Gain-of-Signal Assays for Probing Inhibition of SARS-CoV-2 M(pro)/3CL(pro) in Living Cells

The main protease, M(pro), of SARS-CoV-2 is required to cleave the viral polyprotein into precise functional units for virus replication and pathogenesis. Here, we report quantitative reporters for M(pro) function in living cells in which protease inhibition by genetic or chemical methods results in robust signal readouts by fluorescence (enhanced green fluorescent protein [eGFP]) or bioluminescence (firefly luciferase). These gain-of-signal systems are scalable to high-throughput platforms for quantitative discrimination between M(pro) mutants and/or inhibitor potencies as evidenced by validation of several reported inhibitors. Additional utility is shown by single M(pro) amino acid variants and structural information combining to demonstrate that both inhibitor conformational dynamics and amino acid differences are able to influence inhibitor potency. We further show that a recent variant of concern (Omicron) has an unchanged response to a clinically approved drug, nirmatrelvir, whereas proteases from divergent coronavirus species show differential susceptibility. Together, we demonstrate that these gain-of-signal systems serve as robust, facile, and scalable assays for live cell quantification of M(pro) inhibition, which will help expedite the development of next-generation antivirals and enable the rapid testing of emerging variants.