Application of anti-HPV16 antimicrobial peptide in preparation of antiviral drugs

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an anti-HPV16 antimicrobial peptide in preparation of an antiviral medicine. The amino acid sequence of the antimicrobial peptide Perceptide-TJ-2 is as shown in SEQ ID NO. 1, and the antimicrobial peptide Perceptide-TJ-2 is an amino acid sequence shown in SEQ ID NO. The invention evaluates the antiviral activity of the antimicrobial peptide Perceptide-TJ-2 on HPV16 in vitro, the antimicrobial peptide Perceptide-TJ-2 can effectively inhibit the infection of HPV16 on cells at a lower concentration, the drug selection index reaches 64.64, and the antimicrobial peptide Perceptide-TJ-2 has a certain application prospect and provides a new possibility for the research and development of anti-HPV treatment drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of antimicrobial peptides against HPV16 in the preparation of antiviral drugs. Background Technology

[0002] Human papillomavirus (HPV) is a double-stranded circular DNA virus with a genome of approximately 8,000 base pairs. It encodes six early regulatory proteins (E1, E2, E4, E5, E6, and E7) and two late structural proteins (L1 and L2). More than 400 types of HPV have been identified, of which at least 14 high-risk HPV types can cause cancer. HPV16 is one of the high-risk HPV types closely associated with cervical cancer, with approximately 60% of cervical cancer patients testing positive for it. Furthermore, the detection rate of HPV16 in HPV-induced squamous cell carcinoma of the head and neck exceeds 70%. HPV16 is closely related to the development of HPV-related cancers. HPV infection is limited to the basal cells of epithelial cells. Approximately 90% of HPV-infected patients experience viral clearance mediated by innate and humoral immunity within several months after infection, without causing symptoms or developing into cancer. 10% of patients have persistent infection. Persistent lesions lead to uncontrolled cell proliferation and mutation. It has been confirmed that persistent high-risk HPV infection increases the risk of cervical intraepithelial neoplasia and cancer, eventually leading to cancer.

[0003] HPV infection is one of the challenges facing public health. In clinical practice, HPV vaccination is the main method of prevention. However, there are currently no effective treatments for existing HPV infections. Therefore, it is necessary to develop antiviral agents that can effectively inhibit HPV infection.

[0004] Antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), are found in almost all known life forms, from bacteria to humans. They serve to prevent pathogens from invading the body and are an important component of the host defense system. AMPs typically consist of 10 to 60 amino acids and have diverse structures. The vast majority are cationic due to the presence of Arg and Lys amino acid residues, exhibiting strong inhibitory activity against pathogenic bacteria, multidrug-resistant fungi, and viruses.

[0005] Existing antiviral and antimicrobial peptides reported include HD5, HNP-1, and lactoferrin. As natural AMPs, they generally suffer from poor stability and high synthesis costs. Therefore, new antiviral strategies need to be developed. Summary of the Invention

[0006] To address the problems of poor stability and high synthesis cost of existing antimicrobial peptides against HPV16, this invention provides a novel antiviral strategy. Specifically, it discloses the use of the antimicrobial peptide Perceptide-TJ-2 against HPV16 in the preparation of antiviral drugs. To achieve the above objectives, this invention employs the following technical solution.

[0007] This invention provides the use of the antimicrobial peptide Perceptide-TJ-2 against HPV16 in the preparation of antiviral drugs, the amino acid sequence of which is shown in SEQ ID NO.1: GWKDFKKTIKKLLRGASRLLKF.

[0008] This invention provides the use of the antimicrobial peptide Perceptide-TJ-2, which is effective against HPV16, in the preparation of antiviral drugs, offering a novel antiviral strategy. The antimicrobial peptide Perceptide-TJ-2 exhibits high anti-HPV16 activity (IC50). 50 (0.6685 μM) Compared with existing antimicrobial peptides with anti-HPV16 function, it has better stability and lower synthesis cost, thus solving the problems of poor stability and high synthesis cost of existing antimicrobial peptides against HPV16.

[0009] Preferably, the virus includes HPV virus.

[0010] Preferably, the antimicrobial peptide Perceptide-TJ-2 is used to inhibit the proliferation of HPV virus.

[0011] Preferably, the HPV virus includes HPV16 virus.

[0012] Preferably, the half-maximal inhibitory concentration (IC50) of the antimicrobial peptide Perceptide-TJ-2 against HPV16 virus is 0.6685 µM.

[0013] Preferably, the drug uses the antimicrobial peptide Perceptide-TJ-2 as the active ingredient and is supplemented with pharmaceutically acceptable excipients.

[0014] Preferably, the excipient is selected from at least one of diluents, binders, disintegrants, lubricants, and flow aids.

[0015] Furthermore, the diluent includes any one or more of lactose, sucrose, mannitol, anhydrous dicalcium phosphate, calcium phosphate, powdered cellulose, and microcrystalline cellulose.

[0016] The adhesive comprises any one or more of hydroxypropyl methylcellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, gum arabic, alginate, gelatin, polyvinylpyrrolidone, and ethyl cellulose.

[0017] The disintegrant includes any one or more of starch, pregelatinized starch, croscarmellose sodium, glycolic acid starch sodium, and carboxymethyl cellulose sodium.

[0018] The lubricant includes any one or more of stearic acid, magnesium stearate, calcium stearate, silica, and polyethylene glycol.

[0019] The flow aid includes any one or more of micronized silica gel and talc.

[0020] Preferably, the drug is a solution; the concentration of the antimicrobial peptide Perceptide-TJ-2 in the solution is 0.1 μM to 12.5 μM.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the use of the antimicrobial peptide Perceptide-TJ-2 against HPV16 in the preparation of antiviral drugs, offering a novel antiviral strategy. The antimicrobial peptide Perceptide-TJ-2 against HPV16 provided by this invention exhibits high anti-HPV16 activity (IC50). 50 With a molecular weight of 2634.25 and a molecular weight of 0.6685 μM, this antimicrobial peptide, Perceptide-TJ-2, has an α-helix structure, is free of disulfide bonds and other protein modifications, and exhibits good stability. Compared to existing antimicrobial peptides against HPV16, this peptide represents a significant advancement with better stability and lower synthesis costs.

[0022] This invention evaluated the antimicrobial peptide Perceptide-TJ-2's antiviral activity against HPV16 in vitro. It effectively inhibited HPV16 infection of cells at a low concentration, with a drug selectivity index of 64.64, showing promising application prospects and providing new possibilities for the development of anti-HPV therapeutics. Attached Figure Description

[0023] Figure 1 This invention demonstrates the cytotoxicity of the antimicrobial peptide Perceptide-TJ-2 in 293FT.

[0024] Figure 2 The present invention uses flow cytometry to evaluate the antiviral activity of the antimicrobial peptide Perceptide-TJ-2 against HPV16 Psv; wherein: A represents the FITC positivity rate of 293FT cells that were not infected with HPV16 Psv by flow cytometry. B represents the FITC positivity rate of 293FT cells infected with HPV16 Psv by flow cytometry.

[0025] Figure 3 The present invention uses flow cytometry to evaluate the antiviral activity of the antimicrobial peptide Perceptide-TJ-2 against HPV16 Psv; wherein: After treatment with the antimicrobial peptide Perceptide-TJ-2 at a concentration of 0.1 μM, the FITC positivity rate of 293FT cells infected with HPV16 Psv was detected by flow cytometry. After treatment with the antimicrobial peptide Perceptide-TJ-2 at a concentration of 0.2 μM, the FITC positivity rate of 293FT cells infected with HPV16 Psv was detected by flow cytometry.

[0026] Figure 4 The present invention uses flow cytometry to evaluate the antiviral activity of the antimicrobial peptide Perceptide-TJ-2 against HPV16 Psv; wherein: The figure shows the FITC positivity rate of 293FT cells infected with HPV16 Psv after treatment with 0.39 μM antimicrobial peptide Perceptide-TJ-2 by flow cytometry. After treatment with the antimicrobial peptide Perceptide-TJ-2 at a concentration of 0.78 μM, the FITC positivity rate of 293FT cells infected with HPV16 Psv was detected by flow cytometry.

[0027] Figure 5 The present invention uses flow cytometry to evaluate the antiviral activity of the antimicrobial peptide Perceptide-TJ-2 against HPV16 Psv; wherein: After treatment with the antimicrobial peptide Perceptide-TJ-2 at a concentration of 1.56 μM, the FITC positivity rate of 293FT cells infected with HPV16 Psv was detected by flow cytometry. After treatment with the antimicrobial peptide Perceptide-TJ-2 at a concentration of 3.13 μM, the FITC positivity rate of 293FT cells infected with HPV16 Psv was detected by flow cytometry.

[0028] Figure 6 The present invention uses flow cytometry to evaluate the antiviral activity of the antimicrobial peptide Perceptide-TJ-2 against HPV16 Psv; wherein: After treatment with the antimicrobial peptide Perceptide-TJ-2 at a concentration of 6.25 μM, the FITC positivity rate of 293FT cells infected with HPV16 Psv was detected by flow cytometry. After treatment with 12.5 μM of the antimicrobial peptide Perceptide-TJ-2, the FITC positivity rate of 293FT cells infected with HPV16 Psv was detected by flow cytometry.

[0029] Figure 7 This is a statistical graph illustrating the flow cytometry evaluation of the antimicrobial activity of the antimicrobial peptide Perceptide-TJ-2 against HPV16 Psv in this invention; wherein: A is a statistical graph showing the FITC positivity rate of 293FT cells after treatment with different concentrations of the antimicrobial peptide Perceptide-TJ-2 by flow cytometry (n=3). B is IC 50 The calculated fitted curve.

[0030] Figure 8 The immunofluorescence assay of the antimicrobial peptide Perceptide-TJ-2 against HPV16 Psv was performed in this invention.

[0031] Figure 9 This invention uses molecular docking to simulate the interaction between HPV16 L1 protein and the antimicrobial peptide Perceptide-TJ-2; wherein: A is a molecular docking simulation diagram; B is a magnified view of the binding site.

[0032] Figure 10 The surface plasmon resonance technique used in this invention was used to detect the affinity between HPV16 L1 protein and the antimicrobial peptide Perceptide-TJ-2. Detailed Implementation

[0033] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0034] Example 1 1. Obtaining the antimicrobial peptide Perceptide-TJ-2 The antimicrobial peptide Perceptide-TJ-2 was designed and synthesized using artificial intelligence methods in the early stages, as detailed in Chinese Patent No. CN117024530 B.

[0035] The amino acid sequence of the antimicrobial peptide Perceptide-TJ-2 is shown in SEQ ID NO.1: GWKDFKKTIKKLLRGASRLLKF.

[0036] 2. Effects of the antimicrobial peptide Perceptide-TJ-2 on cytotoxicity Susceptible cells (293FT) of HPV16 pseudovirus were seeded in 96-well plates at a density of 8 × 10⁸ cells per well. 3 24 hours after inoculation, the culture medium was discarded, and different concentrations of the antimicrobial peptide Perceptide-TJ-2 (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM, and 0.78 μM), serially diluted using complete culture medium, were added as the experimental groups. Simultaneously, a culture medium control group (susceptible 293FT cells with culture medium added but not inoculated with HPV16 pseudovirus) and a blank control group (susceptible 293FT cells with HPV16 pseudovirus inoculated but without the addition of antimicrobial peptide Perceptide-TJ-2) were set up.

[0037] The experimental group, culture medium control group, and blank control group were incubated at 37℃ and 5% (v / v) CO2 for 24 h. Then, 10% (v / v) CCK8 reagent was added to each well, and the cells were incubated for 1 h. The absorbance was measured at 450 nm using a microplate reader. Cell viability at 293FT was calculated using GraphPad Prism software. 50 This indicates the half-maximal cytotoxicity concentration (MCC) of the antimicrobial peptide Perceptide-TJ-2.

[0038] The results are as follows Figure 1 The results showed that the antimicrobial peptide Perceptide-TJ-2 had a CC value of 293 FT. 50 The value is 43.21 μM.

[0039] 3. In vitro experiments to evaluate the antiviral activity of the antimicrobial peptide Perceptide-TJ-2 against HPV16 Psv. 3.1 Flow cytometry evaluation of the antimicrobial activity of the antimicrobial peptide Perceptide-TJ-2 against HPV16 Psv In a 48-well plate, 293 FT cells were seeded in each well, with 2 × 10⁶ cells per well. 424 h after inoculation, HPV16 Psv was mixed with serially diluted concentrations of the antimicrobial peptide Perceptide-TJ-2 (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM, and 0.78 μM) at room temperature and incubated for 30 min. Cell culture medium was then added to form the experimental group. Two h after adding the cell culture medium, the medium was replaced with complete medium. An HPV16 Psv group (cells infected with the same titer of HPV16 Psv) and a Mock group were also established. 72 h after HPV16 Psv infection, 293FT cells were digested with trypsin and then collected to obtain a 293FT cell suspension. The 293FT cells were washed once with PBS, resuspended, and the proportion of GFP-positive cells (HPV16 Psv-infected cells) was detected by flow cytometry.

[0040] The cell culture medium was DMEM medium, and the complete culture medium was DMEM medium supplemented with 10% (v / v) FBS.

[0041] The results are as follows Figures 2-7 The results showed that the antimicrobial peptide Perceptide-TJ-2 has antiviral activity against HPV16 Psv in a dose-dependent manner. Within a certain concentration range, the higher the concentration of the antimicrobial peptide Perceptide-TJ-2, the better the inhibitory effect on HPV16 Psv.

[0042] The IC50 of the antimicrobial peptide Perceptide-TJ-2 against HPV16 Psv was calculated using GraphPad Prism. 50 The molecular weight is 0.6685 μM, and the calculated selection index SI is 64.64.

[0043] The above results indicate that the antimicrobial peptide Perceptide-TJ-2 has good prospects for drug development against HPV16.

[0044] 3.2 Immunofluorescence evaluation of the antimicrobial activity of the antimicrobial peptide Perceptide-TJ-2 against HPV16 Psv In a 96-well plate, 293 FT cells were seeded in each well, with 8 × 10⁸ cells per well. 324 h after inoculation, HPV16 Psv was mixed with serially diluted concentrations of the antimicrobial peptide Perceptide-TJ-2 (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM, and 0.78 μM) at room temperature and incubated for 30 min. Cell culture medium was then added, forming the experimental group. Two h after adding the cell culture medium, the medium was replaced with complete medium. Simultaneously, an HPV16 Psv group (cells infected with the same titer of HPV16 Psv) and a mock group were set up. 72 h after HPV16 Psv infection, cell infection at different Perceptide-TJ-2 concentrations was imaged using a cell microplate imaging system.

[0045] The results are as follows Figure 8 The results showed that the antimicrobial peptide Perceptide-TJ-2 has antiviral activity against HPV16 Psv in a dose-dependent manner.

[0046] 4. The antimicrobial peptide Perceptide-TJ-2 binds to HPV16 L1 protein. The interaction between HPV16 capsid protein L1 and the antimicrobial peptide Perceptide-TJ-2 was investigated to explore possible mechanisms against HPV16 infection.

[0047] Molecular docking analysis was performed using AutoDock Vina 1.1.2 software. First, the HPV16 L1 protein structure was hydrogenated using PyMOL Academic Open Source Edition. Then, the processed protein and peptide were converted to PDBQT format using ADFRsuite 1.0 to meet docking requirements. During docking, the processed HPV16 L1 protein and the antimicrobial peptide Perceptide-TJ-2 PDBQT files, along with the docking grid, were used as input files. The global search detail was set to 32, while other parameters remained at their default values. After docking, the conformation with the highest score and optimal binding energy was selected as the final binding mode, and the docking results were visualized and analyzed using PyMOL.

[0048] The results are as follows Figure 9 The results showed that the binding energy between HPV16 L1 protein and the antimicrobial peptide Perceptide-TJ-2 was predicted to be -4.9 kcal / mol, and the two may interact through hydrogen bonds and other mechanisms.

[0049] To detect the interaction between HPV16 L1 protein and the antimicrobial peptide Perceptide-TJ-2, this invention uses surface plasmon resonance technology to immobilize HPV16 L1 on a CM5 chip. Five concentrations (6.25 μM, 3.13 μM, 1.56 μM, 0.78 μM, and 0.39 μM) of the antimicrobial peptide Perceptide-TJ-2 are used as the mobile phase. The binding time is 120 s and the dissociation time is 120 s, and the affinity between the two is detected.

[0050] The results are as follows Figure 10 The results showed that the HPV16 L1 protein interacts with the antimicrobial peptide Perceptide-TJ-2, with a binding constant K. D =5.397×10 -6 M. This binding may block HPV16 from entering cells, thus exerting antiviral activity.

[0051] The experimental results above indicate that the antimicrobial peptide Perceptide-TJ-2 has an inhibitory effect on HPV16, and its effect may be to exert antiviral activity by blocking HPV16 from entering cells.

[0052] This invention provides the use of the antimicrobial peptide Perceptide-TJ-2 against HPV16 in the preparation of antiviral drugs, offering a novel antiviral strategy. The antimicrobial peptide Perceptide-TJ-2 against HPV16 provided by this invention exhibits high anti-HPV16 activity (IC50). 50 With a molecular weight of 2634.25 and a molecular weight of 0.6685 μM, this antimicrobial peptide, Perceptide-TJ-2, has an α-helix structure, is free of disulfide bonds and other protein modifications, and exhibits good stability. Compared to existing antimicrobial peptides against HPV16, this peptide represents a significant advancement with better stability and lower synthesis costs.

[0053] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0054] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. Use of the antimicrobial peptide Perceptide-TJ-2 against HPV16 for the preparation of a medicament against viruses, characterized in that, The amino acid sequence of the antimicrobial peptide Perceptide-TJ-2 is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, The virus includes an HPV virus.

3. Use according to claim 2, characterized in that, The antimicrobial peptide Perceptide-TJ-2 is used for inhibiting the proliferation of the HPV virus.

4. Use according to claim 3, characterized in that, The HPV virus includes an HPV16 virus.

5. Use according to claim 4, characterized in that, The half effective inhibitory concentration of the antimicrobial peptide Perceptide-TJ-2 on the HPV16 virus is 0.6685 µM.

6. Use according to claim 1, characterized in that, The medicine takes the antimicrobial peptide Perceptide-TJ-2 as an active ingredient, and is supplemented with a pharmaceutically acceptable excipient.

7. Use according to claim 6, characterized in that, The excipient is selected from at least one of diluents, binders, disintegrants, lubricants and glidants.

8. Use according to claim 6, characterized in that, The medicine is a solution; the concentration of the antimicrobial peptide Perceptide-TJ-2 in the solution is 0.1 µM~12.5 µM.

Images