A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication and pathogenesis in vivo (preprint)

Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the therapeutic potential of Mac1 inhibition, we generated recombinant viruses and replicons encoding catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced activity by ~10-fold, mutations to aspartic acid (N40D) reduced the catalytic activity by ~100-fold relative to wildtype activity. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, N40D replicated at >1000-fold lower levels while inducing a robust interferon response, and all infected animals survived infection with the mutant, but not the wildtype virus. Our data validate SARS-CoV-2 NSP3 Mac1 domain as a critical viral pathogenesis factor and a reasonable target to develop antivirals, while emphasizing the importance of amino acid identity in viral mutagenesis studies and underscoring the limitations of solely relying on in vitro viral replication studies for target validation.

Structure-based inhibitor optimization for the Nsp3 Macrodomain of SARS-CoV-2 (preprint)

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, few have been described. Here, we report the structure-based development of several chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high resolution X-ray protein crystallography, and binding evaluation with in-solution assays. Potent scaffolds were designed with in silico linkage of previously obtained fragment hits and ultra-large library docking screens of more than 450 million molecules. In total, 160 hits comprising 119 different scaffolds were discovered and 152 Mac1-ligand complex crystal structures were determined, typically to 1 [A] resolution or better. The structure-activity-relationships emerging from this study may template future drug development against Mac1.

The mechanisms of catalysis and ligand binding for the SARS-CoV-2 NSP3 macrodomain from neutron and X-ray diffraction at room temperature (preprint)

The NSP3 macrodomain of SARS CoV 2 (Mac1) removes ADP-ribosylation post-translational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the COVID-19 pandemic. Here, we determined neutron and X-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose-bound states. We characterize extensive solvation in the Mac1 active site, and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a re-evaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.

Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking (preprint)

The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.