PPAR antagonists are ligands that bind their receptor with high affinity without transactivation activity. Recently, they have been demonstrated to maintain insulin-sensitizing and antidiabetic properties, and they serve as an alternative treatment for metabolic diseases. In this work, an affinity-based bioassay was found to be effective for selecting PPAR ligands from the dried extract of an African plant (Diospyros bipindensis). Among the ligands, we identified betulinic acid (BA), a compound already known for its anti-inflammatory, anti-tumour and antidiabetic properties, as a PPARγ and PPARα antagonist. Cell differentiation assays showed that BA inhibits adipogenesis and promotes osteogenesis; either down-regulates or does not affect the expression of a series of adipogenic markers; and up-regulates the expression of osteogenic markers. Moreover, BA increases basal glucose uptake in 3T3-L1 adipocytes. The crystal structure of the complex of BA with PPARγ sheds light, at the molecular level, on the mechanism by which BA antagonizes PPARγ, and indicates a unique binding mode of this antagonist type. The results of this study show that the natural compound BA could be an interesting and safe candidate for the treatment of type 2 diabetes and bone diseases.

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARγ full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARγ, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARγ provides a rationale for the different activation of the ligand towards PPARα and PPARγ, suggesting a novel basis for ligand design.

Resveratrol, a modulator of several signaling proteins, can exert off-target effects involving PPAR transcription factor. However, evidence for the direct interaction between this polyphenol and PPARs is lacking. Here, we addressed the hypothesis that resveratrol and its metabolites control aspects of PPAR transcriptional activity through direct interaction with PPARs. Bioaffinity chromatographic studies with the immobilized ligand-binding domains (LBDs) of PPARγ and PPARα and isothermal titration calorimetry (ITC) allowed the determination of the binding affinities of resveratrol, resveratrol 3-O-glucuronide, resveratrol 4-O-glucuronide and resveratrol 3-O-sulfate to both PPAR-LBDs. Interaction of resveratrol, resveratrol 3-O-glucuronide and resveratrol 4-O-glucuronide with PPARγ-LBD occurred with binding affinities of 1.4, 1.1 and 0.8 μM, respectively, while only resveratrol bound to the PPARα-LBD with a binding affinity of 2.7 μM. Subsequently, X-ray crystallographic studies were carried out to characterize resveratrol binding to the PPARγ-LBD at the molecular level. The electron density map from the crystal structure of the complex between PPARγ-LBD and resveratrol revealed the presence of one molecule of resveratrol bound into the LBD of PPARγ, with the ligand occupying a position close to that of other know PPARγ ligands. Transactivation assays were also performed in HepG2 cells and the results showed that resveratrol was not a PPAR agonist but, instead was able to displace rosiglitazone from PPARγ and Wy-14,643 from PPARα with IC50 values of 27.4 ± 1.8 μM and 31.7 ± 2.5 μM, respectively. We propose that resveratrol acts as a PPAR antagonist through its direct interaction with PPARγ and PPARα.