Competitive Binding Assay with an Umbelliferone-Based Fluorescent Rexinoid for Retinoid X Receptor Ligand Screening

Abstract

Ligands for retinoid X receptors (RXRs), "rexinoids", are attracting interest as candidates for therapy of type 2 diabetes and Alzheimer's and Parkinson's diseases. However, current screening methods for rexinoids are slow and require special apparatus or facilities. Here, we created 7-hydroxy-2-oxo-6-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-2<i>H</i>-chromene-3-carboxylic acid (<b>10</b>, CU-6PMN) as a new fluorescent RXR agonist and developed a screening system of rexinoids using <b>10</b>. Compound <b>10</b> was designed based on the fact that umbelliferone emits strong fluorescence in a hydrophilic environment, but the fluorescence intensity decreases in hydrophobic environments such as the interior of proteins. The developed assay using <b>10</b> enabled screening of rexinoids to be performed easily within a few hours by monitoring changes of fluorescence intensity with widely available fluorescence microplate readers, without the need for processes such as filtration.