A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor
<p dir="ltr">The main protease, M<sup>pro</sup>, 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 ge...
محفوظ في:
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| مؤلفون آخرون: | , , , , , |
| منشور في: |
2022
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إضافة وسم
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| الملخص: | <p dir="ltr">The main protease, M<sup>pro</sup>, 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<sup>pro</sup> proteolytic activity in live cells as well as in vitro. The sensors were generated by sandwiching peptides containing the M<sup>pro</sup> N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and NanoLuc proteins. Co-expression of the sensors with M<sup>pro</sup> in live cells resulted in their cleavage while mutation of the critical C145 residue (C145A) in M<sup>pro</sup> completely abrogated their cleavage. Additionally, the sensors recapitulated the inhibition of M<sup>pro</sup> 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 M<sup>pro</sup> 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<sup>pro</sup> and functional genomics application to determine the effect of sequence variation in M<sup>pro</sup>.</p><p><br></p><h2>Other Information</h2><p dir="ltr">Published in: Communications Chemistry<br>License: <a href="https://creativecommons.org/licenses/by/4.0" target="_blank">https://creativecommons.org/licenses/by/4.0</a><br>See article on publisher's website: <a href="https://dx.doi.org/10.1038/s42004-022-00731-2" target="_blank">https://dx.doi.org/10.1038/s42004-022-00731-2</a></p> |
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