Application of small-molecule inhibitor in preparation of product for resisting Ebola virus infection

Abstract

The invention discloses application of a small-molecule inhibitor in preparation of a product for resisting Ebola virus infection. According to the application, the Ebola virus resistance of the TOFA is determined for the first time, the cytotoxicity is small (the CC50 value is 7.717 mu M), and the TOFA can effectively inhibit the replication of Ebola pseudoviruses (the IC50 value is 1.976 mu M) at low concentration, so that a new candidate small molecule compound is provided for developing a novel medicine for resisting Ebola virus infection, a new technical choice is provided for effectively preventing fulminant infectious diseases, and the application has a wide application prospect. Wide application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a small molecule inhibitor in the preparation of a product for resisting Ebola virus infection. Background Art

[0002] Ebola virus disease (EVD), also known as Ebola hemorrhagic fever (EHF), is a highly contagious hemorrhagic infection caused by the Ebola virus (EOV) infecting humans and non-human primates. Ebola virus is an RNA virus characterized by high infectivity, high mortality, and high pathogenicity. It has a biosafety level 4 rating and is extremely dangerous, with a fatality rate as high as 90%. Currently, the US Food and Drug Administration (FDA) has approved two monoclonal antibodies (mAbs) for the treatment of EVD: Inmazeb and Ebanga. However, in the PALM (Pamoja Tulinde Maisha) study, more than 30% of patients treated with these two drugs died. Therefore, there is an urgent need to develop new EBOV therapeutics to improve EVD treatment outcomes.

[0003] Compared to mAbs, small molecules offer the advantages of ease of production, transportation, and storage, as well as lower production costs. Furthermore, some EVD survivors persist with Ebola virus in their bodies. The large molecular weight of mAbs limits their penetration into immune-protected areas where Ebola virus persists. Small molecules, however, have a smaller molecular weight and greater penetration, helping to completely eliminate the virus and prevent potential transmission and sequelae. The development of small molecules that can protect against Ebola virus infection is underway. Summary of the Invention

[0004] In order to solve the problems in the prior art, the present invention provides a use of a small molecule inhibitor in the preparation of a product for resisting Ebola virus infection.

[0005] The first object of the present invention is to provide a use of a small molecule inhibitor in the preparation of a product for resisting Ebola virus infection.

[0006] The second object of the present invention is to provide a use of a small molecule inhibitor in the preparation of a drug for preventing and / or treating diseases caused by Ebola virus.

[0007] The third object of the present invention is to provide a use of a small molecule inhibitor in the preparation of a product for preventing and / or treating Ebola hemorrhagic fever.

[0008] The fourth object of the present invention is to provide a use of a small molecule inhibitor in the preparation of a product for inhibiting Ebola virus replication.

[0009] A fifth object of the present invention is to provide a use of a small molecule inhibitor in the preparation of a product that inhibits the expression of Ebola virus genes and / or proteins.

[0010] A sixth object of the present invention is to provide a preparation for resisting Ebola virus infection.

[0011] The seventh object of the present invention is to provide a pharmaceutical composition.

[0012] In order to achieve the above object, the present invention is implemented through the following scheme: The present invention uses the Ebola virus replicon system to clarify the anti-Ebola virus effect of acetyl-CoA carboxylase-α (ACCA) allosteric inhibitor (TOFA). TOFA can inhibit the expression of VP40 gene and protein and GP gene and protein, and can inhibit the replication and infection of Ebola pseudovirus.

[0013] The present invention seeks protection for the following: A small molecule inhibitor is used in the preparation of a product for resisting Ebola virus infection, wherein the structural formula of the small molecule inhibitor is , namely TOFA.

[0014] A small molecule inhibitor is used in the preparation of a drug for preventing and / or treating a disease caused by an Ebola virus, wherein the small molecule inhibitor has the structural formula .

[0015] A small molecule inhibitor is used in the preparation of a product for preventing and / or treating Ebola hemorrhagic fever, wherein the structural formula of the small molecule inhibitor is .

[0016] A small molecule inhibitor is used in the preparation of a product for inhibiting Ebola virus replication, wherein the structural formula of the small molecule inhibitor is .

[0017] A small molecule inhibitor is used in the preparation of a product for inhibiting the expression of Ebola virus genes and / or proteins, wherein the small molecule inhibitor has the structural formula .

[0018] Preferably, the Ebola virus gene includes VP40 Genes and / or GP Gene.

[0019] Preferably, the Ebola virus protein includes VP40 Protein and / or GP protein.

[0020] An anti-Ebola virus infection preparation, comprising a structural formula such as Small molecule inhibitors as shown.

[0021] Preferably, the preparation is in the form of injection, tablet, spray, granule, capsule, oral solution or patch.

[0022] Preferably, the content of the small molecule inhibitor in the preparation is 0.1 μM to 12.8 μM.

[0023] More preferably, the content of the small molecule inhibitor in the preparation is 0.1 μM to 2 μM.

[0024] Further preferably, the content of the small molecule inhibitor in the preparation is 0.4 μM.

[0025] A pharmaceutical composition comprising a structural formula The small molecule inhibitor shown and a pharmaceutically acceptable carrier or excipient.

[0026] Preferably, the content of the small molecule inhibitor in the pharmaceutical composition is 0.1 μM to 12.8 μM.

[0027] More preferably, the content of the small molecule inhibitor in the preparation is 0.1 μM to 2 μM.

[0028] Further preferably, the content of the small molecule inhibitor in the pharmaceutical composition is 0.4 μM.

[0029] Compared with the prior art, the present invention has the following beneficial effects: This invention clarifies for the first time the anti-Ebola virus ability of TOFA, which has low cytotoxicity (CC50 value is 7.717 μM). TOFA can effectively inhibit the replication of Ebola pseudovirus at low concentrations (IC50 value is 1.976 μM), providing new candidate small molecule compounds for the development of new drugs against Ebola virus infection, and offering new technical options for effectively preventing severe infectious diseases, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a graph showing the results of the TOFA cytotoxicity experiment in Example 1.

[0031] Figure 2 The figure shows the effect of different concentrations of TOFA on Ebola virus replication in Example 2.

[0032] Figure 3 This is the effect of TOFA on the expression levels of Ebola virus VP40 protein and GP protein in Example 3.

[0033] Figure 4 This is the effect of TOFA on the expression level of the Ebola virus VP40 gene in Example 4.

[0034] Figure 5 This is the effect of TOFA on the expression level of the Ebola virus GP gene in Example 4. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0036] The complete set of EBOV-trVLPs pseudovirus plasmids used in the following examples: p4cis-vRNA-RLuc (encoding proteins: Renilla luciferase, VP40, GP, and VP24), pCAGGS-NP, pCAGGS-VP35, pCAGGS-VP30, pCAGGS-L, pCAGGS-TIM1, pCAGGS-T7, and pCAGGS-firefly luciferase (i.e., pCAGGS-luc2) were kindly provided by Professor Thomas Hoenen of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA. The structural formula of the allosteric inhibitor of acetyl-CoA carboxylase-α (TOFA) is , purchased from MCE Company.

[0037] Example 1 Cytotoxicity experiment of TOFA Cytotoxicity assay was performed using the Biosharp CCK-8 kit (Cat. No. BS350B). The specific steps are as follows: In a 96-well plate, 2000 cells were seeded per well with HEK293T cell suspension (100 μL / well). After 12 h of culture, TOFA was added at final concentrations of 100 nM, 200 nM, 400 nM, 800 nM, 1.6 μM, 3.2 μM, 6.4 μM, and 12.8 μM for drug treatment. Each corresponding well was a drug-addition well, and three replicates were set for each concentration. At the same time, negative control wells without TOFA and blank wells without cells and TOFA were set.

[0038] After 48 h of TOFA treatment, 10 μL of CCK-8 solution was added to each well and incubated in a cell culture incubator for another 4 h. The absorbance of each well at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula: cell viability = [OD(drug-treated well) - OD(blank well)] / [OD(negative control well) - OD(blank well)] × 100%.

[0039] GraphPad Prism was used to fit a nonlinear curve between the logarithm of TOFA concentration and cell viability. The inhibition rate was negatively correlated with cell viability, and the CC50 value was calculated from the resulting curve to determine the TOFA concentration at which the inhibition rate reached 50%.

[0040] like Figure 1 As shown in the figure, the CC50 value of TOFA is 7.717 μM, indicating that the small molecule drug TOFA has low cytotoxicity and high safety when its working concentration is below 7.717 μM.

[0041] Example 2 Effects of different concentrations of TOFA on Ebola virus replication 1. Preparation of the first generation of Ebola pseudovirus (1) Replication-competent pseudovirus system (EBOV virus-like particles) The Ebola virus replicon system used in the present invention is a mature system reported in the prior art (DOI: 10.3389 / fcimb.2017.00479) and recognized. Its working principle is to use reverse genetics technology and eukaryotic expression system to construct multi-life cycle Ebola virus-like particles, and to simulate the Ebola virus adhesion, invasion, transcription, replication, virus particle packaging, virus budding, continuous passage and other multi-life cycles under BSL-2 conditions, thereby providing strong technical support for basic and applied research on Ebola virus.

[0042] The EBOV genome encodes a total of seven structural proteins, including RNA polymerase (L), nucleocapsid protein (NP), polymerase cofactor (VP35) and transcription activator (VP30) that are involved in the viral transcription and replication process, as well as envelope glycoprotein (GP), matrix protein (VP40) and second matrix protein (VP24) that constitute the viral shell structure and participate in viral membrane binding, assembly and budding processes.

[0043] The trVLPs-EBOV pseudovirus system mimics the entire Ebola virus life cycle. The system consists of a tetracistronic minigenome (MG) with initial transcription under the T7 polymerase promoter. The MG contains two non-coding regions on either side and the Renilla luciferase enzyme in the middle. Renilla luciferase Gene, VP40 Gene, GP Genes and VP24 Gene.

[0044] The plasmid encoding MG (p4cis-vRNA-RLuc) and the plasmids encoding NP, VP35, VP30, L and T7 RNA polymerase (pCAGGS-NP, pCAGGS-VP35, pCAGGS-VP30, pCAGGS-L and pCAGGS-T7) were co-transfected into primary cells (Producer cells) to package the primary generation trVLPs-EBOV (i.e., P0 generation trVLPs).

[0045] P0 trVLPs were then used to infect target cells transfected with plasmids encoding TIM1, NP, VP35, VP30, and L, resulting in P1 trVLPs-EBOV. The replication level of trVLPs-EBOV was assessed by measuring Renilla luciferase (Rluc) activity in target cell lysates.

[0046] (2) Preparation of P0 generation trVLPs-EBOV To package multi-lifecycle Ebola virus-like particles (EVLPs), pCAGGS-NP, pCAGGS-VP35, pCAGGS-VP30, pCAGGS-L, pCAGGS-TIM1, and pCAGGS-T7, as well as a eukaryotic expression plasmid for the firefly luciferase Luc2 (pCAGGS-luc2), were prepared using pCAGGS as a backbone. In addition, the p4cis-vRNA-RLuc plasmid, containing a synthetic viral minigenome, was also prepared for subsequent VLP packaging. The construction and sequencing of the plasmids described in this example were jointly completed by the Academy of Military Science, the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Shanghai Jikai Gene Chemistry Technology Co., Ltd., and Jilin Kumei Biotechnology Co., Ltd.

[0047] The specific steps of virus packaging are as follows: revive HEK293T cells, pass them for 3 generations, and then resuspend them in a 5×10 5 / mL were seeded into 6-well plates and cultured in a 37°C cell culture incubator with 5% CO2. After 12 hours of cell culture, plasmid transfection was initiated. The transfection reagent Lipofectamine™ 3000 was removed from the 4°C refrigerator and allowed to stand at room temperature (25°C) until ready to use. 100 µL of Opti-MEM was added to each well of the 6-well plate. To a sterile centrifuge tube, add 300 µL of Opti-MEM and the corresponding plasmid according to the dosage in Table 1. Simultaneously, add 30 µL of P3000 and vortex to mix thoroughly. To another sterile centrifuge tube, add 300 µL of Opti-MEM and 7.5 µL of Lipofectamine™ 3000 transfection reagent to each well, i.e., add 45 µL of Lipofectamine™ 3000 transfection reagent to the sterile centrifuge tube, gently vortex to mix thoroughly, and incubate each well at room temperature for 5 min. Gently vortex the two tubes together and incubate at room temperature (25°C) for 5 min to obtain transfection system 1.

[0048] Table 1 Virus packaging plasmid transfection dose (6-well plate)

[0049] Add 100 μL of transfection system 1 to each well of a 6-well plate, shake the 6-well plate back and forth to evenly disperse the transfection system, and then continue to culture the cells in a 37°C cell culture incubator containing 5% CO2; remove the cell culture supernatant after 24 hours, and add 2 mL of DMEM medium containing 5% v / v fetal bovine serum and 1% v / v double antibody to each well of the 6-well plate; place the 6-well plate after replacing the medium in a 37°C cell culture incubator containing 5% CO2 and continue to culture for 48 hours; collect the cultured cell supernatant into a 15 mL centrifuge tube, centrifuge at 800 g for 5 minutes at room temperature (25°C) to remove cell debris, and obtain P0 generation trVLPs-EBOV; aliquot and freeze at -80°C.

[0050] 2. Transfection of target cells with helper plasmids After 3 generations of continuous passage, the HEK293T cells were revived and cultured in DMEM (containing 10% FBS + 1% double antibody) at a cell density of 4×10 5 / mL were inoculated into 24-well plates and cultured in a 37°C cell culture incubator with 5% CO2. After 12 hours of cell culture, when the cell density reached 80%, plasmid transfection was started.

[0051] To a sterile centrifuge tube, add 500 µL of Opti-MEM and the corresponding plasmid according to the dosage in Table 2. Simultaneously, add 48 µL of P3000 and vortex to mix thoroughly. To another sterile centrifuge tube, add 500 µL of Opti-MEM and 2 µL of Lipofectamine™ 3000 transfection reagent to each well. Add 48 µL of Lipofectamine™ 3000 transfection reagent to the sterile centrifuge tube, gently vortex to mix thoroughly, and incubate each well at room temperature (25°C) for 5 min. Gently vortex the two tubes together and incubate at room temperature (25°C) for 15 min to obtain transfection system 2.

[0052] Table 2 Plasmid transfection dosage for target cells (24-well plate)

[0053] Add 44 µL of transfection system 2 to each well of a 24-well plate, shake the 24-well plate back and forth to evenly disperse the transfection system, and then place the plate in a 37°C cell culture incubator containing 5% CO2 to continue cell culture. The resulting cells are the target cells.

[0054] 3. P0 generation trVLPs-EBOV infection of target cells and drug treatment 24 h after transfection, the culture medium of the target cells was discarded and 300 μL of P0 generation trVLPs-EBOV (1×10 7 The cells were treated with TOFA at final concentrations of 100 nM, 200 nM, 400 nM, 800 nM, 1.6 μM, 3.2 μM, 6.4 μM, and 12.8 μM. Each well was designated as a drug-addition well, and three replicates were set for each concentration. A DMSO-only treatment group served as a negative control. After 24 h of culture, the cell supernatant was discarded and the culture medium was replaced with DMEM supplemented with 4% v / v fetal bovine serum.

[0055] 4. Luciferase activity detection After 48 hours of culture, gently wash the cells twice with pre-chilled PBS and aspirate any remaining liquid. Add 200 μL of pre-prepared 1× Glo Lysis Buffer to each well and incubate at room temperature (25°C) for 15 minutes to ensure complete lysis. Collect the cell lysate.

[0056] Transfer 75 μL of cell lysate to a 96-well, white, light-opaque microplate. Add an equal volume (75 μL) of Dual-Glo® Luciferase Reagent and mix thoroughly by gently pipetting. Incubate at room temperature (25°C) in the dark for 10 minutes. Read the Firefly luciferase signal in each well using a multi-function microplate reader (Synergy H1, BioTek).

[0057] After Firefly signal detection, add an equal volume (75 μL) of Dual-Glo® Stop&Glo® Reagent to the same well, mix thoroughly with a pipette, and incubate at room temperature (25°C) in the dark for 10 minutes. Measure the Renilla luciferase signal in each well.

[0058] The ratio of Firefly to Renilla luciferase activity was used as the normalized result, representing the dual-luciferase activity in each well. A nonlinear curve was fitted between the logarithm of TOFA concentration and viral infectivity using GraphPad Prism 8. The TOFA concentration at which the viral infectivity was 50% was calculated as the IC50 value.

[0059] like Figure 2 As shown, higher TOFA concentrations reduce viral infection rates, with an IC50 value of 1.976 μM. Combined with the cytotoxicity experiments in Example 1, when TOFA was used at a concentration of 400 nM, cell viability approached 100% and viral infection rates decreased by nearly 40%, demonstrating that the small molecule drug TOFA can effectively inhibit Ebola pseudovirus replication at relatively low concentrations, demonstrating its high safety.

[0060] Example 3 Effects of different concentrations of TOFA on the expression levels of Ebola virus VP40 protein and GP protein 1. Preparation of the first generation of Ebola pseudovirus Same as Example 2.

[0061] 2. Transfection of target cells with helper plasmids HEK293T cells were cultured in 12-well plates using the method described in Example 2. When the cell density reached 80%, the helper plasmids were transfected. The corresponding plasmids were added according to the dosages listed in Table 3 to prepare transfection system 3.

[0062] Table 3 Plasmid transfection dose for target cells (12-well plate)

[0063] 3. P0 generation trVLPs-EBOV infection of target cells and drug treatment 24 hours after transfection, the target cell culture medium was discarded and 300 μL of P0 trVLPs-EBOV were inoculated into the wells. The wells treated with TOFA at a final concentration of 400 nM served as the experimental group, while those treated with 0.05% DMSO served as the control group. Three replicates were set up for each group. After an additional 24 hours of culture, the cell supernatant was discarded and the medium was replaced with DMEM supplemented with 4% v / v fetal bovine serum.

[0064] 5. Western Blot After culturing for 48 h, the culture medium was discarded, the cells were washed twice with pre-chilled PBS, 0.1 mL of NP-40 lysis buffer was added to each well, the cells were lysed on ice for 15 min, centrifuged at 12,000 g for 10 min at 4°C, and the supernatant was collected.

[0065] Mix the supernatant with Loading Buffer in a 4:1 ratio and heat in a 95°C metal bath for 10 min to completely denature the proteins. Perform SDS-PAGE electrophoresis, adding 20 μL of protein sample to each well and using a protein marker as a reference. Initially set the voltage at 80 V. Once bromophenol blue has entered the separating gel, adjust the voltage to 120 V and run the gel until the bromophenol blue has migrated to the bottom of the gel. For transfer, soak the NC membrane and filter paper in transfer buffer (containing methanol) to form a "sandwich" structure (filter paper-gel-PVDF membrane-filter paper). Remove any bubbles. Transfer the membrane at a constant voltage of 150 V for 1 h at 4°C. Block the membrane with 5% skim milk powder (in TBST) at room temperature (25°C) for 1 h to reduce nonspecific binding. Dilute VP40-specific antibody (Snio Biological, Catalog No. 40447-MM08), GP-specific antibody (Snio Biological, Catalog No. 40094-R106), and GAPDH-specific antibody (Proteintech, Catalog No. 60004-1-IG) and incubate overnight at 4°C. Wash the membrane three times for 10 minutes each. Incubate with HRP-conjugated secondary antibody (goat anti-rabbit IgG) (ThemoFisher, Catalog No. 31460) diluted in TBST buffer at room temperature (25°C) for 1 hour. Wash the membrane three times for 10 minutes each using TBST buffer. Develop the membrane using chemiluminescence (ECL) reagent (covered at room temperature at 25°C) and incubate for 1 minute. Expose the membrane using a gel imager, adjusting the exposure time to obtain clear bands.

[0066] like Figure 3 As shown in the results, compared with the blank group, the expression levels of VP40 and GP proteins in the control group were significantly increased after Ebola pseudovirus infection; however, after TOFA treatment, the expression of VP40 and GP proteins in the experimental group was significantly reduced compared with the control group. This indicates that TOFA can significantly inhibit the expression of VP40 and GP proteins and inhibit the replication of Ebola virus.

[0067] Example 4 TOFA against Ebola virus VP40 Genes and GP Effects of gene expression levels 1. Preparation of the first generation of Ebola pseudovirus Same as Example 2.

[0068] 2. Transfection of target cells with helper plasmids Same as Example 2.

[0069] 3. P0 generation trVLPs-EBOV infection of target cells and drug treatment 24 h after transfection, the culture medium of target cells was discarded and 300 μL of P0 generation trVLPs-EBOV (1×10 7 The cells were cultured for 24 hours, and the supernatant was discarded. The culture medium was replaced with DMEM supplemented with 4% v / v fetal bovine serum.

[0070] 5. q-PCR detection After 48 hours of culture, the medium was discarded and the cells were washed twice with ice-cold PBS. 1 ml of TRIZOL was added to each well to resuspend the cells. 200 μL of chloroform was added and the cells were shaken vigorously for 15 seconds. The cells were then allowed to stand at room temperature (25°C) for 3 minutes. The cells were centrifuged at 12,000 g for 15 minutes at 4°C. After separation, the upper aqueous phase was carefully aspirated into a new tube. An equal volume of ice-cold isopropanol was added to the aqueous phase, mixed by inversion, and allowed to stand at room temperature (25°C) for 10 minutes. The cells were centrifuged at 12,000 g for 10 minutes at 4°C and the supernatant was discarded. A white RNA precipitate was visible. 1 ml of ice-cold 75% ethanol was added to gently resuspend the precipitate and centrifuged at 7,500 g for 5 minutes at 4°C. The cells were dissolved in 30 μL of DEPC-treated water and the concentration was measured.

[0071] RNA was reverse transcribed into cDNA using the following system: 1 μg template RNA, 4 μL 5× Evo M-MLV RT MasterMix, and RNase-free water to a total of 20 μL. The reverse transcription cycle was: 37°C for 15 min; 85°C for 5 sec.

[0072] The obtained cDNA was used as a template for q-PCR detection with primers (VP40 upstream primer: 5'-AGTTGGACTGGCGGAAGAAC-3'; VP40 downstream primer: 5'-CAGAGTCAATCGGCTGGGTC-3'; GP upstream primer: 5'-CCGGAGACTTTGCCTTCCAT-3'; GP downstream primer: 5'-CTCTCAAGGGGTGTGAGCTG-3'; GAPDH upstream primer: 5'-AATGGGCAGCCGTTAGGAAA-3'; GAPDH downstream primer: 5'-GCCCAATACGACCAAATCAGAG-3').

[0073] The q-PCR reaction system (total volume 25 μL) consisted of 12.5 μL of 2× SYBR Green Pro Taq HS Premix (ROXplus), 0.5 μL each of upstream and downstream primers, and 3 μL of cDNA. Sterile deionized water was added to bring the total volume to 25 μL. The q-PCR reaction program was: 95°C for 30 s; 95°C for 15 s; 60°C for 60 s, 40 cycles.

[0074] After the reaction is completed, collect the amplification data and then use 2 -ΔΔCt Methods The relative expression levels of target genes were calculated.

[0075] like Figure 4 and Figure 5 As shown in the figure, compared with the control group, the mRNA levels of VP40 and GP genes in the experimental group were significantly reduced, indicating that TOFA inhibits the expression of VP40 and GP genes of EBOV. In summary, TOFA can significantly inhibit the gene and protein expression of Ebola virus, inhibit viral replication, and has excellent anti-Ebola virus effect.

[0076] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Use of a small molecule inhibitor in the preparation of a product for resisting Ebola virus infection, characterized in that: The structural formula of the small molecule inhibitor is .

2. Use of a small molecule inhibitor in the preparation of a drug for preventing and / or treating diseases caused by Ebola virus, characterized in that: The structural formula of the small molecule inhibitor is .

3. Use of a small molecule inhibitor in the preparation of a product for preventing and / or treating Ebola hemorrhagic fever, characterized in that: The structural formula of the small molecule inhibitor is .

4. Use of a small molecule inhibitor in the preparation of a product for inhibiting Ebola virus replication, characterized in that: The structural formula of the small molecule inhibitor is .

5. Use of a small molecule inhibitor in the preparation of a product for inhibiting the expression of Ebola virus genes and / or proteins, characterized in that: The structural formula of the small molecule inhibitor is .

6. The use according to claim 5, characterized in that The Ebola virus genes include VP40 gene and / or GP gene.

7. The use according to claim 5, characterized in that The Ebola virus protein includes VP40 protein and / or GP protein.

8. A preparation for resisting Ebola virus infection, characterized in that: Including structural formula such as Small molecule inhibitors as shown.

9. The preparation according to claim 8, characterized in that The preparation is in the form of injection, tablet, spray, granule, capsule, oral solution or patch.

10. A pharmaceutical composition, characterized in that Including structural formula such as The small molecule inhibitor shown and a pharmaceutically acceptable carrier or excipient.

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