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(pro)in vitro. HCV protease inhibitor simeprevir showed the most potency against 3CL(pro) with an EC(50) 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.