Discovery of SARS-CoV-2 Papain-like Protease Inhibitors through a Combination of High-Throughput Screening and a FlipGFP-Based Reporter Assay

Abstract

The papain-like protease (PL^pro) of SARS-CoV-2 is a validated antiviral drug target. Through a fluorescence resonance energy transfer-based high-throughput screening and subsequent lead optimization, we identified several PL^pro inhibitors including <b>Jun9-72-2</b> and <b>Jun9-75-4</b> with improved enzymatic inhibition and antiviral activity compared to <b>GRL0617</b>, which was reported as a SARS-CoV PL^pro inhibitor. Significantly, we developed a cell-based FlipGFP assay that can be applied to predict the cellular antiviral activity of PL^pro inhibitors in the BSL-2 setting. X-ray crystal structure of PL^pro in complex with <b>GRL0617</b> showed that binding of <b>GRL0617</b> to SARS-CoV-2 induced a conformational change in the BL2 loop to a more closed conformation. Molecular dynamics simulations showed that <b>Jun9-72-2</b> and <b>Jun9-75-4</b> engaged in more extensive interactions than <b>GRL0617</b>. Overall, the PL^pro inhibitors identified in this study represent promising candidates for further development as SARS-CoV-2 antivirals, and the FlipGFP-PL^pro assay is a suitable surrogate for screening PL^pro inhibitors in the BSL-2 setting.