Polypeptide medicine for resisting gram-negative bacteria and application

Abstract

The invention discloses a polypeptide drug for resisting gram-negative bacteria and application, and belongs to the technical field of biological medicine. The MIC of the antibacterial peptide obtained through screening on escherichia coli, salmonella typhimurium, pseudomonas aeruginosa, klebsiella pneumoniae, shigella sonnei and stenotrophomonas maltophilia is 16 [mu] g / mL, 2 [mu] g / mL, 16 [mu] g / mL, 4 [mu] g / mL, 2 [mu] g / mL and 4 [mu] g / mL respectively, and the antibacterial peptide has broad-spectrum antibacterial performance. The hemolytic activity and toxicity of the compound are lower than therapeutic concentration threshold values, and the compound can be used for preparing drugs or medical products for treating multi-drug-resistant bacterium infection.

Description

Technical Field

[0001] The present invention relates to a polypeptide drug for anti - Gram - negative bacteria and its application, belonging to the field of biomedical technology. Background Art

[0002] Antibiotic resistance poses a serious threat to global health. Due to intrinsic or acquired resistance mechanisms, the effectiveness of common and important antibiotics is decreasing. It is statistically shown that approximately 1.27 million people died directly from drug - resistant bacterial infections globally in 2019. At the same time, experts predict that 10 million people will die from drug - resistant bacterial infections by 2050. Its severity is at least as great as, and may even be greater than, major diseases such as HIV and malaria. Therefore, there is an increasing need for new antibacterial drugs with different mechanisms of action to treat bacterial infections.

[0003] In recent years, natural antimicrobial peptides, antimicrobial peptide optimization, and biomaterial - assisted delivery of antimicrobial peptides have shown great potential in treating bacterial infections. Antimicrobial peptides are a class of polypeptides with broad - spectrum antibacterial activity, usually produced by organisms in nature, with high specificity and selectivity. At concentrations that inhibit bacterial growth, they are relatively inactive against eukaryotic cells, which makes them considered an attractive alternative to traditional small - molecule drugs, especially important in dealing with drug - resistant bacterial infections. For example, the antimicrobial peptide PL - 5, as a candidate drug with broad - spectrum antibacterial activity, has been shown to be effective against a variety of bacteria, especially suitable for treating bacterial infections of the skin and wounds, especially those caused by stubborn drug - resistant bacteria. Studies have shown that the antimicrobial peptide PL - 5 can not only effectively inhibit the growth of bacteria but also promote wound healing. The results of clinical trials further support this, showing that the antimicrobial peptide PL - 5 spray is a safe and effective drug in treating skin wound infections. In addition, combined with biomaterial - assisted delivery technology, the stability and bioavailability of antimicrobial peptides in vivo can be improved, thus enhancing their therapeutic effects. These characteristics make antimicrobial peptides and their derivatives show broad application prospects in modern medicine, especially in the field of anti - infection treatment.

[0004] Existing polypeptide drugs often face potential cytotoxic risks (such as hemolysis), narrow antibacterial spectra, and insufficient inhibitory efficacy against key pathogenic bacteria: the minimum inhibitory concentration (MIC) against clinically highly harmful strains is generally high. For example, for Klebsiella pneumoniae (carbapenemase - producing strain), MIC > 64 μg / mL, and for Stenotrophomonas maltophilia (naturally multi - drug - resistant bacterium), MIC > 128 μg / mL, resulting in ultra - high doses required for actual treatment and causing serious hemolytic toxicity. These limitations severely restrict the durability of their efficacy, safety, and the feasibility of clinical application. Therefore, it is urgent to break through the existing barriers by screening and designing new polypeptides. Summary of the Invention

[0005] The present invention provides an antimicrobial peptide with the amino acid sequence KWCLRVCYRGACRRRCR (shown in SEQ ID NO.1).

[0006] The present invention also provides a composition containing the above-mentioned antimicrobial peptide.

[0007] In one embodiment, the composition includes but is not limited to drugs, wound care products, medical device coatings, preservatives, cosmetics, skin care products, washing and care products, feed additives, fabric protectants, and environmental disinfectants.

[0008] In one embodiment, the drug contains the antimicrobial peptide and pharmaceutical excipients.

[0009] In one embodiment, the pharmaceutical excipients include but are not limited to solvents, fillers, dispersants, stabilizers, plasticizers, and flavoring agents.

[0010] In one embodiment, the content of the antimicrobial peptide in the drug is ≥ 0.2 mg / g.

[0011] The present invention also provides the application of the above-mentioned antimicrobial peptide in the preparation of drugs for treating bacterial infections.

[0012] In one embodiment, the drug dosage form includes injections, oral tablets, sprays, or topical gels.

[0013] In one embodiment, the bacterial infections include but are not limited to intestinal infections, pneumonia, burn infections, or sepsis caused by one or more bacteria among Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei, and Stenotrophomonas maltophilia.

[0014] The present invention also provides the application of the above-mentioned antimicrobial peptide in the preparation of food preservatives, feed additives, or daily chemical products.

[0015] Beneficial effects: The MICs of the antimicrobial peptide screened in the present invention against Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei, and Stenotrophomonas maltophilia are 16 μg / mL, 2 μg / mL, 16 μg / mL, 4 μg / mL, 2 μg / mL, and 4 μg / mL respectively, showing broad-spectrum antibacterial performance. Its hemolytic activity and toxicity are lower than the therapeutic concentration threshold, and in terms of hemolytic activity and cytotoxicity, the key safety indicators exceed the international standards by more than 10 times (MHC 10 / MIC max = 16, IC 50 / MIC max = 35.2), and it can be used to prepare drugs for treating multi-drug resistant bacterial infections or medical products. Description of the Drawings

[0016] Figure 1 It is a graph showing the results of the erythrocyte hemolysis assay of antimicrobial peptide T.

[0017] Figure 2 It is a graph showing the results of the cytotoxicity assay of antimicrobial peptide T against human embryonic kidney HEK293T cells.

[0018] Figure 3 It is a graph showing the results of the erythrocyte hemolysis assay of antimicrobial peptide D.

[0019] Figure 4 It is a graph showing the results of the cytotoxicity assay of antimicrobial peptide D against human embryonic kidney HEK293T cells. Detailed implementation manners

[0020] 1. Strains 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.

[0021] 2. Culture media MH (Mueller-Hinton) broth culture medium: Add 2 g of beef extract powder, 17.5 g of acid-hydrolyzed casein, and 1.5 g of soluble starch to about 1000 mL of distilled water, heat and stir, and adjust the pH value to 7.2 - 7.4. Autoclave at 121 °C for 15 minutes and set aside.

[0022] DMEM culture medium is purchased from Gibco.

[0023] Example 1 Preparation of polypeptide Entrusted Sheng Gong Bioengineering (Shanghai) Co., Ltd. to prepare the polypeptide. The specific steps include: adopting the Fmoc solid-phase synthesis method to synthesize the peptide segment according to the amino acid sequence shown in SEQ ID NO.1. a) Using Rink Amide MBHA resin as the carrier, coupling Fmoc-protected amino acids with the HBTU / HOBt / DIPEA system; b) Cleavage: Treating with a mixed solution of TFA / TIS / H2O (95:2.5:2.5) for 2.5 hours; c) Precipitation: Adding -20°C methyl tert-butyl ether to the cleavage solution and centrifuging to obtain the crude peptide; d) Purifying by preparative HPLC (C18 reverse-phase column, gradient elution with acetonitrile / 0.1% TFA), performing ESI mass spectrometry detection, and freeze-drying to obtain the final product (5 mg, purity >95%).

[0024] Example 2 Inhibitory effects of the polypeptide on different microorganisms (1) Dissolve the polypeptide prepared in Example 1 with sterile water to prepare a 12.8 mg / mL mother liquor for standby, and adopt the microbroth dilution method to determine the antibacterial activity of the polypeptide.

[0025] (2) Streak the strains Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC700603, Pseudomonas aeruginosa ATCC27853, Shigella sonnei ATCC23875, Salmonella typhimurium ATCC14028, Stenotrophomonas maltophilia ATCC17666 onto blood agar plates respectively. Wait for monoclonal growth, resuspend with sterile normal saline, and adjust the turbidity to the standard of 0.5 McFarland turbidity tube, which corresponds to an initial concentration of about 1×10 8 CFU / mL. Dilute the bacterial suspension of this concentration 1:100 with MH broth to 1×10 6 CFU / mL. All operations are carried out under sterile conditions.

[0026] (3)Add 200 μL of the test drug solutions with 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. Add 100 μL of 6% DMSO to the remaining wells. Subsequently, take 100 μL of the test drug solution from wells A1 - H1 and transfer it to the second set of wells for mixing. Dilute the drug solutions successively to wells A11 - H11 in this manner, and do not add any drug to wells A12 - H12. Finally, add 100 μL of the test bacterial solution to each well, and place the 96 - well plate in an incubator at 37°C for 20 hours. After incubation, add 20 μL of 1% TTC (2,3,5 - triphenyltetrazolium chloride) to each well and let it stand at 37°C for 10 minutes for color development reaction. The results are shown in Table 1, indicating that antibacterial peptide T has good antibacterial activity against the 6 tested Gram - negative bacteria.

[0027] Table 1 Determination results of the minimum inhibitory concentration (MIC) of antibacterial peptide T against six Gram - negative bacteria

[0028] Example 3 Detection of the hemolytic activity of antibacterial peptides Collect fresh rat eyeball blood into an anticoagulant blood collection tube containing sodium heparin, centrifuge at 3000 rpm for 10 minutes at 4°C, discard the supernatant, and wash the red blood cells 3 times with physiological saline until the supernatant is no longer red. Prepare a 10% suspension of the obtained red blood cells with physiological saline for the experiment.

[0029] In the experimental group, dissolve the polypeptide with physiological saline and dilute it to 2048, 1024, 512, 256, 128, and 64 μg / mL respectively. Use physiological saline and TritonX - 100 as negative and positive controls respectively.

[0030] Add 100 μL of the 10% red blood cell suspension to the experimental group, negative control, and positive control groups, and the final reaction volume is 200 μL. Incubate each group of samples in a constant - temperature shaker at 37°C and 200 rpm for 1 hour to ensure sufficient reaction.

[0031] Incubate all samples at 37°C and 200 rpm for 1 hour. After incubation, centrifuge the samples at 4°C and 3000 rpm for 10 minutes, take the supernatant and transfer it to a 96 - well plate, and measure the optical density (OD) value at a wavelength of 540 nm using an enzyme - linked immunosorbent assay (ELISA) reader.

[0032] Hemolysis rate = (OD value of the experimental sample - OD value of the negative control group) ÷ (OD value of the positive control group - OD value of the negative control group) × 100%.

[0033] The results are as follows Figure 1 shown, and the MHC of antimicrobial peptide T 10 is 256 μg / mL, with relatively high safety.

[0034] Example 4 Cytotoxicity determination of antimicrobial peptide Using human embryonic kidney cells HEK293 as the test cell line, the antimicrobial peptide was serially diluted by the double dilution method of DMEM medium, and 8 drug gradients were set, namely 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, 3.90 μg / mL. The above groups containing different concentrations of drugs were used as the experimental groups, the group containing only cells without drugs was used as the control group, and the group containing only cells and adding DMEM medium was used as the blank group.

[0035] The above groups of cells were cultured in an incubator at 37°C and 5% CO2. The cells in the logarithmic growth phase were digested with trypsin to prepare a cell suspension and adjusted to a density of 5×10 4 cells / mL. 100 μL of the cell suspension was added to each well of a 96-well plate, and incubated overnight to allow the cells to adhere. 100 μL of drugs with different concentration gradients was added to each well, with 5 replicates for each gradient. The 96-well plate treated with the antimicrobial peptide was continued to be incubated in the incubator for 24 h. After the incubation, 20 μL of MTT solution (5 mg / mL) was added to each well and cultured for another 4 h.

[0036] After the cell culture was centrifuged at 2000 rpm for 10 min at the end of the culture, the supernatant was discarded, 150 μL of DMSO was added to each well and mixed evenly, and it was placed on a shaker and shaken at a low speed for 10 min to fully dissolve the crystals, and the absorbance at 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The formula for calculating the cell survival rate is: Cell survival rate = (absorbance of experimental wells - absorbance of blank wells) ÷ (absorbance of negative control wells - absorbance of blank wells) × 100%.

[0037] The results are as follows Figure 2 and shown in Table 2, antimicrobial peptide T shows good low cytotoxicity.

[0038] Table 2 Toxicity results of antimicrobial peptide T against human embryonic kidney cells HEK293T

[0039] Example 5 Preparation of antimicrobial peptide products Disperse carbomer 940 (1.0 g) in 80 mL of purified water. After swelling, adjust the pH to 6.5 with triethanolamine; add 10 mL of phosphate buffer solution (PBS, pH 7.0) containing 50 mg of antimicrobial peptide T, mix well, then add 5 g of glycerol and 0.1 g of ethylparaben, and make up the volume to 100 g to prepare a gel containing antimicrobial peptide T. This gel is applicable to skin wound infections, such as burn wounds infected with Pseudomonas aeruginosa.

[0040] Comparative example: Prepare antimicrobial peptide D (KWCLRLCYRGACYRRCR, shown in SEQ ID NO.2) according to the method of Example 1, and verify the inhibitory ability of antimicrobial peptide D against strains according to the method of Example 2. The results are shown in Table 3.

[0041] Table 3 Determination results of the minimum inhibitory concentration of antimicrobial peptide D against six Gram-negative bacteria

[0042] Verify the hemolytic activity of antimicrobial peptide D according to the method of Example 3. The results are as Figure 3 shown, and the MHC 10 of antimicrobial peptide D is 512 μg / mL.

[0043] Verify the cytotoxicity of antimicrobial peptide D according to the method of Example 4. The results are as Figure 4 shown, and antimicrobial peptide D shows relatively high cytotoxicity.

[0044] Table 4 Toxicity results of antimicrobial peptide D against human embryonic kidney HEK293T cells

[0045] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. An antimicrobial peptide, characterized in that, The amino acid sequence is as shown in SEQ ID NO.

1.

2. A composition containing the antimicrobial peptide according to claim 1.

3. The composition according to claim 2, wherein The composition includes drugs, wound care products, medical device coatings, preservatives, cosmetics, skin care products, washing and care products, feed additives, fabric protectants or environmental disinfectants.

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

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

6. The composition according to any one of claims 3 to 5, characterized in that The content of the antimicrobial peptide in the drug is ≥ 0.2 mg / g.

7. Use of the antimicrobial peptide according to claim 1 in the preparation of drugs for anti-bacterial infection.

8. The application according to claim 7, wherein The drug dosage form includes injections, oral tablets, sprays or topical gels.

9. The application according to claim 7, wherein The bacterial infection includes, but is not limited to, intestinal infections, pneumonia, burn infections or sepsis caused by one or more of the bacteria Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei, Stenotrophomonas maltophilia.

10. Use of the antimicrobial peptide according to claim 1 in the preparation of food preservatives, feed additives or daily chemical products.

Images