ala 9 - Leu 10 - Arg 11 - Leu 12 - Trp 13 - a-Lys 14 - e-Lys 15 - Val 16 - Leu 17 - CONH2;

[0028] the derivative peptide IP2-D1 having the amino acid sequence of SEQ ID NO. 7:

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

[0030] the derivative peptide IP2-D2 having the amino acid sequence of SEQ ID NO. 8:

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

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

[0033] Trp 1 -Phe 2 -α-Lys 3 -ε-Lys 4 -Leu 5 - Leu 6 - a-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] a derivative peptide IP2-D4, whose amino acid sequence is shown as SEQ ID NO. 10:

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

[0036] In the sequences of SEQ ID NO. 1-10 Italic underlined letters D represents a D-type amino acid.

[0037] According to the specific embodiment of the present application, a preparation method of the derivative peptide is provided, comprising the following steps:

[0038] Step 1: using Fmoc solid-phase synthesis method, connecting the first amino acid at the C-terminal to the MBHA resin, and sequentially connecting the amino acids from the C-terminal to the N-terminal direction until the completion of the peptide chain, using L-Fmoc-Lys(Boc)-OH as the amino acid for connecting α-Lys, and using L-Boc-Lys(Fmoc)-OH as the amino acid for connecting ε-Lys;

[0039] Step 2: The cleavage solution was prepared according to the volume ratio of TFA:Tis:H2O 95:2.5:2.5, the resin in step 1 was cleaved, and the remaining protecting groups of the peptide chain were removed. The crude polypeptide peptide chain cleavage solution was collected;

[0040] Step 3: The crude polypeptide obtained in step 2 was desalted and purified by RP-HPLC to obtain a derivative peptide with a purity of >99%, and the isolated product was identified by mass spectrometry to confirm the m / z value of the protonated molecular ion peak.

[0041] The application provides application of the derivative peptide in preparation of a medicine for preventing or treating a drug-resistant bacterial infection disease, and specifically comprises:

[0042] The CPF-C1 derivative peptide in the application can be used as a unique effective component or one of effective components in a drug-resistant bacterial infection disease of gram-negative bacteria or / and gram-positive bacteria, and has a therapeutic and preventive effect on the disease.

[0043] The CPF-C1 derivative peptide in the application has a therapeutic and preventive effect on a bacterial biofilm infection-related disease, and can be directly or indirectly used as a unique effective component or one of effective components in a bacterial biofilm infection disease.

[0044] Specific meanings of abbreviations used in the application 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 fluorenylmethoxycarbonyl, Boc is tert-butoxycarbonyl, and Tis is triisopropylsilane.

[0046] The application has the following beneficial effects:

[0047] The application is based on a wild sequence of frog-derived antibacterial peptide CPF-C1, and a series of derivative peptides are designed and synthesized, and the general formula 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 lysines 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 derived peptides designed and synthesized in the present application are comprehensively evaluated in terms of antibacterial activity, toxicity and stability, etc. Specifically, the minimum inhibitory concentration (MIC) detection is performed on standard strains with high clinical isolation rate, the antibiofilm activity and the anti-retention bacteria capacity are studied, and the therapeutic effect on drug-resistant bacteria infected mouse models is investigated. In addition, the serum stability, salt stability and hemolytic activity of the derived peptides are evaluated.

[0050] The results show that the antibacterial activity of the derived peptides obtained by introducing tryptophan and forming isopeptide bonds with ε-Lys is significantly improved, and the hemolytic activity is lower and the safety is increased after forming isopeptide bonds at positions 3, 4, 7 and 8. The antibacterial activity (GM 平均最小抑菌浓度 = 5.8-11.6 μg / mL) of the derived peptides is significantly better than that of the parent peptide CPF-C1 (GM 平均最小抑菌浓度 = 52.9 μg / mL), and the average minimum inhibitory concentration of CPF-IP2, CPF-IP5, CPF-IP6, IP2-D1 and IP2-D4 is 6.2, 7.1, 8.4, 7.6 and 5.8 μg / mL, respectively, which is about 10 times higher. Further research shows that the antibacterial spectrum of the derived peptides is expanded, and the MIC of Pseudomonas aeruginosa, Salmonella typhimurium and Klebsiella pneumoniae is reduced from 32-128 μg / mL to 4-8 μg / mL. Toxicity research shows that the hemolysis rate of CPF-IP2, CPF-IP3, CPF-IP4 and IP2-D4 is less than 10% at the highest detection concentration (128 μg / mL), indicating that they have good safety.

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

[0052] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only aim to explain some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort.

[0053] Figure 1 Mass spectrometry characterization of the derived peptides CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, CPF-IP6 in Example 1.

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

[0055] Figure 3 Minimum inhibitory concentration 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 strains tested in Example 2.

[0056] Figure 4 Hemolytic activity of the test substances CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, CPF-IP6 and IP2-D4 against mouse red blood cells in Example 3.

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

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

[0059] Figure 7 Inhibition rate of the test substance IP2-D4 against bacterial biofilm formation in Example 6.

[0060] Figure 8 Antibacterial activity of the test substance IP2-D4 against bacteria retained in bacterial biofilm in Example 7.

[0061] Figure 9To test the bactericidal ability of 1xMIC, 2xMIC, 4xMIC IP2-D4 and 4xMIC antibiotics against bacteria in Example 8.

[0062] Figure 10 To test the induced drug resistance ability of test substance IP2-D4 and control antibiotics against bacteria in Example 9.

[0063] Figure 11 To test the infection of drug-resistant bacteria on the skin 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) after 3 days of treatment in Example 10. DETAILED DESCRIPTION

[0064] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described in detail below. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0065] Materials and methods

[0066] In the following examples, the experimental conditions and experimental methods are conventional conditions and methods unless otherwise specified, and the reagents or instruments can be obtained from commercial channels.

[0067] Among them, GraphPad Prism 8.0 software is used for data statistics and analysis, and the data is expressed as mean ± standard deviation (Mean ± SD), and the significant difference analysis is carried out by one-way analysis of variance and Tukey test *** P<0.001, ** P<0.01, * P<0.05.

[0068] Example 1 Preparation of derivative peptide

[0069] Step 1: Synthesis of derivative peptide

[0070] Fmoc solid phase synthesis method is used to synthesize from the carboxyl end to the amino end direction, and 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 drying, add DMF to clean 3 times, 3 min each time;

[0072] (2) Indanone test: add indanone reagent (indanone: pyridine: phenol = 1:2:1) into a test tube, dip a small amount of resin into the test tube, and then put the test tube into a boiling water bath for 3 minutes. If the indanone test result is transparent and colorless, it indicates that the resin is normal.

[0073] (3) Resin deprotection: add a DMF solution containing 3% redistilled piperidine to remove the protecting group, and then remove the residual reagent. Repeat the process 4 times, each time for 3 minutes, and then remove the residual reagent.

[0074] (4) Indanone test: add indanone reagent (indanone: pyridine: phenol = 1:2:1) into a test tube, take a small amount of resin into the test tube, and then put the test tube into a boiling water bath for 3 minutes. If the indanone test result is blue-purple, it indicates that the protecting group has been removed.

[0075] (5) Amino acid condensation reaction: in a beaker, 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, and then dissolve them in a small amount of DMF. Immediately add the solution into the resin, stir for 1 hour, remove the solvent, and then wash the resin with DMF for 3 minutes. Repeat the process 3 times.

[0076] (6) Indanone test: if the indanone test result is transparent and colorless, it indicates that the condensation is successful.

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

[0078] For example, when preparing the peptide derived from SEQ ID NO. 1, first, 1-fold amount of MBHA resin is weighed and added to the synthesizer, and 10-15 mL of DCM is added, and oscillated for 30 min to fully wet the resin, and then the solvent is drawn off and the resin is washed with DMF 3 times, each time for 3 min; a small amount of resin is picked from the synthesizer and placed in the pre-prepared indene detection solution, heated in a boiling water bath for 3 min, and the resin is observed for transparency; if transparent, the resin is normal and the operation can continue; 10-15 mL of DMF solution containing 3% of redistilled piperidine is added to the synthesizer to remove the protecting group from the resin; after the reaction is complete, the resin is washed with DMF 4 times, each time for 3 min, and the indene detection is performed again; if the resin is blue-purple, it indicates that the protecting group has been successfully removed, and the condensation reaction can be performed; in a beaker, 3-fold amount of Fmoc-Leu-OH, 3-fold amount of HOBT, 3-fold amount of HBTU, and 6-fold excess of DIEA are weighed and dissolved in a small amount of DMF, and this solution is immediately added to the resin, and after oscillating on a shaker for 1 h, the solvent is drawn off and the resin is washed with DMF and then subjected to indene detection; if the resin is transparent, it indicates that the amino acid has been successfully linked and the deprotection reaction can be performed; the deprotection reaction is performed according to steps (3) and (4), and after success, the condensation reaction of steps (5) and (6) is repeated in the order of the C-N terminal of the compound amino acid; it is noted that when linking α-Lys, L-Fmoc-Lys(Boc)-OH is used, and when linking ε-Lys, L-Boc-Lys(Fmoc)-OH is used; through these steps, the complete peptide derived from SEQ ID NO. 1 can be synthesized, ensuring accurate linking of each amino acid and 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 peptides derived from SEQ ID NO. 2-10 are prepared by the same method, and the structural formula is as follows:

[0082]

[0083]

[0084] Step 2: Cleavage of the polypeptide peptide chain:

[0085] After all the amino acids were coupled, the resin was deprotected by washing with DMF for 3 min, repeated 2 times; methanol for 3 min; DCM for 3 min; and finally methanol for 3 min, repeated 2 times. The solvent was pumped out until the resin became powder. The cleavage solution was prepared by mixing TFA: Tis: H2O = 95: 2.5: 2.5 (V / V / V), and added to the resin for 3 h. The cleavage solution was collected. The solvent was removed by rotary evaporator, and the pre-cooled ethyl ether was added to precipitate the derivative peptide. After completion, deionized water was added to extract, and the water phase was collected and divided into 50 mL beaker, and stored in -80 °C refrigerator overnight for freeze-drying to obtain the crude peptide.

[0086] Step 3: Preparation and purification of the derivative peptide:

[0087] (1) About 40 mg of the crude peptide was dissolved in deionized water to prepare a polypeptide solution. After complete dissolution, the polypeptide solution was filtered through a 0.45 μm filter to remove insoluble substances. The elution solvent (acetonitrile and deionized water) was added with 0.1% TFA.

[0088] (2) High performance liquid chromatography used C18 reverse preparation column, 100% acetonitrile was used to flush until the spectrum was stable. The flow rate gradient was set, and the sample was injected after the initial concentration was balanced.

[0089] (3) After the sample was injected, the absorption peak at 220 nm was detected, and the eluent corresponding to the main peak was collected. After being sealed with plastic wrap and venting, it was stored in -80 °C refrigerator overnight for freeze-drying.

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

[0091] (5) The separated product was characterized and identified by mass spectrometry, and the m / z value of the protonated molecular ion peak was confirmed. The mass spectrum of the compound is shown in Figure 1 、 2 .

[0092] Table 1 Physicochemical properties of the compound 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, Gram-positive bacteria Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 23857, Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591 and Staphylococcus epidermidis ATCC 12228 were selected to detect the minimum inhibitory concentration (MIC) of the derivative peptides obtained in Example 1 to sensitive bacteria, so as to evaluate the in vitro antibacterial activity of the compounds.

[0096] (1) 3 mL of broth medium was added to a sterile test tube, and a single colony was inoculated. The test tube was placed in a 37°C, 180 rpm shaking incubator for 5-7 h to reach the logarithmic growth phase. The turbidity was compared by the McFarland method, and the concentration of the bacterial solution was diluted to 1.0 x 10 6 CFU / mL with MH.

[0097] (2) The derivative peptides obtained in Example 1 were weighed and dissolved in ultrapure water to obtain a stock solution of 2048 μg / mL. A series of concentration gradients of 128, 64, 32, 16, 8 and 4 μg / mL were obtained by double dilution with MH medium.

[0098] (3) Different concentrations of the drug solution obtained in step (2) and an equal volume of the bacterial solution were added to a sterile 96-well plate, and 3 replicate wells were set up for each concentration. The plate was placed in an incubator and incubated at 37°C overnight for 16-18 h.

[0099] (4) The bacterial turbidity was observed, and the minimum concentration of the drug in the clear wells was the minimum inhibitory concentration of the compound. The results are shown in the following table.

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

[0101]

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

[0103]

[0104]

[0105] Note: GM - = the sum of the minimum inhibitory concentrations of the compound to the measured Gram-negative bacteria / the number of measured Gram-negative bacteria; GM + = the sum of the minimum inhibitory concentrations of the compound to the measured Gram-positive bacteria / the number of measured Gram-positive bacteria; GM ALL= The sum of the minimum inhibitory concentrations of the compound against the tested strains / the number of the tested bacteria. When MIC > 128 μg / mL, it is calculated as 256 μg / mL.

[0106] Figure 3 The minimum inhibitory concentrations of the tested compounds 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 are shown in the following table.

[0107] Figure 3 A is the minimum inhibitory concentration of the tested compound against the tested gram-negative bacterial strains;

[0108] Figure 3 B is the minimum inhibitory concentration of the tested compound 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 concentrations of the tested compounds against bacteria show that the above derivative peptides have good antibacterial activity against both gram-positive bacteria 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 are all 4-8 μg / mL, and the antibacterial activity is significantly improved.

[0110] Example 3 Hemolytic activity experiment

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

[0112] (1) Sodium heparin was prepared into a solution with a concentration of 2 mg / mL with PBS, 200 μL per tube was added into a centrifuge tube. Blood was taken out by eyeball blood collection and loaded into a centrifuge tube, and centrifuged at 800 g for 10 min. The supernatant was discarded, and the lower layer of red blood cells was washed with PBS for three times, and diluted 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 with PBS, and through double dilution, 128, 64, 32, 16 and 8 μg / mL drug solutions were obtained.

[0114] (3) Grouped sample loading in 90-hole plate: equal volume of erythrocyte suspension and PBS buffer was added to the negative control group; equal volume of erythrocyte suspension and different concentrations of test substance solution were added to the experimental group; equal volume of erythrocyte suspension and 1% Triton X-100 were added to the positive control group.

[0115] (4) Incubation in a 37°C incubator, removal after 1 h and centrifugation, and transfer of the supernatant to another clean 96-hole plate for detection of the absorbance of the supernatant at 490 nm.

[0116] (5) Calculation of the hemolytic rate of the compound according to the following formula.

[0117] Hemolytic rate (%) = (OD 490 Experimental group - OD 490 negative control group) / (OD 490 positive control group - OD 490 negative control group) x 100%.

[0118] The in vitro toxicity and selectivity of the derived peptides were evaluated by detecting the hemolytic activity of the test substances, and the results are shown in Table 2. Figure 4 The derived peptides all showed low hemolytic activity at the antibacterial concentration (4-8 μg / mL), and in particular, the hemolytic rate of the derived peptides CPF-IP2, CPF-IP3, CPF-IP4 and IP2-D4 was still less than 10% at the highest concentration (128 μg / mL) tested, showing good safety.

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

[0120]

[0121] Note: MHC 20 represents the minimum concentration at which the compound causes 20% hemolysis, and TI = minimum concentration at which the compound causes 20% hemolysis / average minimum inhibitory concentration of the compound.

[0122] From the results, the therapeutic index (TI) of the derived peptides obtained was higher than that of the parent peptide CPF-C1, and in particular, the therapeutic index of CPF-IP2, CPF-IP3, CPF-IP4 and IP2-D4 increased by 5-9 times, with the highest therapeutic index of the derived peptide IP2-D4 being 44.14, indicating that the selectivity of the derived peptides was significantly improved compared with the parent peptide.

[0123] Example 4 Salt stability experiment

[0124] The stability of the test substances CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, CPF-IP6, IP2-D4 in a physiological environment was detected by using 150 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 6 μM NH4Cl, 8 μM ZnCl2, 1 mM MgCl2, 4 μM FeCl3.

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

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

[0127] (3) An equal volume of bacteria solution, salt solution and test substance at a corresponding concentration was added to a 96-well plate, and the plate was incubated at 37°C overnight. The minimum concentration of the drug in the clear wells was recorded, and the change in the minimum inhibitory concentration was calculated.

[0128] The experimental results are shown in Table 1. Figure 5 As shown in Table 1, the antibacterial activity of the test derivative peptides against methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae was almost unchanged in NaCl, KCl, NH4Cl, ZnCl2, MgCl2 and FeCl3 salt solutions, with a change in MIC of 1-2 times, indicating that the derivative peptides have good salt stability.

[0129] Example 5 Serum stability experiment

[0130] The derivative 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) After the Kunming mice were anesthetized, the blood was centrifuged at 8000 rpm / min for 30 min, and the upper serum was aspirated for use.

[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 samples at different time points (0h, 1h, 2h, 3h, 4h, 5h), immediately add an equal volume of ice-cold acetonitrile, vortex, and let stand on ice for 10 min. Centrifuge the sample at 13000g for 15 min and collect the supernatant.

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

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

[0135]

[0136] 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 >0.5 h. In particular, the half-lives of CPF-IP6 and IP2-D4 were 3.304 h and 2.932 h, respectively, showing good in vitro stability.

[0137] Example 6 Bacterial Biofilm Inhibition Experiment

[0138] The derived 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 a nutrient broth medium at 37°C in a constant temperature shaker until the logarithmic growth phase was reached, and then diluted with TSBG medium to a concentration of 1.0 × 10⁻⁶. 6 CFU / mL bacterial suspension.

[0140] (2) The test substance was diluted twice with TSBG medium to a concentration gradient of 64, 32, 16, 8, and 4 μg / mL, with polymyxin B as an antibiotic control. Samples were added in groups to 96-well plates:

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

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

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

[0144] Place in a 37℃ constant temperature incubator and incubate for 24 hours to form a biofilm.

[0145] (3) Take out the 96-well plate, remove the floating bacteria on the surface and gently wash with PBS buffer, fix with anhydrous methanol for 15 min, after drying, add 0.1% crystal violet dye to stain the biofilm for 15 min, remove the crystal violet dye, wash with deionized water for 2-3 times, finally add 95% ethanol for 15 min, detect the absorbance at 595 nm.

[0146] Biofilm formation inhibition rate (%) = [1- (experimental group OD 595 - negative control group OD 595 ) / (positive control group OD 595 - negative control group OD 595 )] x 100%.

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

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

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

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

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

[0152] The experimental results are shown in Table 1. Figure 7 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, can inhibit the formation of 50% of Acinetobacter baumannii biofilm at 2-4 μg / mL, and can inhibit the formation of 50% of Pseudomonas aeruginosa biofilm at 4-8 μg / mL, indicating that the derivative peptide IP2-D4 can inhibit the formation of bacterial biofilm at low concentration, and has strong anti-biofilm activity.

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

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

[0155] Pseudomonas aeruginosa and Acinetobacter baumannii drug-resistant strains were selected to detect the antibacterial activity of IP2-D4 on bacterial biofilm persister bacteria.

[0156] (1) The logarithmic growth phase bacteria were diluted to 1 x 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 planktonic bacteria were removed by washing twice with PBS, and the biofilm was exposed to 200 μL of TSBG medium diluted with high-concentration antibiotics, and incubated at 37°C for 24 h.

[0158] (3) The planktonic bacteria were removed by washing twice with PBS, and the adherent bacteria were transferred to 100 μL of PBS by ultrasonication for 5 min, and then the bacteria were exposed to IP2-D4 at 1 x MIC, 2 x MIC and 4 x MIC, and PBS as a control group. After incubation at 37°C for 0.5 h, 1 h, 2 h, 4 h and 6 h, the number of viable bacteria was determined by plating.

[0159] Figure 8 Example 7 In vitro anti-biofilm persister activity experiment

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

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

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

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

[0164] From the results, a large number of bacteria in the biofilm survived after the biofilm was treated with high concentrations of antibiotics, and the high concentrations of antibiotics could not kill the biofilm-retained bacteria. IP2-D4 had significant bactericidal ability to the retained bacteria. 2xMIC and 4xMIC of IP2-D4 killed the retained bacteria formed by drug-resistant Acinetobacter baumannii NO.91152 and drug-resistant Pseudomonas aeruginosa NO.157 in 1h and 0.5h, respectively, and could kill the retained bacteria formed by drug-resistant Acinetobacter baumannii NO.9336 and drug-resistant Pseudomonas aeruginosa NO.86 in 2h and 1h, respectively, indicating that the derived peptide IP2-D4 has a faster and more significant bactericidal effect on the retained bacteria induced by traditional antibiotics.

[0165] The derived peptides CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, and CPF-IP6 also have a short time to kill the retained bacteria formed by drug-resistant Acinetobacter baumannii NO.9336 and drug-resistant Pseudomonas aeruginosa NO.86, indicating that the derived peptides have a faster and more significant bactericidal effect on the retained bacteria induced by traditional antibiotics.

[0166] Example 8 in vitro bactericidal kinetics

[0167] Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606, Klebsiella pneumoniae ATCC 700603, and MRSA ATCC 33591 were selected to detect the rapid bactericidal ability of IP2-D4 on bacteria.

[0168] (1) The bacteria in the logarithmic growth phase were diluted to 1.0x10 6 CFU / mL in MH liquid medium. The test substance IP2-D4 was diluted to a final concentration of 1xMIC, 2xMIC, and 4xMIC with MH medium. The antibiotic was diluted to a final concentration of 4xMIC.

[0169] (2) IP2-D4 / antibiotic and bacteria were mixed in equal volumes, and cultured in a 37°C constant temperature incubator. Appropriate amounts of bacteria were taken out at different time intervals (0min, 10min, 15min, 30min, 1h, 2h, 3h, 6h, and 24h), diluted with MH medium, and uniformly coated on MH solid medium.

[0170] (3) The MH solid plates were placed in a 37°C constant temperature incubator and cultured overnight. The number of colonies growing on each agar plate was recorded.

[0171] Figure 9 The bactericidal ability of 1xMIC, 2xMIC, and 4xMIC IP2-D4 and 4xMIC antibiotic on bacteria in Example 8;

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

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

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

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

[0176] As can be seen from the figure, the bactericidal time of IP2-D4 against gram-negative bacteria is very fast, and it can kill Pseudomonas aeruginosa ATCC 27853 and Acinetobacter baumannii ATCC 19606 within 5-10 min at a concentration of 4xMIC. In contrast, the bactericidal time against gram-positive bacteria is slower, and it can kill methicillin-resistant Staphylococcus aureus ATCC 33591 within 2 h at a concentration of 4xMIC. The results show that at the same concentration, the bactericidal speed of IP2-D4 is much faster than that of antibiotics.

[0177] Similarly, the bactericidal speed of the derived peptides CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, and CPF-IP6 is faster than that of antibiotics.

[0178] Example 9 Induction of 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 various control antibiotics to induce drug resistance in bacteria.

[0180] (1) Dilute the bacteria in the logarithmic phase to 1.0x10 6 CFU / mL of bacterial suspension with MH liquid medium.

[0181] (2) Double dilute IP2-D4 to 128, 64, 32, 16, 8, and 4 μg / mL with MH liquid medium, and dilute the antibiotics to drug concentrations of 4, 2, 1, 0.5, and 0.125 μg / mL to detect the minimum inhibitory concentration of IP2-D4 and antibiotics.

[0182] (3) Take the sub-inhibitory concentration of bacteria solution, add 3 mL of nutrient broth medium, and culture to the logarithmic growth phase of bacterial growth, and dilute to 1.0x10 6CFU / ml. The minimum inhibitory concentration of each drug was detected again. This process was repeated for 20 consecutive days, and the results were recorded.

[0183] Figure 10 B is the induced drug resistance ability of IP2-D4, polymyxin B, ceftazidime and imipenem to Acinetobacter baumannii ATCC 19606;

[0184] Figure 10 A is the induced drug resistance ability of IP2-D4, polymyxin B, ceftazidime and ciprofloxacin to Pseudomonas aeruginosa ATCC 27853;

[0185] Figure 10 B is the induced drug resistance ability of IP2-D4, polymyxin B, ceftazidime and imipenem to Acinetobacter baumannii ATCC 19606;

[0186] Figure 10 C is the induced drug resistance ability of IP2-D4, polymyxin B, ceftazidime and imipenem to Klebsiella pneumoniae ATCC 700603;

[0187] Figure 10 D is the induced drug resistance ability of IP2-D4, vancomycin and tigecycline to MRSA ATCC 33591;

[0188] As can be seen from the figure, after 20 days of continuous induction, the minimum inhibitory concentration of IP2-D4 to Pseudomonas aeruginosa ATCC 27853 and Acinetobacter baumannii ATCC 19606 did not change, and the minimum inhibitory concentration to 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 bacteria to produce drug resistance. In contrast, antibiotics are easy to induce bacteria to produce drug resistance, and the minimum inhibitory concentration of ceftazidime to Pseudomonas aeruginosa increased by 64 times; the minimum inhibitory concentration of tigecycline to MRSA increased by 128 times. The above results show that IP2-D4 is not easy to induce bacteria to produce drug resistance.

[0189] Similarly, the derived peptides CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5 and CPF-IP6 are not easy to induce bacteria to produce drug 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 Experimental Center of Lanzhou University) were randomly divided into 7 groups, 8 in each group, namely normal group, model group, polypeptide treatment group (derivative peptide, 0.25 mg / kg, 1 mg / kg, 4 mg / kg), polymyxin B experimental group (1 mg / kg), and ceftazidime experimental group (1 mg / kg).

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

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

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

[0195] Figure 11 B is the number of drug-resistant Pseudomonas aeruginosa NO.157 in the wound dressing of normal group, model group and experimental group mice after 3 days of treatment. Similarly with the above results, in the dressing, the bacterial load of the model group was 1.41 x 10 7 CFU / dressing, after treatment, the bacterial amount in the dressing of the IP2-D4 group was reduced to 1.55 x 10 5 CFU / dressing, 2.29 x 10 5 CFU / dressing and 4.57 x 10 4 CFU / dressing, significantly reducing the bacterial load in the wound dressing of mice, and inhibiting the growth of bacterial biofilm in the dressing.

[0196] Figure 11 C is the degree of wound healing of normal group, model group and experimental group mice after 3 days of treatment. As can be seen from the figure, the wound of the model group is severely inflamed and has pus exudation, after treatment with IP2-D4 at doses of 0.25 mg / kg, 1 mg / kg and 4 mg / kg, the wound is scabbed on the 7th day, and the wound is 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] Figure 11 D is the content of inflammatory factors in the wound of normal group, model group and experimental group mice 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 wound site of the model group mice was significantly higher than that of the normal group, and after treatment with IP2-D4, the content of these two inflammatory factors in the wound was significantly reduced, indicating that IP2-D4 has anti-inflammatory activity.

[0198] In vivo activity experiments show that IP2-D4 can effectively reduce the bacterial load in the skin wound and dressing of mice, 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, CPF-IP6 can also effectively reduce the bacterial load in the skin wound and dressing of mice, and have the ability to promote wound healing and anti-inflammatory.

[0199] In summary, the derived peptides CPF-IP1, CPF-IP2, CPF-IP3, CPF-IP4, CPF-IP5, CPF-IP6, IP2-D1, IP2-D2, IP2-D3 and IP2-D4 have significantly improved antibacterial activity compared to the parent peptides, show good stability in various physiological salt environments, and the derived peptide IP2-D4 has strong inhibitory effect on bacterial biofilm and persister bacteria, and has fast bactericidal speed and is not easy to induce bacterial resistance. Further research shows that IP2-D4 shows good therapeutic effect on mouse skin wound infection and has anti-inflammatory activity.

[0200] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A frog-derived antimicrobial peptide, CPF-C1, characterized in that, The amino acid sequence of the derived peptide is shown in SEQ ID No.

10.

2. The use of the frog-derived antimicrobial peptide CPF-C1 as described in claim 1 in the preparation of antimicrobial drugs or compositions for treating bacterial infections, characterized in that, The bacteria include Gram-negative bacteria and / or Gram-positive bacteria; The Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhimurium, or Acinetobacter baumannii. The Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, or methicillin-resistant Staphylococcus aureus (MRSA).

3. The use of the frog-derived antimicrobial peptide CPF-C1 as described in claim 1 in the preparation of antibacterial biofilm drugs or compositions, characterized in that, The bacteria include Gram-negative bacteria and / or Gram-positive bacteria; The Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhimurium, or Acinetobacter baumannii. The Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, or methicillin-resistant Staphylococcus aureus (MRSA).

4. An antibacterial drug for treating bacterial infections, characterized in that, The drug uses the frog-derived antimicrobial peptide CPF-C1 as the active ingredient as described in claim 1, and the bacteria include Gram-negative bacteria and / or Gram-positive bacteria; The Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhimurium, or Acinetobacter baumannii. The Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, or methicillin-resistant Staphylococcus aureus (MRSA).

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