Estrogen receptors (ERs), are members of the super family of ligand-regulated nuclear transcription factors, mediate the action of estrogens, including its primary endogenous ligand 17b-estradiol (E2). ERs have two isoforms, namely estrogen receptor α (ERα) and estrogen receptor β (ERβ). One of the functional domains of ER—ligand binding domain (LBD) has two binding sites; the ligand binding cleft (LBC) directly interacts with the ligands, and the second binding cleft—action function 2 (AF-2) hosts a coactivator or corepressor protein, which up- or down-regulate ER-mediated transcription. ERs are promiscuous, this allows many xenobiotic compounds that have structural similarities with E2 can bind at the LBC. These compounds are xenoestrogens (e.g., genistein in soy foods, bisphenol-A in plastics). Despite the crucial role of ERs in human health, little is known about the details of the communication between their two binding sites and the precision of interactions between the LBC with enormous number of xenoestrogens presenting in environment and foods. These complex interactions were studied in the research described in this thesis using molecular modelling system (Schrödinger).

The first part of this thesis investigates the architectural communication between the two binding sites (LBC and AF-2); this provides a basis for understanding the different levels of ER’s biological activities. In this work, Schrödinger (a molecule computational platform) is used to study the interactions between ligands (i.e., in this thesis, flavonoids) and the LBC; this indicates the potential effects of structural features of ligands on their binding energies and binding affinities with ERs, thus differentiating ligands’ ERs-driven bioactivities. The second part of this thesis describes studies on structure-activity relationships for dietary xenoestrogens-isoflavones with ERa using a gene reporter bioassay (MELN assay). The third part of the thesis uses a cell proliferation study (Caco-2 cell line) to study the structureactivity relationships of selected isoflavones with ERβ. In this part, gallic acid, an inhibitor of UDP-glucose dehydrogenase, is used to interfere with the conjugation of isoflavones; this investigates the effect of intestinal phase II metabolism on isoflavone’s bioactivity (i.e., ERdriven activity). The last part of the thesis uses an in vitro gut fermentation system to investigate the effects of selected isoflavones gut bacterial populations, and uses a Caco-2 monolayer system to study the absorption and metabolism of the isoflavones.

The studies show that, the communication between the two binding sites (LBC and AF-2) via sharing helix components is triggered by ligands docked at the LBC. Ligands with different structural properties can initiate different degrees of conformational changes in the LBC, resulting in different knock-on effects on AF-2, this facilities different amino acid residue orientations change in AF-2, which would form correspondingly different noncovalent interactions with the regulatory protein. This, in turn, likely determines the bioactivity of ERs. In addition, the precision of interactions between ligands (i.e., flavonoids) and LBC indicates the polar substitution arrangements (i.e., hydroxyl groups) might affect the binding energy and binding affinity, and thus, likely results in different ER-mediated bioactivities. This finding is supported by increased ligand output from isoflavones-exposed MELN cells and increase proliferation of ERβ-expressing Caco-2 cells exposed to isoflavones. In addition, the UDP-glucose 6-dehydrogenase inhibitor, gallic acid increases the proliferative ERβ-driven effect of isoflavones on Caco-2 cells, because it prevented phase II metabolism of isoflavones. In the in vitro gut fermentation experiments, isoflavones change gut bacterial populations, and these changes are likely positive in a human health setting. Finally, in vivo cell culture experiment shows isoflavones are taken up by a Caco-2 cell monolayer model system which mimics the gut mucosa; this indicates that isoflavones might be absorbed and metabolised by gut mucosa.