Broad-spectrum antibacterial combination peptide with low hemolysis and application thereof

Abstract

The application relates to the technical field of biological materials, and discloses a broad-spectrum antibacterial combined peptide with low hemolysis and an application thereof. The amino acid sequence is one of the following: Meta211: X2IRRLWRX3X2RLIR, Meta201: IWRX3X1RX3LRLX1RR, and Meta185: X2LRRX1LTRLIRX2I; wherein X1, X2 and X3 are random sites, X1 is L or I, X2 is V or L, and X3 is I or V; Meta211, Meta201 and Meta185 are all mixtures selected from random sites. The antibacterial combined peptide has strong antibacterial effects on various bacteria, especially has significant bacteriostatic effects on bacillus subtilis, exhibits excellent low hemolysis, provides a sustainable, economic and efficient solution for the upgrading of antibacterial drugs, and has important social significance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to a broad-spectrum antibacterial combined peptide with low hemolysis and its application. BACKGROUND

[0002] Antibiotics are one of the greatest drug inventions, which has saved the lives of countless patients. However, as antibiotics are used more and more, the number of drug-resistant strains also increases, and the choice of clinical treatment is difficult, so the development of new antibacterial drugs is imminent. Antimicrobial peptides are a kind of polypeptide substances widely existing in organisms in nature, and their sources are very extensive. At present, more than 1700 kinds of antimicrobial peptides have been found in plants, insects, mammals, bacteria, viruses and other organisms. Antimicrobial peptides are important innate immune molecules, which have membrane-destroying bactericidal mechanism and are not easy to produce drug resistance.

[0003] CN 117004590 A discloses an antibacterial polypeptide and its application. The amino acid sequence is LKKVITKIIKGVNNWLKQLGKV or LKKIVTKIGKIVNNWLKQLGKV. The antibacterial polypeptide has strong antibacterial activity against common clinical pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii. It also has strong antibacterial activity against various multi-drug resistant bacteria, such as methicillin-resistant Staphylococcus aureus, carbapenem-resistant Escherichia coli, and carbapenem-resistant Klebsiella pneumoniae. Especially, carbapenem-resistant Acinetobacter baumannii has a MIC as low as 4 μM for Cov6 polypeptide. It also has the advantages of low hemolysis and low cytotoxicity, and has the conditions to be developed as a drug and a food additive, and has a wide application prospect.

[0004] CN 119285707 A discloses an antibacterial polypeptide, a composition and its application. The amino acid sequence of the antibacterial polypeptide is KWKIKWPVKWFKMNH2. Experiments show that the antibacterial polypeptide has broad-spectrum antibacterial activity against Gram-positive bacteria and / or Gram-negative bacteria, and also has low cytotoxicity and low hemolysis. The antibacterial polypeptide consists of 14 amino acids, the peptide chain is short, the molecular weight is small, and it is easy to be artificially synthesized. It is a small molecule polypeptide with high application value, which can be used for preparing a new antibacterial agent and has a broad application prospect.

[0005] Although the existing antibacterial peptides have biological activity, some still have side effects such as hemolysis, cytotoxicity, poor in-vivo stability and other defects, and it is difficult to achieve clinical application. Different antibacterial peptides usually show single bacteriostatic effect on bacteria or specific several bacteria, so it is still necessary to develop antibacterial peptides targeting different bacteria, especially polypeptides with bacteriostatic effect on multiple bacteria, which have important significance in the fields of medicine, food and biology. SUMMARY

[0006] This invention provides a class of broad-spectrum antibacterial combination peptides with low hemolytic activity. These combination peptides have a strong antibacterial effect against a variety of bacteria, especially Bacillus subtilis, and exhibit excellent low hemolytic activity.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A class of broad-spectrum antimicrobial combination peptides with low hemolytic activity, wherein the amino acid sequence of the broad-spectrum antimicrobial combination peptide is one of the following:

[0009] Meta211: X2IRRLWRX3X2RLIR

[0010] Meta201: IWRX3X1RX3LRLX1RR

[0011] Meta185: X2LRRX1LTRLIRX2I

[0012] Among them, X1, X2 and X3 is a random site, X1 is L or I, X2 is V or L, and X3 is I or V; Meta211, Meta201, and Meta185 are mixtures of all selected random sites.

[0013] The present invention also provides the use of the aforementioned broad-spectrum antimicrobial combination peptide in the preparation of medical products for the treatment and / or prevention of bacterial infections.

[0014] The medical products include coatings for medical devices, coatings for implantable medical devices, dressings, and one or more of the following. The antimicrobial combination peptides of this invention possess excellent broad-spectrum antimicrobial properties and can be used to prepare anti-infective drugs, such as injections and topical sprays, for burn / wound infections caused by drug-resistant bacteria; they can also be used to prepare medical device coatings, such as catheter and implant surface modification, to inhibit biofilm formation; or to prepare eye drops and wound dressings to achieve antimicrobial effects.

[0015] The present invention also provides the application of the aforementioned broad-spectrum antibacterial combination peptide in the preparation of daily-use products for preventing bacterial infections; the daily-use products include one or more of the following: chemical preservatives, food preservatives, and pet disinfectants.

[0016] The bacteria are Gram-positive and / or Gram-negative.

[0017] The bacteria include one or more of Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Enterococcus faecalis, and Bacillus subtilis. The broad-spectrum antibacterial peptide of the present application has significant antibacterial efficacy against Bacillus subtilis, and the minimum inhibitory concentration is as low as 2 μg / mL. The broad-spectrum antibacterial peptide can be used to prepare food preservatives, and can inhibit spoilage bacteria such as Bacillus subtilis in cold-chain meat products (addition amount ≤ 0.1 mg / kg).

[0018] The present application also provides a nucleotide sequence encoding the broad-spectrum antibacterial combination peptide, which includes DNA and RNA.

[0019] The present application also provides a composition containing the broad-spectrum antibacterial combination peptide.

[0020] The composition contains any one or more of a pharmaceutically or food-acceptable carrier, excipient, diluent, adjuvant, or vehicle.

[0021] The present application also provides an antibacterial product containing the broad-spectrum antibacterial combination peptide, the nucleotide sequence, or the composition.

[0022] Preferably, the antibacterial product includes a drug for preventing and / or treating diseases caused by bacterial infection, an implantable medical device, etc.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] The antibacterial combination peptide provided by the present application has broad-spectrum antibacterial properties, excellent antibacterial effect, low biological toxicity and hemolysis, and particularly has significant bacteriostatic effect against low hemolysis and Bacillus subtilis, and can be used to prepare antibacterial products or drugs, such as anti-infection injection or dressing, daily chemical preservative, food preservative, etc. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The ability of Meta211, Meta201, and Meta185 at different concentrations to penetrate the outer membrane of Acinetobacter baumannii pathogen.

[0026] Figure 2 The ability of Meta211, Meta201, and Meta185 at different concentrations to depolarize the plasma membrane of Acinetobacter baumannii pathogen. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below with examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application. Those skilled in the art can make modifications or equivalent replacements based on the technical scheme of the present application without departing from the spirit and scope of the present application, which should be covered within the protection scope of the present application.

[0028] The raw materials used in the following specific embodiments are all purchased from the market.

[0029] Example 1

[0030] The synthesis of the antibacterial combination peptide specifically includes:

[0031] 1. Swelling of the resin: 2-chlorotrityl chloride resin (2-CTC) resin is weighed and soaked in dichloromethane (DCM) for 1 hour to swell the resin. Then the resin is washed with dimethyl sulfoxide (DMF) for 3 times to remove residual solvents and impurities.

[0032] 2. Coupling of the first amino acid: 1 or 2 protected amino acids (e.g., 1 eq of amino acid A and 1 eq of amino acid B, the molar ratio of the two amino acids is 1:1) are reacted with 1.5 eq of diisopropylethylamine (DIEA) in DMF for 2 hours to link these amino acids to the resin.

[0033] 3. Capping: After the resin is drained, it is washed with DMF for 3 times, then methanol and DIEA are added for capping treatment, and the reaction is carried out for 1 hour to protect the amino group on the resin.

[0034] 4. Removal of Fmoc protecting group: 20% piperidine in DMF is used to remove the Fmoc protecting group, and the reaction is carried out for 10 minutes, repeated 2 times to ensure complete removal of the protecting group.

[0035] 5. Coupling of new amino acid: After washing the resin, 3 eq of 1 or 2 amino acids (e.g., amino acid C and amino acid D, the molar ratio of the two amino acids is 1:1) and 3 eq of HOBT and 3 eq of DIC are added, and the reaction is carried out in DMF for 1.5 hours to link these amino acids to the peptide chain.

[0036] 6. Repeat steps 4 and 5 until all the desired amino acids are coupled and the Fmoc protecting group at the N-terminus is removed. The resin is washed and drained to prepare for the subsequent deprotection step.

[0037] 7. Deprotection and peptide chain cleavage: 95% trifluoroacetic acid (TFA), 2% Tis (thiazolone), 2% EDT (ethylenediaminetetraacetic acid) and 1% water are used for the reaction, and the reaction is carried out for 2 hours to cleave the protecting groups of the resin and the side chains of the polypeptide.

[0038] 8. Filtration and precipitation: The resin was filtered, and the filtrate was washed with ice-ethanol, and the precipitate was reserved after centrifugation, which was the crude product. The final random peptide mixture was obtained by freeze-drying treatment.

[0039] Example 2 Analysis of the antibacterial activity of three AMPCs (antibacterial combination peptides)

[0040] Step 1: Seven bacterial solutions (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 15442, Acinetobacter baumannii ATCC 19606, Staphylococcus aureus ATCC 25923, Salmonella typhimurium ATCC 14028, Enterococcus faecalis ATCC 29212, and Bacillus subtilis ATCC 6633) were inoculated into sterilized MHB medium and cultured at 37°C, 250 rpm for 18 hours overnight.

[0041] Step 2: Three antibacterial combination peptides were respectively prepared into 128 µg / mL with PBS, and were diluted into 128, 64, 32, 16, 8, 4, 2, and 1 µg / mL with PBS by 2 times of continuous dilution. 50 µL of each concentration was added to the A-H wells of a 96-well plate. Each antibacterial combination peptide was repeated for 3 groups, and was added to the 1-9 column wells.

[0042] Step 3: The seven bacteria after culture were respectively diluted into 5×105 CFU / mL with PBS. (Because there are many types of bacteria, one bacterial solution was used for each plate, and Staphylococcus aureus was taken as an example) 50 µL of the bacterial dilution was added to the A-F wells with the antibacterial combination peptide dilution. At this time, the peptide concentration (µg / mL) to be tested is shown in Table 1.

[0043] Table 1: The final concentration of antibacterial combination peptides Meta211, Meta201, and Meta185 in the MIC determination experiment, unit: µg / mL

[0044]

[0045] Step 4, 100 μL PBS solution was added into A10-H10 well as negative control, 100 μL bacteria solution of 5 x 105 CFU / mL was added into A11-H11 well as positive control. The 96-well plate was sealed with sealing film and then put into a self-sealing bag and placed in a 37°C constant temperature incubator for overnight culture. The OD600 value of A1-G9 well was measured by microplate reader, the minimum concentration corresponding to the minimum OD600 value in each antibacterial peptide group was the minimum inhibitory concentration (MIC) value of the antibacterial peptide corresponding to the bacteria, and the results are shown in Table 2.

[0046] Table 2 MIC of antibacterial peptide Meta211, Meta201 and Meta185 against 7 pathogens, unit: μg / mL

[0047]

[0048] The antibacterial peptide Meta211, Meta201 and Meta185 are compositions of a plurality of sequences of antibacterial peptides, which are directly obtained by synthesis, rather than being synthesized and then mixed. In the amino acid sequence of the polypeptide in the table, the amino acids in the brackets are a random one, such as (LV) indicating L or V. For example, the specific sequence of the antibacterial peptide can be as follows: Meta211 is a composition of antibacterial peptides with amino acid sequences as shown in SEQ ID NO. 1-8, specifically:

[0049] SEQ ID NO. 1: LIRRLWRILRLIR

[0050] SEQ ID NO. 2: LIRRLWRIVRLIR

[0051] SEQ ID NO. 3: LIRRLWRVLRLIR

[0052] SEQ ID NO. 4: LIRRLWRVVRLIR

[0053] SEQ ID NO. 5: VIRRLWRILRLIR

[0054] SEQ ID NO. 6: VIRRLWRIVRLIR

[0055] SEQ ID NO. 7: VIRRLWRVLRLIR

[0056] SEQ ID NO. 8: VIRRLWRVVRLIR

[0057] Meta201 is a composition of antibacterial peptides with amino acid sequences as shown in SEQ ID NO. 9-24, specifically:

[0058] SEQ ID NO. 9: IWRVLRILRLLRR

[0059] SEQ ID NO. 10: IWRVLRILRLIRR

[0060] SEQ ID NO. 11: IWRVLRVLRLLRR

[0061] SEQ ID NO. 12: IWRVLRVLRLIRR

[0062] SEQ ID NO. 13: IWRVIRILRLLRR

[0063] SEQ ID NO. 14: IWRVIRILRLIRR

[0064] SEQ ID NO. 15: IWRVIRVLRLLRR

[0065] SEQ ID NO. 16: IWRVIRVLRLIRR

[0066] SEQ ID NO. 17: IWRILRILRLLRR

[0067] SEQ ID NO. 18: IWRILRILRLIRR

[0068] SEQ ID NO. 19: IWRILRVLRLLRR

[0069] SEQ ID NO. 20: IWRILRVLRLIRR

[0070] SEQ ID NO. 21: IWRIIRILRLLRR

[0071] SEQ ID NO. 22: IWRIIRILRLIRR

[0072] SEQ ID NO. 23: IWRIIRVLRLLRR

[0073] SEQ ID NO. 24: IWRIIRVLRLIRR

[0074] Meta185 is a composition of an antibacterial peptide having an amino acid sequence as shown in SEQ ID NO. 25~32, specifically:

[0075] SEQ ID NO. 25: LLRRLLTRLIRLI

[0076] SEQ ID NO. 26: LLRRLLTRLIRVI

[0077] SEQ ID NO. 27: LLRRILTRLIRLI

[0078] SEQ ID NO. 28: LLRRILTRLIRVI

[0079] SEQ ID NO. 29: VLRRLLTRLIRLI

[0080] SEQ ID NO. 30: VLRRLLTRLIRVI

[0081] SEQ ID NO. 31: VLRRILTRLIRLI

[0082] SEQ ID NO. 32: VLRRILTRLIRVI

[0083] Table 2 shows that the three antibacterial combination peptides of the present application all exhibit good bacteriostatic ability against seven different bacteria, have strong efficacy, and have wide universality. The three polypeptides are particularly effective against Bacillus subtilis (Bacillus subtilis ATCC6633), and all exhibit strong bacteriostatic effects, with a MIC of only 2 μg / mL.

[0084] Example 3 Analysis of hemolytic ability of AMPCs

[0085] I. The person being swabbed performs venous blood collection.

[0086] II. Precool the centrifuge (2000 rpm), and divide the collected blood into 1.5 mL ep tubes, centrifuge at 2000 rpm and 4°C for 5 min, discard the supernatant, and collect the blood cells. Wash the collected blood cells three times with PBS (washed at a ratio of PBS: blood plasma = 0.35:0.65), then dilute to 2% of the original concentration with PBS, and mix slowly on a decolorizing bed after dilution.

[0087] III. Place 50 μL of the polypeptides prepared in Example 1 (the final concentration of the polypeptides is 128-1 μg / mL, 2-fold gradient dilution) and the same volume of red blood cell suspension in each well of a 96-well plate, and add 50 μL of PBS + 50 μL of red blood cell suspension as a negative control; 50 μL of red blood cell suspension + 50 μL of 0.2% Triton x-100 as a positive control, and place the 96-well plate in a 37°C constant temperature incubator for incubation for 1 hour.

[0088] IV. After 1 hour of incubation, remove the 96-well plate, centrifuge at 1000 rpm and 4°C for 5 min.

[0089] V. Absorb the supernatant of the solution after centrifugation, transfer to a clean 96-well plate, and use an enzyme marker to measure the OD at 570 nm (OD570) and 655 nm (OD655). 570The hemolysis rate is calculated by measuring the absorbance value at a certain point.

[0090] Hemolysis rate (%) = [(sample OD)] 570 - Negative control OD 570 ) / (Positive control OD 570 - Negative control OD 570 )]×100%.

[0091] The minimum hemolytic concentration (MHC) is the concentration at which the antimicrobial combination peptide causes a 10% hemolysis rate. This experiment was independently repeated three times, and the average value of the three tests was used to obtain Table 3.

[0092] Table 3. HC50 of AMPCs for human erythrocytes (hRBCs), in mg / L

[0093]

[0094] As can be seen from Table 3, the three antibacterial peptides synthesized in this invention have good biocompatibility.

[0095] Example 4: Investigation of the antibacterial mechanism of AMPCs

[0096] I. Effect of NPN (1-naphthylaminobenzene) uptake assay on the effect of antimicrobial combination peptides on the extracellular membrane permeability of Acinetobacter baumannii.

[0097] Logarithmically growing microbial cells were harvested (5,000 rpm, 5 min) and diluted to OD 600 = 0.2 in 5 mM HEPES buffer (pH = 7.4, containing 5 mM glucose). The bacterial suspension was further incubated with 10 μM NPN in the dark for 30 min. Subsequently, different concentrations of peptides prepared in Example 1 were added (final peptide concentrations were 128–1 µg / mL, serially diluted 2-fold), and NPN fluorescence was detected (excitation λ = 350 nm, emission λ = 420 nm). The results are as follows: Figure 1 As shown, the fluorescence signal detected after treatment with AMPCs increased significantly, indicating that the permeability of the bacterial outer membrane was significantly enhanced, and that AMPCs exerted their bactericidal effect through membrane interaction mechanisms.

[0098] II. Effect of antimicrobial combination peptides on the depolarization of the inner membrane of Acinetobacter baumannii by staining with the cell fluorescent probe DiSC3-5.

[0099] Harvest bacteria in mid-log phase and wash three times with 5 mM HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) buffer, then resuspend in buffer containing 0.1% glucose to OD. 600The concentration was 0.07. DiSC3-5 was added to a final concentration of 0.4 μM, and the mixture was incubated at 37 °C in the dark for 90 minutes. Subsequently, 300 mM KCl was added and incubated for 20 minutes, followed by treatment with different concentrations of peptide. Fluorescence (excitation wavelength 622 nm, emission wavelength 670 nm) was monitored until the fluorescence signal stabilized, and membrane potential changes were assessed. The results were recorded, background fluorescence was subtracted, and the data were normalized. The results are as follows: Figure 2 As shown, the fluorescence signal detected after treatment with AMPCs increased significantly, indicating that the inner membrane was significantly depolarized and lost its potential. AMPCs exerted their bactericidal effect through membrane interaction mechanisms.

Claims

1. A class of low hemolytic broad-spectrum antibacterial combination peptides, characterized in that, The amino acid sequence of the broad-spectrum antibacterial combined peptide is one of the following: Meta211: X2IRRLWRX3X2RLIR Meta201: IWRX3X1RX3LRLX1RR Meta185: X2LRRX1LTRLIRX2I Wherein, X1, X2 and X3 are random sites, X1 is L or I, X2 is V or L, X3 is I or V; Meta211, Meta201 and Meta185 are mixtures of all selected random sites.

2. The broad-spectrum antibacterial combined peptide according to claim 1 is used for preparing medical products for treating and / or preventing bacterial infection. The bacteria include one or more of Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Enterococcus faecalis, Bacillus subtilis.

3. Use according to claim 2, characterized in that, The medical products include one or more of the coating of medical devices, the coating of implantable medical devices, and dressings.

4. Use of the broad-spectrum antibacterial combination peptide according to claim 1 for the manufacture of a daily product for the prevention of bacterial infections, characterized in that, The daily products include one or more of pet disinfectants.

5. A nucleotide encoding the broad-spectrum antibacterial combinatorial peptide of claim 1, characterized in that, The nucleotides include DNA and RNA.

6. A composition containing the broad-spectrum antibacterial combined peptide according to claim 1.

7. The composition of claim 6, wherein, The composition contains one or more of pharmaceutically acceptable carriers, excipients, diluents, adjuvants or vehicles.

8. An antibacterial product, characterized by The broad-spectrum antibacterial combined peptide according to claim 1, the nucleotide according to claim 5 or the composition according to claim 6.

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