Application of small molecule compound in preparation of antiviral drugs

Abstract

The invention relates to the technical field of animal virology, in particular to application of a small molecule compound in preparation of antiviral drugs. The specific technical scheme is as follows: the small molecule compound is Y206 or pharmaceutically acceptable salt thereof, and the virus comprises Zika virus; the compound disclosed by the invention has a very good inhibition effect on replication of Zika virus, dengue virus and hepatitis C virus, provides a basis for further design and optimization of drugs for inhibiting infection of the Zika virus, creates good conditions for research and development of novel efficient broad-spectrum anti-flavivirus infection drugs, and has broad application prospects. Important application prospects and innovative significance are realized in the medical field.

Description

Technical Field

[0001] The present invention relates to the technical field of animal virology, and in particular to the application of a small molecule compound in the preparation of an antiviral drug. Background Art

[0002] Zika virus is a small enveloped positive-sense single-stranded RNA virus, about 11kb in size, belonging to the genus Flavivirus of the family Flaviviridae. Other viruses in the genus Flavivirus include dengue virus, yellow fever virus, West Nile virus, tick-borne encephalitis virus, Japanese encephalitis virus, etc. Zika virus is an insect-borne virus, mainly transmitted by Aedes aegypti mosquitoes, and other modes of transmission include sexual transmission, mother-to-child transmission, and blood transmission. Zika virus was first discovered in the Zika Forest in the Ugan region in 1947 and was detected in a rhesus monkey. Zika virus was then detected in sub-Saharan Africa and spread to Central and South America through Southeast Asia. For most adults, infection with Zika virus may cause fever, rash, conjunctivitis, headache, muscle weakness, etc., and may even cause serious neurological complications such as Guillain-Barré syndrome and microcephaly in newborns.

[0003] There is currently no vaccine or specific anti-Zika virus drug for clinical use, and there is no specific method to treat Zika virus infection. Therefore, it is necessary to find new treatment options. In the past few years, some studies have discovered many candidate drugs through drug repurposing, high-throughput screening of compound libraries, and de novo design. The research on antiviral drugs for Zika virus mainly includes drugs targeting the structural proteins and non-structural proteins of the virus. Therefore, it is urgent to find antiviral drugs against Zika virus, and it is expected to be able to treat patients with acute Zika infection and pregnant women infected with Zika virus.

[0004] Zika virus and dengue virus have overlapping geographical distributions and similar clinical manifestations. The antigenic conservation of Zika virus and dengue virus and the cross-reactivity of antibodies have greatly hindered the research of vaccines for the two viruses. Most monoclonal antibodies against dengue virus cannot neutralize Zika virus infection and even increase the infection of Zika virus and dengue virus, leading to antibody-dependent infection enhancement effect, which worsens Zika virus infection and increases the spread of Zika virus in areas where Zika virus and dengue virus are prevalent. Finding small molecule drugs that inhibit Zika virus is extremely important for the prevention and control of Zika epidemics.

[0005] Zika virus enters host cells through receptor-mediated endocytosis. After the viral envelope protein membrane fuses with the cell endosome membrane, the viral genome is released into the host. Viral RNA is translated by host ribosomes to produce polyproteins. Polyproteins produce non-structural proteins and structural proteins under the action of host cell proteases and viral proteases. Non-structural proteins include NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 proteins. Structural proteins include envelope E protein, membrane prM protein, and nucleocapsid C protein. Non-structural proteins play an important role in the life cycle and reproduction of the virus. NS3 protein has polymerase and helicase activities, and plays an important role in protein hydrolysis and maturation. NS2B is an auxiliary factor for NS3. NS5 includes methyltransferase and RNA-dependent RNA polymerase functions, and plays an important role in the process of viral replication. E protein plays an important role in virus invasion, prM protein plays a role in the regulation of host immune defense, and protects cell membrane proteins during virus synthesis. C protein binds to the cell membrane and plays a role in the budding stage of the virus.

[0006] The present invention aims to find a small molecule compound targeting viral polymerase that has a good inhibitory effect on Zika virus. Nucleoside analogs are used as inhibitors of viral polymerase in clinic, which can compete with natural adenosine triphosphate, participate in the synthesis of RNA chains, terminate the extension of RNA chains, and thus play a role in inhibiting viral infection. NS5 plays an important role in genome replication, and there is no similar enzyme in the human body. The inhibitor can specifically target the virus, making it a good target for studying antiviral drugs. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides an application of a small molecule compound in the preparation of an antiviral drug.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] The present invention discloses an application of a small molecule compound in the preparation of an antiviral drug, wherein the small molecule compound is Y206 or a pharmaceutically acceptable salt thereof;

[0010] The structural formula of the Y206 is as follows:

[0011]

[0012] The viruses include Zika virus.

[0013] Preferably, the antiviral effect is at least one of the following: inhibiting the replication of Zika virus; inhibiting Zika virus infection of cells.

[0014] Preferably, the virus further includes any one of dengue virus and hepatitis C virus.

[0015] Preferably, the antiviral effect is at least one of the following: inhibiting the replication of dengue virus; inhibiting dengue virus infection of cells; inhibiting the replication of hepatitis C virus; inhibiting hepatitis C virus infection of cells.

[0016] Correspondingly, a use of a small molecule compound in the preparation of a drug or a pharmaceutical composition, the drug or pharmaceutical composition comprising Y206 or a pharmaceutically acceptable salt thereof; the function of the drug or pharmaceutical composition is at least one of the following: inhibiting the replication of Zika virus; inhibiting Zika virus infection of cells; treating central nervous system diseases infected by Zika virus; inhibiting the replication of dengue virus; inhibiting dengue virus infection of cells; inhibiting the replication of hepatitis C virus; inhibiting hepatitis C virus infection of cells;

[0017] The structural formula of the Y206 is as follows:

[0018]

[0019] The present invention has the following beneficial effects:

[0020] The present invention takes the screening of small molecule compound libraries and the Zika virus infection system as the entry point, makes full use of advanced virology research tools such as live virus in cell culture and immunofluorescence, and discovers small molecule compounds that resist the replication of Zika virus through methods such as virology, biochemistry, and structural biology. It has been verified that the compounds have a good inhibitory effect on the replication of Zika virus, dengue virus, and hepatitis C virus, providing a basis for further designing and optimizing drugs that inhibit Zika virus infection, and creating good conditions for the research and development of new, highly effective, broad-spectrum drugs against flavivirus infection. It has important application prospects and innovative significance in the medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results are from western blotting. After the compound and Zika virus were mixed to infect cells, the amount of virus was detected by western blotting.

[0022] Figure 2 The inhibitory effect of Y206 on Zika virus; after the compound and Zika virus were mixed to infect cells, the inhibitory effect of the compound on viral infection was detected by WB and In cell western.

[0023] Figure 3 The results show that Y206 inhibits dengue virus. The compound was mixed with DENV and then infected into cells. The inhibitory effect of the compound on virus infection was detected by In cell western method.

[0024] Figure 4The results are as follows: the inhibition of Y206 on live hepatitis C virus; the compound was mixed with HCV and then infected into cells, and the inhibitory effect of the compound on viral infection was detected by In cell western. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0027] The present invention discloses an application of a small molecule compound in the preparation of an antiviral drug. The small molecule compound is Y206 or a pharmaceutically acceptable salt thereof. The virus comprises Zika virus. The Zika virus comprises any one of the three strains of MR766, PRVABC59 and SZ01.

[0028] The structural formula of the Y206 is as follows:

[0029]

[0030] Furthermore, the antiviral activity is at least one of the following: inhibiting the replication of Zika virus; inhibiting Zika virus infection of cells. The cells are mammalian cells, specifically Vero cells or SNB-19 cells.

[0031] Furthermore, the virus includes any one of dengue virus and hepatitis C virus. The antiviral performance is at least one of the following: inhibiting the replication of dengue virus; inhibiting dengue virus infection of cells; inhibiting the replication of hepatitis C virus; inhibiting hepatitis C virus infection of cells.

[0032] The present invention discloses an application of a small molecule compound in preparing a medicine or a pharmaceutical composition. The medicine or the pharmaceutical composition comprises Y206 or a pharmaceutically acceptable salt thereof. The function of the medicine or the pharmaceutical composition is at least one of the following: inhibiting the replication of Zika virus; inhibiting Zika virus infection of cells; inhibiting the replication of dengue virus; inhibiting dengue virus infection of cells; inhibiting the replication of hepatitis C virus; and inhibiting hepatitis C virus infection of cells.

[0033] The structural formula of the Y206 is as follows:

[0034]

[0035] The present invention will be further described below in conjunction with specific embodiments.

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative experiments in the following examples are repeated three times, and the results are averaged.

[0037] In the following examples, the drug (Y206 or its salt) was first prepared with DMSO (dimethyl sulfoxide) as a solvent to obtain a mother solution with a concentration of 10 mM. When used, the mother solution was added to DMEM culture medium to obtain dilutions containing different concentrations of the drug. Compound Y206 was purchased from Shanghai Taosu Biochemical Technology Co., Ltd., item number E518-0961.

[0038] The cell lines used in the following examples are as follows:

[0039] 1. Huh7.5.1 cells are described in the following literature: Jin Z, Pablo G, Guofeng C, et al. Robusthepatitis C virus infection in vitro. [J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(26): 9294-9299. The public can obtain them from the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences.

[0040] 2. Vero cells: National Laboratory Cell Resource Sharing Platform, resource number: 1101MON-PUMC000060.

[0041] 3. A549 cells: National Experimental Cell Resource Sharing Platform, resource number: 1101HUM-PUMC000002.

[0042] 4. SNB-19 cells are described in the following literature: Gross JL, Behrens DL, Mullins DE, Kornblith PL, Dexter DL. Plasminogen activator and inhibitor activity in human glioma cells and modulation by sodium butyrate. Cancer Res. 1988 Jan 15; 48(2): 291-6. PMID: 3121170. The public can obtain them from the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences.

[0043] The above Huh7.5.1, Vero, A549, and SNB-19 cells were cultured in DMEM medium. All cells needed to be cultured in a medium supplemented with 10% FBS and 1% penicillin-streptomycin mixture (company: Gibco, catalog number: 15140122). The cell culture conditions were 37°C, 5% CO 2 .

[0044] The JFH-1 HCVcc virus strain used in the following examples is recorded in the following literature: Si Y, Liu S, Liu X, Jacobs JL, Cheng M, Niu Y, Jin Q, Wang T, Yang WA human claudin-1-derived peptide inhibits hepatitis C virus entry. Hepatology. 2012 Aug; 56 (2): 507-15. doi: 10.1002 / hep.25685. Epub 2012 Jun 11. PMID: 22378192; PMCID: PMC3406249., and the public can obtain it from the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences. The normal concentration of JFH-1 HCVcc used is MOI = 0.1 (if there is a special mark, it will be marked).

[0045] The ZIKV SZ01 virus strain used in the following examples is recorded in the following literature: Deng YQ, Zhao H, Li XF, Zhang NN, Liu ZY, Jiang T, Gu DY, Shi L, He JA, Wang HJ, Sun ZZ, Ye Q, Xie DY, Cao WC, Qin CF. Isolation, identification and genomic characterization of the Asian lineage Zika virus imported to China. Sci China Life Sci. 2016Apr;59(4):428-30.doi:10.1007 / s11427-016-5043-4.Epub 2016Mar 18.PMID:26993654., and the public can obtain it from the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences. The normal concentration of ZIKV SZ01 is MOI=1 (if there is a special mark, it will be marked).

[0046] The DENV-2 virus strain used in the following examples is recorded in the following document: Miagostovich MP, Sequeira PC, Dos Santos FB, Maia A, Nogueira RM, Schatzmayr HG, Harris E, Riley LW. Molecular typing of dengue virus type 2 in Brazil. Rev Inst Med Trop Sao Paulo. 2003 Jan-Feb; 45(1): 17-21. doi: 10.1590 / s0036-46652003000100004. PMID: 12751317., and is stored in the laboratory of Professor Li Yiping of Sun Yat-sen University.

[0047] The antibodies used in the following examples and where they were purchased are as follows:

[0048] Anti-HCV core mouse monoclonal antibody was purchased from Thermo Company, catalog number 39-6900. Horseradish peroxidase-labeled goat anti-mouse secondary antibody was purchased from Jackson ImmunoResearch; DyLight 488-labeled donkey anti-mouse secondary antibody was purchased from Jackson ImmunoResearch; IRDye 800-labeled donkey anti-mouse secondary antibody was purchased from Li-COR. Mouse ZIKV E protein antibody was purchased from BioFront Technologies, catalog number BF-1176-56. Mouse ZIKV NS1 antibody was purchased from BioFront Technologies, catalog number BF-1225-36. Rabbit DENV-2 antibody was from GeneTex, catalog number GTX127277.

[0049] The solutions used in the following examples were derived from:

[0050] 1. PBS buffer: Beijing Solebow Technology Co., Ltd., product number: P1020, pH 7.4, 0.01 M.

[0051] 2. 4% paraformaldehyde: Solebao Company, product number: P1110.

[0052] 3. Triton x-100: Solebao Company, item number: T8200.

[0053] The data processing and analysis software in the following examples are as follows: ABPRISM Primer Express 2.0, Roche Applied Science and other software tools for primer design and analysis. Experimental data were analyzed and processed using GraphPad Prism 6.0, Photoshop 6.0, and Image J software. The experimental results were expressed as mean ± standard deviation, and the independent sample t test was used for difference significance analysis. p < 0.05 was significantly different, indicated by *; p < 0.01 was extremely significantly different, indicated by **; p < 0.001 was indicated by ***.

[0054] Experimental Example 1: Y206 inhibits Zika virus infection

[0055] 1. Amplification of Zika virus and detection of its virulence.

[0056] 1. When the growth density of Vero cells is about 70% to 80%, take out the virus suspension frozen in the -80℃ refrigerator, put it on ice or at 4℃ to revive, put it in a microcentrifuge for a few seconds, take the virus supernatant and inoculate it into Vero cells, and place it in 5% CO 2 , incubate in a 37°C cell culture incubator to allow the virus particles to fully adsorb to the cell surface. After 2 hours, discard the supernatant and add 10% FBSDMEM culture medium. Observe cell growth. When Vero cells fill the culture dish, they can continue to be subcultured at a ratio of 1:3. After 5 days of culture, when cytopathic effects (CPE) caused by viral infection are observed in the culture dish, the cell supernatant can be collected, centrifuged at 450g for 3 minutes at 4°C, the virus supernatant is filtered with a 0.45μM filter, and stored in a -80°C refrigerator for use.

[0057] 2. After the virus is prepared, the virus titer is analyzed by FFU / ml and the virulence of the obtained ZIKV is determined. The method is as follows: SNB-19 cells are inoculated into a 96-well plate, and when the cell growth density reaches 70% to 80%, the ZIKV virus solution is taken and diluted 10 times with DMEM medium containing 10% FBS and then inoculated into the cells, and the group not infected with ZIKV virus is used as the control group, and In Cell Western (ICW) detection is performed 48 hours later.

[0058] In cell western experimental steps: Take out the treated 96-well cells, discard the supernatant, rinse the cells twice with 100μl PBS per well, then add 100μl of tissue cell fixative (4% paraformaldehyde) per well, and fix on a low-speed shaker at room temperature. After 30 minutes, discard the tissue fixative, rinse the cells 3 times with 100μl PBS buffer per well on a low-speed shaker, 5 minutes each time. After discarding PBS, add 0.1% TritonX-100 per well at 100μl, and permeabilize the membrane for 15 minutes at room temperature. TritonX-100 is prepared with PBS. After 15 minutes, discard the supernatant, and rinse the cells 3 times with 100μl PBS per well at low speed, 5 minutes each time. During this period, prepare a 3% BSA solution with PBS, wait for the rinsing process to end, add 100μl of 3% BSA to each well of the cells, and place on a shaker at room temperature for 1 hour. Then, the ZIKV NS1 antibody was diluted 1:500 with 3% BSA, and 50 μl of NS1 primary antibody was added to each well after discarding the blocking solution, and placed on a shaker at 4°C overnight. The next day, the cells were removed and the supernatant was discarded. They were rinsed with PBS three times as described above, and then the PBS was discarded. Under light-proof conditions, the fluorescent secondary antibody (800CW, mouse antibody) was diluted with PBS at 1:800, and 50 μl was added to each well. Incubated on a low-speed shaker at room temperature for 1 hour in the dark. After 1 hour, the cells were rinsed with PBS three times in the same way, each for 5 minutes, and light was avoided throughout the process. Finally, the Odyssey system was used for imaging and processing. After cleaning the scanning panel of the system with alcohol and lint-free paper, the 800nm ​​scanning channel was selected, the high quality and 84μm resolution were set, and other options were the system defaults. The cell plate was scanned to detect the fluorescence intensity and analyzed.

[0059] 2. Y206 inhibits Zika virus infection

[0060] SNB-19 cells were inoculated into 96-well plates and 12-well plates, cultured to a cell density of 70%, and the gradient-diluted drugs were mixed with the virus solution of MOI=1 and added to the cells. The highest drug concentration was 10μM, and the drug was diluted twice in a gradient, and remdesivir was added as a positive control. An equal volume of DMSO was set as a negative control group. Three replicate wells were set up for each drug concentration.

[0061] After 48 hours, the amount of virus was detected by In cell western and protein immunoblotting. The steps of protein immunoblotting were as follows: when collecting total cell protein, the supernatant was discarded, 4× protein loading buffer was added to the cells in the well plate, 100 μl per well, and the sample was transferred to a 1.5 ml EP tube after repeated blowing and grinding of the cells with a gun tip, and placed in a 95°C metal bath to cook the sample for 10 minutes, and then centrifuged at 12000r for 10 minutes at 4°C. Then, protein electrophoresis was performed using a pre-prepared SDS-PAGE gel stored at 4°C, and 80V electrophoresis was changed to 120V electrophoresis for about 1 hour after 30 minutes of electrophoresis. Then, the membrane was transferred to electrophoresis at a constant voltage of 100V for 90 minutes, and the gel protein was transferred to the PVDF membrane. At the same time, a 5% blocking solution was prepared with skim milk powder. After the transfer, the PVDF membrane was placed in the blocking solution and incubated on a low-speed shaker at room temperature for 1 hour. Then, ZIKV E antibody was diluted with 5% skim milk at a ratio of 1:3000 as the primary antibody and incubated overnight on a shaker at 4°C. The next day, the PVDF membrane incubated with the primary antibody was removed and the primary antibody was recovered. Then, the membrane was washed 3 times with TBST membrane washing buffer on a shaker at room temperature for 5 minutes each time. After the washing solution was poured out, the HRP-labeled mouse secondary antibody prepared with 5% skim milk was incubated at room temperature for 1 hour. Then, the membrane was washed 3 times with TBST in the same way for 5 minutes each time. Then, the PVDF membrane was treated with ECL luminescent solution for 1 to 2 minutes under light-proof conditions, and then X-ray film exposure, development and fixing were performed in a dark room. After the film was dried naturally, the image was scanned and saved.

[0062] The results are as follows Figure 1 , 2 shown. Figure 1 The results of Western blotting are shown in Figure 2. Figure 2 This is the result of In cell western quantification. Figure 2 In the figure, the horizontal axis is the logarithm of the drug concentration with a base of 10, and the vertical axis is the infection efficiency of Zika virus. The results show that Y206 inhibits the infection of Zika virus in a dose-dependent manner. The positive control group, remdesivir, can inhibit the infection of Zika virus in a dose-dependent manner, and the blank control group, DMSO, has no significant effect on the infection of Zika virus.

[0063] Experimental Example 2: Y206 inhibits HCV infection

[0064] 1. Cultivation of live HCV virus and detection of virus titer

[0065] Huh7.5.1 cells were inoculated into a 10 cm culture dish, and when the cells grew to a density of 70%, they were infected with live HCV virus at MOI = 0.1. Six hours later, the cells were replaced with 10% FBSDMEM. After 48 to 72 hours, when the cells showed obvious pathological changes, the supernatant was collected and aliquoted and frozen in a -80 refrigerator. The virus titer was detected by In cell western.

[0066] 2. Y206 inhibits HCV live virus infection

[0067] Huh7.5.1 cells were inoculated into 96-well plates and grown to a density of 70%. The cells were infected with a mixture of live HCV virus and drugs. The highest drug concentration was 10 μM. The cells were diluted two-fold. After 48 hours, the virus content was detected by In cell western. The results are shown in Figure 2. Figure 4 As shown, the horizontal axis is the logarithmic value of the drug concentration with the base 10, and the vertical axis is the infection efficiency of the HCV live virus, such as Figure 4 As shown, Y206 can inhibit the infection of live HCV virus in a dose-dependent manner.

[0068] Experimental Example 3Y206 inhibits DENV infection

[0069] 1. Amplification and toxicity detection of dengue virus DENV-2

[0070] Vero cells were inoculated into a 10 cm dish. When the cell density grew to 70%, DENV-2 with an MOI of 10 was added. Virus dilution was prepared with serum-free DMEM medium. After 6 hours of infection, the medium was changed to 2% serum and culture was continued. The cell status was observed. After about 48 to 72 hours, obvious pathological changes of the cells appeared. The supernatant was collected, centrifuged to remove the cell precipitate, and the cells were divided and frozen in a -80 refrigerator.

[0071] A549 cells were evenly spread in a 96-well plate in advance. When the cell density was about 70% to 90%, the virus solution was diluted in DMEM medium containing 10% FBS. The group not infected with DENV-2 virus was used as the control group and placed at 37°C and 5% CO. 2 After 48 hours in a cell culture incubator under the same conditions, the virus titer was detected by In Cell Western.

[0072] 2. Y206 inhibits DENV infection

[0073] A549 cells were inoculated into 96-well plates. When the cell density reached 70%, the cells were infected with a mixture of drugs and viruses. After 48 hours of culture, the virus titer was detected using In Cell Western. Figure 3 As shown, Y206 can inhibit the infection of DENV virus in a dose-dependent manner.

[0074] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a small molecule compound in the preparation of an antiviral drug, characterized in that: The small molecule compound is Y206 or a pharmaceutically acceptable salt thereof; The structural formula of the Y206 is as follows: The viruses include Zika virus.

2. The use according to claim 1, characterized in that: The antiviral effect is manifested as at least one of the following: inhibiting the replication of Zika virus; inhibiting Zika virus infection of cells.

3. The use according to claim 1, characterized in that: The virus also includes any one of dengue virus and hepatitis C virus.

4. The use according to claim 3, characterized in that: The antiviral effect is at least one of the following: inhibiting the replication of dengue virus; inhibiting dengue virus infection of cells; inhibiting the replication of hepatitis C virus; inhibiting hepatitis C virus infection of cells.

5. Use of a small molecule compound in the preparation of a drug or a pharmaceutical composition, characterized in that: The drug or pharmaceutical composition comprises Y206 or a pharmaceutically acceptable salt thereof; the function of the drug or pharmaceutical composition is at least one of the following: inhibiting the replication of Zika virus; inhibiting Zika virus infection of cells; treating central nervous system diseases infected by Zika virus; inhibiting the replication of dengue virus; inhibiting dengue virus infection of cells; inhibiting the replication of hepatitis C virus; inhibiting hepatitis C virus infection of cells; The structural formula of the Y206 is as follows:

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