Comparative analyses of mechanistic differences among antiestrogens.

Antiestrogens such as tamoxifen are one of the most effective methods of treating estrogen receptor (ERalpha) positive breast cancers; however, the effectiveness of this therapy is limited by the almost universal development of resistance to the drug. If antiestrogens are recognized differently by the cell as it has been suggested, then in disease conditions where tamoxifen fails to function effectively, a mechanistically different antiestrogen might yield successful results. Although many antiestrogens have been developed, a direct comparison of their mechanisms of action is lacking, thus limiting their utility. Therefore, to determine if there are mechanistic differences among available antiestrogens, we have carried out a comprehensive analysis of the molecular mechanisms of action of 4-hydroxy-tamoxifen (40HT), idoxifene, raloxifene, GW7604, and ICI 182,780. Using a novel set of peptides that recognize different surfaces on ERalpha, we have found that following binding to ERalpha, each ligand induces a distinct ERalpha-ligand conformation. Furthermore, transcriptional assays indicate that each ERalpha-ligand complex is recognized distinctly by the transcription machinery, and consequently, antiestrogens vary in their ability to inhibit estradiol- and 40HT-mediated activities. Relative binding assays have shown that the affinity of these ligands for ERalpha is not always representative of their inhibitory activity. Using this assay, we have also shown that the pharmacology of each antiestrogen is influenced differently by hormone binding proteins. Furthermore, GW7604, like ICI 182,780, but unlike the other antiestrogens evaluated, decreases the stability of the receptor. Overall, our results indicate that there are clear mechanistic distinctions among each of the antiestrogens studied. However, GW7604 and ICI 182,780 differ more significantly from tamoxifen than idoxifene and raloxifene. These data, which reveal differences among antiestrogens, should assist in the selection of compounds for the clinical regulation of ERalpha function.