SARS-CoV-2 nsP3 Macrodomain Characterization and Inhibitor Screening

Viral macrodomains have mono-ADP-ribose hydrolytic activity, which can reverse the cellular post-translational modification mediated by different poly(ADP-ribose) polymerases (PARPs) that can attach a single ADP-ribose molecule or poly(ADP-ribose) chain to its target substrate. The viral macrodomain enzymatic activity for the removal of mono-ADP-ribose from protein is critical for viral replication, contributes to virulence and helps to combat host innate immunity. SARS-CoV-2, the causative agent for COVID-19, has an ADP-ribosylhydrolase macrodomain (Mac1) within non-structural protein 3 (nsP3), which is a promising therapeutic target for COVID-19 drug development. By deploying a high-throughput luciferase assay previously developed in our lab, we characterized the SARS-CoV-2 nsP3 macrodomain and screened its inhibitors. In this thesis, we show that the SARS-CoV-2 macrodomain was capable of hydrolyzing mono-ADP-ribose from a model ADP-ribosylated substrate with a Km of 178.48 ± 53.24 µM and kcat of 2.07 ± 0.64 sec-1 at 0.86 nM. By screening the LOPAC and SELLECK FDA small-molecule libraries, we identified dasatinib as a selective inhibitor that can suppress the enzymatic activity of the SARS-CoV-2 nsP3 macrodomain with an IC50 of 59.21 ± 9.54 µM without an inhibitory effect on the human counterpart MacroD2. Compared with other published compounds that bind to the ADP-ribose hydrolyzing pocket at 100 µM, we found dasatinib is the most potent compound that has been discovered and has a selective inhibitory pattern