Conformational modulation of the farnesoid X receptor by prenylflavonoids: Insights from hydrogen deuterium exchange mass spectrometry (HDX-MS), fluorescence titration and molecular docking studies

Abstract

We report on the molecular interactions of the Farnesoid X Receptor (FXR) with prenylflavonoids, an emerging class of FXR modulators. FXR is an attractive therapeutic target for mitigating metabolic syndromes (MetS) because FXR activates the inhibitory nuclear receptor, small heterodimer partner (SHP), thereby inhibiting both gluconeogenesis and de novo lipogenesis. We and others have shown that xanthohumol (XN), the principal prenylflavonoid of the hop plant (Humulus lupulus L.), is a FXR agonist based on its ability to affect lipid and glucose metabolism in vivo and to induces FXR target genes in biliary carcinoma cells and HEK293 cells. However, studies are currently lacking to rationalize the molecular mechanisms of FXR modulation by prenylflavonoids. We addressed this deficiency and report the first systematic study of FXR prenylflavonoid interactions. We combined Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) with computational studies for dissecting molecular recognition and conformational impact of prenylflavonoid interactions on the ligand binding domain (LBD) of human FXR. Four prenylflavonoids were tested: xanthohumol, a prenylated chalcone, two prenylated flavonones, namely isoxanthohumol (IX) and 8-prenylnaringenin (8PN), and a semisynthetic prenylflavonoid derivative, tetrahydroxanthohumol (TX). Enhancement of the HDX protection profile data by in silico predicted models of FXR prenylflavonoid complexes resulted in mapping of the prenylflavonoid interactions within the canonical ligand binding pocket. Our findings provide a foundation for the exploration of the chemical scaffolds of prenylated chalcones and flavanones as leads for future structure activity studies of this important nuclear receptor with potential relevance for ameliorating lipid metabolic disorders associated with obesity and MetS.

Protein–ligand binding with the coarse-grained Martini model

“…This finding together with the different SASA values of FXR in the holo and apo form, suggests an induced fit binding mechanism of obeticholic acid. Previous studies with FXR 64 , 65 and other type II nuclear receptors 66 , 67 also indicate stabilization upon ligand binding of the region around the binding site, including helices 2, 5, and 6.…”

“…This finding together with the different SASA values of FXR in the holo and apo form, suggests an induced fit binding mechanism of obeticholic acid. Previous studies with FXR 64,65 and other type II nuclear receptors 66,67 also indicate stabilization upon ligand binding of the region around the binding site, including helices 2, 5, and 6. Together, our results demonstrate the ability of the reparametrized Martini CG model to reproduce experimental binding modes for a variety of receptors, even in challenging cases as GPCRs where ligand binding occurs in environments of different polarity and the nuclear receptor FXR where moderate conformational rearrangement of the binding pocket is induced by ligand binding.…”

Non-estrogenic Xanthohumol Derivatives Mitigate Insulin Resistance and Cognitive Impairment in High-Fat Diet-induced Obese Mice

“…XN treatment can lower LDL-c levels by upregulating CYP7A1 35 (the rate-limiting enzyme that mediates the conversion of cholesterol into bile acids (BAs)) and by farnesoid X receptor (FXR)-mediated downregulation of PCSK9 37 . XN binds to the ligand-binding domain of FXR 38 and appears to activate farnesoid X receptors (FXR) in vivo 35 . FXR is thought to be an attractive therapeutic target for MetS because when activated it inhibits gluconeogenesis and DNL 39 .…”

Xanthohumol microbiome and signature in healthy adults (the XMaS trial): a phase I triple-masked, placebo-controlled clinical trial

“…These effects come from known influences of xanthohumol on transcription factors that may provide upstream effects on inflammatory processes [ 6 ]. For instance, transcription factors that regulate gene expression known to be involved in the pathogenesis and progression of a broad array of human diseases are modulated by xanthohumol, including nuclear factor-kappa B (NFκB) [ 7 – 10 ], nuclear factor erythroid 2-related factor 2 (NRF2) [ 11 , 12 ], and the farnesoid X receptor (FXR) [ 13 , 14 ] (see Table 1 ).…”

“…Using in vitro binding studies and in silico models, xanthohumol and its metabolites are ligands for the FXR and preliminary in vitro data indicates xanthohumol activates FXR-regulated genes at low concentrations, supporting xanthohumol functions as a pharmacologic agonist of FXR [ 4 , 13 ]. The FXR receptor is known to regulate intestinal permeability as well as innate immunity including production of the pro-inflammatory cytokines: interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70, tumor necrosis factor alpha (TNF-α), and interferon-gamma (IFN-γ) [ 15 , 16 ].…”