| Nuclear receptors (NRs) represent a large class of ligand-inducible protein transcription factors that regulate important signalling mechanisms in higher organisms. NRs are essential for normal cellular function, but they also play roles in numerous diseases, including cancer and diabetes. As transcription factors, NRs exert their effects by controlling the expression of specific genes. Upon binding of specific, lipophilic, small molecules, many NRs undergo conformational changes that lead to dissociation of corepressors, translocation to the nucleus, binding to specific DNA sequences, recruitment of coactivators, and activation of gene expression.;The androgen receptor is a nuclear receptor implicated in prostate cancer. In an effort to discover antiandrogens with low steroid receptor cross-reactivity, we designed and evaluated novel 11beta- and 17alpha-substituted 19-nortestosterone analogues. 11beta-alkyl-19-nortestosterone analogues bind tightly to the AR ligand binding domain and exhibit minimal antiglucocorticoid activity. The 11beta-pentyl and 11beta-octyl analogues are excellent inhibitors of dimerization in an AR two hybrid assay, and the 11beta-octyl and 11beta-decyl analogues are potent partial agonists in an AR reporter gene assay. Studies of additional analogues revealed that appending the side chain of the antiestrogen raloxifene to the 11beta position of a nortestosterone derivative yielded an excellent inhibitor of dimerization in an AR two hybrid assay and a potent partial agonist in an AR reporter gene assay. This analogue was able to antagonize hormone-mediated gene expression much more effectively than the initial 11beta-alkyl analogues studied.;In an effort to modulate estrogen receptor action by altering the chaperone pathway, we evaluated small molecule chemical inducers of dimerization (CID) composed of estrone linked to geldanamycin, an inhibitor of the chaperone protein Hsp90. This CID, termed E1-G, was shown to heterodimerize ER and Hsp90 protein in living cells. This compound induces dimerization between the Hsp90 N-terminal region and the ERbeta ligand binding domain in a yeast three hybrid assay. In mammalian cells, the E1-G dimer is less toxic than geldanamycin, but it also displays less effective inhibition of ER-mediated gene expression as compared to geldanamycin. Confocal microscopy experiments with orthogonally-labeled fluorescent fusion proteins demonstrated colocalization of the ER and Hsp90 that can be inhibited by excess estradiol. Although we believe these effects are due to heterodimerization of ER and Hsp90 in the cytoplasm of living cells, this mechanism requires further investigation.;Because nuclear receptors are major drug targets, screening to identify novel NR ligands is an important research objective. To pursue this objective, we used a mammalian whole cell fluorescent sensor to screen a 4000 molecule library for novel antiestrogens. We also evaluated this strategy as an approach for the discovery of ligands of other nuclear receptors.;In an unrelated line of research, we investigated small molecule alkylimidazoles and dialkylimidazolium salts as antimicrobial agents. We also studied polyallylamine polymers with imidazolium salt substituents. Alkyl and dialkylimidazoles with longer alkyl side chains were more effective antimicrobial agents, with octyl, decyl and dodecyl the most effective. Although the polymers were less potent antimicrobial agents than the parent small molecules, we determined that the length of the linker between the polymer backbone and the imidazole ring is crucial for antimicrobial activity. |
