Repurposing of HIV/HCV protease inhibitors against SARS-CoV-2 3CL^pro

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen that caused the global COVID-19 outbreak. The 3C-like protease (3CL^pro) of SARS-CoV-2 plays a key role in virus replication and has become an ideal target for antiviral drug design. In this work, we have employed bioluminescence resonance energy transfer (BRET) technology to establish a cell-based assay for screening inhibitors against SARS-CoV-2 3CL^pro, and then applied the assay to screen a collection of known HIV/HCV protease inhibitors. Our results showed that the assay is capable of quantification of the cleavage efficiency of 3CL^pro with good reproducibility (Z' factor is 0.59). Using the assay, we found that 9 of 26 protease inhibitors effectively inhibited the activity of SARS-CoV-2 3CL^pro in a dose-dependent manner. Among them, four compounds exhibited the ability to bind to 3CL^proin vitro. HCV protease inhibitor simeprevir showed the most potency against 3CL^pro with an EC₅₀ vale of 2.6 μM, bound to the active site pocket of 3CL^pro in a predicted model, and importantly, exhibited a similar activity against the protease containing the mutations P132H in Omicron variants. Taken together, this work demonstrates the feasibility of using the cell-based BRET assay for screening 3CL^pro inhibitors and supports the potential of simeprevir for the development of 3CL^pro inhibitors.