SARS-CoV-2 Protease Inhibition and Biosensor Development

Infections caused by a novel strain of coronaviruses, Severe Acute Respiratory Syndrome Coronavirus – 2 (SARS-CoV-2), causing COVID-19 has already caused more than 5 million deaths and greater than 400 million infections worldwide. COVID-19 involves severe respiratory ailments and significant endothelial injuries and multi-organ failure. Although the development of vaccines against the disease does help in reducing the disease burden, the emergence of variants with increased infectivity and/or disease severity has made it critical for development of the alternative therapeutic approaches. In this regard, the discovery of antiviral drugs to block the activity of viral proteins has been proposed. One such strategy is the inhibition of one of the viral proteases, the main protease (M^pro), because of its indispensable role in viral polyprotein processing necessary for viral replicase complex formation and other processes. This report describes the screening for SARS-CoV-2 M^pro inhibitors using computational and biophysical assays and the development of a biosensor for monitoring the activity of SARS-CoV-2 proteases using the Bioluminescence Resonance Energy transfer (BRET) technology. For drug repurposing, a phosphodiesterase 5 (PDE5) inhibitor, tadalafil, was tested for its ability to inhibit M^pro activity and one M^pro allosteric site was determined. A BRET-based, genetically encoded M^pro and PL^pro (dual protease) biosensor was engineered containing cognate M^pro and PL^pro cleavage sites with the aim to aid SARS-CoV-2 protease inhibitors. The findings suggested that tadalafil failed to inhibit Mpro while in silico screenings using drug libraries revealed some potential allosteric inhibitors of M^pro. In vitro and live cell experiments revealed that a dual sensor construct containing the mNeonGreen and mScarlet fluorescent proteins as acceptors can report the cleavage of M^pro and PL^pro with high sensitivity and specificity. Overall, results reported here will be useful in the development of COVID-19 therapy through targeting SARS-CoV-2 proteases.