Synthesis and biological evaluation of nuclear hormone receptor ligands

Nuclear receptors (NRs) are transcription factors that control gene expression in cells of higher organisms. Those receptors are regulated by ligand binding, which controls interactions on DNA with specific coactivators and corepressors. These interactions engage the transcriptional machinery to regulate the expression of mRNA and the downstream production of proteins. Steroid hormone receptors are a subfamily of NRs regulated by lipophilic steroid hormones. To detect ligands of steroid hormone receptors, we investigated ligand-mediated protein stabilization to develop novel fluorescent cellular sensors. These sensors involved expression of yellow fluorescent protein (YFP) fused to the ligand binding domains (LBDs) of estrogen receptors (ERalpha, ERbeta), the androgen receptor (AR), and the glucocorticoid receptor (GR). Expression of these proteins in S. cerevisiae yeast and treatment with cognate steroid receptor ligands resulted in dose-dependent cellular fluorescence enhancements that correlated with known relative receptor binding affinity values and paralleled ligand-mediated receptor dimerization quantified with analogous yeast two hybrid transcriptional assays. These sensors provide a novel non-transcriptional and high-throughput method to identify and analyze ligand, of nuclear hormone receptors.; To antagonize functions of the AR in cells, we synthesized novel, potent, and selective steroidal antiandrogens comprising 11beta-alkyl-substituted analogues of nortestosterone. Nortestosterone analogues bearing an 11beta-pentyl/octyl side-chain were found to bind tightly to recombinant AR protein, potently block AR-mediated gene expression in a mammalian two-hybrid assay, and inhibit proliferation of LNCaP prostate cancer cells in vivo. These and related compounds may provide interesting candidates for cellular proliferation studies in animal models of human prostate cancer.; To enforce interactions between steroid hormone receptors and other proteins, we synthesized novel chemical inducers of protein dimerization (CIDs). Compounds were synthesized that heterodimerize estrogen receptors and glucocorticoid receptors with streptavidin in living yeast cells. These compounds provide tools for the identification of protein targets of biologically active small molecules and provide novel probes of nuclear hormone receptors.