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

Abstract

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 noncovalent smallmolecule 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 pro inhibitor with an inhibition constant (K i ) of 2.9 μM (95% CI 2.2, 4.0). Furthermore, using room-temperature X-ray crystallography, we show that MCULE-5948770040 binds to a cleft in the primary binding site of M pro forming stable hydrogen bond and hydrophobic interactions. We then used multiple μs-time scale 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 pro and offers a springboard for further therapeutic design.

Neoechinulin A as a Promising SARS-CoV-2 Mpro Inhibitor: In Vitro and In Silico Study Showing the Ability of Simulations in Discerning Active from Inactive Enzyme Inhibitors

“…Regarding neoechinulin A ( 1 ) and echinulin ( 2 ), they both achieved stable binding inside the M pro active site with average RMSDs of 2.16 Å and 2.21 Å, respectively. The dynamics of these two related structures (i.e., compounds 1 and 2 ) over the course of the simulation were also convergent to that of the reported co-crystallized ligand (RMSD ~2 Å) [ 25 ] ( Figure 4 B).…”

“…As shown in Figure 7 , the unbinding of eurocristatine ( 3 ) (i.e., the inactive compound) was achieved by a force not higher than ~108 pN, while active compounds neoechinulin A ( 1 ) and echinulin ( 2 ) along with the co-crystalized inhibitor YD1 required an unbinding force ranging from 399 pN to 909 pN. The force profile plotted in Figure 7 clearly indicated that neoechinulin A ( 1 ) (IC 50 = 0.47 µM) has the highest binding stability inside the M pro active site (i.e., needed unbinding force ~909 pN), followed by the co-crystalized inhibitor YD1 ( K i = 2.9 µM) [ 25 ] (i.e., needed unbinding force ~685 pN) followed by echinulin ( 2 ) (IC 50 = 3.9 µM) (i.e., needed unbinding force ~399 pN). These differences in the unbinding forces between neoechinulin A ( 1 ), echinulin ( 2 ), and the co-crystalized inhibitor YD1 may be attributed to the degree of ligand–protein H-bonds’ stability during the unbinding process in addition to the degree of hydrophobic interactions with hydrophobic residues inside the enzyme active site, particularly with HIS-41, where neoechinulin A ( 1 ) was able to establish a significant stable hydrophobic interaction with this hydrophobic residue ( Figure 6 A).…”

Covalent narlaprevir- and boceprevir-derived hybrid inhibitors of SARS-CoV-2 main protease

“…For example, several research groups have focused their attention on structure-based drug design of noncovalent M pro inhibitors starting from the anti-epileptic drug perampanel 28 , 29 , or from ML188 and ML300 previously designed to inhibit SARS-CoV-1 M pro 30 – 33 . We have recently performed a structure-activity relationship study 34 on a noncovalent compound newly discovered by high-throughput virtual screening 35 . Others have designed and evaluated a series of peptidomimetic reversible or irreversible covalent inhibitors 36 – 38 , or nonpeptidic small molecules with a novel chemical scaffold 39 .…”

Large library docking for novel SARS-CoV-2 main protease non-covalent and covalent inhibitors

“…21,284,498 compounds scored lower than −35 kcal/mol and the poses of top scoring 5,004,192 compounds were extracted. 214,580 compounds formed favorable interactions with key residues such as His163, Glu166 and the P2 subpocket, 181,866 of which obtained ECFP4-based TC coefficients of less than 0.35 to the 1,716 known SARS-CoV and SARS-CoV-2 MPro inhibitors reported in the literature 2–4, 8, 9, 11, 12, 14, 17, 20, 26, 28, 59–75 . Finally, roughly 9,000 top-ranking compounds were visually inspected, and 167 molecules were ordered from Enamine Ltd., 146 of which (87.4%) were successfully synthesized.…”