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

Poly-ADP-ribosylation
is an important protein post-translational
modification with diverse biological consequences. After binding poly-ADP-ribose
on axis inhibition protein 1 (AXIN1) through its WWE domain, RING
finger protein 146 (RNF146) can ubiquitinate AXIN1 and promote its
proteasomal degradation and thus the oncogenic WNT signaling. Therefore,
inhibiting the RNF146 WWE domain is a potential antitumor strategy.
However, due to a lack of suitable screening methods, no inhibitors
for this domain have been reported. Here, we developed a fluorescence
polarization (FP)-based competition assay for the screening of RNF146
WWE inhibitors. This assay relies on a fluorescently tagged iso-ADP-ribose
tracer compound, TAMRA-isoADPr. We report the design
and synthesis of this tracer compound and show that it is a high-affinity
tracer for the RNF146 WWE domain. This provides a convenient assay
and will facilitate the development of small-molecule inhibitors for
the RNF146 WWE domain.