Abstract
The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 152 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated protein dynamics within the active site, and key inhibitor motifs that will template future drug development against Mac1.
Significance Statement SARS-CoV-2 encodes a viral macrodomain protein (Mac1) that hydrolyzes ribo-adenylate marks on viral proteins, disrupting the innate immune response to the virus. Catalytic mutations in the enzyme make the related SARS-1 virus less pathogenic and non-lethal in animals, suggesting that Mac1 will be a good antiviral target. However, no potent inhibitors of this protein class have been described, and pharmacologically the enzyme remains an orphan. Here, we computationally designed potent inhibitors of Mac1, determining 150 inhibitor-enzyme structures to ultra-high resolution by crystallography. In silico fragment linking and molecular docking of > 450 million virtual compounds led to inhibitors with submicromolar activity. These molecules may template future drug discovery efforts against this crucial but understudied viral target.
Competing Interest Statement
A.A. is a co-founder of Tango Therapeutics, Azkarra Therapeutics, and Ovibio Corporation; a consultant for SPARC, Bluestar, ProLynx, Earli, Cura, GenVivo, and GSK; a member of the SAB of Genentech, GLAdiator, Circle, and Cambridge Science Corporation; receives grant/research support from SPARC and AstraZeneca; and holds patents on the use of PARP inhibitors held jointly with AstraZeneca, which he has benefited financially (and may do so in the future). J.E.G is consultant for Protego BioPharma and Contour Therapeutics as well as founder of Kaizen Therapeutics. B.K.S. is co-founder of BlueDolphin, LLC, a molecular docking contract research organization, Epiodyne, and Deep Apple Therapeutics, Inc., both drug discovery companies, has recently consulted for Umbra, Abbvie, and Dice Therapeutics, and is on the SAB of Schrodinger. J.J.I. co-founded Deep Apple Therapeutics, Inc., and BlueDolphin, LLC. J.S.F. is a consultant for, has equity in, and receives research support from Relay Therapeutics. I.A. is a consultant for Dark Blue Therapeutics.
Footnotes
Competing Interest Statement: A.A. is a co-founder of Tango Therapeutics, Azkarra Therapeutics, and Ovibio Corporation; a consultant for SPARC, Bluestar, ProLynx, Earli, Cura, GenVivo, and GSK; a member of the SAB of Genentech, GLAdiator, Circle, and Cambridge Science Corporation; receives grant/research support from SPARC and AstraZeneca; and holds patents on the use of PARP inhibitors held jointly with AstraZeneca, which he has benefited financially (and may do so in the future). J.E.G is consultant for Protego BioPharma and Contour Therapeutics as well as founder of Kaizen Therapeutics. B.K.S. is co-founder of BlueDolphin, LLC, a molecular docking contract research organization, Epiodyne, and Deep Apple Therapeutics, Inc., both drug discovery companies, has recently consulted for Umbra, Abbvie, and Dice Therapeutics, and is on the SAB of Schrodinger. J.J.I. co-founded Deep Apple Therapeutics, Inc., and BlueDolphin, LLC. J.S.F. is a consultant for, has equity in, and receives research support from Relay Therapeutics. I.A. is a consultant for Dark Blue Therapeutics.
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