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.