Importance of the Proximity and Orientation of Ligand-Linkage to the Design of Cinnamate-GW9662 Hybrid Compounds as Covalent PPARγ Agonists.

Covalent agonists of PPARγ cause unique receptor conformational changes and behave as selective PPARγ modulators, whereas there are few covalent agonists other than endogenous unsaturated fatty acids metabolites. Previously, we established a cell-based strategy to identify new PPARγ ligands and synthesized a new-type of covalent agonist that possesses the hybrid structure of a plant-derived cinnamic acid derivative and GW9662, a covalent antagonist. Herein, we report six analogues that differ in how the two fragments are linked together. Compounds with a simplified linker showed potent agonistic activity with improved EC₅₀ values (less than 5 nM), indicating that close proximity between the two fragments improves binding affinity. When the position of cinnamic acid moiety was placed at 4' carbon of aniline ring, PPARγ agonist activity was completely abolished. Docking studies suggested that the activation profile likely depends on interaction with the cavity around helix 3, β-sheet, and Ω-loop region in the ligand-binding domain. Furthermore, a cell-based assay revealed that agonist-type compounds activate PPARγ transcription in a manner dependent on covalent linkage with the Cys285 residue leading to prolonged transactivation. This activation feature reflects pharmacological benefits of covalent drugs, suggesting that these hybrid compounds may serve as potential leads for a new-class of covalent PPARγ ligands.