Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CL(pro) Inhibitors

The 3C-like protease (3CL(pro)) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CL(pro) has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CL(pro) inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CL(pro) in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CL(pro) and two luciferase fragments linked together by a 3CL(pro) cleavage site. 3CL(pro)-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CL(pro) results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CL(pro) inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CL(pro), including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CL(pro). Of these, only five exhibited significant inhibition of 3CL(pro) in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CL(pro).