The objective of this investigation is to determine the molecular distinctions between estrogen agonism and antagonism in hormone dependent tissues and cancers and to develop and characterize novel compounds that have desired tissue-selective estrogenic or antiestrogenic properties. Because these effects are mediated by one or both of two known mammalian estrogen receptors, ERalpha and ERbeta, knowledge of the differentially induced conformational changes of receptor-ligand complexes and analysis of behavior profiles for novel receptor ligands is essential for developing selective estrogen receptor modulators (SERMs). Therefore, a major goal of this project is to correlate compound behavior in vitro and in vivo with the crystallographic structures of ERalpha and ERbeta complexed with novel steroidal and nonsteroidal SERMs. This information will be used to help modify or design compounds with predictable, altered pharmacological properties. To accomplish these goals we will: 1) Determine the detailed crystallographic structures of ERalpha and ERbeta LBDs complexed with known and/or newly synthesized SERMs with unique tissue-selective and receptor-selective properties. Structure information will be used in conjunction with mutagenic analyses to define important contacts within the ligand binding pocket and to modify and/or design ligands with altered pharmacological properties. 2) Characterize novel ERalpha and/or ERbeta-selective ligands in in vitro and in hormone sensitive in vivo animal tumor models, including athymic mouse xenografts and rat mammary tumor models. To determine potential therapeutic utility, we will study the in vivo pharmacology of candidate ligands in rats and mice by measuring utertrophic response, cholesterol levels, and bone density. This behavior will be compared with the activities of both ERs in reporter assays of estrogen responsive promoters in both transiently and stably transfected cells. It is anticipated that the utilization of ER subtype-specific interactions in ligand design will allow the creation of new compounds that act differently on the two ERs and possess novel therapeutic properties. In addition, with the design and/or natural occurrence of compounds that selectively target ERalpha or ERbeta, structure information should help reveal the molecular basis for such behavior.
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