Use of embonic acid or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for combating ebola virus

Abstract

The application discloses application of embelin or a pharmaceutically acceptable salt thereof in preparation of an anti-Ebola virus drug. The application finds that the embelin can effectively inhibit growth or reproduction of the Ebola virus, inhibit transcription and replication of the Ebola virus, target an "active pocket" of a binding interface of Ebola virus VP30 protein and NP protein, and inhibit RNA synthesis of the Ebola virus. The application expands the use range of the embelin, and especially in the case that specific anti-Ebola virus drugs are seriously scarce, provides a new drug and a new treatment mode for inhibiting Ebola virus disease caused by the Ebola virus.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of embenic acid or its pharmaceutically acceptable salts in the preparation of anti-Ebola virus drugs. Background Technology

[0002] Ebola virus belongs to the Filoviridae family. It is transmitted from wild animals to humans and spreads between humans, causing Ebola virus disease (EVD), also known as Ebola viral hemorrhagic fever. Symptoms include fever, headache, abdominal pain, severe weakness and fatigue; diarrhea, bloody stools, internal and external bleeding, multiple organ failure, and ultimately death. Currently, there is no specific antiviral drug for Ebola; clinical treatment primarily focuses on supportive care to improve survival rates.

[0003] Ebola virus is a single-stranded, negative-sense RNA virus whose genome encodes seven structural proteins and one non-structural protein. The gene sequence is: 3' end - NP - VP35 - VP40 - GP - VP30 - VP24 - L - 5' end. The VP30, NP, VP35, and L proteins of Ebola virus form a large complex responsible for Ebola virus RNA synthesis. Research indicates that the VP30 protein of Ebola virus participates in Ebola virus RNA transcription and replication, and that VP30 can directly interact with NP to regulate RNA synthesis. High-resolution crystal structures of the VP30-NP binding key peptide complex were resolved using X-ray diffraction crystallography. Analysis of this crystal structure revealed that VP30 binds to the NP-binding peptide through a specific "molecular pocket," and this binding interface represents a potential target for anti-Ebola virus drugs.

[0004] Embelin is a small-molecule natural product of the phenylkun class isolated from *Ardisia crenata*, a plant in the genus *Ardisia*. Its traditional use is for expelling intestinal parasites. Modern pharmacological studies have found that embelin is a novel XIAP cell permeability inhibitor. It specifically inhibits the cytochrome P450 isoenzyme CYP2C9 and possesses various biological activities, including anti-inflammatory, antibacterial, antioxidant, and induction of solid tumor growth inhibition and tumor cell apoptosis. Literature reports that embelin has outstanding pharmacological activity against human leukemia and various solid tumors. It can bind to the XIAP BIR3 domain with Smac protein and caspsase-9, inhibiting the growth of human pancreatic cancer cells and prostate cancer cells; and inducing apoptosis in human leukemia cells by downregulating XIAP. Currently, there is no application of embelin in the prevention or treatment of Ebola virus disease. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide the use of embenic acid or a pharmaceutically acceptable salt thereof in the preparation of anti-Ebola virus drugs.

[0006] Enbenic acid has the molecular formula C 17 H 26 O4, with a molecular weight of 294.39, has the following structural formula:

[0007]

[0008] This invention provides the use of embenic acid or a pharmaceutically acceptable salt thereof in the preparation of anti-Ebola virus drugs.

[0009] This invention provides a drug for treating Ebola virus, the active ingredient of which includes embenic acid or a drug-acceptable salt thereof.

[0010] This invention provides the use of embenic acid or a pharmaceutically acceptable salt thereof in the preparation of drugs that target the binding interface between the Ebola virus VP30 protein and the NP protein.

[0011] Furthermore, the targeting effect is a targeted inhibition effect.

[0012] This invention provides a drug that targets the binding interface between the VP30 protein and the NP protein of the Ebola virus, the active ingredient of which includes embenic acid or a drug-acceptable salt thereof.

[0013] This invention provides the use of embenic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting Ebola virus RNA synthesis.

[0014] This invention provides a drug for inhibiting Ebola virus RNA synthesis, the active ingredient of which includes embenic acid or a drug-acceptable salt thereof.

[0015] This invention provides the use of embenic acid or a pharmaceutically acceptable salt thereof in the preparation of medicaments for inhibiting Ebola virus genome transcription and / or replication.

[0016] The present invention provides a medicament for inhibiting the transcription and / or replication of the Ebola virus genome, the active ingredient of which includes embenic acid or a pharmaceutically acceptable salt thereof.

[0017] The beneficial effects of this invention are as follows:

[0018] This invention discovers the application of embenic acid or its pharmaceutically acceptable salts in the preparation of anti-Ebola virus drugs. This application expands the scope of use of embenic acid, especially in the context of a severe shortage of specific anti-Ebola virus drugs, providing a new drug and a new treatment for inhibiting Ebola virus-induced Ebola virus disease. As an antiviral drug, experiments show that embenic acid can effectively inhibit Ebola virus in Huh7-4P cells of the Ebola virus minigenome system. Embenic acid inhibits the transcription and replication of the Ebola virus genome, inhibiting the growth or reproduction of Ebola virus. It can inhibit the synthesis of the corresponding genome in Huh7-4P cells of the Ebola minigenome system, with a half-maximal inhibitory concentration (IC50). 50 The concentration was 1.31 μM; embenic acid can target the "active pocket" at the binding interface between the Ebola virus VP30 protein and NP protein, and exhibits good inhibitory activity in fluorescence polarization experiments, with an IC50 value of 1.31 μM. 50 The concentration was 55.37 μM. Enbecic acid showed no significant cytotoxicity within its effective concentration range. Therefore, it can be used to prepare anti-Ebola virus drugs. Attached Figure Description

[0019] Figure 1 This is a fluorescence polarization inhibition rate curve of embenic acid at different concentrations inhibiting the interaction interface between Ebola virus VP30 protein and NP at different concentrations in Example 1 of the present invention. The horizontal axis represents the concentration of embenic acid, and the vertical axis represents the polarization fluorescence value. The half-maximal inhibitory concentration (IC50) of embenic acid for inhibiting Ebola virus VP30 protein was calculated based on the inhibition rate. 50 value.

[0020] Figure 2 This is a graph showing the inhibition rate of embezzic acid at different concentrations on Ebola virus mini-genome system Huh7-4P cells in Example 2 of the present invention. The horizontal axis represents the concentration of embezzic acid, and the vertical axis represents the fluorescence value of the luciferase reporter gene of the mini-genome system with the solvent group as the control. The half-maximal inhibitory concentration (IC50) of embezzic acid on the Ebola virus mini-genome system was also determined. 50 value.

[0021] Figure 3 This is a cytotoxicity curve of embezzic acid at different concentrations on Ebola virus mini-genome system Huh7-4P cells in Example 3 of the present invention. The horizontal axis represents the concentration of embezzic acid, and the vertical axis represents the survival rate of Huh7-4P cells with the solvent group as the control. The median lethal concentration (Cd) of embezzic acid for cells was also determined. 50 value.

[0022] Figure 4This is a graph showing the affinity of embenic acid at different concentrations with Ebola virus VP30 protein in Example 4 of the present invention. The horizontal axis represents the binding and dissociation time of VP30 protein with embenic acid, and the vertical axis represents the binding response value of embenic acid with VP30 protein with the solvent group as a control. The affinity constant K between embenic acid and Ebola VP30 protein was also calculated. D value.

[0023] Figure 5 This is a graph showing the change in Tm value of VP30 protein after embenic acid binds to Ebola virus VP30 protein under different pH conditions in Example 5 of the present invention. The horizontal axis represents different pH environments, and the vertical axis represents the Tm value of VP30 protein with the solvent group as a control. The binding ability of embenic acid to Ebola VP30 protein under different pH conditions is determined based on the change in Tm value before and after binding. Detailed Implementation

[0024] To better understand the content of this invention, the following description, in conjunction with the accompanying drawings and specific implementation methods, will further illustrate the content of this invention. However, the scope of protection of this invention is not limited to the following embodiments.

[0025] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in this specification is for the purpose of describing particular embodiments and is not intended to limit the scope of the invention.

[0026] The following examples mainly evaluate the anti-Ebola virus activity of embenic acid by constructing a fluorescence polarization method for the interaction between Ebola virus VP30 protein and NP-binding peptide and an Ebola virus minigenome system, and confirm that embenic acid has the effect of inhibiting Ebola virus genome transcription and replication, and also inhibiting Ebola virus RNA synthesis.

[0027] The Ebola virus minigenome Huh7-4P cells used in this invention were kindly provided by Professor Zhong Jin of the Shanghai Pasteur Institute.

[0028] In this invention, Ebola virus VP30 is stably overexpressed. 110-272 The protein plasmid was constructed and stored within our unit. The VP30 plasmid was used. 110-272 The protein samples were expressed and purified in our unit, with a purity >95%.

[0029] The FITC-eNP fluorescent peptide used in the embodiments of this invention was synthesized by Nanjing Genscript Biotech Co., Ltd., with a purity >95%.

[0030] The cell growth culture medium used in this embodiment of the invention consists of: DMEM basal medium supplemented with 10% fetal bovine serum, 1% ampicillin / streptomycin, and 0.1 mg / mL hygromycin. The culture medium is stored at 4°C and preheated to 37°C before use.

[0031] The embenic acid used in this embodiment of the invention was purchased from Shanghai Taosu Biochemical Technology Co., Ltd., with a purity >99%.

[0032] The dual-luciferase detection kit used in this embodiment of the invention was purchased from Shanghai Beyotime Biotechnology Co., Ltd., and includes cell lysis buffer and luciferase reaction substrate.

[0033] Example 1

[0034] Detection of the inhibitory activity of embenic acid against the "active pocket" at the binding interface between Ebola virus VP30 protein and NP (e.g.) Figure 1 ).

[0035] 1. Establish a system based on Ebola virus VP30 110-272 Fluorescence polarization method for protein-NP interaction:

[0036] 1) VP30 110-272 Protein concentration determination: The FITC-eNP fluorescent peptide was dissolved in a buffer solution consisting of 10 mM HEPES (pH 7.0), 150 mM NaCl, and 2 mM DTT, to a concentration of 1 μM. VP30 110-272 The protein was diluted to an initial concentration of 240 μM using the same buffer, and then serially diluted 2.5 times. The total reaction volume was 100 μL. First, 50 μL of VP30 at different concentrations was added... 110-272 Protein solutions were added to 96-well black plates, with three replicates for each concentration, resulting in 10 drug gradients and final concentrations of (120, 48, 19.2, 7.68, 3.07, 1.23, 0.49, 0.20, 0.079, 0.031 μM). Then, 50 μL of 1 μM FITC-eNP fluorescent peptide solution was added to the system, bringing the final concentration to 500 mM. The mixture was vortexed for 5 seconds and incubated at room temperature for 30 minutes. Polarization fluorescence values ​​were then read using a microplate reader. The instrument settings were: excitation wavelength 485 nm, emission wavelength 528 nm, bandpass 20 nm, automatic gain, reading height 8.5 mm, and G factor 1.26. VP30 was determined by concentration titration. 110-272 Protein concentration.

[0037] 2) Determination of the optimal concentration of FITC-eNP fluorescent peptide: The FITC-eNP fluorescent peptide was dissolved in a buffer solution containing 10 mM HEPES (pH 7.0), 150 mM NaCl, and 2 mM DTT, bringing the concentration to 1 μM. The VP30 was determined based on the titration experiment described above. 110-272 To determine the optimal concentration, different concentrations of FITC-eNP fluorescent peptide were set within a protein concentration range. Different concentrations of VP30 were then titrated with the FITC-eNP fluorescent peptide at different concentrations. 110-272 Protein concentration. The total volume of the reaction system is 100 μL. First, 50 μL of VP30 at different concentrations is added. 110-272 Protein solutions were added to 96-well microplates, with three replicates for each concentration, resulting in 10 drug gradients and final concentrations of (120, 48, 19.2, 7.68, 3.07, 1.23, 0.49, 0.20, 0.079, 0.031 μM). Then, 50 μL of FITC-eNP fluorescent peptide solutions at different concentrations were added to the system, with three replicates for each concentration, resulting in 5 drug gradients and final concentrations of (500, 400, 300, 200, 100, 50 mM). The mixture was shaken for 5 seconds and incubated at room temperature for 30 minutes. Polarization fluorescence values ​​were then read using a microplate reader. The instrument settings were: excitation wavelength 485 nm, emission wavelength 528 nm, bandpass 20 nm, automatic gain, reading height 8.5 mm, and G factor 1.26. The optimal concentration of the FITC-eNP fluorescent peptide was determined by titration.

[0038] 3) Determine the IC of the established method 50 The optimal VP30 was determined based on the concentration titration method described above. 110-272 Protein concentration range and FITC-eNP fluorescent peptide concentration were used to validate the method and calculate IC50. 50 The total volume of the reaction system is 100 μL. First, 50 μL of VP30 at different concentrations is added. 110-272 Protein solutions were added to 96-well black plates, with three replicates for each concentration, resulting in 10 drug gradients and final concentrations of (120, 48, 19.2, 7.68, 3.07, 1.23, 0.49, 0.20, 0.079, 0.031 μM). Then, 50 μL of 0.6 μM FITC-eNP fluorescent peptide solution was added to the system, bringing the final concentration to 300 mM. The mixture was vortexed for 5 seconds and incubated at room temperature for 30 minutes. The polarization fluorescence values ​​were then read using a microplate reader. The instrument settings were: excitation wavelength 485 nm, emission wavelength 528 nm, bandpass 20 nm, automatic gain, readout height 8.5 mm, and G factor 1.26.

[0039] 2. Determination of the half-maximal inhibitory concentration (IC50) of embezzic acid by fluorescence polarization method. 50 ):

[0040] The optimal VP30 determined based on the established fluorescence polarization method described above 110-272 Protein concentration and FITC-eNP fluorescent peptide concentration were used to validate the method and calculate IC50. 50 The total volume of the reaction system is 100 μL. First, 50 μL of VP30 with a concentration of 15.36 μM is added. 110-272 Protein solution was added to 96-well microplates. Then, 1 μL of embezzled acid (DMSO solvent) at different concentrations was added to the system, ensuring the same proportion of DMSO in all wells. The initial drug concentration was set at 200 μM, with 12 drug gradients, each concentration in triplicate, resulting in final drug concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.195, and 0.098 μM. The drug and protein were co-incubated for 30 minutes. Finally, 50 μL of 0.6 μM FITC-eNP fluorescent peptide solution was added to the system, bringing the final concentration to 300 mM. The mixture was vortexed for 5 seconds and incubated at room temperature for 60 minutes. The polarization fluorescence values ​​were then read using a microplate reader. The instrument was set to: excitation wavelength 485nm, emission wavelength 528nm, bandpass 20nm, automatic gain, readout height 8.5mm, and G-factor 1.26.

[0041] 3. Results: such as Figure 1 As shown;

[0042] 1) VP30 110-272 Determination of protein concentration: Based on the fluorescence polarization signal measured by the microplate reader, an inhibition curve was fitted using Prism 8.0 software. The concentration was then determined based on the fitted curve and IC50. 50 Value, determine VP30 110-272 The protein concentration was 7.68 μM.

[0043] 2) Determination of FITC-eNP fluorescent peptide concentration: Based on the fluorescence polarization signal measured by the microplate reader, an inhibition curve was fitted using Prism 8.0 software. The trend of the fitted curve and IC50 were then analyzed for different concentrations. 50 The optimal concentration of FITC-eNP fluorescent peptide was determined to be 300 nM.

[0044] 3) Determine the IC of the established method 50 Based on the fluorescence polarization signal measured by the microplate reader, the established fluorescence polarization method was verified. Using Prism 8.0 software, an inhibition curve was fitted, and the IC50 was calculated based on the fitted curve. 50 The value is 2.92 μM.

[0045] 4) Half-maximal inhibitory concentration (IC50) of embezzled acid 50 The inhibition rate was calculated based on the correlation between fluorescence value and drug concentration. Using Prism 8.0 software, a curve was plotted, and the half-maximal inhibitory concentration (IC50) of embezzic acid was calculated. 50 The value is 55.37 μM.

[0046] Example 2

[0047] Assay of the antiviral activity of embenic acid against the Ebola virus minigenome (e.g.) Figure 2 ).

[0048] 1. Method:

[0049] 1) Antiviral activity experiments on a minigenome system:

[0050] Antiviral activity assays were performed using the Ebola virus minigenome system. The antiviral activity of embelin was evaluated using this internationally recognized anti-Ebola virus evaluation system. Huh7-4P cells from the logarithmically growing Ebola virus minigenome system were seeded at a rate of 1*10^4 cells / well in 96-well cell culture plates, with 100 μL of cell culture medium per well. Cells were incubated at 37°C for 24 hours.

[0051] The drug was dissolved in DMSO to prepare a high-concentration stock solution, which was then diluted to 400 μM with DMEM medium containing 2% fetal bovine serum (FBS). This was repeated two-fold serially to create 12 concentration gradients: 200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.195, and 0.098 μM. 100 μL of drug was added to each well, for a total system volume of 200 μL. Each concentration was tested in triplicate. A control group was prepared with an equal volume of DMSO solvent, and a blank group was prepared with an equal volume of complete DMEM medium. After drug addition, the cells were incubated at 37°C for 24 hours. The 96-well plates were then removed, and the cell culture supernatant was aspirated and discarded. Cells were washed once with 100 μL / well of sterile PBS (pH 7.4), and 100 μL of 1X cell lysis buffer was added to each well. Cells were lysed at room temperature with shaking for 15 minutes. Transfer 50 μL / well of the lysis supernatant to a 96-well white ELISA plate. Add an equal volume of diluted luciferase substrate according to the instructions of the dual-luciferase assay kit. Immediately detect the fluorescence value using an ELISA reader. Determine the activity of embezzlediate in inhibiting viral genome synthesis based on the fluorescence value. Calculate the inhibition rate based on the correlation between fluorescence value and drug concentration, plot a curve, and calculate the half-maximal inhibitory concentration (IC50) of embezzlediate. 50 .

[0052] 2. Results: such as Figure 2 As shown;

[0053] Using the DMSO solvent group as a blank control, the inhibition rate of the drug-treated groups was calculated based on fluorescence values. Then, based on the inhibition rates of different drug concentrations, Prism 8.0 software was used to fit drug inhibition rate curves, and the half-maximal inhibitory concentration (IC50) of embelin for inhibiting Ebola virus RNA synthesis was calculated. 50 The value is 1.31 μM.

[0054] Example 3

[0055] Cytotoxicity assay of embezzled acid (e.g.) Figure 3 ).

[0056] 1. Method:

[0057] 1) Cell seeding:

[0058] Huh7-4P cells in the logarithmic growth phase were adjusted to a cell density of 1*10^4 cells / well and seeded at 100 μL / well in 96-well plates and cultured overnight.

[0059] 2) Drug concentration design:

[0060] Huh7-4P cells: Before drug administration, the drug was diluted to 200 μM in DMEM medium containing 2% fetal bovine serum. 100 μL of the diluted drug was added to each well of the 96-well Huh7-4P cells in step 1), with a final volume of 200 μL per well. The drug was serially diluted 2-fold to obtain 9 concentration gradients, with total concentrations of 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, and 0.39 μM, respectively. Each drug concentration was tested in triplicate, with the DMSO solvent treatment group serving as a blank control.

[0061] 3) Detect absorbance:

[0062] After culturing in an incubator for 48 hours, add 10 μL of CCK-8 working solution to each well and continue incubation for another 3 hours. Measure the absorbance at 450 nm using a microplate reader.

[0063] 4) Based on the measured OD values, calculate the survival rate of Huh7-4P cells under the action of various drug concentrations compared with the control group.

[0064] 2. Results: such as Figure 3 As shown;

[0065] Embelin (100 μM) has an effect on Huh7-4P cells ( ) within the effective concentration range. Figure 3 No obvious toxic effects, CC 50 >100μM.

[0066] Example 4

[0067] Enbecic acid and Ebola virus VP30 110-272 Protein affinity assay (e.g.) Figure 4 ).

[0068] method

[0069] 1) Desalination:

[0070] Desalting column was used for VP30 prior to biotin labeling. 110-272 The protein was subjected to buffer replacement, with the original TrisHCl buffer replaced by sodium phosphate buffer. The desalting column was first washed with 12 mL of PBS at VP30. 110-272 After thawing the protein on ice, centrifuge at 20000×g for 15 minutes. The supernatant was then added to a desalting column. Once the protein solution was completely incorporated into the column, PBS was added to maintain a total volume of 500 μL. Subsequent fractions of PBS (400 μL, 400 μL, 200 μL, and 200 μL) were added sequentially, collecting each fraction individually for a total of five protein fractions. The A280 value of the protein after buffer replacement was measured, and multiplied by its absorbance coefficient to obtain the protein concentration.

[0071] 2) Biotin labeling:

[0072] The tube with the highest protein concentration after desalting was selected for biotin labeling. NHS-PEG12-Biotin was dissolved in DMSO to prepare a 10 mM stock solution. The required amount of 10 mM NHS-PEG12-Biotin was calculated using the formula:

[0073]

[0074] After adding biotin and mixing well, incubate at room temperature for 30 minutes. During incubation, continuously wash the desalting column with PBS. After incubation, remove free biotin (NHS-PEG12-Biotin) using the desalting column again. First, wash the desalting column with 12 mL of PBS. Add the labeled protein solution to the desalting column. After the protein solution has completely entered the column, add PBS to maintain a total volume of 500 μL. Then, add 400 μL, 400 μL, 200 μL, and 200 μL of PBS sequentially, collecting each fraction for a total of five protein fractions. Measure the A280 value of each fraction and multiply it by its absorbance coefficient to obtain the concentration of the labeled protein. Store the labeled protein at -80℃.

[0075] 3) Biomembrane Interference Experiment (BLI):

[0076] This experiment used the Fortebio Octect molecular interaction instrument. Preliminary experiments were conducted to determine the initial binding affinity and concentration range of the compounds, with a VP30 setup. 110-272 The protein concentration was set at 50 μg / mL, and the embezzled acid concentration was set at 500 μM. Before adding samples, a 96-well black plate was prepared, buffer was added, and the Fortebio streptavidin (SA) sensor used in this experiment was placed in it, and it was soaked for at least ten minutes to equilibrate the sensor. Another 96-well black plate was prepared, and equilibration wells, curing wells, binding wells, and dissociation wells were set up, with 200 μL of the drug added to each well. Tris-HCl buffer (pH 7.5) was added to the equilibration wells. VP30 was added to the curing wells at a final concentration of 50 μg / mL. 110-272 The protein was treated with a final drug concentration of 500 μM. The Octet system software was programmed as follows: Baseline 120 s; Loading 300 s; Baseline 180 s; Association 180 s; Dissociation 180 s. The signal acquisition frequency was set to 2 Hz. An untreated SA sensor was used as a control, and the program was executed according to the settings. After the program completed, the data was processed in the Octet system's analysis software according to the software manual to preliminarily determine the drug's affinity for the protein and the drug concentration range.

[0077] Affinity constant determination: VP30 was set up 110-272 The protein concentration was 50 μg / mL, and the initial concentration of embezzled acid was set at 1000 μM. Before adding samples, a 96-well black plate was prepared, buffer was added, and the Fortebio streptavidin (SA) sensor used in this experiment was placed in it, and it was soaked for at least ten minutes to equilibrate the sensor. Another 96-well black plate was prepared, and equilibration wells, curing wells, binding wells, and dissociation wells were set up, with 200 μL of the drug added to each well. Tris-HCl buffer (pH 7.5) was added to the equilibration wells. VP30 was added to the curing wells at a final concentration of 50 μg / mL. 110-272Proteins were added to wells with an equal concentration gradient of drug. The initial and initial drug concentration was 1000 μM, with 2-fold serial dilutions resulting in six concentration gradients: 1000, 500, 250, 125, 62.5, and 31.25 μM. The program was set on the Octet system software as follows: Baseline 120s; Loading 300s; Baseline 180s; Association 180s; Dissociation 180s. The signal acquisition frequency was set to 2Hz. An un-curedated SA sensor was used as a control, and the program was executed according to the settings. After the program completed, the data was processed on the Octet system analysis software according to the software manual. Curve fitting was selected as follows: binding and dissociation, global fitting, and 1:1 model.

[0078] 2. Results: such as Figure 4 As shown;

[0079] After the program finishes execution, process the data in the Octet system's analysis software according to the software manual. For curve fitting, select: binding and dissociation, global fitting, or a 1:1 model. Fit the drug inhibition rate curve and calculate the ratio of embezzic acid to VP30. 110-272 Protein affinity constant K D It is 0.97±0.09mM.

[0080] Example 5

[0081] Enbecic acid and Ebola virus VP30 110-272 Thermal drift experiments of proteins (e.g.) Figure 5 ).

[0082] 1. Method:

[0083] To achieve a final protein concentration of 0.1-1 mg / mL, first dilute the protein to 13 μM using a universal buffer (150 mM NaCl, 20 mM Tris, pH 7.5). Then dilute embezzled acid to 15.6 μM using DMSO. The drug concentration should be 1.2 times the protein concentration. Add VP30... 110-272 The protein was placed in different pH buffers (pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5), and then 10 μL of VP30 in each pH buffer was taken. 110-272The protein was mixed with 10 μL of the drug or an equal volume of DMSO in the appropriate pH buffer to adjust the final protein concentration to 6.5 μM and the final drug concentration to 7.8 μM. Two control groups were included: one group without protein (drug and different pH buffers), and the other group without compound (protein and DMSO in different pH buffers). After incubation for 10 minutes, 1 μL of SYPRO Orange solution was added to each sample well or blank well, and incubation was continued for another 30 minutes. All samples and controls were prepared in triplicate. All samples and controls were placed in 8-tube PCR instruments and heated from 25°C to 95°C at a rate of 1°C / min on a Bio-Rad CFX1000 instrument, with the ROX channel used for fluorescence signal monitoring.

[0084] 2. Results: such as Figure 5 As shown;

[0085] In thermal drift experiments, SYPRO Orange, a fluorescent dye that binds to hydrophobic residues, is used to monitor VP30 as temperature increases. 110-272 Denaturation was performed and the melting temperature (Tm) was calculated. When a drug binds to a protein, the protein's stability may change (i.e., Tm shift). For embenic acid, under six different pH conditions, 6.5 μM VP30 was used at a 1:1.2 molar ratio. 110-272 Thermal drift assays were performed using a mixture of 7.8 μM embenic acid and protein samples without embenic acid as controls. VP30 110-272 and VP30 110-272 The mixtures with embezzic acid all exhibited the highest thermal stability at pH 7.5. At pH 7.0, the mixtures with VP30 showed... 110-272 In comparison, VP30 110-272 and VP30 110-272 When mixed with embenic acid, the Tm increases by 2°C. The Tm values ​​change most significantly at pH 8.5, compared to VP30. 110-272 In comparison, VP30 110-272 and VP30 110-272 The mixture with embezzled acid resulted in a 6°C decrease in Tm. This indicates that the drug exhibits the strongest affinity for proteins and the best binding ability at pH 8.5.

[0086] The above description is only a specific embodiment of the present invention and not all embodiments. Any equivalent modifications made by those skilled in the art to the technical solutions of the present invention by reading the present invention specification shall be covered by the claims of the present invention.

Claims

1. The use of embenic acid or its pharmaceutically acceptable salts in the preparation of anti-Ebola virus drugs.

2. Use of embenic acid or its pharmaceutically acceptable salt in the preparation of drugs that inhibit Ebola virus RNA synthesis.

3. Use of embenic acid or its pharmaceutically acceptable salts in the preparation of drugs that inhibit Ebola virus genome transcription and / or replication.

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