WO2021143806A1 - Hsp70抑制剂广谱抗黄病毒活性的应用 - Google Patents

Hsp70抑制剂广谱抗黄病毒活性的应用 Download PDF

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WO2021143806A1
WO2021143806A1 PCT/CN2021/072027 CN2021072027W WO2021143806A1 WO 2021143806 A1 WO2021143806 A1 WO 2021143806A1 CN 2021072027 W CN2021072027 W CN 2021072027W WO 2021143806 A1 WO2021143806 A1 WO 2021143806A1
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virus
cell
flaviviridae
cells
flavivirus
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钟武
曹瑞源
杨晶晶
李薇
李松
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中国人民解放军军事科学院军事医学研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4174—Arylalkylimidazoles, e.g. oxymetazolin, naphazoline, miconazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to the field of biomedicine, in particular to the application of HSP70 inhibitors with broad-spectrum antiflavivirus activity.
  • HSP70 is a series of highly conserved proteins that participate in cell responses to changes in temperature and nutritional environment, pathogen invasion, and oxidative stress. According to reports, HSP70 in the cytoplasm can participate in different stages of DENV infection, including virus entry, RNA replication, and virus particle biosynthesis. The co-localization study of HSP70 and ZIKV protein found that HSP70 is involved in ZIKV infection and plays an important role.
  • HSP70 can also interact with JEV envelope protein domain III and its interaction, and play a role in virus entry, replication and protein synthesis; participate in YFV replication and NS3/4A cleavage; bind to HCV NS5A protein Participate in viral RNA replication and viral particle assembly.
  • HSP70 protein has a variety of domains with drug binding functions, so that many small molecule compounds with different structures have HSP70 inhibitory activity, which is very beneficial to the discovery of small molecule drugs with HSP70 inhibitory activity.
  • the HSP70 inhibitors that have been reported include: ATP analogs that act on the NBD domain, benzothiazines and flavonoids, Geellin derivatives that bind to the C-terminal EEVD sequence, and benzene ethyl sulfonate that binds to SBD. Amides and so on.
  • the inventors selected a variety of HSP70 inhibitors with different mechanisms of action to evaluate their anti-flavivirus activity, hoping to select small molecular compounds with broad-spectrum anti-flavivirus activity for druggability studies.
  • the HSP70 inhibitor compound Apotozole (the compound represented by formula I) has a broad-spectrum anti-flavivirus activity, and has a broad spectrum of anti-flavivirus activity against 4 flaviviruses, including Zika virus, Leather virus, Japanese encephalitis virus and yellow fever virus all show good pathological protection effects and have good druggability.
  • the present invention provides the use of the compound represented by formula I (Apoptozole) in the preparation of a medicine for the treatment or prevention of viral infectious diseases of the Flaviviridae virus,
  • the medicament is used to treat or prevent a viral infectious disease of the flaviviridae in a non-immune-deficient mammal.
  • the non-immune deficient mammal is a non-IFN receptor deficient mammal.
  • the non-immune-deficient mammal is a non-RIG-I-deficient mammal.
  • the mammal is a human or a mouse.
  • the flaviviridae virus is selected from at least one of Zika virus, dengue virus, Japanese encephalitis virus, yellow fever virus, flavivirus, enterovirus, rhinovirus, and herpes virus.
  • the present invention provides the use of the compound represented by formula I (Apoptozole) in anti-flavivirus activity, or in enhancing or improving the cell viability or cell survival rate of cells infected with flaviviridae.
  • the compound For non-therapeutic purposes, such as for scientific research,
  • the present invention provides the use of the compound represented by formula I (Apoptozole) in the preparation of reagents for anti-flavivirus activity, or to enhance or increase the cell viability of flaviviridae virus-infected cells Or cell survival rate,
  • the cell is selected from at least one of African green monkey kidney cells (Vero), hamster kidney cells (BHK), human liver cancer cells (Huh7), and human liver cancer cells (Huh7.5).
  • Vero African green monkey kidney cells
  • BHK hamster kidney cells
  • Human liver cancer cells Huh7
  • Human liver cancer cells Huh7.5
  • the cell is a non-IFN receptor deficient cell.
  • the cell is a non-RIG-I deficient cell.
  • the flaviviridae virus is selected from at least one of Zika virus, dengue virus, Japanese encephalitis virus, yellow fever virus, flavivirus, enterovirus, rhinovirus, and herpes virus.
  • the anti-flavivirus activity, or enhancement or improvement of the cell viability or cell survival rate of cells infected with a Flaviviridae virus is achieved by at least one of the following:
  • flaviviridae viruses preferably Zika virus, dengue virus, Japanese encephalitis virus and yellow fever virus
  • Flaviviridae virus preferably Zika virus
  • F Up-regulate the expression of IFN of Flaviviridae virus (preferably Zika virus) to exert antiviral effect.
  • Flaviviridae virus preferably Zika virus
  • Apoptozole can reduce the cellular CPE levels of Zika virus, Dengue virus, Japanese encephalitis virus and yellow fever virus at micromolar concentrations in in vitro antiviral experiments; this protective effect is in the RIG-I-deficient Huh7 .5 Decrease in cells;
  • Apoptozole can reduce the viral nucleic acid load level in the cells of different Zika virus isolates and the number of infectious virus particles secreted in the supernatant after different Zika virus isolates infect different cell lines at micromolar concentrations.
  • Apoptozole is in RIG- The inhibitory effect of I-deficient Huh7.5 cells on the formation of viral RNA and infectious virus particles is reduced;
  • Apoptozole can reduce the production of Zika virus E protein and NS1 protein at micromolar concentrations. This effect is also reduced in RIG-I-deficient Huh7.5 cells, suggesting that the antiviral effect of Apoptozole is related to innate immunity. ;
  • transcriptome analysis shows that Apotozole mainly acts by regulating the lipid metabolism and natural immunity of Huh7 cells, which is consistent with the previous experimental conclusions;
  • Apoptozole can provide about 60% protection to 1-day-old ICR mice infected with Zika virus at a concentration of 1 mg/kg, and reduce the viral blood caused by Zika virus infection by up-regulating IFN expression at a concentration of 10 mg/kg. disease.
  • Apoptozole has no obvious protection against Zika virus infection in immunodeficient mice, which further confirms that Apoptozole may exert an antiviral effect by regulating innate immunity.
  • Figure 1 shows that HSP70 inhibitors exhibit broad-spectrum antiflavivirus activity in different cell lines.
  • Apoptozole shows a dose-dependent protective effect against 4 flavivirus infections in different cell lines (BHK, Vero, Huh7), and can significantly reduce the cytopathic effects (CPE) caused by viral infections.
  • CPE cytopathic effects
  • A, B, and C are the protective effects of Apoptozole on BHK, Vero and Huh7 infected with Zika virus SMGC-1; D, E, and F are the protective effects of Apoptozole on four different flaviviruses, including ZIKV (ZIKV).
  • ZIKV ZIKV
  • -SMGC-1 Japanese encephalitis virus
  • JEV-SA14 Japanese encephalitis virus
  • YFV-17D yellow fever virus
  • DEV-NGC dengue virus
  • D is BHK cells
  • E Vero cell
  • F Huh7 cell
  • G, H, I in turn are the toxicity of Apotozole to BHK, Vero and Huh7 cells at working concentrations.
  • FIG. 2 shows that Apoptozole inhibits Zika virus RNA replication and infectious virus particle formation.
  • Apoptozole in Vero, BHK, A549, Huh7 and Huh7.5 cells has a significant inhibitory effect on the viral RNA replication and the production of infectious virus particles of two different Zika virus isolates SMGC-1 and MR766 strains, and has shown Dose dependent.
  • AE is the inhibitory effect of Apoptozole on the RNA synthesis of Zika virus SMGC-1 and MR766 in Vero, BHK, A549, Huh7 and Huh7.5 cell lines
  • FJ is Apoptozole in Vero, BHK, A549, The inhibitory effect of Huh7 and Huh7.5 cell lines on Zika virus SMGC-1 strain and MR766 strain infectious virus particles
  • KO in turn is the effect of Apoptozole on Vero, BHK, A549, Huh7 and Huh7.5 cells at working concentrations toxicity.
  • FIG 3 shows that Apoptozole inhibits the production of Zika virus E protein and NS1 protein.
  • Apoptozole can effectively inhibit the production of Zika virus envelope protein (E-protein, IFA) and non-structural protein 1 (NS1, WB) in different cell lines (A549, Huh7, Huh7.5).
  • E-protein, IFA Zika virus envelope protein
  • NS1, WB non-structural protein 1
  • DAPI is the nuclear channel labeled with nuclear dye in IF imaging
  • MERGE is the co-localization (merged) picture of the nuclear channel and the fluorescent signal of the target protein.
  • A, C, E are the inhibitory effects of Apoptozole on Zika virus E protein in A549, Huh7 and Huh7.5 cell lines
  • B, D, F are the corresponding quantitative analysis results
  • G is Apoptozole in Vero
  • HJ is the corresponding quantitative data.
  • Figure 4 shows that Apoptozole exerts an antiviral effect in the post-adsorption stage of the process of Zika virus infecting cells. After adding Apoptozole to different cell lines at different stages of Zika virus infection, the test showed that Apoptozole mainly plays a role in the virus replication stage, and has no obvious inhibitory effect on the adsorption and entry of the virus.
  • DAPI is the nuclear channel labeled with nuclear dye in IF imaging
  • MERGE is the co-localization (merged) picture of the nuclear channel and the fluorescent signal of the target protein.
  • A is a schematic diagram of the time points when Apotozole is added
  • B, C, and D are the inhibition of viral RNA replication by Apotozole in Vero, Huh7 and Huh7.5 cells in order
  • E, F, G are Apotozole in Vero in order
  • Sequential experiments performed in Huh7 and Huh7.5 cells inhibit the expression of viral proteins.
  • 2'-CMA (2'-C-Methyladenosine, 2-C-methyladenosine) is a positive compound that has been reported to specifically act on the stage of virus replication.
  • Figure 5 shows that transcriptome analysis reveals that Apoptozole exerts antiviral effects by regulating lipid metabolism and natural immunity.
  • a and B are the heat maps and corresponding numbers of significantly changed genes caused by Apoptozole; C is the list of all genes with significant changes; D is the GO (Gene Ontology) function annotation analysis results of all significantly changed genes; E is KEGG (Kyoto Encyclopedia of Genes and Genomes) functional enrichment analysis results for all significantly changed genes.
  • FIG. 6 shows that Apoptozole protects against Zika virus infection by up-regulating innate immunity in wild-type mice.
  • Apoptozole has a dose-dependent protective effect on ICR suckling mice inoculated with a lethal dose of Zika virus; at the same time, Apoptozole can reduce the viremia of Zika virus-infected Balb/c mice by up-regulating the expression of interferon.
  • A is the protective effect of Apoptozole on Zika virus infection in the ICR suckling mouse lethal model
  • B is the corresponding body weight change
  • C is the inhibition of Apoptozole on viremia in Balb/c mice
  • D, E, F is the effect of Apoptozole on the change of IFN expression level.
  • FIG 7 shows that Apoptozole loses its protective effect against Zika virus infection in immunodeficient mice. In the lethal A129 and AG6 mouse models of interferon receptor deficiency, Apoptozole cannot exert antiviral effects.
  • AC is the survival curve, weight change curve and viremia of A129 mice with Zika virus infection type I interferon receptor deficiency after administration of Apoptozole;
  • DF Zika virus infection type I/II in sequence Survival curve, weight change curve and viremia of AG6 mice deficient in interferon receptor combination after administration of Apoptozole.
  • the main purpose of the present invention is to find anti-flavivirus drugs with broad-spectrum anti-flavivirus activity for the treatment of multiple flavivirus infections.
  • the present invention discovers through creative research that a variety of HSP70 inhibitors can protect cells infected by flaviviruses, among which Apotozole is the most prominent. Apoptozole has shown a good pathological protection effect against 4 flaviviruses in 3 different cell lines, including Zika virus, dengue virus, Japanese encephalitis virus and yellow fever virus.
  • Example 1 HSP70 inhibitor Apoptozole reduces the CPE of Zika virus-infected cells
  • the African green monkey kidney cells (Vero), hamster kidney cells (BHK), and human liver cancer cells (Huh7 and Huh7.5) used in the experiment were preserved in this room, and the source and the number of passages were clear.
  • the cells were cultured in a cell culture incubator saturated with humidity at 37°C and 5% CO 2.
  • pass 1:3-1:6 change the medium every 48h during the culture process, about 2-5 days (cells cover the monolayer), trypsinize with 0.25% EDTA for 2min for passaging.
  • the complete medium used for cell growth is DMEM high glucose medium supplemented with 10% FBS and penicillin double antibody
  • the maintenance medium is DMEM high glucose medium supplemented with 2% FBS and penicillin double antibody.
  • the experimental plan is as follows:
  • the cells covering the bottom of the flask were trypsinized with 0.25% EDTA and resuspended in complete medium to prepare a single cell suspension. After counting, the cells were inoculated into 96-well plates at a density of 10,000 cells per well at 37°C and 5% humidity saturated Cultivate for 24 hours under CO 2 conditions. Dilute the ZIKV virus stock solution with maintenance medium and add it to a 96-well plate to make its final concentration 100TCID50; at the same time, dilute the HSP70 inhibitor with maintenance medium and add it to a 96-well plate with final concentrations of 10 ⁇ M, 3 ⁇ M, and 1 ⁇ M, respectively. 0.3 ⁇ M, 0.1 ⁇ M, 0.03 ⁇ M, set cell control group and virus control group.
  • Luminescent Cell Viability detection solution protected from light and lysed for 5 minutes, allowed to stand for 3 minutes, and finally measured the fluorescence signal intensity with Molecular Devices M5.
  • the cell viability calculation formula is:
  • Example 2 The HSP70 inhibitor Apotozole reduces the CPE of cells infected with flavivirus, enterovirus, rhinovirus, herpes virus, Zika virus, dengue virus, Japanese encephalitis virus, and yellow fever virus
  • the African green monkey kidney cells (Vero), baby hamster kidney cells (BHK), and human liver cancer cells (Huh7 and Huh7.5) used in the experiment were preserved in this room, and the source and the number of passages were clear.
  • the cells were cultured in a cell culture incubator saturated with humidity at 37°C and 5% CO 2. Usually pass 1:3-1:6 and cultivate
  • the complete medium used for cell growth is DMEM high glucose medium supplemented with 10% FBS and penicillin double antibody
  • the maintenance medium is DMEM high glucose medium supplemented with 2% FBS and penicillin double antibody.
  • Vero cells Take Vero cells as an example to illustrate the experimental protocol, that is, the Vero cells that cover the bottom of the bottle are trypsinized in 0.25% EDTA and resuspended in complete medium to prepare a single cell suspension. After counting, inoculate at a density of 10,000 cells per well. In 96-well plate, incubate for 24 hours at 37°C and 5% CO 2 saturated with humidity.
  • Example 3 Experiments for Apoptozole to reduce viral RNA replication in Zika virus-infected cells and viral load in the supernatant
  • ZIKV-ASF GGTCAGCGTCCTCTCTAATAAACG
  • ZIKV-ASR GCACCCTAGTGTCCACTTTTTCC
  • ZIKV Probe AGCCATGACCGACACCACACCGT
  • 2Vero cells were seeded in a 12-well plate with the same seeding density as step 1. After the cells are fully attached, take the supernatant of each treatment group obtained in 1, respectively dilute with 2% cell maintenance solution to 10 -3 , 10 -4 , and 10 -5 , inoculate 500 ⁇ L per well in a 12-well plate, incubate for 2 hours Discard the supernatant, mix 2% low melting point agarose with 2 ⁇ DMEM medium 1:1, and add it to a 12-well plate, 1 mL per well after it has cooled to an appropriate temperature. Place it at room temperature until it solidifies and put it back into the incubator to continue incubating for 96 hours.
  • the treated 12-well plate is fixed with 1mL 4% formaldehyde for 4 hours, rinsed with running water to remove the upper medium, add 500 ⁇ L 1% crystal violet staining solution to each well for 15min, rinse with running water, dry upside down on absorbent paper, and count each Pfu number of holes.
  • Adherent cells were fully lysed (80 ⁇ L/well) with cell lysate (RIPA lysate: 5 ⁇ SDS Loading Buffer) to obtain whole cell protein, boiled at 100°C for 5 minutes to fully denature, and then used 10% SDS-PAGE Electrophoresis at 80V/120V for 2h separates proteins of different sizes.
  • the protein and NC membrane were transferred to the membrane under 200mA current for 100min to make the membrane fully transferred.
  • the membrane is cut to a suitable size according to the molecular weight of the target protein.
  • Use 5% skimmed milk powder diluted with TBST to block at room temperature for 1 hour add the primary antibody diluted in the blocking solution: anti-ZIKV NS1 protein (Zika virus non-structure protein 1 (Non-structure protein I)) and GAPDH, 4°C shaker Incubate overnight.
  • TBST was washed 3 times, 5min each time, and then the color was developed and photographed by the ultra-sensitive chemiluminescence method.
  • the absorbance was analyzed with BioRad ChemiDox (BioRad's chemiluminescence imager). The relative density is determined by the absolute density of ZIKV NS1 protein/GAPDH.
  • ANOVA Analysis of variance
  • African green monkey kidney cells (Vero) and hamster kidney cells-21 stably transformed ZIKV replicon cell line (BHK-21Rep) were preserved in this room, and the source and the number of passages were clear.
  • the cultivation conditions and methods are the same as above.
  • the sequence experiment was carried out with reference to the reported method (Taguwa, et al., 2015).
  • the concentration of Apoptozole used in the experiment was 10 ⁇ M
  • 2'-C-methyladenosine (2'-C- Methyladenosine, abbreviated as 2'-CMA,) is the positive control.
  • the replicon activity detection was carried out with reference to the reported method (Jia-Qi Li, et al., 2018), and heparin (Heparin, abbreviated as HP) and 2'-CMA were selected as positive controls.
  • BHK-21Rep cells were seeded with 10,000 cells/well in 96-well bottom-permeable white plates, and the final concentrations of Apotozole were 10 ⁇ M, 3 ⁇ M, 1 ⁇ M, 0.3 ⁇ M and 0.1 ⁇ M, respectively. The luminescence signal was detected after 48h.
  • ANOVA Analysis of variance
  • the human hepatocellular carcinoma cells (Huh7) used in the experiment were preserved in this room, with a clear source and passage number.
  • the cultivation conditions and methods are the same as above.
  • RNA sample obtained in step (2) is sent to Megabio for transcriptome sequencing and analysis, and the RNA-seq analysis result is obtained.
  • Example 7 Apoptozole can exert anti-ZIKV activity by up-regulating IFN expression in wild-type mice
  • the 1-day-old ICR suckling mice and 3-4-week-old Balb/c mice used in the experiment were SPF grade, purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd., with clear sources and qualified by inspection.
  • mice Three litters of 1-day-old ICR suckling mice were inoculated intraperitoneally with a lethal dose of ZIKV virus, and the mother mice were intraperitoneally administered at the doses of placebo, 0.5 mg/kg and 1 mg/kg, for 10 consecutive days. Record the weight and mortality of the suckling mice, and draw the weight curve and survival curve.
  • mice Twenty 3-4 weeks old Balb/c mice were randomly divided into two groups, one group was the control group, and the other group was the administration group (10mg/kg). And 12 hours after the challenge, were administered 3 times respectively. At 24 hours after the challenge, 50 ⁇ l of whole blood was taken to extract total RNA to detect the ZIKV RNA content in the blood. At the same time, the serum was separated, and the serum IFN- ⁇ , IFN- ⁇ was measured using a commercial ELISA kit. IFN- ⁇ and MCP-1 content detection.
  • mice and 7-8 week old AG6 mice used in the experiment were SPF grade, kept in this room, with clear sources, and qualified by inspection.
  • Appropriate number of A129 and AG6 mice were randomly divided into 3 groups. After lethal dose challenge, placebo, 0.5mg/kg Apoptozole and 1mg/kg Apoptozole were given intraperitoneally, respectively, for 10 consecutive days. Body weight, morbidity and mortality, and weight were plotted Curve and survival curve.
  • mice Sixteen 3-4 weeks old A129 mice or 7-8 weeks old AG6 mice were randomly divided into two groups, one group was the control group, the other group was the administration group (1mg/kg), and the administration group was administered ip 2 After that, 50 ⁇ l of whole blood was taken to extract total RNA to test the ZIKV RNA content in the blood.
  • Log-Rank was used to calculate the statistical significance of survival curve; analysis of variance (ANOVA) was used to calculate the statistical significance of viremia, and the data were represented in the form of mean ⁇ standard deviation. p ⁇ 0.05 indicates statistical difference.

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Abstract

一种HSP 70抑制剂广谱抗黄病毒活性的应用，具体为式I所示化合物在制备药物中的用途，所述药物用于治疗或预防黄病毒科病毒的病毒性传染病。该化合物具有广谱的抗黄病毒活性，在3种不同细胞系中对4种黄病毒，包括寨卡病毒、登革病毒、乙脑病毒和黄热病毒均表现出良好的病变保护效应，具有良好的成药性。

Description

HSP70抑制剂广谱抗黄病毒活性的应用 技术领域
本发明涉及生物医药领域，具体涉及HSP70抑制剂广谱抗黄病毒活性的应用。
背景技术
目前，日益增多的病毒性传染病称为公共卫生健康的主要威胁。其中，黄病毒科病毒在过去几十年间的多次流行使其成为各国科学家研究的焦点。2015-2017年间亚太地区发生的寨卡病毒大流行引起数千例小头症和格林-巴利综合征；登革病毒感染人数每年超过3.9亿，造成约21亿美元的经济损失；日本乙型脑炎每年新增感染人数约6.79万人次，死亡率高达20-30％，而幸存者中有30-50％承受着病毒感染造成的神经损伤所引发的后遗症。黄热病毒在南美洲与撒哈拉沙漠以南非洲地区造成相当高的死亡率。在这一严峻背景下，临床上仍无获批的抗病毒药物，因此开发具有良好抗黄病毒活性的小分子药物迫在眉睫。
目前，经过验证的具有广谱抗黄病毒活性的药物靶标十分有限，HSP70作为新近发现的一个具有良好潜力的药物靶标引起了发明人的关注。HSP70是一系列高度保守的蛋白质，参与细胞对温度和营养环境变化，病原体侵袭，以及氧化应激等反应。据报道，包浆中的HSP70可以参与DENV感染的不同阶段，包括病毒的进入、RNA复制与病毒粒子的生物合成。HSP70与ZIKV蛋白的共定位研究发现，HSP70参与ZIKV感染，并起重要作用。此外，HSP70还能够与JEV包膜蛋白结构域III及其相互作用，并在病毒的进入、复制和蛋白合成过程中发挥作用；参与YFV的复制与NS3/4A剪切；与HCV的NS5A蛋白结合参与病毒RNA复制与病毒颗粒的组装。
除此之外，HSP70蛋白表面有多种具有药物结合功能的结构域，使得许多具有不同结构的小分子化合物都具有HSP70抑制活性，对发掘具有HSP70抑制活性的小分子药物十分有利。目前已报道的HSP70抑制剂包括：作用于NBD结构域的ATP类似物、苯并噻嗪类与黄酮类化合物，与C-末端EEVD序列结合的格埃林衍生物以及与SBD结合的苯乙磺酰胺等。在此背景下，发明人选择了多种作用机制不同的HSP70抑制剂进行抗黄病毒活性评价，希望能够优选出具有广谱抗黄病毒活性的小分子化合物进行成药性研究。
发明内容
本申请是基于发明人对以下事实和问题的发现和认识作出的：
发明人通过大量的实验筛选后发现，HSP70抑制剂化合物Apoptozole(式I所示化合物)具有广谱的抗黄病毒活性，在3种不同细胞系中对4种黄病毒，包括寨卡病毒，登革病毒，乙脑病毒和黄热病毒均表现出良好的病变保护效应，具有良好的成药性。
为此，在本发明的第一方面，本发明提供了式I所示化合物(Apoptozole)在制备药物中的用途，所述药物用于治疗或预防黄病毒科病毒的病毒性传染病，
Figure PCTCN2021072027-appb-000001
在一些实施方案中，所述药物用于治疗或预防非免疫缺陷型哺乳动物黄病毒科病毒性传染病。
在一些实施方案中，所述非免疫缺陷型哺乳动物为非IFN受体缺陷型哺乳动物。
在一些实施方案中，所述非免疫缺陷型哺乳动物为非RIG-I缺陷型哺乳动物。
在一些实施方案中，所述哺乳动物为人或小鼠。
在一些实施方案中，所述黄病毒科病毒选自寨卡病毒、登革病毒、乙脑病毒、黄热病毒、黄病毒、肠道病毒、鼻病毒、疱疹病毒的至少之一。
在本发明的第二方面，本发明提供了式I所示化合物(Apoptozole)在抗黄病毒活性，或者增强或提高黄病毒科病毒感染细胞的细胞活力或细胞存活率中的用途，所述化合物用于非治疗目的，如用于科学研究，
Figure PCTCN2021072027-appb-000002
在本发明的第三方面，本发明提供了式I所示化合物(Apoptozole)在制备试剂中的用途，所述试剂用于抗黄病毒活性，或者增强或提高黄病毒科病毒感染细胞的细胞活力或细胞存活率，
Figure PCTCN2021072027-appb-000003
在一些实施方案中，所述细胞选自非洲绿猴肾细胞(Vero)、仓鼠肾细胞(BHK)、人肝癌细胞(Huh7)、人肝癌细胞(Huh7.5)的至少之一。
在一些实施方案中，所述细胞为非IFN受体缺陷型细胞。
在一些实施方案中，所述细胞为非RIG-I缺陷型细胞。
在一些实施方案中，所述黄病毒科病毒选自寨卡病毒、登革病毒、乙脑病毒、黄热病毒、黄病毒、肠道病毒、鼻病毒、疱疹病毒的至少之一。
在一些实施方案中，所述抗黄病毒活性，或者增强或提高黄病毒科病毒感染细胞的细胞活力或细胞存活率是通过下列的至少之一实现的：
A、降低黄病毒科病毒(优选寨卡病毒、登革病毒、乙脑病毒和黄热病毒)的细胞CPE水平；
B、降低黄病毒科病毒感染细胞中的病毒核酸载量水平和病毒颗粒数量；
C、降低黄病毒科病毒(优选寨卡病毒)E蛋白和NS1蛋白的产生；
D、抑制黄病毒科病毒(优选寨卡病毒)RNA复制；
E、在病毒吸附后阶段发挥抗病毒作用；
调节细胞的脂代谢和天然免疫发挥抗病毒作用；
F、上调黄病毒科病毒(优选寨卡病毒)IFN表达发挥抗病毒作用。
有益效果
本发明的发明人在经过长期研究后，发现了Apoptozole在细胞内的一些新作用特点：
第一，Apoptozole在体外抗病毒实验中，可以在微摩尔级浓度下降低寨卡病毒、登革病毒、乙脑病毒和黄热病毒的细胞CPE水平；这一保护效应在RIG-I缺陷的Huh7.5细胞中有所降低；
第二，Apoptozole可以在微摩尔级浓度下降低不同寨卡病毒分离株感染不同细胞系后细胞中的病毒核酸载量水平及上清中分泌出的感染病病毒颗粒数量，同时，Apoptozole在RIG-I缺陷的Huh7.5细胞中对病毒RNA和感染性病毒颗粒形成的抑制效果有所降低；
第三，Apoptozole可以在微摩尔级浓度下降低寨卡病毒E蛋白和NS1蛋白的产生，该效应在RIG-I缺陷的Huh7.5细胞中也有所降低，提示了Apoptozole抗病毒效果与天然免疫相关；
第四，Apoptozole在时序实验中显示，其发挥抗病毒作用主要在病毒吸附后阶段，与现有文献报道有所区别，提示了HSP70抑制剂抗黄病毒的新机制；
第五，转录组分析表明Apoptozole主要通过调节Huh7细胞的脂代谢和天然免疫发挥作用，与前期实验结论相符；
第六，Apoptozole可以在1mg/kg浓度下对感染寨卡病毒的1日龄ICR小鼠提供约60％的保护，并在10mg/kg浓度下通过上调IFN表达降低寨卡病毒感染引起的病毒血症。
第七，Apoptozole在免疫缺陷小鼠中对寨卡病毒感染没有明显保护，以此进一步证实Apoptozole可能通过调节天然免疫发挥抗病毒作用。
附图说明
图1表示HSP70抑制剂在不同细胞系中表现出广谱抗黄病毒活性。Apoptozole在不同细胞系(BHK,Vero，Huh7)中对4种黄病毒感染表现出剂量依赖的保护效果，可显著减少病毒感染引起的细胞病变效应(Cytopathic effects，CPE)。
图1中A，B，C依次为Apoptozole对寨卡病毒SMGC-1株感染的BHK、Vero和Huh7保护效果；D，E，F依次为Apoptozole对4种不同黄病毒，包括寨卡病毒(ZIKV-SMGC-1)，日本乙型脑炎病毒(JEV-SA14)，黄热病毒(YFV-17D)和登革病毒(DENV-NGC)在3种不同细胞系的抑制效果(D为BHK细胞，E为Vero细胞，F为Huh7细胞)；G，H，I依次为Apoptozole在工作浓度下对BHK，Vero和Huh7细胞的毒性。
图2表示Apoptozole抑制寨卡病毒RNA复制与感染性病毒颗粒形成。Apoptozole在Vero，BHK，A549，Huh7与Huh7.5细胞中对两种不同寨卡病毒分离株SMGC-1和MR766株的病毒RNA复制与感染性病毒颗粒的产生均有明显抑制效果，并表现出剂量依赖性。同时发现RIG-1失活突变的Huh7.5细胞中Apoptozole的抗病毒效果有所降低。
图2中A-E依次为Apoptozole在Vero，BHK，A549，Huh7和Huh7.5细胞系中对寨卡病毒SMGC-1株与MR766株RNA合成的抑制效果；F-J依次为Apoptozole在Vero，BHK，A549，Huh7和Huh7.5细胞系中对寨卡病毒SMGC-1株与MR766株感染性病毒颗粒产生的抑制效果；K-O依次为Apoptozole在工作浓度下对Vero，BHK，A549，Huh7和Huh7.5细胞的毒性。
图3表示Apoptozole抑制寨卡病毒E蛋白和NS1蛋白产生。Apoptozole在不同细胞系(A549，Huh7，Huh7.5)中均能有效抑制寨卡病毒包膜蛋白(E-protein，IFA)与非结构蛋白1(NS1，WB)的产生。
图3中DAPI为IF成像中核染料标记的细胞核通道，MERGE为细胞核通道与目 的蛋白荧光信号共定位(合并)图片。图中A，C，E依次为Apoptozole在A549，Huh7和Huh7.5细胞系中对寨卡病毒E蛋白产生的抑制效果，B，D，F依次为对应的定量分析结果；G为Apoptozole在Vero，Huh7和Huh7.5细胞系中对寨卡病毒NS1蛋白产生的抑制效果，H-J为对应的定量数据。
图4表示Apoptozole在寨卡病毒感染细胞过程的吸附后阶段发挥抗病毒作用。在不同细胞系感染寨卡病毒的不同阶段加入Apoptozole后检测显示，Apoptozole主要在病毒复制阶段发挥作用，对病毒的吸附、进入无明显抑制作用。
图4中DAPI为IF成像中核染料标记的细胞核通道，MERGE为细胞核通道与目的蛋白荧光信号共定位(合并)图片。图中A为Apoptozole加入的时间点示意图，B，C，D依次为Apoptozole在Vero，Huh7和Huh7.5细胞中进行的时序实验对病毒RNA复制的抑制；E，F，G依次为Apoptozole在Vero，Huh7和Huh7.5细胞中进行的时序实验对病毒蛋白表达的抑制效果。2’-CMA(2’-C-Methyladenosine,2-C-甲基腺苷)为已报道特异性作用于病毒复制阶段的阳性化合物。
图5表示转录组分析揭示Apoptozole通过调节脂代谢与天然免疫发挥抗病毒作用。
图5中A,B为Apoptozole引起的显著性变化基因热图与相应数量；C为具有显著变化的全部基因列表；D为对全部显著变化基因的GO(Gene Ontology)功能注释分析结果；E为对全部显著变化基因的KEGG(Kyoto Encyclopedia of Genes and Genomes)功能富集分析结果。
图6表示Apoptozole在野生型小鼠中通过上调天然免疫保护抵抗寨卡病毒感染。Apoptozole对致死剂量寨卡病毒接种的ICR乳鼠变现出剂量依赖的保护效果；同时Apoptozole可通过上调干扰素的表达降低寨卡病毒感染的Balb/c小鼠的病毒血症。
图6中A为Apoptozole在ICR乳鼠致死模型中对寨卡病毒感染的保护效果，B为对应的体重变化情况；C为Apoptozole对Balb/c小鼠病毒血症的抑制，D，E，F，G为Apoptozole对IFN表达水平变化的影响。
图7表示Apoptozole在免疫缺陷鼠中失去对寨卡病毒感染的保护效应。在干扰素受体缺陷的A129与AG6小鼠致死模型中，Apoptozole不能发挥抗病毒作用。
图7中A-C依次为寨卡病毒感染的Ⅰ型干扰素受体缺陷的A129小鼠在给予Apoptozole后的生存曲线，体重变化曲线及病毒血症；D-F依次为寨卡病毒感染的Ⅰ/Ⅱ型干扰素受体联合缺陷的AG6小鼠在给予Apoptozole后的生存曲线，体重变化曲线及病毒血症。
具体实施方式
下面详细描述本发明的实施例，下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
本发明主要目的是寻找具有广谱抗黄病毒活性的抗黄病毒药物，用于多种黄病毒感染的救治。本发明通过创造性的研究发现多种HSP70抑制剂能够保护黄病毒感染的细胞，其中Apoptozole最为突出。Apoptozole在3种不同细胞系中对4种黄病毒，包括寨卡病毒，登革病毒，乙脑病毒和黄热病毒均表现出良好的病变保护效应。
实施例1 HSP70抑制剂Apoptozole降低寨卡病毒感染细胞CPE实验
本发明中用到的实验材料和实验方法：
(1)细胞培养和细胞系
实验过程中所用非洲绿猴肾细胞(Vero)，仓鼠肾细胞(BHK)，人肝癌细胞(Huh7和Huh7.5)为本室保存，来源及传代次数明确。细胞培养于37℃，5％CO 2的湿度饱和细胞培养箱中。通常按1:3-1:6传代，培养过程中每48h换液一次，约2-5天(细胞铺满单层)用0.25％的EDTA胰酶消化2min进行传代。细胞生长所用完全培养基为添加10％FBS和青链霉素双抗的DMEM高糖培养基，维持培养基为添加2％FBS和青 链霉素双抗的DMEM高糖培养基。
(2)细胞活力检测
细胞活力用CellTiter
Figure PCTCN2021072027-appb-000004
Luminescent Cell Viability Assay测定。
实验方案如下：
铺满瓶底的细胞在0.25％EDTA胰酶消化后用完全培养基重悬，制备单细胞悬液，计数后以每孔10000细胞的密度接种96孔板，在37℃、湿度饱和的5％CO 2条件下培养24小时。用维持培养基稀释ZIKV病毒原液，加入96孔板，使其终浓度为100TCID50；同时，将HSP70抑制剂用维持培养基倍比稀释加入96孔板，其终浓度分别为10μM，3μM，1μM，0.3μM，0.1μM，0.03μM，设置细胞对照组和病毒对照组。处理6天后弃上清，每孔加入用PBS缓冲液2倍稀释的CellTiter
Figure PCTCN2021072027-appb-000005
Luminescent Cell Viability检测液，避光震荡裂解5min，静置3min，最后用Molecular Devices M5测定荧光信号强度。细胞活力计算公式为：
Figure PCTCN2021072027-appb-000006
实验结果如图1中A、B、C、G、H、I所示。
实施例2 HSP70抑制剂Apoptozole降低黄病毒、肠道病毒、鼻病毒、疱疹病毒、寨卡病毒、登革病毒、乙脑病毒、黄热病毒感染细胞CPE实验
本发明中用到的实验材料和实验方法：
(1)细胞培养和细胞系，病毒株
实验过程中所用非洲绿猴肾细胞(Vero)、幼地鼠肾细胞(BHK)、人肝癌细胞(Huh7和Huh7.5)为本室保存，来源及传代次数明确。细胞培养于37℃，5％CO 2的湿度饱和细胞培养箱中。通常按1:3-1:6传代，培养
过程中每48h换液一次，约2-5天(细胞铺满单层)用0.25％的EDTA胰酶消化2min进行传代。细胞生长所用完全培养基为添加10％FBS和青链霉素双抗的DMEM高糖培养基，维持培养基为添加2％FBS和青链霉素双抗的DMEM高糖培养基。
Figure PCTCN2021072027-appb-000007
(2)细胞活力检测
细胞活力用CellTiter
Figure PCTCN2021072027-appb-000008
Luminescent Cell Viability Assay测定。
以Vero细胞为例进行实验方案说明，即：铺满瓶底的Vero细胞在0.25％EDTA胰酶消化后用完全培养基重悬，制备单细胞悬液，计数后以每孔10000细胞的密度接种96孔板，在37℃、湿度饱和的5％CO 2条件下培养24小时。用维持培养基稀释ZIKV病毒原液，加入96孔板，使其终浓度为100TCID50；同时，将Apoptozole化合物用维持培养基倍比稀释加入96孔板，其终浓度分别为10μM，3μM，1μM，0.3μM，0.1μM和0.03μM，设置 细胞对照组和病毒对照组。处理6天后弃上清，每孔加入用PBS缓冲液2倍稀释的CellTiter
Figure PCTCN2021072027-appb-000009
Luminescent Cell Viability检测液，避光震荡裂解5min，静置3min，最后用Molecular Devices M5测定荧光信号强度。CPE计算公式为：
Figure PCTCN2021072027-appb-000010
Figure PCTCN2021072027-appb-000011
实验结果如图1中D、E、F所示。
实施例3 Apoptozole降低寨卡病毒感染细胞内的病毒RNA复制与上清中病毒载量实验
本发明中用到的实验材料和实验方法：
(1)一步法实时定量RT-PCR实验与转录组分析
①Vero，BHK,A549，Huh7和Huh7.5细胞于75cm 2培养瓶中培养，待其铺满瓶底后按1:3接种12孔板，贴壁培养过夜。细胞完全贴壁后，用2％细胞维持培养基将ZIKV病毒稀释成相应浓度，然后加入6孔板中使每孔含有病毒量为100TCID50(MOI＝0.01)，再用2％细胞维持液将Apoptozole和HSP70系列化合物分别稀释成相应浓度，加入到对应的孔中，使药物最终浓度分别为10μM，3μM和1μM，继续培养72h，待细胞开始出现病变即收集上清，8000rpm 5min离心、分装，于-80℃保存待用。
②RNA提取
1)取经不同浓度Apoptozole和HSP70系列化合物处理过的细胞及病毒对照组的细胞，加入350μl Buffer RLT，用移液枪吹吸混匀使其充分裂解；
2)加入等体积的70％乙醇，混匀；
3)将上述混合液转入无RNA酶的2ml收集管中，12000rpm离心15s；
4)加入700μBuffer RW1,12000rpm离心15s，弃废液；
5)加入500μBuffer RPE,12000rpm离心15s，弃废液；
6)加入500μBuffer RPE,12000rpm离心2min，弃废液；
7)换新的无RNA酶的2ml收集管，12000rpm离心1min，使滤柱干燥；
8)换上新的1.5ml收集管，每管加入50μl不含RNA酶的水，12000rpm离心2min，洗脱液即含有相应的RNA，用Nano Drop检测各RNA浓度。
③Real-time PCR
1)用重组ZIKV线性化质粒制备标准品：根据线性化质粒浓度和分子量计算标准品拷贝数，并将其精确稀释为-1，-2，-3，-4，-5，-6，-7，-8，-9.。。。直至拷贝数低于荧光定量PCR仪检测限。标准品制备完成后将各浓度标准品分装，于-80℃冻存待用。
2)用Takara公司的One Step PrimeScript TM RT-PCR Kit(Perfect Real Time)，检测样品中ZIKV RNA载量，每个样品进行三次独立重复试验。实验过程中所用引物和探针序列如下：
ZIKV-ASF：GGTCAGCGTCCTCTCTAATAAACG
ZIKV-ASR：GCACCCTAGTGTCCACTTTTTCC
ZIKV Probe：AGCCATGACCGACACCACACCGT
(2)pfu检测
①Vero细胞于75cm 2培养瓶中培养，待其铺满瓶底后按1:3接种12孔板，贴壁培养过夜。细胞完全贴壁后，用2％细胞维持培养基将ZIKV病毒稀释成相应浓度，然后加入6孔板中使每孔含有病毒量为100TCID50(MOI＝0.01)，再用2％细胞维持液将Apoptozole和HSP70系列化合物分别稀释成相应浓度，加入到对应的孔中，使药物最终浓度分别为10μM，3μM和1μM，继续培养72h，待细胞开始出现病变即收集上清，8000rpm 5min离心、分装，于-80℃保存待用。
②Vero细胞接种12孔板，接种密度同步骤①。待细胞完全贴壁后，取①所得各处理组 上清，分别用2％细胞维持液稀释为10 -3,10 -4,10 -5，每孔500μL接种于12孔板，孵育2小时后弃上清，将2％低熔点琼脂糖与2×DMEM培养基1:1混匀，待其降到适当温度后加入12孔板，每孔1mL。室温放置至凝固，放回培养箱继续培养96小时。
③处理好的12孔板加1mL 4％甲醛固定4小时，流水冲洗去掉上层培养基，每孔加500μL 1％结晶紫染色液固定15min，流水冲洗，倒扣于吸水纸上晾干，计数各孔pfu数。
实验结果如图2所示。
实施例4 Apoptozole降低寨卡病毒感染细胞内病毒蛋白合成实验
本发明中用到的实验材料和实验方法：
(1)细胞培养和细胞系
实验过程中所用非洲绿猴肾细胞(Vero)、人肝癌细胞(Huh7)、人肝癌细胞RIG-Ⅰ失活突变单克隆(Huh7.5)为本室保存，来源及传代次数明确。培养条件与方法同上。
(2)IFA
A549，Huh7，Huh7.5细胞按10000Cells/孔接种于96孔底透黑板，过夜贴壁培养。待细胞完全贴壁后，用2％细胞维持培养基将ZIKV病毒稀释成相应浓度，接入96孔板中(MOI＝0.5)，孵育2小时后弃液，加入预先用2％细胞维持液稀释好的Apotozole和HSP70系列化合物，使其终浓度分别为10μM，3μM和1μM，继续培养48小时后弃液，PBS洗涤2次，每孔加100μL 4％甲醛固定30min，弃固定液，加PBS缓冲液，进行抗体孵育标记后成像。
(3)Western blot蛋白分析
Vero，Huh7，Huh7.5细胞于75cm 2培养瓶中培养，待其铺满瓶底后按1:3接种12孔板，贴壁培养过夜。细胞完全贴壁后，用2％细胞维持培养基将ZIKV病毒稀释成相应浓度，然后加入6孔板中使每孔含有病毒量为100TCID50(MOI＝0.01)，再用2％细胞维持液将Apoptozole稀释成相应浓度，加入到对应的孔中，使药物最终浓度分别10μM，3μM，1μM,0.3μM和0.1μM，继续培养72h，待细胞开始出现病变即收集上清。贴壁细胞用细胞裂解液(RIPA裂解液：5×SDS Loading Buffer)充分裂解(80μL/孔)，获得全细胞蛋白，100℃煮沸5min，使其充分变性，然后用10％的SDS-PAGE在80V/120V下电泳2h使不同大小的蛋白分离。蛋白与NC膜在200mA电流下转膜100min，使其充分转膜。
转膜完成后，根据目的蛋白分子量将膜裁剪成合适大小。用TBST稀释的5％脱脂奶粉室温摇床封闭1小时，加入封闭液稀释的一抗：抗ZIKV NS1蛋白(寨卡病毒非结构蛋白1(Non-structure protein I))和GAPDH，4℃摇床孵育过夜。，加入二抗：HRP标记的山羊抗小鼠IgG抗体(1:5000稀释)，室温摇床孵育1小时。TBST洗3次，每次5min，然后通过超敏化学发光法进行显色拍照。最后用BioRadChemiDox(BioRad的化学发光成像仪)分析吸光度。相对密度由ZIKV NS1 protein/GAPDH的绝对密度决定。
(4)统计学分析
利用方差分析(ANOVA)进行统计学显著性的计算。数据以平均值±标准差的形式来体现。p<0.05表示具有统计学差异。
实验结果如图3所示。
实施例5 Apoptozole抗寨卡病毒作用阶段发现
本发明中用到的实验材料和实验方法：
(1)细胞培养和细胞系
实验过程中所用非洲绿猴肾细胞(Vero)、仓鼠肾细胞-21稳转ZIKV复制子细胞系(BHK-21Rep)为本室保存，来源及传代次数明确。培养条件与方法同上。
(2)时序实验
时序实验参考已报道的方法进行(Taguwa,et al.,2015)，实验中所用Apoptozole浓 度为10μM，ZIKV感染剂量为MOI＝1，选用2’-C-甲基腺苷(2’-C-Methyladenosine，缩写为2’-CMA,)为阳性对照。
(3)复制子实验
复制子活性检测参考已报道的方法进行(Jia-Qi Li,et al.,2018)，选用肝素(Heparin，缩写为HP)与2’-CMA为阳性对照。BHK-21Rep细胞按10000细胞/孔接种96孔底透白板，Apotozole终浓度分别为10μM，3μM，1μM,0.3μM和0.1μM，48h后检测发光信号。
(4)统计学分析
利用方差分析(ANOVA)进行统计学显著性的计算。数据以平均值±标准差的形式来体现。p<0.05表示具有统计学差异。
实验结果如图4所示。
实施例6 基于RNA-seq技术的Apoptozole抗寨卡病毒机制探索
本发明中用到的实验材料和实验方法：
(1)细胞培养和细胞系
实验过程中所用人肝癌细胞(Huh7)为本室保存，来源及传代次数明确。培养条件与方法同上。
(2)RNA提取
1)取10μM Apoptozole处理24h的病毒感染细胞与病毒对照和细胞对照收集液，加入350μl Buffer RLT，用移液枪吹吸混匀使其充分裂解；
2)加入等体积的70％乙醇，混匀；
3)将上述混合液转入无RNA酶的2ml收集管中，12000rpm离心15s；
4)加入700μBuffer RW1,12000rpm离心15s，弃废液；
5)加入500μBuffer RPE,12000rpm离心15s，弃废液；
6)加入500μBuffer RPE,12000rpm离心2min，弃废液；
7)换新的无RNA酶的2ml收集管，12000rpm离心1min，使滤柱干燥；
8)换上新的1.5ml收集管，每管加入50μl不含RNA酶的水，12000rpm离心2min，洗脱液即含有相应的RNA，用Nano Drop检测各RNA浓度。
(3)RNA seq
步骤(2)所得RNA样品送美吉生物进行转录组测序与分析，得出RNA-seq分析结果。
实验结果如图5所示。
实施例7 Apoptozole在野生型小鼠体内可通过上调IFN表达发挥抗ZIKV活性
本发明中用到的实验材料和实验方法：
(1)小鼠品系
实验过程中所用1日龄ICR乳鼠与3-4周龄Balb/c小鼠为SPF级，由维通利华实验动物技术有限公司购入，来源明确，经检验合格。
(2)ICR乳鼠致死性感染保护
整窝1日龄ICR乳鼠3窝，腹腔接种致死剂量ZIKV病毒，同时对母鼠进行腹腔给药，给药剂量分别为安慰剂，0.5mg/kg和1mg/kg，连续给药10天，记录乳鼠体重及发病死亡情况，绘制体重曲线和生存曲线。
(3)Balb/c小鼠病毒血症及细胞因子与趋化因子检测
20只3-4周龄Balb/c小鼠随机分为两组，一组为对照组，一组为给药组(10mg/kg)，给药组通过i.p.在攻毒前12h，攻毒同时及攻毒后12h分别给药3次，攻毒后24h取50μl全血提取总RNA检测血液中ZIKV RNA含量，同时分离血清，利用商品化ELISA试剂盒进行血清中IFN-α，IFN-β，IFN-γ与MCP-1含量检测。
(4)统计学分析
利用Log-Rank进行生存曲线统计学显著性的计算。p<0.05表示具有统计学差异。
实验结果如图6所示。
实施例8 Apoptozole在IFN受体缺陷小鼠体内不能发挥抗ZIKV活性
本发明中用到的实验材料和实验方法：
(1)小鼠品系
实验过程中所用3-4周龄A129小鼠与7-8周龄AG6小鼠为SPF级，为本室保存，来源明确，经检验合格。
(2)干扰素受体缺陷鼠致死性感染保护实验
适当数量的A129与AG6小鼠随机分为3组，致死剂量攻毒后分别腹腔给予安慰剂，0.5mg/kg Apoptozole和1mg/kg Apoptozole，连续给药10天，体重及发病死亡情况，绘制体重曲线和生存曲线。
(3)干扰素受体缺陷鼠病毒血症检测
16只3-4周龄A129小鼠或7-8周龄AG6小鼠随机分为两组，一组为对照组，一组为给药组(1mg/kg)，给药组i.p.给药2天，之后取50μl全血提取总RNA检测血液中ZIKV RNA含量。
(4)统计学分析
利用Log-Rank进行生存曲线统计学显著性的计算；利用方差分析(ANOVA)进行病毒血症统计学显著性的计算，数据以平均值±标准差的形式来体现。p<0.05表示具有统计学差异。
实验结果如图7所示。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (8)

  1. 式I所示化合物在制备药物中的用途，所述药物用于治疗或预防黄病毒科病毒的病毒性传染病，
    Figure PCTCN2021072027-appb-100001
  2. 权力要求1所述的用途，其中，所述药物用于治疗或预防非免疫缺陷型哺乳动物黄病毒科病毒性传染病；
    优选地，所述非免疫缺陷型哺乳动物为非IFN受体缺陷型哺乳动物；
    优选地，所述非免疫缺陷型哺乳动物为非RIG-I缺陷型哺乳动物；
    优选地，所述哺乳动物为人或小鼠。
  3. 权利要求1所述的用途，其中，所述黄病毒科病毒选自寨卡病毒、登革病毒、乙脑病毒、黄热病毒、黄病毒、肠道病毒、鼻病毒、疱疹病毒的至少之一。
  4. 式I所示化合物在抗黄病毒活性，或者增强或提高黄病毒科病毒感染细胞的细胞活力或细胞存活率中的用途，所述化合物用于非治疗目的，
    Figure PCTCN2021072027-appb-100002
  5. 式I所示化合物在制备试剂中的用途，所述试剂用于抗黄病毒活性，或者增强或提高黄病毒科病毒感染细胞的细胞活力或细胞存活率，
    Figure PCTCN2021072027-appb-100003
    Figure PCTCN2021072027-appb-100004
  6. 权力要求4或5所述的用途，其中，所述细胞选自非洲绿猴肾细胞(Vero)、仓鼠肾细胞(BHK)、人肝癌细胞(Huh7)、人肝癌细胞(Huh7.5)的至少之一；
    优选地，所述细胞为非IFN受体缺陷型细胞；
    优选地，所述细胞为非RIG-I缺陷型细胞；
  7. 权利要求4或5所述的用途，其中，所述黄病毒科病毒选自寨卡病毒、登革病毒、乙脑病毒、黄热病毒、黄病毒、肠道病毒、鼻病毒、疱疹病毒的至少之一。
  8. 权利要求4或5所述的用途，其中，所述抗黄病毒活性，或者增强或提高黄病毒科病毒感染细胞的细胞活力或细胞存活率是通过下列的至少之一实现的：
    A、降低黄病毒科病毒(优选寨卡病毒、登革病毒、乙脑病毒和黄热病毒)的细胞CPE水平；
    B、降低黄病毒科病毒感染细胞中的病毒核酸载量水平和病毒颗粒数量；
    C、降低黄病毒科病毒(优选寨卡病毒)E蛋白和NS1蛋白的产生；
    D、抑制黄病毒科病毒(优选寨卡病毒)RNA复制；
    E、在病毒吸附后阶段发挥抗病毒作用；
    调节细胞的脂代谢和天然免疫发挥抗病毒作用；
    F、上调黄病毒科病毒(优选寨卡病毒)IFN表达发挥抗病毒作用。
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