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 tracerTAMRA-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 validatedZ8539(IC506.4 μM) andGS441524(IC5015.2 μM), two literature-reported small-molecule inhibitors
of SARS-CoV-2 Macro1. Our data suggest thatGS441524is
highly selective for SARS-CoV-2 Macro1 over other human and viral
macrodomains. Furthermore, using this assay, we identifiedpNP-ADPr(ADP-ribosylatedp-nitrophenol, IC50370 nM) andTFMU-ADPr(ADP-ribosylated trifluoromethyl
umbelliferone, IC50590 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.