Effects of small molecule ligands on the conformational dynamics of the Farnesoid X Receptor ligand binding domain (FXR-LBD)

Graduation date: 2014The Farnesoid X Receptor (FXR) is a member of the nuclear receptor superfamily of transcription factors that plays a key role in the regulation of bile acids, lipid and glucose metabolisms. The regulative function of FXR is governed by conformational changes of the ligand binding domain (LBD) upon ligand binding. Although FXR is a highly researched potential therapeutic target, only a limited number of FXR-agonist complexes have been successfully crystallized and subsequently yielded high resolution structures. There is currently no structural information of any FXR-antagonist complexes publically available. This dissertation focuses on the development and application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) to characterize the impact of various ligands on the structural dynamics of the FXR-LBD. Additionally, fluorescence spectroscopy is used to complement the HDX studies.\ud Firstly, we explored the use of amide HDX coupled with mass spectrometry for characterizing conformational changes in the FXR-LBD upon binding of three well known ligands: GW4064, a synthetic non-steroidal high affinity agonist; the bile acid chenodeoxycholic acid (CDCA), the endogenous low affinity agonist of FXR; and Z-guggulsterone (GG), an in vitro antagonist of the steroid chemotype. Ligand-specific deuterium incorporation profiles were obtained for the FXR LBD upon interaction with the diverse ligand chemotypes. Comparison of the HDX profiles of the ligand-bound FXR-LBD complexes revealed a unique mode of interaction for GG. The conformational features of the FXR-LBD-antagonist interaction are discussed.\ud Secondly, several functional studies focusing on the health promoting effects of prenylflavonoids have suggested that prenylflavonoids, including xanthohumol (XN), isoxanthohumol (IX), and 8-prenylnaringenin (8-PN), are capable of modulating lipid metabolism possibly via modulating expression of FXR target genes. Therefore, we hypothesize that prenylflavonoid-type molecules might be FXR ligands that exert their regulatory effects by binding to the FXR-LBD thereby inducing changes in structural dynamics in a ligand-specific manner. Under identical experimental solution-phase conditions, we determined the effects of binding of prenylflavonoid-type compounds on the structural dynamics of FXR-LBD. Deuterium incorporation profiles were obtained for the four prenylflavonoids: XN, IX, 8-PN, and one derivative of XN, tetrahydroxanthohumol (TX). The HDX protection profiles of the FXR-LBD in presence of the diverse prenylflavonoids indicated partial conformational stabilization of the LBD in particular in regions that would be consistent with a model in which the prenylflavonoids interact with the canonical ligand binding cavitiy. In silico molecular docking studies predicted that the series of prenylflavonoids tested can act indeed as ligands of the FXR-LBD, in agreement with our HDX data. We discuss the prenylflavonoid-induced conformational changes in the context of the conformational changes that were observed with the known agonist (GW4064, CDCA) and in vitro antagonist (Z-GG).\ud Finally, to complement our HDX studies, intrinsic fluorescence titration experiments were conducted to investigate the interactions between the FXR-LBD and the seven ligands studied. The analysis of the quenching data suggested that GG and the prenylflavonoids have a strong ability to quench fluorescence of the tryptophan residues in the hFXR-LBD through a static quenching mechanism. The apparent dissociate constants K[subscript d] of the three prenylflavonoids with hFXR-LBD were 2.7±0.1 μM for XN, 5.9±0.3 μM for IX, and 2.2±0.0 μM for 8-PN, vindicating genereally low affinity interactions between the prenylflavonoids and FXR. The analysis of the Scatchard plots and Hill plots suggested that hFXR-LBD seems to have more than one ligand binding site for IX and 8-PN. The comparison of the molecular volume between the small molecule compounds studied and eight well known typical FXR agonists suggested that hFXR-LBD has a tendency to offering more than one ligand binding cavity for compounds with smaller volume (