Peptide derived from antimicrobial peptide thanatin and medical use thereof

Abstract

Provided are a peptide derived from an antimicrobial peptide Thanatin and a medical use thereof. The antimicrobial peptide demonstrates different antimicrobial activities on various clinical multi-drug-resistant bacteria without any hemolytic activity and cytotoxicity and can be used for preparing a drug for resisting infection caused by multi-drug-resistant bacteria. Peptides having such structures are used alone or in combination as an auxiliary material for other drugs or as an additive for food, cosmetics, and fodders, so that infection caused by drug-resistant bacteria can be treated without inducing generation of drug-resistant bacteria.

Description

An antimicrobial peptide Thanatin derivative peptide and its medical use Technical Field

[0001] The present invention relates to an antimicrobial peptide Thanatin derivative peptide and its medical application, belonging to the field of polypeptides. Background Art

[0002] The discovery of antibiotics from the 1940s to the 1960s brought a significant breakthrough in the treatment of bacterial infections and led to their widespread use in food, agriculture, and other sectors. However, the long-term and overuse of antibiotics has led to the emergence of bacterial resistance. The emergence of multidrug-resistant bacteria has become a major threat to human health and life worldwide. In 2019, the WHO reported that 700,000 people died worldwide from drug-resistant bacterial infections and estimated that this number would increase to 10 million by 2050, becoming the leading cause of death worldwide. The antibiotic era has ended, and we are gradually entering a post-antibiotic era. The lack of strong antibiotic candidates and a generally weak antibiotic pipeline have created bottlenecks in the development of new antibiotics. Therefore, there is an urgent need to develop new treatments to combat infections caused by multidrug-resistant pathogens and multidrug-resistant bacteria. New antimicrobial drugs can mitigate the emergence of bacterial resistance and contribute to prevention, treatment, and improvements in human life.

[0003] Antimicrobial peptides (AMPs), also known as host defense peptides, not only possess a wide range of biological activities against bacteria, fungi, and viruses, but some also possess immunomodulatory effects, indirectly helping to eliminate pathogens. Since the 1880s, AMPs have been isolated from prokaryotes and eukaryotes. To date, there are 4,500 naturally occurring and synthetic peptides with antimicrobial activity in nature. Due to their key role in innate immunity, they have long been considered the greatest hope for addressing antibiotic resistance.

[0004] Thanatin is a cationic antimicrobial peptide isolated from the Hemipteran insect Podisus maculiventris. It exhibits broad-spectrum activity against Gram-negative and Gram-positive bacteria, as well as various fungi. It also exhibits potent inhibitory effects on bacterial and fungal growth at relatively low concentrations. Thanatin has a very low hemolytic rate and is nontoxic to human cells. In animal models, it has demonstrated a highly effective neutralizing effect against lipopolysaccharide-mediated sepsis and infections caused by Gram-negative multidrug-resistant pathogens. Thanatin is composed of 21 amino acids (GSKKPVPIIYCNRRTGKCQRM). I8-M21 form two antiparallel β-sheets, while C11 and C18 form an intramolecular disulfide bond. Thanatin carries six net positive charges and is amphipathic, soluble in water and partitioning into lipid environments. The hydrophilic and hydrophobic amino acids are spatially separated, and the hydrophobic residues are generally believed to play a significant role in its antimicrobial activity. Its broad-spectrum antimicrobial activity, combined with its low cytotoxicity and high in vivo stability, can be used to treat infections caused by MDR pathogens.

[0005] Despite the advantages of antimicrobial peptides, such as broad-spectrum bactericidal activity, rapid onset of action, and low resistance, the success rate of their clinical application as antimicrobial drugs is low. Currently, the clinical application of antimicrobial peptides faces four major challenges. First, toxicity. Generally speaking, cationic antimicrobial peptides exert their antimicrobial activity by binding to negatively charged bacterial membranes with their positive charge. However, studies have also shown that antimicrobial peptides can also act on some mammalian cells, meaning that they can directly bind to host cells, which can have adverse effects on the human body. Second, stability is a concern. Antimicrobial peptides are potentially unstable to proteases, serum, salt, and pH. Antimicrobial peptides are easily hydrolyzed by proteases and peptidases in serum, resulting in a very short half-life and difficulty in achieving rapid efficacy in vivo via intravenous injection. This explains why antimicrobial peptides are currently typically used for local rather than systemic treatments. Third, there is the issue of in vitro and in vivo activity discrepancies. Many antimicrobial peptides may exhibit good antimicrobial activity in vitro, but once in the body, they are affected by factors such as salt concentration and pH, causing their activity to change and preventing effective bactericidal activity. Fourth, there's the issue of production costs. While natural resources are available, the extraction, separation, and purification of antimicrobial peptides are technically difficult. Compared to small-scale chemical drugs, chemical synthesis of antimicrobial peptides is quite expensive, making industrial production difficult. Genetically engineered antimicrobial peptides exhibit unstable activity and low yields.

[0006] Therefore, there is an urgent need to transform natural peptides with large molecular weight and high production cost into artificial peptides with small molecular weight and relatively low production cost, and to improve their own stability, control toxicity, and enhance the clinical application potential of antimicrobial peptides.

[0007] Summary of the Invention

[0008] Purpose of the invention: The technical problem to be solved by the present invention is to provide a novel antimicrobial peptide Thanatin derivative peptide and its pharmaceutically acceptable salt, a preparation method and application in the preparation of drugs against drug-resistant bacteria.

[0009] Technical Solution: To solve the above technical problems, the present invention provides Thanatin derivative peptides and pharmaceutically acceptable salts. Based on the peptide chain length, the present invention provides the following five Thanatin derivative peptides of different lengths, the sequences of which are as follows:

[0010] 1.21 peptide.

[0011] GSKKPVPIIYCNRRTGKCQRM

[0012] GSKKPVPIIYCNRRT{β-Ala}KCQRM

[0013] GSKKPVPIIYCNRRS{β-Ala}KCQRM

[0014] GSKKPVPIIYCNRRT{β-Ala}KCQRF

[0015] GSKKPVPIIYCNRRT{β-Ala}KCQRW

[0016] 2. 20 peptides.

[0017] GSKKPVPIIYCNRRGKCQRM

[0018] 3. 18 peptides.

[0019] KPVPIIYCNRRT{β-Ala}KCQRM

[0020] KPVPIIYCNRRS{β-Ala}KCQRM

[0021] KPVPIIYCNRRT{β-Ala}KCQRF

[0022] KPVPIIYCNRRT{β-Ala}KCQRW

[0023] KPVPIIYCNRRT{β-Ala}KCQRY

[0024] KPVPIIYCNRRT{β-Ala}KCQR{L-Homophenylalanine}

[0025] KPVPIIYCNRRTGKCQRM

[0026] VPVPIIYCNRRTGKCQRM

[0027] LPVPIIYCNRRTGKCQRM

[0028] WPVPIIYCNRRTGKCQRM

[0029] FPVPIIYCNRRTGKCQRM

[0030] KPVPIIYCNRRT{β-Ala}KCQRM-NH2

[0031] {Aceticacid}PVPIIYCNRRTGKCQRM

[0032] {Cyclopropanecarboxylicacid}PVPIIYCNRRTGKCQRM

[0033] {Cyclohexanecarboxylicacid}PVPIIYCNRRTGKCQRM

[0034] {Benzoicacid}PVPIIYCNRRTGKCQRM

[0035] {4-Phenylbutyricacid}PVPIIYCNRRTGKCQRM

[0036] KPVPII{D-Tyr}CNRRT{β-Ala}KCQR{L-Homophenylalanine}

[0037] KPVPII{L-Homotyrosine}CNRRT{β-Ala}KCQR{L-Homophenylalanine}

[0038] KPVPII{L-m-Tyrosine}CNRRT{β-Ala}KCQR{L-Homophenylalanine}

[0039] KPVPIIYCNR{D-Lys}T{β-Ala}KCQR{L-Homophenylalanine}

[0040] KPVPIIYCNR{L-Orn}T{β-Ala}KCQR{L-Homophenylalanine}

[0041] KPVPIIYCNR{Dab}T{β-Ala}KCQR{L-Homophenylalanine}

[0042] KPVPIIYCNR{D-Orn}T{β-Ala}KCQR{L-Homophenylalanine}

[0043] KPVPIIYCNRRT{β-Ala}KC{D-Lys}R{L-Homophenylalanine}

[0044] KPVPIIYCNRRT{β-Ala}KC{L-Orn}R{L-Homophenylalanine}

[0045] KPVPIIYCNRRT{β-Ala}KC{D-Orn}R{L-Homophenylalanine}

[0046] KPVPIIYCNRRT{β-Ala}KC{Dab}R{L-Homophenylalanine}

[0047] 4. 17 - peptide.

[0048] KPVPIIYCNRRGKCQRM

[0049] KPVPIIYCNRRSKCQRM

[0050] KPVPIIYCNRR{β-Ala}KCQRM

[0051] KPVPIIYC{β-Ala}RRGKCQRM

[0052] PVPIIYCNRRT{β-Ala}KCQRF

[0053] PVPIIYCNRRTGKCQRM

[0054] PVPIIYCNRRTGKCQRM - NH2

[0055] 5. 16 - peptide.

[0056] VPIIYCNRRT{β-Ala}KCQRF

[0057] V{Hyp}IIYCNRRT{β-Ala}KCQRF

[0058] VPIIYCNRRT{β-Ala}KCQR{L-Homophenylalanine}

[0059] VPII{D-Tyr}CNRRT{β-Ala}KCQR{L-Homophenylalanine}

[0060] VPII{L-Homotyrosine}CNRRT{β-Ala}KCQR{L-Homophenylalanine}

[0061] VPII{Lm-Tyrosine}CNRRT{β-Ala}KCQR{L-Homophenylalanine}

[0062] VPIIYCNR{D-Lys}T{β-Ala}KCQR{L-Homophenylalanine}

[0063] VPIIYCNR{L-Orn}T{β-Ala}KCQR{L-Homophenylalanine}

[0064] VPIIYCNR{Dab}T{β-Ala}KCQR{L-Homophenylalanine}

[0065] VPIIYCNR{D-Orn}T{β-Ala}KCQR{L-Homophenylalanine}

[0066] VPIIYCNRRT{β-Ala}KC{D-Lys}R{L-Homophenylalanine}

[0067] VPIIYCNRRT{β-Ala}KC{L-Orn}R{L-Homophenylalanine}

[0068] VPIIYCNRRT{β-Ala}KC{D-Orn}R{L-Homophenylalanine}

[0069] VPIIYCNRRT{β-Ala}KC{Dab}R{L-Homophenylalanine}

[0070] Among them, two cysteines in the peptide chain form a disulfide bond.

[0071] Among them, the C-termini of all peptides form amide structures.

[0072] The Thanatin-derived peptide contains one or more of amino acids, acetic acid, cyclopropanecarboxylic acid, benzoic acid, 4-phenylbutyric acid or diaminobenzidine.

[0073] Wherein, the amino acids include β-amino acids.

[0074] Among them, the amino acids constituting all the above peptides are L-type or D-type isomers.

[0075] The present invention also provides a pharmaceutical composition containing the Thanatin-derived peptide or a pharmaceutically acceptable salt or a pharmaceutically acceptable excipient.

[0076] The present invention also provides use of the Thanatin-derived peptide or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing drugs for treating sepsis and inflammation caused by Gram-positive bacteria, Gram-negative bacteria and fungi.

[0077] The present invention also provides the use of the Thanatin derivative peptide or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of drugs, cosmetics or feed for inhibiting drug-resistant bacteria.

[0078] The present invention also provides the use of the Thanatin derivative peptide or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of medicines, cosmetics or feed for treating infections caused by drug-resistant bacteria.

[0079] Wherein, when the drug-resistant bacteria are Gram-negative bacteria, the drug-resistant bacteria include one or more of Klebsiella pneumoniae, Pseudomonas aeruginosa or Escherichia coli.

[0080] Wherein, when the drug-resistant bacteria are Gram-positive bacteria, the drug-resistant bacteria include Staphylococcus aureus.

[0081] The drug can be prepared into different dosage forms by adding pharmaceutically acceptable excipients.

[0082] Among them, the pharmaceutically acceptable excipients refer to various conventional excipients required for the preparation of different dosage forms, such as diluents, adhesives, disintegrants, glidants, lubricants, flavoring agents, inclusion materials, adsorption materials, etc., which are prepared by conventional preparation methods into any commonly used oral preparations, such as granules, powders, tablets, capsules, pills, oral liquids, decoctions, and pellets.

[0083] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0084] 1. The present invention provides a group of Thanatin-derived peptides with high antimicrobial activity. These peptides are derived from the original Thanatin sequence by truncations, mutations, deletions, and the introduction of non-natural amino acids and non-natural components. In vitro activity tests show that these antimicrobial peptides exhibit different antimicrobial activities against a variety of clinical multidrug-resistant bacteria. In vitro hemolysis and cytotoxicity tests also demonstrate that these peptides are non-hemolytic and non-cytotoxic. The antimicrobial peptides synthesized by the present invention can be used to prepare drugs for treating multidrug-resistant bacterial infections.

[0085] 2. A number of small peptides with high antibacterial activity against clinical multidrug-resistant bacteria were synthesized by solid-phase synthesis. The preparation process is stable and simple, suitable for large-scale industrial production;

[0086] 3. The peptides with the above structures are used alone or in combination as excipients for other drugs, or as additives for food, cosmetics, and feed; they can treat infections caused by drug-resistant bacteria without inducing the production of drug-resistant bacteria; after being hydrolyzed by enzymes in the body, the peptides can be converted into amino acids that can be used by the human body, without residue problems, and are environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 is a mass spectrum of target peptide 1;

[0088] Figure 2 is a chromatogram of target peptide 1;

[0089] FIG3 is a mass spectrum of target peptide 13;

[0090] FIG4 is a chromatogram of target peptide 13;

[0091] FIG5 is a mass spectrum of target peptide 14;

[0092] FIG6 is a chromatogram of target peptide 14;

[0093] FIG7 is a mass spectrum of target peptide 15;

[0094] FIG8 is a chromatogram of target peptide 15;

[0095] FIG9 is a mass spectrum of target peptide 16;

[0096] FIG10 is a chromatogram of target peptide 16. DETAILED DESCRIPTION

[0097] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0098] Example 1 Solid Phase Synthesis of Target Peptide

[0099] The target peptide was synthesized using Fmoc solid-phase synthesis.

[0100] 1. Synthesis of target peptides 1-17, target peptides 19-40, and target peptides 42-55

[0101] Step 1: Resin swelling

[0102] Before the synthesis reaction, soak the reaction tube in dichloromethane (DCM) overnight to prevent resin adhesion during the reaction. Wang resin (Tianjin Nankai Hecheng Technology Co., Ltd.) (500 mg, 0.8 mmol / g) was weighed and added to the soaked reaction tube. DCM (2 / 3 of the column volume) was added along the tube wall to swell the resin for 1 hour to fully swell. After swelling, the DCM was removed using a vacuum pump.

[0103] Step 2: Linking of the first amino acid (taking target peptide 1 as an example)

[0104] ① Weigh methionine Fmoc-Met-OH (Gill Biochemical (Shanghai) Co., Ltd.) (497.808 mg, 1.6 mmol), HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, Gill Biochemical (Shanghai) Co., Ltd., 606.784 mg, 1.6 mmol), and 1-hydroxybenzotriazole (HOBT, Gill Biochemical (Shanghai) Co., Ltd., 216.208 mg, 1.6 mmol) into a 25 mL eggplant-shaped flask. Then, add 5 mL of N,N-dimethylformamide (DMF, Anaiji Chemical) and allow it to fully dissolve. Then, add 793.35 μL of N,N-diisopropylethylamine (DIPEA, Anaiji Chemical) and vortex to mix. After fully activating the alanine, transfer the mixture to a reaction tube. Place the reaction tube on a shaker at 200 rpm for 1 h. After completion of the reaction, drain the reaction solution under reduced pressure and wash the resin three times with DMF, DCM, and DMF, respectively, for 1 min each. ② Add 5 mL of the prepared blocking solution (DIPEA: acetic anhydride: DMF = 1:2:7, v / v) to block the end, shake and react for 30 minutes, then remove the reaction solution and wash the resin three times with DMF, DCM, and DMF, respectively, for 1 minute each time.

[0105] Step 3: Connecting the remaining amino acids

[0106] Following the amino acid sequence of each target peptide, ligate each amino acid sequentially from the C-terminus to the N-terminus using the same ligation method as the first. For sterically hindered natural amino acids and difficult-to-link unnatural amino acids, a second coupling is required. For this second coupling, a different and more potent condensing agent, HATU (2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate, manufactured by Gill Biochemical (Shanghai) Co., Ltd.), should be used. After each condensation step, test with ninhydrin. If the resin remains colorless after two condensations, proceed to the next condensation step. If color is still present after testing with ninhydrin, cap the ends before proceeding to the next condensation step.

[0107] Step 4: Cleavage and purification of target peptide

[0108] ① Cleavage of the target peptide: After all amino acids on the target peptide have been linked, add twice the volume of resin to the reaction tube with a pre-prepared, ice-cooled cleavage buffer (volume ratio: trifluoroacetyl TFA: phenol: HO: trifluoromethanesulfonate (TIPS) = 88:5:5:2). Shake the reaction at room temperature for 3 h. After the reaction, collect the filtrate and add it to 20 mL of pre-chilled icy ether. Slowly add dropwise until a white flocculent precipitate completely precipitates. The precipitate is then collected, the supernatant removed by centrifugation, and washed three times with icy ether to remove as many impurities as possible. During cleavage, trifluoroacetic acid also removes the side chain protecting groups. The collected precipitate is freeze-dried to obtain a crude product. ② 80 mg of the crude target peptide obtained above was accurately weighed and added to a sample dissolution vial. 8 mL of the prepared sample dissolution solvent (acetonitrile: water = 1:3, v / v) was then added and ultrasonicated to dissolve it completely. Once fully dissolved, the product was purified using reverse-phase high-performance liquid chromatography (chromatographic conditions were the same as in Example 3). According to the chromatographic peak conditions, fractions were collected at different time periods, and the molecular weight of each collected fraction was confirmed by ESI-MS. The fractions whose actual molecular weight was consistent with the theoretical target peptide molecular weight were collected, and then freeze-dried to obtain the pure target peptide products.

[0109] 2. Target peptides 18 and 41 were synthesized using Rink resin.

[0110] For Rink resin, the Fmoc protecting group on the resin needs to be removed before condensation. Add 1.5 times the volume of the resin to the swollen resin and prepare a 20% piperidine solution (DMF: piperidine = 4:1, v / v). Then place the peptide synthesis tube on an orbital shaker and shake at 200r / min at room temperature for 5 minutes to remove the Fmoc protecting group. Then, drain the deprotection solution under reduced pressure and wash the resin three times with DMF, DCM, and DMF, respectively, for 1 minute each time to ensure that impurities are fully removed. Repeat the above experimental process and remove it again, the second time for 15 minutes. Other steps are as above.

[0111] Example 2 Confirmation of the molecular weight of the target peptide

[0112] In order to further verify the molecular weight of each target peptide obtained by reverse HPLC separation and freeze-drying, the molecular weight of each target peptide was confirmed by ESI-MS. The specific mass spectrometry conditions were as follows: Nebulizing Gas Flow: 1.5 L / min; CDL Temp: 250; Block Temp: 200; Interface Bias: +4.5 kV; Drying Gas Flow: 5 L / min; T.Flow: 0.2 ml / min; B.conc: 50% H2O / 50% MeOH.

[0113] The mass spectrometry results of some target peptides are shown in the accompanying figures: Figure 1 (SEQ ID NO. 1), Figure 3 (SEQ ID NO. 3), Figure 5 (SEQ ID NO. 14), Figure 7 (SEQ ID NO. 15), and Figure 9 (SEQ ID NO. 16). The results demonstrate that target peptides 1 to 55 were successfully synthesized in Example 1. The synthesized target peptides are shown in Table 1:

[0114] Table 1 Target peptide structure sequence

[0115] Example 3 Purity Identification of Target Peptide

[0116] 300 mg of each freeze-dried target peptide was accurately weighed and dissolved in 5 mL of sample solvent (acetonitrile:water = 1:3, v / v). Ultrasonication was performed to fully dissolve the target peptide, and the purity was confirmed by reverse-phase high-performance liquid chromatography. The chromatographic conditions for the purity of each target peptide were as follows: column model: Inertsil ODS-3 4.6 x 250 mm; detection wavelength: 220 nm; flow rate: 1 mL / min; injection volume: 30 μL; column temperature: 4°C.

[0117] The specific mobile phase ratios are shown in Table 2.

[0118] Table 2 HPLC analysis mobile phase ratios for each target peptide

[0119] The results showed that the purity of all target peptides was ≥98%. The mass spectra and chromatograms of some target peptides are shown in the accompanying figures: Figure 2 (SEQ ID NO.1), Figure 4 (SEQ ID NO.13), Figure 6 (SEQ ID NO.14), Figure 8 (SEQ ID NO.15), and Figure 10 (SEQ ID NO.16).

[0120] Experimental Example 4: Antibacterial activity test of target peptide against clinical drug-resistant bacteria

[0121] 1. Source of strain

[0122] All clinical drug-resistant bacteria used in this experiment were isolated from the Bacteria Laboratory of Zhongda Hospital Affiliated to Southeast University.

[0123] 2. Minimum inhibitory concentration (MIC) determination

[0124] MIC was determined according to the broth microdilution method (Document M38-A2) of the National Committee for Clinical Laboratory Standards (NCCLS). 55 peptide sample solutions were prepared at a concentration of 5.12 mg / mL using sterile saline. The test bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus) were inoculated into nutrient broth (10 g of peptone, 5.0 g of sodium chloride, beef broth to 1000 ml), cultured at 37°C for 24 hours, and diluted 1:10 with sterile saline before use. 5 Dilution was performed by fold, 12 bacterial culture tubes were taken and numbered, 1.6 mL of nutrient broth was added to the first tube, and 0.8 mL of nutrient broth was added to the remaining 11 tubes. 0.2 mL of each polypeptide solution was added to the first tube, 1.0 mL was taken out after mixing and added to the second tube, and so on and so forth, diluted to the 12th tube in sequence, 0.2 mL of bacterial solution was added to each tube, shaken gently and evenly, and cultured at 37 ° C for 24 h. The lowest polypeptide concentration for sterile growth was the minimum sample concentration (MIC) for inhibiting bacterial growth. All experiments were repeated 3 times. Table 3 shows the minimum inhibitory concentration of the anti-clinical multi-drug resistant peptides prepared in Example 1 against several bacteria.

[0125] Table 3 Minimum inhibitory concentration (MIC) of each target peptide

[0126] As shown in Table 3, most of the target peptides showed strong anti-Klebsiella pneumoniae and Escherichia coli activities, and their activities were better than those of the natural peptide Thanatin. Some peptides showed excellent anti-Pseudomonas aeruginosa and anti-Staphylococcus aureus activities.

[0127] Example 5 Target peptide hemolysis rate test

[0128] Dissolve the target peptide in ultrapure water or DMSO to prepare a target peptide stock solution at a concentration of 48 mg / mL. Prepare a positive control Triton-X-100 stock solution (set to 1%). Add 198 μL of PBS to a 96-well plate (add 196 μL of PBS to the highest concentration of the positive control). Take 2 μL of the target peptide stock solution and 4 μL of the positive control stock solution and add them to the prepared PBS (100 mM, pH = 7.2-7.4) and mix thoroughly (this results in a test drug concentration of 480 μg / mL and a positive control concentration of 2%). Then, aspirate 100 μL of each mixed solution and add it to another well containing 100 μL of PBS and mix thoroughly. Repeat this process for five test drug dilution points and 11 positive control dilution points. Add 100 μL of a 4% red blood cell suspension to each well, so that the sample solution concentrations, from highest to lowest, are 240, 120, 60, 30, and 15 μg / mL, respectively. The wells containing only cell suspension served as negative control groups. The 96-well plate was placed in an incubator and incubated for 1 hour at 37°C and 60 rpm.

[0129] Qualitative hemolysis analysis: After incubation, centrifuge at 1000g for 3 minutes. Visually inspect for hemolysis. If the solution in the wells is clear red with no cells or a small amount of red blood cells remaining at the bottom of the tube, hemolysis is present. If all red blood cells sink and the supernatant is clear and colorless, hemolysis is absent. Results showed that no hemolysis occurred at the highest concentration of 240 μg / mL for any target peptide; no hemolysis was observed at other lower concentrations.

[0130] Quantitative study of hemolysis: Carefully pipette 100 μL of supernatant into a new 96-well plate and measure the absorbance at 540 nm (OD 540 ).

[0131] Calculation of hemolysis rate:

[0132] Z: red blood cell hemolysis rate; At: absorbance of the test drug group; Anc: absorbance of the negative control group; Apc: absorbance of the positive control group.

[0133] The hemolysis rate results of the target peptides are shown in Table 4. The results showed that all target peptides did not undergo hemolysis at a concentration of 0.24 g / L.

[0134] Example 6 Target Peptide Toxicity Test

[0135] The cytotoxicity of the target peptide was tested using the MTT method. Macrophages iBMDM and lung epithelial cells BEAS-2B were seeded in 96-well plates at a density of 8,000 cells per well and cultured at 37°C for 24 hours. After 24 hours, each well was treated with 0.1, 1, 10, and 100 μM of the peptide, respectively, and cultured for another 24 hours. After the treatment time, 20 μL of MTT was added and incubated for 3 hours. After the incubation period, DMSO (100 μl) was added to each well to dissolve the purple formazan crystals (MTT metabolites). The plate was shaken for 10 minutes, and the absorbance was finally measured at 570 nm. The results of the target peptide toxicity test are shown in Table 4.

[0136] Table 4 Hemolysis rate and cytotoxicity information of each target peptide

[0137] As shown in Table 4, the half-maximal inhibitory concentrations of all target peptides against macrophages and lung epithelial cells were greater than 100 μM, indicating that the target peptides did not produce cytotoxicity or had very low cytotoxicity.

Claims

1. A Thanatin derivative peptide or a pharmaceutically acceptable salt thereof, characterized in that: The amino acid sequence of the Thanatin-derived peptide is any one of SEQ ID NOs. 1 to 55.

2. The Thanatin derivative peptide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that The Thanatin derivative peptide contains one or more of amino acids, acetic acid, cyclopropanecarboxylic acid, benzoic acid, 4-phenylbutyric acid or diaminobenzidine.

3. The Thanatin derivative peptide or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The amino acids include β-amino acids.

4. The Thanatin derivative peptide or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The amino acid is an L-type or D-type isomer.

5. A pharmaceutical composition, characterized in that The invention contains the Thanatin derivative peptide according to any one of claims 1 to 4 or a pharmaceutically acceptable salt or a pharmaceutically acceptable excipient thereof.

6. Use of the Thanatin derivative peptide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of drugs, cosmetics or feed for inhibiting drug-resistant bacteria.

7. Use of the Thanatin derivative peptide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a medicament, cosmetic or feed for treating infection caused by drug-resistant bacteria.

8. The use according to any one of claims 6 to 7, characterized in that: When the drug-resistant bacteria are Gram-negative bacteria, the drug-resistant bacteria include one or more of Klebsiella pneumoniae, Pseudomonas aeruginosa or Escherichia coli.

9. The use according to any one of claims 6 to 7, characterized in that: When the drug-resistant bacteria are Gram-positive bacteria, the drug-resistant bacteria include Staphylococcus aureus.

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