Fluorescence polarization studies of xenoestrogen interactions with human estrogen receptors, design and synthesis of mechanism-based inhibitors of cytochrome P450 enzymes
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Description
Part I. Epidemiological and experimental studies support the hypothesis that dietary estrogens from plant sources (phytoestrogens) may play a role in the prevention of breast and prostate cancer. The molecular mechanisms for such chemopreventive effect are still unclear. We have investigated the possibility that phytoestrogens may bind differentially to estrogen receptor proteins (ERalpha and ERbeta) and affect the interactions of the ligand-ER complexes with different ERE sequences. Fluorescence polarization was used to measure the binding affinities of genistein, coumestrol, daidzein, glyceollin and zearalenone for human ERalpha and ERbeta. Competition binding experiments revealed higher affinity of the phytoestrogens; for ERbeta than ERalpha. Genistein (IC50 12nM) is the most potent and has the same relative binding affinity for ERbeta as E2. We have also studied the effect of these phytoestrogens on the ability of ERalpha and ERbeta to associate with specific DNA sequences (EREs). Direct binding of human recombinant estrogen receptors to fluorescein-labeled estrogen response elements (EREs) indicate that phytoestrogens can cause conformational changes in both human estrogen receptors which result in altered affinities of the complexes for the ERE from the Xenopus vit A2 gene and an ERE from the human pS2 gene Synthetic estrogens have diverse chemical structures and may either positively or negatively affect the estrogenic signaling pathways through interactions with the estrogen receptors. Modeling studies suggest that 4-(1-adamantyl)phenol and 4,4'(1,3-adamantanediyl)diphenol can bind in the ligand binding site of ERalpha. We used fluorescence polarization to compare the binding affinities of 4-(1-adamantyl)phenol (AdP), 4,4'-(1,3-adamantanediyl)diphenol (AdDP), and 2-(1-Adamantyl)-4-methylphenol (AdMP) for human ERalpha and ERbeta with the binding affinities of the known ER ligands, diethylstilbestrol and 4-hydroxytamoxifen. Competition binding experiments show that AdDP has greater affinity for both ERs than AdP, while AdMP does not bind the receptor proteins. We also found that AdDP and AdP can cause conformational changes in ERalpha and ERbeta, which result in altered affinities of the ERs for fluorescein-labeled EREs Part II. Cytochrome P450 monooxygenases are one of the most catalytically versatile enzyme system in nature and have been detected m essentially all animals, plants, fungi, and bacteria studied. Physiological functions are broad and diverse, including, detoxification of foreign chemicals (drugs, carcinogens, etc) and biosynthesis of prostaglandins, leukotreins, and steroids This part of the thesis is focused on the design and synthesis of specific acetylenic mechanism-based inhibitors for the hepatic cytochrome P450 2D6 and the plant P450 73A1. Ethynyl phenethylamines and propynyloxyphenylamines have been synthesized as putative mechanism-based inhibitors of P450 2D6. Several propynyloxybenzoic and propynyoxycinnamic; acids mechanism-based inhibitors of the plant P450 73A1 have been also prepared. Enzyme inhibition studies show that 4-propynyloxybenzoic acid is the most potent mechanism-based inhibitor of P450 73A1 among the compounds synthesized