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, 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-2oxo-6-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-2H-chromene-3-carboxylic acid (10, CU-6PMN) as a new fluorescent RXR agonist and developed a screening system of rexinoids using 10. Compound 10 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 10 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.

Synthesis of a Coumarin-Based PPARγ Fluorescence Probe for Competitive Binding Assay

“…Recently, a coumarin-containing nuclear receptor RXR agonist was reported. The authors demonstrated its utility in a competitive binding assay for several RXR ligands [52]. Coumarins, thus, appear suitable as nuclear receptor ligands.…”

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

“…1 H-NMR (400 MHz, CDCl 3 ): δ 8.88 (1H, d, J = 2.2 Hz), 8.03 (1H, s), 7.96 (1H, dd, J = 9.1, 2.2 Hz), 6.68 (1H, s), 6.23 (1H, d, J = 9.1 Hz), 3.78 (2H, d, J = 6.3 Hz), 3.36 (1H, sep, J = 6.9 Hz), 2.17 (1H, sep, J = 6.6 Hz), 1.32−1.26 (11H, m), 1.07 (6H, d, J = 6.6 Hz). 13 (14). To a solution of 13 (476 mg, 1.20 mmol) in anhydrous DMF (8 mL) were added chloromethyl methyl ether (0.1 mL, 1.3 mmol) and triethylamine (0.18 mL, 1.3 mmol) under an Ar atmosphere.…”

“…22 IC 50 Determination of Test Compounds Using 7. The competitive binding assay using 7 was performed as described 14 with some modifications (see Figure S5). Black 384-well plates purchased from Griner were used (4 wells per sample).…”

“…On the other hand, the reporter gene assay requires 3−4 days, the TR-FRET assay requires special equipment, and the [ 3 H]9cis-RA assay also requires special precautions. 14 Thus, there is a great need for a screening method that can detect rexinoids quickly using a standard plate reader, as is currently being done for RAR. 15 We have already applied a fluorescent rexinoid (4, CU-6PMN) for this purpose, 14 developing a rapid and nonhazardous assay method without the need for bound-free (B/F) separation.…”

“…14 Thus, there is a great need for a screening method that can detect rexinoids quickly using a standard plate reader, as is currently being done for RAR. 15 We have already applied a fluorescent rexinoid (4, CU-6PMN) for this purpose, 14 developing a rapid and nonhazardous assay method without the need for bound-free (B/F) separation. However, this suffers from the drawback that 4 binds not only at the ligand-binding domain (LBD) but also at the dimer− dimer interface of hRXRα, and we found that the interaction with the dimer−dimer interface induced a conformational change of the LBD, leading to a 1.5-fold increase of [ 3 H]9cis-RA binding.…”