Insights into Dynamic Mechanism of Ligand Binding to Peroxisome Proliferator-Activated Receptor γ toward Potential Pharmacological Applications.

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily, which regulates the transcription of a variety of genes involved in lipid and glucose metabolism, inflammation, and cell proliferation. These functions correlate with the onset of type-2 diabetes, obesity, and immune disorders, which makes PPARγ a promising target for drug development. The majority of PPARγ functions are regulated by binding of small molecule ligands, which cause conformational changes of PPARγ followed by coregulator recruitment. The ligand-binding domain (LBD) of PPARγ contains a large Y-shaped cavity that can be occupied by various classes of compounds such as full agonists, partial agonists, natural lipids, and in some cases, a combination of multiple molecules. Several crystal structure studies have revealed the binding modes of these compounds in the LBD and insight into the resulting conformational changes. Notably, the apo form of the PPARγ LBD contains a highly mobile region that can be stabilized by ligand binding. Furthermore, recent biophysical investigations have shed light on the dynamic mechanism of how ligands induce conformational changes in PPARγ and result in functional output. This information may be useful for the design of new and repurposed structures of ligands that serve a different function from original compounds and more potent pharmacological effects with less undesirable clinical outcomes. This review provides an overview of the peculiar characteristics of the PPARγ LBD by examining a series of structural studies focused on the dynamic mechanism of binding and the potential applications of strategies for ligand screening and chemical labeling.

Structural Insights into the Loss-of-Function R288H Mutant of Human PPARγ.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor and the molecular target of thiazolidinedione-class antidiabetic drugs. It has been reported that the loss of function R288H mutation in the human PPARγ ligand-binding domain (LBD) may be associated with the onset of colon cancer. A previous in vitro study showed that this mutation dampens 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2, a natural PPARγ agonist)-dependent transcriptional activation; however, it is poorly understood why the function of the R288H mutant is impaired and what role this arginine (Arg) residue plays. In this study, we found that the apo-form of R288H PPARγ mutant displays several altered conformational arrangements of the amino acid side chains in LBD: 1) the loss of a salt bridge between Arg288 and Glu295 leads to increased helix 3 movement; 2) closer proximity of Gln286 and His449 via a hydrogen bond, and closer proximity of Cys285 and Phe363 via hydrophobic interaction, stabilize the helix 3-helix 11 interaction; and 3) there is steric hindrance between Cys285/Gln286/Ser289/His449 and the flexible ligands 15d-PGJ2, 6-oxotetracosahexaenoic acid (6-oxoTHA), and 17-oxodocosahexaenoic acid (17-oxoDHA). These results suggest why Arg288 plays an important role in ligand binding and why the R288H mutation is disadvantageous for flexible ligand binding.

Structural Basis for Anti-non-alcoholic Fatty Liver Disease and Diabetic Dyslipidemia Drug Saroglitazar as a PPAR α/γ Dual Agonist.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor-type transcription factors that consist of three subtypes (α, γ, and ß/δ) with distinct functions and PPAR dual/pan agonists are expected to be the next generation of drugs for metabolic diseases. Saroglitazar is the first clinically approved PPARα/γ dual agonist for treatment of diabetic dyslipidemia and is currently in clinical trials to treat non-alcoholic fatty liver disease (NAFLD); however, the structural information of its interaction with PPARα/γ remains unknown. We recently revealed the high-resolution co-crystal structure of saroglitazar and the PPARα-ligand binding domain (LBD) through X-ray crystallography, and in this study, we report the structure of saroglitazar and the PPARγ-LBD. Saroglitazar was located at the center of "Y"-shaped PPARγ-ligand-binding pocket (LBP), just as it was in the respective region of PPARα-LBP. Its carboxylic acid was attached to four amino acids (Ser289/His323/His449/Thr473), which contributes to the stabilization of Activating Function-2 helix 12, and its phenylpyrrole moiety was rotated 121.8 degrees in PPARγ-LBD from that in PPARα-LBD to interact with Phe264. PPARδ-LBD has the consensus four amino acids (Thr253/His287/His413/Tyr437) towards the carboxylic acids of its ligands, but it seems to lack sufficient space to accept saroglitazar because of the steric hindrance between the Trp228 or Arg248 residue of PPARδ-LBD and its methylthiophenyl moiety. Accordingly, in a coactivator recruitment assay, saroglitazar activated PPARα-LBD and PPARγ-LBD but not PPARδ-LBD, whereas glycine substitution of either Trp228, Arg248, or both of PPARδ-LBD conferred saroglitazar concentration-dependent activation. Our findings may be valuable in the molecular design of PPARα/γ dual or PPARα/γ/δ pan agonists.

Definition of functionally and structurally distinct repressive states in the nuclear receptor PPARγ.

The repressive states of nuclear receptors (i.e., apo or bound to antagonists or inverse agonists) are poorly defined, despite the fact that nuclear receptors are a major drug target. Most ligand bound structures of nuclear receptors, including peroxisome proliferator-activated receptor γ (PPARγ), are similar to the apo structure. Here we use NMR, accelerated molecular dynamics and hydrogen-deuterium exchange mass spectrometry to define the PPARγ structural ensemble. We find that the helix 3 charge clamp positioning varies widely in apo and is stabilized by efficacious ligand binding. We also reveal a previously undescribed mechanism for inverse agonism involving an omega loop to helix switch which induces disruption of a tripartite salt-bridge network. We demonstrate that ligand binding can induce multiple structurally distinct repressive states. One state recruits peptides from two different corepressors, while another recruits just one, providing structural evidence of ligand bias in a nuclear receptor.