A group of antimicrobial peptides targeting Gram-negative bacteria

Abstract

This invention discloses a group of antimicrobial peptides targeting Gram-negative bacteria, belonging to the field of biomedical technology. This invention screened and obtained a group of antimicrobial peptides that exhibit good broad-spectrum antibacterial properties against *Escherichia coli*, *Salmonella typhimurium*, *Pseudomonas aeruginosa*, *Klebsiella pneumoniae*, *Shigella sonnei*, and *Stenotrophomonas maltophilia*. Among them, antimicrobial peptides 11A-1, 11A-4, and 11A-5 have hemolytic and toxic properties below the therapeutic concentration threshold, and can be used to prepare drugs or medical products for treating multidrug-resistant bacterial infections.

Description

Technical Field

[0001] This invention relates to a group of antimicrobial peptides targeting Gram-negative bacteria, belonging to the field of biomedical technology. Background Technology

[0002] Since the advent of penicillin, antibiotics, as commonly used clinical drugs, have saved countless lives. However, with their widespread use and even abuse, bacteria have developed drug resistance and spread widely. Among them, multidrug-resistant "superbugs" pose a serious threat to life. In response, the World Health Organization has highlighted the urgent need for new antibacterial drugs and published a list of key pathogens. The list includes multidrug-resistant bacteria, with Gram-negative bacteria occupying a significant position. Carbapenem-resistant Acinetobacter baumannii is ranked first, and it currently faces a situation where no effective treatment is available.

[0003] Existing antimicrobial drugs are insufficient to meet clinical needs, while the development of new antibiotics is slow, posing a major challenge in defeating "superbugs." In recent years, the emergence of antimicrobial peptides has brought a glimmer of hope to this problem. Antimicrobial peptides, also known as host defense peptides, are an important component of the innate immune system of multicellular organisms. Currently, over 2000 antimicrobial peptides from eukaryotes have been discovered. Most of them possess broad-spectrum antimicrobial activity, are characterized by small molecular weight, positive charge, and amphiphilicity. Their widely accepted mechanism of action is through multi-target membrane hydrolysis, thus minimizing the likelihood of inducing drug resistance. Gram-negative bacteria possess a double membrane barrier, making it difficult for many conventional drugs to penetrate the bacterial cell and exert their effects. This characteristic gives antimicrobial peptides an inherent advantage in killing Gram-negative bacteria. Currently, clinically used antimicrobial peptides include daptomycin, vancomycin, and teicoplanin for treating Gram-positive bacterial infections, and polymyxin B for treating Gram-negative bacterial infections. However, although antimicrobial peptides act on cell membranes, their affinity for prokaryotic cell membranes rich in negatively charged phospholipids is higher than that for eukaryotic cell membranes which are predominantly electrically neutral, they still have the potential for high cytotoxicity. This is a major factor hindering their translation into clinically developed drugs.

[0004] Existing peptide drugs often face potential cytotoxic risks (such as hemolysis), narrow antibacterial spectrum, and insufficient inhibitory efficacy against key pathogens. Their minimum inhibitory concentrations (MICs) against clinically hazardous strains are generally high; for example, the MIC for Klebsiella pneumoniae (a carbapenemase-producing strain) is >64 μg / mL, and for Stenotrophomonas maltophilia (a naturally occurring multidrug-resistant bacterium) is >128 μg / mL. This necessitates extremely high doses for actual treatment, leading to severe hemolytic toxicity. These limitations severely restrict their efficacy persistence, safety, and clinical feasibility. Therefore, there is an urgent need to overcome these existing barriers through the screening and design of novel peptides. Summary of the Invention

[0005] This invention provides an antimicrobial polypeptide with an amino acid sequence of any one of the following (1) to (5):

[0006] (1) KWCFRVCYRGACYYRCR (shown in SEQ ID NO.1);

[0007] (2) KWCFRVCYRGACRRRCR (shown in SEQ ID NO.2);

[0008] (3) KWCFYVCYRGACRRRCR (shown in SEQ ID NO.3);

[0009] (4) KWCFRLCYRGACYRRCR (shown as SEQ ID NO.4);

[0010] (5) KWCLRVCYRGACYRRCR (shown in SEQ ID NO.5).

[0011] In one embodiment, the amino acid sequence of the antimicrobial polypeptide is shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.5.

[0012] The present invention also provides compositions containing the said antimicrobial peptide.

[0013] In one embodiment, the composition includes, but is not limited to, pharmaceuticals, wound care products, medical device coatings, and environmentally friendly disinfectants.

[0014] In one embodiment, the drug contains the antimicrobial peptide and pharmaceutical excipients.

[0015] In one embodiment, the pharmaceutical excipients include, but are not limited to, solvents, fillers, dispersants, stabilizers, plasticizers, and flavoring agents.

[0016] In one embodiment, the content of antimicrobial peptides in the drug is ≥0.2 mg / g.

[0017] The present invention also provides the use of the antimicrobial peptide in the preparation of antibacterial infection drugs.

[0018] In one embodiment, the drug dosage form includes an injection, an oral tablet, a spray, or a topical gel.

[0019] In one embodiment, the bacterial infection includes, but is not limited to, intestinal infection, pneumonia, burn infection, or sepsis caused by one or more of the following bacteria: Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei, and Stenotrophomonas maltophilia.

[0020] Beneficial effects:

[0021] The peptides screened in this invention possess broad-spectrum antibacterial properties. Specifically, the MICs (microindices) of antimicrobial peptide 11A-1 against *Escherichia coli*, *Salmonella typhimurium*, *Pseudomonas aeruginosa*, *Klebsiella pneumoniae*, *Shigella sonnei*, and *Stenotrophomonas maltophilia* are 2 μg / mL, 2 μg / mL, 4 μg / mL, 4 μg / mL, 2 μg / mL, and 4 μg / mL, respectively. Its hemolytic activity and toxicity are below the therapeutic concentration threshold. In terms of hemolytic activity and cytotoxicity, key safety indicators exceed international standards by more than 10 times (MHC). 10 / MIC max =16,IC 50 / MIC max =35.2), which can be used to prepare drugs for the treatment of multidrug-resistant bacterial infections or medical products. Attached Figure Description

[0022] Figure 1 This is a graph showing the results of the erythrocyte hemolysis assay for the polypeptide.

[0023] Figure 2 The figure shows the results of the toxicity assay of the peptide on human embryonic kidney cells HEK293T cells. Detailed Implementation

[0024] 1. Strains

[0025] Escherichia coli ( Escherichia coli ATCC25922, Klebsiella pneumoniae ( Klebsiella pneumoniae ATCC700603, Pseudomonas aeruginosa ( Pseudomonas aeruginosa ATCC27853, Shigella sonnei ( Shigella sonnei ATCC23875, Salmonella typhimurium ( Salmonella Typhimurium ATCC14028, Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia ATCC17666 are all commercial strains.

[0026] 2. Culture medium

[0027] MH (Mueller-Hinton) broth medium: Add 2 g of beef extract powder, 17.5 g of acid-hydrolyzed casein, and 1.5 g of soluble starch to approximately 1000 mL of distilled water, heat and stir, and adjust the pH to 7.2-7.4. 121 o Autoclave for 15 minutes and set aside.

[0028] DMEM medium was purchased from Gibco.

[0029] Example 1 Preparation of polypeptides

[0030] The preparation of the peptides was commissioned to Sangon Biotech (Shanghai) Co., Ltd. The specific steps included: synthesizing peptides using the Fmoc solid-phase synthesis method according to the amino acid sequences shown in Table 1; a) using Rink Amide MBHA resin as a carrier, and coupling Fmoc to protect the amino acids using an HBTU / HOBt / DIPEA system; b) cleavage: treating with a TFA / TIS / H2O (95:2.5:2.5) mixture for 2.5 hours; c) precipitation: adding -20°C cleavage fluid... o C. Methyl tert-butyl ether, centrifuged to obtain crude peptide; d) purified by preparative HPLC (C18 reversed-phase column, acetonitrile / 0.1% TFA gradient elution), detected by ESI mass spectrometry, and lyophilized to obtain the final product (5 mg, purity >95%).

[0031] Table 1. Peptides and sequences involved in this application

[0032]

[0033] Here, "-" represents a disulfide bond formed between amino acids.

[0034] Example 2: Inhibitory effects of polypeptides on different microorganisms

[0035] (1) Dissolve the polypeptides prepared in Example 1 in sterile water to obtain a stock solution of 12.8 mg / mL for later use. The antibacterial activity of the polypeptides was determined by the micro-broth dilution method.

[0036] (2) Streak strains of Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC700603, Pseudomonas aeruginosa ATCC27853, Shigella sonnei ATCC23875, Salmonella typhimurium ATCC14028, and Stenotrophomonas maltophilia ATCC17666 onto blood agar plates. After monoclonal growth, resuspend in sterile physiological saline and adjust the turbidity to 0.5 McFarland standard, which corresponds to approximately 1×10⁻⁶. 8 The initial concentration of CFU / mL was determined. This bacterial suspension was then diluted 1:100 with MH broth to a concentration of 1×10⁻⁶. 6 CFU / mL, all operations were performed under aseptic conditions.

[0037] (3) Add 200 μL of the test peptide or drug solution at concentrations of 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, and 1 μg / mL to wells A1-H1 of a sterile 96-well plate, respectively. Add 100 μL of 6% DMSO to the remaining wells. Then, transfer 100 μL of the test drug solution from wells A1-H11 to the second well and mix well. Dilute the peptide or drug solution sequentially to wells A11-H11 using this method. Do not add any drug to wells A12-H12. Finally, add 100 μL of the test bacterial culture to each well and incubate the 96-well plate at 37°C for 20 hours. After incubation, 20 μL of 1% TTC (2,3,5-triphenyltetrazolium chloride) was added to each well, and the mixture was incubated at 37°C for 10 minutes to allow for colorimetric reaction. The results are shown in Table 2. The peptides exhibited good antibacterial activity against all six Gram-negative bacteria tested.

[0038] Table 2. Results of minimum inhibitory concentration (MIC) determination of peptides against six Gram-negative bacteria (unit: μg / mL)

[0039]

[0040] Example 3: Detection of hemolytic activity of peptides

[0041] Fresh blood from rat eyeballs was collected into an anticoagulant blood collection tube containing sodium heparin. The tube was centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant was discarded, and the red blood cells were washed three times with physiological saline until the supernatant no longer appeared red. The resulting red blood cells were then prepared into a 10% suspension with physiological saline for experimental use.

[0042] In the experimental group, the peptides were dissolved in physiological saline and diluted to 2048, 1024, 512, 256, 128 and 64 μg / mL, respectively. Physiological saline and Triton X-100 were used as negative and positive controls, respectively.

[0043] 100 μL of 10% erythrocyte suspension was added to each of the experimental group, negative control, and positive control group, with a final reaction volume of 200 μL. Each sample was incubated at 37°C and 200 rpm for 1 hour to ensure a complete reaction.

[0044] All samples were incubated at 37°C and 200 rpm for 1 hour. After incubation, the samples were centrifuged at 4°C and 3000 rpm for 10 minutes. The supernatant was collected and transferred to a 96-well plate. The optical density (OD) value was measured at 540 nm using a microplate reader.

[0045] Hemolysis rate = (OD value of experimental sample - OD value of negative control group) ÷ (OD value of positive control group - OD value of negative control group) × 100%.

[0046] The results are shown in Table 3. The MHC values ​​of peptides 11A-1 and 11A-4 were both 250 μg / mL, indicating a low probability of hemolysis and high safety.

[0047] Table 3 MHC values ​​of peptides

[0048]

[0049] MHC refers to the drug concentration that causes 10% hemolysis.

[0050] Example 4: Polypeptide cytotoxicity assay

[0051] Human embryonic kidney cells HEK293T were used as the test cell line. The peptides prepared in Example 1 were serially diluted using the two-fold dilution method with DMEM medium. Eight drug gradients were set up: 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL, 7.8125 μg / mL, and 3.90 μg / mL. Cells with different drug concentrations were used as experimental groups, cells without drugs were used as control groups, and cells with only DMEM medium were used as blank groups.

[0052] The cells from each group were cultured in a 37°C, 5% CO2 incubator. Logarithmic growth phase cells were digested with trypsin to prepare cell suspensions, and the density was adjusted to 5 × 10⁶ cells / mL. 4 Cells / mL: Add 100 μL of cell suspension to each well of a 96-well plate and incubate overnight to allow cell adhesion. Add 100 μL of different concentration gradients of drugs to each well, with 5 replicates for each gradient. Incubate the 96-well plates with each peptide treatment in an incubator for another 24 h. After incubation, add 20 μL of MTT (thiazolyl blue) solution (5 mg / mL) to each well and continue incubating for another 4 h.

[0053] After the cell culture was completed, it was centrifuged at 2000 rpm for 10 min, the supernatant was discarded, 150 μL of DMSO was added to each well and mixed well. The mixture was then placed on a shaker and shaken at low speed for 10 min to fully dissolve the crystals. The absorbance at 490 nm was measured using a microplate reader. The cell viability was calculated using the following formula: Cell viability = (Absorbance of experimental wells - Absorbance of blank wells) ÷ (Absorbance of negative control wells - Absorbance of blank wells) × 100%.

[0054] The results are as follows Figure 2As shown in Table 4, peptides 11A-4 and 11A-5 exhibited good low cytotoxicity.

[0055] Table 4. Results of toxicity of peptides or drugs to human embryonic kidney cells HEK293T cells.

[0056]

[0057] Example 5: Preparation of antimicrobial peptide products

[0058] Carbomer 940 (1.0 g) was dispersed in 80 mL of purified water and swollen. The pH was adjusted to 6.5 with triethanolamine. 10 mL of phosphate-buffered saline (PBS, pH 7.0) containing 50 mg of the peptide prepared in Example 1 was added, and the mixture was stirred. Then, 5 g of glycerol and 0.1 g of ethylparaben were added, and the volume was brought to 100 g to prepare a peptide-containing gel. This gel is suitable for skin wound infections, such as burns infected with Pseudomonas aeruginosa.

[0059] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An antimicrobial peptide, characterized in that, The amino acid sequence is any one of the following (1) to (3): (1) KWCFRVCYRGACYYRCR; (2) KWCFRLCYRGACYRRCR; (3) KWCLRVCYRGACYRRCR.

2. A composition containing the antimicrobial peptide of claim 1.

3. The composition according to claim 2, characterized in that, The composition includes, but is not limited to, pharmaceuticals, wound care products, medical device coatings, or environmentally friendly disinfectants.

4. The composition according to claim 3, characterized in that, The drug contains the antimicrobial peptide and pharmaceutical excipients.

5. The composition according to claim 4, characterized in that, The pharmaceutical excipients include, but are not limited to, solvents, fillers, dispersants, stabilizers, plasticizers, or flavoring agents.

6. The composition according to any one of claims 3 to 5, characterized in that, The drug contains ≥0.2 mg / g of antimicrobial peptides.

7. The use of the antimicrobial peptide according to claim 1 in the preparation of antibacterial infection drugs, characterized in that, The bacterial infection is an intestinal infection, pneumonia, burn infection, or sepsis caused by one or more of the following bacteria: Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei, and Stenotrophomonas maltophilia.

8. The application according to claim 7, characterized in that, The dosage forms of the drug include injections, oral tablets, sprays, or topical gels.

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