Identification of a New Type of Covalent PPARγ Agonist using a Ligand-Linking Strategy

Abstract

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor that plays an important role in adipogenesis and glucose metabolism. The ligand-binding pocket (LBP) of PPARγ has a large Y-shaped cavity with multiple subpockets where multiple ligands can simultaneously bind and cooperatively activate PPARγ. Focusing on this unique property of the PPARγ LBP, we describe a novel two-step cell-based strategy to develop PPARγ ligands. First, a combination of ligands that cooperatively activates PPARγ was identified using a luciferase reporter assay. Second, hybrid ligands were designed and synthesized. For proof of concept, we focused on covalent agonists, which activate PPARγ through a unique activation mechanism regulated by a covalent linkage with the Cys285 residue in the PPARγ LBP. Despite their biological significance and pharmacological potential, few covalent PPARγ agonists are known except for endogenous fatty acid metabolites. With our strategy, we determined that plant-derived cinnamic acid derivatives cooperatively activated PPARγ by combining with GW9662, an irreversible antagonist. GW9662 covalently reacts with the Cys285 residue. A docking study predicted that a cinnamic acid derivative can bind to the open cavity in GW9662-bound PPARγ LBP. On the basis of the putative binding mode, structures of both ligands were linked successfully to create a potent PPARγ agonist, which enhanced the transactivation potential of PPARγ at submicromolar levels through covalent modification of Cys285. Our approach could lead to the discovery of novel high-potency PPARγ agonists.

Modification of the Orthosteric PPARγ Covalent Antagonist Scaffold Yields an Improved Dual-Site Allosteric Inhibitor

“…A recent study demonstrated that attaching a large lipophilic moiety to GW9662, which can occupy more space in the large orthosteric pocket, can convert the orthosteric covalent antagonist scaffold into a covalent agonist that activates PPARγ transcription. 38 Another study expanded the orthosteric covalent antagonist scaffold into the allosteric site to demonstrate that occupancy of the allosteric site can block phosphorylation of PPARγ. 21 Here, we desired to develop a covalent dual-site PPARγ antagonist that induces no or low transcriptional activation on its own and inhibits both orthosteric and allosteric activation by other PPARγ-binding ligands.…”

Ligand Activation of PPARγ by Ligustrazine Suppresses Pericyte Functions of Hepatic Stellate Cells via SMRT-Mediated Transrepression of HIF-1α

“…Furthermore, molecular simulation evidence could also explain why GW9662 antagonized ligustrazine effects. It has been delineated that GW9662 covalently reacted with the Cys285 residue in the PPARγ ligand-binding domain, which is closely adjacent to the Ser289 residue 42 . Accordingly, we speculated that GW9662 could competitively interrupt the formation of hydrogen bonds between the pyrazine nitrogen of ligustrazine and the Ser289 residue in the active cavity of PPARγ, resulting in the loss of ligustrazine suppression of HSC pericyte functions.…”

A compound-based proteomic approach discloses 15-ketoatractyligenin methyl ester as a new PPARγ partial agonist with anti-proliferative ability

“…Here we clearly demonstrate that the binding to PPARγ is strictly dependent on this cysteine residue at position 285, as a site-directed mutation impairs the transactivation ability (Figs 3 and 5). This residue has been shown to be essential for the activity and covalent binding of some PPARγ agonists such as 15d-PGJ 2 25, indicating that the same amino acid residue may be crucial to the binding of both full and partial agonists5051. Further investigations are necessary to clarify this aspect.…”