A polypeptide drug for resisting Gram-negative bacteria and its application

Abstract

The present invention discloses a polypeptide drug for use against Gram-negative bacteria and its application, belonging to the field of biomedicine. The antimicrobial peptide screened and obtained by the present invention has broad-spectrum antibacterial properties, with MICs of 16 μg / mL, 2 μg / mL, 16 μg / mL, 4 μg / mL, 2 μg / mL, and 4 μg / mL against Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei, and Stenotrophomonas maltophilia, respectively. Its hemolytic and toxic properties are below the therapeutic concentration threshold, and it can be used to prepare drugs or medical products for treating multidrug-resistant bacterial infections.

Description

Technical Field

[0001] The present invention relates to a polypeptide drug for resisting Gram-negative bacteria and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Antibiotic resistance poses a serious threat to global health. The effectiveness of common and important antibiotics is decreasing due to intrinsic or acquired resistance mechanisms. According to statistics, approximately 1.27 million people died worldwide in 2019 as a direct result of drug-resistant bacterial infections, and some experts predict that 10 million people will die by 2050. Its severity is at least as great as, and potentially even greater than, major diseases such as HIV and malaria. Therefore, there is a growing need for new antimicrobial drugs with different mechanisms of action to treat bacterial infections.

[0003] In recent years, natural antimicrobial peptides, their optimization, and biomaterial-assisted delivery have shown great potential in treating bacterial infections. Antimicrobial peptides are a class of peptides with broad-spectrum antimicrobial activity, typically produced by organisms in nature, and exhibit high specificity and selectivity. Their relative inactivity against eukaryotic cells at concentrations that inhibit bacterial growth makes them attractive alternatives to traditional small molecule drugs, particularly in combating drug-resistant bacterial infections. For example, the antimicrobial peptide PL-5, a candidate drug with broad-spectrum antimicrobial activity, has been shown to be effective against a wide range of bacteria and is particularly well-suited for treating bacterial infections of the skin and wounds, particularly those caused by recalcitrant, drug-resistant bacteria. Studies have shown that the antimicrobial peptide PL-5 not only effectively inhibits bacterial growth but also promotes wound healing. This finding is further supported by clinical trial results demonstrating that the antimicrobial peptide PL-5 spray is a safe and effective treatment for skin wound infections. Furthermore, incorporating biomaterial-assisted delivery technologies can improve the in vivo stability and bioavailability of antimicrobial peptides, thereby enhancing their therapeutic efficacy. 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 peptide drugs often face potential cytotoxicity risks (such as hemolysis), a narrow antibacterial spectrum, and insufficient inhibitory efficacy against key pathogens. Minimum inhibitory concentrations (MICs) against clinically hazardous strains are generally high, for example, MICs > 64 μg / mL against Klebsiella pneumoniae (a carbapenemase-producing strain) and > 128 μg / mL against Stenotrophomonas maltophilia (an inherently multidrug-resistant bacterium). This results in the need for ultra-high doses for actual treatment, leading to severe hemolytic toxicity. These limitations severely restrict their long-lasting efficacy, safety, and feasibility of clinical application. Therefore, there is an urgent need to overcome these barriers through the screening and design of novel peptides. Summary of the Invention

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

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

[0007] In one embodiment, the composition includes, but is not limited to, a drug, a wound care product, a medical device coating, or an environmentally friendly disinfectant.

[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 antimicrobial peptide in the drug is ≥0.2 mg / g.

[0011] The present invention also provides the use of the antimicrobial peptide in preparing antibacterial infection drugs.

[0012] In one embodiment, the pharmaceutical dosage form includes an injection, an oral tablet, a spray, or a topical gel.

[0013] In one embodiment, the bacterial infection includes but is not limited to intestinal infection, pneumonia, burn infection or sepsis caused by one or more bacteria selected from the group consisting of Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei and Stenotrophomonas maltophilia.

[0014] Beneficial effects:

[0015] The antimicrobial peptides screened and obtained by the present invention have MICs of 16μg / mL, 2μg / mL, 16μg / mL, 4μg / mL, 2μg / mL, and 4μg / mL against Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei, and Stenotrophomonas maltophilia, respectively, showing broad-spectrum antibacterial properties. Their hemolytic activity and toxicity are below the therapeutic concentration threshold, and their key safety indicators for hemolytic activity and cytotoxicity 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 or medical products for treating multidrug-resistant bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the result of the red blood cell hemolysis assay of antimicrobial peptide T.

[0017] Figure 2This is a graph showing the results of the toxicity test of antimicrobial peptide T on human embryonic kidney HEK293T cells.

[0018] Figure 3 This is the result of red blood cell hemolysis assay of antimicrobial peptide D.

[0019] Figure 4 This is a graph showing the results of the toxicity test of antimicrobial peptide D on human embryonic kidney HEK293T cells. DETAILED DESCRIPTION

[0020] 1. Strains

[0021] 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 commercial strains.

[0022] 2. Culture medium

[0023] Mueller-Hinton (MH) broth: Add 2 g beef extract powder, 17.5 g acid-hydrolyzed casein, and 1.5 g soluble starch to approximately 1000 mL of distilled water. Heat with stirring and adjust the pH to 7.2-7.4. Autoclave at 121°C for 15 minutes and set aside.

[0024] DMEM culture medium was purchased from Gibco.

[0025] Example 1 Preparation of polypeptide

[0026] Sangon Biotech (Shanghai) Co., Ltd. was commissioned to prepare the peptide. The specific steps included: synthesizing a peptide based on the amino acid sequence of SEQ ID NO. 1 using the Fmoc solid-phase synthesis method. a) Fmoc-protected amino acids were coupled to the peptide using the HBTU / HOBt / DIPEA system using Rink Amide MBHA resin as a support; b) cleavage: treatment with a mixture of TFA / TIS / H2O (95:2.5:2.5) for 2.5 hours; c) precipitation: addition of -20°C methyl tert-butyl ether to the cleavage solution, followed by centrifugation to obtain the crude peptide; d) purification by preparative HPLC (C18 reverse-phase column, acetonitrile / 0.1% TFA gradient elution), detection by ESI mass spectrometry, and lyophilization to obtain the final product (5 mg, >95% purity).

[0027] Example 2 Inhibitory effects of peptides on different microorganisms

[0028] (1) The polypeptide prepared in Example 1 was dissolved in sterile water to a 12.8 mg / mL stock solution for later use. The antibacterial activity of the polypeptide was determined by the microbroth dilution method.

[0029] (2) Streak the strains Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC700603, Pseudomonas aeruginosa ATCC27853, Shigella sonnei ATCC23875, Salmonella typhimurium ATCC14028, and Stenotrophomonas maltophilia ATCC17666 onto blood agar plates, wait for single colonies to grow, resuspend in sterile saline, and adjust the turbidity to 0.5 McFarland turbidimetric tube standard, corresponding to approximately 1×10 8 The initial concentration of CFU / mL was calculated. The bacterial suspension at this concentration was diluted with MH broth at 1:100 to 1×10 6 CFU / mL, and all operations were performed under sterile conditions.

[0030] (3) Add 200 μL of the test drug solution 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 each of the sterile 96-well plates A1-H1, and add 100 μL of 6% DMSO to the remaining wells. Subsequently, transfer 100 μL of the test drug solution from A1-H1 to the second well and mix thoroughly. Follow this method to dilute the drug solution to A11-H11 in sequence. No drug is added to wells A12-H12. Finally, add 100 μL of the test bacterial solution to each well and incubate the 96-well plate at 37°C for 20 hours. After the incubation period, add 20 μL of 1% TTC (2,3,5-triphenyltetrazolium chloride) to each well and incubate at 37°C for 10 minutes for color development. The results are shown in Table 1. Antimicrobial peptide T has good antibacterial activity against the six Gram-negative bacteria tested.

[0031] Table 1 Minimum inhibitory concentration (MIC) determination results of antimicrobial peptide T against six Gram-negative bacteria

[0032]

[0033] Example 3 Detection of Hemolytic Activity of Antimicrobial Peptides

[0034] Fresh rat eyeball blood was collected into an anticoagulant blood collection tube containing sodium heparin. Centrifuge at 3000 rpm for 10 minutes at 4°C. Discard the supernatant. Wash the red blood cells three times with saline until the supernatant no longer appears red. Prepare a 10% suspension of the resulting red blood cells in saline for experimental use.

[0035] 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 TritonX-100 were used as negative and positive controls, respectively.

[0036] 100 μL of a 10% red blood cell suspension was added to each of the experimental, negative, and positive control groups, for a final reaction volume of 200 μL. Each sample was incubated at 37°C, 200 rpm on a constant temperature shaker for 1 hour to ensure adequate reaction.

[0037] 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 a wavelength of 540 nm using a microplate reader.

[0038] 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%.

[0039] The results are as follows Figure 1 As shown, the MHC of antimicrobial peptide T 10 It is 256 μg / mL, which is relatively safe.

[0040] Example 4 Antimicrobial Peptide Cytotoxicity Assay

[0041] Human embryonic kidney HEK293 cells were used as the test cell line, and the antimicrobial peptide was gradiently diluted using the two-fold dilution method of DMEM medium. Eight drug gradients were set up, 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, and 3.90 μg / mL. The above-mentioned groups containing different concentrations of drugs were used as the experimental group, the group containing only cells without drugs was used as the control group, and the group containing only cells and DMEM medium was used as the blank group.

[0042] The cells in the above groups were cultured in a 37°C, 5% CO2 incubator. The cells in the logarithmic growth phase were digested with trypsin, and the cell suspension was prepared and the density was adjusted to 5×10 4 cells / mL, 100 μL of cell suspension was added to each well of a 96-well plate and incubated overnight to allow cells to adhere. 100 μL of different concentration gradients of drug were then added to each well, with five replicates per gradient. The 96-well plate treated with antimicrobial peptides was incubated in an incubator for another 24 h. After the incubation period, 20 μL of MTT solution (5 mg / mL) was added to each well and cultured for another 4 h.

[0043] After incubation, the cell culture was centrifuged at 2000 rpm for 10 minutes. The supernatant was discarded, and 150 μL of DMSO was added to each well, mixed thoroughly, and shaken at low speed for 10 minutes to fully dissolve the crystals. The absorbance at 490 nm was measured using a microplate reader. The cell viability was calculated as follows: Cell viability = (absorbance of experimental well - absorbance of blank well) ÷ (absorbance of negative control well - absorbance of blank well) × 100%.

[0044] The results are as follows Figure 2 As shown in Table 2, antimicrobial peptide T showed good low cytotoxicity.

[0045] Table 2 Toxicity of antimicrobial peptide T to human embryonic kidney HEK293T cells

[0046]

[0047] Example 5 Preparation of antimicrobial peptide products

[0048] Carbomer 940 (1.0 g) was dispersed in 80 mL of purified water. After swelling, the pH was adjusted to 6.5 with triethanolamine. 10 mL of phosphate buffered saline (PBS, pH 7.0) containing 50 mg of antimicrobial peptide T was added and mixed thoroughly. 5 g of glycerol and 0.1 g of ethylparaben were added to the final volume of 100 g to prepare a gel containing antimicrobial peptide T. This gel is suitable for treating skin wound infections, such as burn wounds infected with Pseudomonas aeruginosa.

[0049] Comparative Example:

[0050] Antimicrobial peptide D (KWCLRLCYRGACYRRCR, shown in SEQ ID NO. 2) was prepared according to the method of Example 1. The inhibitory ability of antimicrobial peptide D against the strain was verified according to the method of Example 2. The results are shown in Table 3.

[0051] Table 3 Minimum inhibitory concentration of antimicrobial peptide D against six Gram-negative bacteria

[0052]

[0053] The hemolytic activity of antimicrobial peptide D was verified according to the method of Example 3. Figure 3 As shown, the MHC of antimicrobial peptide D 10 It is 512 μg / mL.

[0054] The cytotoxicity of antimicrobial peptide D was verified according to the method of Example 4. The results are as follows Figure 4 As shown, antimicrobial peptide D showed higher cytotoxicity.

[0055] Table 4 Toxicity of antimicrobial peptide D to human embryonic kidney HEK293T cells

[0056]

[0057] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

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

1.

2. A composition containing the antimicrobial peptide according to claim 1, characterized in that: The composition is a medicine, a wound care product, a medical device coating or an environmentally friendly disinfectant.

3. The composition according to claim 2, characterized in that The medicine contains the antimicrobial peptide and pharmaceutical excipients.

4. The composition according to claim 3, characterized in that The pharmaceutical excipients include but are not limited to solvents, fillers, dispersants, stabilizers, plasticizers, and flavoring agents.

5. The composition according to any one of claims 2 to 4, characterized in that The content of antimicrobial peptide in the medicine is ≥0.2 mg / g.

6. Use of the antimicrobial peptide according to claim 1 in the preparation of antibacterial infection drugs, characterized in that: The bacteria are selected from the group consisting of: Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei, and Stenotrophomonas maltophilia.

7. The use according to claim 6, characterized in that The pharmaceutical dosage forms include injections, oral tablets, sprays or external gels.

8. The use according to claim 6, characterized in that The bacterial infection is intestinal infection, pneumonia, burn infection or sepsis caused by one or more bacteria selected from the group consisting of Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei and Stenotrophomonas maltophilia.

Images