Convenient Retinoid X Receptor Binding Assay Based on Fluorescence Change of the Antagonist NEt-C343

Retinoid
X receptor (RXR) modulators (rexinoids) are considered
to have therapeutic potential for multiple diseases, such as Alzheimer’s
disease and Parkinson’s disease. To overcome various disadvantages
of prior screening methods, we previously developed an RXR binding
assay using a fluorescent RXR ligand, CU-6PMN (4). However,
this ligand binds not only at the ligand-binding domain (LBD) but
also at the dimer–dimer interface of hRXRα. Here, we
present a new fluorescent RXR antagonist 6-[N-ethyl-N-(5-isobutoxy-4-isopropyl-2-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano­[2,3-f]­pyrido­[3,2,1-ij]­quinoline-10-carboxamido)­phenyl)­amino]­nicotinic
acid (NEt-C343, 7), which emits strong fluorescence only
when bound to the RXR-LBD. It allows us to perform a rapid, simple,
and nonhazardous binding assay that does not require bound/free separation
and uses a standard plate reader. The obtained Ki
values of known compounds were correlated with the Ki values obtained using the standard [3H]­9cis-retinoic acid assay. This assay should be useful for drug discovery
as well as for research on endocrine disruptors, functional foods,
and natural products.