Development of potent and selective inhibitors targeting the papain-like protease of SARS-CoV-2

The COVID-19 pandemic has been disastrous to society and effective drugs are urgently needed. The papain-like protease domain (PLpro) of SARS-CoV-2 (SCoV2) is indispensable for viral replication and represents a putative target for pharmacological intervention. In this work, we describe the development of a potent and selective SCoV2 PLpro inhibitor, 19. The inhibitor not only effectively blocks substrate cleavage and immunosuppressive function imparted by PLpro, but also markedly mitigates SCoV2 replication in human cells, with a submicromolar IC50. We further present a convenient and sensitive activity probe, 7, and complementary assays to readily evaluate SCoV2 PLpro inhibitors in vitro or in cells. In addition, we disclose the co-crystal structure of SCoV2 PLpro in complex with a prototype inhibitor, which illuminates their detailed binding mode. Overall, these findings provide promising leads and important tools for drug discovery aiming to target SCoV2 PLpro. [Display omitted] •Development of a sensitive and affordable PLpro activity probe•Identification of potential SCoV2 PLpro inhibitors through HTS•Co-crystal structure determination and lead optimization•Characterization of a submicromolar inhibitor of SCoV2 PLpro represents a putative druggable target for SCoV2. Utilizing multiple approaches, including fluorogenic activity probes, HTS, co-crystallization, structure-based design, and other optimized cellular assays, Shan et al. successfully develop a potent and selective inhibitor of SCoV2 PLpro.