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

The papain-like protease (PL) 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 inhibitors including and with improved enzymatic inhibition and antiviral activity compared to , which was reported as a SARS-CoV PL inhibitor. Significantly, we developed a cell-based FlipGFP assay that can be applied to predict the cellular antiviral activity of PL inhibitors in the BSL-2 setting. X-ray crystal structure of PL in complex with showed that binding of to SARS-CoV-2 induced a conformational change in the BL2 loop to a more closed conformation. Molecular dynamics simulations showed that and engaged in more extensive interactions than . Overall, the PL inhibitors identified in this study represent promising candidates for further development as SARS-CoV-2 antivirals, and the FlipGFP-PL assay is a suitable surrogate for screening PL inhibitors in the BSL-2 setting.