An antibacterial peptide cAMP-1116nc with broad-spectrum antibacterial effect and its application

Abstract

The present invention relates to the technical field of antibacterial peptide applications, and particularly relates to an antibacterial peptide cAMP-1116nc with a broad-spectrum bacteriostatic effect and its applications. The antibacterial peptide cAMP-1116nc is obtained by N-terminal acetylation modification and C-terminal amidation modification of the polypeptide shown in SEQ ID NO.1. The antibacterial peptide cAMP-1116nc of the present invention exhibits significant bacteriostatic activity against a variety of Gram-positive and Gram-negative bacteria. The antibacterial peptide cAMP-1116nc can efficiently kill the clinically common Gram-negative pathogenic bacterium Acinetobacter baumannii under the conditions of pH = 2, pH = 7.4, and pH = 8.4, and has low cytotoxicity, and has the potential to prepare antibacterial drugs, and is suitable for developing antibacterial products for various uses, including antibacterial drugs, disinfectants, and biological preservatives. The present invention provides a new functional polypeptide in the field of antibacterial peptides, which has important practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptide applications, and particularly relates to an antimicrobial peptide cAMP-1116nc with broad-spectrum antibacterial effects and its applications. Background Art

[0002] In recent years, with the exacerbation of the problem of antibiotic resistance, antimicrobial peptides have gradually become an important direction in the research of new anti-infective drugs due to their broad antibacterial spectrum, strong antibacterial activity, and low tendency to generate drug resistance. Different from traditional antibiotics, antimicrobial peptides exhibit high antibacterial ability against a variety of pathogenic microorganisms through unique mechanisms of action such as disrupting the integrity of cell membranes.

[0003] Although antimicrobial peptides have good antibacterial properties, they still face several challenges in practical applications, such as insufficient environmental stability, high toxicity, and low selectivity for certain pathogenic bacteria. To address these issues, in recent years, scientists have developed various antimicrobial peptide modification techniques, including N-terminal acetylation, C-terminal amidation, cyclization modification, introduction of unnatural amino acids, etc. These techniques have achieved remarkable results in enhancing the environmental stability, reducing toxicity, and enhancing targeting of antimicrobial peptides. However, most of the existing antimicrobial peptides have a relatively limited antibacterial spectrum. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides an antimicrobial peptide cAMP-1116nc with broad-spectrum antibacterial effects and its applications.

[0005] The present invention adopts the following technical solutions:

[0006] First, the present invention provides an antimicrobial peptide cAMP-1116nc with broad-spectrum antibacterial effects, and the antimicrobial peptide cAMP-1116nc is obtained by N-terminal acetylation modification and C-terminal amidation modification of the polypeptide shown in SEQ ID NO.1. The molecular formula of the antimicrobial peptide cAMP-1116nc is C 75 H 125 O 13 N 21 S1, with a molecular weight of 1559.95.

[0007] The present invention provides an antimicrobial peptide cAMP-1116nc. This antimicrobial peptide not only has broad-spectrum antibacterial activity but also exhibits excellent stability under various extreme environments. The antimicrobial peptide cAMP-1116nc provided by the present invention has a significant antibacterial effect against a variety of Gram-positive and Gram-negative pathogenic bacteria, and the minimum inhibitory concentrations against clinically common drug-resistant strains such as Staphylococcus aureus and Acinetobacter baumannii are as low as 8 μg / mL and 16 μg / mL respectively. At the same time, this antimicrobial peptide has good stability under pepsin conditions and shows high antibacterial activity in environments with pH = 2, pH = 7.4, and pH = 8.4. In addition, the antimicrobial peptide of the present invention has low cytotoxicity to human skin keratinocytes HaCat and bronchial epithelial cells BEAS-2B, demonstrating excellent safety.

[0008] Secondly, the present invention provides the use of the antimicrobial peptide cAMP-1116nc in the preparation of an antibacterial product for inhibiting Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, multidrug-resistant Acinetobacter baumannii, and Klebsiella pneumoniae.

[0009] Furthermore, the antibacterial product is a drug or a bacteriostatic agent.

[0010] Specifically, the antimicrobial peptide can be used as a broad-spectrum antibacterial drug for the treatment of various diseases caused by drug-resistant bacterial infections, especially suitable for combating multidrug-resistant strains, including Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, multidrug-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The bacteriostatic agent can be used in the fields of medical and health, food preservation, personal care products, and environmental disinfection.

[0011] Furthermore, the antibacterial product takes the antimicrobial peptide cAMP-1116nc as the only active ingredient.

[0012] Furthermore, the minimum inhibitory concentrations of the antimicrobial peptide cAMP-1116nc against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, multidrug-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae are 8 μg / mL, 16 μg / mL, 16 μg / mL, 16 μg / mL, 64 μg / mL, and 32 μg / mL in sequence.

[0013] Furthermore, the bacteriostatic agent is obtained by dissolving the antimicrobial peptide cAMP-1116nc in sterile water.

[0014] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0015] Furthermore, the dosage form of the drug is one of water infusion, powder, lotion, tincture, oil, emulsion, ointment, plaster or aerosol.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] Broad-spectrum and high-efficiency: The antibacterial peptide cAMP-1116nc has significant antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria, especially has remarkable effects on the clinical multi-drug resistant strains Staphylococcus aureus and Acinetobacter baumannii.

[0018] Environmental stability: The antibacterial peptide cAMP-1116nc can maintain excellent activity under pepsin conditions and in different pH environments.

[0019] Low cytotoxicity: Toxicity experiments show that the antibacterial peptide of the present invention has low toxicity to human skin keratinocytes and bronchial epithelial cells and higher safety.

[0020] Broad application prospects: The antibacterial peptide cAMP-1116nc is not only suitable for the development of anti-infective drugs, but also can be used for the preparation of food preservatives, disinfectants and other antibacterial products.

[0021] In summary, the present invention provides a functional polypeptide with novel source and excellent performance in the field of antibacterial peptides, providing important technical support and alternative resources for the development of antibacterial drugs and the treatment of multi-drug resistant bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the amino acid composition and structure of the antibacterial peptide cAMP-1116nc, A is the amino acid composition, and B is the amino acid structure.

[0023] Figure 2 It is the MIC determination results of the antibacterial peptide cAMP-1116nc against various pathogenic bacteria. In the figure, Staph 1 is Staphylococcus aureus 1, Staph 2 is Staphylococcus aureus 2, Staph 3 is Staphylococcus aureus 3, Pseudomonas is Pseudomonas aeruginosa, Acinetobacter 1 is Acinetobacter baumannii 1, Acinetobacter 2 is Acinetobacter baumannii 2, and Klebsiella is Klebsiella pneumoniae.

[0024] Figure 3 It is the stability experiment results of the antibacterial peptide cAMP-1116nc.

[0025] Figure 4 It is the cytotoxicity experiment results of the antibacterial peptide cAMP-1116nc. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0027] The pathogenic bacteria numbers and sources used in the following examples:

[0028] Staphylococcus aureus 1: ATCC12600.

[0029] Staphylococcus aureus 2: ATCC33591, a multi-drug resistant strain.

[0030] Staphylococcus aureus 3: ATCC33592, a multi-drug resistant strain.

[0031] Klebsiella pneumoniae: ATCC17978.

[0032] Acinetobacter baumannii 1: ATCC19606.

[0033] Acinetobacter baumannii 2: bio-53272, a multi-drug resistant strain.

[0034] Staphylococcus aureus 1, Staphylococcus aureus 2, Staphylococcus aureus 3, Klebsiella pneumoniae, and Acinetobacter baumannii 1 were purchased from Beijing NaChuangLian Biotechnology Co., Ltd., Acinetobacter baumannii 2 was purchased from Beijing BioWin Biotech Co., Ltd., and Pseudomonas aeruginosa was from the laboratory.

[0035] The present invention provides a broad-spectrum antibacterial peptide cAMP-1116nc against multi-drug resistant bacteria, and its amino acid sequence is shown in SEQ ID NO.1. The antibacterial peptide cAMP-1116nc exhibits high antibacterial activity under the conditions of Ph = 7.4 and Ph = 2 simulating gastric acid, and can kill a variety of Gram-positive bacteria and Gram-negative bacteria including Staphylococcus aureus, such as Acinetobacter baumannii. The minimum inhibitory concentration value MIC of the antibacterial peptide against Staphylococcus aureus is 8 μg / mL. The antibacterial peptide cAMP-1116nc has extremely high antibacterial value for medical drugs and can provide alternative medicinal resources for clinical confrontation against multi-drug resistant pathogenic bacteria.

[0036] Example 1: An antibacterial peptide cAMP-1116nc and its preparation method.

[0037] The antibacterial peptide cAMP-1116nc is obtained by N-terminal acetylation modification and C-terminal amidation modification of the polypeptide shown in SEQ ID NO.1, and its molecular formula is C 75 H 125 O 13 N 21S1 has a molecular weight of 1559.95 Da.

[0038] SEQ ID NO.1: ILKFPRKVFRCI.

[0039] The amino acid structure and related schematic diagrams are from the website of Allpeptide: https: / / www.allpeptide.com / jiegoutu.html. Since the online tool AlphaFold3 cannot predict the structure of peptides with modifications, the protein structure of the unmodified peptide of cAMP-1116nc was predicted to reflect the structure of cAMP-1116nc indirectly. The prediction results are as Figure 1 shown, indicating that this peptide is a typical α-helical peptide with amphiphilic characteristics, which helps its interaction with the bacterial membrane. The website of AlphaFold3 is: https: / / golgi.sandbox.google.com / about. Preparation method: The antimicrobial peptide cAMP-1116nc was prepared by Shanghai Sangon Biotech Co., Ltd. using solid-phase chemical synthesis method, and the purity reached >95%.

[0040] Example 2: Determination of the MIC of the antimicrobial peptide cAMP-1116nc.

[0041] The MIC of the antimicrobial peptide was determined by the broth microdilution method, referring to the guidelines of the Clinical and Laboratory Standards Institute: Wayne, P.A. Performance Standards for Antimicrobial Disk Susceptibility Tests, Clinical and Laboratory Standards Institute, 1991. The above-mentioned pathogenic bacteria were inoculated into sterile LB medium and cultured overnight at 37°C with shaking. Each pathogenic bacterium was inoculated into fresh LB medium at an inoculation amount of 1%, and cultured until the exponential phase. The cell concentration was adjusted to 1×10 5 cfu / mL to obtain the bacterial suspension. Then, 180 μL of the bacterial suspension was transferred to a 96-well plate. The antimicrobial peptide powder was dissolved in sterile water and diluted into an antimicrobial peptide solution with serial two-fold dilutions. 20 μL was taken and added to the bacterial suspension in the 96-well plate to make the antimicrobial peptide concentration range in the bacterial suspension from 0.5 μg / mL to 256 μg / mL. After the 96-well plate was incubated at 37°C for 24 hours, the growth of bacteria was detected by an enzyme-linked immunosorbent assay reader. The MIC was defined as the minimum antimicrobial peptide concentration for detecting bacterial growth. The experiment was set with 3 replicates.

[0042] As Figure 2As shown, the MICs of the antimicrobial peptide cAMP-1116nc against Staphylococcus aureus 1, Staphylococcus aureus 2, Staphylococcus aureus 3, Pseudomonas aeruginosa, Acinetobacter baumannii 1, Acinetobacter baumannii 2, and Klebsiella pneumoniae were detected to be 8 μg / mL, 16 μg / mL, 16 μg / mL, 64 μg / mL, 16 μg / mL, 16 μg / mL, and 32 μg / mL, respectively.

[0043] Example 3: Determination of the stability of the antimicrobial peptide cAMP-1116nc.

[0044] Sterile PBS solution was added to 1 mol / L HCl and NaOH solutions to prepare PBS solutions with pH = 2, pH = 7.4, and pH = 8.4. Then, the antimicrobial peptide was dissolved in PBS solutions with different pH values and incubated at 37 °C for 1 h. Subsequently, the change in the MIC of the antimicrobial peptide under different pH treatments was determined according to the method described in Example 2. The results showed that, as Figure 2 shown, pH had little effect on the antimicrobial peptide. When pH = 2 and pH = 7.4, the MIC of the peptide did not change. When pH = 8.4, the MIC of the peptide only became twice the MIC, with a small change range.

[0045] For the determination of pepsin stability, pepsin was dissolved in sterile PBS solution with pH = 2 at a final concentration of 100 μg / ml. Then, the peptide was dissolved in the above pepsin solution and treated at 37 °C for 1 h. Subsequently, it was boiled at 100 °C for 15 min to inactivate pepsin. Then, the change in the MIC of the antimicrobial peptide against Acinetobacter baumannii 1 under different pH treatments was determined according to the method described in Example 2. The results showed that, as Figure 3 shown, when the pepsin concentration was as high as 100 μg / ml, the MIC of the antimicrobial peptide did not change and it was very stable.

[0046] Example 4: Toxicity determination of the antimicrobial peptide cAMP-1116nc.

[0047] Cytotoxicity experiments of the antimicrobial peptide cAMP-1116nc were performed using human bronchial epithelial cells BEAS-2B and human keratinocytes HaCat. Cells BEAS-2B and HACAT were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and cultured in a cell incubator at 37°C with 5% CO2. Cell subculture: When the cells grew to 80% - 90%, the medium was removed, and the cells were washed 1 - 2 times with PBS. After removing the PBS, 1 ml of trypsin was added to digest the cells for 1 - 3 minutes, and then 3 ml of complete medium was added to neutralize the trypsin to terminate the digestion. The digested cells were transferred to a 15 ml centrifuge tube and centrifuged at 1000 rpm for 5 minutes. After pouring off the supernatant, 3 ml of medium was added to resuspend the cells, and the cells were passaged at a ratio of 1:3 into a culture dish for cultivation. The BEAS-2B and HACAT cells that grew to 80% - 90% in the culture dish were digested with trypsin, centrifuged at 1000 rpm, the supernatant was removed, and the cells were resuspended with complete medium. The cells were seeded into 96-well plates, with 5000 cells seeded in each well. After the cells adhered, drugs with concentrations of 0 μg / mL, 64 μg / mL, 96 μg / mL, and 128 μg / mL were added respectively. After gently mixing, the plates were placed in the incubator for 24 h. Then 10 μl of MTT with a concentration of 5 mg / ml was added, and the cells were continued to be cultured in the cell incubator for about 3 h. The medium was removed, 150 μl of DMSO was added to each well, shaken evenly, and the absorbance was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The OD values of each well were read at a wavelength of 570 nm using the ELISA reader, and the cell survival rate was calculated using the following formula:

[0048] Cell survival rate (%) = (OD of sample group / OD of blank control group) × 100.

[0049] After detection, the cytotoxicity results of the antimicrobial peptide cAMP-1116nc are as Figure 4 shown. When the concentration of the antimicrobial peptide was as high as 96 μg / ml, the survival rates of human bronchial epithelial cells BEAS-2B and human keratinocytes HaCat were still greater than 90%, indicating that the antimicrobial peptide cAMP-1116nc has extremely low cytotoxicity and great potential for medical treatment.

[0050] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they learn the basic creative concepts. Therefore, the appended claims are intended to be construed as including the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

Claims

1. An antibacterial peptide cAMP-1116nc with broad-spectrum antibacterial effect, characterized in that, The antibacterial peptide cAMP-1116nc is obtained by N-terminal acetylation modification and C-terminal amidation modification of the polypeptide shown in SEQ ID NO.

1.

2. Use of the antimicrobial peptide cAMP-1116nc according to claim 1 in the preparation of antimicrobial products, characterized in that, The antibacterial product is used to inhibit Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

3. Use of the antibacterial peptide cAMP-1116nc according to claim 2 in the preparation of antibacterial products, characterized in that, The antibacterial product is a drug.

4. Use of the antibacterial peptide cAMP-1116nc according to claim 3 in the preparation of antibacterial products, characterized in that, The drug is a bacteriostatic agent.

5. Use of the antibacterial peptide cAMP-1116nc according to claim 2 in the preparation of antibacterial products, characterized in that, The antibacterial product takes the antibacterial peptide cAMP-1116nc as the only active ingredient.

6. Use of the antimicrobial peptide cAMP-1116nc according to claim 4 in the preparation of antimicrobial products, characterized in that, The bacteriostatic agent is obtained by dissolving the antibacterial peptide cAMP-1116nc in sterile water.

7. Use of the antibacterial peptide cAMP-1116nc according to claim 3 in the preparation of antibacterial products, characterized in that, The drug also includes pharmaceutically acceptable excipients.

8. Use of the antibacterial peptide cAMP-1116nc according to claim 7 in the preparation of antibacterial products, characterized in that, The dosage form of the drug is one of aqueous infusion, powder, lotion, tincture, oil, emulsion, ointment, plaster, or aerosol.

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