A rapid, homogeneous, fluorescence polarization binding assay for peroxisome proliferator-activated receptors alpha and gamma using a fluorescein-tagged dual PPARα/γ activator

Peroxisome proliferator-activated receptors (PPARs) and other members of the nuclear hormone receptor family are important drug targets for the treatment of metabolic diseases. PPARα and PPARγ play crucial roles in lipid and glucose metabolism, respectively. Therefore, screening methods that help to rapidly identify activators of these receptors should be of considerable value. A homogeneous fluorescence polarization (FP) ligand binding assay capable of rapidly identifying ligands that bind to both PPARα and PPARγ has been developed using purified PPARα or PPARγ ligand binding domains and a fluorescein-labeled analog (FLA) of a potent dual PPARα/γ activator. FLA activator showed good binding affinity toward both PPARα (Ki = 0.7 μM) and PPARγ (Ki = 0.4 μM). The binding of FLA activator was rapid and reached a plateau within 10 min. The resulting FP signal was stable for at least 18 h. The FP binding assay performed robustly in a 384-well format, and the average Z′ value was 0.77. There was a good correlation between the binding potency (IC50 values) and rank order of binding potency for a panel of standard PPAR ligands obtained in FP binding assay and scintillation proximity assay or gel filtration binding assays using 3H-labeled PPARα (r2 = 0.99) and PPARγ (r2 = 0.99) ligands. There was also a good correlation of IC50 values obtained by FP binding assay and scintillation proximity assay for the clinically used PPAR activators. Thus, the FP binding assay with a single fluorescein-labeled PPARα/γ dual activator offers a homogeneous nonradioactive, sensitive, robust, and less expensive high-throughput assay for detecting compounds that bind to both PPARγ and PPARα. Using this FP binding assay, we have identified a large number of PPARα/γ dual activators. A similar assay platform may be easily adapted to other members of the nuclear hormone receptor family.