Method for predicting and calculating efficacy of inhibitor of TMPRSS2 protein

Abstract

The invention discloses a TMPRSS2 protein inhibitor efficacy prediction calculation method. A scoring function is constructed through a logP value, a virtual docking score and living cell inhibitor effect data to predict a drug inhibition effect. The method specifically comprises the following steps: constructing a TMPRSS2 overexpression cell model to screen drugs; the cell viability of various potential inhibitors is detected, and high-toxicity inhibitors are eliminated through a CCK-8 experiment; using flow cytometry to detect the cell average fluorescence intensity through a TMPRSS2 fluorescent probe to evaluate the enzyme activity influence of the inhibitor, and calculating the IC30 value; measuring the logP value of an effective inhibitor, and predicting the binding capacity of the effective inhibitor and TMPRSS2 by using Schrodinger software to obtain a virtual docking score; and analyzing the relationship among the IC30 value, the logP value and the virtual docking score, and constructing a scoring function to predict the drug effect. According to the method, the drug effect prediction can be simply, conveniently and quickly carried out, the drug screening consumption is reduced, and the drug screening period is shortened.

Description

Technical Field

[0001] The present invention relates to the field of assisted drug screening, and particularly relates to a method for predicting the efficacy of inhibitors of TMPRSS2 protein by calculation. Background Art

[0002] TMPRSS2 (transmembrane serine protease 2) is an important host cell factor for proteolytic activation of 7 known coronavirus proteins including SARS-COV-2, SARS-COV, MERS, CoV229E, OC43, NL63, and HKU1. Knocking out or inhibiting TMPRSS2 can reduce the infection in SARS and MERS mouse models.

[0003] The fusion gene of TMPRSS2 and erythroblastosis virus E26 oncogene (ERG) is specific in prostate cancer and also plays an important role in the occurrence and development of prostate cancer. However, the deletion of TMPRSS2 does not affect development or homeostasis, which indicates that specific inhibitors of TMPRSS2 may exhibit broad antiviral activity and help in the diagnosis and treatment of prostate cancer without causing a large number of adverse side effects. Therefore, it is of great significance to predict the efficacy of inhibitors that inhibit the enzymatic activity of TMPRSS2. However, currently, common techniques for predicting the efficacy of inhibitors have problems such as high screening consumption, cumbersome operation, and long screening cycle.

[0004] Therefore, to solve the above problems, a method for predicting the efficacy of inhibitors of TMPRSS2 protein by calculation is needed, which can simply and quickly predict the efficacy of drugs, reduce the drug screening consumption, and shorten the drug screening cycle. Summary of the Invention

[0005] In view of this, the object of the present invention is to overcome the defects in the prior art and provide a method for predicting the efficacy of inhibitors of TMPRSS2 protein by calculation, which can simply and quickly predict the efficacy of drugs, reduce the drug screening consumption, and shorten the drug screening cycle.

[0006] The method for predicting the efficacy of inhibitors of TMPRSS2 protein of the present invention includes:

[0007] Constructing a model cell with overexpression of TMPRSS2;

[0008] Respectively detecting the influence of several inhibitors to be screened on the viability of the model cells, eliminating the inhibitors with cytotoxicity exceeding the set value, and taking the remaining inhibitors as the first inhibitors;

[0009] Detecting the inhibitory effect of the first inhibitors on enzymatic activity to obtain the second inhibitors, and judging whether the inhibitory effect of the second inhibitors on enzymatic activity reaches the set effect, and taking the inhibitors that reach the set effect as the screened inhibitors.

[0010] Further, a model cell with overexpressed TMPRSS2 was constructed, specifically including:

[0011] Construct a recombinant plasmid of TMPRSS2: The recombinant plasmid of TMPRSS2 includes the amino acids at positions 106 - 492 and the coding sequence of a 6×His tag at the N-terminus, and BamHI and HindIII restriction site sequences are added to both sides of the coding sequence, and inserted into the pcDNA 3.1 vector to express the recombinant protein of TMPRSS2;

[0012] Process the recombinant plasmid: Transform the recombinant plasmid into competent Escherichia coli DH5α cells, after screening with an agar plate containing ampicillin, pick a monoclonal colony and transfer it to a liquid LB medium for culture. After 12 hours of growth, use an endotoxin-free plasmid miniprep kit to extract the plasmid;

[0013] Cell culture and transfection: The human embryonic kidney cell line HEK293T was passaged in high-glucose DMEM medium containing 1% penicillin-streptomycin sulfate and 10% fetal bovine serum under the culture conditions of 37°C and 5% CO2; 4×10 5 cells were seeded in each well of a 6-well plate. After culturing for about 20 hours, when the cell density reached 70% confluence, recombinant plasmid transfection was carried out.

[0014] Further, the recombinant plasmid transfection was carried out according to the following method:

[0015] Remove the cell growth medium, add 2 mL of fresh pre-warmed complete medium to each well. For each well of cells, dilute 2 μg of DNA with 100 μL of serum-free medium, mix well to form a DNA dilution, and immediately add 4 μL of PEI transfection reagent to the 100 μL of DNA dilution, and mix gently;

[0016] Incubate at room temperature for 10 - 15 min to form a DNA-PEI cationic nucleic acid transfection reagent complex; add the transfection complex to the cells, and gently mix by shaking the culture plate; after transfection, place it in a 37°C, 5% CO2 incubator for culture.

[0017] Further, the effect of the inhibitor to be screened on the viability of the model cells was detected, specifically including:

[0018] Prepare 100 μL of cell suspension in a 96-well plate; place the culture plate in an incubator at 37 °C and 5% CO2 for 24 hours; prepare dilutions of the inhibitor in culture medium at concentrations of 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM respectively. After aspirating the old culture medium, add 100 μL of the inhibitor dilutions with different concentration gradients to each well. Only add culture medium to the blank wells, and add 0.1% DMSO to the control wells. After culturing for 24 hours, add 10 μL of CCK-8 solution to each well, incubate in the incubator for 2 hours, and then measure the absorbance at 450 nm using a microplate reader.

[0019] Further, to detect the inhibitory effect of the first inhibitor on enzyme activity, specifically including:

[0020] Cell suspension preparation: 24 hours after cell transfection, add a dilution of the inhibitor with a final concentration of 100 μM to the 6-well plate. 48 hours after cell transfection, add a probe solution with a final concentration of 60 μM to the 6-well plate; incubate in an incubator at 37 °C and 5% CO2 for 2 hours, remove the old culture medium, wash 3 times with PBS, after trypsin digestion and centrifugation, resuspend the cell pellet at the bottom with 1 mL of PBS, centrifuge the obtained cell suspension at 1000 rpm for 3 min, and repeat three times; finally, filter the cell suspension through a 40-μm cell strainer, and place the prepared cell suspension sample in a 5-ml flow tube for flow cytometry analysis;

[0021] Flow cytometry analysis: Before analysis, mix the cell suspension well, set the excitation wavelength to 405 nm, and collect data from the fluorescence signal channel between 531 nm and 598 nm; collect a total of 2×10 4 cells for analysis of each sample; calculate the fluorescence intensity of a single cell, that is, the mean fluorescence intensity MFI, and compare the fluorescence intensity between different samples; Mean Fluorescence Intensity MFI = Total fluorescence intensity of the fluorescence channel of the positive cell population / Total number of cells in the positive cell population.

[0022] Further, judge whether the inhibitory effect on enzyme activity reaches the set effect according to the following method:

[0023] Take the logarithm of the inhibitor concentration as the abscissa and the inhibition rate as the ordinate, calculate the linear regression equation, and then substitute the inhibition rate of 30% into the linear regression equation to obtain the corresponding abscissa value, which is the logIC30 value;

[0024] According to the logIC 30 value of the inhibitor, calculate the IC 30 value of the inhibitor, compare the IC 30 value with the effective range of the set IC 30 value. If the IC 30 value is within the effective range, the inhibitory effect on enzyme activity reaches the set effect; otherwise, it does not reach the set effect.

[0025] Furthermore, the logIC value can also be determined according to the following method: 30 Value:

[0026] log IC 30 = -0.1919S + 0.3202log P - 0.5488;

[0027] Among them, the inhibitor is prepared into a stock solution with a concentration of 5 mg / mL in methanol, and then diluted with methanol to prepare a working solution of 50 μg / mL. After filtering through a 0.22 mm filter membrane, it is bottled, and 1 mL is taken for injection for high-performance liquid chromatography detection; logP = a + b*logk; a and b are constants, k is the retention factor, k = (t r -t0) / t0, t r is the chromatographic retention time, t0 is the retention time of the methanol solvent; S is the binding ability of the inhibitor to TMPRSS2.

[0028] Furthermore, the Schrödinger software is used to predict the binding ability of the inhibitor to TMPRSS2:

[0029] Inhibitor conformation optimization: Download the inhibitor structure from the PubChem database, perform three-dimensional conformation optimization on the inhibitor in the Ligprep module, set the maximum number of atoms of the ligand to 500, the force field to OPLS4, the ionization state search condition limit to pH 7.0 ± 2.0, select Epik to search for tautomers, and keep the remaining parameters default. After running the program, the potential conformations are obtained as the final screening set for subsequent protein molecule docking;

[0030] Protein crystal data preprocessing: Download the human TMPRSS2 protein file from the PDB protein database, import it into the Schrödinger software, and perform water molecule removal, hydrogen addition, side chain completion, redundant ligand deletion, and energy minimization processing in the Protein Preparation Wizard panel, and retain the original ligand in the protein file for control analysis of the docking results;

[0031] Molecular docking: Prepare the docking pocket, establish a protein docking box file with a size of at the spatial position where the original ligand is located in the Receptor Grid Generation panel and save it, keeping all parameters default; perform software simulation docking of the above optimized inhibitor screening set with the docking box in the Ligand Docking module, and perform docking scoring to obtain the docking scoring result.

[0032] The beneficial effects of the present invention are as follows: A method for predicting the efficacy of an inhibitor of TMPRSS2 protein disclosed by the present invention constructs a scoring function to predict the drug inhibitory effect through logP value, virtual docking score and live cell inhibitor effect data. Specifically, a TMPRSS2 overexpression cell model is constructed to screen drugs; the cell viability of 16 potential inhibitors is detected, and the highly toxic ones are eliminated through the CCK-8 experiment; flow cytometry is used to detect the average fluorescence intensity of cells through a TMPRSS2 fluorescent probe to evaluate the influence of the inhibitor on enzyme activity, and IC 30 value is calculated; the logP value of the effective inhibitor is measured, and its binding ability to TMPRSS2 is predicted using Schrödinger software to obtain the virtual docking score, and the relationship between the IC 30 value, logP value and virtual docking score is analyzed to construct a scoring function to predict the drug effect. The prediction method of the present invention is simple and efficient, can reduce the consumption of drug screening, and shorten the drug screening cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described below with reference to the drawings and embodiments:

[0034] Figure 1 Schematic diagram of the recombinant protein expression vector of the present invention;

[0035] Figure 2 Schematic diagram of the plasmid map of the present invention;

[0036] Figure 3 Schematic diagram of the immunoblotting result of the present invention;

[0037] Figure 4 Schematic diagram of the immunofluorescence result of the present invention;

[0038] Figure 5 Schematic diagram of the inhibitor screening flow chart of the present invention;

[0039] Figure 6 Schematic diagram of the influence of the inhibitor on cell viability of the present invention;

[0040] Figure 7 Schematic diagram of the inhibitory effect of the TMPRSS2 enzyme activity of the present invention;

[0041] Figure 8 Schematic diagram of the inhibitory effect of different concentrations of the inhibitor of the present invention;

[0042] Figure 9 Schematic diagram of the retention time of 5 standards of the present invention;

[0043] Figure 10 Schematic diagram of the standard curve of the present invention;

[0044] Figure 11Schematic diagram of the retention times of six inhibitors of the present invention;

[0045] Figure 12 Schematic diagram of the spatial conformation of the human TMPRSS2 protein document of the present invention;

[0046] Figure 13 Docking effect diagram of six inhibitors of the present invention with TMPRSS2. Detailed implementation manners

[0047] The following further describes the present invention in conjunction with the accompanying drawings of the specification, as shown in the figures:

[0048] This embodiment discloses a method for predicting the efficacy of inhibitors of the TMPRSS2 protein, including the following steps:

[0049] Construct a model cell with overexpression of TMPRSS2;

[0050] Detect the effects of several inhibitors to be screened on the viability of the model cells respectively, eliminate the inhibitors with excessive set cytotoxicity, and use the remaining inhibitors as the first inhibitors;

[0051] Detect the enzyme activity inhibition effect of the first inhibitor to obtain the second inhibitor, judge whether the enzyme activity inhibition effect of the second inhibitor reaches the set effect, and use the inhibitor that reaches the set effect as the screened inhibitor.

[0052] In this embodiment, constructing a model cell with overexpression of TMPRSS2 specifically includes:

[0053] Construct a TMPRSS2 recombinant plasmid: The TMPRSS2 recombinant plasmid includes the amino acids at positions 106-492 and the 6×His tag coding sequence at the N-terminus, and BamHI and HindIII restriction site sequences are added to both sides of the coding sequence, and inserted into the pcDNA 3.1 vector to express the TMPRSS2 recombinant protein; among them, pcDNA3.1 is a mammalian expression vector, and the recombinant protein expression vector is as Figure 1 shown, and the plasmid map is as Figure 2 shown.

[0054] Process the recombinant plasmid: Transform the recombinant plasmid into Escherichia coli DH5α competent cells, after screening with an agar plate containing ampicillin (100 μg / mL), select a single colony and transfer it to a liquid LB medium for culture. After 12 hours of bacteria growth, use an endotoxin-free plasmid miniprep kit to extract the plasmid, and send it to BGI for sequencing verification;

[0055] Cell culture and transfection: The human embryonic kidney cell line HEK293T was passaged in high-glucose DMEM medium containing 1% penicillin-streptomycin sulfate and 10% fetal bovine serum under the culture conditions of 37°C and 5% CO2; 4×10 5 cells were seeded in each well of a 6-well plate. After culturing for approximately 20 hours, when the cell density reached 70% confluence, recombinant plasmid transfection was performed.

[0056] In this example, the recombinant plasmid transfection was performed according to the following method:

[0057] Remove the cell growth medium, and add 2 mL of freshly preheated complete medium to each well. For each well of cells, dilute 2 μg of DNA with 100 μL of serum-free medium, mix well to form a DNA dilution, and immediately add 4 μL of PEI transfection reagent to 100 μL of the DNA dilution, and mix gently;

[0058] Incubate at room temperature for 10 - 15 min to form a DNA-PEI cationic nucleic acid transfection reagent complex; add the transfection complex to the cells, and gently mix by shaking the culture plate; after transfection, place it in a 37°C, 5% CO2 incubator for culture.

[0059] The same transfection method can be used to transfect the pcDNA3.1 empty vector as an experimental control, and the expression of the recombinant protein was detected 48 hours after transfection.

[0060] Furthermore, immunoblotting detection can be performed:

[0061] After 48 hours of cell overexpression, the cells were collected by centrifugation at 2,000×g for 5 min, lysed thoroughly with RIPA, and total cell protein was extracted. Protein samples were separated by SDS-PAGE. After transferring and blocking the membrane, it was incubated with the primary antibody and the secondary antibody successively. The primary antibodies were rabbit anti-TMPRSS2 antibody (1:500) and rabbit anti-His antibody (1:1000), and incubated overnight at 4°C. The secondary antibody was HRP-labeled goat anti-rabbit IgG secondary antibody (1:5000). Finally, it was developed by ECL method and imaged with a gel imager. The immunoblotting results are as Figure 3 shown, and the expression of the TMPRSS2 recombinant protein was verified by immunoblotting detection.

[0062] In addition, immunofluorescence detection can also be performed:

[0063] After 48 hours of transfection, the culture medium was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 15 min, followed by washing with PBS. The cells were blocked with 5% BSA at room temperature for 30 min, incubated with goat anti-His primary antibody (diluted 1:100) in a wet box at 37 °C for 2 hours, and then washed 3 times with PBS. Diluted rabbit anti-goat secondary antibody labeled with Alexa Fluor488 (diluted 1:250) was added, and the cells were incubated in a wet box at 37 °C for 1 hour, followed by washing 3 times with PBS. DAPI was added, and the cells were allowed to stand at room temperature for 10 min, followed by washing 3 times with PBS. After adding the mounting medium, the cells were placed under a confocal microscope for imaging. The immunofluorescence results are as Figure 4 shown, and the overexpression of TMPRSS2 recombinant protein on the cell membrane was verified by immunofluorescence.

[0064] In this example, the present invention screened for effective inhibitors by detecting cell viability and detecting enzyme activity based on flow cytometry, and further explored the IC 30 value, measured the logP value based on HPLC method, and based on Schrödinger software to study the relationship among the molecular docking score value docking score of the three, providing certain guidance for subsequent drug screening.

[0065] The flow chart for inhibitor screening is as Figure 5 shown. Among them, Group A: 16 potential inhibitors; Group B: inhibitors with cell viability ≥ 75% at a concentration of 100 μM; Group C: inhibitors with significant inhibitory effect on TMPRSS2 enzyme activity at a concentration of 100 μM.

[0066] Detecting the effect of the inhibitor to be screened on the viability of the model cells specifically includes:

[0067] Sixteen potential inhibitors that can effectively inhibit TMPRSS2 enzyme activity in vitro were selected, and the CCK8 method was used to detect their effects on the viability of 293T cells. Among them, 293T cells overexpressing TMPRSS2 were used as model cells.

[0068] 100 μL of cell suspension was prepared in a 96-well plate; the culture plate was placed in an incubator at 37 °C and 5% CO2 for 24 hours; the inhibitors were respectively prepared into dilution solutions with concentrations of 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM with culture medium. After aspirating the old culture medium, 100 μL of inhibitor dilution solutions with different concentration gradients were added to each well. Only culture medium was added to the blank well, and 0.1% DMSO was added to the control well. After 24 hours of culture, 10 μL of CCK-8 solution was added to each well, and after incubating in the incubator for 2 hours, the absorbance at 450 nm was measured with an enzyme-linked immunosorbent assay reader.

[0069] The basic information of the 16 inhibitors is shown in Table 1, and the effect of the inhibitors on cell viability is as Figure 6 shown.

[0070] Table 1

[0071]

[0072]

[0073] Through the above vitality detection, the first inhibitors obtained are 10 inhibitors with a cell vitality ≥ 75% at a concentration of 100 μM, including inhibitors No. 1, 2, 4, 5, 10, 13, 11, 14, 15, and 16, namely Acyclovir, Penciclovir, Ebselen, Entecavir, Lamivudine, Ribavirin, Molnupiravir, AEBSF, Stavudine, and Nafamostat Mesylate, which are Group B inhibitors.

[0074] In this example, to detect the inhibitory effect of the first inhibitors on enzyme activity, it specifically includes:

[0075] Cell suspension preparation: 24 hours after cell transfection, add a dilution of the inhibitor with a final concentration of 100 μM to the 6-well plate. 48 hours after cell transfection, add a probe solution with a final concentration of 60 μM to the 6-well plate. Incubate in a 37°C, 5% CO2 incubator for 2 hours, remove the old medium, wash 3 times with PBS, after trypsin digestion and centrifugation, resuspend the bottom cell pellet with 1 mL of PBS. Centrifuge the obtained cell suspension at 1000 rpm for 3 minutes, and repeat three times. Finally, filter the cell suspension through a 40-μm cell filter, and place the prepared cell suspension sample in a 5-ml flow tube for flow cytometry detection;

[0076] Flow cytometry detection: Mix the cell suspension well before detection. Set the excitation wavelength to 405 nm, and collect data from the fluorescence signal channel between 531 nm and 598 nm. A total of 2×10 4 cells are collected for analysis for each sample; calculate the fluorescence intensity of a single cell, that is, the mean fluorescence intensity MFI, and compare the fluorescence intensity between different samples; Mean fluorescence intensity MFI = total fluorescence intensity of the fluorescence channel of the positive cell population / total number of cells in the positive cell population.

[0077] Among them, the inhibitory effect of Group B inhibitors on TMPRSS2 enzyme activity at a concentration of 100 μM is as Figure 7 shown.

[0078] Through the above detection of the enzyme activity inhibition effect, a second inhibitor with a significant inhibition effect at a concentration of 100 μM was obtained. The second inhibitor includes 6 inhibitors numbered 1, 2, 10, 11, 14, and 16, namely Acyclovir, Penciclovir, Lamivudine, Molnupiravir, AEBSF, and Nafamostat Mesylate, which are defined as Group C inhibitors.

[0079] In this example, further, concentration gradients were set: 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM. Flow cytometry was used to detect the inhibition effect of different concentrations of Group C inhibitors on TMPRSS2 enzyme activity. It was found in the detection that the inhibition effects of Acyclovir, Penciclovir, and Nafamostat Mesylate did not show a good trend with the change of concentration. After linear fitting, R 2 < 0.9, and the corresponding IC30 values were too large or the IC 30 values could not be effectively obtained. However, the inhibition effects of 3 inhibitors, Lamivudine, Molnupiravir, and AEBSF, showed a good trend, and the IC 30 values of Lamivudine, Molnupiravir, and AEBSF could be well calculated, which were 4.665 μM, 7.380 μM, and 5.476 μM respectively. For these 3 inhibitors, the inhibition effects of inhibitors at different concentrations are as Figure 8 shown.

[0080] Therefore, the following method can be used to judge whether the enzyme activity inhibition effect reaches the set effect:

[0081] Taking the logarithm of the inhibitor concentration as the abscissa and the inhibition rate as the ordinate, calculate the linear regression equation, and then substitute the inhibition rate of 30% into the linear regression equation to obtain the corresponding abscissa value as logIC 30 value;

[0082] According to the logIC 30 value of the inhibitor, calculate the IC 30 value of the inhibitor. Compare the IC 30 value with the effective range of the set IC 30 value. If the IC 30 value is within the effective range, the enzyme activity inhibition effect reaches the set effect; otherwise, it does not reach the set effect. Among them, the effective range includes a relatively effective range and a medium-effective range, and the range values are 1–5 μM and 5–10 μM respectively. Or the effective range includes a relatively effective range, a medium-effective range, and a weak-effective range, and the range values are 1–5 μM, 5–10 μM, and >10 μM respectively.

[0083] Among them, the effectiveness grading of the inhibition effect is shown in Table 2 as follows:

[0084] Table 2

[0085]

[0086] In this embodiment, the logIC value can also be determined according to the following method to form a scoring function: 30 value, forming a scoring function:

[0087] log IC 30 = -0.1919S + 0.3202log P - 0.5488;

[0088] Among them, the logP value is detected by HPLC:

[0089] Sample preparation: The standard product and the Group C inhibitor are prepared into a stock solution with a concentration of 5 mg / mL in methanol, and then diluted with methanol to prepare a working solution with a concentration of 50 μg / mL. All the above samples are filtered through a 0.22 mm filter membrane and bottled, and 1 mL is taken for injection for high-performance liquid phase detection.

[0090] Liquid phase parameter setting: ZORBAX RX-C18 chromatographic column (4.6×250 mm 5 mm); mobile phase A is water, mobile phase B is methanol, isocratic elution with 15% A and 75% B; column temperature is 30°C; flow rate is 1 mL / min; injection volume is 5 μL, and the detection wavelength is 210 nm.

[0091] Result analysis: For the standard product and the inhibitor to be tested, the retention factor (k) is calculated according to the chromatographic retention time (t r ), and the calculation formula is as follows. The retention time of the methanol solvent is denoted as t0, and the retention factors of the standard product and the inhibitor are calculated.

[0092] k = (t r -t0) / t0.

[0093] The retention times of 5 standard products are as Figure 9 shown; the retention factors are shown in Table 3:

[0094] Table 3

[0095]

[0096] According to the OECD guidelines, logP can be experimentally calculated according to the logarithm of the retention factor (logk) under a fixed percentage of organic modifier. The calculation formula is as follows:

[0097] logP = a + b*logk;

[0098] Among them, the logarithm logk of the retention factor k of the standard product is used as the abscissa, and the standard curve is plotted with the standard product logP as the ordinate, as Figure 10 shown. Therefore, the values of a and b can be obtained.

[0099] The retention times of 6 inhibitors (Group C inhibitors) are as Figure 11 shown; substituting the obtained constants a and b into the logP calculation formula corresponding to the inhibitor according to the standard curve, the logP values corresponding to the inhibitor are obtained, and the results are shown in Table 4:

[0100] Table 4

[0101]

[0102]

[0103] Use Schrödinger software to predict the binding ability S of the inhibitor to TMPRSS2:

[0104] Inhibitor conformational optimization: Download the inhibitor structure from the PubChem database, perform three-dimensional conformational optimization on the inhibitor in the Ligprep module, set the maximum number of atoms of the ligand to 500, the force field to OPLS4, the ionization state search condition limit to pH 7.0 ± 2.0, select Epik to search for tautomers, and keep the remaining parameters default. After running the program, the potential conformations are obtained as the final screening set for subsequent protein molecule docking;

[0105] Protein crystal data preprocessing: Download the human TMPRSS2 protein file (humanTMPRSS2, PDB ID: 7MEQ, whose spatial conformation is as Figure 12 shown) from the PDB protein database, import it into Schrödinger software, and perform water molecule removal, hydrogen addition, side chain completion, deletion of redundant ligands, and energy minimization processing in the ProteinPreparation Wizard panel, and retain the original ligand in the protein file for control analysis of the docking results;

[0106] Molecular docking: Prepare the docking pocket, establish a protein docking box file with a size of at the spatial position of the original ligand in the Receptor Grid Generation panel and save it, keeping all parameters default; perform software simulation docking of the above optimized inhibitor screening set with the docking box in the Ligand Docking module, select SP for the docking accuracy, and perform docking scoring to obtain the docking scoring result.

[0107] The docking score result is used as the binding ability S of the inhibitor to TMPRSS2. The more negative the docking score result is, that is, the larger the absolute value is, the better the docking effect represents. The docking effect diagrams of 6 inhibitors and TMPRSS2 are as shown in Figure 13 shown, and the docking score and key docking amino acid residues are shown in Table 5:

[0108] Table 5

[0109]

[0110]

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A method for calculating the efficacy prediction of a TMPRSS2 protein inhibitor, characterized in that: include: Constructing TMPRSS2 overexpression model cells; respectively detecting the effects of several inhibitors to be screened on the viability of the model cells, eliminating inhibitors exceeding the set cytotoxicity, and using the remaining inhibitors as the first inhibitor; The enzyme activity inhibition effect of the first inhibitor is detected to obtain a second inhibitor, and it is determined whether the enzyme activity inhibition effect of the second inhibitor reaches the set effect, and the inhibitor that reaches the set effect is used as the screened inhibitor.

2. The method for calculating the efficacy prediction of TMPRSS2 protein inhibitors according to claim 1, characterized in that: Constructing a TMPRSS2 overexpression model cell, specifically including: Construction of TMPRSS2 recombinant plasmid: The TMPRSS2 recombinant plasmid includes amino acids 106-492 and a 6×His tag coding sequence at the N-terminus, and BamHI and HindIII restriction site sequences are added on both sides of the coding sequence, respectively, and inserted into a pcDNA3.1 vector to express TMPRSS2 recombinant protein; Treatment of recombinant plasmid: transform the recombinant plasmid into E. coli DH5α competent cells, select monoclonal colonies after screening on agar plates containing ampicillin, transfer and culture to liquid LB medium, and extract the plasmid using an endotoxin-free plasmid extraction kit after 12 hours of bacterial growth; Cell culture and transfection: HEK293T cells were cultured at 37°C and 5% CO2 in high-glucose DMEM medium containing 1% penicillin-streptomycin sulfate and 10% fetal bovine serum. 4 × 10 cells were seeded per well of a 6-well plate. 5 After culturing for about 20 hours, the recombinant plasmid was transfected when the cell density reached 70% confluence.

3. The method for calculating the efficacy prediction of the TMPRSS2 protein inhibitor according to claim 2, characterized in that: Perform recombinant plasmid transfection as follows: Remove the cell growth medium and add 2 mL of fresh pre-warmed complete medium to each well. For each well of cells, dilute 2 μg of DNA with 100 μL of serum-free medium and mix thoroughly to make DNA dilution solution. Immediately add 4 μL of PEI transfection reagent to 100 μL of DNA dilution solution and mix gently. Incubate at room temperature for 10-15 minutes to form a DNA-PEI cationic nucleic acid transfection reagent complex; add the transfection complex to the cells and shake the culture plate to gently mix; after transfection, place in a 37°C, 5% CO2 incubator for culture.

4. The method for calculating the efficacy prediction of TMPRSS2 protein inhibitors according to claim 1, characterized in that: Detecting the effect of the inhibitor to be screened on the viability of the model cells, specifically including: Prepare 100 μL of cell suspension in a 96-well plate; place the culture plate in a 37°C, 5% CO2 incubator for 24 hours; prepare the inhibitor with culture medium into dilutions of 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM concentrations, respectively. After removing the old culture medium, add 100 μL of inhibitor dilutions with different concentration gradients to each well, add only culture medium to the blank wells, and add 0.1% DMSO to the control wells. After culturing for 24 hours, add 10 μL of CCK-8 solution to each well, and incubate in the incubator for 2 hours, measure the absorbance at 450 nm with an enzyme reader.

5. The method for calculating the efficacy prediction of TMPRSS2 protein inhibitors according to claim 1, characterized in that: Detecting the enzyme activity inhibition effect of the first inhibitor, specifically including: Preparation of cell suspension: 24 hours after cell transfection, add inhibitor dilution with a final concentration of 100 μM to the 6-well plate, and 48 hours after cell transfection, add probe solution with a final concentration of 60 μM to the 6-well plate; incubate at 37°C, 5% CO2 incubator for 2 hours, remove the old culture medium, wash 3 times with PBS, digest with trypsin and centrifuge, resuspend the bottom cell pellet with 1 mL PBS, centrifuge the obtained cell suspension at 1000 rpm for 3 minutes, repeat three times; finally, filter the cell suspension with a 40 μm cell filter, and place the prepared cell suspension sample in a 5 ml flow tube for flow cytometry detection; Flow cytometry: Before flow cytometry, mix the cell suspension, set the excitation wavelength to 405 nm, and collect data from the fluorescence signal channel between 531 nm and 598 nm. A total of 2 × 10 4 The fluorescence intensity of a single cell, namely the mean fluorescence intensity (MFI), was calculated to compare the fluorescence intensity between different samples; the mean fluorescence intensity (MFI) = total fluorescence intensity of the fluorescence channel of the positive cell population / total number of cells in the positive cell population.

6. The method for calculating the efficacy prediction of TMPRSS2 protein inhibitors according to claim 1, characterized in that: Determine whether the enzyme activity inhibition effect has reached the set effect according to the following method: The linear regression equation was calculated with the logarithm of the inhibitor concentration as the horizontal axis and the inhibition rate as the vertical axis. Then, the inhibition rate of 30% was substituted into the linear regression equation to obtain the corresponding horizontal axis value, logIC 30 value; According to the logIC of the inhibitor 30 The IC value of the inhibitor was calculated 30 value, the IC 30 Value and setting IC 30 The value is compared within the valid range. If IC 30 If the value is within the effective range, the enzyme activity inhibition effect reaches the set effect, otherwise, the set effect is not achieved.

7. The method for calculating the efficacy prediction of the TMPRSS2 protein inhibitor according to claim 6, characterized in that: The logIC can also be determined as follows 30 value: log IC 30 =-0.1919S+0.3202log P-0.5488 The inhibitor was prepared into a stock solution with a concentration of 5 mg / mL in methanol, and then diluted with methanol to prepare a working solution of 50 ug / mL, filtered through a 0.22 mm filter membrane and bottled, and 1 mL was injected for HPLC detection; logP = a + b * logk; a and b are constants, k is the retention factor, k = (t r -t0) / t0,t r is the chromatographic retention time, t0 is the retention time of methanol solvent; S is the binding ability of the inhibitor to TMPRSS2.

8. The method for calculating the efficacy prediction of the TMPRSS2 protein inhibitor according to claim 7, characterized in that: The Schrödinger software was used to predict the binding ability of inhibitors to TMPRSS2: Inhibitor conformation optimization: The inhibitor structure was downloaded from the PubChem database, and the inhibitor was optimized in three dimensions in the Ligprep module. The maximum number of atoms of the ligand was set to 500, the force field was set to OPLS4, the ionization state search condition was limited to pH 7.0±2.0, and Epik was selected to search for tautomers. The other parameters remained default. After running the program, the potential conformations were obtained as the final screening set for subsequent protein molecule docking. Protein crystal data preprocessing: Download the human TMPRSS2 protein file from the PDB protein database, import it into the Schrödinger software, remove water molecules, add hydrogen, complete the side chain, delete redundant ligands, and minimize energy in the Protein Preparation Wizard panel, and retain the original ligand in the protein file for comparison analysis of docking results; Molecular docking: Prepare the docking pocket and create a grid with the original ligand size of The protein docking box file is saved, and all parameters remain default; the optimized inhibitor screening set is docked with the docking box in the Ligand Docking module for software simulation docking, and docking scoring is performed to obtain the docking scoring result.

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