A genetically encoded BRET-based SARS-CoV-2 M^pro protease activity sensor

The main protease, M^pro, is critical for SARS-CoV-2 replication and an appealing target for designing anti-SARS-CoV-2 agents. Therefore, there is a demand for the development of improved sensors to monitor its activity. Here, we report a pair of genetically encoded, bioluminescence resonance energy transfer (BRET)-based sensors for detecting M^pro proteolytic activity in live cells as well as in vitro. The sensors were generated by sandwiching peptides containing the M^pro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and NanoLuc proteins. Co-expression of the sensors with M^pro in live cells resulted in their cleavage while mutation of the critical C145 residue (C145A) in M^pro completely abrogated their cleavage. Additionally, the sensors recapitulated the inhibition of M^pro by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based M^pro sensors revealed a molecular crowding-mediated increase in the rate of M^pro activity and a decrease in the inhibitory potential of GC376. The sensors developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 M^pro and functional genomics application to determine the effect of sequence variation in M^pro.