Anti-acinetobacter baumannii antibacterial peptide as well as preparation method and application thereof

Abstract

The invention provides an antibacterial peptide for resisting acinetobacter baumannii, which is a) a polypeptide with an amino acid sequence of SEQ ID NO: 1 (KRIXRRIXIFLR), or b) a derivative peptide obtained by replacing and / or deleting and / or adding 1-5 amino acid residues on the polypeptide with the amino acid sequence of SEQ ID NO: 1, wherein X is Fmoc-S < 5 >-OH total hydrocarbon stapled amino acid. The invention also provides a preparation method and application of the antibacterial peptide. The protease degradation tolerant derivative is screened out through total hydrocarbon stapled amino acid replacement and structure-effect relationship research, and the protease degradation tolerant derivative is easy to prepare, high in biological safety and good in clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide synthesis, and in particular to an anti-multi-drug resistant Acinetobacter baumannii polypeptide capable of withstanding protease degradation and a preparation method thereof. Background Art

[0002] Overuse of antibiotics and the lag in the development of new drugs have led to an increasingly severe situation of antibiotic resistance worldwide. According to the current development trend, by 2050, antibiotic resistance will cause 10 million deaths worldwide and a loss of $100 trillion per year. Recent global research data show that antibiotic resistance caused 4.71 million deaths in 2021, of which resistant bacteria including Acinetobacter baumannii (hereinafter referred to as Baumann) caused 1.14 million deaths (Lancet.2024, 404(10459):1199-1226). In the latest list of priority bacterial pathogens, the World Health Organization has listed resistant Baumannii as a critical group of pathogens, and it is urgent to develop new antibacterial drugs.

[0003] Antimicrobial peptides are a class of small molecule polypeptides with different degrees of antimicrobial effects. They are important components for animals and plants to resist invasion by exogenous pathogenic microorganisms. Most antimicrobial peptides are rich in basic amino acids, such as arginine (Arg) and lysine (Lys), and exhibit positive charge properties. This enables them to easily bind to bacterial membranes through electrostatic interactions, thereby achieving a powerful membrane-destroying bactericidal effect (Antimicrob Agents Chemother. 2018, 62). Since it is difficult for bacteria to change the negative charge characteristics of the membrane surface, antimicrobial peptides are difficult to be tolerated by bacteria, and therefore are regarded as a new generation of candidate antibiotics. Among all species, endogenous antimicrobial peptides in the human body have attracted much attention due to their low immunogenicity. Previous studies have found that the human cathelicidin antimicrobial peptide LL37 has highly efficient anti-Bauman activity (PLoS One 2015.10:e0141107), providing a template for the design of anti-resistant Bowman peptide antibiotics.

[0004] Although LL37 has a highly effective anti-Bauman effect, natural LL37 still has structural defects that limit drug conversion. First, LL37 has obvious hemolytic and cytotoxic effects (Int Endod J2019; 52: 1327-43). Secondly, LL37 is composed of 37 natural amino acids, and the cost of preparing high-purity polypeptides, especially through large-scale chemical synthesis of polypeptides, is relatively high. In order to solve these defects, previous studies have found through structure-effect relationship studies that the KR12 fragment from Lys18 to Arg29 is the antibacterial active region of LL37 (J.Biol.Chem.283(47)(2008)32637-32643). The twelve-peptide KR12 is not only easy to synthesize, but also has the advantage of good biosafety. However, due to the simplified structure, the antibacterial activity of KR12 is significantly weakened relative to LL37. In addition, KR12 is easily degraded by proteases and has poor in vivo stability, which is also not conducive to drug development.

[0005] Based on the site mutation scheme (J.Med.Chem 58(7)(2015)3083-3093), the present invention modifies the Gln of KR12 5 and Asp 9 Replacing with Arg and Ile, respectively, resulted in the derivative peptide Q5RD9I-KR12 (hereinafter referred to as d12). The anti-Bauman effect of this linear short peptide was significantly enhanced, solving the defect of decreased activity of KR12. Under this premise, how to improve the ability of d12 to resist protease degradation has become a bottleneck limiting drug development.

[0006] In summary, the increasing number of drug-resistant Bowman infections threatens human health, and the development of new anti-Bauman drugs is urgent. Antimicrobial peptides have the advantage of not being easily tolerated by bacteria and are a candidate for curbing superbug infections. The human antimicrobial peptide LL37 has the characteristics of strong anti-Bauman effects, but its cytotoxicity and high preparation cost are not conducive to drug development. New drug design based on the antibacterial core region KR12 of LL37 is expected to solve the above drawbacks. After site modification, the antibacterial effect of d12 is enhanced relative to KR12, but there is still the disadvantage of poor enzyme stability. Summary of the invention

[0007] The purpose of the present invention is to provide an anti-multi-drug resistant Acinetobacter baumannii polypeptide which is easy to artificially synthesize and can withstand protease degradation, so as to solve the defect of shortage of existing drugs.

[0008] To achieve the above objectives, the present application provides an anti-multi-drug resistant Acinetobacter baumannii polypeptide that can withstand protease degradation, an antimicrobial peptide against Acinetobacter baumannii, which is:

[0009] a) a polypeptide having an amino acid sequence of SEQ ID NO: 1 (KRIXRRIXIFLR), or,

[0010] b) a derivative peptide obtained by replacing and / or deleting and / or adding 1 to 5 amino acid residues of the polypeptide having the amino acid sequence of SEQ ID NO: 1;

[0011] Wherein, X is Fmoc-S5-OH all-hydrocarbon-stapled amino acid.

[0012] In one embodiment of the present invention, the antimicrobial peptide is in a random curl in an aqueous solution and in a β-folded structure in a lipid environment.

[0013] In one embodiment according to the present invention, the antimicrobial peptide is resistant to protease degradation.

[0014] Another aspect of the present invention provides a method for preparing an antimicrobial peptide, comprising:

[0015] 1) Weigh an appropriate amount of 2-Cl(Trt)-Cl resin into a reactor and soak it in dichloromethane (DCM) for 30 minutes;

[0016] 2) Weigh an appropriate amount of Fmoc-Arg(PBF)-OH (C-terminus) into a centrifuge tube, add anhydrous DCM and 0.5 mmol of N,N-diisopropylethylamine (DIEA), and shake well; then draw the solution into the reactor of step 1), react for 90 minutes with nitrogen bubbling, add methanol and DCM mixture after the reaction, and react for another 20 minutes; wherein, in terms of g:mmol, the ratio of 2-Cl(Trt)-Cl resin to Fmoc-Arg(PBF)-OH (C-terminus) is 10:3; in terms of g:mL, the ratio of 2-Cl(Trt)-Cl resin to initial DCM and coupling DCM is 1:20:20.

[0017] 3) Washing: drain the liquid in the reactor, then add industrial-grade dimethylformamide (DMF) to the reactor to completely soak the resin in the solution, wash for 30 seconds, and then use a vacuum pump to drain the liquid in the reactor. The draining time is about 30 seconds. Repeat this operation several times;

[0018] 4) Fmoc removal: Add enough piperidine / DMF solution to the reactor to completely soak the resin in the solution, and react with nitrogen bubbling for 20 minutes;

[0019] 5) Washing: refer to 3), and replace the industrial grade DMF with analytical grade DMF during the fifth washing process;

[0020] 6) Secondary amino acid condensation: Weigh appropriate amounts of Fmoc-Leu-OH (second position from the C-terminus) and 1-hydroxybenzotriazole (HOBT) in a centrifuge tube and fully dissolve them in DMF; then add N, N ，- diisopropylcarbodiimide (DIC), mixed for 1 min, added to the dried resin, and reacted by nitrogen bubbling for 1 h; wherein the molar ratio of Fmoc-Leu-OH, HOBT and DIC is 1:1:5; in terms of mmol:ml, the ratio of Fmoc-Leu-OH to DMF is 1:10;

[0021] Wash the resin several times with industrial pure DMF;

[0022] 7) Condensing amino acids in sequence: remove the Fmoc group according to step 4), repeat step 6), and condense Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-S5-OH, Fmoc-Ile-OH, Fmoc-Arg-OH, Fmoc-Arg-OH, Fmoc-S5-OH, Fmoc-Ile-OH, Fmoc-Arg-OH, Fmoc-Lys-OH in sequence according to the order of amino acids;

[0023] 8) washing, refer to step 5);

[0024] 9) Stapling cyclization: Add appropriate amount of Grubbs catalyst and use DCM as solvent for stapling cyclization. The cyclization time is 4h.

[0025] 10) Resin drying: Wash the resin with methanol 3 times and vacuum dry it into dry particles;

[0026] 11) Peptide cleavage:

[0027] Pour the dried 2-Cl(Trt)-Cl resin into a centrifuge tube, add the cutting solution, and cut at room temperature for 1.5 h;

[0028] 12) Crude product collection: filter out the resin to obtain a filtrate, add ice ether to the filtrate for washing and centrifugation to obtain a crude peptide chain precipitate;

[0029] 13) Peptide purification: Liquid chromatography is used to purify the crude product obtained in step 12) to obtain a pure antimicrobial peptide;

[0030] Preferably, the washing step further includes resin detection: taking a small amount of 2-Cl(Trt)-Cl resin, adding 2-3 drops of ninhydrin solution, and heating at 100° C. until the resin turns blue to verify that Fmoc removal is successful.

[0031] In one embodiment of the present invention, the ninhydrin solution is obtained by dissolving ninhydrin in anhydrous ethanol, and the concentration thereof is 0.05 g / ml.

[0032] In one embodiment of the present invention, the piperidine / DMF solution in step 4) is prepared by mixing piperidine and DMF in a volume ratio of 1:4.

[0033] In one embodiment according to the present invention, the cutting fluid is formed by mixing trifluoroacetic acid (TFA), triisopropylsilane, 1,2-ethanedithiol and ultrapure water in a volume ratio of 95:2:2:1; preferably, the cutting fluid: resin = 10 mL: 1 g;

[0034] Preferably, the amount of glacial ether added is 10 times the volume of the cutting fluid;

[0035] In one embodiment of the present invention, in the liquid chromatography of step 13), the chromatographic column is daisogel chromatographic filler; mobile phase A is a TFA aqueous solution with a volume fraction of 0.1%; mobile phase B is a mixture of TFA and acetonitrile, wherein the volume fraction of TFA is 0.1%.

[0036] Another aspect of the present invention provides a pharmaceutical composition for preventing and treating Acinetobacter baumannii infection, comprising the above-mentioned antimicrobial peptide.

[0037] Another aspect of the present invention further provides the use of the antimicrobial peptide in the preparation of a drug for combating multidrug-resistant Acinetobacter baumannii.

[0038] The beneficial effects of the above technical solution of the present invention are as follows:

[0039] The polypeptide of the present invention kills multidrug-resistant Acinetobacter baumannii at a minimum inhibitory concentration (MIC) of 5 μg / mL.

[0040] The polypeptide of the present invention can resist proteinase K degradation.

[0041] The polypeptide of the present invention has low cytotoxicity and hemolytic activity.

[0042] The short peptide D24 of the present invention consists of only 12 amino acids, of which two sites are all-hydrocarbon stapled amino acids. D24 can effectively kill multi-drug resistant Acinetobacter baumannii, and has a low antibacterial MIC value. Compared with the linear peptide D12 of the same sequence length, D24 has a strong ability to resist protease degradation and has good medicinal value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is a liquid phase chromatogram of the d24 polypeptide of the present invention;

[0044] Figure 2 This is a mass spectrum identification diagram of the d24 polypeptide of the present invention;

[0045] Figure 3 d24 polypeptide of the present invention in aqueous solution and sodium dodecyl sulfate (SDS) solution circular dichroism scanning curve;

[0046] Figure 4 is a line graph showing that the d24 polypeptide and the d12 polypeptide of the present invention are degraded by proteinase K;

[0047] Figure 5 The toxicity results of the d24 polypeptide and LL37 of the present invention on human vascular endothelial cells HUVEC are shown in the bar graph;

[0048] Figure 6 It is a bar graph showing the hemolysis results of the d24 polypeptide of the present invention and LL37. DETAILED DESCRIPTION

[0049] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0050] Unless otherwise specified, all reagents used in this example were of analytical grade, and the progress of all chemical reactions was detected by thin layer chromatography.

[0051] Example 1 Preparation of d24 ​​short peptide

[0052] 1) Weigh 0.5 g of 2-Cl(Trt)-Cl resin (Kanglong Biotechnology, Changzhou) using an electronic balance, put it into a reactor (25×250 mm), add dichloromethane (DCM, Tianquan Chemical, Chengdu) and soak for 30 minutes.

[0053] 2) Weigh 0.15mmol of Fmoc-Arg(PBF)-OH (C-terminal, Jier Biochemical, Shanghai) into a centrifuge tube, add anhydrous DCM and 0.5mmol of N,N-diisopropylethylamine (DIEA, Aladdin Biochemical, Shanghai), and shake well. Use a disposable pipette to draw the solution into the reactor in the previous step, and react with nitrogen bubbling for 90min. After the reaction is completed, add 1ml of methanol (Aladdin Biochemical) and 5mL of DCM mixture, and react for another 20min.

[0054] 3) Washing: After draining the liquid in the reactor with a circulating water vacuum pump, add industrial-grade dimethylformamide (DMF) to the reactor with a wash bottle to completely soak the resin in the solution. Wash for 30 seconds, then drain the liquid in the reactor with a circulating water vacuum pump. The draining time is about 30 seconds. Repeat this operation 4 times.

[0055] 4) Fmoc removal: Use a wash bottle to add 20% piperidine (Sino-pharmaceutical reagent, Shanghai) / DMF solution into the reactor to completely soak the resin in the solution, and allow nitrogen to bubble for 20 min.

[0056] 5) Washing: refer to 3), and replace the industrial grade DMF with analytical grade DMF during the fifth washing process;

[0057] 6) Resin detection: Take 5-10 2-Cl(Trt)-Cl resins, add 2-3 drops of ninhydrin solution, and heat at 100°C until the resin turns blue, indicating that Fmoc removal is successful.

[0058] 7) Condensation of secondary amino acids: Weigh 0.5 mmol Fmoc-Leu-OH (second position from the C-terminus, Gill Biochemical) and 0.5 mmol 1-hydroxybenzotriazole (HOBT, Aladdin Biochemical) in a centrifuge tube and dissolve them in 5 ml DMF; then add 2.5 mmol N, N ， -Diisopropylcarbodiimide (DIC, Aladdin Biochemical), mixed for 1 min, added to the dried resin, and reacted by nitrogen bubbling for 1 h.

[0059] Wash the resin with industrial pure DMF for 4 times, take 5-10 2-Cl(Trt)-Cl resins and add them to the ninhydrin solution, heat at 100°C for 2 minutes until the resin becomes colorless and the amino acid condensation is completed.

[0060] 8) Amino acids were sequentially condensed: the Fmoc group was removed according to step 4), and step 7) was repeated, and 0.5 mmol Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-S5-OH, Fmoc-Ile-OH, Fmoc-Arg-OH, Fmoc-Arg-OH, Fmoc-S5-OH, Fmoc-Ile-OH, Fmoc-Arg-OH, and Fmoc-Lys-OH were sequentially condensed according to the amino acid sequence. The amino acid raw materials were all purchased from Jier Biochemical.

[0061] 9) Resin detection: Refer to 7) and observe the color of the resin. If it is colorless, it indicates that the connection is complete.

[0062] 10) Washing, same as 5).

[0063] 11) Stapling cyclization: Add 0.15 mmol of Grubbs' catalyst (Aladdin Biochemical) and use DCM as solvent for stapling cyclization. The cyclization time is 4 h.

[0064] 12) Resin drying: Wash the resin with methanol three times and vacuum dry it into dry particles.

[0065] 13) Peptide cleavage:

[0066] Pour the dried 2-Cl(Trt)-Cl resin into a centrifuge tube, add the cutting solution, and cut at room temperature for 1.5 h.

[0067] 14) Crude product collection: Filter to remove the resin to obtain a filtrate, add ice ether (Chuandong Chemical, Chongqing) to the filtrate for washing and centrifugation to obtain a crude antimicrobial peptide precipitate.

[0068] 15) Peptide purification: Liquid chromatography was used to purify the crude product obtained in step 14) to obtain a pure antimicrobial peptide (amino acid sequence SEQ ID NO: 1KRIXRRIXIFLR). In the liquid chromatography, the chromatographic column was a daisogel chromatographic filler; the mobile phase A was a TFA aqueous solution with a volume fraction of 0.1%; the mobile phase B was a mixture of TFA and analytically pure acetonitrile (Aladdin Biochemical), wherein the volume fraction of TFA was 0.1%.

[0069] The purity of the pure d24 peptide was analyzed by SHIMADZU high performance liquid chromatography. The analytical column was Inertsil ODS-SP (4.6×250 mm×5 μm), the injection volume was 30 μL, the mobile phase A was 0.1% TFA / water, B was 0.1% TFA / acetonitrile, the flow rate was 1 mL / min, and the detection wavelength was 220 nm. Figure 1 As shown, the polypeptide of the present invention peaks at 11.362 min, with a purity of 98.421%. The molecular weight of the peak product analyzed by SHIMADZU LC-MS2010 liquid chromatography-mass spectrometry is 1620.5 Da ( Figure 2 ), which differs from the theoretical molecular weight of 1621.03 Da by less than 1.

[0070] Example 2d24 Antibacterial test of polypeptide microbroth dilution method

[0071] (1) Prepare MHB (OXIOD, CM0405B) plates by dissolving 1.5 g agar powder (Beijing Solebow, Cat#A8190) in 100 mL of culture medium. After the culture medium solidifies, seal the plates with sealing film and store at 4°C until use.

[0072] (2) Pipette 10 μL of multidrug-resistant Acinetobacter baumannii bacterial suspension and inoculate the bacteria onto the MHB plate using the three-line method. Invert the plate and incubate it in a 37°C bacterial incubator overnight.

[0073] (3) Pick up a single clone of bacteria with a sterile pipette tip and inoculate it into 10 mL of sterile MHB medium. Incubate overnight at 37°C in a constant temperature shaker at 200 rpm.

[0074] (4) Inoculate 100 μL of bacterial suspension into 10 mL of sterile MHB medium at a ratio of 1:100 (v / v) and culture the bacteria in a 37°C constant temperature shaker at 200 rpm until the logarithmic growth phase. Pipette 1 mL of bacterial suspension and centrifuge at 4°C at 8000 rpm for 5 min.

[0075] (5) Discard the supernatant, resuspend the bacteria in 1 mL of MHB medium, centrifuge again at 4°C, 8000 rpm, 5 min, and repeat the washing three times.

[0076] (6) Use MHB to adjust the bacterial concentration to 2×10 5CFU / mL and place on ice for later use.

[0077] (7) 0.02% acetic acid and 0.4% bovine serum albumin (BSA) were used as diluents to prepare peptide and antibiotic stock solutions, and then 0.01% acetic acid (containing 0.2% BSA) was used to dilute the peptide and antibiotic stock solutions in multiple ratios to adjust the drug concentrations to 25, 50, 100, 200, 400, 800, 1600, and 3200 μg / mL.

[0078] (8) 100 μL of bacterial solution (2×10 5 CFU / mL) and 11 μL of different concentrations of antimicrobial drugs, mix well, cover the well plate, and culture in a 37°C constant temperature incubator for 24 h.

[0079] (9) The plate was read at 600 nm absorbance by an ELISA reader, and the plate was shaken in parallel for 5 seconds before reading. According to the reading standard for antimicrobial peptide MIC modified by Hancock laboratory (Wu M, et al. J Biol Chem, 1999, 274: 29-35), the peptide concentration corresponding to a bactericidal rate exceeding 70% was the MIC. The MIC judgment standard for cefotaxime (Shanghai Biotech, A601276) and ciprofloxacin (Shanghai Biotech, A600310) was a bactericidal rate exceeding 99%.

[0080] The results of the microbroth dilution method antibacterial experiment showed that the MIC of the d24 polypeptide of the present invention against multidrug-resistant Acinetobacter baumannii was 5.0 μg / mL, which was the same as the antibacterial effect of the parent peptide d12, and significantly better than the LL37 and KR12 polypeptides (MICs were 10 and 160 μg / mL, respectively) (Table 1). Compared with traditional antibiotics cefotaxime and ciprofloxacin, the d24 polypeptide has obvious antibacterial advantages.

[0081] In the present invention, multi-drug resistant Acinetobacter baumannii includes cephalosporins (such as ceftazidime or cefepime), carbapenems (such as imipenem), β-lactamase inhibitors (such as cefoperazone / sulbactam), fluoroquinolones (such as ciprofloxacin) and aminoglycosides (such as amikacin). If it is resistant to all the above antibacterial drugs, including cefepime, ceftazidime, imipenem, meropenem, piperacillin / tazobactam, ciprofloxacin, and levofloxacin, it is called pan-drug resistant Acinetobacter baumannii (PDR-AB).

[0082] Table 1. Minimum inhibitory concentration (MIC, μg / mL) of antimicrobial peptides and traditional antibiotics against Acinetobacter baumannii

[0083]

[0084] Example 3d24 Polypeptide Secondary Structure Analysis

[0085] (1) Prepare 200 μg / mL polypeptide and 200 mM sodium dodecyl sulfate (SDS) solution (simulating lipid environment, see Wang C. et al, Sci Rep, 2016, 6: 22875) with sterile ultrapure water.

[0086] Pipette 20 μL SDS into 200 μL peptide to obtain a peptide-SDS solution with a final SDS concentration of 20 mM.

[0087] (2) Take 200 μL of peptide or peptide-SDS solution, transfer it to a quartz cell with a 1 mm optical path, and remove bubbles by ultrasound.

[0088] (3) The measurement temperature of the British Applied Photophysics circular dichroism spectrometer (Chirascan) was set to 25°C, the optical path length was 0.05 cm, and the ellipticity of the sample was recorded at 190-260 nm, with a reading interval of 1 nm.

[0089] (4) The raw data obtained from three independent experiments were processed using Pro-Data Chirascan software (version 4.1). The secondary structure of the peptides in solution was analyzed using CDNN software (version 2.1).

[0090] like Figure 3 As shown in the figure, the circular dichroism scanning curves of d24 ​​polypeptide in aqueous solution and SDS lipid environment. The d24 polypeptide is a random curl in aqueous solution, and a β-folded structure in SDS lipid globules. This shows that the d24 polypeptide structure is relatively hard and stable.

[0091] Example 4d24 Polypeptide protease tolerance test

[0092] (1) The d12 and d24 peptides were prepared into 2 mg / mL stock solutions with 50 mM Tris-HCl buffer (pH 7.4). 50 μL of the peptides were incubated with 150 μL of 50 mM Tris-HCl buffer containing 10 ng of proteinase K (Shanghai Bio-Tech, ST535) at 37°C for 10, 20, 30 and 60 minutes. PMSF (Shanghai Bio-Tech, ST507) was added to terminate the enzymatic reaction. After centrifugation at 12,000 rpm for 15 minutes at 4°C, the supernatant was taken for sample detection using an Orbitrap Exploris 120 high-resolution LC-MS instrument (Thermo Fisher Scientific).

[0093] (2) Inject 2 μL, flow rate 0.3 mL, run time 20 min, add 10-65% buffer B (acetonitrile) to buffer A (0.1% trifluoroacetic acid aqueous solution) to form a linear gradient eluent to separate the peptides. Use an Agilent column (ZORBAX300Extend-C18) with a mass spectrometry scan range of 130-1000 (m / z). Repeat the experiment three times independently.

[0094] like Figure 4 As shown, the degradation rate of d24 ​​polypeptide and proteinase K after 20 minutes of co-incubation was only 17.8%, while d12 was degraded by more than 89%, which shows that the introduction of all-hydrocarbon staple amino acids can significantly enhance the proteolytic resistance of d24. The d24 polypeptide of the present invention can withstand protease degradation and has good stability.

[0095] Example 5d24 polypeptide cytotoxicity and hemolysis experiments

[0096] (1) Cytotoxicity evaluation: Human vascular endothelial cell line HUVEC (ATCC, USA) was cultured in 1640 cell culture medium (Gibco) at 37°C and 5% CO2. Adherent cells were digested with 0.25% trypsin and 1×10 4 The cells were inoculated in a sterile 96-well plate at a density of CFU / mL, with 100 μL per well. After the cells adhered again, the supernatant was discarded, 100 μL of fresh culture medium was replaced, d24 and LL37 polypeptides were added to the final concentrations of 25, 50, and 100 μg / mL, mixed, and cultured for 24 hours. The supernatant was discarded, the plate was washed three times with PBS, and 10 μL of CCK-8 reagent (Dojindo, Shanghai) was added to each well. Incubate at 37°C and 5% CO2 for 1 hour. The suspension OD was detected by an enzyme-labeled instrument. 450 Absorbance value. After peptide incubation, the cell absorbance is measured as Abs peptide , the absorbance of the control group without treatment was Abs control , calculate cell viability: cell survival rate (%) = (Abs peptide -Abs blank ) / (Abs control -Abs blank ) × 100%. One well was set up for the experiment, and the independent experiment was repeated 3 times.

[0097] (2) Hemolytic activity assessment: 8-week-old male BALB / c mice were purchased from the Experimental Animal Center of the Army Medical University. The eyeballs were removed to collect blood, and the blood was quickly collected into anticoagulant tubes. The red blood cells were collected by centrifugation at 12,000 rpm at 4°C for 10 min. The precipitated red blood cells were resuspended with 4 times the volume of cooled PBS, and the mixture was centrifuged at 12,000 rpm at 4°C for 10 min, and the supernatant was discarded. The mixture was centrifuged again at 600 rpm at low temperature for 10 min, and the supernatant was discarded to remove platelets. The precipitated red blood cells were resuspended with 4 times the volume of cooled PBS to prepare a 20% red blood cell working solution, which was placed on ice for later use. PBS was prepared to a polypeptide concentration of 50 and 100 μg / mL. 300 ul of the red blood cell working solution and 1.2 ml of the polypeptide dissolved in PBS were taken into a sterile EP tube, mixed by pipetting, and incubated at 37°C for 2 hours. Centrifuged at 3,500 rpm at room temperature for 5 min, 100 μL of the supernatant was aspirated into a 96-well plate, and the OD was measured using an enzyme reader. 405 Absorbance value.

[0098] The absorbance after the polypeptide and red blood cells are co-incubated is A pep The absorbance of the negative control group without drug treatment was A blank , ultrapure water 100% hemolysis absorbance meter is A tot . Peptide hemolytic activity (A pep -A blank ) / (A tot -A blank )×100.

[0099] like Figure 5 , 6 As shown, LL37 has obvious hemolysis and cytotoxicity, while the biosafety of the d24 polypeptide of the present invention is significantly improved. Specifically, the hemolysis rate of 100 μg / mL LL37 polypeptide can reach 18%, and the cell survival rate is only 43%; the hemolysis and cytotoxicity of the d24 polypeptide of the same concentration are not obvious. Therefore, the d24 polypeptide of the present invention has better drug development and application value.

[0100] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An antimicrobial peptide against Acinetobacter baumannii, comprising: a) a polypeptide having an amino acid sequence of SEQ ID NO: 1 (KRIXRRIXIFLR), wherein X is a Fmoc-S5-OH all-hydrocarbon stapled amino acid; or, b) a derivative peptide obtained by replacing and / or deleting and / or adding 1 to 5 amino acid residues of the polypeptide having the amino acid sequence of SEQ ID NO: 1; in, X is Fmoc-S5-OH all-hydrocarbon-stapled amino acid.

2. The antimicrobial peptide according to claim 1, characterized in that The antimicrobial peptide is in a random curling state in an aqueous solution and in a β-folding structure in a lipid environment.

3. The antimicrobial peptide according to claim 1, characterized in that The antimicrobial peptide is resistant to protease degradation.

4. The method for preparing the antimicrobial peptide according to any one of claims 1 to 3, comprising: 1) Weigh an appropriate amount of 2-Cl(Trt)-Cl resin into a reactor and soak it in dichloromethane (DCM) for 30 minutes; 2) Weigh an appropriate amount of Fmoc-Arg(PBF)-OH (C-terminus) into a centrifuge tube, add anhydrous DCM and 0.5 mmol of N,N-diisopropylethylamine (DIEA), and shake well; then draw the solution into the reactor of step 1), react with nitrogen bubbling for 90 minutes, add methanol and DCM mixture after the reaction, and react for another 20 minutes; wherein, in terms of g:mmol, the ratio of 2-Cl(Trt)-Cl resin to Fmoc-Arg(PBF)-OH (C-terminus) is 10:3; in terms of g:mL, the ratio of 2-Cl(Trt)-Cl resin to initial DCM and coupling DCM is 1:20:20; 3) Washing: drain the liquid in the reactor, then add industrial-grade dimethylformamide (DMF) to the reactor to completely soak the resin in the solution, wash for 30 seconds, and then use a vacuum pump to drain the liquid in the reactor. The draining time is about 30 seconds. Repeat this operation several times; 4) Fmoc removal: Add enough piperidine / DMF solution to the reactor to completely soak the resin in the solution, and react with nitrogen bubbling for 20 minutes; 5) Washing: refer to 3), and replace the industrial grade DMF with analytical grade DMF during the fifth washing process; 6) Secondary amino acid condensation: Weigh appropriate amounts of Fmoc-Leu-OH (second position from the C-terminus) and 1-hydroxybenzotriazole (HOBT) in a centrifuge tube and fully dissolve them in DMF; then add N, N ， - diisopropylcarbodiimide (DIC), mixed for 1 min, added to the dried resin, and reacted by nitrogen bubbling for 1 h; wherein the molar ratio of Fmoc-Leu-OH, HOBT and DIC is 1:1:5; in terms of mmol:ml, the ratio of Fmoc-Leu-OH to DMF is 1:10; Wash the resin several times with industrial pure DMF; 7) Condensing amino acids in sequence: remove the Fmoc group according to step 4), repeat step 6), and condense Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-S5-OH, Fmoc-Ile-OH, Fmoc-Arg-OH, Fmoc-Arg-OH, Fmoc-S5-OH, Fmoc-Ile-OH, Fmoc-Arg-OH, Fmoc-Lys-OH in sequence according to the order of amino acids; 8) washing, refer to step 5); 9) Stapling cyclization: Add appropriate amount of Grubbs catalyst and use DCM as solvent for stapling cyclization. The cyclization time is 4h; 10) Resin drying: Wash the resin with methanol 3 times and vacuum dry it into dry particles; 11) Peptide cleavage: Pour the dried 2-Cl(Trt)-Cl resin into a centrifuge tube, add the cutting solution, and cut at room temperature for 1.5 h; 12) Crude product collection: filter out the resin to obtain a filtrate, add ice ether to the filtrate for washing and centrifugation to obtain a crude peptide chain precipitate; 13) Peptide purification: Liquid chromatography is used to purify the crude product obtained in step 12) to obtain a pure antimicrobial peptide; Preferably, the washing step further includes resin detection: taking a small amount of 2-Cl(Trt)-Cl resin, adding 2-3 drops of ninhydrin solution, and heating at 100° C. until the resin turns blue to verify that Fmoc removal is successful.

5. The preparation method according to claim 4, wherein The ninhydrin solution is obtained by dissolving ninhydrin in anhydrous ethanol, and its concentration is 0.05 g / ml.

6. The preparation method according to claim 4 or 5, wherein: Step 4) The piperidine / DMF solution is prepared by mixing piperidine and DMF in a volume ratio of 1:

4.

7. The preparation method according to any one of claims 4 to 6, wherein The cutting fluid is prepared by mixing trifluoroacetic acid (TFA), triisopropylsilane, 1,2-ethanedithiol and ultrapure water in a volume ratio of 95:2:2:1; preferably, the cutting fluid: resin = 10 mL: 1 g; Preferably, the amount of glacial ether added is 10 times the volume of the cutting fluid.

8. The preparation method according to any one of claims 4 to 7, wherein In the liquid chromatography of step 13), the chromatographic column is filled with daisogel; the mobile phase A is a TFA aqueous solution with a volume fraction of 0.1%; and the mobile phase B is a mixture of TFA and acetonitrile, wherein the volume fraction of TFA is 0.1%.

9. A pharmaceutical composition for resisting infection by Acinetobacter baumannii, comprising the antimicrobial peptide according to any one of claims 1 to 3.

10. Use of the antimicrobial peptide according to any one of claims 1 to 3 in the preparation of a medicament for combating multidrug-resistant Acinetobacter baumannii infection.

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