A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain
Ananya Anmangandla, Sadhan Jana, Kewen Peng, Shamar D. Wallace, Saket R. Bagde, Bryon Drown, Jiashu Xu, Paul J. Hergenrother, J. Christopher Fromme, Hening Lin
Abstract
High Resolution Image Download MS PowerPoint Slide Viral macrodomains, which can bind to and/or hydrolyze adenine diphosphate ribose (ADP-ribose or ADPr) from proteins, have been suggested to counteract host immune response and be viable targets for the development of antiviral drugs. Therefore, developing high-throughput screening (HTS) techniques for macrodomain inhibitors is of great interest. Herein, using a novel tracer TAMRA-ADPr, an ADP-ribose compound conjugated with tetramethylrhodamine, we developed a robust fluorescence polarization assay for various viral and human macrodomains including SARS-CoV-2 Macro1, VEEV Macro, CHIKV Macro, human MacroD1, MacroD2, and PARP9 Macro2. Using this assay, we validated Z8539 (IC 50 6.4 μM) and GS441524 (IC 50 15.2 μM), two literature-reported small-molecule inhibitors of SARS-CoV-2 Macro1. Our data suggest that GS441524 is highly selective for SARS-CoV-2 Macro1 over other human and viral macrodomains. Furthermore, using this assay, we identified pNP-ADPr (ADP-ribosylated p -nitrophenol, IC 50 370 nM) and TFMU-ADPr (ADP-ribosylated trifluoromethyl umbelliferone, IC 50 590 nM) as the most potent SARS-CoV-2 Macro1 binders reported to date. An X-ray crystal structure of SARS-CoV-2 Macro1 in complex with TFMU-ADPr revealed how the TFMU moiety contributes to the binding affinity. Our data demonstrate that this fluorescence polarization assay is a useful addition to the HTS methods for the identification of macrodomain inhibitors.