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).