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 .
【저자키워드】 COVID-19, SARS-CoV-2, Antiviral, protease, inhibitor, luciferase, 3CLpro, 【초록키워드】 HIV, cytotoxicity, 3CL pro, in vitro, protease inhibitors, HCV, antiviral drug, specificity, viral replication, cleavage, small molecule, Proteases, reporter, False positive, compounds, inhibitors of SARS-CoV-2, effort, cleavage site, luciferase activity, replication-competent virus, limitations, FIVE, Cell, current, BSL-2, BSL-3 facility, identify, performed, lack, reported, screened, facilitate, exhibited, reduce, lentiviral, inhibit SARS-CoV-2, other compound, Z-FA-FMK, 【제목키워드】 3CL pro, development, identification, Complementation,