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• W4210279980 abstract "Abstract The SARS-CoV-2 main protease, M pro , is critical for its replication and is an appealing target for designing anti-SARS-CoV-2 agents. In this regard, a number of assays have been developed based on its cleavage sequence preferences to monitor its activity. These include the usage of Fluorescence Resonance Energy Transfer (FRET)-based substrates in vitro and a FlipGFP reporter, one which fluoresces after M pro -mediated cleavage, in live cells. Here, we have engineered a pair of genetically encoded, Bioluminescence Resonance Energy Transfer (BRET)-based sensors for detecting SARS-CoV-2 M pro proteolytic activity in living host cells as well as in vitro assays. The sensors were generated by sandwiching M pro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and nanoLuc proteins. Co-expression of the sensor with the M pro in live cells resulted in its cleavage in a dose- and time-dependent manner while mutation of the critical C145 residue (C145A) in M pro completely abrogated the sensor cleavage. Importantly, the BRET-based sensors displayed increased sensitivities and specificities as compared to the recently developed FlipGFP-based M pro sensor. 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 sensor 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 ." @default.
• W4210279980 created "2022-02-08" @default.
• W4210279980 creator A5004334168 @default.
• W4210279980 creator A5011860224 @default.
• W4210279980 creator A5020620079 @default.
• W4210279980 creator A5066367543 @default.
• W4210279980 creator A5080114733 @default.
• W4210279980 creator A5088322519 @default.
• W4210279980 creator A5089417735 @default.
• W4210279980 date "2022-02-01" @default.
• W4210279980 modified "2023-09-27" @default.
• W4210279980 title "A Genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor" @default.
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