The objective of this investigation is to determine the molecular and structural distinctions between estrogen agonism and antagonism in hormone dependent tissues and cancers and to identify, develop and characterize novel compounds that have desired tissue-selective estrogenic or antiestrogenic properties. Because these effects are mediated by one or both of the two estrogen receptors, ERa and ER(J, knowledge of the 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 ERa and ER(3 bound to 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 the human ERa and ER(5 proteins, including the LBDs and additional functional domains up to full-length receptor, in combination with known and novel SERMs with unique tissue-selective and receptor-selective properties. Crystallizations will be assisted by the inclusion of coregulator peptides and/or crystallization chaperones (Fab and FN3 monobody) to stabilize ER complexes. Structure guided mutagenesis will be used to define important contacts within and between functional domains. This information will be used to modify and/or design ligands with altered pharmacological properties. 2) Characterize novel ERa and/or ER0- selective ligands in in vitro and in hormone-sensitive animal tumor models, especially SCID mouse xenografts and C3(1)/SV40 T-antigen transgenic mice that develop spontaneous mammary tumors. To determine potential therapeutic utility, we will also study the in vivo pharmacology of candidate ligands in rats and mice by measuring uterotrophic 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 transfected cells. The characterization of ER subtype-specific interactions will facilitate the identification and/or creation of new compounds that act differently on ERa and ER3 and possess novel therapeutic properties. Detailed structure information will also help reveal the molecular basis for such behavior. It is anticipated that compounds derived from this study may have application in the treatment and/or prevention of hormone sensitive cancers, especially breast cancer, and may also have utility as agents for hormone replacement therapy.
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