Dual-Reporter System for Real-Time Monitoring of SARS-CoV-2 Main Protease Activity in Live Cells Enables Identification of an Allosteric Inhibition Path.

The SARS-CoV-2 pandemic is an ongoing threat to global health, and the continuing emergence of contagious variants highlights the urgent need for additional antiviral therapy to attenuate COVID-19 disease. The SARS-CoV-2 main protease (3CL pro ) presents an attractive target for such therapy due to its high sequence conservation and key role in the viral life cycle. In this study, we designed a fluorescent-luminescent cell-based reporter for the detection and quantification of 3CL pro intracellular activity. Employing this platform, we examined the efficiency of known protease inhibitors against 3CL pro and further identified potent inhibitors through high-throughput chemical screening. Computational analysis confirmed a direct interaction of the lead compounds with the protease catalytic site and identified a prototype for efficient allosteric inhibition. These developments address a pressing need for a convenient sensor and specific targets for both virus detection and rapid discovery of potential inhibitors.