Iterative computational design and crystallographic screening identifies potent inhibitors targeting the Nsp3 macrodomain of SARS-CoV-2

Significance

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. There are currently no well-validated inhibitors for this protein. Here, we discovered and optimized several different classes of ligands that bind to Mac1 with low- to sub-micromolar affinity. Ligands were designed by linking together small-molecule fragments and by ultra-large library docking of 450 million molecules. Overall, we discovered 160 ligands in 119 different scaffolds, and 152 Mac1-ligand complex crystal structures were determined. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated protein dynamics within the active site, and key structural information that will guide future drug development for this important antiviral target.

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 153 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 conformational changes within the active site, and key inhibitor motifs that will template future drug development against Mac1.