A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain
Citations Over TimeTop 10% of 2023 papers
Abstract
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 (IC50 6.4 μM) and GS441524 (IC50 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, IC50 370 nM) and TFMU-ADPr (ADP-ribosylated trifluoromethyl umbelliferone, IC50 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.
Related Papers
- → Fluorescence Polarization and Anisotropy in High Throughput Screening: Perspectives and Primer(2000)297 cited
- → Displacement of protein-bound aptamers with small molecules screened by fluorescence polarization(2008)78 cited
- → Ligand Discovery: High-Throughput Binding: Fluorescence Polarization (Anisotropy)(2021)8 cited
- → An Ultra-High-Throughput Screen for Catalytic Inhibitors of Serine/Threonine Protein Phosphatases Types 1 and 5 (PP1C and PP5C)(2016)8 cited
- → A high-throughput screening assay for pyruvate carboxylase(2018)7 cited