Drug-Repurposing Screening Identified Tropifexor as a SARS-CoV-2 Papain-like Protease Inhibitor.

The global COVID-19 pandemic underscores the dire need for 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). However, the development of papain-like protease (PL) inhibitors faces several obstacles. Nevertheless, PL represents a high-profile drug target given its multifaceted roles in viral replication. PL is involved in not only the cleavage of viral polyprotein but also the modulation of host immune response. In this study, we conducted a drug-repurposing screening of PL against the MedChemExpress bioactive compound library and identified three hits, EACC, KY-226, and tropifexor, as potent PL inhibitors with IC values ranging from 3.39 to 8.28 μM. The three hits showed dose-dependent binding to PL in the thermal shift assay. In addition, tropifexor inhibited the cellular PL activity in the FlipGFP assay with an IC of 10.6 μM. Gratifyingly, tropifexor showed antiviral activity against SARS-CoV-2 in Calu-3 cells at noncytotoxic concentrations. Overall, tropifexor represents a novel PL inhibitor that can be further developed as SARS-CoV-2 antivirals.