Increased Molecular Flexibility Widens the Gap between K i and K d values in Screening for Retinoid X Receptor Modulators.

Screening for small-molecule modulators targeting a particular receptor is frequently based on measurement of K d, i.e., the binding constant between the receptor and the compound of interest. However, K d values also reflect binding at receptor protein sites other than the modulatory site. We designed derivatives of retinoid X receptor (RXR) antagonist CBTF-EE (1) with modifications that altered their conformational flexibility. Compounds 6a,b and 7a,b showed quite similar K d values, but 7a,b exhibited 10-fold higher K i values than those of 6a,b. Further, 6a,b showed potent RXR-antagonistic activity, while 7a,b were inactive. These results suggest that increased conformational flexibility promotes binding at nontarget receptor sites. In this situation, conventional determination of K d is less effective for screening purposes than the determination of K i using a ligand that binds specifically to the site regulating transcriptional activity. Thus, the use of K i values for orthosteric ligands may increase the hit rate in screening active regulatory molecules.

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.