Increased Molecular Flexibility Widens the Gap between K_iand K_dvalues in Screening for Retinoid X Receptor Modulators

Screening for small-molecule modulators targeting a particular receptor is frequently based on measurement of Kd, i.e., the binding constant between the receptor and the compound of interest. However, Kd 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 Kd values, but 7a,b exhibited 10-fold higher Ki 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 Kd is less effective for screening purposes than the determination of Ki using a ligand that binds specifically to the site regulating transcriptional activity. Thus, the use of Ki values for orthosteric ligands may increase the hit rate in screening active regulatory molecules.