TY - JOUR T1 - Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and FlipGFP-based reporter assay JF - bioRxiv DO - 10.1101/2021.03.15.435551 SP - 2021.03.15.435551 AU - Zilei Xia AU - Michael Dominic Sacco AU - Chunlong Ma AU - Julia Alma Townsend AU - Naoya Kitamura AU - Yanmei Hu AU - Mandy Ba AU - Tommy Szeto AU - Xiujun Zhang AU - Xiangzhi Meng AU - Fushun Zhang AU - Yan Xiang AU - Michael Thomas Marty AU - Yu Chen AU - Jun Wang Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/03/16/2021.03.15.435551.abstract N2 - 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 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 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 µM, respectively, which were 3-fold more potent than GRL0617 (EC50 = 25.1 µM). 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.Competing Interest StatementA patent was filed to claim the compounds described herein as potential SARS-CoV-2 antivirals. ER -