Application of SARMS compounds containing ester group aromatic propionamide and their metabolites in the preparation of anti-SARS-CoV-2 drugs

Abstract

The present invention belongs to the field of pharmaceutical technology, and specifically discloses the application of a SARMs compound EG-017 containing an ester group aromatic propanamide and its metabolite EG-2 in the preparation of anti-SARS-CoV-2 drugs, inhibiting multi-organ damage such as lung and heart caused by SARS-CoV-2, and treating pulmonary fibrosis. The in vitro anti-SARS-CoV-2 activity and cytotoxicity of the compound were evaluated by IFA, which well verified that EG-017 has obvious inhibitory activity on the replication of SARS-CoV-2 βCoV / KOR / KCDC03 / 2020 strain in Vero cells, indicating its application prospect in the preparation of anti-SARS-CoV-2 drugs; the experiment also showed that EG-017 can well inhibit the cytotoxicity of high-concentration rapamycin to cells; the MTT method was used to verify that EG-017 can significantly improve the survival rate of rat cardiomyocytes. The potential application value of EG-017 and its metabolite in the treatment of lung lesions caused by SARS-CoV-2 and other lung diseases such as pulmonary fibrosis caused by other reasons was verified through the experiment on the effect of EG-017 on the fibrosis process of TGF-β1-induced A549 lung cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the use of a SARMs compound containing an ester aromatic propionamide and its metabolites in the preparation of anti-new coronavirus drugs. Background Art

[0002] Initial symptoms of COVID-19 include fever, dry cough, and fatigue. A small number of patients experience upper respiratory and gastrointestinal symptoms, such as nasal congestion, runny nose, and diarrhea, which can progress to severe cases. Initially, the lungs are the primary lesion, which can then spread to the heart and other organs throughout the body. Clinical symptoms include dyspnea, and in severe cases, the disease can rapidly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis, coagulopathy, and multiple organ failure. The novel coronavirus is primarily transmitted through respiratory droplets and contact. Epidemiological surveys have shown that many cases can be traced back to close contact with confirmed cases. Currently, there is no effective medication to control the disease. Summary of the Invention

[0003] The purpose of the present invention is to provide a SARMs (Selective Androgen Receptor Modulators) compound containing an ester aromatic propionamide and its metabolites for use in the preparation of anti-new coronavirus drugs.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] The application of SARMs compounds containing ester aromatic propionamide and their metabolites in the preparation of anti-new coronavirus drugs.

[0006] Preferably, the SARMs compound containing ester aromatic propionamide is EG-017, whose chemical formula is C 25 H 17 F3N4O4.

[0007] Preferably, the metabolite of the ester-containing aromatic propionamide compound is EG-2.

[0008] Preferably, the ester-containing aromatic propionamide compounds and their metabolites are used to inhibit the cytotoxicity of rapamycin to cells.

[0009] Preferably, EG-017 and its metabolites are used in the preparation of drugs for treating lung lesions caused by the new coronavirus.

[0010] Preferably, EG-017 and its metabolites are potentially used in the preparation of drugs for treating pulmonary fibrosis lung diseases.

[0011] Preferably, the use of EG-017 and its metabolites in the preparation of a medicament for treating diseases caused by adriamycin-induced myocardial cell injury.

[0012] The advantages of the present invention are as follows: The immunofluorescence assay (IFA) was used to evaluate the in vitro anti-SARS-CoV-2 activity and cytotoxicity of the compound. It was well verified that EG-017 had significant inhibitory activity against the replication of SARS-CoV-2 βCoV / KOR / KCDC03 / 2020 strain in Vero cells, indicating its application prospect in the preparation of anti-SARS-CoV-2 drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Histogram of the cytotoxicity of EG-017 at different concentrations.

[0014] Figure 2 : Histogram of cell injury induced by EG-017 at different concentrations.

[0015] Figure 3 : Effect of EG-017 at different concentrations on the expression of Vim protein in A549 cells.

[0016] Figure 4 : Effect of EG-017 at different concentrations on the proliferation of TGF-β1-induced hepatic stellate cells. DETAILED DESCRIPTION OF THE INVENTION

[0017] EG017 mentioned in the text is a new non-steroidal SARM with the chemical formula C 25 H 17 F3N4O4, and the Chinese chemical name is (S)-1-((4-cyano-3-(trifluoromethyl)phenyl)amino)-3-(4-cyanophenoxy)-2-methyl-1-oxopropan-2-yl nicotinate. The preparation method and structural formula of the said EG017 refer to the content recorded in the patent document with the patent number CN201410033958.0.

[0018] Mechanism of action of EG-017

[0019] Preliminary studies have shown that the new coronavirus causes damage to the human body through the ACE2 target; ACE2 is present in the epithelium of the nose, mouth and lungs, as well as in most major tissues and organs of the human body, and is the entry point for the new coronavirus to infect the human body; the new coronavirus initially causes lung infection, causing severe damage to the lung epithelium and endothelial tissue, and then spreads through the blood to cause lesions in the heart and other important vessels; ACE2 contains androgen receptors, and the target of SARMs is the androgen receptor. Using large doses of exogenous androgen receptor modulators SARMs can quickly activate the normal biological functions of lung organs containing ACE2, regulate the lung immune system, and avoid damage to the lungs caused by the immune storm caused by the new coronavirus; at the same time, exogenous sarms can also compete with the virus to bind to ACE2 of related cells (thereby reducing the chance of viral invasion of cells and reducing the damage of the virus to the body), which can indirectly enhance the body's tolerance to the virus, and at the same time promote the body's rapid recovery from the fight against the virus.

[0020] 1) Use a large amount of exogenous non-steroidal androgen receptor modulators to regulate the activity of lung macrophages (containing androgen receptors) and enhance the lung tissue's ability to clear viruses; 2) Use exogenous non-steroidal androgen modulators to bind to ACE2 (containing androgen receptors) in the body, change ACE2 activity and spatial structure, block the binding site between the virus and ACE2, and thus reduce the damage caused by the virus to the human body through the ACE2 channel; 3) Use exogenous androgen receptor modulators to regulate the body's cell nuclear activity, enhance the body's own ability to fight viruses, and enhance the body's self-repair ability.

[0021] Specifically, research has shown that humans possess a specific protein that enables the virus to infect human cells. This protein, called angiotensin-converting enzyme 2, or ACE2, provides a "receptor" for the coronavirus, providing an entry point for the coronavirus to enter and infect various human cells. ACE2 is present in many cell types and tissues, including the lungs, heart, blood vessels, kidneys, liver, and gastrointestinal tract. It is found in epithelial cells, which line certain tissues and form a protective barrier. The exchange of oxygen and carbon dioxide between the lungs and blood vessels occurs in the epithelial lining of the lungs. ACE2 is present in the epithelium lining the nose, mouth, and lungs. In the lungs, ACE2 is highly abundant on type 2 pneumocytes, a key cell type found in the lung's cavities where oxygen is absorbed and waste carbon dioxide is released.

[0022] The ACE enzyme converts angiotensin I into angiotensin II. The primary role of ACE2 is to break down angiotensin II into molecules that counteract its harmful effects. That is, ACE2 helps regulate many of the activities of a protein called angiotensin II (ANG II), which can increase blood pressure and inflammation, damage to blood vessel linings, and various types of tissue damage. ACE2 converts ANG II into other molecules that counteract its effects.

[0023] At the same time, studies have shown that what is most relevant to COVID-19 is that ANG II can increase inflammation and the death of Alviollicular cells, which are critical for bringing oxygen into the body. In summary, these harmful effects of ANG II are reduced by ACE2.

[0024] In vitro cell experiments showed that the maximum dose efficacy of EG02 and EG017 in HEK293 cells, which had been exogenously transfected with the androgen receptor, exceeded 70%. In human prostate cancer cells (LNCaP) endogenously expressing the androgen receptor, EG02 and EG017 demonstrated excellent tissue selectivity, with the maximum dose efficacy of EG02 reaching 66.7% and EG017 47.2%. These results demonstrate that both EG017 and EG02 exhibit tissue selectivity, demonstrating highly selective androgen receptor regulation, with EG017 showing a more pronounced effect.

[0025] Human metabolism studies have shown that EG-017 can be metabolized into EG-2 in the body, and the time to reach maximum concentration in the body is 1.5-3 hours and 6-7 hours, respectively.

[0026] In order to verify the effectiveness of EG-017 in treating lesions of organs such as the lungs caused by the novel coronavirus, the following specific examples are used to illustrate and verify the beneficial effects of EG-017.

[0027] Test compounds and control compounds

[0028] (1) Test compound

[0029] Name: EG-017

[0030] Source: Yaoyuan Pharmaceutical Chemistry (Shanghai) Co., Ltd.

[0031] Batch number: R191095

[0032] Preparation method: Prepare 120mM stock solution with 100% DMSO

[0033] (2) Test compound 2

[0034] Name: rapamycin

[0035] Source: MedChemExpress

[0036] Batch number: 61925

[0037] Preparation method: Prepare 40mM stock solution with 100% DMSO

[0038] (3) Control compound 1

[0039] Name: Remdesivir

[0040] Source: MedChemExpress

[0041] Batch number: CS-0028115

[0042] Preparation method: Prepare 10mM stock solution with 100% DMSO

[0043] (4) Control compound 2

[0044] Name: Lopinavir

[0045] Source: Selleckchem

[0046] Batch number: S138003

[0047] Preparation method: Prepare 10mM stock solution with 100% DMSO

[0048] (5) Control compound 3

[0049] Name: Chloroquine diphosphate salt, Chloroquine, Chloroquine diphosphate, Chloroquine phosphate

[0050] Source: Sigma-Aldrich

[0051] Batch number: 109M4003V

[0052] Preparation method: Prepare 30mM stock solution with 100% DMSO

[0053] Virus strain

[0054] The novel coronavirus βCoV / KOR / KCDC03 / 2020 strain was provided by the Korea Centers for Disease Control and Prevention (KCDC), sequence number NCCP43326.

[0055] cell

[0056] African green monkey kidney (Vero) cells were obtained from the American Type Culture Collection (ATCC), catalog number CCL-81. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, WelGene) supplemented with 10% fetal bovine serum (Gibco) and 1% anti-antibody (Gibco). DMEM supplemented with 2% fetal bovine serum (Gibco) and 1% anti-antibody (Gibco) was used as the experimental culture medium.

[0057] Main instruments and reagents

[0058] (1) Main instruments: The main instruments used in this project are a high-content imaging analyzer (PerkinElmer, model Operetta), an automatic dispenser (ThermoFisher, model Multidrop Combi), a plate washer (BioTek, model ELx406), and a liquid workstation (CyBio, model CyBi-HummingWell).

[0059] (2) Main reagents: The main reagents used in this project include the primary antibody against the novel coronavirus N protein Human SARS-CoV-2N protein antibody (Beijing Sino Biological Technology Co., Ltd., catalog number 40143-T62), AlexaFluor 488-labeled goat anti-rabbit IgG secondary antibody (Molecular probe, catalog number MOP-A-11034), and cell nucleus staining solution Hoechst 33342 (Molecular Probes, catalog number MOP-H-3570).

[0060] Test methods

[0061] This study used IFA to evaluate the in vitro antiviral activity of test compounds against the novel coronavirus (COVID-19) strain βCoV / KOR / KCDC03 / 2020 in Vero cells. The antiviral activity of the test compounds EG-017 and rapamycin was tested individually and in combination with EG-017 in the presence of 100 μM rapamycin. Remdesivir, lopinavir, and chloroquine phosphate served as positive controls. Ten concentrations of the test and control compounds were tested. The tested concentrations are shown in Table 1.

[0062] Table 1. Test concentrations of test compounds and control compounds

[0063]

[0064] The average antiviral inhibition rate and average cell viability of the above compounds at different concentrations are shown in the table below:

[0065]

[0066]

[0067]

[0068] Cell plating

[0069] Vero cells were trypsinized and diluted to 480,000 cells per ml in assay medium. Using an automated dispenser, the diluted cells were added to a 384-well assay plate, with 25 μl of culture medium (12,000 cells per well). The cells were cultured overnight in a 5% CO2, 37°C incubator.

[0070] Compound treatment and viral infection

[0071] The compound was diluted with DMSO and then added to the test cell wells using a liquid workstation. 25 μl of SARS-CoV-2 virus diluted in the experimental culture medium was then added to each well at an MOI of 0.0125. A cell control (cells without compound treatment or virus infection) and a no-compound-treated control (cells infected with virus, no compound treatment, and 0.5% DMSO added) were set up. The final volume of cell culture medium in each well was 50 μl. The cells were cultured in a 5% CO2, 37°C incubator for 24 hours.

[0072] Immunofluorescence staining

[0073] (1) 24 hours after viral infection, add 17 μl of 16% paraformaldehyde to each well and incubate at room temperature for 30 minutes.

[0074] (2) Aspirate the supernatant and wash the plate twice with DPBS;

[0075] (3) Add 25 μl of 0.25% Triton X-100 to each well and incubate at room temperature for 20 minutes;

[0076] (4) Aspirate 0.25% TritonX-100 and wash the plate twice with DPBS;

[0077] (5) Add 25 μl of diluted primary antibody (1:3000 dilution) to each well and incubate at 37°C for 1 hour;

[0078] (6) Remove the primary antibody and wash the plate twice with DPBS;

[0079] (7) Add 25 μl of diluted secondary antibody, goat anti-rabbit IgG labeled with Alexa Fluor 488 (diluted 1:2000) and 2.5 μg / ml (diluted 1:4000) of Hoechst 33342 to each well, and incubate at 37 °C for 1 hour;

[0080] (8) Aspirate the secondary antibody and Hoechst, and wash the plate twice with DPBS;

[0081] (9) Read the plate using a high-content imaging analyzer, Operetta. Instrument settings: 488 / 405 emission, 20x objective, 5 fields of view per well.

[0082] Data analysis

[0083] Use Columbus software to quantitatively analyze the total number of cells (number of Hoechst-stained cells) and the number of cells infected with the novel coronavirus (number of cells labeled with Alexa Fluor 488) in the images obtained by reading the plate with the high-content imaging analyzer. The data of the proportion of infected cells and the total number of cells are used for the analysis of the antiviral activity and cytotoxicity of the compound.

[0084] The calculation formula is as follows:

[0085] Inhibition rate (%) = 100 - (proportion of infected cells in the test well - average proportion of infected cells in the cell control well) / (average proportion of infected cells in the control well without compound treatment - average proportion of infected cells in the cell control well) x 100

[0086] Cell viability (%) = total number of cells in the test well / average total number of cells in the control well without compound treatment x 100

[0087] Use XLfit 4 software to perform non-linear fitting analysis on the inhibition viability and cell viability of the compound and calculate the IC 50 and CC 50 values. The fitting method is "Sigmoidal dose-response". IC 50 and CC 50 The calculation formula for is: Y = Bottom + (Top - Bottom) / (1 + (IC 50 / X)^Hillslope). Among them, IC 50 represents the 50% inhibitory concentration.

[0088] Results and conclusions

[0089] The results of the in vitro inhibitory activity of the test compound and the control compound against the replication of the novel coronavirus are shown in Table 2. The experiment was conducted once.

[0090] The results showed that the control compounds chloroquine phosphate, lopinavir and remdesivir all showed anti-new coronavirus activity, IC 50 The values were 4.3μM, 14.1μM and 5.82μM, respectively, which are consistent with the data reported in the literature (Sangeun J., et al. 2020). The three control compounds did not show obvious cytotoxicity within the tested concentrations. 50 The values were greater than the highest tested concentration, see Table 2.

[0091] The test compounds EG-017 and rapamycin showed significant inhibitory activity against the replication of the novel coronavirus βCoV / KOR / KCDC03 / 2020 strain in Vero cells, with IC50 values of 17.09 μM and 9.34 μM, respectively. The test compound EG-017 showed no significant cytotoxicity in Vero cells, with a CC50 value greater than the highest tested concentration, >300 μM. Rapamycin showed significant cytotoxicity, with a CC50 value of 29.5 μM.

[0092] In the presence of 100 μM rapamycin, combined antiviral activity of EG-017 and rapamycin was tested. Rapamycin exhibited significant cytotoxicity at a concentration of 100 μM (Table 7), making it impossible to distinguish the inhibitory activity of the combined drug against viruses. However, the results of the combined drug use indicated that EG-017 could inhibit the cytotoxicity of rapamycin at concentrations ranging from 300 μM to 11.11 μM. The antiviral efficacy of the combined drug requires further evaluation at a lower rapamycin concentration.

[0093] Table 2. Anti-novel coronavirus activity of test compounds and control compounds

[0094] Compound Unit <![CDATA[IC 50 ]]> <![CDATA[CC 50 > EG-017 μM 17.09 >300 Rapamycin μM 9.34 29.5 Chloroquine μM 4.30 >150 Lopinavir μM 14.10 >50 Remdesivir μM 5.82 >50

[0095] The following MTT assay was used to further verify that EG-017 and its metabolites could repair the cytotoxicity of doxorubicin on cardiomyocytes.

[0096] Experimental subjects: H9C2 rat cardiomyocytes

[0097] Experimental method: MTT assay

[0098] Experimental principle:

[0099] The MTT method is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-violet crystalline formazan and deposit it in the cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells. By measuring its light absorption value at a wavelength of 550 nm with an enzyme-linked immunosorbent detector, the number of living cells can be indirectly reflected. Within a certain range of cell numbers, the amount of MTT crystals formed is proportional to the number of cells. This method has been widely used in anti-tumor drug screening and cytotoxicity tests.

[0100] Experimental procedures

[0101] 1. Cytotoxicity of different concentrations of EG-017

[0102] (1) Collect H9C2 cells in the logarithmic phase, adjust the cell suspension concentration to 5×10 4 cells / mL, add 100 μL to each well, and plate to adjust the density of the cells to be tested to 5000 cells / well. The edge wells are filled with sterile PBS.

[0103] (2) Incubate at 5% CO2 and 37 °C until the cell monolayer covers the bottom of the well (96-well flat bottom plate). Add drugs with concentration gradients. Plate the cells the previous afternoon and add the drugs the next morning. Six gradients of EG-017 are set at 0, 1, 10, 50, 100, and 500 μM, 100 μL per well, and 4 - 6 replicate wells are set. At the same time, set a zero-adjustment well (culture medium, MTT, dimethyl sulfoxide), and a control well (cells, drug dissolution medium with the same concentration, culture solution, MTT, dimethyl sulfoxide).

[0104] (3) Incubate at 5% CO2 and 37 °C for 24 hours, and observe under an inverted microscope.

[0105] (4) Add 20 μL of MTT solution (5 mg / mL, i.e., 0.5% MTT) to each well, and continue to culture in an incubator at 5% CO2 and 37 °C for 4 h.

[0106] (5) Terminate the culture and carefully aspirate the culture solution in the well.

[0107] (6) Add 150 μL of dimethyl sulfoxide to each well, place it on a shaker and shake at low speed for 10 min to fully dissolve the crystals. Measure the absorbance value of each well at OD 490 nm with an enzyme-linked immunosorbent detector.

[0108] The experimental results are as Figure 1As shown in the figure, where A: The cell viability of EG-017 at concentrations of 1, 10, 50, 100, and 500 μM was detected by the MTT method. *P < 0.05 compared with the control group; **P < 0.01 compared with the control group; ***P < 0.001 compared with the control group; ****P < 0.0001 compared with the control group. The results showed that A. EG-017 could significantly promote the proliferation of cardiomyocytes at concentrations of 100 and 500 μM.

[0109] 2. Induction of cell damage by different concentrations of EG-017

[0110] (1) Collect H9C2 cells in the logarithmic phase and adjust the cell suspension concentration to 5×10 4 cells / mL. Add 100 μL to each well and plate to adjust the cell density to 5000 cells / well. The edge wells are filled with sterile PBS.

[0111] (2) Incubate at 5% CO2 and 37°C until the cell monolayer covers the bottom of the well (96-well flat bottom plate). Add drugs with concentration gradients. Plate the cells the afternoon before, and add the drugs the next morning. Six gradients of EG-017 are set at 0, 1, 10, 50, 100, and 500 μM, 100 μL per well, and 4 - 6 replicates are set. After pretreatment for 2 hours, change to the medium containing DOX (1 μM) and continue to culture. At the same time, set the zero adjustment wells (medium, MTT, dimethyl sulfoxide), and the control wells (cells, the same concentration of drug dissolution medium, culture solution, MTT, dimethyl sulfoxide).

[0112] (3) Incubate at 5% CO2 and 37°C for 48 hours and observe under an inverted microscope.

[0113] (4) Add 20 μL of MTT solution (5 mg / mL, that is, 0.5% MTT) to each well and continue to culture in an incubator at 5% CO2 and 37°C for 4 hours.

[0114] (5) Terminate the culture and carefully aspirate the culture solution in the wells.

[0115] (6) Add 150 μL of dimethyl sulfoxide to each well, place it on a shaker and shake at low speed for 10 minutes to fully dissolve the crystals. Measure the absorbance of each well at OD 490 nm with an enzyme-linked immunosorbent assay detector.

[0116] The results are as Figure 2As shown, where A: The cell viability was detected by MTT method under the condition of 1 μM adriamycin with the acting concentrations of EG-017 being 1, 10, 50, 100, and 500 μM. *P < 0.05 compared with the control group C; **P < 0.01 compared with the control group C; ***P < 0.001 compared with the control group C; ****P < 0.0001 compared with the control group C. #P < 0.05 compared with the control group D; ##P < 0.01 compared with the control group D; P < 0.001 compared with the control group D; #P < 0.0001 compared with the control group D. In the figure, C: control; D: control + DOX (1 μM).

[0117] The results showed that A. EG-017 at the concentrations of 100 and 500 μM could significantly increase the survival rate of cardiomyocytes under DOX treatment.

[0118] To further verify the effectiveness of EG-017 in the preparation of anti-SARS-CoV-2 drugs, the following experiments were carried out to verify the effect of EG-017 on the fibrosis process of TGF-β1-induced A549 lung cancer cells.

[0119] Experimental subjects: A549 lung cancer cells

[0120] Experimental method: WB method

[0121] Experimental principle:

[0122] Western blot, namely protein blotting, is a protein detection technique that transfers the total proteins of cells or tissues separated by electrophoresis from the gel to a solid support such as NC membrane or PVDF membrane, and then uses specific antibodies to detect a specific antigen. By analyzing the position and depth of coloring of the specific protein band, the expression of the protein in cells or tissues can be known.

[0123] Transforming growth factor-β (TGF-β1) is the most critical cytokine in liver fibrosis. TGF-β1 is used to induce fibrosis of A549 lung cancer cells in vitro. Vimentin (Vim) is significantly expressed in the fibrotic lung cancer cells, while the expression of E-cadherin (E-Cad) is significantly decreased. The protein expression levels of Vim and E-Cad are detected by WB technology to verify the fibrosis level of A549 cells.

[0124] Experimental steps

[0125] Western blot was used to detect the effects of different concentrations of EG-017 on the protein expression of Vim and E-Cad in cells.

[0126] 1. Protein sample preparation

[0127] (1) Cultivate A549 cells to the logarithmic phase, digest them and seed them in six-well plates. Set up a blank control group and a drug administration group. After 24 h, when the cell monolayer evenly covers about 80% of the bottom of the plate, add 2 ml of medium containing TGF-β1 at a concentration of 20 ng / ml to each well. The serum concentration of the medium is 2%. After 48 h, aspirate and discard the original medium, add medium containing different concentrations of EG-017, and place it in an incubator for 48 h.

[0128] (2) Extraction of total cellular proteins: After 48 h, take out the six-well plates, aspirate the medium, wash twice with PBS, add trypsin to digest the cells, collect the cells, centrifuge and discard the supernatant, wash twice with PBS, then add the prepared RIPA lysis buffer, shake back and forth or pipette several times to make the lysis buffer fully contact with the cells, and lyse on ice for 30 min, shaking every 5 min. After lysis, place the EP tube in a low-temperature centrifuge and centrifuge at 12,000 rpm for 5 min. The colorless and transparent supernatant is the total cellular protein. Determine the concentration of the centrifuged supernatant by BCA protein quantification method. Add the diluted protein sample and 5×SDS loading buffer in a ratio of 4:1, and boil in boiling water for 5 min to completely denature the protein, and store at -70 °C for later use.

[0129] 2. SDS-PAGE electrophoresis for protein separation

[0130] Prepare the separating gel and stacking gel according to the instructions of the gel preparation kit and the molecular weight of the target protein. Add the extracted proteins to the sample wells with a pipette, add 10 μL for each sample and 3 μL for the protein marker. Assemble the electrophoresis apparatus, then adjust the electrophoresis apparatus to 80 V. When the sample bands run near the boundary between the stacking gel and the separating gel, adjust the electrophoresis apparatus to 120 V and continue running the gel. Stop running the gel when it is about 1 cm from the bottom. After electrophoresis, cut out the required bands according to the marker indication.

[0131] 3. Transfer of proteins

[0132] (1) Prepare enough transfer buffer (100 mL of 10× transfer buffer + 200 mL of methanol + 700 mL of ultrapure water) for standby.

[0133] (2) After electrophoresis, take out the glass plate and gently pry it open with forceps. After wetting with transfer buffer, cut out the target band according to the position of the protein marker, mark it and put it into the transfer buffer. Transfer the gel to the transfer buffer. Cut out a PVDF membrane with the same size as the cut gel, soak it in methanol for 5 min to activate it, and then put it into the transfer buffer to equilibrate for 2 min for standby.

[0134] (3) Fabricate the sandwich structure: Open the clip in the electrotransfer solution, place it horizontally, pad a layer of sponge on the black clip, and use a glass rod to roll and drive away the bubbles; then place a thick filter paper, fix it and use a glass rod to drive away the bubbles, then place the gel, cover the PVDF membrane on the gel, place three layers of filter paper and sponge upwards in sequence, and finally close and clamp the white clip.

[0135] (4) Place the clip into the transfer tank, with the gel at the negative electrode and the membrane at the positive electrode.

[0136] (5) Transfer membrane conditions: Constant current of 200 mA, for 100 min. Heat will be generated during electrotransfer, and it should be transferred on ice bath.

[0137] 4. Immune reaction

[0138] (1) Milk blocking: After the transfer is completed, take out the PVDF membrane and place it in an incubation box containing 5% skim milk prepared with 5 mL TBST on a shaker, and block it at room temperature for 4 h.

[0139] (2) Primary antibody incubation: Dilute GAPDH, Vim, and E-Cad antibodies with 1×TBST at a ratio of 1:1000. After blocking, pour out the blocking solution, add the prepared primary antibody dilution solution to cover the PVDF membrane, incubate overnight at 4°C, take it out the next day, and rinse it 3 times with 1×TBST, 15 min each time.

[0140] (3) Secondary antibody incubation: Dilute the secondary antibody with 1×TBST at a ratio of 1:10000. After overnight incubation, recover the primary antibody dilution solution, add the secondary antibody dilution solution and place it on a shaker, incubate at room temperature for 2 h, and then rinse it 3 times with TBST, 15 min each time.

[0141] 5. ECL luminescence detection

[0142] (1) Mix equal volumes of reagent A and reagent B in the luminescence kit in a centrifuge tube.

[0143] (2) Place the PVDF membrane on the sample tray, and then evenly drip the mixed luminescence solution on the PVDF membrane.

[0144] (3) Place it in the instrument and operate the instrument to make it expose.

[0145] The results of the effects of different concentrations of EG-017 on the expression of Vim protein in A549 cells are as Figure 3 shown. It detected the expression of Vim protein in cells after treating cells with EG-017 at concentrations of 0.4, 2, and 10 μM for 48 h by WB method. The results showed that after treating cells for 48 h, EG-017 inhibited the expression of Vim protein. The results of the effects of different concentrations of EG-017 on the proliferation of TGF-β1-induced hepatic stellate cells are as Figure 4As shown, the WB method was used to detect the expression of E-cad protein in cells after treatment with EG-017 at concentrations of 50, 100, and 200 for 48 hours. The results showed that EG-017 promoted the expression of E-cad protein.

[0146] Finally, the above experiments showed that EG-017 could inhibit TGF-β1-induced fibrosis in A549 cells and showed a certain dose-dependence.

[0147] There are still various implementation manners of the present invention. All technical solutions formed by using equivalent transformations or equivalent substitutions fall within the protection scope of the present invention.

Claims

1. Use of EG-017 in the preparation of a medicament for treating lung lesions caused by the novel coronavirus, wherein the chemical formula of EG-017 is C 25 H 17 F3N4O4, and the chemical name is (S)-1-((4-cyano-3-(trifluoromethyl)phenyl)amino)-3-(4-cyanophenoxy)-2-methyl-1-oxopropan-2-yl nicotinate.

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