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Updated: May 14, 2025
A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
Published on: January 9, 2019
A phycobiliprotein-based reporter assay for the evaluation of SARS-CoV-2 main protease activity
Le Cao1, Shuyuan Shi1, Chaofeng Zhang2
1College of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan, 030024, People's Republic of China; College of Artificial Intelligence, Taiyuan University of Technology, Taiyuan, 030024, People's Republic of China.
Abstract:
SARS-CoV-2 Mpro is crucial for viral replication and transcription and is highly conserved. Therefore, it is an ideal target for developing broad-spectrum antiviral drugs. To address resistance to existing drugs caused by mutations, a simple and sensitive method for detecting the activity of Mpro is needed. Considering the excellent fluorescence properties of phycobiliproteins, this study developed a phycobiliprotein-based reporter assay to evaluate Mpro activity. An engineered lyase was generated by inserting the Mpro recognition sequence between the phycobiliprotein lyases CpcF and CpcE. To ensure that the binding of CpcE and CpcF depended on the linker, a series of truncated forms were constructed. Among them, the activity of CpcE/F-10 was significantly reduced in the presence of Mpro; however, both genetic and chemical inhibition of Mpro activity reversed these results. These data indicated that the fluorescence of phycobiliproteins was negatively correlated with Mpro activity. The reporter assay developed here will contribute to determining the impact of Mpro mutations and screening for new inhibitors.
