Characterization of all SARS-CoV-2 Mpro & PLpro protease cleavage sites and variants for identifying lead dual protease inhibitor compounds using novel dual color, BRET-based biosensors

Here we propose to utilize a new class of dual-color, Bioluminescence Resonance Energy Transfer (BRET)-based, dual protease biosensor (DuProSense; invention disclosure D2023-0029 under review at QF-IDKT) to determine the kinetics of all Mpro and PLpro cleavage sites in SARS-CoV-2 polyproteins, determine the impact of variant associated mutations in the proteases, and identify lead chemical compounds targeting both proteases towards COVID-19 therapy. Coronavirus Disease of 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), remains a serious health and economic threat due to emergence of newer variants with increased infectivity and resistance to vaccine-induced immune response. Additionally, the emergence of variants with mutations making Mpro resistant to the currently available anti-COVID-19 drug, nirmatrelvir (part of Pfizer’s Paxlovid) further aggravates the situation. While the proteolytic processing of polyproteins, pp1a and pp1ab, by Mpro and PLpro, which is essential for viral replication, are known, a comprehensive analysis of each one of Mpro and PLpro cleavage sites in living cells and the impact of mutations on the activity of Mpro and PLpro and their inhibition by known inhibitors is lacking. To address this lacuna, we plan to use our newly developed, BRET-based biosensor platform (DuProSense) to understand the cleavage kinetics of all Mpro and PLpro cleavage sites in living cells and combine it with molecular dynamics (MD) simulations analysis to determine differences across the sites in terms of their interaction with catalytic sites. Additionally, we plan to characterize mutations in Mpro and PLpro associated with newly emerging variants in a similar manner. Finally, insights gained from above will be used to screen for lead compounds (deep learning-based in-silico docking and functional assays) that can inhibit both Mpro and PLpro (dual protease inhibitors) using both in vitro as well as in live viral infection assays using DuProSense. Primary outcomes of the project include a detailed information on the proteolytic processing of SARS-CoV-2 polyproteins, impact of mutations in Mpro and PLpro, identification of lead dual protease inhibitors with potential towards anti-COVID-19 therapy, and validation of DuProSense for COVID-19 research such as functional genomics and drug discovery (multibillion-dollar industry spread across the globe) applications. Importantly, insights gained from the project will be useful in understanding the infection mechanism of other coronaviruses. These outcomes will be exploited through patenting that can be either licensed to industrial partners or a QF-private partnership start-up, publication of a high impact manuscript, certainly aid in the local infectious disease research capacity building in Qatar through the establishment of the enabling biosensor technology and personnel training as well as Qatar’s responsiveness to future coronavirus threats.

Hamad Bin Khalifa University (HBKU)