Group of antibacterial polypeptides aiming at gram-negative bacteria

Abstract

The invention discloses a group of antibacterial polypeptides aiming at gram-negative bacteria, and belongs to the technical field of biological medicines. According to the invention, a group of antibacterial peptides is obtained through screening, and the antibacterial peptides have good broad-spectrum antibacterial performance on escherichia coli, salmonella typhimurium, pseudomonas aeruginosa, klebsiella pneumoniae, shigella sonnei and stenotrophomonas maltophilia. Wherein the hemolytic activity and toxicity of the antibacterial peptides 11A-1, 11A-4 and 11A-5 are lower than therapeutic concentration threshold values, and the antibacterial peptides 11A-1, 11A-4 and 11A-5 can be used for preparing drugs or medical products for treating multi-drug-resistant bacterial infection.

Description

TECHNICAL FIELD

[0001] The present application relates to a group of antibacterial polypeptides against gram-negative bacteria, belonging to the field of biological medicine. BACKGROUND

[0002] Since the advent of penicillin, antibiotics have been used as a common clinical drug to save countless lives, but with its widespread use even abuse, bacteria have developed drug resistance and spread widely, among which the "superbugs" showing multiple drug resistance pose a serious threat to life safety. In response to this, the World Health Organization has proposed the urgent need for new antibacterial drugs and published a list of priority pathogens, all of which are multiple drug-resistant bacteria, among which gram-negative bacteria occupy an important position, and carbapenem-resistant Acinetobacter baumannii ranks first, and is currently facing the dilemma of no available drugs.

[0003] Existing antibacterial drugs have failed to meet clinical needs, and the development of new antibiotics is slow, which is a major obstacle to defeating "superbugs". In recent years, the emergence of antibacterial peptides has brought a glimmer of hope to this difficult problem. Antimicrobial peptides, also known as host defense peptides, are an important part of the innate immune system of multicellular organisms. More than 2000 kinds of antimicrobial peptides from eukaryotes have been found. They mostly have broad-spectrum antibacterial activity, with characteristics of small molecular weight, positive charge, and amphiphilicity. The currently widely accepted mechanism of action is to exert an antibacterial effect through a multi-target membrane hydrolysis mechanism, so it is not easy to induce drug resistance. Gram-negative bacteria have a double-membrane barrier, making it difficult for many conventional drugs to enter the bacterial intracellular to exert their effects, and this feature gives antimicrobial peptides an innate advantage in killing gram-negative bacteria. Currently used antimicrobial peptides in clinical practice include daptomycin, vancomycin, and teicoplanin for treating gram-positive bacterial infections, and polymyxin B for treating gram-negative bacterial infections. However, antimicrobial peptides act on the cell membrane, although their affinity for prokaryotic cell membranes rich in negatively charged phospholipids is higher than that for eukaryotic cell membranes dominated by electrically neutral, but there is still a possibility of high cytotoxicity, which is a major factor hindering their conversion to clinical drug development.

[0004] However, existing polypeptide drugs often face potential cytotoxicity risks (such as hemolysis), narrow antibacterial spectrum, and insufficient inhibition of key pathogenic bacteria: the minimum inhibitory concentration (MIC) for clinically high-risk strains is generally high, for example: MIC > 64 μg / mL for Klebsiella pneumoniae (carbapenemase-producing strain), and MIC > 128 μg / mL for Stenotrophomonas maltophilia (naturally multiple drug-resistant bacteria), resulting in the need for high doses for actual treatment, which can cause serious hemolytic toxicity. These limitations severely restrict the durability, safety, and clinical applicability of their efficacy, so there is an urgent need to screen and design new polypeptides to break through existing barriers. SUMMARY

[0005] The present application provides antibacterial polypeptides, the amino acid sequence is as follows (1) ~ (5) any one of: (1) KWCFRVCYRGACYYRCR (as shown in SEQ ID NO. 1); (2) KWCFRVCYRGACRRRCR (as shown in SEQ ID NO. 2); (3) KWCFYVCYRGACRRRCR (as shown in SEQ ID NO. 3); (4) KWCFRLCYRGACYRRCR (as shown in SEQ ID NO. 4); (5) KWCLRVCYRGACYRRCR (as shown in SEQ ID NO. 5).

[0006] In one embodiment, the amino acid sequence of the antibacterial polypeptide is as shown in SEQ ID NO. 1, SEQ ID NO. 4 or SEQ ID NO. 5.

[0007] The present application also provides a composition containing the antibacterial peptide.

[0008] In one embodiment, the composition includes but is not limited to drugs, wound care products, medical device coatings, preservatives, cosmetics, skin care products, hair care products, feed additives, fabric protectants, environmentally friendly disinfectants.

[0009] In one embodiment, the drug contains the antibacterial peptide and pharmaceutical excipients.

[0010] In one embodiment, the pharmaceutical excipients include but are not limited to solvents, fillers, dispersants, stabilizers, plasticizers, flavorings.

[0011] In one embodiment, the content of antibacterial peptide in the drug is ≥0.2 mg / g.

[0012] The present application also provides the use of the antibacterial peptide in the preparation of antibacterial infection drugs.

[0013] In one embodiment, the drug dosage form includes injection, oral tablet, spray or external gel.

[0014] 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.

[0015] The present application also provides the use of the antibacterial peptide in the preparation of food preservatives, feed additives or daily chemical products.

[0016] Beneficial effects: The polypeptide screened by the application has broad-spectrum bacteriostatic performance, wherein the MIC of the antibacterial peptide 11A-1 to Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei and Stenotrophomonas maltophilia is 2 μg / mL, 2 μg / mL, 4 μg / mL, 4 μg / mL, 2 μg / mL and 4 μg / mL respectively. The hemolytic property and toxicity thereof are lower than the therapeutic concentration threshold, and in terms of hemolytic activity and cytotoxicity, the key safety indicators are more than 10 times higher than the international standard (MHC 10 / MIC max = 16, IC 50 / MIC max = 35.2), and can be used for preparing a multiple drug-resistant bacterial infection treatment drug or a medical product. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The figure is a result of red blood cell hemolysis determination of the polypeptide.

[0018] Figure 2 The figure is a result of toxicity determination of the polypeptide on human embryonic kidney cells HEK293T. DETAILED DESCRIPTION

[0019] 1. Strains Escherichia coli (E. coli) ATCC25922, Klebsiella pneumoniae (K. pneumoniae) ATCC700603, Pseudomonas aeruginosa (P. aeruginosa) ATCC27853, Shigella sonnei (S. sonnei) ATCC23875, Salmonella typhimurium (S. typhimurium) ATCC14028 and Stenotrophomonas maltophilia (S. maltophilia) ATCC17666 are all commercialized strains. Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Shigella sonnei Salmonella Typhimurium Stenotrophomonas maltophilia

[0020] 2. Culture medium MH (Mueller-Hinton) broth medium: add beef infusion powder 2 g, acid hydrolyzed casein 17.5 g and soluble starch 1.5 g into about 1000 mL of distilled water, heat and stir and adjust the pH value to 7.2-7.4.121 o C autoclave for 15 minutes and reserve.

[0021] DMEM medium is purchased from Gibco.

[0022] Example 1 Preparation of polypeptide ​​​​​​The polypeptide was prepared by commissioning of Zhejiang Hisun Biomedicine Pharmacy Co., Ltd. The specific steps included: using Fmoc solid-phase synthesis method, according to the amino acid sequence shown in Table 1, the peptide segment was synthesized, a) using Rink Amide MBHA resin as carrier, coupling Fmoc protected amino acid with HBTU / HOBt / DIPEA system; b) cutting: using TFA / TIS / H2O (95:2.5:2.5) mixed solution for 2.5 hours; c) elution: the cutting solution was added to -20 o C methyl tert-butyl ether, centrifugation to obtain crude peptide; d) purification by preparative HPLC (C18 reversed-phase column, acetonitrile / 0.1 %TFA gradient elution), ESI mass spectrometry detection, freeze-drying to obtain the final product (5 mg, purity >95 %).

[0023] Table 1 Polypeptide and sequence involved in the application

[0024] Wherein, “-” represents the disulfide bond formed between the amino acids.

[0025] Example 2 Inhibition of polypeptide on different microorganisms (1) The polypeptide prepared in Example 1 was dissolved in sterile water to 12.8 mg / mL stock solution for use, and the microdilution method was used to determine the antibacterial activity of the polypeptide.

[0026] (2) The strains Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC700603, Pseudomonas aeruginosa ATCC27853, Shigella sonnei ATCC23875, Salmonella typhimurium ATCC14028, and Stenotrophomonas maltophilia ATCC17666 were streaked onto blood agar plates, and when single colonies grew, they were resuspended with sterile normal saline and adjusted to a turbidity of 0.5 McFarland turbidity tube standard, which corresponded to an initial concentration of about 1×10 8 CFU / mL. The bacterial suspension at this concentration was diluted 1:100 with MH broth to 1×10 6 CFU / mL, and all operations were carried out under sterile conditions.

[0027] (3) In the sterile 96-well plate Al-Hl, 200 μL of the polypeptide or drug solution with the concentration 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 was added into each well, and 100 μL of 6% DMSO was added into the rest of the wells. Then, 100 μL of the drug solution in Al-Hl was transferred to the second well, and mixed. In this way, the polypeptide or drug solution was diluted to Al 1-Hl 1, and A12-H12 well was not added with any drug. Finally, 100 μL of the tested bacteria solution was added into each well, and the 96-well plate was placed at 37°C for 20 hours. After the incubation, 20 μL of 1% TTC (2,3,5-triphenyltetrazolium chloride) was added into each well, and incubated at 37°C for 10 minutes for color development. The results are shown in Table 2, and the polypeptide has good antibacterial activity against the six tested gram-negative bacteria.

[0028] Table 2. The results of the minimum inhibitory concentration (MIC) determination of the polypeptide against six gram-negative bacteria (unit: μg / mL)

[0029] Example 3. Hemolytic activity detection of the polypeptide The fresh eyeball blood of the rat was taken into an anticoagulation blood collection tube containing sodium heparin, centrifuged at 3000 rpm for 10 minutes at 4°C, and the supernatant was discarded. The red blood cells were washed with physiological saline for 3 times until the supernatant was not red. The obtained red blood cells were prepared into a 10% suspension solution with physiological saline for use in the test.

[0030] The polypeptide was dissolved in physiological saline and diluted to 2048, 1024, 512, 256, 128 and 64 μg / mL for the experimental group, and physiological saline and Triton X-100 were used as the negative and positive controls, respectively.

[0031] 100 μL of the red blood cell suspension solution with the concentration of 10% was added into each well of the experimental group, the negative control and the positive control, and the final volume was 200 μL. Each sample was incubated at 37°C in a constant temperature shaker at 200 rpm for 1 hour to ensure sufficient reaction.

[0032] All samples were incubated at 37°C at 200 rpm for 1 hour, and after the incubation, the samples were centrifuged at 4°C at 3000 rpm for 10 minutes. The supernatant was transferred to a 96-well plate, and the optical density (OD) value was determined at 540 nm wavelength using a microplate reader.

[0033] Hemolysis rate = (OD value of the experimental sample - OD value of the negative control) ÷ (OD value of the positive control - OD value of the negative control) x 100%.

[0034] The results are shown in Table 3. The MHC values of polypeptides 11A-1 and 11A-4 are both 250 μg / mL, which indicates low hemolysis possibility and high safety.

[0035] Table 3 MHC values of polypeptides

[0036] wherein MHC is the drug concentration causing 10% hemolysis.

[0037] Example 4 Polypeptide cytotoxicity assay Human embryonic kidney cells HEK293T were used as the test cell strain, and the polypeptides prepared in Example 1 were diluted by gradient dilution using DMEM medium. Eight drug gradients were set, i.e. 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. The cells with the above-mentioned different concentrations of drugs were used as the experimental group, the cells without drugs were used as the control group, and the cells with only DMEM medium were used as the blank group.

[0038] The cells in each group were cultured in a 37°C, 5% CO2 incubator. Logarithmic growth phase cells were trypsinized, and cell suspensions were prepared 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 the cells were incubated overnight to adhere. 100 μL of the drug at different concentrations was added to each well, with 5 replicate wells for each gradient. The 96-well plate treated with each polypeptide was further incubated in the incubator for 24 h. After incubation, 20 μL of MTT (thiazolyl blue) solution (5 mg / mL) was added to each well, and the cells were further incubated for 4 h.

[0039] The cell culture after incubation was centrifuged at 2000 rpm for 10 min, and the supernatant was discarded. 150 μL of DMSO was added to each well, mixed well, and placed on a shaker for low-speed shaking for 10 min to fully dissolve the crystals. The absorbance at 490 nm was measured using a microplate reader. The cell survival rate was calculated according to the formula: cell survival rate = (absorbance of experimental well - absorbance of blank well) ÷ (absorbance of negative control well - absorbance of blank well) × 100%.

[0040] The results are shown in Table 4 and Table 4. Figure 1 Polypeptides 11A-4 and 11A-5 showed good low cytotoxicity.

[0041] Table 4 Toxicity results of polypeptides or drugs on human embryonic kidney cells HEK293T

[0042] Example 5 Preparation of antibacterial polypeptide product Carbomer 940 (1.0 g) was dispersed in 80 mL purified water, after swelling, pH was adjusted to 6.5 with triethanolamine; 10 mL of polypeptide prepared in Example 1 containing 50 mg polypeptide in phosphate buffer (PBS, pH 7.0) was added, after mixing, 5 g of glycerol and 0.1 g of hydroxyphenyl ethyl ester were added, and the volume was made up to 100 g, to prepare a polypeptide-containing gel. The gel is suitable for skin wound infection, such as burn wound infected with Pseudomonas aeruginosa.

[0043] Although the present application has been disclosed in its preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. An antimicrobial polypeptide, characterized in that, The amino acid sequence is any one of the following (1) to (5): (1) KWCFRVCYRGACYYRCR; (2) KWCFRVCYRGACRRRCR; (3) KWCFYVCYRGACRRRCR; (4) KWCFRLCYRGACYRRCR; (5) 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, preservatives, cosmetics, skin care products, toiletries, feed additives, fabric protectants, 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.

8. The application according to claim 7, characterized in that, The bacterial infections include, but are not limited to, intestinal infections, pneumonia, burn infections, or sepsis caused by one or more of the following bacteria: Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei, and Stenotrophomonas maltophilia.

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

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

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