The estrogen receptor (ER) is a hormone-regulated transcription factor. In order to explore at a fundamental level the geometric and topological features of whether a bivalent ligand could bind to the ER dimer, crystal structures of ER dimers were used to rationally design nonsteroidal bivalent
estrogen ligands for the ER. Guided by this structure-based ligand design, we prepared by modular synthetic routes three series of bivalent ligands tethered by flexible spacers of varying lengths, and we evaluated their ER-binding affinities for the two ER subtypes and their biological activities in different cell lines. Bivalent ligands based on the antagonist hydroxytamoxifen (OHT) were well suited for intensive study. Binding affinities of the OHT-based bivalent ligands were related to the length of the spacer in a distinctive fashion, reaching two maximum values at 14 and 29 A in both ER subtypes. These results, supplemented by molecular modeling of bivalent OHT ligand interactions with dimeric ERs, demonstrate that the bivalent concept can operate in determining ER-ligand binding affinity, and they suggest that there are two distinct modes for the binding of bivalent estrogen ligands to the ER dimers, an intermolecular mode as well as an intramolecular mode. Our insights, particularly the possibility of intramolecular bivalent binding on a single ER monomer, may provide an alternative strategy to prepare more selective and active ER antagonists for endocrine therapy of breast cancer.