Hit Expansion of a Noncovalent SARS-CoV-2 Main Protease Inhibitor

[Image: see text] Inhibition of the SARS-CoV-2 main protease (M(pro)) is a major focus of drug discovery efforts against COVID-19. Here we report a hit expansion of non-covalent inhibitors of M(pro). Starting from a recently discovered scaffold (The COVID Moonshot Consortium. Open Science Discovery of Oral Non-Covalent SARS-CoV-2 Main Protease Inhibitor Therapeutics. bioRxiv 2020.10.29.339317) represented by an isoquinoline series, we searched a database of over a billion compounds using a cheminformatics molecular fingerprinting approach. We identified and tested 48 compounds in enzyme inhibition assays, of which 21 exhibited inhibitory activity above 50% at 20 μM. Among these, four compounds with IC(50) values around 1 μM were found. Interestingly, despite the large search space, the isoquinolone motif was conserved in each of these four strongest binders. Room-temperature X-ray structures of co-crystallized protein–inhibitor complexes were determined up to 1.9 Å resolution for two of these compounds as well as one of the stronger inhibitors in the original isoquinoline series, revealing essential interactions with the binding site and water molecules. Molecular dynamics simulations and quantum chemical calculations further elucidate the binding interactions as well as electrostatic effects on ligand binding. The results help explain the strength of this new non-covalent scaffold for M(pro) inhibition and inform lead optimization efforts for this series, while demonstrating the effectiveness of a high-throughput computational approach to expanding a pharmacophore library.