key: cord-0782927-5y88k9a0
authors: Geethakumari, Anupriya M; Ahmed, Wesam S; Rasool, Saad; Fatima, Asma; Uddin, S.M. Nasir; Aouida, Mustapha; Biswas, Kabir H
title: A Genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor
date: 2022-02-01
journal: bioRxiv
DOI: 10.1101/2022.01.31.478460
sha: 39cbb026472db0482468460ed3292d0a06b4d66e
doc_id: 782927
cord_uid: 5y88k9a0

The SARS-CoV-2 main protease, Mpro, 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 Mpro-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 Mpro proteolytic activity in living host cells as well as in vitro assays. The sensors were generated by sandwiching Mpro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and nanoLuc proteins. Co-expression of the sensor with the Mpro in live cells resulted in its cleavage in a dose- and time-dependent manner while mutation of the critical C145 residue (C145A) in Mpro completely abrogated the sensor cleavage. Importantly, the BRET-based sensors displayed increased sensitivities and specificities as compared to the recently developed FlipGFP-based Mpro sensor. 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 sensor 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.

. Genetically encoded, BRET-based live cell SARS-CoV-2 M pro protease activity sensor. A schematic representation of the genetically encoded, BRET-based SARS-CoV-2 M pro protease activity sensor expressed in live cells. Close positioning of the NLuc and mNG proteins result in a significant resonance energy transfer in the absence of the SARS-CoV-2 M pro protease activity. Activity of the SARS-CoV-2 M pro results in the cleavage of the sensor resulting in a decrease in the resonance energy transfer between NLuc and mNG leading to a decrease in the green fluorescence of the sensor. Shown in grey is the surface representation of M pro structure (SARS-CoV; PDB: 2Q6G 65 ) highlighting the critical C145 residue (green) required for proteolytic activity and the peptide substrate peptide (TSAVLQSGFRK; red). loop modelling using very-slow loop MD refining mode to generate 100 refined 156 models. The same scoring functions were used to assess the refined models. The We note that while BRET comes 396 with several advantages including a higher signal-to-noise ratio and an extended 397 dynamic range compared to some other methods 89 , the presence of the acceptor and propensity by both the short as well as the long peptide (Supp. Fig. 1 ). In order to 411 assess structural flexibility and secondary structure formation by the two peptides, we 412 generated structural models of the peptides using the substrate peptide co- (Fig. 6A,B) . Percentage proteolytic cleavage activity determined from the 644 BRET ratio indicate that GC376 starts to inhibit M pro at 33.3 μM concentration and 645 continued to do so until a concentration of 333 μM (Fig. 6A,B; insets) . The data has 646 showed that the IC50 values for the short sensor is 127.4 ± 23.33 μM and that for long 

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