Luciferase complementation assay for identification of SARS-CoV- 2 3CLPRO inhibitors

ABSTRACT

Background: The 3C-like protease (3CLpro) of SARS-CoV-2 has been widely pursued as a target for COVID-19 anti-viral drug development because it is essential for viral replication and lacks significant homology to 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 the cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility and are not amenable to high-throughput screening. Methods: To address these limitations, we explored the use of a cell-based luciferase complementation reporter to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. We constructed lentiviral vectors that co-express 3CLpro and a split reporter in which two luciferase fragments were linked by a 3CLpro cleavage site. 3CLpro-mediated cleavage of the reporter was expected to result in loss of complementation and low luciferase activity, whereas inhibition of 3CLpro was expected to result in significantly higher levels of luciferase activity. Results: In the absence of inhibitors, we found that most of the luciferase reporter was cleaved by 3CLpro, resulting in low luciferase activity. However, inhibition of 3CLpro, either with the small molecule GC376 or an inactivating mutation (C145A), prevented cleavage and resulted in an ∼10-fold increase in luciferase reporter activity. We also found that our reporter assay can easily distinguish between cytotoxicity and true inhibition of 3CLpro. With this assay, we screened 31 additional 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 3CLpro. Of these, only four compounds exhibited significant activity against SARS-CoV-2 3CLpro in cells boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. Conclusion: We have developed a novel luciferase complementation reporter assay for identification of SARS-CoV-2 3CLpro inhibitors in living cells. The assay is sensitive, rapid, easy to perform, and can readily differentiate cytotoxicity from 3CLpro inhibition, a powerful feature that should reduce false positives during screening. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.