Determinants of ligand-induced nuclear receptor activation

Abstract: The nuclear receptors (NRs) constitute a highly conserved superfamily of transcription factors restricted to metazoans. In general, these receptors are regulated by small lipophilic molecules such as steroids, thyroid hormones and retinoids and play an important role in cellular regulation. Several of the NRs are implicated in different medical conditions ranging from metabolic and inflammatory disorders to cancer and are therefore considered as important drug targets. In the development of high affinity ligands targeting the ligand binding domain (LBD) of NRs, knowledge about the molecular mechanisms behind transcriptional activation of the NRs is of great importance. In this thesis the molecular determinants of ligand-induced modulation of NR activity are investigated for PXR, ERR, LXR and PPAR. The pregnane X receptor (PXR) is implicated in drug metabolism and used as a negative filter in the development of drugs. The molecular mechanisms behind the species specific activation observed for human and mouse PXR was studied. We identified a critical amino acid residue, Gln285/Ile282, involved in the species specific activation observed for PXRs. Further, we suggest amino acid residue His407/Glu4O4 as important for the transcriptional activity of human and mouse PXR, respectively. The estrogen-related receptors (ERRs) are involved in metabolism and women's health. Recently, an invertebrate member of the NR3 subfamily, drosophila estrogen-related receptor (dERR), was identified. We wanted to characterize dERR in terms of transcriptional activation, ligand dependent regulation and structure of the LBD. dERR was shown to bind estrogen response elements (EREs) and ERR response elements (ERREs) suggesting evolutionary conserved DNA binding properties of ERRs. In cell-based reporter gene assays, dERR activity was not suppressed by the known ERR inverse agonists, 4-hydroxytamoxifen, tamoxifen or diethylstilbestrol. To elucidate possible structural differences in the ligand binding pocket of dERR compared to ERRs, a model of the dERR LBP was built. Based on this model mutants were designed and a dERR triple mutant was shown to display ligandinduced suppression of activity in a cell-based reporter gene assay. The liver X receptors (LXRs) are implicated in reverse cholesterol transport, intestinal cholesterol absorption and lipoprotein remodeling. We determined the crystal structure of the LXRalpha LBD, in order to gain insights into the molecular mechanisms behind LXR activation. LXRalpha was crystallized as a heterodimer with the retinoid X receptor beta (RXRbeta) in complex with their respective ligands T0901317 and methoprene acid, and a glucocorticoid receptor interacting protein 1 (GRIP-1) peptides. The complex is in a fully agonistic conformation. Based on structural and mutational studies, we suggest that the observed hydrogen bonds with His421 and Trp443 serve as the molecular basis for ligand dependent activation of LXRs. Further, the absence of interaction between helix 12 of LXR and amino acid residues of helix 7 of RXR indicates an alternative mechanism for permissiveness other than earlier described. The peroxisome proliferator-activated receptors (PPARs) are important regulators of lipid and glucose homeostasis and established molecular targets for the treatment of type 2 diabetes and cardiovascular disease. We present the crystal structures of PPARgamma in complex with a novel class of PPARalpha/gamma modulators, the 5-substituted 2-benzoylaminobenzoic acids (2BABAs), displaying a unique binding epitope. This class of compounds was found to interact distinct from the conserved network of hydrogen bonds, previously suggested to serve as the molecular basis for ligand-induced transcriptional activation of PPARs. This suggests an alternative mechanism for ligand dependent activation of PPARs. Furthermore, one of these compounds was tested in ob/ob mice and displayed antidiabetic effects in vivo, likely to result from PPARgamma activation by the 2-BABA compounds.