A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain

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.

Iso-ADP-Ribose Fluorescence Polarization Probe for the Screening of RNF146 WWE Domain Inhibitors

“…The IC 50 of isoADPr (Figure 3B) was calculated from the FP binding curve to be 623 nM, which is comparable to the reported K d value of 370 nM. In our previous study on FP assay for macrodomains, 16 we found the ADPr-N 3 precursor is more potent than ADPr for SARS-CoV-2 Macro1. Here, we also tested the 3′-azido-isoADPr precursor and found it is 2fold more potent than isoADPr for RNF146 WWE.…”

“…It should be noted that significant amounts of 3′-azido-AMP and monophosphorylated byproducts were also formed, and normal-phase purification in the water–ammonia–isopropanol system was conducted to achieve good separation. Finally, click reaction of 9 with the previously reported TAMRA-alkyne furnished the target molecule TAMRA-isoADPr ( 10 ).…”

“…The procedure is adapted from our previous work . Briefly, the RNF146 WWE protein (200 nM, 2X) was mixed with TAMRA-isoADPr (40 nM, 2X) in the assay buffer to give the protein–tracer mixture.…”

GS-441524-Diphosphate-Ribose Derivatives as Nanomolar Binders and Fluorescence Polarization Tracers for SARS-CoV-2 and Other Viral Macrodomains

“…Procedures and assay conditions were previously described . mP values were calculated using the equation below: mP = F / / − G * F ⊥ F / / + G * F ⊥ × 1000 where F // and F ⊥ are the parallel and perpendicular fluorescence intensities, respectively, and G is the grating factor of the instrument, which was calibrated so that 20 nM 5-TAMRA in water has an mP shift of 50.…”

“…The general procedure and assay conditions were same as previously described . The final protein and tracer concentrations for different protein–tracer pairs used in this study are listed in Table .…”