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TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS
Danielle Cicka1,2,† , Qiankun Niu1,† , Min Qui1,3 , Kun Qian1 , Eric Mille1,4 , Dacheng Fan1 , Xiulei Mo1,4 , Andrey A. Ivanov1,3,4 , Stefan G. Sarafianos5 , Yuhong Du1,3,4 , Haian Fu1,3,4,*
1Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
2Graduate Program in Molecular and Systems Pharmacology, Laney Graduate School of Emory University, Atlanta, GA 30322, USA
3Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, GA 30322, USA
4Winship Cancer Institute of Emory University, Atlanta, GA 30322, USA
5Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA
These authors contributed equally to this work
*Correspondence to:Haian Fu ,
J Mol Cell Biol, Volume 15, Issue 3, March 2023, mjad017,
Keyword: SARS-CoV-2, COVID-19, protein–protein interaction, TMPRSS2, SPIKE, high-throughput screening, TR-FRET

SARS-CoV-2, the coronavirus that causes the disease COVID-19, has claimed millions of lives over the past 2 years. This demands rapid development of effective therapeutic agents that target various phases of the viral replication cycle. The interaction between host transmembrane serine protease 2 (TMPRSS2) and viral SPIKE protein is an important initial step in SARS-CoV-2 infection, offering an opportunity for therapeutic development of viral entry inhibitors. Here, we report the development of a time-resolved fluorescence/Förster resonance energy transfer (TR-FRET) assay for monitoring the TMPRSS2–SPIKE interaction in lysate from cells co-expressing these proteins. The assay was configured in a 384-well-plate format for high-throughput screening with robust assay performance. To enable large-scale compound screening, we further miniaturized the assay into 1536-well ultrahigh-throughput screening (uHTS) format. A pilot screen demonstrated the utilization of the assay for uHTS. Our optimized TR-FRET uHTS assay provides an enabling platform for expanded screening campaigns to discover new classes of small-molecule inhibitors that target the SPIKE and TMPRSS2 protein–protein interaction.