Use of zinc pyrithione in the preparation of a drug for treating Japanese encephalitis virus infection

Abstract

The application provides application of zinc pyrithione in preparation of a medicine for treating Japanese encephalitis virus infection. The application has the beneficial effect that the zinc pyrithione has significant inhibitory effect on NS2B-NS3 protease activity in the Japanese encephalitis virus, can be used as an inhibitor of the NS2B-NS3 protease in the Japanese encephalitis virus, and is expected to become a potential medicine for resisting Japanese encephalitis virus infection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of anti-Japanese encephalitis medicine, and particularly relates to application of zinc pyrithione in preparation of a medicine for treating Japanese encephalitis virus infection. BACKGROUND

[0002] Japanese encephalitis (JE) is an acute infectious disease caused by Japanese encephalitis virus (JEV). The mortality and disability rate of Japanese encephalitis are high, and Japanese encephalitis is one of the main infectious diseases threatening the health of people, especially children. The peak season of Japanese encephalitis is summer and autumn, and the distribution of Japanese encephalitis is closely related to the distribution of the vector mosquitoes. China is a high-incidence area of Japanese encephalitis. In the 1960s and early 1970s, a large epidemic of Japanese encephalitis occurred in China. With the increase of the vaccination rate, the incidence of Japanese encephalitis has been greatly reduced, but nearly ten thousand cases are still reported every year.

[0003] JEV is mainly transmitted through mosquito bites, and Culex tritaeniorhynchus is the main transmission medium. Since mosquitoes can carry viruses over winter and can be transmitted through eggs, mosquitoes are not only transmission media, but also long-term storage hosts. Pigs and waterfowl are amplification hosts of JEV, and can develop into high viral blood after being infected with JEV, and then infect mosquitoes again. Most of the infected patients have no obvious symptoms at the beginning, and then may have high fever, accompanied by symptoms such as consciousness, nausea and vomiting. Severe cases can develop into convulsions, coma, paralysis and the like. JEV can cause extensive lesions in the brain parenchyma, and the lesions in the cerebral cortex, brainstem and basal nucleus are most obvious, which brings great challenges to the recovery of patients. Since there is no specific drug for treating Japanese encephalitis at present, doctors can only treat the symptoms, such as antiviral, anti-infection, cooling, dehydration and the like, so the research on the inhibitors of JEV is an important task at present.

[0004] JEV belongs to the flavivirus genus and is an RNA enveloped virus with a single-stranded positive-sense RNA genome of approximately 11 kb in length. The open reading frame in the genomic RNA of JEV encodes a polyprotein precursor of approximately 3432 amino acids, which is subsequently cleaved into three structural proteins and seven non-structural proteins by its own NS3 protease or host proteases. The JEV NS3 protease is highly conserved among JEV genotypes and has two domains, the N-terminal protease domain and the C-terminal RNA helicase domain. The JEV NS2B protein is a cofactor of the NS3 protein and is related to the stability of NS3 and substrate recognition. The N-terminal protease domain (180 residues) of the NS3 protein directly interacts with the central hydrophilic domain of NS2B, which is about 40 amino acids in length, to form an active serine protease. The active NS2B-NS3 protease is essential for the processing of viral polyprotein and plays a crucial role in viral replication and pathogenesis. Therefore, NS2B-NS3 can be an important target for studying inhibitors of Japanese encephalitis virus.

[0005] Zinc pyrithione, also known as Zinc pyrithione, is an excellent anti-scaling agent and anti-overflow agent, can effectively kill the fungi that produce dandruff, has the effects of relieving itching, removing dandruff, reducing hair loss and delaying the production of white hair, is a high-efficiency and safe anti-itching and anti-dandruff agent, and can also be used as an excellent, broad-spectrum, low-toxicity and environmentally-friendly fungicide and bacteriostatic agent, and is widely used in fields such as civilian coatings, adhesives and carpets. However, so far, the application of zinc pyrithione in the treatment of Japanese encephalitis virus infection has not been reported. SUMMARY

[0006] The first object of the present application is to provide the application of zinc pyrithione in the preparation of a drug for treating Japanese encephalitis virus infection, which can be used as a small molecule inhibitor of NS2B-NS3 protease in Japanese encephalitis virus. At the molecular level, by setting up a negative control, it is found that zinc pyrithione has good inhibitory activity on NS2B-NS3 protease in Japanese encephalitis virus, so the compound has the potential to become a potential drug for inhibiting Japanese encephalitis virus infection.

[0007] Another object of the present application is to provide a drug for treating Japanese encephalitis virus infection.

[0008] To solve the above technical problems, the technical scheme adopted by the present application is that zinc pyrithione is applied in the preparation of a drug for treating Japanese encephalitis virus infection.

[0009] Preferably, the zinc pyrithione is a small molecule inhibitor of NS2B-NS3 protease in Japanese encephalitis virus.

[0010] Preferably, the structure of the zinc pyrithione is as follows:

[0011]

[0012] A medicine for treating Japanese encephalitis virus infection, comprising the zinc pyrithione of claim 1 and one or more pharmaceutically acceptable carriers.

[0013] Preferably, the carrier comprises one or more of diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorptive carriers, lubricants and synergists.

[0014] Preferably, the preparation of the medicine is an injection, a tablet, a pill, a capsule, a suspension or an emulsion.

[0015] According to the above technical solution, it can be concluded that zinc pyrithione can be used as a small molecule inhibitor of NS2B-NS3 protease in Japanese encephalitis virus, and zinc pyrithione has a significant inhibitory effect on the activity of Japanese encephalitis virus NS2B-NS3 protease, thereby enabling the development of potential drugs for treating Japanese encephalitis virus through a new approach. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a schematic diagram of the inhibitory effect of zinc pyrithione of the embodiment of the present application on Japanese encephalitis virus NS2B-NS3 protease

[0017] Figure 2 is the IC of the inhibitory effect of zinc pyrithione of the embodiment of the present application on Japanese encephalitis virus NS2B-NS3 protease 50 determination schematic diagram DETAILED DESCRIPTION

[0018] The present application will be further described below in conjunction with the embodiments and the accompanying drawings:

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in the description herein is for describing specific embodiments and the exemplary examples only and is not intended to be limiting of the present application. It should be specially noted that there can be multiple names for the same organic structure, as long as the structure is within the scope of the present patent, it is within the protection scope of the present patent.

[0020] Unless otherwise defined, the raw materials, reagents, etc. in the following examples and comparative examples can be obtained from the market or prepared according to the reported methods.

[0021] First, the expression and purification of Japanese encephalitis virus NS2B-NS3 protease are carried out:

[0022] S1: Obtain the gene sequence of JEV NS2B and NS3 from NCBI website, and use G4S G4 linker to connect the two protein genes, construct into pET-15b vector, obtain the recombinant plasmid for sequencing identification, then transform the plasmid with correct sequence into E. coli BL21 (DE3) cells.

[0023] S2: Spread the transformed BL21 (DE3) cells on plates containing ampicillin, and incubate in an incubator overnight. Then pick single colonies into small test tubes of LB medium for activation culture for 5-6 h, and transfer to large bottles of LB medium for expansion culture. Incubate at 37℃ until the OD 600 of the bacterial solution is about 0.6. Then reduce the temperature of the incubator to 16℃, and add isopropyl thiogalactoside for induction expression for 16-18 h.

[0024] S3: Transfer the induced bacterial solution to a collection barrel, and use a large floor centrifuge to centrifuge and collect the bacterial cells. Discard the supernatant, and resuspend the precipitated bacterial cells in a bacterial disruption buffer (50 mM Tris, 300 mM NaCl, pH 7.5). Use an ultrasonic cell disrupter to disrupt the bacteria, and finally use a low-temperature high-speed centrifuge to centrifuge at 4℃ and 10,000 rpm.

[0025] S4: Pour the obtained supernatant into an affinity chromatography column containing Ni-NTA filler, and use low-concentration and high-concentration imidazole buffer to wash and elute the impurities, obtain the target protein, and perform polyacrylamide gel electrophoresis verification.

[0026] S5: Further purify using anion exchange chromatography and size exclusion chromatography to obtain high-purity target protein with uniform charge.

[0027] Obtain the purified Japanese encephalitis NS2B-NS3 protein, and establish a stable inhibitor screening system for Japanese encephalitis NS2B-NS3 protein based on the principle of fluorescence resonance energy transfer:

[0028] Preparation of fluorescent substrate: use a 95% pure Bz-Nle-Lys-Arg-Arg-AMC polypeptide compound (purchased from Shanghai Jier Biochemical Co., Ltd.) as the fluorescent substrate; then determine the enzyme activity buffer composition as 20 mM Tris, 30% glycerol, 0.5 mM CHAPS, pH 9.0, for diluting the above purified Japanese encephalitis NS2B-NS3 protein;

[0029] The drug screening system consisted of 100 μL, including 89 μL of JEV NS2B-NS3 pro solution (final concentration 2 μM), 1 μL of compound (final concentration 20 μM), and 10 μL of fluorescent substrate (final concentration 20 μM). The fluorescence intensity was measured using an Infinite M1000 Pro full-wavelength multi-functional microplate detector, with incident and emission wavelengths of 355 nm and 460 nm, respectively.

[0030] The screening steps are as follows:

[0031] S1: First, add 89 μL of protein solution and 1 μL of compound to a 96-well black microplate, incubate at 37°C for 5 min, then quickly add 10 μL of fluorescent substrate and monitor the fluorescence using a microplate reader.

[0032] A negative control experiment was also designed, replacing 1 μL of the compound with 1 μL of 95% DMSO, while keeping other experimental conditions unchanged.

[0033] S2: The enzyme kinetic curve was measured using a microplate reader. The slope of the first 300 seconds was analyzed as the initial velocity of the enzyme-catalyzed reaction. V0 was set as the initial velocity without inhibitors. i The initial rate of inhibitor addition was used; based on the initial rate, the inhibition rate (Ir) of each compound against JEVNS2B-NS3 pro was calculated (1-V). i / V0) and residual activity Ra (ResidualActivity, Ra)(V i / V0).

[0034] S3: For compounds with an inhibition rate (Ir) > 70%, a second screening and fluorescence quenching experiment are required to eliminate false positive results caused by operational errors. First, add 89 μL of protein solution to a 96-well black ELISA plate and incubate at 37°C for 5 min. Then, quickly add 10 μL of fluorescent substrate and monitor the fluorescence using an ELISA reader until the fluorescence intensity no longer changes significantly. Record the fluorescence value at this point as Q1. Next, add 1 μL of inhibitor to the ELISA plate and record the current fluorescence value as Q2. Then, use the formula:

[0035] Q r = (Q1-Q2) / Q 2* 100%

[0036] The fluorescence quenching rate Q was calculated. r If the fluorescence quenching rate is less than 20%, a false positive can be ruled out; if the fluorescence quenching rate is greater than or equal to 20%, it is a false positive.

[0037] like Figure 1As shown, the pyrithione zinc is substituted, the inhibition rate Ir of pyrithione zinc to JEV NS2B-NS3 pro is more than 90%, and the fluorescence quenching rate in the fluorescence quenching experiment is less than 20%. It can be determined that pyrithione zinc can be used as an inhibitor to inhibit Japanese encephalitis protein, and the inhibition rate is high, which can play a good role.

[0038] The IC 50 of pyrithione zinc was determined:

[0039] First, the protein JEV NS2B-NS3 pro required for the experiment was diluted to a final concentration of 2 μM, and the substrate Bz-Nle-Lys-Arg-Arg-AMC was prepared to a final concentration of 20 μM.

[0040] According to the results of the primary screening of the drug screening system, the final concentration of the inhibitor was roughly set to 17, which were 320 μM, 160 μM, 80 μM, 40 μM, 20 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.312 μM, 0.156 μM, 0.078 μM, 0.039 μM, 0.019 μM, 0.010 μM, 0.005 μM, 0.002 μM, respectively.

[0041] JEV NS2B-NS3 pro was mixed with pyrithione zinc at various concentrations and incubated at 37°C for 10 min, then 10 μL of fluorescent substrate was added, and the fluorescence value-time curve was recorded by the enzyme marker. The initial rate of JEV NS2B-NS3 pro reaction was analyzed using Graphpad prism 8.0 software, and the relationship curve of pyrithione zinc concentration reciprocal value and inhibition rate Ir was obtained, and finally the IC 50 value was calculated.

[0042] As Figure 2 shown, the IC 50 was 0.53±0.21 μM when the substrate concentration was 20 μM. It has great application potential in the preparation of small molecule inhibitors of NS2B-NS3 protease of Japanese encephalitis virus, and has the hope of becoming a potential drug for anti-Japanese encephalitis virus infection.

[0043] According to the above experiment, pyrithione zinc can be used as an inhibitor of Japanese encephalitis virus NS2B-NS3 protease, so a drug for preventing or treating Japanese encephalitis virus infection can be prepared, and the active ingredient is pyrithione zinc. The drug contains pyrithione zinc and one or more pharmaceutically acceptable carriers.

[0044] The carrier includes diluents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surface active agents, adsorptive carriers, lubricants, synergists, which are conventional in the field of pharmacy. The medicine can be used in the form of injection, tablet, pill, capsule, suspension or emulsion. The administration route can be oral, transdermal, intravenous or intramuscular injection.

[0045] The above detailed description of the embodiments of the present application is only a preferred embodiment of the present application, and cannot be considered as limiting the scope of the present application. Any equivalent changes and improvements made within the scope of the present application should still belong to the patent scope of the present application. Moreover, the professional terms and other materials involved in the present application are only used to clearly describe the advantages and effects of the present application, and should not be considered as a limitation of the innovation of the present application. The above embodiments are only part of the practical application effects of the present application, and are not used to limit the patent scope of the present application. Any improvements and substitutions made by the person skilled in the art on the basis of the present application should still belong to the protection scope of the present application.

Claims

1. Use of zinc pyrithione in the preparation of a medicament for the treatment of Japanese encephalitis virus infection.

2. Use according to claim 1, characterized in that: The zinc pyrithione is a small molecule inhibitor of NS2B-NS3 protease in Japanese encephalitis virus.

3. Use according to claim 1, characterized in that: The structural formula of the zinc pyrithione is: 。

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