Fluorescent retinoid X receptor ligands for fluorescence polarization assay

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

“…Compared to CRBPII, less space is available in the ligand-binding site of RXR 25 , and therefore we considered that the acidic domain of RXR agonists would have to be converted to a fluorophore.…”

“…Pohl et al reported fluorescent retinoids targeting cellular retinoid binding protein II (CRABPII). 26,27 Compared to CRBPII, less space is available in the ligand-binding site of RXR, 25 and therefore, we considered that the acidic domain of RXR agonists will have to be converted to a fluorophore. Umbelliferone (7) is a fluorophore that can be detected with a widely available fluorescent filter set (Ex 360 nm/Em 465 nm).…”

“…2-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)benzene-1,3-diol (25). To a solution of 24 (108 mg, 0.27 mmol) was added 4 M HCl/EtOAc (2 mL).…”

“…We previously reported the fluorescent RXR agonist 5 (Figure ), which has a fluorescent carbostyril structure 6 in place of the 1,1,4,4-tetramethyltetralin structure of 3 . Compound 5 has been used in a fluorescence polarization-based assay targeting RXRs . However, the RXR agonist activity of 5 was much weaker than that of 3 (the EC 50 values were 1 μM and 20 nM, respectively), and its relatively poor binding ability to RXRs was a significant drawback.…”

“…Compound 5 has been used in a fluorescence polarization-based assay targeting RXRs. 25 However, the RXR agonist activity of 5 was much weaker than that of 3 (the EC 50 values were 1 μM and 20 nM, respectively), and its relatively poor binding ability to RXRs was a significant drawback. Therefore, in this study, we aimed to create a new fluorescent RXR agonist that would be suitable for highly sensitive RXR ligand screening using a standard fluorescence plate reader.…”