Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and FlipGFP-based reporter assay

Abstract

The papain-like protease (PLpro) of SARS-CoV-2 is a validated antiviral drug target. PLpro is 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 PLpro inhibitors with IC50 values at the single-digit micromolar range. Subsequent lead optimization led to potent inhibitors with IC50 values ranging from 0.56 to 0.90 {micro}M. 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 PLpro inhibitors in the BSL-2 setting. Two compounds selected from the FlipGFP-PLpro assay, Jun9-53-2 and Jun9-72-2, inhibited SARS-CoV-2 replication in Caco-2 hACE2 cells with EC50 values of 8.89 and 8.32 {micro}M, respectively, which were 3-fold more potent than GRL0617 (EC50 = 25.1 {micro}M). The X-ray crystal structures of PLpro in 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 PLpro inhibitors identified in this study represent promising starting points for further development as SARS-CoV-2 antivirals, and FlipGFP-PLpro assay might be a suitable surrogate for screening PLpro inhibitors in the BSL-2 setting.