High Throughput Virtual Screening and Validation of a SARS-CoV-2 Main Protease Non-Covalent Inhibitor

Despite the recent availability of vaccines against the acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the search for inhibitory therapeutic agents has assumed importance especially in the context of emerging new viral variants. In this paper, we describe the discovery of a novel non-covalent small-molecule inhibitor, MCULE-5948770040, that binds to and inhibits the SARS-Cov-2 main protease (M^pro) by employing a scalable high throughput virtual screening (HTVS) framework and a targeted compound library of over 6.5 million molecules that could be readily ordered and purchased. Our HTVS framework leverages the U.S. supercomputing infrastructure achieving nearly 91% resource utilization and nearly 126 million docking calculations per hour. Downstream biochemical assays validate this M^proinhibitor with an inhibition constant (K_i) of 2.9µM [95% CI 2.2, 4.0]. Further, using room-temperature X-ray crystallography, we show that MCULE-5948770040 binds to a cleft in the primary binding site of M^proforming stable hydrogen bond and hydrophobic interactions. We then used multipleµs-timescale molecular dynamics (MD) simulations, and machine learning (ML) techniques to elucidate how the bound ligand alters the conformational states accessed by M^pro, involving motions both proximal and distal to the binding site. Together, our results demonstrate how MCULE-5948770040 inhibits M^proand offers a springboard for further therapeutic design.