Low-toxicity antibacterial combined peptide with broad-spectrum antibacterial activity and application thereof

Abstract

The invention relates to the technical field of biological materials, and discloses a low-toxicity antibacterial combined peptide with broad-spectrum antibacterial activity and application of the low-toxicity antibacterial combined peptide, the amino acid sequence of the low-toxicity antibacterial combined peptide is one of Meta222: KX2FIX3X1LLX3RLLX1, Meta231: X1X1X3RLTKWLLKKI and Meta202: X1X1X1RX1LRWX1RRRX1, X1, X2 and X3 are random sites, X1 is L or I, X2 is I or V, and X3 is R or K; the Meta222, the Meta231 and the Meta202 are respectively a mixture of all selected sites in the random sites. The antibacterial combined peptide constructed by the invention has a good antibacterial effect on drug-resistant strains, the antibacterial broad-spectrum property is remarkably improved, meanwhile, the preparation cost is greatly reduced on the basis of process optimization, and a feasible solution is provided for wide popularization and application of the antibacterial combined peptide.

Description

Technical Field

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

[0002] With the widespread use of antibiotics, bacterial resistance has become an increasingly serious problem, posing a significant challenge to global public health. Developing novel antimicrobial agents has become an urgent need in the scientific research field. Antimicrobial peptides, as a class of short peptide molecules, whether natural or synthetic, have shown great potential in controlling bacterial infections due to their broad-spectrum antimicrobial activity and rapid bacterial membrane lysis mechanism. Furthermore, their low tendency to induce resistance provides new treatment options for clinical practice. However, the high cost of synthesis severely limits their industrialization and large-scale application.

[0003] CN116874613A discloses a broad-spectrum and highly effective antibacterial peptide APH143, its preparation method, and its applications. The amino acid sequence of the antibacterial peptide APH143 is LWKKFKLKKKFLWLWKKF-NH2. This invention starts with the hybrid peptide P18 of cephalosporin and melitrix venom peptide, and obtains the imperfect amphiphilic α-helical antibacterial peptide APH143 through structure-activity relationship studies. The prepared peptide APH143 exhibits good broad-spectrum in vitro antibacterial activity against Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa, and also shows good stability. The prepared peptide APH143 is readily available and can be produced on a large scale, showing potential application value in the treatment of bacterial and fungal infectious diseases.

[0004] CN117003832A discloses a broad-spectrum antimicrobial peptide, Pan28, and its applications, belonging to the fields of biotechnology and biomedicine. The provided broad-spectrum antimicrobial peptide, Pan28, with the sequence EEEDKKEDVGTVVGIDLGTTYSCVGVFK, can significantly inhibit the growth of *Escherichia coli*, *Bacillus subtilis*, carbapenem-resistant *Pseudomonas aeruginosa*, and methicillin-resistant *Staphylococcus aureus*. Therefore, the antimicrobial peptide Pan28 shows promise as a novel antimicrobial drug for treating infections caused by various bacteria, including drug-resistant bacteria, and has good application prospects.

[0005] It is evident that different antimicrobial peptides exhibit antimicrobial effects against different bacteria, and achieving effective antimicrobial activity against multiple bacteria simultaneously is quite rare. Antimicrobial peptides with broad-spectrum antimicrobial effects and low toxicity have important guiding significance for biomedicine and warrant further research. Summary of the Invention

[0006] This invention constructs an antimicrobial combination peptide that has good antimicrobial effects against drug-resistant strains, significantly improving the broad spectrum of antimicrobial activity. At the same time, based on process optimization, the synthesis cost is reduced to 1 / 30 of that of a single antimicrobial peptide, greatly improving the economy and practicality of antimicrobial peptide preparations and providing a practical solution for their widespread application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A class of low-toxicity antimicrobial combination peptides with broad-spectrum antimicrobial activity, wherein the amino acid sequence of the low-toxicity antimicrobial combination peptide is one of the following: Meta222: KX2FIX3X1LLX3RLLX1 Meta231: X1X1X3RLTKWLLKKI Meta202: X1X1X1RX1LRWX1RRRX1 Among them, X1, X2 and X3 is a random site, X1 is L or I, X2 is I or V, and X3 is R or K; Meta222, Meta231, and Meta202 are mixtures of all selected random sites.

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

[0009] The products include one or more of the following: coatings for medical devices, coatings for implantable medical devices, 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.

[0012] The present invention also provides a nucleotide sequence encoding the aforementioned low-toxicity antimicrobial combination peptide, the nucleotide sequence 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 low-toxicity antimicrobial 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, characterized in that it comprises the aforementioned low-toxicity antibacterial combination peptide, the aforementioned nucleotide sequence, or the aforementioned composition.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The three antimicrobial combination peptides provided by this invention have broad-spectrum antimicrobial activity and low toxicity. They can be safely used in clinical applications to treat systemic or local infections. They also have good tolerability in long-term medication scenarios such as chronic infections and implant-related infections. Furthermore, they are safer for sensitive populations such as those with weakened immune systems, infants, and the elderly. In addition, they can significantly reduce the risk of hemolysis and improve the biocompatibility of materials in contact applications such as medical device coatings and anti-corrosion packaging. Attached Figure Description

[0018] Figure 1 The ability of Meta222, Meta231 and Meta202 to target the outer membrane permeability of Acinetobacter baumannii pathogen at different concentrations.

[0019] Figure 2 The ability of Meta222, Meta231 and Meta202 to depolarize the plasma membrane of Acinetobacter baumannii pathogen at different concentrations. Detailed Implementation

[0020] 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.

[0021] All raw materials used in the following specific implementation methods were purchased from the market.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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 the combined peptide side chains.

[0029] 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.

[0030] 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.

[0031] Step 2: Prepare the three antimicrobial combination peptides to 128 µg / mL with PBS, and then serially dilute them with PBS at 2-fold 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. Perform three replicates for each antimicrobial peptide, adding to wells 1-9.

[0032] 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.

[0033] Table 1. Final concentrations of the antimicrobial combination peptides Meta222, Meta231, and Meta202 in the MIC assay, in µg / mL.

[0034] 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 peptide group is the minimum inhibitory concentration (MIC) of that antimicrobial peptide for the corresponding bacteria. The results are shown in Table 2.

[0035] Table 2. MICs (µg / mL) of the antimicrobial combination peptides Meta222, Meta231, and Meta202 against seven pathogens.

[0036] The antimicrobial combination peptides Meta222, Meta231, and Meta202 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 combination 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: Meta222 is a composition of antimicrobial peptides with amino acid sequences as shown in SEQ ID NO. 1~32, specifically: SEQ ID NO.1: KVFIRILLKRLLL SEQ ID NO.2: KVFIRILLKRLLI SEQ ID NO.3: KVFIRILLRRLLL SEQ ID NO.4:KVFIRILLRRLLI SEQ ID NO.5:KVFIRLLLKRLLL SEQ ID NO.6:KVFIRLLLKRLLI SEQ ID NO.7:KVFIRLLLRRLLL SEQ ID NO.8:KVFIRLLLRRLLI SEQ ID NO.9:KVFIKILLKRLLL SEQ ID NO.10:KVFIKILLKRLLI SEQ ID NO.11:KVFIKILLRRLLLL SEQ ID NO.12:KVFIKILLRRLLI SEQ ID NO.13:KVFIKLLLKRLLL SEQ ID NO.14:KVFIKLLLKRLLI SEQ ID NO.15:KVFIKLLLRRLLL SEQ ID NO.16:KVFIKLLLRRLLI SEQ ID NO.17:KIFIRILLKRLLL SEQ ID NO.18: KIFIRILLKRLLI SEQ ID NO.19:KIFIRILLRRLLL SEQ ID NO.20: KIFIRILLRRLLI SEQ ID NO.21: KIFIRLLLKRLLL SEQ ID NO.22: KIFIRLLLKRLLI SEQ ID NO.23: KIFIRLLRRLLL SEQ ID NO.24: KIFIRLLRRLLLI SEQ ID NO.25:KIFIKILLKRLLL SEQ ID NO.26: KIFIKILLKRLLI SEQ ID NO.27: KIFIKILLRRLL SEQ ID NO.28: KIFIKILLRRLLI SEQ ID NO.29: KIFIKLLLKRLLL SEQ ID NO.30: KIFIKLLLKRLLI SEQ ID NO.31: KIFIKLLLRRLLL SEQ ID NO.32: KIFIKLLLRRLLI Meta231 is a composition of antimicrobial peptides with amino acid sequences as shown in SEQ ID NO. 33~40, specifically: SEQ ID NO.33: ILKRLTKWLLKKI SEQ ID NO.34: ILRRLTKWLLKKI SEQ ID NO.35: IIKRLTKWLLKKI SEQ ID NO.36: IIRRLTKWLLKKI SEQ ID NO.37: LLKRLTKWLLKKI SEQ ID NO.38: LLRRLTKWLLKKI SEQ ID NO.39: LIKRLTKWLLKKI SEQ ID NO.40: LIRRLTKWLLKKI Meta202 is a composition of antimicrobial peptides with amino acid sequences as shown in SEQ ID NO.41~104, specifically: SEQ ID NO.41: LLIRRLLRRWIRRRI SEQ ID NO.42: LLIRRLLRRWIRRRL SEQ ID NO.43: LLIRRLLRWLRRRI SEQ ID NO.44: LLIRRLLLRWLRRRL SEQ ID NO.45: LLIRILRWIRRRI SEQ ID NO.46: LLIRILRWIRRRL SEQ ID NO.47: LLIRILRWLRRRI SEQ ID NO.48: LLIRILRWLRRRL SEQ ID NO.49: LLLLRLLRWIRRRI SEQ ID NO.50: LLLLRLLRWIRRRL SEQ ID NO.51: LLLLRLLRWLRRRI SEQ ID NO.52:LLLRLLRWLRRRL SEQ ID NO.53:LLLRILRWIRRRI SEQ ID NO.54:LLLRILRWIRRRL SEQ ID NO.55:LLLRILRWLRRRI SEQ ID NO.56:LLLRILRWLRRRL SEQ ID NO.57:LIIRLLRWIRRRI SEQ ID NO.58:LIIRLLRWIRRRL SEQ ID NO.59:LIIRLLRWLRRRI SEQ ID NO.60:LIIRLLRWLRRRL SEQ ID NO.61:LIIRILRWIRRRI SEQ ID NO.62:LIIRILRWIRRRL SEQ ID NO.63:LIIRILRWLRRRI SEQ ID NO.64:LIIRILRWLRRRL SEQ ID NO.65:LILRLLRWIRRRI SEQ ID NO.66:LILRLLRWIRRRL SEQ ID NO.67:LILRLLRWLRRRI SEQ ID NO.68:LILRLLRWLRRRL SEQ ID NO.69:LILRILRWIRRRI SEQ ID NO.70:LILRILRWIRRRL SEQ ID NO.71:LILRILRWLRRRI SEQ ID NO.72:LILRILRWLRRRL SEQ ID NO.73:ILIRLLRWIRRRI SEQ ID NO.74:ILIRLLRWIRRRL SEQ ID NO.75:ILIRLLRWLRRRI SEQ ID NO.76:ILIRLLRWLRRRL SEQ ID NO.77:ILIRILRWIRRRI SEQ ID NO.78:ILIRILRWIRRRL SEQ ID NO.79:ILIRILRWLRRRI SEQ ID NO.80:ILIRILRWLRRRL SEQ ID NO.81:ILLRLLRWIRRRI SEQ ID NO.82:ILLRLLRWIRRRL SEQ ID NO.83:ILLRLLRWLRRRI SEQ ID NO.84:ILLRLLRWLRRRL SEQ ID NO.85:ILLRILRWIRRRI SEQ ID NO.86:ILLRILRWIRRRL SEQ ID NO.87:ILLRILRWLRRRI SEQ ID NO.88:ILLRILRWLRRRL SEQ ID NO.89:IIIRLLRWIRRRI SEQ ID NO.90:IIIRLLRWIRRRL SEQ ID NO.91:IIIRLLRWLRRRI SEQ ID NO.92:IIIRLLRWLRRRL SEQ ID NO.93:IIIRILRWIRRRI SEQ ID NO.94:IIIRILRWIRRRL SEQ ID NO.95:IIIRILRWLRRRI SEQ ID NO.96:IIIRILRWLRRRL SEQ ID NO.97:IILRLLRWIRRRI SEQ ID NO.98:IILRLLRWIRRRL SEQ ID NO.99:IILRLLRWLRRRI SEQ ID NO.100:IILRLLRWLRRRL SEQ ID NO.101:IILRILRWIRRRI SEQ ID NO.102: IILRILRWIRRRL SEQ ID NO.103: IILRILRWLRRRI SEQ ID NO.104: IILRILRWLRRRL Table 2 shows that the three antimicrobial combination peptides of the present invention all exhibit good antibacterial activity against seven different bacteria, demonstrating strong efficacy and broad applicability. Among them, Meta231 and Meta202 have more significant antibacterial effects against Bacillus subtilis ATCC6633.

[0037] Example 3: Hemolytic activity analysis of AMPCs 1. Blood samples are collected from the recipients via vein.

[0038] 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.

[0039] 3. Place 50 µL of the combined peptides at different concentrations prepared in Example 1 (the final concentration of the combined peptides was 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.

[0040] 4. After culturing for 1 hour, remove the 96-well plate and centrifuge at 1000 rpm and 4℃ for 5 min.

[0041] 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.

[0042] Hemolysis rate (%) = [(sample OD)] 570 - Negative control OD 570 ) / (Positive control OD 570 - Negative control OD 570 )]×100%.

[0043] 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.

[0044] Table 3. HC50 of AMPCs for human erythrocytes (hRBCs), in mg / L

[0045] As shown in Table 3, the HC50 values ​​of the three antimicrobial combination peptides Meta222, Meta231, and Meta202 synthesized in this invention are 267.20 μg / mL, 176.00 μg / mL, and 168.30 μg / mL, respectively, all significantly higher than their corresponding minimum inhibitory concentrations (MICs). This indicates that at concentrations where effective antimicrobial activity is achieved, they exhibit almost no significant hemolytic activity against human erythrocytes, demonstrating extremely low cytotoxicity and high cell selectivity. Therefore, the antimicrobial combination peptides of this invention maintain broad-spectrum and highly effective antimicrobial activity while also possessing excellent safety.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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 was significantly increased after treatment with AMPCs, indicating that the inner membrane was significantly depolarized and lost its potential. AMPCs exerted their bactericidal effect through membrane interaction mechanism.

Claims

1. A class of low-toxicity antimicrobial combination peptides with broad-spectrum antimicrobial activity, characterized in that, The amino acid sequence of the low-toxicity antimicrobial combination peptide is one of the following: Meta222: KX2FIX3X1LLX3RLLX1 Meta231: X1X1X3RLTKWLLKKI Meta202: X1X1X1RX1LRWX1RRRX1 Among them, X1, X2 and X3 are random sites, X1 is L or I, X2 is I or V, and X3 is R or K; Meta222, Meta231 and Meta202 are mixtures of all selected random sites.

2. The use of the low-toxicity antimicrobial combination peptide according to claim 1 in the preparation of products or medicines 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, 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 low-toxicity antimicrobial combinatorial 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 low-toxicity antimicrobial 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 low-toxicity antimicrobial combination peptide of claim 1, the nucleotide sequence of claim 7, or the composition of claim 8.

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