Computer-aided Virtual Screening Of TMPRSS2 Inhibitors That Potentially Block SARS-CoV-2 S Protein Activation

Coronavirus disease 2019（COVID-19）,caused by severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）,continues to spread worldwide.COVID-19 has become an epidemic,so the search for antiviral compounds is essential.SARS-CoV-2 requires the participation of viral proteins and host proteins in the process of infecting the body.SARS-CoV-2 infected cells begin when the S1 subunit of the SARS-CoV-2 spike protein（S）binds to the cell receptor angiotensin converting enzyme 2（ACE2）,after which the fusion peptide（FP）of the S2 subunit is responsible for membrane fusion to release genomic RNA into the cell.Transmembrane protease serine 2（TMPRSS2）cleaves the S2’site of the S2 subunit of S protein,exposing the fusion peptide.It can be seen that TMPRSS2 is a key protease in the process of virus invasion of the body,so researchers have discovered many TMPRSS2 inhibitors to fight COVID-19,but there is currently no TMPRSS2 inhibitor in clinical trials that can significantly improve the symptoms of COVID-19.Therefore,the exploration of new TMPRSS2 inhibitors has important application value.The main research contents and results of this paper are as follows:（1）TMPRSS2 three-dimensional structure acquisition and virtual screening of potential inhibitors of TMPRSS2The TMPRSS2 protein structure used in this study is based on the human derived TMPRSS2 crystal structure（PDB ID:7MEQ）.Since there is a loss of 18 amino acids in the crystal structure of TMPRSS2,we use the SWISS-MODEL homologous modeling method to reconstruct the protein structure,and the Ramachandran plot under the Procheck module and the Align analysis in Pymol prove that the reconstructed protein structure is highly reliable.The reconstructed protein structure was virtually screened by the FDA-approved drug database in the ZINC15 database by molecular docking method through the Auto Dock Vina v.1.2.0 program,and 10 potential TMPRSS2 inhibitors were screened according to the size of the protein and small molecule binding capacity.（2）In vitro effect detection of potential inhibitors of TMPRSS2The expression of exogenous proteases by E.coli is well established.In this experiment,by constructing p ET-28a（+）-6×His-TMPRSS2 plasmid,E.coli BL21（DE3）was used for TMPRSS2 exogenous expression,protein denaturation purification and protein gradient dialysis renaturation,and finally TMPRSS2 protease with high activity was obtained with a specific activity of 4.2 U/μg.The inhibitory effect of virtually screened potential inhibitors of TMPRSS2 was tested by in vitro enzyme bioassay experiments.The substrate for the in vitro enzyme bioassay experiment was the trypsin fluorescent substrate Boc-Gln-Ala-Arg-AMC.The results showed that Raltegravir could effectively inhibit the activity of TMPRSS2 within the concentration range we detected,and the half maximal inhibitory concentration(IC₅₀)was 67 n M,and the inhibition effect reached n M level.The remaining small molecules did not detect good inhibitory activity.（3）Molecular dynamics simulation preliminarily explored the interaction between TMPRSS2 inhibitors and themThe stability of complex systems in molecular dynamics simulation is an important reference for mechanism exploration.In this study,the RMSD/RMSF/R_g analysis of the Raltegravir and TMPRSS2 composite system was first analyzed by Gromacs software to determine the stability of the system,and Camostat and TMPRSS2simulation system were used as positive controls,and TMPRSS2 protein systems without small molecules were used as negative controls.The RMSD values of proteins and small molecules in Raltegravir and TMPRSS2 mimicry systems were 0.22±0.02nm and 0.19±0.02 nm,respectively.The R_gvalue was 2.15±0.02 nm;RMSF values did not fluctuate significantly compared with the negative control group.From the above data,it can be seen that the Raltegravir complex system is very stable.Next,the mechanism by which Raltegravir exerts inhibitory effects is explored by analyzing the interaction of Raltegravir with TMPRSS2.Through hydrogen bond analysis,it is known that Raltegravir and TMPRSS2 maintain 1-3 hydrogen bonds during the simulation process of 100 ns.Through cluster analysis,it can be seen that Raltegravir has hydrogen bond interaction with Lys342 and Gly462 amino acids.Among them,the amino acid residue Gly462 has hydrogen bond interaction between a variety of known TMPRSS2 inhibitors and TMPRSS2 simulation,which may be a key amino acid that exerts an inhibitory effect.In summary,this paper obtains potential inhibitors of TMPRSS2 by virtual screening of the FDA-approved drug database in the ZINC15 database.Raltegravir was verified as an inhibitor of TMPRSS2 with an IC₅₀ of 67 n M by in vitro enzyme activity assay.By molecular dynamics simulation,it was found that Raltegravir has a strong hydrogen bond interaction with TMPRSS2,and Gly462 may be a key amino acid that exerts an inhibitory effect.This paper provides a new option for finding novel drugs to treat COVID-19 and has important implications for the development of drugs to treat COVID-19.