High Throughput Virtual Screening and Validation of a SARS-CoV-2 Main Protease Non-Covalent 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 non-covalent small-molecule inhibitor, MCULE-5948770040, that binds to and inhibits the SARS-Cov-2 main protease (Mpro) 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 Mpro inhibitor with an inhibition constant (Ki) of 2.9 uM [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 Mpro forming stable hydrogen bond and hydrophobic interactions. We then used multiple μs-timescale molecular dynamics (MD) simulations, and machine learning (ML) techniques to elucidate how the bound ligand alters the conformational states accessed by Mpro, involving motions both proximal and distal to the binding site. Together, our results demonstrate how MCULE-5948770040 inhibits Mpro and offers a springboard for further therapeutic design.

Structural, Electronic, and Electrostatic Determinants for Inhibitor Binding to Subsites S1 and S2 in SARS-CoV-2 Main Protease

“…The aromatic dichlorobenzene P2 group orchestrates itself into the hydrophobic S2 subsite by displacing Met49 and S2 helix (residues 46–52) and rearranging His41 and Gln189 to create π–π stacking interactions. 40 P1 and P2 groups of 1 are connected by a saturated heterocyclic piperazine–amide linker that includes a carbonyl aimed toward the oxyanion hole and a potentially ionizable tertiary amine preceding P2. Analysis of the nuclear density demonstrated that the latter amine nitrogen is protonated with the D atom directed away from His41 and into the bulk solvent.…”

“…The significant accuracy of K i measurements indicates that the potencies of HL-3-68 and Mcule-CSR-494190-S1 are 3-fold and 2-fold better than the K i of 2.9 μM of the previously reported compound 1 . 40 Of note, none of the compounds demonstrated antiviral activities against SARS-CoV-2 in cell-based assays ( Figure S2 ).…”

“…MD simulations of M pro (ligand-free), the M pro -HL-3-68, and M pro - 1 complexes reveal a consistent picture of how the HL-3-68 ligand is more stable within the primary binding site of M pro compared to compound 1 reported in our previous study. 40 We quantified the conformational changes using the root-mean-square deviation (RMSD) analysis across each trajectory ( Figures 4 and S6 ). The distribution of the RMSDs determined from at least three replicates of the simulations (shown in Figure 4 A as a histogram of all conformers from MD trajectories) further reveals that HL-3-68 stabilizes the binding pocket of M pro more than compound 1 .…”

“…For each system, we did observe slightly different fluctuations in chain A and chain B, which agrees with the previous simulation results. 13 , 40 Across the three simulation systems, the M pro -HL-3-68 complex had the lowest average RMSD from both chains when compared with M pro and the M pro - 1 system ( Figure S6 ). Per-residue fluctuations were characterized by calculating the root-mean-square fluctuations (RMSF) of the Cα-atoms using the average conformation of each trajectory as the reference structure.…”

“… 36 Here, we report a structure–activity relationship (SAR) study performed on a competitive noncovalent inhibitor Mcule-5948770040 (compound 1 ) of a novel scaffold that we recently discovered through a large-scale virtual screening and validated using in vitro enzyme inhibition assays and X-ray crystallography. 40 The aim of our SAR study was to chemically modify compound 1 that binds across the M pro catalytic site in the substrate-binding subsites S1 and S2 to reveal structural, electronic, and electrostatic determinants of these ligand-binding sites. Compound 1 has a general architecture of P1–linker–P2 ( Scheme 1 ).…”