Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors

• Published in: Viruses 2021-01, Vol.13 (2), p.173
• Main Authors: Rawson, Jonathan M. O., Duchon, Alice, Nikolaitchik, Olga A., Pathak, Vinay K., Hu, Wei-Shau
• Format: Article
• Language: English
• Subjects: 3CLpro; antiviral; Calpain; Cell permeability; Clinical trials; Coronaviruses; COVID-19; Cytotoxicity; Drug development; HIV; Human immunodeficiency virus; luciferase; Plasmids; protease; Proteinase inhibitors; Proteins; Reagents; Replication; RNA polymerase; SARS-CoV-2; Severe acute respiratory syndrome coronavirus 2
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v13020173

The 3C-like protease (3CLpro) 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 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro 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 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro 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 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro 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 3CLpro.