Drug repurposing screening identified tropifexor as a SARS-CoV-2 papain-like protease inhibitor

The global COVID-19 pandemic underscores the dire need of effective antivirals. Encouraging progress has been made in developing small molecule inhibitors targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and main protease (M ^pro ). However, the development of papain-like protease (PL ^pro ) inhibitors faces several obstacles. Nevertheless, PL ^pro represents a high-profile drug target given its multifaceted roles in viral replication. PL ^pro is involved in not only the cleavage of viral polyprotein but also modulation of host immune response. In this study, we conducted a drug-repurposing screening of PL ^pro against the MedChemExpress bioactive compound library and identified three hits, EACC, KY-226, and tropifexor, as potent PL ^pro inhibitors with IC ₅₀ values ranging from 3.39 to 8.28 µM. The three hits showed dose-dependent binding to PL ^pro in the thermal shift assay. In addition, tropifexor inhibited the cellular PL ^pro activity in the FlipGFP assay with an IC ₅₀ of 10.6 µM. Gratifyingly, tropifexor showed antiviral activity against SARS-CoV-2 in Calu-3 cells with an EC ₅₀ of 4.03 µM, a 7.8-fold increase compared to GRL0617 (EC ₅₀ = 31.4 µM). Overall, tropifexor represents a novel PL ^pro inhibitor that can be further developed as SARS-CoV-2 antivirals.