Establishment and application of a cell-based high-throughput screening model for TMPRSS2 inhibitors
Transmembrane serine protease 2(TMPRSS2)is a cell surface protease widely present in the human body.It is involved in the infection of various viruses such as severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),and in the cell invasion,tumor growth and metastasis processes of prostate cancer.This study used Boc-Gln-Ala-Arg-AMC as the fluorescent substrate to determine the cleavage activity of TMPRSS2 towards SARS-CoV-2 S protein.Then cell-based screening model for TMPRSS2 inhibitors was established in Vero E6 cells overexpressing TMPRSS2(Vero E6/TMPRSS2).Seven compounds exhibiting TMPRSS2 inhibitory activities with low toxicity were obtained through high-throughput screening(HTS)from natural and synthetic compound pure product library of National Center for Screening Novel Microbial Drugs.Surface plasmon resonance(SPR)has shown that the obtained inhibitors could bind to TMPRSS2 with moderate affinity in a dose dependent manner.Cell-cell fusion experiments have shown that the obtained inhibitors can inhibit the occurrence of S protein mediated cell-cell fusion by inhibiting TMPRSS2 cleavage of SARS-CoV-2 S protein in a concentration dependent manner.Preliminary pseudovirus experiment showed that the inhibitors may reduce the pseudovirus infection into Opti-HEK-293T-ACE2 cells to varying degrees.In a word,this study successfully established a cell-based HTS model for TMPRSS2 inhibitor and preliminarily confirmed that the seven screened inhibitors possessed in vitro anti-TMPRSS2 activities,providing new structural scaffolds for the development of new drugs against SARS-CoV-2.
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