T0070907, a selective ligand for peroxisome proliferator-activated receptor γ, functions as an antagonist of biochemical and cellular activities

Abstract

The nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ (NR1C3)) plays a central role in adipogenesis and is the molecular target for the thiazolidinedione (TZD) class of antidiabetic drugs. In a search for novel non-TZD ligands for PPARγ, T0070907 was identified as a potent and selective PPARγ antagonist. With an apparent binding affinity (concentration at 50% inhibition of [3H]rosiglitazone binding or IC50) of 1 nM, T0070907 covalently modifies PPARγ on cysteine 313 in helix 3 of human PPARγ2. T0070907 blocked PPARγ function in both cell-based reporter gene and adipocyte differentiation assays. Consistent with its role as an antagonist of PPARγ, T0070907 blocked agonist-induced recruitment of coactivator-derived peptides to PPARγ in a homogeneous time-resolved fluorescence-based assay and promoted recruitment of the transcriptional corepressor NCoR to PPARγ in both glutathione S-transferase pull-down assays and a PPARy/retinoid X receptor (RXR) a-dependent gel shift assay. Studies with mutant receptors suggest that T0070907 modulates the interaction of PPARγ with these cofactor proteins by affecting the conformation of helix 12 of the PPARγ ligand-binding domain. Interestingly, whereas the T0070907-induced NCoR recruitment to PPARγ/RXRα heterodimer can be almost completely reversed by the simultaneous treatment with RXRα agonist LGD1069, T0070907 treatment has only modest effects on LGD1069-induced coactivator recruitment to the PPAR7γRXRα heterodimer. These results suggest that the activity of PPARγ antagonists can be modulated by the availability and concentration of RXR agonists. T0070907 is a novel tool for the study of PPARγ/RXRα heterodimer function.