Application of cetylpyridinium chloride in preparation of medicine for treating or preventing silkworm baculovirus infection

Abstract

The invention discloses application of cetylpyridinium chloride in preparation of medicine for treating or preventing silkworm baculovirus infection, the molecular formula of cetylpyridinium chloride is C21H38N. Cl, at the cellular level, when the final use concentration of a small molecule compound is 5 mu M or above, replication of BmNPV can be completely inhibited, replication of BmNPV can be continuously inhibited after infection of BmNPV to cells is completed, and the application of cetylpyridinium chloride in preparation of medicine for treating or preventing silkworm baculovirus infection is achieved. A certain treatment effect is achieved; after 10-50 mu M of medicine is sprayed to the surfaces of mulberry leaves and acts on silkworm individuals through a feeding way, the medicine has a good prevention and control effect on BmNPV, and proliferation of viruses in hosts can be effectively inhibited. The method can be used for research, development and production of drugs for preventing and controlling silkworm blood pyosis, and has application and popularization values.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and particularly relates to the application of cetylpyridinium chloride in the preparation of a drug for treating or preventing Bombyx mori baculovirus infection. Background Art

[0002] Bombyx mori nucleopolyhedrovirus (BmNPV) can cause septicemia in Bombyx mori, which is the most common and serious infectious disease in the current sericulture industry. According to incomplete statistics, the annual reduction in cocoon production due to the harm of silkworm diseases is about 15%-20%. At present, there is still no effective drug to prevent and control septicemia in Bombyx mori. In production, the preventive effect is mostly achieved by chemical disinfectants (chlorine preparations, aldehyde preparations, surfactants and lime), etc. However, these disinfectants are highly irritating to sericulturists and will cause environmental pollution. In recent years, it has been found that chemicals such as β-propiolactone, alkylated 5'-guanylic acid, nalidixic acid and fenamidone have certain preventive effects on BmNPV, but the prevention and control effects are not ideal. How to reduce the investment in drug research and development and accelerate the development and screening of antiviral drugs is of great significance for antiviral drugs for silkworms.

[0003] Traditional drug R & D has a long cycle, high cost, high risk, and high blindness. Virtual screening technology reduces the cost and time of drug discovery, can reduce a huge number of chemical molecules (millions of compounds) to a manageable potential quantity, and improves the effectiveness of drugs obtained through virtual screening according to the properties of drugs (water solubility, acid-base property, molecular weight, etc.). This technology can quickly screen out small molecules in the compound library that can fit the target protein to a large extent through complex multi-directional computational simulations, molecular docking, kinetic scoring, etc., and finally give a series of candidate antiviral drugs based on the scores. Budded virus (BV) can quickly infect among cell tissues and then cause systemic infection, and its infection speed is more than a thousand times that of occlusion derived virus (ODV). BV is mainly related to membrane fusion proteins (such as GP64, PKIP, F protein, LEF-6, etc.) during invasion. GP64 protein is a typical type I membrane fusion protein and is the key membrane fusion protein mediating the fusion of baculovirus BV and the host cell membrane. Spiky protrusions composed of GP64 protein can be observed inside the envelope of BV through electron microscopy, and these protrusions are necessary for the effective budding of BV. When GP64 is deleted, the virus cannot bud to produce infectious progeny viruses. Searching for antiviral drugs through GP64 protein can directly block infection at the virus invasion stage and is an ideal target protein for inhibiting virus proliferation. Therefore, based on virtual screening technology, the R & D and screening of anti-BmNPV drugs can be realized, which can greatly reduce the cost and cycle of drug R & D and provide a strong guarantee for the virus prevention and control and treatment in the sericulture industry. Summary of the Invention

[0004] In view of this, one of the purposes of the present invention is to provide the use of cetylpyridinium chloride in the preparation of a drug for treating or preventing baculovirus infection in silkworms; the second purpose of the present invention is to provide a drug composition for anti-BmNPV virus infection.

[0005] To achieve the above purposes, the present invention provides the following technical solutions:

[0006] 1. The use of cetylpyridinium chloride in the preparation of a drug for treating or preventing baculovirus infection in silkworms, characterized in that the molecular formula of the cetylpyridinium chloride is C 21 H 38 N·Cl, and the structural formula is as shown in formula (Ⅰ), and the baculovirus is nucleopolyhedrovirus.

[0007]

[0008] In some embodiments of the present invention, the cetylpyridinium chloride binds to the membrane fusion protein GP64 of the silkworm nucleopolyhedrovirus to block the virus from invading host cells.

[0009] In some embodiments of the present invention, the cetylpyridinium chloride is obtained by virtual screening technology, and the screening process includes the following steps:

[0010] a. Obtain the three-dimensional structure of the GP64 protein;

[0011] b. Use molecular docking software to screen candidate molecules in the compound library with a binding energy to GP64 ≤ -20 kcal / mol;

[0012] c. Verify the inhibition rate of the candidate molecules against BmNPV through in vitro antiviral experiments to obtain cetylpyridinium chloride.

[0013] In some embodiments of the present invention, the safe concentration of cetylpyridinium chloride at the individual level of silkworms is 1 μM - 100 μM.

[0014] In some embodiments of the present invention, the effective concentration of cetylpyridinium chloride at the cell level of silkworms is 1 μM - 10 μM.

[0015] In some embodiments of the present invention, the therapeutically effective concentration of cetylpyridinium chloride at the individual level of silkworms is 5 μM - 50 μM.

[0016] In some embodiments of the present invention, the prophylactically effective concentration of cetylpyridinium chloride at the individual level of silkworms is 5 μM - 10 μM.

[0017] In some embodiments of the present invention, by spraying the cetylpyridinium chloride solution onto the surface of mulberry leaves, it acts on silkworms through the ingestion pathway.

[0018] In some embodiments of the present invention, the cetylpyridinium chloride is applied to the 4th or 5th instar larval stage of silkworms.

[0019] 2. An anti-BmNPV virus infection pharmaceutical composition, comprising cetylpyridinium chloride and a pharmaceutically acceptable carrier, and the dosage form of the composition is an aqueous solution or a soluble powder.

[0020] The beneficial effects of the present invention are as follows:

[0021] The small molecule compound involved in the present invention is derived from the mainstream virtual screening technology, uses the GP64 protein of Bombyx mori nucleopolyhedrovirus as the target protein, obtains the three-dimensional structure through PDB, AlphaFold protein structure database, etc., uses the small molecule database as the ligand, obtains a series of candidate antiviral drugs through molecular docking and binding free energy evaluation, and conducts the effectiveness and safety detection of the candidate antiviral drugs, and finally screens the effective small molecule compound (cetylpyridinium chloride) in the present invention.

[0022] The molecular formula of the target small molecule of the present invention is C 21 H 38N·Cl, the structural formula is shown in formula (Ⅰ). There is no evidence in the prior art showing its therapeutic effect on Bombyx mori nucleopolyhedrovirus or similar viruses. The present invention explores its new use for preparing drugs for preventing and treating septicemia of Bombyx mori. The half cytotoxic concentration (Concentration of cytotoxicity 50%; CC 50 ) of the small molecule compound is 10 μM, and the half effective concentration (Concentration for 50% of maximal effect; EC 50 ) is 1.28 μM, and it significantly reduces the virus titer (Tissue culture infective dose 50%; TCID 50 ). When the final concentration of the small molecule compound is 7.5 μM, it can completely inhibit the replication of Bombyx mori nucleopolyhedrovirus (MOI = 1). Incubating the drug with the virus in advance and then infecting the cells can inhibit the replication of Bombyx mori nucleopolyhedrovirus, showing a preventive effect. When the final concentration of the small molecule compound is 10 μM - 50 μM when used on the silkworm body, it can inhibit the proliferation and replication of the virus in the silkworm body, so it has a prevention and control effect. When the final concentration of the small molecule compound is 5 μM - 10 μM when used on the silkworm body, it can effectively prevent the proliferation and replication of baculovirus in the silkworm body. The present invention can be used for the research and development and production of drugs for preventing and treating septicemia of Bombyx mori, and has the value of application and promotion. The present invention can be used for the research and development and production of drugs for preventing and controlling septicemia of Bombyx mori, and has the value of application and promotion.

[0023] The usage mode of the target small molecule of the present invention at the individual level is spraying. Spray it on clean mulberry leaves, and after drying, it can have a prevention and control or treatment effect. Spraying twice a day can effectively inhibit the proliferation of baculovirus in the silkworm body. Since the maximum safe concentration at the individual level is 200 μM - 300 μM, spraying 10 μM when the silkworm is in the molting stage of the fifth instar can effectively prevent the occurrence of BmNPV without affecting the economic traits of the silkworm. Through individual experiments, the present invention has effectively improved the onset situation of silkworm viruses, can effectively prevent the occurrence of virus diseases, and has important application value for the development of the sericulture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the following drawings are provided for illustration:

[0025] Figure 1For the virtual screening of BmNPV GP64 protein; A: Flow chart of virtual screening; B: Schematic diagram of the structure of GP64 protein and the drug screening area; C: Virtual screening results of FDA library and European Pharmacopoeia library; D: Virtual screening results of antiviral compound library 1; E: Virtual screening results of antiviral compound library 2; F: Virtual screening results of antiviral compound library 3; G: Binding analysis of small molecule and GP64 protein.

[0026] Figure 2 For the exploration of the effectiveness of candidate drugs; A: Analysis of the antiviral effect of 1 μM candidate drug; B: Effect of small molecule (1 μM) on the expression of viral fluorescent protein.

[0027] Figure 3 For the analysis of the antiviral performance of small molecule drugs; A: CC 50 analysis; B: EC 50 analysis; C: Analysis of the inhibition of viral nucleocapsid protein VP39 expression by small molecule; D: Analysis of the inhibition effect of different concentrations of small molecule on virus replication (gp41); E: Analysis of virus copy number; F: Analysis of the inhibition effect of small molecule (5 μM) on virus replication (gp41) (*p < 0.05, **p < 0.01).

[0028] Figure 4 For the analysis of the toxicity of small molecule drugs (200 μM, 300 μM) at the individual level; A: Statistics of the body weight of silkworms; B: Statistics of the survival rate of silkworms; C: Statistics of the economic traits of silkworms (*p < 0.05, **p < 0.01).

[0029] Figure 5 Analysis of the prevention and control effect of small molecule drugs on virus (1×10 6 per head) at the individual level; A: Statistics of the survival rate of the 5th and 4th instar larvae of silkworms infected with polyhedra treated with 10 μM small molecule; B: Analysis of the inhibition effect of small molecule on virus replication; C: Analysis of the inhibition effect of 10 μM small molecule on virus replication; D: Analysis of the inhibition effect of 50 μM small molecule on virus replication (*p < 0.05, **p < 0.01).

[0030] Figure 6 Analysis of the prevention effect of small molecule drugs (5 μM, 10 μM) on virus (1×10 5 per head) at the individual level; A: Analysis of the inhibition effect of small molecule on virus replication; B: Statistics of the clustering rate of silkworms; C: Statistics of the survival rate of silkworms; D: Statistics of the total weight of pupae and cocoons of silkworms; E: Statistics of the weight of cocoons of silkworms; F: Statistics of the weight of pupae of silkworms (*p < 0.05, **p < 0.01). Detailed implementation method

[0031] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0032] In the present invention, the molecular formula of the small molecule drug is C 21 H 38 N·Cl, and the chemical name is cetylpyridinium chloride, and the structural formula is shown in Formula (I).

[0033]

[0034] Example 1. Virtual screening to obtain candidate target drugs

[0035] The above background technology shows that antiviral drugs can be screened and studied by using the GP64 protein as the target protein. The virtual screening process is as shown in Figure 1 , A. The three-dimensional structure of the GP64 protein ([[]] Figure 1 Figure 1 , B) was obtained by software such as PDB and alphafold. The amino acid sequence of the GP64 protein is shown in SEQ ID NO.1. Small molecule drugs in the compound library (FDA library, European Pharmacopoeia library, and related antiviral compound libraries) were used as ligands for virtual screening. The GP64 protein and the ligand were optimized by software such as Autoduck and Discovery Studio (dehydration, hydrogenation, adding chemical bonds, and optimizing the structure, etc.). A series of candidate antiviral compounds ([[]] Figure 1 Figure 1 , C-F) were obtained by affinity scoring and evaluating the free binding energy of the protein and the molecule. The interaction mode between the GP64 protein and the small molecule drug of the present invention is as shown in Figure 1 , G.

[0036] The amino acid sequence of the GP64 protein is as follows (SEQ ID NO.1):

[0037] MLLVNQSYQGFDKKHTSEMVGAIVLYVLLAAAHSAFAAEHCNAQMKTGPYKIKNLDITPPKETLQKDVEITIVETDYNENVIIGYKGYYQAYAYNGGSLDPNTRVEESMKTLTVGKEDLLMWGIRQQCEVGEELIDRWGSDSEECFRDNEGRGQWVKGKELVKRQNNNHFAYHTCNKSWRCGVSTSKMYSRLECHDDTDECQVYILDAEGNPINVTVDTALHRDGVSMILKQKSTFTTRQVKAACLLIKDDKNNPESVTREHCLIDNDIYDLSKNTWNCRFNRCIKRKVEHQVKKRPPTWRHNVRAKYTEGDTATKGDLMHIQEELMYENDLLKMNIELMHAHINKINNMLHDLIVSVAKVDERLIGNLMNNSVSSTFLSDDTFLLMPCTNPPAHTSNCYNNSIYKEGRWVANTDSSQCIDFSNYKELAIDDDVEFWIPTIGNTTYHDSWKDASGWSFIAQQKSNLITTMENTKFGGVGTSLNDITSMAEGELAAKLTSFMFGHVATFVIVFIVILFLYCMVRNRNSRQY

[0038] Example 2. Efficacy of candidate drugs, CC 50 , EC 50 and TCID50 analysis

[0039] 1. Efficacy analysis of candidate drugs

[0040] The vBm-EGFP virus recombinant bacmid constructed in our laboratory previously (reference: Nachuan Liao, Zhanqi Dong, Xinling Zhang, Qi Qin, Yan Luo, Liang Huang, Peng Chen, Cheng Lu, Minhui Pan, Construction of a CRISPR / FnCas12a multi-sites editing system for inhibiting proliferation of Bombyx mori nuclear polyhedrosis virus, International Journal of Biological Macromolecules, Volume 193, Part A, 2021, Pages 585 - 591, ISSN 0141 - 8130.) was used to analyze the effectiveness of candidate drugs.

[0041] (1) Approximately 1×10 4 BmN cells were seeded in each well of a 48-well plate, and 0.6 mL of TC-100 insect cell medium containing 10% FBS (fetal bovine serum) was added to each well.

[0042] (2) After culturing at 27 °C for 48 h, it was used for subsequent analysis of the effectiveness of drugs.

[0043] (3) The stock solutions (10 mM) of all small molecule drugs were prepared using DMSO for subsequent dilution in experiments.

[0044] (4) The stock solutions were diluted to a final concentration of 1 μM, mixed with BV (MOI = 1), and then added to the 48-well plate for analyzing the effectiveness of candidate drugs.

[0045] (5) After 24 h, the plate was placed under a fluorescence microscope to observe the expression of green fluorescent protein, and it was measured by flow cytometry.

[0046] The results showed that the small molecule drug of the present invention could significantly inhibit the proliferation of the virus at 1 μM, and the inhibition rate was 36.6% ( Figure 2 , A - B).

[0047] 2. CC of small molecule drugs 50 Analysis

[0048] (1) Approximately 5×10 4 BmN cells were seeded in each well of a 24-well plate, and 1 mL of TC-100 insect cell medium containing 10% FBS (fetal bovine serum) was added to each well.

[0049] (2) After culturing at 27 °C for 48 h, small molecule drugs with final concentrations of 0, 0.01, 0.1, 1, 2.5, 5, and 10 μM were added to the cells respectively.

[0050] (3) After culturing for 24 h, CCK-8 was added to the well plate. After incubating at 27 °C for 2 h, detection was performed using a microplate reader at a wavelength of 450 nm, and CC was analyzed by Graphpad. 50 。

[0051] The experimental results showed that the small molecule of the present invention was CC 50 was 10 μM ( Figure 3 , A).

[0052] 3. EC of small molecule drug 50 Analysis

[0053] (1) Approximately 5×10 4 BmN cells were seeded in each well of a 24-well plate, and 1 mL of TC-100 insect cell medium containing 10% FBS (fetal bovine serum) was added to each well.

[0054] (2) After culturing at 27 °C for 48 h, small molecule drugs with final concentrations of 0, 0.1, 1, 2.5, 5, 10 μM and BV (MOI = 1) were added to the cells respectively.

[0055] (3) After culturing for 24 h and 48 h, the expression levels of EGFP fluorescent protein, BmNPV proliferation and replication-related gene (gp41), and nucleocapsid protein (VP39) were analyzed.

[0056] The experimental results showed that the small molecule of the present invention was EC 50 was 1.28 μM, and the small molecule drug significantly reduced the expression of gp41 and VP39 ( Figure 3 , B-D).

[0057] 4. TCID of small molecule drug 50 Analysis

[0058] (1) Approximately 5×10 4 BmN cells were seeded in each well of a 24-well plate, and 1 mL of TC-100 insect cell medium containing 10% FBS (fetal bovine serum) was added to each well.

[0059] (2) After culturing at 27 °C for 48 h, a small molecule drug with a final concentration of 2.5 μM and BV (MOI = 1) were added to the cells respectively.

[0060] (3) After culturing for 24 h, 48 h, 72 h, and 96 h, samples were collected and the virus titer change was measured by the end point method and the gp41 expression was analyzed.

[0061] The experimental results showed that the infectivity of the virus was significantly reduced after treatment with the small molecule drug, and the replication of the virus in cells was decreased ( Figure 3 , E-F). Therefore, the small molecule drug of the present invention can significantly inhibit the proliferation and replication of the virus, and has good cell safety, and the performance of antiviral drugs for silkworms can be explored at the individual level.

[0062] Example 3. Exploration of the safety of the drug in individuals

[0063] The final concentrations of the diluted small molecules were 200 and 300 μM to explore the safety of the drug in individuals

[0064] (1) Spray the small molecule drug with the above final concentration on the leaves, and feed the silkworms (Dazao strain, starting from the 4th instar) after natural drying, twice a day. The weight change and mortality of the silkworms were counted daily, and the final mortality was counted.

[0065] The experimental results showed that the 200 μM small molecule drug did not significantly change the economic traits of silkworms ( Figure 4 , C). However, the 300 μM small molecule drug significantly reduced the weight of silkworms and caused all silkworms to die and unable to spin cocoons ( Figure 4 , A-B). Therefore, it is recommended that the maximum concentration of the small molecule used subsequently be 100 μM (200 μM may be the critical value).

[0066] Example 4. Efficacy of small molecules in preventing and controlling viruses in vivo

[0067] Dilute the above small molecule mother liquor to a final concentration of 10 μM to analyze the efficacy of small molecules in preventing and controlling viruses in vivo

[0068] (1) Mix the above small molecule with virus inclusion bodies (10 6 per head) and then feed the silkworms (Dazao strain).

[0069] (2) After 6 h, spray the small molecule on the leaves, and feed after natural drying, twice a day.

[0070] (3) Count the mortality of silkworms and the expression level of gp41

[0071] The experimental results showed that the small molecule (10 μM) significantly reduced the mortality of silkworms and decreased the expression level of gp41 ( Figure 5 , A-B). The optimal prevention and control concentration of the small molecule for 4th instar silkworms is 10 μM. If the concentration of virus inclusion bodies is too high, the proliferation of the virus cannot be effectively inhibited ( Figure 5 , B). The appropriate prevention and control concentration of the small molecule for 5th instar silkworms is 10-50 μM ( Figure 5, C-D). It shows that small molecules have the effect of preventing and controlling viruses.

[0072] Example 5. Analysis of the preventive effect of small molecules on viruses

[0073] Dilute the above-mentioned small molecule mother liquor to a final concentration of 5 μM and 10 μM, and analyze the preventive effect of small molecules on viruses.

[0074] (1) After diluting the above-mentioned small molecules to a final concentration of 5 μM and 10 μM, feed them to silkworms (Dazao strain, 5th instar newly molted larvae).

[0075] (2) After 24 h, give the silkworms virus inclusion bodies to lick (10 5 per head). Spray the corresponding small molecule drug on mulberry leaves twice a day, let it dry naturally and then feed it to the silkworms. After 3 days, count the mortality rate, analyze the expression level of gp41 and the changes in the most economic traits.

[0076] The experimental results show that the small molecule drug (5 μM) has a good preventive effect and the effect is better than that of 10 μM ( Figure 6 , A). The small molecule drug (5 μM) inhibits the replication of virus gp41 and improves the survival rate of silkworms ( Figure 6 , C). At the same time, it increases the cocooning rate of silkworms by 34.7% ( Figure 6 , B), and significantly improves the economic traits of silkworms such as the total weight of cocoons and pupae, pupal weight, and cocoon weight ( Figure 6 , D-F). It shows that small molecules have a good preventive effect on virus diseases at low concentrations.

[0077] In summary, the small molecule drug of the present invention is obtained by virtual screening and has a good preventive and control effect. Especially, the preventive effect on BmNPV is better at low concentrations (5 - 10 μM). Small molecules at higher concentrations (10 - 50 μM) have a certain preventive and control effect and can inhibit the proliferation of viruses in the host body.

[0078] The above-mentioned embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Use of cetylpyridinium chloride in the preparation of a medicament for treating or preventing baculovirus infection in silkworms, characterized in that: The molecular formula of cetylpyridinium chloride is C 21 H 38 N·Cl, the structural formula is shown in formula (I), the baculovirus is a nuclear polyhedrosis virus, 2. The use according to claim 1, characterized in that: The cetylpyridinium chloride blocks the virus from invading host cells by binding to the membrane fusion protein GP64 of the Bombyx mori nuclear polyhedrosis virus.

3. The use according to claim 1, characterized in that: The cetylpyridinium chloride is obtained by virtual screening technology, and the screening process comprises the following steps: a. Obtain the three-dimensional structure of GP64 protein; b. Use molecular docking software to screen candidate molecules in the compound library with binding energy to GP64 ≤-20 kcal / mol; c. The inhibition rate of the candidate molecule on BmNPV was verified through in vitro antiviral experiments, and cetylpyridinium chloride was obtained.

4. The use according to claim 1, characterized in that: The safe concentration of cetylpyridinium chloride at the individual level of silkworms is 1 μM-100 μM.

5. The use according to claim 1, characterized in that: The effective concentration of cetylpyridinium chloride on the silkworm cell level is 1 μM-10 μM.

6. The use according to claim 1, characterized in that: The therapeutic effective concentration of cetylpyridinium chloride on individual silkworm level is 5 μM-50 μM.

7. The use according to claim 1, characterized in that: The effective concentration of cetylpyridinium chloride for preventing silkworms at the individual level is 5 μM-10 μM.

8. The use according to claim 6 or 7, characterized in that: By spraying cetylpyridinium chloride solution on the surface of mulberry leaves, it acts on silkworms through the feeding pathway.

9. The use according to claim 6 or 7, characterized in that: The cetylpyridinium chloride is applied to the 4th or 5th instar larvae of silkworm.

10. A pharmaceutical composition for resisting BmNPV virus infection, characterized in that: The composition comprises cetylpyridinium chloride and a pharmaceutically acceptable carrier, and the dosage form of the composition is an aqueous solution or a soluble powder.

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