Increased Molecular Flexibility Widens the Gap between Ki and Kd 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.