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A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor

journal contribution
submitted on 2024-04-22, 07:41 and posted on 2024-04-22, 07:42 authored by Anupriya M. Geethakumari, Wesam S. Ahmed, Saad Rasool, Asma Fatima, S. M. Nasir Uddin, Mustapha Aouida, Kabir H. Biswas

The main protease, Mpro, 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 Mpro proteolytic activity in live cells as well as in vitro. The sensors were generated by sandwiching peptides containing the Mpro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and NanoLuc proteins. Co-expression of the sensors with Mpro in live cells resulted in their cleavage while mutation of the critical C145 residue (C145A) in Mpro completely abrogated their cleavage. Additionally, the sensors recapitulated the inhibition of Mpro by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based Mpro sensors revealed a molecular crowding-mediated increase in the rate of Mpro 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 Mpro and functional genomics application to determine the effect of sequence variation in Mpro.


Other Information

Published in: Communications Chemistry
License: https://creativecommons.org/licenses/by/4.0
See article on publisher's website: https://dx.doi.org/10.1038/s42004-022-00731-2

History

Language

  • English

Publisher

Springer Nature

Publication Year

  • 2022

License statement

This Item is licensed under the Creative Commons Attribution 4.0 International License.

Institution affiliated with

  • Hamad Bin Khalifa University
  • College of Health and Life Sciences - HBKU