ORGN 531 |
Synthesis of a high-affinity PPARgamma ligand for high-throughput fluorescence polarization assays
| Michael J. DeGrazia1, Jerry Thompson2, John P. Vanden Heuvel2, and Blake R. Peterson1. (1) Department of Chemistry, The Pennsylvania State University, 152 Davey Lab, University Park, PA 16802, (2) Center for Molecular Toxicology and Department of Veterinary Science, Pennsylvania State University, 226 Fenske Laboratory, University Park, PA 16802 |
The peroxisome proliferator activated receptor (PPAR) family of transcription factors is implicated in numerous diseases including Alzheimer’s, asthma, atherosclerosis, inflammation, multiple sclerosis, cancer, and diabetes. We employed the X-ray crystal structure of the PPARg isoform complexed with the potent small molecule agonist GI262570 to design and synthesize a novel fluorescent and high-affinity probe for homogeneous and high-throughput fluorescent polarization (FP) assays. The fluorescent ligand 1 was synthesized based on the structure of GI262570 with the addition of a propargylamine-derived side chain coupled to fluorescein. The recombinant PPARg 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 PPARg ligands provided Ki values that were highly correlated with analogous values obtained by scintillation proximity (SP) assays. However, values obtained by FP more consistently reflected compound activities from whole cell assays. This fluorescent PPARg probe enables the first high throughput and homogenous FP assays for the identification of novel endogenous and exogenous PPARg ligands. |
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Bioorganic, Molecular Recognition, Asymmetric Reactions and Syntheses
11:00 AM-1:00 PM, Wednesday, September 10, 2003 Javits Convention Center -- Hall 1B/1C, Poster
Division of Organic Chemistry
The 226th ACS National Meeting, New York, NY, September 7-11, 2003
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