ABSTRACT
SARS-CoV-2, the coronavirus that causes the disease COVID-19, has claimed millions of lives over the past two 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 a 1536-well ultra-high 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.