AbstractThe papain-like protease (PLpro) of SARS-CoV-2 is a validated antiviral drug target. PLprois involved in the cleavage of viral polyproteins and antagonizing host innate immune response through its deubiquitinating and deISG15ylating activities, rendering it a high profile antiviral drug target. Through a FRET-based high-throughput screening, several hits were identified as PLproinhibitors with IC50values at the single-digit micromolar range. Subsequent lead optimization led to potent inhibitors with IC50values ranging from 0.56 to 0.90 µM. To help prioritize lead compounds for the cellular antiviral assay against SARS-CoV-2, we developed the cell-based FlipGFP assay that is suitable for quantifying the intracellular enzymatic inhibition potency of PLproinhibitors in the BSL-2 setting. Two compounds selected from the FlipGFP-PLproassay, Jun9-53-2 and Jun9-72-2, inhibited SARS-CoV-2 replication in Caco-2 hACE2 cells with EC50values of 8.89 and 8.32 µM, respectively, which were 3-fold more potent than GRL0617 (EC50= 25.1 µM). The X-ray crystal structures of PLproin complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to the more closed conformation. Overall, the PLproinhibitors identified in this study represent promising starting points for further development as SARS-CoV-2 antivirals, and FlipGFP-PLproassay might be a suitable surrogate for screening PLproinhibitors in the BSL-2 setting.