Synthesis of a high-affinity fluorescent PPARgamma ligand for high-throughput fluorescence polarization assays.

Members of the peroxisome proliferator activated receptor (PPAR) family of transcription factors are under investigation as molecular targets for the treatment of numerous diseases including Alzheimer's, asthma, atherosclerosis, inflammation, multiple sclerosis, cancer, and diabetes. We employed the X-ray crystal structure of the PPARgamma subtype complexed with the potent small molecule agonist GI262570 (farglitazar) to design and synthesize a novel fluorescent and high-affinity probe for homogeneous and high-throughput fluorescent polarization (FP) assays. Examination of this X-ray structure revealed that the phenyl carbon atom meta to the oxazole moiety of GI262570 is exposed to solvent at the bottom of a narrow protein cavity. A derivative of GI262570 was synthesized bearing a linear phenylacetylene-derived side chain comprising propargylamine coupled to fluorescein. This fluorescent analogue was designed to project the fluorophore into the adjacent protein cavity with minimal effects on receptor affinity and maximal effects on fluorescence polarization properties. The recombinant PPARgamma ligand binding domain protein bound tightly and specifically to this probe with Kd=61+/-14 nM as determined by FP measurements. Competition binding assays with known PPARgamma ligands provided Ki values that were highly correlated with analogous values obtained by scintillation proximity (SP) assays. This fluorescent PPARgamma probe enables high-throughput and homogenous FP assays for the identification of novel endogenous and exogenous PPARgamma ligands, and this rational ligand design approach may be applied to other therapeutically important members of the nuclear hormone receptor superfamily.