A high-throughput screen for TMPRSS2 expression identifies FDA-approved compounds that can limit SARS-CoV-2 entry

Yanwen Chen,Daniel P Camarco,Bo Lin,Jie Liu,Yuan Liu,Ferhan Tuncer,Jason R Kennerdell,Mads B Larsen,Bill B Chen,Travis B Lear,John F Mcdyer,Michael M Myerburg,John W Evankovich,Toren Finkel,Irene Alfaras,Karina C Lockwood,Laura Salminen

doi:10.1038/s41467-021-24156-y

Abstract

SARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease. The Spike (S) protein of SARS-CoV-2 attaches to host lung epithelial cells through the cell surface receptor ACE2, a process dependent on host proteases including TMPRSS2. Here, we identify small molecules that reduce surface expression of TMPRSS2 using a library of 2,560 FDA-approved or current clinical trial compounds. We identify homoharringtonine and halofuginone as the most attractive agents, reducing endogenous TMPRSS2 expression at sub-micromolar concentrations. These effects appear to be mediated by a drug-induced alteration in TMPRSS2 protein stability. We further demonstrate that halofuginone modulates TMPRSS2 levels through proteasomal-mediated degradation that involves the E3 ubiquitin ligase component DDB1- and CUL4-associated factor 1 (DCAF1). Finally, cells exposed to homoharringtonine and halofuginone, at concentrations of drug known to be achievable in human plasma, demonstrate marked resistance to SARS-CoV-2 infection in both live and pseudoviral in vitro models. Given the safety and pharmacokinetic data already available for the compounds identified in our screen, these results should help expedite the rational design of human clinical trials designed to combat active COVID-19 infection.

Highlights

SARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease
We identify the E3 ligase component DDB1- and CUL4-associated factor 1 (DCAF1) as critical for regulating TMPRSS2 stability and show the effect of halofuginone to reduce TMPRSS2 abundance is DCAF1-dependent
In order to assess agents that might alter TMPRSS2 expression, we first engineered full-length human TMPRSS2 to express a Cterminal 11 amino acid tag (HiBiT), which produces a bioluminescent signal when combined with a complementary protein (LgBiT) and a furimazine substrate[18]

Summary

Introduction

SARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease. We identify homoharringtonine and halofuginone as the most attractive agents, reducing endogenous TMPRSS2 expression at sub-micromolar concentrations These effects appear to be mediated by a drug-induced alteration in TMPRSS2 protein stability. Based on our screening strategy and already published pharmacokinetic and toxicology profiles, we identified compounds halofuginone and homoharringtonine as agents that reduce cell surface expression of TMPRSS2. Both halofuginone and homoharringtonine reduced entry of pseudotyped SARS-CoV-2 in TMPRSS2-expressing lung epithelial cells, and significantly reduced Calu-3 infection with authentic SARS-CoV-2. We identify the E3 ligase component DDB1- and CUL4-associated factor 1 (DCAF1) as critical for regulating TMPRSS2 stability and show the effect of halofuginone to reduce TMPRSS2 abundance is DCAF1-dependent

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