Molecular basis of estrogen receptor antagonism

Abstract: Estrogen Receptors (ERs), ERa and ERb, are responsible for mediating the physiological effects of the steroid hormone 17b-estradiol and structurally related compounds. ER signaling is a complex process where the combined effects of accessory proteins, known as coregulators, promoter characteristics and influence from other signaling systems, determine the cell-specific transcriptional response in a given situation. Central to the regulatory mechanism is the ability of ERs to adopt distinct conformations in response to ligands and thereby determine the relative affinity of coregulators. Dysregulation of ER signaling leads to increased cell proliferation and reproductive diseases. Synthetic anti-estrogens can antagonize inappropriate ER signaling constituting the rationale behind their clinical use in cancer therapy. The mechanisms that govern anti-estrogen action are poorly understood with several of the currently applied drugs having unwanted side effects in non-target tissues, and after prolonged treatment tumor cells become resistant to anti-estrogen therapy. Recent findings extend the traditional view of ER antagonists as compounds that solely block estrogen effects to include “active antagonist receptor signaling”. The aim of this thesis was to better understand the molecular details involved in mediating the effects of antagonist-bound ER complexes. To this end we have investigated coregulator interaction surfaces on ERs using peptides that mimic coregulator interactions. A major finding of our studies is that the ER-ligand-binding domains harbor surfaces capable of recruiting coregulators in the presence of antagonists, which may represent bona fide control surfaces involved in mediating antagonist activity. We identified peptide interactions that can serve as models for coregulator mediated subtype and antagonist specific effects (Paper I) and a peptide interaction (Paper II) that serve as a model for coregulator mediated agonist and antagonist action. In addition, a peptide interaction-mechanism overlapping with a known nuclear receptor (NR) corepressor surface was characterized with detailed structural analysis (Paper III). Moreover, the peptide-receptor interaction system turned out to be a very useful system for monitoring receptor conformation after binding of uncharacterized ligands. Phytoestrogens, such as genistein, are being extensively investigated in search of new therapeutic agents for estroge n-related pathologies. 8-prenylnaringenin is a potent plantderived estrogenic compound, whereas naringenin lacking a prenyl-group is not. We therefore specifically investigated the impact of alkyl chain length and degree of branching at C(8) of the naringenin skeleton on estrogenic effects (Paper IV). Taken together, the in vitro bioassays of the 10 novel naringenin derivatives, accessed by a regioselective alkylation scheme at C(8), showed a very interesting relationship between the nature of the alkyl substituent and the established bioactivity. They span an activity spectrum ranging from full agonists to partial agonists to antagonists. Our systematic approach to investigate the impact of the nature of the substituent provides further evidence that subtle modifications to ligands greatly influence their pharmacological properties. Delineation of the biological activity elicited by compounds with various chemical properties together with characterization of the complex ER antagonist signaling is expected to increase our understanding of how to target estrogen-dependent tumors in a more optimal manner.