Antibacterial random combined peptide and application thereof

Abstract

The invention relates to the technical field of biology, and provides an antibacterial random combined peptide and application thereof, and the amino acid sequence of the antibacterial random combined peptide is one of the following: Meta183: X1RX2X3LRRLLRRLI; meta209: X < 4 > IRRX < 4 > WX < 1 > RX < 4 > LRLI; meta217: RLX1RLLRX4WRX3X1X1, where X1, X2, X3 and X4 are random sites, X1 is L or I, X2 is Q or S, X3 is V or L, and X4 is I or V; the Meta183, the Meta209 and the Meta217 are mixtures selected from all the random sites respectively. The antibacterial combined peptide can be used for various clinical drug-resistant strains, not only provides a new direction for solving the drug resistance problem, but also provides a theoretical basis and technical support for large-scale and standardized production of antibacterial peptides, and has important strategic significance and clinical value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a class of antibacterial random combination peptides and their applications. Background Technology

[0002] Antibiotic resistance has become a core challenge in global medical strategy. Existing antibiotics are gradually becoming ineffective, necessitating novel antimicrobial strategies. Antimicrobial peptides have attracted widespread attention due to their unique membrane disruption mechanisms and low resistance rates, but their high synthesis costs severely limit their widespread adoption.

[0003] CN115433258A discloses a method for screening antimicrobial peptides from self-fermented walnut meal and their applications. The method involves fermenting walnut meal with a compound probiotic strain to enhance its antimicrobial activity against dominant bacteria causing prickly pear spoilage. Changes in antimicrobial peptides in the fermented walnut meal were investigated, and peptides with antimicrobial activity, including FGGDSTHP, ALGGGY, YVVPW, and PLLRW, were screened. The interaction mechanism between the antimicrobial peptides and phenolic compounds was further analyzed, and the optimal antimicrobial combination YVVPW-SA was obtained, enhancing the synergistic antimicrobial activity against *P. victoriae*.

[0004] CN118005741A discloses an antimicrobial polypeptide AP16A, its preparation method, and its applications, belonging to the field of biopeptides. The sequence of the antimicrobial polypeptide AP16A is: ValLysValArgLysLeuIleArgArgLeuArgArgIleArgIleAlaArgLeuIle. The prepared antimicrobial polypeptide AP16A exhibits better broad-spectrum antimicrobial activity, showing significant activity against Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, and Escherichia coli. It also significantly enhances antimicrobial activity against drug-resistant Klebsiella pneumoniae and drug-resistant Pseudomonas aeruginosa. Furthermore, it exhibits minimal cytotoxicity to normal cells, extremely low erythrocyte lysis, and good stability, making it a promising novel drug for treating bacterial infections.

[0005] However, due to their complex synthesis process and high cost, they are difficult to widely use in clinical and public health systems. To overcome this bottleneck, simplifying the synthesis process of antimicrobial peptides, reducing synthesis costs, and improving the practicality and accessibility of antimicrobial peptides will provide a sustainable, cost-effective solution for the upgrading of antimicrobial drugs, which has significant social implications. Summary of the Invention

[0006] This invention addresses the problem of antibiotic resistance by providing a novel class of random antibacterial combination peptides. These combination peptides possess broad-spectrum antibacterial activity and low toxicity, offering a sustainable, cost-effective solution for the upgrading of antibacterial drugs and having significant social implications.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A class of antimicrobial random combination peptides, wherein the amino acid sequence of the antimicrobial random combination peptide is one of the following: Meta183: X1RX2X3LRRLLRRLI Meta209: X4IRRX4WX1RX4LRLI Meta217: RLX1RLLRX4WRX3X1X1 Among them, X1, X2, X3 and X4 are random sites, X1 is L or I, X2 is Q or S, X3 is V or L, and X4 is I or V; Meta183, Meta209 and Meta217 are mixtures of all selected random sites.

[0008] The present invention also provides the use of the aforementioned antimicrobial random combination peptide in the preparation of products or medicaments for treating and / or preventing bacterial infections.

[0009] The products include one or more of the following: medical devices, implantable medical devices, drugs, dressings, food preservatives, and pet disinfectants.

[0010] The bacteria are Gram-positive and / or Gram-negative.

[0011] The bacteria include one or more of Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Enterococcus faecalis, and Bacillus subtilis. The random combination peptides of the present invention have significant antibacterial effects against Acinetobacter baumannii, which is beneficial for the treatment of serious diseases such as epidermal infections and even internal infections such as sepsis.

[0012] The present invention also provides nucleotide sequences encoding the aforementioned antimicrobial random combination peptides, said nucleotide sequences comprising DNA and RNA.

[0013] The present invention also provides an expression vector containing the nucleotide sequence described above.

[0014] The present invention also provides a class of compositions containing the aforementioned antimicrobial random 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 antibacterial random combination peptide, the aforementioned nucleotide sequence, or the aforementioned composition.

[0017] Preferably, the antibacterial products include drugs for the prevention and / or treatment of diseases caused by bacterial infections, such as epidermal infections, internal infections, and sepsis caused by Acinetobacter baumannii infection. The preparation of related drugs or implantable medical devices using the antibacterial peptides of the present invention can achieve significant antibacterial efficacy and play an important clinical role in the treatment of diseases.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention, with improved practicality as its core objective, constructs an antimicrobial combo peptide that targets multiple clinically resistant bacterial strains. It retains the highly effective bactericidal properties of traditional antimicrobial peptides while significantly reducing manufacturing costs through peptide co-design and production optimization; the synthesis cost is approximately 30 times lower than that of single-peptide products. This achievement not only provides a new direction for solving the drug resistance problem but also offers a theoretical foundation and technical support for the large-scale, standardized production of antimicrobial peptides, possessing significant strategic importance and clinical value. Attached Figure Description

[0019] Figure 1 The ability of Meta183, Meta209 and Meta217 to target the outer membrane permeability of Acinetobacter baumannii pathogen at different concentrations.

[0020] Figure 2 The ability of Meta183, Meta209 and Meta217 to depolarize the plasma membrane of Acinetobacter baumannii pathogen at different concentrations. 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 random 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: Antibacterial activity analysis of three AMPCs (antimicrobial random 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 Meta183, Meta209, and Meta217 in the MIC assay, in µg / mL. Step 4: Add 100µL of PBS solution to wells A10-H10 as a negative control, and add 100µL of bacterial suspension (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.

[0035] Table 2. MICs (µg / mL) of the antimicrobial combination peptides Meta183, Meta209, and Meta217 against seven pathogens. The antimicrobial random combination peptides Meta183, Meta209, and Meta217 are compositions of antimicrobial peptides with multiple sequences. These compositions are obtained directly through synthesis, rather than by synthesizing and mixing multiple antimicrobial peptides. In the table, the amino acid sequences of the peptides are represented by amino acids in parentheses, indicating a random amino acid, such as (LV) representing L or V. For example, the specific sequence of the antimicrobial random combination peptides might be as follows: Meta183 is a composition of antimicrobial peptides with amino acid sequences as shown in SEQ ID NO. 1~8, specifically: SEQ ID NO.1: IRQLLRRLLRRLI SEQ ID NO.2: IRQVLRRLLRRLI SEQ ID NO.3: IRSLLRRLLRRLI SEQ ID NO.4: IRSVLRRLLRRLI SEQ ID NO.5: LRQLLRRLLRRLI SEQ ID NO.6: LRQVLRRLLRRLI SEQ ID NO.7: LRSLLRRLLRRLI SEQ ID NO.8: LRSVLRRLLRRLI Meta209 is a composition of antimicrobial peptides with amino acid sequences as shown in SEQ ID NO. 9~24, specifically: SEQ ID NO.9: IIRRIWIRILRLI SEQ ID NO.10: IIRRIWIRVLRLI SEQ ID NO.11: IIRRIWLRILRLI SEQ ID NO.12: IIRRIWLRVLRLI SEQ ID NO.13: IIRRVWIRILRLI SEQ ID NO.14: IIRRVWIRVLRLI SEQ ID NO.15: IIRRVWLRILRLI SEQ ID NO.16: IIRRVWLRVLRLI SEQ ID NO.17: VIRRIWIRILRLI SEQ ID NO.18: VIRRIWIRVLRLI SEQ ID NO.19: VIRRIWLRILRLI SEQ ID NO.20: VIRRIWLRVLRLI SEQ ID NO.21: VIRRVWIRILRLI SEQ ID NO.22: VIRRVWIRVLRLI SEQ ID NO.23: VIRRVWLRILRLI SEQ ID NO.24: VIRRVWLRVLRLI Meta217 is a composition of antimicrobial peptides with amino acid sequences shown in SEQ ID NOs. 25 to 56, specifically: SEQ ID NO.25: RLIRLLRIWRLLI SEQ ID NO.26: RLIRLLRIWRLLL SEQ ID NO.27: RLIRLLRIWRLII SEQ ID NO.28: RLIRLLRIWRLIL SEQ ID NO.29: RLIRLLRIWRVLI SEQ ID NO.30: RLIRLLRIWRVLL SEQ ID NO.31: RLIRLLRIWRVII SEQ ID NO.32: RLIRLLRIWRVIL SEQ ID NO.33: RLIRLLRVWRLLI SEQ ID NO.34: RLIRLLRVWRLLL SEQ ID NO.35: RLIRLLRVWRLII SEQ ID NO.36: RLIRLLRVWRLIL SEQ ID NO.37: RLIRLLRVWRVLI SEQ ID NO.38: RLIRLLRVWRVLL SEQ ID NO.39: RLIRLLRVWRVII SEQ ID NO.40: RLIRLLRVWRVIL SEQ ID NO.41: RLLRLLRIWRLLI SEQ ID NO.42: RLLRLLRIWRLLL SEQ ID NO.43: RLLRLLRIWRLII SEQ ID NO.44: RLLRLLRIWRLIL SEQ ID NO.45: RLLRLLRIWRVLI SEQ ID NO.46: RLLRLLRIWRVLL SEQ ID NO.47: RLLRLLRIWRVII SEQ ID NO.48: RLLRLLRIWRVIL SEQ ID NO.49: RLLRLLRVWRLLI SEQ ID NO.50: RLLRLLRVWRLLL SEQ ID NO.51: RLLRLLRVWRLII SEQ ID NO.52: RLLRLLRVWRLIL SEQ ID NO.53: RLLRLLRVWRVLI SEQ ID NO.54: RLLRLLRVWRVLL SEQ ID NO.55: RLLRLLRVWRVII SEQ ID NO.56: RLLRLLRVWRVIL Table 2 shows that the three antimicrobial peptides of the present invention exhibit good antibacterial activity against seven different bacteria, demonstrating high efficacy and broad applicability.

[0036] Example 3: Hemolytic activity analysis of AMPCs 1. Blood samples are collected from the recipients via vein.

[0037] 2. Pre-cool the centrifuge (2000 rpm), aliquot the collected blood into 1.5 mL ep tubes, centrifuge at 2000 rpm and 4˚C 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.

[0038] 3. Place 50 µL of the combined peptides of different concentrations prepared in Example 1 (final peptide concentration of 128-1 µg / mL, serially diluted 2-fold) 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.

[0039] 4. After culturing for 1 hour, remove the 96-well plate and centrifuge at 1000 rpm and 4℃ for 5 min.

[0040] 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.

[0041] Hemolysis rate (%) = [(sample OD)] 570 - Negative control OD 570 ) / (Positive control OD 570 - Negative control OD 570 )]×100%.

[0042] 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.

[0043] Table 3. HC50 of AMPCs for human erythrocytes (hRBCs), in mg / L As can be seen from Table 3, the three antibacterial combination peptides synthesized in this invention have low toxicity, and the hemolytic concentration is more than 50 times that of the working concentration, making them very safe to use at the working concentration.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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 the membrane potential change was assessed. The results were recorded, background fluorescence was subtracted, and the data were normalized. The results are shown below. Figure 2 As shown, the fluorescence signal detected after treatment with AMPCs is significantly increased, indicating that the inner membrane is significantly depolarized and loses its potential. The AMPCs of this invention generate bactericidal effect through membrane interaction mechanism.

Claims

1. A class of antibacterial random combination peptides, characterized in that, The amino acid sequence of the antimicrobial random combination peptide is one of the following: Meta183: X1RX2X3LRRLLRRLI Meta209: X4IRRX4WX1RX4LRLI Meta217: RLX1RLLRX4WRX3X1X1 Among them, X1, X2, X3 and X4 are random sites, X1 is L or I, X2 is Q or S, X3 is V or L, and X4 is I or V; Meta183, Meta209 and Meta217 are mixtures of all selected random sites.

2. The use of the antimicrobial random combination peptide according to claim 1 in the preparation of products or medicaments for treating and / or preventing bacterial infections.

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

4. The application according to claim 2, characterized in that, The bacteria are Gram-positive and / or Gram-negative.

5. The application according to claim 2, 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.

6. The nucleotide sequence encoding the antimicrobial random combination peptide of claim 1, characterized in that, The nucleotide sequence includes DNA and RNA.

7. An expression vector containing the nucleotide sequence of claim 6.

8. A composition comprising the antimicrobial random 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 antimicrobial random combination peptide of claim 1, the nucleotide sequence of claim 7, or the composition of claim 8.

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