Low-hemolytic broad-spectrum antibacterial combined peptide and application thereof

Abstract

The invention relates to the technical field of biological materials, and discloses a low-hemolytic broad-spectrum antibacterial combined peptide and application thereof. The amino acid sequence of the amino acid sequence is one of the following sequences: Meta211: X2IRRLWRX3X2RLIR, Meta201: IWRX3X1RX3LRLX1RR, Meta185: X2LRRX1LTRLIRX2I, MetaLRRX1LTRLIRX2I, MetaLRRX1LTRLIRX2I, MetaLRRX1LTRLRX2I, MetaLRRX1LTRLRX2I Wherein X1, X2 and X3 are random sites, X1 is L or I, X2 is V or L, and X3 is I or V; the Meta211, the Meta201 and the Meta185 are mixtures selected from all the random sites respectively. The antibacterial combined peptide can have a powerful antibacterial effect on various bacteria, particularly has a remarkable antibacterial effect on bacillus subtilis, shows excellent low hemolysis, provides a sustainable, economical and efficient solution for upgrading of antibacterial drugs, and has important social significance.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a class of low-hemolytic, broad-spectrum antibacterial combination peptides and their applications. Background Technology

[0002] Antibiotics are one of the greatest pharmaceutical inventions, saving countless lives. However, with their increasing use, the number of drug-resistant strains is also rising, making drug selection for clinical treatment difficult. Therefore, the development of new antimicrobial drugs is urgently needed. Antimicrobial peptides are polypeptides widely found in organisms in nature. Their sources are very diverse; more than 1700 antimicrobial peptides have been discovered in plants, insects, mammals, bacteria, viruses, and other organisms. Antimicrobial peptides are important innate immune molecules, possessing membrane-breaking bactericidal mechanisms and being less prone to inducing drug resistance.

[0003] CN 117004590 A discloses an antimicrobial polypeptide and its applications. Its amino acid sequence is LKKVITKIIKGVNNWLKQLGKV or LKKIVTKIGKIVNNWLKQLGKV. This antimicrobial polypeptide exhibits strong antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus, and common clinical pathogens such as Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. It also shows strong antimicrobial activity against various multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus, carbapenem-resistant Escherichia coli, and carbapenem-resistant Klebsiella pneumoniae, all of which are sensitive to it. In particular, the MIC of the carbapenem-resistant Acinetobacter baumannii against the Cov6 polypeptide is as low as 4 μM. Furthermore, it has the advantages of extremely low hemolysis and cytotoxicity, making it suitable for development as a drug and food additive, and possessing broad application prospects.

[0004] CN 119285707 A discloses an antimicrobial peptide, a composition thereof, and its application. The amino acid sequence of the antimicrobial peptide is KWKIKWPVKWFKMNH2. Experiments show that this antimicrobial peptide has broad-spectrum antimicrobial activity against Gram-positive and / or Gram-negative bacteria, while also exhibiting low cytotoxicity and low hemolytic activity. This antimicrobial peptide is composed of 14 amino acids, has a short peptide chain, small molecular weight, and is easy to synthesize artificially. It is a small molecule peptide with great application value and can be used to prepare novel antimicrobial agents, showing broad application prospects.

[0005] While existing antimicrobial peptides possess biological activity, some still suffer from side effects such as hemolysis and cytotoxicity, as well as poor in vivo stability, making clinical application difficult. Furthermore, different antimicrobial peptides typically exhibit antibacterial effects against single bacteria or specific bacteria. Therefore, research continues on antimicrobial peptides targeting different bacteria, particularly peptides that can inhibit multiple bacteria, which is of great significance to the medical, food, and biological fields. Summary of the Invention

[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: 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: Meta211: X2IRRLWRX3X2RLIR Meta201: IWRX3X1RX3LRLX1RR Meta185: X2LRRX1LTRLIRX2I 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.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] The bacteria are Gram-positive and / or Gram-negative.

[0012] 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 agent of this invention exhibits significant antibacterial efficacy against Bacillus subtilis, with a minimum inhibitory concentration as low as 2 μg / mL. It can be used to prepare food preservatives, inhibiting spoilage bacteria such as Bacillus subtilis in cold chain meat products (addition amount ≤ 0.1 mg / kg).

[0013] The present invention also provides a nucleotide sequence encoding the aforementioned broad-spectrum antimicrobial combination peptide, the nucleotide sequence comprising DNA and RNA.

[0014] The present invention also provides a class of compositions containing the aforementioned broad-spectrum antibacterial combination peptides.

[0015] The composition contains one or more of a pharmaceutically or food-acceptable carrier, excipient, diluent, adjuvant, or medium.

[0016] The present invention also provides an antibacterial product comprising the aforementioned broad-spectrum antibacterial combination peptide, the aforementioned nucleotide sequence, or the aforementioned composition.

[0017] Preferably, the antimicrobial product includes drugs for the prevention and / or treatment of diseases caused by bacterial infections, implantable medical devices, etc.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The antimicrobial combination peptides provided by this invention have broad-spectrum antimicrobial activity, excellent antimicrobial effect, low biotoxicity and hemolytic activity, and have significant antimicrobial effect against low hemolytic and Bacillus subtilis. They can be used to prepare antimicrobial products or drugs, such as anti-infective injections or dressings, daily chemical preservatives, food preservatives, etc. Attached Figure Description

[0019] Figure 1 The ability of Meta211, Meta201 and Meta185 at different concentrations to target the outer membrane permeability of Acinetobacter baumannii pathogen.

[0020] Figure 2 The ability of Meta211, Meta201 and Meta185 to depolarize the plasma membrane of Acinetobacter baumannii pathogen at different concentrations was investigated. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should be covered within the protection scope of this invention.

[0022] All raw materials used in the following specific implementation methods were purchased from the market.

[0023] Example 1 The synthesis of antimicrobial combination peptides specifically includes: 1. Swelling of dendrites: Weigh 2-chlorotriphenylmethyl chloride resin (2-CTC) and soak it in dichloromethane (DCM) to swell the resin for 1 hour. Then wash the resin three times with dimethyl thionamide (DMF) to remove residual solvent and impurities.

[0024] 2. Connecting the first amino acid: Take one or two protected amino acids (e.g., 1 eq of amino acid A and 1 eq of amino acid B in a 1:1 molar ratio) and react them with 1.5 eq of diisopropylethylamine (DIEA) in DMF for 2 hours to connect these amino acids to the resin.

[0025] 3. End-capping: After the resin is dried, it is washed three times with DMF, and then methanol and DIEA are added for end-capping treatment. The reaction is carried out for 1 hour to protect the amino groups on the resin.

[0026] 4. Removal of Fmoc protecting group: Use 20% piperidine in DMF to remove the Fmoc protecting group, react for 10 minutes, and repeat twice to ensure complete removal of the protecting group.

[0027] 5. Linking new amino acids: After washing the resin, add 3 eq of one or two amino acids (e.g., amino acid C and amino acid D in a 1:1 molar ratio) and 3 eq of HOBT and 3 eq of DIC, and react in DMF for 1.5 hours to link these amino acids to the peptide chain.

[0028] 6. Repeat steps 4 and 5 until all the required amino acids are added, and remove the N-terminal Fmoc protecting group. Wash the resin and dry it to prepare for the subsequent deprotection step.

[0029] 7. Deprotection and peptide chain cleavage: The reaction was carried out using 95% trifluoroacetic acid (TFA), 2% Tis (thiazolidinone), 2% EDT (ethylenediaminetetraacetic acid), and 1% water for 2 hours to cleave the protecting groups of the resin and peptide side chains.

[0030] 8. Filtration and precipitation: Filter the resin, wash the filtrate with ice-cold ether, centrifuge and retain the precipitate, which is the crude product. After freeze-drying, the final random peptide mixture is obtained.

[0031] Example 2: Antimicrobial activity analysis of three AMPCs (antimicrobial combination peptides) Step 1: Inoculate the seven bacterial cultures [Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 15442, Acinetobacter baumannii ATCC 19606, Staphylococcus aureus ATCC 25923, Salmonella typhimurium ATCC 14028, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633] into sterile MHB medium and incubate overnight at 37˚C and 250 rpm for 18 hours.

[0032] Step 2: Prepare the three antimicrobial combination peptides to 128 µg / mL with PBS, and then serially dilute them with PBS at concentrations of 128, 64, 32, 16, 8, 4, 2, and 1 µg / mL. Add 50 µL of each concentration to the AH wells of a 96-well plate. Repeat each antimicrobial combination peptide preparation three times, adding to wells 1-9.

[0033] Step 3: Dilute the seven cultured bacteria to 5 × 10⁵ CFU / mL with PBS. (Due to the large number of bacterial species, only one bacterial culture is used per plate; Staphylococcus aureus will be used as an example later.) Add 50 µL of bacterial dilution to each of the AF wells containing the existing antimicrobial peptide dilution. The peptide concentrations (µg / mL) at this point are shown in Table 1 below.

[0034] Table 1. Final concentrations of the antimicrobial combination peptides Meta211, Meta201, and Meta185 in the MIC assay, in µg / mL.

[0035] Step 4: Add 100µL of PBS solution to wells A10-H10 as a negative control, and add 100µL of bacterial culture (5×10⁵ CFU / mL) to wells A11-H11 as a positive control. Seal the 96-well plate with sealing film, place it in a self-sealing bag, and incubate overnight at 37℃ for 18 hours. Measure the OD600 values ​​of wells A1-G9 using a microplate reader. The minimum concentration corresponding to the lowest OD600 value in each antimicrobial combination peptide group is the minimum inhibitory concentration (MIC) of the corresponding bacteria. The results are shown in Table 2.

[0036] Table 2. MICs (µg / mL) of the antimicrobial combination peptides Meta211, Meta201, and Meta185 against seven pathogens.

[0037] The antimicrobial combination peptides Meta211, Meta201, and Meta185 are compositions of antimicrobial peptides with multiple sequences. These compositions are obtained directly through synthesis, rather than by synthesizing multiple antimicrobial peptides and then mixing them. In the table, the amino acid sequences of the peptides are represented by one random amino acid within parentheses, such as (LV) indicating L or V. For example, the specific sequence of the antimicrobial combination peptides might be as follows: Meta211 is a composition of antimicrobial peptides with amino acid sequences as shown in SEQ ID NO. 1~8, specifically: SEQ ID NO.1: LIRRLWRILRLIR SEQ ID NO.2: LIRRLWRIVRLIR SEQ ID NO.3: LIRRLWRVLRLIR SEQ ID NO.4: LIRRLWRVVRLIR SEQ ID NO.5: VIRRLWRILRLIR SEQ ID NO.6: VIRRLWRIVRLIR SEQ ID NO.7: VIRRLWRVLRLIR SEQ ID NO.8: VIRRLWRVVRLIR Meta201 is a composition of antimicrobial peptides with amino acid sequences as shown in SEQ ID NO. 9~24, specifically: SEQ ID NO.9: IWRVLRILRLLRR SEQ ID NO.10: IWRVLRILRLIRR SEQ ID NO.11: IWRVLRVLRLLRR SEQ ID NO.12: IWRVLRVLRLIRR SEQ ID NO.13: IWRVIRILRLLRR SEQ ID NO.14: IWRVIRILRLIRR SEQ ID NO.15: IWRVIRVLRLLRR SEQ ID NO.16: IWRVIRVLRLIRR SEQ ID NO.17: IWRILRILRLLRR SEQ ID NO.18: IWRILRILRLIRR SEQ ID NO.19: IWRILRVLRLLRR SEQ ID NO.20: IWRILRVLRLIRR SEQ ID NO.21: IWRIIRILRLLRR SEQ ID NO.22: IWRIIRILRLIRR SEQ ID NO.23: IWRIIRVLRLLRR SEQ ID NO.24: IWRIIRVLRLIRR Meta185 is a composition of antimicrobial peptides with amino acid sequences as shown in SEQ ID NO. 25~32, specifically: SEQ ID NO.25: LLRRLLTRLIRLI SEQ ID NO.26: LLRRLLTRLIRVI SEQ ID NO.27: LLRRILTRLIRLI SEQ ID NO.28: LLRRILTRLIRVI SEQ ID NO.29: VLRRLLTRLIRLI SEQ ID NO.30: VLRRLLTRLIRVI SEQ ID NO.31: VLRRILTRLIRLI SEQ ID NO.32: VLRRILTRLIRVI Table 2 shows that the three antimicrobial combination peptides of the present invention exhibit good antibacterial activity against seven different bacteria, demonstrating strong efficacy and broad applicability. The three peptides, in particular, show strong antibacterial effects against Bacillus subtilis ATCC6633, with a MIC of only 2 µg / mL.

[0038] Example 3: Hemolytic activity analysis of AMPCs 1. Blood samples are collected from the recipients via venous swab.

[0039] 2. Pre-cool the centrifuge (2000 rpm), aliquot the collected blood into 1.5 mL ep tubes, centrifuge at 2000 rpm and 4℃ for 5 min, discard the supernatant, wash the collected blood cells three times with PBS (washing ratio, PBS: plasma = 0.35: 0.65), then dilute with PBS to 2% of the original concentration, and slowly mix on a decolorizing shaker.

[0040] 3. Place 50 µL of peptides at different concentrations (final peptide concentration of 128-1 µg / mL, serially diluted 2-fold) prepared in Example 1 and an equal volume of red blood cell suspension into each well of a 96-well plate. Add 50 µL of PBS + 50 µL of red blood cell suspension as a negative control; add 50 µL of red blood cell suspension + 50 µL of 0.2% Tritonx-100 as a positive control. Incubate the 96-well plate in a 37°C incubator for 1 hour.

[0041] 4. After culturing for 1 hour, remove the 96-well plate and centrifuge at 1000 rpm and 4℃ for 5 min.

[0042] 5. After centrifugation, aspirate the supernatant from the solution and transfer it to a clean 96-well plate. Then, use a microplate reader to analyze the solution at 570 nm (OD500). 570 The hemolysis rate is calculated by measuring the absorbance value at a certain point.

[0043] Hemolysis rate (%) = [(sample OD)] 570 - Negative control OD 570 ) / (Positive control OD 570 - Negative control OD 570 )]×100%.

[0044] 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.

[0045] Table 3. HC50 of AMPCs for human erythrocytes (hRBCs), in mg / L

[0046] As can be seen from Table 3, the three antibacterial peptides synthesized in this invention have good biocompatibility.

[0047] Example 4: Investigation of the antibacterial mechanism of AMPCs I. Effect of NPN (1-naphthylaminobenzene) uptake assay on the effect of antimicrobial combination peptides on the extracellular membrane permeability of Acinetobacter baumannii.

[0048] 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.

[0049] 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.

[0050] 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. 600 The 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 broad-spectrum antibacterial combination peptides with low hemolytic activity, characterized in that, The amino acid sequence of the broad-spectrum antibacterial combination peptide is one of the following: Meta211: X2IRRLWRX3X2RLIR Meta201: IWRX3X1RX3LRLX1RR Meta185: X2LRRX1LTRLIRX2I Among them, 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 mixtures of all selected random sites.

2. The use of the broad-spectrum antibacterial combination peptide according to claim 1 in the preparation of medical products for treating and / or preventing bacterial infections.

3. The application according to claim 2, characterized in that, The medical products include one or more of the following: coatings for medical devices, coatings for implantable medical devices, and dressings.

4. The application of the broad-spectrum antibacterial combination peptide according to claim 1 in the preparation of daily-use products for preventing bacterial infections, characterized in that, The daily-use products include one or more of the following: chemical preservatives, food preservatives, and pet disinfectants.

5. The application according to any one of claims 2-4, characterized in that, The bacteria are Gram-positive and / or Gram-negative.

6. The application according to any one of claims 2-4, characterized in that, The bacteria include one or more of Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Enterococcus faecalis, and Bacillus subtilis.

7. The nucleotide sequence encoding the broad-spectrum antibacterial combination peptide of claim 1, characterized in that, The nucleotide sequence includes DNA and RNA.

8. A composition comprising the broad-spectrum antibacterial combination peptide of claim 1.

9. The composition according to claim 8, characterized in that, The composition contains one or more of a pharmaceutically or food-acceptable carrier, excipient, diluent, adjuvant, or medium.

10. An antibacterial product, characterized in that, It includes the broad-spectrum antimicrobial combination peptide of claim 1, the nucleotide sequence of claim 7, or the composition of claim 8.

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