BIVALENT LIGANDS AS PROBES OF ESTROGEN-RECEPTOR ACTION

The estrogen receptor (ER) is a hormone-regulated transcription factor which is thought to bind to specific DNA sequences as a homodimer. In order to better understand structural requirements for dimerization a nd its functional role in ER action, we synthesized a series of bivale nt ligands based on the non-steroidal estrogen hexestrol. These molecu lar probes join two hexestrol molecules of the erythro (E, active) con figuration with either 4 or 8 carbon linkers (designated E-4-E and E-8 -E series, respectively), or with longer linkers comprised of ethylene glycol units (E-eg-E series). Several other bi- and monovalent contro l compounds were prepared. The bivalent ligands bind to ER with a rela tive affinity 1-7% that of estradiol. While most of the ligands demons trated normal monophasic displacement curves in competitive binding as says with [H-3]estradiol, uncharacteristic biphasic competitive bindin g curves were seen for some of the ligands, indicating possible struct ure-specific, negative site-site interaction. In ER-deficient Chinese hamster ovary (CHO) cells transfected with an expression vector encodi ng ER, one series of bivalent ligands (E-4-E) had little stimulatory a ctivity and inhibited transcription stimulated by hexestrol, as determ ined by a transient transfection assay using an estrogen-responsive re porter gene construct [(ERE)(2)-TATA-CAT, containing two estrogen resp onse elements linked to a TATA promoter and the chloramphenicol acetyl transferase reporter gene]. Monovalent or control bivalent ligands fa iled to antagonize hexestrol-stimulated activity and were as fully act ive as hexestrol itself. Studies performed in MCF-7 human breast cance r cells, which contain endogenous ER, yielded similar bioactivity prof iles for the E-4-E bivalent inhibitory ligands, showing them to be eff ective estrogen antagonists, when using either induction of progestero ne receptor or (ERE)(2)-TATA-CAT transcriptional activation as the end point. The E-8-E ligand, however, acted as a partial agonist/antagonis t of ERE-reporter gene transactivation and a full agonist of progester one receptor induction in MCF-7 cells, thus showing cell- and response -specific differences in the effects of this bivalent ligand. These bi valent ligands for ER do not show enhanced potency or receptor binding affinity; however, some of them display binding properties that sugge st the possibility of structure-specific negative site-site interactio n, and some of them function as quite effective estrogen antagonists.