Mechanistic elucidation guided by covalent inhibitors for the development of anti-diabetic PPARγ ligands

Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a ligand-regulated transcription factor that plays crucial roles in adipogenesis, lipid metabolism, and glucose homeostasis. Several PPAR gamma ligands possess anti-diabetic activity and they commonly inhibit the phosphorylation of PPAR gamma at serine 273 (Ser273). The recently reported PPAR gamma ligand SR1664, which selectively blocks the phosphorylation of PPARg without classical agonism, has potent anti-diabetic activity, indicating that the inhibition of Ser273 phosphorylation is sufficient to provoke anti-diabetic effects. In this study, we revealed the X-ray structure of PPAR gamma co-crystallized with SR1664 bound to the alternate binding site of PPAR gamma and confirmed that the alternate site binding of SR1664 blocks the phosphorylation of Ser273. Furthermore, using covalent inhibitors as chemical tools, we demonstrated that the inhibition of phosphorylation is attributed to the occupation of a specific site which is a hydrophobic region between helix 3 and beta 3-beta 4 at the binding pocket of PPAR gamma. In high-fat diet-induced obese mice, we confirmed the anti-diabetic activity of our covalent inhibitor SB1453 that was designed to bind at the specific site in PPAR gamma for blocking the phosphorylation of Ser273. Lastly, the target selectivity of SB1453 was demonstrated by fluorescence-based visualization of target proteins complexed with the covalent probe 11 containing a bioorthogonal functional group.