Application of a small molecule compound in the preparation of antiviral drugs

Abstract

This invention relates to the field of animal virology, specifically to the application of a small molecule compound in the preparation of antiviral drugs. The specific technical solution is as follows: the small molecule compound is Y206 or a pharmaceutically acceptable salt thereof, and the virus includes Zika virus; the structural formula of Y206 is as follows: The compound disclosed in this invention exhibits excellent inhibitory effects on the replication of Zika virus, dengue virus, and hepatitis C virus, providing a foundation for further designing and optimizing drugs to inhibit Zika virus infection, and creating favorable conditions for the development of novel, highly effective, and broad-spectrum antiviral drugs. It has significant application prospects and innovative value in the medical field.

Description

Technical Field

[0001] This invention relates to the field of animal virology technology, specifically to the application of a small molecule compound in the preparation of antiviral drugs. Background Technology

[0002] Zika virus is a small, enveloped, positive-sense, single-stranded RNA virus, approximately 11 kb in size, belonging to the genus Flaviviridae in the family Flaviviridae. Viruses in the genus Flaviviridae also include dengue virus, yellow fever virus, West Nile virus, tick-borne encephalitis virus, and Japanese encephalitis virus. Zika virus is an arbovirus, primarily transmitted by Aedes aegypti mosquitoes, with other modes of transmission including sexual contact, mother-to-child transmission, and blood transmission. Zika virus was first discovered in 1947 in the Zika Forest in Uganda, detected in a rhesus monkey. It was subsequently detected in sub-Saharan Africa and spread through Southeast Asia to Central and Southern America. In most adults, Zika virus infection may cause fever, rash, conjunctivitis, headache, and muscle weakness; it can even lead to serious neurological complications such as Guillain-Barré syndrome and microcephaly in newborns.

[0003] Currently, there are no vaccines or specific anti-Zika virus drugs available for clinical use, and no specific treatment for Zika virus infection. Therefore, finding new treatment options is essential. In recent years, studies have identified many candidate drugs through approaches such as repurposing drugs, high-throughput screening of compound libraries, and de novo design. Research on antiviral drugs against Zika virus mainly focuses on drugs targeting both structural and non-structural proteins of the virus. Therefore, there is an urgent need to find antiviral drugs against Zika virus, which may offer treatment for 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 and dengue viruses and the cross-reactivity of their antibodies have significantly hindered vaccine research for both viruses. Most monoclonal antibodies against dengue virus cannot neutralize Zika virus infection and may even increase the infection rate of both Zika and dengue viruses, leading to antibody-dependent enhancement of infection, worsening Zika virus infection, and increasing the spread of Zika virus in areas where both viruses are prevalent. The search for small-molecule drugs to inhibit Zika virus is extremely important for the control of Zika epidemics.

[0005] Zika virus enters host cells via receptor-mediated endocytosis. After the viral envelope protein membrane fuses with the cellular endosomal membrane, the viral genome is released into the host. Viral RNA is translated by host ribosomes to produce polyproteins. These polyproteins, under the action of host cellular proteases and viral proteases, produce non-structural and structural proteins. Non-structural proteins include NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 proteins, while structural proteins include the envelope E protein, the membrane prM protein, and the nucleocapsid C protein. Non-structural proteins play important roles in the viral life cycle and replication. The NS3 protein has polymerase and helicase activities, playing a crucial role in proteolysis and maturation. NS2B is a cofactor for NS3. NS5 includes methyltransferase and RNA-dependent RNA polymerase activities, playing a vital role in viral replication. The E protein plays an important role in viral invasion, while the prM protein plays a role in regulating host immune defense and protecting cell membrane proteins during viral synthesis. The C protein binds to the cell membrane and plays a role during viral budding.

[0006] This invention aims to identify small molecule compounds that target the viral polymerase and exhibit good inhibitory effects against Zika virus. Nucleoside analogs, used clinically as inhibitors of viral polymerases, compete with natural adenosine triphosphate (ATP) to participate in RNA chain synthesis, terminating RNA chain elongation and thus inhibiting viral infection. NS5 plays a crucial role in genome replication, and since no similar enzyme exists in humans, its inhibitors can specifically target the virus, making it a promising target for antiviral drug research. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides an application of small molecule compounds in the preparation of antiviral drugs.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention discloses the application of a small molecule compound in the preparation of antiviral drugs, wherein the small molecule compound is Y2O6 or a pharmaceutically acceptable salt thereof;

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

[0011]

[0012] The virus mentioned includes the Zika virus.

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

[0014] Preferably, the virus also includes dengue virus or hepatitis C virus.

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

[0016] Accordingly, the use of a small molecule compound in the preparation of a drug or pharmaceutical composition, said drug or pharmaceutical composition comprising Y2O6 or a pharmaceutically acceptable salt thereof; said drug or pharmaceutical composition having the function of at least one of the following: inhibiting Zika virus replication; inhibiting Zika virus infection of cells; treating central nervous system diseases caused by Zika virus infection; inhibiting dengue virus replication; inhibiting dengue virus infection of cells; inhibiting hepatitis C virus replication; inhibiting hepatitis C virus infection of cells;

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

[0018]

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

[0020] This invention takes small molecule compound library screening and Zika virus infection system as its starting point, making full use of advanced virological research tools such as cell culture of live viruses and immunofluorescence. Through virology, biochemistry, and structural biology methods, small molecule compounds that inhibit Zika virus replication have been discovered. After verification, the compounds have shown good inhibitory effects on the replication of Zika virus, dengue virus, and hepatitis C virus. This provides a foundation for further designing and optimizing drugs to inhibit Zika virus infection and creates favorable conditions for the development of novel, highly effective, and broad-spectrum drugs against flavivirus infection. It has important application prospects and innovative significance in the medical field. Attached Figure Description

[0021] Figure 1 The results are for Western blotting; after the compound was mixed with Zika virus to infect cells, the viral load was detected by Western blotting.

[0022] Figure 2 To investigate the inhibitory effect of Y206 on Zika virus, the inhibitory effect of the compound on viral infection was detected by Western blotting and in-cell western blotting after the compound was mixed with Zika virus to infect cells.

[0023] Figure 3 The results of Y206's inhibition of dengue virus were shown; the compound was mixed with DENV and then used to infect cells, and the inhibitory effect of the compound on viral infection was detected by in-cell western spectroscopy.

[0024] Figure 4The results show the inhibitory effect of Y206 on live hepatitis C virus. After mixing the compound with HCV, cells were infected, and the inhibitory effect of the compound on viral infection was detected by in-cell western spectroscopy. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

[0027] This invention discloses the application of a small molecule compound in the preparation of antiviral drugs, wherein the small molecule compound is Y206 or a pharmaceutically acceptable salt thereof; the virus includes Zika virus; and the Zika virus includes any one of the three strains MR766, PRVABC59, and SZ01.

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

[0029]

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

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

[0032] This invention discloses the use of a small molecule compound in the preparation of a drug or pharmaceutical composition, wherein the drug or pharmaceutical composition comprises Y2O6 or a pharmaceutically acceptable salt thereof; the drug or pharmaceutical composition functions to 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 Y206 is as follows:

[0034]

[0035] The present invention will be further described below with reference to specific embodiments.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0037] In the following examples, the drug (Y206 or its salt) was first prepared into a 10 mM stock solution using DMSO (dimethyl sulfoxide) as a solvent. The stock solution was then added to DMEM culture medium to obtain diluted solutions containing different concentrations of the drug. Compound Y206 was purchased from Shanghai Taosu Biochemical Technology Co., Ltd., catalog 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. They can be obtained by the public from the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences.

[0040] 2. Vero cells: National Experimental Cell Resource Sharing Platform, Resource No.: 1101MON-PUMC000060.

[0041] 3. A549 cells: National Experimental Cell Resource Sharing Platform, Resource No.: 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. They can be obtained by the public from the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences.

[0043] The Huh7.5.1, Vero, A549, and SNB-19 cells were all cultured in DMEM medium. All cell cultures were performed with the addition of a mixture of 10% FBS and 1% penicillin-streptomycin (Gibco, catalog number: 15140122) to the medium. Cell culture conditions were 37°C and 5% CO2.

[0044] The JFH-1HCVcc virus strain used in the following examples is described 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 2012Jun 11.PMID:22378192; PMCID:PMC3406249. It can be obtained by the public from the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences. The normal concentration for use of JFH-1HCVcc is MOI = 0.1 (if any is special, it will be marked).

[0045] The ZIKV SZ01 virus strain used in the following examples is described 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 Asianlineage Zika virus imported to China. Sci China Life Sci. 2016 Apr;59(4):428-30. doi:10.1007 / s11427-016-5043-4. Epub 2016 Mar 18. PMID:26993654. It can be obtained by the public from the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences. The normal concentration of ZIKV SZ01 is MOI=1 (if any special concentration is indicated).

[0046] The DENV-2 virus strain used in the following examples is described in the following literature: 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, Sun Yat-sen University.

[0047] The antibodies used in the following examples and their sources of purchase are as follows:

[0048] The anti-HCV core mouse monoclonal antibody was purchased from Thermo Fisher Scientific, catalog number 39-6900. Horseradish peroxidase-labeled goat anti-mouse secondary antibody was purchased from Jackson Immuno Research; DyLight 488-labeled donkey anti-mouse secondary antibody was purchased from Jackson Immuno Research; and IRDye 800-labeled donkey anti-mouse secondary antibody was purchased from Li-COR. The mouse-derived ZIKV E protein antibody was purchased from BioFront Technologies, catalog number BF-1176-56. The mouse-derived ZIKV NS1 antibody was purchased from BioFront Technologies, catalog number BF-1225-36. The rabbit-derived DENV-2 antibody was obtained from GeneTex, catalog number GTX127277.

[0049] The solutions used in the following examples are from the following sources:

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

[0051] 2.4% Paraformaldehyde: Solarbio, Product No.: P1110.

[0052] 3. Triton x-100: Solarbio, part number: T8200.

[0053] The data processing and analysis software used in the following examples is as follows: ABPRISM Primer Express 2.0, Roche Applied Science, and other software tools were used for primer design and analysis. Experimental data were analyzed and processed using GraphPad Prism 6.0, Photoshop 6.0, and ImageJ software. Experimental results are expressed as mean ± standard deviation, and independent samples t-tests were used to analyze the significance of differences. p < 0.05 was considered significant (*); p < 0.01 was considered highly significant (**); and p < 0.001 was indicated by ***.

[0054] Experiment 1: Y206 inhibits Zika virus infection

[0055] I. Zika virus amplification and virulence level detection.

[0056] 1. When the Vero cell density reaches approximately 70%–80%, remove the virus suspension frozen at -80°C and thaw it on ice or at 4°C. After a few seconds of brief centrifugation, inoculate the virus supernatant into Vero cells and incubate in a 5% CO2, 37°C cell culture incubator to allow virus particles to fully adhere to the cell surface. After 2 hours, discard the supernatant and add medium containing 10% FBSDMEM. Observe cell growth. When the Vero cells have reached confluence with the culture dish, continue passage at a 1:3 ratio. After 5 days of culture, when cytopathic effect (CPE) caused by viral infection is observed in the culture dish, collect the cell supernatant, centrifuge at 450g for 3 minutes at 4°C, filter the virus supernatant through a 0.45μM filter, aliquot, and store at -80°C for later use.

[0057] 2. After virus preparation, the viral titer was analyzed by FFU / ml, and the virulence of the obtained ZIKV was determined. The method was as follows: SNB-19 cells were seeded into 96-well plates, and when the cell density reached 70%–80%, ZIKV virus solution was taken and serially diluted 10-fold with DMEM medium containing 10% FBS before being seeded into cells. The uninfected group was used as the control group. In-Cell Western (ICW) detection was performed after 48 hours.

[0058] In-cell Western blotting procedure: After treatment, remove cells from each of the 96-well cells, discard the supernatant, and rinse twice with 100 μl of PBS per well. Then, add 100 μl of cell fixative (4% paraformaldehyde) per well and fix on a low-speed shaker at room temperature. After 30 minutes, discard the fixative and rinse three times with 100 μl of PBS buffer per well on a low-speed shaker for 5 minutes each time. After discarding the PBS, add 100 μl of 0.1% Triton X-100 per well and rupture the membrane at room temperature for 15 minutes. Triton X-100 should be prepared using PBS. After 15 minutes, discard the supernatant and rinse three times with 100 μl of PBS per well on a low-speed shaker for 5 minutes each time. During this time, prepare a 3% BSA solution in PBS. After the rinsing process is complete, add 100 μl of 3% BSA to each well and incubate on a shaker at room temperature for 1 hour. Then, ZIKV NS1 antibody was diluted 1:500 with 3% BSA. After discarding the blocking solution, 50 μl of NS1 primary antibody was added to each well, and the cells were incubated overnight on a shaker at 4°C. The next day, the cells were removed, the supernatant was discarded, and the cells were washed three times with PBS as described above, then the PBS was discarded. Under light-protected conditions, fluorescent secondary antibody (800CW, murine antibody) was diluted 1:800 with PBS, and 50 μl was added to each well. The cells were incubated for 1 hour on a low-speed shaker at room temperature in the dark. After 1 hour, the cells were washed three times with PBS for 5 minutes each time, avoiding light exposure throughout the process. Finally, imaging and processing were performed on the Odyssey system. After cleaning the scanning panel of the system with alcohol and lint-free paper, the 800nm ​​scanning channel was selected, and high quality and 84μm resolution were set. Other options were left at system defaults. The cell plate was scanned to detect fluorescence intensity and analyzed.

[0059] II. Y206 inhibits Zika virus infection

[0060] SNB-19 cells were seeded into 96-well and 12-well plates and cultured until the cell density reached 70%. Serially diluted drug solutions were mixed with viral buffer at an MOI of 1 and added to the cells, with a maximum drug concentration of 10 μM. Two-fold serial dilutions were performed, and remdesivir was added as a positive control. An equal volume of DMSO was used as a negative control. Three replicates were performed for each drug concentration.

[0061] Forty-eight hours later, viral load was detected using In-Cell Western blotting and Western blotting, respectively. Western blotting procedure: When collecting total cellular protein, the supernatant was discarded. 100 μl of 4× protein loading buffer was added to each well of the plate. The cells were repeatedly pipetted and ground with a pipette tip before transferring the sample to a 1.5 ml EP tube. The tube was then boiled in a 95°C metal bath for 10 minutes, followed by centrifugation at 12000 rpm for 10 minutes at 4°C. Protein electrophoresis was then performed using a pre-prepared SDS-PAGE gel stored at 4°C. Electrophoresis was performed at 80V for 30 minutes, then switched to 120V for approximately 1 hour. Finally, the gel was transferred to a PVDF membrane under constant voltage (100V) for 90 minutes. Simultaneously, a 5% blocking buffer was prepared using skim milk powder. After transfer, the PVDF membrane was placed in the blocking buffer and incubated on a low-speed shaker at room temperature for 1 hour. The ZIKV E antibody was then diluted 1:3000 with 5% skim milk 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. The membrane was then washed three times with TBST washing buffer on a shaker at room temperature for 5 minutes each time. After discarding the washing solution, the membrane was incubated for 1 hour at room temperature with HRP-labeled mouse secondary antibody prepared with 5% skim milk. The membrane was then washed three more times with TBST in the same manner for 5 minutes each time. The PVDF membrane was then treated with ECL chemiluminescence solution for 1–2 minutes under light-protected conditions, followed by X-ray exposure, development, and fixing in a dark room. After the film was air-dried, the images were scanned and saved.

[0062] The results are as follows Figure 1 , 2 As shown. Figure 1 For the results of Western blot analysis of proteins, Figure 2 The results are from In-cell western quantization. Figure 2 In the graph, the horizontal axis represents the logarithm of the drug concentration to the base 10, and the vertical axis represents the Zika virus infection efficiency. The results show that Y206 inhibits Zika virus infection in a dose-dependent manner. In the positive control group, remdesivir inhibited Zika virus infection in a dose-dependent manner, while the blank control group, DMSO, had no significant effect on Zika virus infection.

[0063] Experimental Example 2: Y206 inhibits HCV infection

[0064] I. Culture and Virus Titer Detection of Live HCV

[0065] Huh7.5.1 cells were seeded into 10cm culture dishes and allowed to grow to a density of 70%. The cells were then infected with live HCV virus at an MOI of 0.1. Six hours later, the medium was replaced with 10% FBSDMEM. After 48–72 hours, when significant cytopathic effects were observed, the supernatant was collected, aliquoted, and cryopreserved at -80°C. Viral titer was determined using in-cell Western blotting.

[0066] II. Y206 inhibits infection by live HCV virus.

[0067] Huh7.5.1 cells were seeded into 96-well plates. When the cell density reached 70%, they were infected with a mixture of live HCV virus and drug. The maximum drug concentration was 10 μM, serially diluted twofold. Viral content was detected by In-Cell Western Spectroscopy after 48 hours. Results are as follows: Figure 4 As shown, the horizontal axis represents the logarithmic value of the drug concentration to the base 10, and the vertical axis represents the infection efficiency of live HCV virus. Figure 4 As shown, Y206 can inhibit HCV infection in a dose-dependent manner.

[0068] Experimental Example 3: Y206 inhibits DENV infection

[0069] I. Amplification and Viral Titer Detection of Dengue Virus DENV-2

[0070] Vero cells were seeded into 10 cm dishes. When the cell density reached 70%, DENV-2 with an MOI of 10 was added. A virus dilution was prepared using serum-free DMEM medium. After 6 hours of infection, the medium was replaced with medium containing 2% serum and cultured for another 6 hours. The cell status was observed. After about 48 to 72 hours, obvious lesions appeared in the cells. The supernatant was collected, the cell pellet was removed by centrifugation, and the cells were aliquoted and frozen in a -80°C freezer.

[0071] A549 cells were evenly seeded into 96-well plates in advance. When the cell density was about 70% to 90%, the virus solution was serially diluted with DMEM medium containing 10% FBS. The uninfected DENV-2 virus group was used as the control group. After 48 hours in a cell culture incubator at 37°C and 5% CO2, the viral titer was detected by In Cell Western blotting.

[0072] II. Y206 inhibits DENV infection

[0073] A549 cells were seeded into 96-well plates. When the cell density reached 70%, the cells were co-infected with a mixture of drug and virus. After 48 hours of culture, the viral titer was detected using In Cell Western blotting. The results are as follows: Figure 3 As shown, Y206 can inhibit DENV virus infection in a dose-dependent manner.

[0074] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a small molecule compound in the preparation of antiviral drugs, characterized in that: The small molecule compound is Y2O6 or a pharmaceutically acceptable salt thereof; The structural formula of Y206 is as follows: The virus mentioned includes the Zika virus.

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

3. The application according to claim 1, characterized in that: The virus also includes dengue virus and hepatitis C virus.

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

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

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