Biology subjects

Brust, R.

Publications and source records attributed to Brust, R..

3 recordsLinked to original sources

bioRxiv2018

Structure-function analysis of 1-hydroxypioglitazone, a major in vivo metabolite of the PPARγ agonist pioglitazone

The thiazolidinedione (TZD) pioglitazone (Pio) is an FDA-approved drug for type 2 diabetes mellitus that binds and activates the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR{gamma}). Although TZDs have potent antidiabetic effects, they also display harmful side effects that have necessitated a better understanding of their mechanisms of action. In particular, little is known about the effect of in vivo TZD metabolites on the structure and function of PPAR{gamma}. Here, we present a structure-function comparison of Pio and a major in vivo metabolite, 1-hydroxypioglitazone (PioOH). PioOH displayed a lower binding affinity and reduced potency in coregulator recruitment assays compared to Pio. To determine the structural basis of these findings, we solved an X-ray crystal structure of PioOH bound to PPAR{gamma} ligand-binding domain (LBD) and compared it to a published Pio-bound crystal structure. PioOH exhibited an altered hydrogen bonding network that could underlie its reduced affinity and potency compared to Pio. Solution-state structural analysis using NMR spectroscopy and hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis revealed that PioOH stabilizes the PPAR{gamma} activation function-2 (AF-2) coactivator binding surface better than Pio. In support of AF-2 stabilization, PioOH displayed stabilized coactivator binding in biochemical assays and better transcriptional efficacy (maximal transactivation response) in a cell-based assay that reports on the activity of the PPAR{gamma} LBD. These results, which indicate that Pio hydroxylation affects both its potency and efficacy as a PPAR{gamma} agonist, contribute to our understanding of PPAR{gamma}-binding drug metabolite interactions and may assist in future PPAR{gamma} drug design efforts.

biochemistry↗

bioRxiv2018

Coincident binding of synthetic and natural ligands to the nuclear receptor PPARγ

Crystal structures of peroxisome proliferator-activated receptor gamma (PPAR{gamma}) have revealed overlapping binding modes for synthetic and natural/endogenous ligands, indicating competition for the orthosteric pocket. Here we show that cobinding of a synthetic ligand to the orthosteric pocket can push natural and endogenous PPAR{gamma} ligands (fatty acids) out of the orthosteric pocket towards an alternate ligand-binding site near the functionally important omega ({Omega}) loop. X-ray crystallography, NMR spectroscopy, all-atom molecular dynamics simulations, and mutagenesis coupled to quantitative functional assays reveal that synthetic ligand and fatty acid cobinding can form a \"ligand link\" to the {Omega} loop and synergistically affect the structure and function of PPAR{gamma}. These findings contribute to a growing body of evidence indicating ligand binding to nuclear receptors can be more complex than the classical one-for-one orthosteric exchange of a natural or endogenous ligand with a synthetic ligand.

biochemistry↗

bioRxiv2018

A structural mechanism for directing inverse agonism of PPARγ

Small chemical modifications can have significant effects on ligand efficacy and receptor activity, but the underlying structural mechanisms can be difficult to predict from static crystal structures alone. Here we show how a simple phenyl-to-pyridyl substitution between two common covalent orthosteric ligands targeting peroxisome proliferator-activated receptor gamma (PPAR{gamma}) converts a transcriptionally neutral antagonist (GW9662) into an inverse agonist (T0070907). X-ray crystallography, molecular dynamics simulations, and mutagenesis coupled to activity assays reveal a water-mediated hydrogen bond network linking the T0070907 pyridyl group to Arg288 that is essential for inverse agonism. NMR spectroscopy reveals that PPAR{gamma} exchanges between two long-lived conformations when bound to T0070907 but not GW9662, including a conformation that prepopulates a corepressor-bound state, priming PPAR{gamma} for high affinity corepressor binding. Our findings demonstrate that ligand engagement of Arg288 may provide new routes for developing PPAR{gamma} inverse agonist.

biochemistry↗