A phycobiliprotein-based reporter assay for the evaluation of SARS-CoV-2 main protease activity

SARS-CoV-2 M^pro 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 M^pro is needed. Considering the excellent fluorescence properties of phycobiliproteins, this study developed a phycobiliprotein-based reporter assay to evaluate M^pro activity. An engineered lyase was generated by inserting the M^pro 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 M^pro; however, both genetic and chemical inhibition of M^pro activity reversed these results. These data indicated that the fluorescence of phycobiliproteins was negatively correlated with M^pro activity. The reporter assay developed here will contribute to determining the impact of M^pro mutations and screening for new inhibitors.