An antimicrobial peptide against Acinetobacter baumannii, its preparation method and application

Abstract

This invention provides an antimicrobial peptide against Acinetobacter baumannii, which is: a) a polypeptide having the amino acid sequence SEQ ID NO:1 (KRIXRRIXIFLR), or b) a derived peptide obtained by substituting and / or deleting and / or adding 1-5 amino acid residues of the polypeptide having the amino acid sequence SEQ ID NO:1; wherein X is a Fmoc-S5-OH all-hydrocarbon-bound amino acid. This invention also provides a method for preparing this antimicrobial peptide and its applications. Derivatives resistant to protease degradation were screened through all-hydrocarbon-bound amino acid substitution and structure-effect relationship studies. The peptide is easy to prepare, has high biosafety, and shows promising clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of polypeptide synthesis technology, specifically to a polypeptide resistant to multidrug-resistant Acinetobacter baumannii that can withstand protease degradation and its preparation method. Background Technology

[0002] The 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 current trends, by 2050, antibiotic resistance will cause 10 million deaths globally, resulting in an annual loss of $100 trillion. Recent global research data shows that antibiotic resistance caused 4.71 million deaths in 2021, of which 1.14 million were caused by drug-resistant bacteria, including Acinetobacter baumannii (hereinafter referred to as Baumann) (Lancet. 2024, 404(10459):1199-1226). In the latest list of priority bacterial pathogens, the World Health Organization has listed drug-resistant Baumannii as a critical pathogen, highlighting the urgent need to develop new antibacterial drugs.

[0003] Antimicrobial peptides are a class of small-molecule polypeptides with varying degrees of antibacterial activity, serving as important components for the defense of animals and plants against exogenous pathogenic microorganisms. Most antimicrobial peptides are rich in basic amino acids, such as arginine (Arg) and lysine (Lys), exhibiting a positive charge. This allows them to easily bind to bacterial membranes via electrostatic interactions, thereby achieving a powerful membrane-disrupting bactericidal effect (Antimicrob Agents Chemother. 2018, 62). Because bacteria have difficulty altering the negative charge on their membrane surfaces, antimicrobial peptides are rarely tolerated by bacteria and are therefore considered potential next-generation antibiotics. Among all species, human endogenous antimicrobial peptides have attracted significant attention due to their low immunogenicity. Previous studies have found that the human cathelicidin antimicrobial peptide LL37 possesses potent anti-Baumann activity (PLoS One 2015.10:e0141107), providing a template for designing antibiotics targeting drug-resistant Baumann peptides.

[0004] Despite its potent antibacterial activity against Baumann, natural LL37 suffers from structural defects that limit its drug conversion. First, LL37 exhibits significant hemolytic activity and cytotoxicity (Int Endod J2019;52:1327-43). Second, LL37 is composed of 37 natural amino acids, making the preparation of high-purity peptides, especially through large-scale chemical synthesis, relatively costly. To address these limitations, previous studies have identified the KR12 fragment (Lys18 to Arg29) as the antibacterial active region of LL37 (J. Biol. Chem. 283(47)(2008)32637-32643). The dodecapeptide KR12 is not only easy to synthesize but also possesses good biocompatibility. However, due to its simplified structure, the antibacterial activity of KR12 is significantly weaker than that of LL37. Furthermore, KR12 is easily degraded by proteases and has poor in vivo stability, which also hinders drug development.

[0005] Based on the site mutation scheme (J.Med.Chem 58(7)(2015)3083-3093), this invention will modify the Gln of KR12. 5 and Asp 9 Replacing Arg and Ile with Arg and Ile respectively yielded the derived peptide Q5RD9I-KR12 (hereinafter referred to as d12). This linear short peptide exhibited significantly enhanced anti-Baumann activity, overcoming the deficiency of decreased KR12 activity. Under these circumstances, improving the resistance of d12 to protease degradation became a bottleneck limiting its drug development.

[0006] In summary, the increasing prevalence of drug-resistant Baumannii infections threatens human health, making the development of novel anti-Baumannii drugs an urgent priority. Antimicrobial peptides, with their advantage of being less susceptible to bacterial tolerance, are candidate solutions for curbing superbug infections. The human antimicrobial peptide LL37 exhibits strong anti-Baumannii activity, but its cytotoxicity and high preparation cost hinder its drug development. Drug design based on the KR12 antimicrobial core region of LL37 holds promise for overcoming these drawbacks. After site modification, d12 exhibits enhanced antimicrobial activity compared to KR12, but still suffers from poor enzyme stability. Summary of the Invention

[0007] The purpose of this invention is to provide a polypeptide against multidrug-resistant Acinetobacter baumannii that is easy to synthesize artificially and can withstand protease degradation, in order to address the shortage of existing drugs.

[0008] To achieve the above objectives, this application provides a polypeptide against multidrug-resistant Acinetobacter baumannii that is resistant to protease degradation, an antimicrobial peptide against Acinetobacter baumannii, which is as follows:

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

[0010] b) Derived peptides obtained by replacing and / or deleting and / or adding 1-5 amino acid residues of a polypeptide with the amino acid sequence SEQ ID NO:1;

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

[0012] In one embodiment of the invention, the antimicrobial peptide exhibits a random coiled structure in aqueous solution and a β-sheet structure in a lipid environment.

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

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

[0015] 1) Weigh an appropriate amount of 2-Cl(Trt)-Cl resin into the 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 add the solution to the reactor in step 1), bubble the reaction under nitrogen for 90 min, add methanol and DCM mixture after the reaction is complete, and react for another 20 min; wherein, in g:mmol, the ratio of 2-Cl(Trt)-Cl resin to Fmoc-Arg(PBF)-OH (C-terminus) is 10:3; in g:mL, the ratio of 2-Cl(Trt)-Cl resin to initial DCM and coupled DCM volume is 1:20:20.

[0017] 3) Washing: Drain the liquid in the reactor, then add industrial-grade dimethylformamide (DMF) to the reactor to completely immerse the resin in the solution. Wash for 30 seconds, then use a vacuum pump to drain the liquid in the reactor for about 30 seconds. Repeat this operation several times.

[0018] 4) De-Fmoc: Add sufficient piperidine / DMF solution to the reactor to completely immerse the resin in the solution, and bubble the reaction with nitrogen for 20 minutes;

[0019] 5) Washing: Refer to 3), and replace the industrial-grade DMF with analytical-grade DMF during the 5th wash;

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

[0021] The resin was washed several times with industrial-grade DMF.

[0022] 7) Sequential amino acid condensation: Remove the Fmoc group as in 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, and Fmoc-Lys-OH in sequence according to the amino acid order.

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

[0024] 9) Staple cyclization: Add an appropriate amount of Grubb catalyst and use DCM as solvent to carry out staple cyclization for 4 hours.

[0025] 10) Resin drying: Wash the resin three times with methanol and then vacuum dry it into dry granules.

[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 hours.

[0028] 12) Crude product collection: Filter to remove resin to obtain filtrate, add ice-cold ether to the filtrate, wash and centrifuge to obtain crude peptide chain precipitate;

[0029] 13) Peptide purification: The crude product obtained in step 12) was purified by liquid chromatography to obtain pure antimicrobial peptide;

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

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

[0032] In one embodiment of the 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 of the invention, the cutting fluid is composed of trifluoroacetic acid (TFA), triisopropylsilane, 1,2-ethylenedithiol 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 ice-cold 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 a Daisogel chromatographic packing; the mobile phase A is a TFA aqueous solution with a volume fraction of 0.1%; the 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 treating Acinetobacter baumannii infection, comprising the above-described antimicrobial peptide.

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

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

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

[0040] The polypeptide described in this invention is resistant to proteinase K degradation.

[0041] The polypeptides described in this invention have low cytotoxicity and hemolytic activity.

[0042] The d24 short peptide described in this invention consists of only 12 amino acids, two of which are all-hydrocarbon bound amino acids. d24 can effectively kill multidrug-resistant Acinetobacter baumannii with a low antibacterial MIC value. Compared to the linear polypeptide d12 of the same sequence length, d24 has strong resistance to protease degradation and good medicinal value. Attached Figure Description

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

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

[0045] Figure 3 The circular dichroism chromatographic scans of the d24 polypeptide of the present invention in aqueous solution and sodium dodecyl sulfonate (SDS) solution are shown.

[0046] Figure 4 Line graphs showing the degradation of the d24 and d12 polypeptides of the present invention by proteinase K.

[0047] Figure 5 The bar chart shows the toxicity results of the d24 peptide and LL37 of the present invention on human vascular endothelial cells (HUVECs).

[0048] Figure 6 This is a bar chart showing the hemolysis results of the d24 polypeptide and LL37 of the present invention. Detailed Implementation

[0049] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0050] Unless otherwise specified, all reagents used in this embodiment are of analytical grade, and the progress of all chemical reactions is detected by thin-layer chromatography.

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

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

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

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

[0055] 4) De-Fmoc: Add 20% piperidine (Sinopharm Reagent, Shanghai) / DMF solution to the reactor using a wash bottle to completely immerse the resin in the solution, and bubble the reaction with nitrogen for 20 minutes.

[0056] 5) Washing: Refer to 3), and replace the industrial-grade DMF with analytical-grade DMF during the fifth wash;

[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, then Fmoc removal is successful.

[0058] 7) Secondary amino acid condensation: Weigh 0.5 mmol Fmoc-Leu-OH (C-terminal second position, Jier Biochemistry) and 0.5 mmol 1-hydroxybenzotriazole (HOBT, Aladdin Biochemistry) into a centrifuge tube, dissolve thoroughly with 5 ml DMF; then add 2.5 mmol N,N ， - Diisopropylcarbodiimide (DIC, Aladdin Biochemical), mix for 1 min, add to the dried resin, and react with nitrogen bubbling for 1 h.

[0059] The resin was washed four times with industrial pure DMF. Five to ten 2-Cl(Trt)-Cl resin particles were added to a ninhydrin solution and heated at 100°C for 2 minutes until the resin became colorless, indicating that the amino acid condensation was complete.

[0060] 8) Sequential amino acid condensation: Following step 4), remove the Fmoc group, repeat step 7), and sequentially condense 0.5 mmol of 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 according to the amino acid sequence. All amino acid raw materials were purchased from Jier Biochemical.

[0061] 9) Resin test: 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) Staple cyclization: Add 0.15 mmol of Grubb catalyst (Aladdin Biochemical) and use DCM as solvent for staple cyclization. Cyclization time: 4 h.

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

[0065] 13) Peptide cleavage:

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

[0067] 14) Crude product collection: Filter to remove resin to obtain filtrate, add ice-cold ether (Chuandong Chemical, Chongqing) to the filtrate, wash and centrifuge to obtain crude antimicrobial peptide precipitate.

[0068] 15) Peptide purification: The crude product obtained in step 14) was purified by liquid chromatography to obtain the pure antimicrobial peptide (amino acid sequence SEQ ID NO: 1KRIXRRIXIFLR). In the liquid chromatography method, the chromatographic column was Daisogel chromatographic packing material; mobile phase A was a 0.1% (v / v) TFA aqueous solution; mobile phase B was a mixture of TFA and analytical grade acetonitrile (Aladdin Biochemical), wherein the volume fraction of TFA was 0.1%.

[0069] SHIMADZU high-performance liquid chromatography (HPLC) was used to analyze the purity of pure d24 peptide. The analytical column was an Inertsil ODS-SP (4.6 × 250 mm × 5 μm), the injection volume was 30 μL, the mobile phase A was 0.1% TFA / water, and the mobile phase 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 this invention elutes at 11.362 min, with a purity of 98.421%. Analysis using a SHIMADZU LC-MS2010 liquid chromatography-mass spectrometry system revealed the molecular weight of the eluting product to be 1620.5 Da. Figure 2 The difference between the theoretical molecular weight of 1621.03 Da and the actual molecular weight is less than 1.

[0070] Example 2: Antibacterial Experiment Using the Microbroth Dilution Method for Dysbiosis of Dyspeptides

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

[0072] (2) Take 10 μL of multidrug-resistant Acinetobacter baumannii bacterial suspension and inoculate the bacteria onto an MHB plate using the triple-line method. Invert the plate and incubate overnight at 37°C.

[0073] (3) Pick 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 incubate the bacteria at 37°C with a shaker at 200 rpm until the logarithmic growth phase. Take 1 mL of the bacterial solution 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 wash three times.

[0076] (6) Adjust the bacterial concentration to 2×10 using MHB. 5CFU / mL, keep on ice for later use.

[0077] (7) Prepare peptide and antibiotic stock solutions using 0.02% acetic acid and 0.4% bovine serum albumin (BSA) as diluents, and then serially dilute the peptide and antibiotic stock solutions with 0.01% acetic acid (containing 0.2% BSA) to adjust the drug concentration to 25, 50, 100, 200, 400, 800, 1600, and 3200 μg / mL.

[0078] (8) 100 μL of bacterial suspension (2 × 10⁻⁶) was added to each well of a sterilized polypropylene 96-well plate (Corning, USA, 3365). 5 Add 11 μL of different concentrations of antibacterial drug (CFU / mL) and mix well. Cover the well plate and incubate at 37°C for 24 hours.

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

[0080] The antibacterial experiment using the micro-broth dilution method showed that the MIC of the d24 peptide of this invention in killing 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 LL37 and KR12 peptides (MICs of 10 and 160 μg / mL, respectively) (Table 1). Compared with traditional antibiotics cefotaxime and ciprofloxacin, the d24 peptide has a significant antibacterial advantage.

[0081] In this invention, multidrug-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 resistance to all of the above antibiotics, including cefepime, ceftazidime, imipenem, meropenem, piperacillin / tazobactam, ciprofloxacin, and levofloxacin, it is termed pan-drug-resistant Acinetobacter baumannii (PDR-AB).

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

[0083]

[0084] Example 3: Analysis of the secondary structure of the d24 peptide

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

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

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

[0088] (3) Set the measurement temperature of the Applied Photophysics Chirascan to 25℃ and the optical path to 0.05cm, record the ellipticity of the sample from 190 to 260nm, and take readings at 1nm intervals.

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

[0090] like Figure 3 The image shows the circular dichroism chromatographic scans of peptide d24 in aqueous solution and SDS lipid environments. Peptide d24 exhibits a random coil structure in aqueous solution, while it displays a β-sheet structure in SDS lipoglobules. This indicates that peptide d24 has a relatively rigid and stable structure.

[0091] Example 4: d24 Peptide Protease Tolerance Test

[0092] (1) Prepare a 2 mg / mL stock solution of d12 and d24 peptides using 50 mM Tris-HCl buffer (pH 7.4). Take 50 μL of peptides and incubate with 150 μL of 50 mM Tris-HCl buffer containing 10 ng of proteinase K (Shanghai Beyotime, ST535) at 37 °C for 10, 20, 30 and 60 minutes. Add PMSF (Shanghai Beyotime, ST507) to terminate the enzymatic reaction. After centrifugation at 12000 rpm for 15 min at 4 °C, use an Orbitrap Exploris 120 high-resolution LC-MS instrument (Thermo Fisher Scientific) to collect the supernatant for sample detection.

[0093] (2) Inject 2 μL of sample at a flow rate of 0.3 mL for 20 min. Add 10-65% buffer B (acetonitrile) to buffer A (0.1% trifluoroacetic acid aqueous solution) to form a linear gradient elution buffer for peptide separation. Use an Agilent column (ZORBAX 300 Extend-C18) with a mass spectrometry scan range of 130-1000 m / z. Repeat the experiment independently three times.

[0094] like Figure 4 As shown, the degradation rate of d24 ​​peptide after co-incubation with proteinase K for 20 minutes was only 17.8%, while that of d12 was over 89%. This demonstrates that the introduction of all-hydrocarbon amino acids significantly enhances the proteolytic resistance of d24. The d24 peptide of this invention is resistant to protease degradation and exhibits good stability.

[0095] Example 5: D24 Peptide Cytotoxicity and Hemolysis Assay

[0096] (1) Cytotoxicity assessment: 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 at a concentration of 1×10⁻⁶. 4 Cells were seeded at a density of CFU / mL into sterile 96-well plates, 100 μL per well. After cell re-attachment, the supernatant was discarded, and 100 μL of fresh culture medium was added. D24 and LL37 peptides were added to final concentrations of 25, 50, and 100 μg / mL, respectively. The mixture was then incubated for 24 h. The supernatant was discarded, and the cells were washed three times with PBS. 10 μL of CCK-8 reagent (Dojindo, Shanghai) was added to each well. The cells were incubated at 37℃ and 5% CO2 for 1 h. The OD of the suspension was measured using a microplate reader. 450 Absorbance value. The absorbance of cells after peptide incubation is measured as Abs. peptide The absorbance of the control group (without treatment) was Abs. control Calculate cell viability: Cell viability (%) = (Abs) peptide -Abs blank ) / (Abs control -Abs blank )×100%. The experiment was set up with one auxiliary well, and the independent experiment was repeated 3 times.

[0097] (2) Hemolytic activity assessment: Eight-week-old male BALB / c mice were purchased from the Experimental Animal Center of Army Medical University. Blood was collected by enucleation and quickly transferred to anticoagulant tubes. Red blood cells were collected by centrifugation at 12,000 rpm for 10 min at 4°C. The precipitated red blood cells were resuspended in 4 volumes of cooled PBS and centrifuged at 12,000 rpm for 10 min at 4°C, discarding the supernatant. The cells were then centrifuged again at 600 rpm for 10 min at 4°C, discarding the supernatant and removing platelets. The precipitated red blood cells were resuspended in 4 volumes of cooled PBS to prepare a 20% red blood cell working solution, which was kept on ice for later use. Peptide concentrations of 50 and 100 μg / mL were prepared in PBS. 300 μL of the red blood cell working solution and 1.2 mL of the peptide dissolved in PBS were transferred to sterile EP tubes, mixed by pipetting, and incubated at 37°C for 2 hours. The cells were centrifuged at 3500 rpm for 5 min at room temperature, and 100 μL of the supernatant was transferred to a 96-well plate. OD was measured using a microplate reader. 405 Absorbance value.

[0098] The absorbance of the polypeptide after co-incubation with red blood cells is A. pep The absorbance of the negative control group without drug treatment was A. blank The ultrapure water 100% hemolysis absorbance meter is A. tot Polypeptide hemolytic activity (A) pep -A blank ) / (A tot -A blank Calculate by multiplying by 100.

[0099] like Figure 5 , 6 As shown, LL37 exhibits significant hemolysis and cytotoxicity, while the biocompatibility of the d24 peptide of this invention is significantly improved. Specifically, the hemolysis rate of 100 μg / mL LL37 peptide can reach 18%, and the cell viability is only 43%; at the same concentration, d24 peptide shows no significant hemolysis or cytotoxicity. Therefore, the d24 peptide of this invention has better value for drug development and application.

[0100] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide against Acinetobacter baumannii, comprising: The polypeptide has the amino acid sequence SEQ ID NO:1 (KRIXRRIXIFLR), where X is a Fmoc-S5-OH whole-hydrocarbon amino acid.

2. The antimicrobial peptide according to claim 1, characterized in that, The antimicrobial peptide exhibits random coiling in aqueous solution and a β-sheet structure in a lipid environment, and is resistant to protease degradation.

3. The method for preparing the antimicrobial peptide according to any one of claims 1-2, comprising: 1) Weigh an appropriate amount of 2-Cl(Trt)-Cl resin into the reactor and soak it in dichloromethane (DCM) for 30 minutes; 2) Weigh an appropriate amount of Fmoc-Arg(PBF)-OH (C-end) into a centrifuge tube, add anhydrous DCM and 0.5 mmol of N,N-diisopropylethylamine (DIEA), and shake well; then add the solution to the reactor in step 1), bubble under nitrogen for 90 min, and after the reaction is complete, add methanol and DCM mixture, and react for another 20 min; wherein, in g:mmol, the ratio of 2-Cl(Trt)-Cl resin to Fmoc-Arg(PBF)-OH (C-end) is 10:3; in g:mL, the volume ratio of 2-Cl(Trt)-Cl resin to initial DCM and coupled DCM is 1:20:20; 3) Washing: Drain the liquid in the reactor, then add industrial-grade dimethylformamide (DMF) to the reactor to completely immerse the resin in the solution. Wash for 30 seconds, then use a vacuum pump to drain the liquid in the reactor for 30 seconds. Repeat this operation several times. 4) De-Fmoc: Add sufficient piperidine / DMF solution to the reactor to completely immerse the resin in the solution, and bubble the reaction with nitrogen for 20 minutes; 5) Washing: Refer to 3), and replace the industrial-grade DMF with analytical-grade DMF during the 5th wash; 6) Secondary amino acid condensation: Weigh appropriate amounts of Fmoc-Leu-OH (C-terminal second position) and 1-hydroxybenzotriazole (HOBT) into a centrifuge tube, dissolve them thoroughly with DMF, and then add N, N ， - Diisopropylcarbodiimide (DIC), mixed for 1 min, added to the dried resin, and reacted under nitrogen bubbling for 1 h; wherein, the molar ratio of Fmoc-Leu-OH, HOBT and DIC is 1:1:5; and the ratio of Fmoc-Leu-OH to DMF is 1:10 in mmol:ml. The resin was washed several times with industrial-grade DMF. 7) Sequential amino acid condensation: Remove the Fmoc group as in 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, and Fmoc-Lys-OH in sequence according to the amino acid order. 8) Washing, refer to step 5). 9) Staple cyclization: Add an appropriate amount of Grubb catalyst and use DCM as solvent to carry out staple cyclization for 4 hours; 10) Resin drying: Wash the resin three times with methanol and then vacuum dry it into dry granules. 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 to remove resin to obtain filtrate, add ice-cold ether to the filtrate, wash and centrifuge to obtain crude peptide chain precipitate; 13) Peptide purification: The crude product obtained in step 12) was purified by liquid chromatography to obtain pure antimicrobial peptide.

4. The preparation method according to claim 3, characterized in that, The washing step is followed by resin testing: take a small amount of 2-Cl(Trt)-Cl resin, add 2-3 drops of ninhydrin solution, and heat at 100°C until the resin turns blue to verify successful removal of Fmoc.

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

6. The preparation method according to claim 3, 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 claim 3, wherein, Step 11) The cutting fluid is composed of trifluoroacetic acid (TFA), triisopropylsilane, 1,2-ethylenedithiol and ultrapure water in a volume ratio of 95:2:2:

1.

8. The preparation method according to claim 3, characterized in that, Step 11) The cutting fluid: resin = 10 mL: 1 g.

9. The preparation method according to claim 3, characterized in that, Step 12) The amount of ice ether added is 10 times the volume of the cutting fluid.

10. The preparation method according to claim 3, wherein, In the liquid chromatography method of step 13), the chromatographic column is a Daisogel chromatographic packing; the mobile phase A is a TFA aqueous solution with a volume fraction of 0.1%; the mobile phase B is a mixture of TFA and acetonitrile, wherein the volume fraction of TFA is 0.1%.

11. A pharmaceutical composition for treating Acinetobacter baumannii infection, comprising the antimicrobial peptide as described in any one of claims 1-2.

12. Use of the antimicrobial peptide as described in any one of claims 1-2 in the preparation of a medicament for treating multidrug-resistant Acinetobacter baumannii infection.

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