SR-2595

The nuclear receptor PPARγ regulates adipogenesis and plays a central role in lipid and glucose homeostasis, and is the molecular target of the glitazones (TZDs), therapeutics used to treat insulin resistance and type-2 diabetes (T2D). Although the TZDs, which are PPARγ agonists, demonstrated robust clinical efficacy in T2D, their use has been hampered by an array of untoward side effects. Paradoxically, partial agonists (e.g. MRL24), antagonists (e.g. SR1664), and inverse agonists (e.g. SR10171 and SR2595), possess similar insulin-sensitizing efficacy as the TZDs in obese diabetic mice. Given the unique pharmacology of these modulators, we sought to identify the components of the PPARγ transcriptional complex that is regulated by these ligands. To achieve this, we employed subcellular fractionation of adipocytes combined with either trapping of the receptor complex on biotinylated DNA oligonucleotide, or classical immunoprecipitation. Tandem mass spectrometry analysis revealed unique, partially overlapping, compound- and subcellular compartment-specific complexes. Components of these interactomes are putative coregulators of PPARγ. Interestingly, complexes isolated in the cytosol contain sets of proteins involve in cellular assembly and extracellular matrix. Furthermore, the interactome observed for cytosolic non-DNA bound receptor was distinct from that observed from nuclear chromatin associated PPARγ, suggesting cellular compartment-specific roles for this receptor.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulator of adipogenesis and the pharmacological target of the thiazolidinedione (TZD) class of insulin sensitizers. Activation of PPARγ by TZDs promotes adipogenesis at the expense of osteoblast formation, contributing to their associated adverse effects on bone. Recently, we reported the development of PPARγ antagonist SR1664, designed to block the obesity-induced phosphorylation of serine 273 (S273) in the absence of classical agonism, to derive insulin-sensitizing efficacy with improved therapeutic index. Here we identify the structural mechanism by which SR1664 actively antagonizes PPARγ, and extend these findings to develop the inverse agonist SR2595. Treatment of isolated bone marrow-derived mesenchymal stem cells with SR2595 promotes induction of osteogenic differentiation. Together these results identify the structural determinants of ligand-mediated PPARγ repression, and suggest a therapeutic approach to promote bone formation.