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Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors.
Rawson, Jonathan M O; Duchon, Alice; Nikolaitchik, Olga A; Pathak, Vinay K; Hu, Wei-Shau.
  • Rawson JMO; Viral Recombination Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA.
  • Duchon A; Viral Recombination Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA.
  • Nikolaitchik OA; Viral Recombination Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA.
  • Pathak VK; Viral Mutation Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA.
  • Hu WS; Viral Recombination Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA.
Viruses ; 13(2)2021 01 24.
Article in English | MEDLINE | ID: covidwho-1052508
ABSTRACT
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.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Antiviral Agents / Protease Inhibitors / Coronavirus 3C Proteases / SARS-CoV-2 / Luciferases Type of study: Prognostic study Topics: Traditional medicine Limits: Humans Language: English Year: 2021 Document Type: Article Affiliation country: V13020173

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Antiviral Agents / Protease Inhibitors / Coronavirus 3C Proteases / SARS-CoV-2 / Luciferases Type of study: Prognostic study Topics: Traditional medicine Limits: Humans Language: English Year: 2021 Document Type: Article Affiliation country: V13020173