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

The papain-like protease (PL^pro) of SARS-CoV-2 is a validated antiviral drug target. PL^prois 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 PL^proinhibitors 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 PL^proinhibitors in the BSL-2 setting. Two compounds selected from the FlipGFP-PL^proassay, 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 PL^proin 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 PL^proinhibitors identified in this study represent promising starting points for further development as SARS-CoV-2 antivirals, and FlipGFP-PL^proassay might be a suitable surrogate for screening PL^proinhibitors in the BSL-2 setting.