A fluorescence-based, gain-of-signal, live cell system to evaluate SARS-CoV-2 main protease inhibition

The likelihood of continued circulation of COVID-19 and its variants, and novel coronaviruses due to future zoonotic transmissions, combined with the current paucity of coronavirus antivirals, emphasize the need for improved screening in developing effective antivirals for the treatment of infection by SARS-CoV-2 (CoV2) and other coronaviruses. Here we report the development of a live-cell based assay for evaluating the intracellular function of the critical, highly-conserved CoV2 target, the Main 3C-like protease (Mpro). This assay is based on expression of native wild-type mature CoV2 Mpro, the function of which is quantitatively evaluated in living cells through cleavage of a biosensor leading to loss of fluorescence. Evaluation does not require cell harvesting, allowing for multiple measurements from the same cells facilitating quantification of Mpro inhibition, as well as recovery of function upon removal of inhibitory drugs. The pan-coronavirus Mpro inhibitor, GC376, was utilized in this assay and effective inhibition of intracellular CoV2 Mpro was found to be consistent with levels required to inhibit CoV2 infection of human lung cells. We demonstrate that GC376 is an effective inhibitor of intracellular CoV2 Mpro at low micromolar levels, while other predicted Mpro inhibitors, bepridil and alverine, are not. Results indicate this system can provide a highly effective high-throughput coronavirus Mpro screening system. •Expression vector for fully functional mature SARS-CoV-2 (CoV2) Main Protease (Mpro) in human cells.•Expression vector for an intracellular red-fluorescent protein biosensor that loses fluorescence when cleaved by CoV2 Mpro.•Treatment of dually-transfected cells with an effective CoV2 Mpro inhibitor, GC-376, results in recovery of red fluorescence.•This is a positive-signal assay for expeditious screening of CoV2 Mpro inhibitors functioning intracellularly in real-time.•Removal of SARS-CoV-2 Main Protease inhibitor allows for recovery of protease function and loss of fluorescence in real-time.