Abstract
The main protease (M-pro) of SARS-CoV-2 is an important target for COVID-19 therapy. Here, a pair of genetically encoded BRET-based sensors for detecting M-pro activity are generated by sandwiching N-terminal autocleavage sites in between the mNeonGreen and NanoLuc proteins.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.
| Original language | English |
|---|---|
| Article number | 117 |
| Number of pages | 13 |
| Journal | Communications Chemistry |
| Volume | 5 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 28 Sept 2022 |
Keywords
- Accelerated molecular-dynamics
- Bioluminescence
- Cleavage
- Cov m-pro
- Identification
- Main protease
- Respiratory syndrome coronavirus
- Sars-coronavirus
- Sequence alignment
- Substrate-specificity