High-throughput Activity Assay for Screening Inhibitors of the SARS-CoV-2 Mac1 Macrodomain

Dasovich, Morgan; Zhuo, Junlin; Goodman, Jack A.; Thomas, Ajit; McPherson, Robert Lyle; Jayabalan, Aravinth Kumar; Busa, Veronica F.; Cheng, Shang-Jung; Murphy, Brennan A.; Redinger, Karli R.; Tsukamoto, Takashi; Slusher, Barbara; Bosch, Jürgen; Wei, Huijun; Leung, Anthony K. L.

Macrodomains are a class of conserved ADP-ribosylhydrolases expressed by viruses of pandemic concern, including coronaviruses and alphaviruses. Viral macrodomains are critical for replication and virus-induced pathogenesis; therefore, these enzymes are a promising target for antiviral therapy. However, no potent or selective viral macrodomain inhibitors currently exist, in part due to the lack of a high-throughput assay for this class of enzymes. Here, we developed a high-throughput ADP-ribosylhydrolase assay using the SARS-CoV-2 macrodomain Mac1. We performed a pilot screen which identified dasatinib and dihydralazine as ADP-ribosylhydrolase inhibitors. Importantly, dasatinib does not inhibit MacroD2, the closest Mac1 homolog in humans. Our study demonstrates the feasibility of identifying selective inhibitors based on ADP-ribosylhydrolase activity, paving the way for screening large compound libraries to identify improved macrodomain inhibitors and explore their potential as antiviral therapies for SARS-CoV-2 and future viral threats.