Antibacterial peptide camp502nc3 with high stability and application thereof

Abstract

The present application relates to the technical field of antibacterial peptide application, and particularly relates to an antibacterial peptide CAMP502NC3 with high stability and application thereof.The antibacterial peptide CAMP502NC3 not only exhibits good antibacterial activity on staphylococcus aureus, but also can maintain certain bactericidal capacity in a wide pH environment of pH 2-8, a variety of protease environments, for example, pepsin, trypsin, chymotrypsin, and a salt concentration condition of up to 250 mM, and shows high stability and has the potential to prepare environment-stable antibacterial drugs.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide application technology, specifically relating to a highly stable antimicrobial peptide CAMP502NC3 and its applications. Background Technology

[0002] Staphylococcus aureus is a Gram-positive pathogen widely found on human skin and mucous membranes. It is one of the leading pathogens causing community and hospital-acquired infections, leading to a range of diseases from mild skin and soft tissue infections to life-threatening pneumonia, bacteremia, and endocarditis. Studies show that Staphylococcus aureus often exhibits strong drug resistance, making its clinical treatment more complex. This pathogen can adapt to various harsh environments, including high salinity and low oxygen, and can enhance its adhesion and survival capabilities within the host and on the surface of medical devices by forming biofilms, resulting in persistent infections that are difficult to eradicate.

[0003] Antimicrobial peptides, as a class of small molecule polypeptides with broad-spectrum antibacterial activity, have been regarded as a highly promising alternative to traditional antibiotics in recent years. These innate immune molecules have advantages such as unique mechanisms of action and low susceptibility to inducing drug resistance. However, most natural antimicrobial peptides suffer from poor stability. In the complex in vivo environment, they are easily inactivated by various factors: for example, under extreme pH conditions such as gastric acid, the spatial conformation of antimicrobial peptides may change, leading to decreased activity; more importantly, abundant proteases in the body (such as trypsin and pepsin) can rapidly degrade the linear structure of antimicrobial peptides; in addition, high concentrations of salt ions in the physiological environment can neutralize the positive charge of antimicrobial peptides, interfering with their electrostatic interaction with bacterial cell membranes, thereby significantly weakening their bactericidal efficacy.

[0004] Therefore, it is of great significance to develop a novel antimicrobial peptide that has highly efficient killing activity against Staphylococcus aureus and can maintain structural stability and functional integrity under different physiological conditions. Summary of the Invention

[0005] This application addresses the problems of poor stability of existing antimicrobial peptides in complex physiological environments, susceptibility to protease degradation, and significant decrease in activity under high salt or extreme pH conditions. It provides a highly stable antimicrobial peptide and its applications. An antimicrobial peptide, CAMP502NC3, has been developed, which not only exhibits good basic antimicrobial activity against Staphylococcus aureus, but also maintains a certain bactericidal ability in a wide pH range of 2-8, under various protease environments (pepsin, trypsin, chymotrypsin), and at salt concentrations up to 250 mM.

[0006] The first aspect of the present invention provides an antimicrobial peptide with high stability, the amino acid sequence of which is shown in SEQ ID NO: 1, wherein the N atom of the main chain of the lysine residue at position 3 in the sequence is methylated, the N-terminus of the antimicrobial peptide is acetylated, and the C-terminus is amidated.

[0007] Specifically, the amino acid sequence SEQ ID NO: 1 is ILKLSRFCKKLI.

[0008] Furthermore, its minimum inhibitory concentration against Staphylococcus aureus is 8 μg / mL.

[0009] Furthermore, its minimum inhibitory concentration against Staphylococcus aureus is no higher than 16 μg / mL in the pH range of 2.0 to 8.0.

[0010] Furthermore, after incubation at 37°C for 1 hour in pepsin, trypsin, or chymotrypsin at a final concentration of 100 μg / mL, the minimum inhibitory concentration against Staphylococcus aureus is no higher than 32 μg / mL.

[0011] Furthermore, its minimum inhibitory concentration against Staphylococcus aureus in a culture medium supplemented with 150 mM to 250 mM NaCl is no higher than 32 μg / mL.

[0012] A second aspect of the invention provides the use of the antimicrobial peptides described above in the preparation of bacteriostatic or bactericidal agents.

[0013] An antibacterial drug, wherein the drug has the antimicrobial peptide CAMP502NC3 as its sole active ingredient.

[0014] Furthermore, the antibacterial agent or bactericide is used to inhibit or kill Staphylococcus aureus.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] This invention provides a highly stable antimicrobial peptide and its applications. The peptide is specifically modified by N-methylation of the 3rd lysine residue in the main chain, followed by N-terminal acetylation and C-terminal amidation of the peptide backbone. The resulting antimicrobial peptide, CAMP502NC3, not only exhibits good basic antimicrobial activity against Staphylococcus aureus (MIC as low as 8 μg / mL), but more importantly, it maintains a certain bactericidal ability under a wide pH range of 2-8, various protease environments (pepsin, trypsin, chymotrypsin), and salt concentrations up to 250 mM, demonstrating high stability. Attached Figure Description

[0017] Figure 1The 2D structures of the antimicrobial peptide CAMP502NC3 and its propeptide CAMP502NC are shown.

[0018] Figure 2 The 3D structures of the antimicrobial peptide CAMP502NC3 and its propeptide CAMP502NC are shown.

[0019] Figure 3 The effects of the antimicrobial peptide CAMP502NC3 and its propeptide CAMP502NC on Staphylococcus aureus were studied; among them, Figure 3 In this context, A represents the MIC measurement result. Figure 3 B in the figure represents bacterial growth during MIC determination.

[0020] Figure 4 The results show the MIC determination of the antimicrobial peptide CAMP502NC3 and its propeptide CAMP502NC against Staphylococcus aureus under different environmental conditions; among them, Figure 4 In Figure A, the MIC measurement results are shown under different pH conditions. Figure 4 B represents the MIC determination results under different protease conditions. Figure 4 C represents the MIC measurement results under different salinity conditions.

[0021] Figure 5 The bacterial growth of antimicrobial peptides CAMP502NC, CAMP502NC3, and CAMP502NC10 was determined at MICs under pH conditions of 2, 7.4, and 8.

[0022] Figure 6 This serves as a control group for measuring the MIC of antimicrobial peptides CAMP502NC, CAMP502NC3, and CAMP502NC10 at pH=2, pH=7.4, and pH=8.

[0023] Figure 7 The bacterial growth of antimicrobial peptides CAMP502NC, CAMP502NC3, and CAMP502NC10 was determined under pepsin, trypsin, and chymotrypsin conditions.

[0024] Figure 8 This serves as a control group for measuring the MIC of antimicrobial peptides CAMP502NC, CAMP502NC3, and CAMP502NC10 under conditions of pepsin, trypsin, and chymotrypsin.

[0025] Figure 9Bacterial growth of antimicrobial peptides CAMP502NC, CAMP502NC3, and CAMP502NC10 was determined at MIC under conditions of 150 mM, 200 mM, and 250 mM NaCl.

[0026] Figure 10 This serves as a control group for determining the MIC of antimicrobial peptides CAMP502NC, CAMP502NC3, and CAMP502NC10 under conditions of 150 mM, 200 mM, and 250 mM NaCl. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0028] The first aspect of this embodiment provides an antimicrobial peptide with high stability, the amino acid sequence of which is shown in SEQ ID NO:1, and the N atom of the main chain of the lysine residue at position 3 in the sequence is modified by methylation, the N-terminus of the antimicrobial peptide is modified by acetylation, and the C-terminus is modified by amidation.

[0029] Specifically, the amino acid sequence SEQ ID NO: 1 is ILKLSRFCKKLI.

[0030] The amino acid sequence of the formed antimicrobial peptide CAMP502NC3 is: AC-IL(MLysMe)KLSRFCKKLI-NH2.

[0031] In some embodiments, the minimum inhibitory concentration against Staphylococcus aureus is not higher than 16 μg / mL in the pH range of 2.0 to 8.0. Specifically, the pH is 2, 3, 4, 5, 6, 7.4, or 8.

[0032] In some embodiments, after incubation at 37°C in pepsin, trypsin or chymotrypsin at a final concentration of 100 μg / mL, the minimum inhibitory concentration against Staphylococcus aureus is not higher than 32 μg / mL.

[0033] In some embodiments, the minimum inhibitory concentration against Staphylococcus aureus in a culture medium supplemented with 150 mM to 250 mM (150 mM, 200 mM, 250 mM) NaCl is not higher than 32 μg / mL.

[0034] A second aspect of the invention provides the use of the antimicrobial peptides described above in the preparation of bacteriostatic or bactericidal agents.

[0035] Specifically, the drug uses the antimicrobial peptide CAMP502NC3 as its sole active ingredient.

[0036] In some embodiments, the antibacterial agent or bactericide is used to inhibit or kill Staphylococcus aureus.

[0037] To better understand the technical solutions of the above embodiments, the following more detailed experimental examples are provided for further explanation.

[0038] Example 1: Physicochemical properties and preparation of antimicrobial peptide CAMP502NC3

[0039] The amino acid sequence of the antimicrobial peptide CAMP502NC3 is AC-IL(MLysMe)KLSRFCKKLI-NH2. This antimicrobial peptide contains 12 amino acids. The N-methylation modification of the main chain N atom of amino acid K at position 3 is performed, replacing a hydrogen atom (-H) on the N atom of amino acid K with a methyl group (-CH3). Simultaneously, acetylation modification is performed at the N-terminus, and amidation modification is performed at the C-terminus. The molecular formula of CAMP502NC3 is C... 72 H 129 N 19 O 14 S, with a molecular weight of 1516.90 g / mol. Since the methyl group is an uncharged group, CAMP502NC3 should have similar physicochemical properties to its original peptide CAMP502NC, with a net charge of 4, carrying 4 positive charges, and an isoelectric point of 11. The antimicrobial peptide CAMP502NC10 has similar physicochemical properties to CAMP502NC3, the only difference being the position of the N-methylated lysine residue in the sequence; the modification site for antimicrobial peptide CAMP502NC10 is position 10.

[0040] The amino acid structures and related schematic diagrams are derived from ChemDraw software. The 2D and 3D structures of CAMP502NC and CAMP502NC3 were drawn using ChemDraw software, as shown below. Figure 1 and Figure 2 As shown. Since the online tool AlphaFold3 cannot predict the structure of peptides with main-chain methylation modifications, the protein structure of the unmodified peptide CAMP502 (IKLSRFCKKLI) was predicted. The prediction results showed that CAMP502 is a typical α-helical peptide. Because (MLysMe)K is structurally similar to K, the conformation of CAMP502NC3 should also be a similar α-helical shape. In summary, the antimicrobial peptide CAMP502NC3 is an amphiphilic α-helical-like cationic peptide.

[0041] In this embodiment, the antimicrobial peptides CAMP502NC3, CAMP502NC10, and their proteptide CAMP502NC were synthesized by Sangon Biotech (Shanghai) Co., Ltd. using solid-phase chemical synthesis, with a purity greater than 95%.

[0042] Example 2: Determination of the bactericidal effect of antimicrobial peptide CAMP502NC3 against Staphylococcus aureus

[0043] 2.1 Determination of Minimum Inhibitory Concentration (MIC)

[0044] The Staphylococcus aureus used in the experiment was the Staphylococcus aureus standard strain ATCC12600. The MIC of antimicrobial peptides was determined using the broth microdilution method, following the guidelines of the Clinical and Laboratory Standards Institute (Wayne, PA Performance Standards for Antimicrobial Disk Susceptibility Tests (Clinical and Laboratory Standards Institute, 1991)). Staphylococcus aureus was inoculated into sterile CAMHB medium and cultured overnight at 37°C with shaking. A 1% inoculum of Staphylococcus aureus was then inoculated into fresh CAMHB medium, and after reaching the exponential phase, the cell concentration was adjusted to 1 × 10⁻⁶ cells / year. 5 180 μL of bacterial culture was transferred to a 96-well plate at a concentration of cfu / mL. The antimicrobial peptide powder was dissolved in sterile water and diluted to a serially diluted 2-fold antimicrobial peptide solution. 20 μL of this solution was added to the bacterial culture in the 96-well plate to achieve an antimicrobial peptide concentration range of 4-64 μg / mL. After incubating the 96-well plate at 37°C for 20 hours, bacterial growth was detected using a microplate reader. The MIC was defined as the minimum antimicrobial peptide concentration required to detect bacterial growth. The experiment was performed in triplicate. The test results are as follows: Figure 3 As shown.

[0045] The MIC of the antimicrobial peptide CAMP502NC3 and its propeptide CAMP502NC against Staphylococcus aureus was determined to be 8 μg / mL.

[0046] 2.2 Determination of the bactericidal effect of antimicrobial peptide CAMP502NC3 against Staphylococcus aureus under different pH conditions

[0047] The Staphylococcus aureus standard strain ATCC12600 was inoculated into sterile CAMHB medium and cultured overnight at 37°C with shaking. Then, 1% of the above Staphylococcus aureus standard strain ATCC12600 was inoculated into fresh CAMHB medium and cultured until the exponential phase. The cell concentration was then adjusted to 1×10⁻⁶. 5CFU / mL bacterial culture was obtained and set aside. Then, 180 μL of the bacterial culture was transferred to a 96-well plate. Antimicrobial peptide powder was dissolved and diluted with sterile PBS at different pH values ​​(pH=2, 7.4, and 8) to prepare serially diluted 2-fold antimicrobial peptide solutions. 20 μL of each solution was added to the bacterial culture in the 96-well plate to achieve an antimicrobial peptide concentration range of 4-32 μg / mL. The 96-well plate was incubated at 37°C for 12 hours, and bacterial growth (OD600) was detected using a microplate reader. The experiment was performed in triplicate. The test results are as follows: Figure 4 , Figure 5 and Figure 6 As shown.

[0048] At pH 2, the MIC of the antimicrobial peptide CAMP502NC against Staphylococcus aureus was 8 μg / mL, while the MIC of CAMP502NC3 against Staphylococcus aureus increased slightly to 16 μg / mL; at pH 7.4 and pH 8, the MICs of both antimicrobial peptides CAMP502NC and CAMP502NC3 against Staphylococcus aureus were 8 μg / mL. Figure 4 As shown, the results indicate that the N-methylated antimicrobial peptide CAMP502NC3 exhibits good pH stability, and its antimicrobial activity is only slightly affected under acidic conditions.

[0049] 2.3 Determination of the bactericidal effect of antimicrobial peptide CAMP502NC3 against Staphylococcus aureus under different protease conditions

[0050] To clarify the effect of modification sites on activity, the antimicrobial peptide CAMP502NC10 was used as a control for CAMP502NC3. Both are derived peptides with N-methylation modification on the N atom of the main chain at specific lysine residues (positions 10 and 3, respectively). Except for the modification sites, their modification types are consistent with the parent peptide CAMP502NC. The Staphylococcus aureus standard strain ATCC12600 was inoculated into sterile CAMHB medium and cultured overnight at 37°C with shaking. The above Staphylococcus aureus standard strain ATCC12600 was then inoculated into fresh CAMHB medium at a 1% inoculum and cultured to the exponential phase. The cell concentration was then adjusted to 1×10⁻⁶ cells / year. 5CFU / mL bacterial culture was obtained for later use. Pepsin (pH=2), trypsin (pH=8), and chymotrypsin (pH=8) were dissolved in sterile PBS solutions at their respective suitable pH values ​​to a final concentration of 100 μg / mL. The peptide was then dissolved in the protease solutions to a concentration of 640 μg / mL. The mixture was incubated at 37°C for 1 hour, followed by boiling at 100°C for 15 minutes to inactivate the proteases. 180 μL of the bacterial culture was then transferred to a 96-well plate. 20 μL of an antimicrobial peptide-enzyme-PBS mixture was added to the bacterial culture in the 96-well plate to achieve an antimicrobial peptide concentration ranging from 4 to 64 μg / mL. The 96-well plate was incubated at 37°C for 12 hours, and bacterial growth (OD600) was detected using a microplate reader. The experiment was performed in triplicate. The test results are as follows: Figure 4 , Figure 7 , Figure 8 As shown.

[0051] like Figure 4 As shown, under pepsin hydrolysis conditions, the MIC of the antimicrobial peptide CAMP502NC against Staphylococcus aureus was 8 μg / mL, while the MIC of CAMP502NC3 against Staphylococcus aureus increased slightly to 16 μg / mL, possibly due to the slight influence of the aforementioned acidic conditions on the antibacterial activity of CAMP502NC3. The MIC of the antimicrobial peptide CAMP502NC10 against Staphylococcus aureus was 16 μg / mL, consistent with the results for CAMP502NC3.

[0052] Under trypsin digestion conditions, the MIC of antimicrobial peptide CAMP502NC against Staphylococcus aureus was >64 μg / mL, while the MIC of antimicrobial peptide CAMP502NC3 against Staphylococcus aureus was 16 μg / mL, indicating that CAMP502NC3 was more stable to trypsin than CAMP502NC. Trypsin typically recognizes basic amino acids (such as lysine K and arginine R), but the methylation modification at the N-terminus of lysine (K) at position 3 in the CAMP502NC3 sequence may create steric hindrance, enhancing hydrophobic interactions between residues and weakening the recognition and cleavage efficiency of trypsin at this site. The MIC of antimicrobial peptide CAMP502NC10 against Staphylococcus aureus was greater than 64 μg / mL, consistent with CAMP502NC, indicating that this modification did not improve its trypsin stability.

[0053] Under chymotrypsin digestion conditions, the MICs of antimicrobial peptides CAMP502NC and CAMP502NC3 against Staphylococcus aureus were both 16 μg / mL. The MIC of antimicrobial peptide CAMP502NC10 against Staphylococcus aureus was greater than 64 μg / mL, indicating that this site modification did not improve its chymotrypsin stability.

[0054] In summary, this study confirms that the site-specific enhancement effect of N-methylation modification on the stability of antimicrobial peptides against proteases varies. Modification of the antimicrobial peptide CAMP502NC3 significantly enhanced its stability against pepsin, trypsin, and chymotrypsin, maintaining good antibacterial activity after enzymatic hydrolysis, and exhibiting significantly better tolerance to trypsin than the original peptide CAMP502NC. However, the same modification at the CAMP502NC10 site did not produce a similar stabilizing effect. These results clearly demonstrate that N-methylation modification can effectively improve the stability of antimicrobial peptides and exhibits significant site specificity.

[0055] 2.4 Determination of the bactericidal effect of antimicrobial peptide CAMP502NC3 against Staphylococcus aureus under different salinity conditions

[0056] In this experiment, the antimicrobial peptide CAMP502NC10 was used as a control for CAMP502NC3. Staphylococcus aureus was inoculated into sterile CAMHB medium and cultured overnight at 37°C with shaking. At a 1% inoculum size, the above Staphylococcus aureus was inoculated into fresh CAMHB medium containing different salinities (different concentrations of NaCl), and cultured until the exponential phase. The cell concentration was then adjusted to 1 × 10⁻⁶ cells / year. 5 Bacterial culture was obtained at CFU / mL and prepared for use. NaCl concentration gradients were set at 150 mM (8.766‰), 200 mM (11.688‰), and 250 mM (14.610‰). Then, 180 μL of the bacterial culture was transferred to a 96-well plate. Antimicrobial peptide powder was dissolved in sterile water and diluted to a serially 2-fold dilution. 20 μL of this solution was added to the bacterial culture in the 96-well plate to maintain an antimicrobial peptide concentration range of 4-32 μg / mL. The 96-well plate was incubated at 37°C for 12 hours, and bacterial growth (OD600) was detected using a microplate reader. The experiment was performed in triplicate. The test results are as follows: Figure 4 , Figure 9 , Figure 10 As shown.

[0057] like Figure 4 As shown, when NaCl (150mM-250mM NaCl) is added to the culture medium, the MIC values ​​of antimicrobial peptides CAMP502NC3, CAMP502NC10, and their proteptide CAMP502NC against Staphylococcus aureus all increase with increasing salinity, indicating that the antibacterial effects of antimicrobial peptides CAMP502NC3, CAMP502NC10, and their proteptide CAMP502NC are affected by salinity.

[0058] At 150 mM, the MIC of antimicrobial peptide CAMP502NC3 and its proteptide CAMP502NC against Staphylococcus aureus standard strain was 16 μg / mL, while the MIC of antimicrobial peptide CAMP502NC10 against Staphylococcus aureus standard strain was greater than 32 μg / mL. At 200 mM, the MIC of antimicrobial peptide CAMP502NC3 and its proteptide CAMP502NC against Staphylococcus aureus standard strain was 16 μg / mL, while the MIC of antimicrobial peptide CAMP502NC10 against Staphylococcus aureus standard strain was greater than 32 μg / mL. At 250 mM, the MIC of both proteptide CAMP502NC and antimicrobial peptide CAMP502NC10 against Staphylococcus aureus standard strain was greater than 32 μg / mL, while the MIC of antimicrobial peptide CAMP502NC3 against Staphylococcus aureus standard strain was 32 μg / mL. That is, the antimicrobial peptide CAMP502NC3 has stronger salt stability than the original peptide CAMP502NC, while the antimicrobial peptide CAMP502NC10 has poorer salt stability.

[0059] In summary, the effect of N-methylation modification on salt stability varies depending on the site. The salt stability of the antimicrobial peptide CAMP502NC3 is significantly better than that of the original peptide CAMP502NC, while no similar improvement in salt stability was observed for the antimicrobial peptide CAMP502NC10.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A highly stable antimicrobial peptide, CAMP502NC3, characterized in that, The amino acid sequence is shown in SEQ ID NO: 1, and the N atom of the main chain of the lysine residue at position 3 in the sequence is modified by methylation, the N-terminus of the antimicrobial peptide is modified by acetylation, and the C-terminus is modified by amidation.

2. The application of the antimicrobial peptide CAMP502NC3 according to claim 1 in the preparation of antibacterial agents, characterized in that, The antimicrobial peptide CAMP502NC3 is used to inhibit or kill Staphylococcus aureus.

3. The application as described in claim 2, characterized in that, Its minimum inhibitory concentration against the standard strain of Staphylococcus aureus ATCC12600 is 8 μg / mL.

4. The application as described in claim 2, characterized in that, Its minimum inhibitory concentration against the standard strain of Staphylococcus aureus ATCC12600 is no higher than 16 μg / mL in the pH range of 2.0 to 8.

0.

5. The application as described in claim 2, characterized in that, After incubation at 37°C for 1 hour in pepsin, trypsin or chymotrypsin at a final concentration of 100 μg / mL, the minimum inhibitory concentration against the standard strain of Staphylococcus aureus ATCC12600 is no higher than 32 μg / mL.

6. The application as described in claim 2, characterized in that, Its minimum inhibitory concentration against the standard strain of Staphylococcus aureus ATCC12600 in a culture medium with an additional 150 mM to 250 mM NaCl is no higher than 32 μg / mL.

7. The use of the antimicrobial peptide CAMP502NC3 according to claim 1 in the preparation of antibacterial agents or bactericides, characterized in that, The antibacterial agent is used to inhibit Staphylococcus aureus, and the bactericide is used to kill Staphylococcus aureus.

8. The application of the antimicrobial peptide CAMP502NC3 according to claim 7 in the preparation of antibacterial agents or bactericides, characterized in that, The only active ingredient is the antimicrobial peptide CAMP502NC3.

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