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

Inhibition of the SARS-CoV-2 main
protease (Mpro) is
a major focus of drug discovery efforts against COVID-19. Here we
report a hit expansion of non-covalent inhibitors of Mpro. 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 IC50 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 Mpro inhibition and inform lead optimization efforts for this series,
while demonstrating the effectiveness of a high-throughput computational
approach to expanding a pharmacophore library.