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.