The main protease (Mpro) of SARS-CoV-2 is crucial for viral replication and a promising target for antiviral drug development. This paper reports the creation of two genetically encoded bioluminescence resonance energy transfer (BRET)-based sensors for detecting Mpro activity in live cells and in vitro. These sensors utilize peptides containing Mpro's N-terminal autocleavage sites (short: AVLQSGFR; long: KTSAVLQSGFRKME) sandwiched between mNeonGreen and NanoLuc proteins. Cleavage of the sensors in cells co-expressing Mpro was observed, while a catalytically inactive Mpro mutant (C145A) prevented cleavage. The sensors also accurately reflected Mpro inhibition by GC376. In vitro studies revealed that molecular crowding enhances Mpro activity and reduces GC376's inhibitory effect. These sensors are valuable tools for drug discovery targeting SARS-CoV-2 Mpro and functional genomics studies investigating Mpro sequence variations.

Anupriya M. Geethakumari, Wesam S. Ahmed, Saad Rasool, Asma Fatima, S. M. Nasir Uddin, Mustapha Aouida, Kabir H. Biswas