Antibiotic peptides against acinetobacter baumannii and uses thereof

Abstract

The application discloses three kinds of anti-A.baumannii peptides and application thereof, and the amino acid sequences of the three kinds of anti-A.baumannii peptides are R-R-W-W-R-I-W-NH2, R-W-W-R-W-I-NH2 and R-R-W-I-R-W-I-T-NH2 respectively. The three kinds of anti-A.baumannii peptides have strong bactericidal performance on A.baumannii, guarantee small cytotoxicity and hemolysis, and have low actual cost, and can be used for preparing medicines for treating and / or preventing bacterial infections such as A.baumannii.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to three kinds of anti-Acinetobacter baumannii peptides and application thereof. BACKGROUND

[0002] Acinetobacter baumannii is the leading cause of nosocomial infections, known as the "nightmare of intensive care units". Due to its characteristics of easy drug resistance, resistance to chemical disinfectants, resistance to heat and moisture, resistance to low temperature, resistance to dryness, strong resistance to ultraviolet rays, and long survival time on dry surfaces, it is listed as the first "CRITICAL" bacteria by the World Health Organization (WHO). Polymyxin is a classic drug for clinical treatment of Acinetobacter baumannii, but more and more studies have found that Acinetobacter baumannii has gradually developed resistance to polymyxin, so it is urgent to develop new antibacterial drugs to cope with Acinetobacter baumannii infection.

[0003] Antibacterial peptides are a kind of polypeptide substances with antibacterial activity, which have strong killing effect on bacteria. Antibacterial peptides carrying positive charges can be adsorbed on the surface of bacteria with negative charges and self-organize on the surface, thereby cracking the bacterial cell membrane. A large number of studies have proved that this bactericidal mechanism is not easy to produce drug resistance. Therefore, antibacterial peptides are also considered as the ideal alternative drugs of the next generation of antibiotics to cope with bacterial drug resistance. The inventors have previously found a variety of antibacterial peptides, such as antibacterial nonapeptide, antibacterial octapeptide, antibacterial heptapeptide, and antibacterial hexapeptide, which can be specifically referred to CN202210794110.4, CN202210794146.2, CN202111458874.8, CN202110696847.8, and other patent technologies.

[0004] However, it is difficult to kill Acinetobacter baumannii, and it is also very difficult to develop antibacterial peptides against this kind of bacteria. SUMMARY

[0005] In a first aspect, the present application provides three kinds of anti-Acinetobacter baumannii peptides, which have strong bactericidal performance against Acinetobacter baumannii, ensure small cytotoxicity and hemolyticity, and have low actual cost.

[0006] The anti-Acinetobacter baumannii peptide has an amino acid sequence as follows:

[0007] Antibacterial peptide 1: R-R-W-W-R-I-W-NH2 (SEQ ID NO: 1),

[0008] Antibacterial peptide 2: R-W-W-R-W-I-NH2 (SEQ ID NO: 2),

[0009] Antibacterial peptide 3: R-R-W-I-R-W-I-T-NH2 (SEQ ID NO: 3).

[0010] The three kinds of anti-A.baumannii peptides have strong anti-bacterial activity on A.baumannii and other bacteria, and low cytotoxicity and hemolytic activity at high concentrations, and can be applied to drugs for treating diseases caused by A.baumannii and other bacteria.

[0011] In a second aspect, the present application provides the use of the anti-A.baumannii peptide in the first aspect in the preparation of a drug for treating and / or preventing bacterial infection.

[0012] Further, in the use, the bacteria are gram-negative bacteria.

[0013] Further, in the use, the bacteria are A.baumannii.

[0014] In a third aspect, the present application provides a drug for treating and / or preventing bacterial infection, wherein the drug contains at least one of the anti-A.baumannii peptides in the first aspect.

[0015] Further, in the drug for treating and / or preventing bacterial infection, the bacteria are gram-negative bacteria.

[0016] Further, in the drug for treating and / or preventing bacterial infection, the bacteria are A.baumannii.

[0017] The drug for treating and / or preventing bacterial infection can further comprise a pharmaceutically acceptable carrier.

[0018] The three kinds of anti-A.baumannii peptides provided by the present application have universal anti-bacterial activity on A.baumannii and other bacteria, and the main mechanism of action is to destroy the cell membrane surface of A.baumannii and other bacteria. The net charge of the three kinds of anti-A.baumannii peptides is positive, and they can be adsorbed to the cell membrane of A.baumannii and other bacteria with negative surface. When the anti-A.baumannii peptides adsorbed on the cell membrane surface reach a certain concentration, the amphiphilic peptides will insert into the phospholipid layer of the cell membrane and self-assemble to form pores, thereby destroying the cell membrane of A.baumannii and other bacteria and killing them.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] 1) The present application accidentally found that the three kinds of anti-A.baumannii peptides with the above-mentioned specific sequences have strong anti-bacterial activity on A.baumannii and other bacteria, and low cytotoxicity and hemolytic activity at high concentrations, and can be applied to drugs for treating or preventing diseases caused by A.baumannii and other bacteria.

[0021] 2) The three anti-A.baumannii peptides of the present application can be artificially synthesized (conventional techniques can be used, such as solid-phase synthesis, etc.), which is easy to operate, has a short peptide chain, and has very low preparation cost, raw material cost and application cost, and has very good application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 1 is a high performance liquid chromatography (HPLC) (a) and mass spectrum (b) result diagram of the anti-bacterial peptide 1.

[0023] Figure 2 Figure 2 is a HPLC (a) and mass spectrum (b) result diagram of the anti-bacterial peptide 2.

[0024] Figure 3 Figure 3 is a HPLC (a) and mass spectrum (b) result diagram of the anti-bacterial peptide 3. DETAILED DESCRIPTION

[0025] The present application will be further described below in conjunction with the drawings and specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application.

[0026] The operation methods not specified in the following examples are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

[0027] Example 1

[0028] Solid-phase synthesis of the anti-bacterial peptide 1-anti-bacterial peptide 3:

[0029] I. Swelling of the resin

[0030] 0.6 g of 2-Chlorotrityl Chloride Resin with a substitution degree of 0.4 mmol / g was weighed, the resin was placed in a reaction tube, dichloromethane (DCM) (15 mL / g) was added, and oscillation was carried out for 30 minutes.

[0031] II. Coupling of the first amino acid

[0032] The solvent was removed by suction filtration through a sand core, 3-fold molar excess of Fmoc-L-Leu-OH amino acid was added, 10-fold molar excess of diisopropylethylamine (DIEA) was added, a small amount of dimethylformamide (DMF) was added for dissolution, and oscillation was carried out for 1 hour. Washing was carried out 6 times alternately with DMF and DCM.

[0033] III. Deprotection

[0034] 15 mL of 20% piperidine DMF solution (15 mL / g) was added for 5 minutes, and 15 mL of 20% piperidine DMF solution (15 mL / g) was added again for 15 minutes.

[0035] IV. Detection

[0036] Pipette off the piperidine solution, wash the resin with ethanol (10 mL / g) three times, add one drop of ninhydrin, KCN, and phenol solution, heat at 105-110°C for 5 minutes, and a dark blue color indicates a positive reaction.

[0037] V. Wash

[0038] DMF (10 mL / g) twice, methanol (10 mL / g) twice, DMF (10 mL / g) twice.

[0039] VI. Coupling

[0040] Protecting amino acid (Fmoc-L-Gly-OH) in three-fold excess, O-benzotriazole- tetramethyluronium hexafluorophosphate (HBTU) in three-fold excess, both dissolved in as little DMF as possible, added to the reaction tube, and N-methylmorpholine (NMM) added immediately in ten-fold excess. Reaction time is 30 minutes.

[0041] VII. Wash

[0042] DMF (10 mL / g) once, methanol (10 mL / g) twice, DMF (10 mL / g) twice.

[0043] VIII. Repeat steps II through VI to sequentially couple the amino acids in the sequence from right to left.

[0044] IX. After the last amino acid is coupled, deprotect the resin by washing according to the following procedure.

[0045] DMF (10 mL / g) twice, methanol (10 mL / g) twice, DMF (10 mL / g) twice, DCM (10 mL / g) twice, and aspirate for 10 minutes.

[0046] X. Cleavage of the polypeptide from the resin

[0047] Prepare the cleavage solution (10 / g) TFA 94.5%; water 2.5%; EDT 2.5%; TIS 1%.

[0048] Load the resin into a flask or centrifuge tube, and the ratio of resin to cleavage solution is 10 mL / g. Incubate with constant shaking for 120 minutes.

[0049] XI. Blow dry and wash

[0050] Blow the cleavage solution dry with nitrogen, chromatograph the residue with ether, wash the residue with ether six times, and then evaporate at room temperature. The crude peptide is obtained.

[0051] XII. Purification of the polypeptide by HPLC

[0052] Specific procedure steps:

[0053] 1. Take the crude peptide 200 mg and place in a vessel. Dissolve in 2-5 mL of 50% aqueous acetonitrile. Sonicate for 2 minutes if necessary.

[0054] 2. Filter the dissolved solution through a 0.45 μm filter.

[0055] 3. Analysis: Take 3 μL of the crude peptide and analyze by analytical HPLC. The mobile phase is water and acetonitrile, time 30 minutes, gradient elution, first equilibrate the HPLC with the starting gradient for 5 minutes then inject the sample, starting gradient water 95%, acetonitrile 5%, ending gradient water 5%, acetonitrile 95%.

[0056] 4. Preparation: Prepare the sample for injection. Equilibrate the preparative HPLC for 10 minutes, starting gradient water 95%, acetonitrile 5%, ending gradient water 25%, acetonitrile 75%, gradient time 40 minutes. Collect the sample from the detector.

[0057] 5. Identification: Take samples from the collected sample for purity and MS identification.

[0058] Thirteen, finally lyophilize the purified solution to obtain the finished product.

[0059] Fourteen, seal the white powder polypeptide, and store at -20 °C.

[0060] Figures 1-3 The HPLC (a) and mass spectrum (b) results of the antibacterial peptides 1-3 are shown in the following figures, respectively.

[0061] Example 2

[0062] Detection of antibacterial activity of antibacterial peptides.

[0063] The following standard strains used are purchased from the Guangdong Microbial Culture Collection Center.

[0064] The antibacterial activity of the antibacterial peptides synthesized in Example 1 was detected by the 96-well plate method, and the FDA-approved antibacterial peptide polymyxin was used as a control for gram-negative bacteria to evaluate the antibacterial activity of antibacterial peptides 1-3.

[0065] The antibacterial activity of the antibacterial peptides was tested according to the following steps.

[0066] I. A. baumannii was cultured overnight on sterilized MH medium plates, and a single colony was inoculated into sterilized MH medium and incubated at 37 °C, 150 rpm, for 18 hours overnight.

[0067] II. The antibacterial peptides 1-3 were respectively diluted with PBS to 1024 μg / mL, and then diluted with PBS to 1024, 512, 256, 128, 64, 32, 16, 8 μg / mL by 2 times of continuous dilution, and 100 μL of each solution was added to the 2-9 column holes of a 96-hole plate, and repeated 6 groups to add B-G rows. The cultured bacteria were diluted with MH to 5x10^5 CFU / mL, and 100 μL of the diluted bacterial solution was added to the B2-D9 holes as the experimental group. At this time, the concentration (μg / mL) of the peptide to be tested was as follows:

[0068] Column number 2 3 4 5 6 7 8 9 Concentration 512 256 128 64 32 16 8 4

[0069] 100 μL of MH solution was added to the E2-G9 holes as the control group, and 100 μL of PBS and MH was respectively added to the B10-D10 holes as the negative control, and 100 μL of PBS and the diluted bacterial solution was respectively added to the E10-G10 holes as the positive control. The 96-hole plate was sealed with sealing film and placed in a self-sealing bag at 37°C, 150 rpm, and incubated overnight. The OD600 value of B2-D9 holes was measured by an enzyme marker, and the minimum OD600 value corresponded to the minimum concentration, which was the MIC value of the antibacterial peptide to the bacteria.

[0070] Table 1 shows the minimum inhibitory concentration (MIC, μg / mL, 0-32: +, 32-512: ++) of antibacterial peptides 1-3 and polymyxin to Acinetobacter baumannii.

[0071] Table 1

[0072] Antibacterial peptide 1 Antibacterial peptide 2 Antibacterial peptide 3 Polymyxin A. baumannii + + + +

[0073] The smaller the MIC in Table 1, the stronger the antibacterial ability. Table 1 shows that the three antibacterial peptides of the present application exhibit good antibacterial ability to Acinetobacter baumannii.

[0074] Example 3

[0075] Cytotoxicity detection of antibacterial peptides 1-3.

[0076] This example is used to detect the cytotoxicity of the three antibacterial peptides against Acinetobacter baumannii synthesized in Example 1 to human 3T3 cells, and a short bacillus peptide approved by FDA is used as a comparison.

[0077] Cytotoxicity detection steps:

[0078] I. NIH-3T3 cells were grafted into a 96-hole plate at a density of 5000 / hole and cultured for 24 hours to fully adhere.

[0079] II. Prepare 100 μg / mL of antibacterial peptide 1-3 in PBS solution, and dilute with DMEM medium to 50 μg / mL. After removing the culture medium in the 96-well plate, add 200 μL of 50 μg / mL drug-containing medium, and continue to culture for 24 hours, with 5 parallel samples in each group.

[0080] III. After removing the drug medium, wash 3 times with PBS, and add 20 μL of CCK-8 and 100 μL of DMEM medium in each well, and incubate for 2 hours. Remove the supernatant, and use an enzyme label instrument to read the OD 490 nm absorption value.

[0081] The cytotoxicity calculation formula is:

[0082] Cell survival rate = sample pool absorption value / blank pool absorption value x 100%.

[0083] Table 2 shows the antibacterial peptide 1-3 cytotoxicity test results (drug concentration 50 μg / mL), with short bacitracin and PBS as controls, where the cell survival rate is 80-100%: +, <80%: ++.

[0084] Table 2

[0085] Antibacterial peptide 1 Antibacterial peptide 2 Antibacterial peptide 3 Gramicidin Polymyxin PBS Cell survival rate + + + ++ + +

[0086] In Table 2, the greater the cell survival rate represents the smaller the cytotoxicity of the antibacterial peptide. Table 2 shows that antibacterial peptides 1-3 have significantly smaller cytotoxicity than short bacitracin, providing strong support for the development of drugs.

[0087] Example 4

[0088] In vitro hemolytic test of antibacterial peptide 1-3

[0089] This example is used to detect the hemolysis of the three antibacterial peptides synthesized in Example 1 on red blood cells, and uses FDA-approved short bacitracin as a comparison. The blood sample used is taken from a sterile mouse.

[0090] The steps of the hemolysis test are:

[0091] I. Take 1 mL of whole blood, centrifuge at 1500 rpm for 5 minutes, remove the supernatant and repeat 3 times, and add 20 mL of PBS to prepare a blood cell solution.

[0092] II. Antibacterial peptide 1-3 was prepared at 100 μg / mL, and 100 μL was added to a 96-well plate as an experimental group. Triton-X100 (1 wt%) 100 μL and PBS 100 μL were added to a 96-well plate as a positive control and a negative control, respectively, and each sample was repeated 5 groups. 100 μL of blood cell solution was added to the well plate containing the sample (at this time, the concentration of all samples was halved, and the concentration of all experimental groups was 50 μg / mL), the well plate was sealed in a self-sealing bag, and incubated at 37°C and 150 rpm for 1 hour.

[0093] III. After incubation, the mixed solution was aspirated into a 1.5 mL centrifuge tube, and after centrifugation at 1500 rpm for 8 minutes, the supernatant was aspirated and tested for OD 570 nm value by an enzyme marker.

[0094] Hemolysis rate = (A m -A n ) / (A p -A n )

[0095] wherein A m is the OD value of the experimental group, A p is the OD value of the positive control, and A n is the OD value of the negative control.

[0096] Table 3 shows the hemolytic detection results of antibacterial peptide 1-3 (drug concentration 100 μg / mL), with short bacitracin and Triton-X100 as controls, wherein the hemolysis rate 0-30%: +, >30%: ++.

[0097] Table 3

[0098] Antibacterial peptide 1 Antibacterial peptide 2 Antibacterial peptide 3 Gramicidin Triton-X100 PBS Hemolysis rate + + + ++ ++ +

[0099] The greater the hemolysis rate represents the greater the toxicity of the antibacterial peptide to blood cells. Table 3 shows that antibacterial peptide 1-3 exhibits significantly less hemolysis than short bacitracin, providing strong support for the development of a drug.

[0100] In addition, it should be understood that, after reading the above description of the present application, those skilled in the art can make various modifications or modifications to the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. An anti-Acinetobacter baumannii peptide, characterized in that, The amino acid sequence of the anti-Acinetobacter baumannii peptide is one of the following: Antimicrobial peptide 1: RRWWRIW-NH2, Antimicrobial peptide 2: RWWRWI-NH2, Antimicrobial peptide 3: RRWIRWIT-NH2.

2. The use of the anti-Acinetobacter baumannii peptide according to claim 1 in the preparation of medicaments for treating and / or preventing bacterial infections, characterized in that, The bacteria in question is Acinetobacter baumannii.

3. A medicine suitable for treating and / or preventing bacterial infections, characterized in that, The drug contains at least one of the anti-Acinetobacter baumannii peptides according to claim 1, wherein the bacterium is Acinetobacter baumannii.

4. The drug according to claim 3, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

Images