title: A rapid, homogeneous, fluorescence polarization binding assay for peroxisome proliferator-activated receptors alpha and gamma using a fluorescein-tagged dual PPARalpha/gamma activator.
abstract: Peroxisome proliferator-activated receptors (PPARs) and other members of the nuclear hormone receptor family are important drug targets for the treatment of metabolic diseases. PPARalpha and PPARgamma 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 PPARalpha and PPARgamma has been developed using purified PPARalpha or PPARgamma ligand binding domains and a fluorescein-labeled analog (FLA) of a potent dual PPARalpha/gamma activator. FLA activator showed good binding affinity toward both PPARalpha (K(i)=0.7microM) and PPARgamma (K(i)=0.4microM). The binding of FLA activator was rapid and reached a plateau within 10 min. The resulting FP signal was stable for at least 18h. 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 (IC(50) 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 (3)H-labeled PPARalpha (r(2)=0.99) and PPARgamma (r(2)=0.99) ligands. There was also a good correlation of IC(50) 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 PPARalpha/gamma dual activator offers a homogeneous nonradioactive, sensitive, robust, and less expensive high-throughput assay for detecting compounds that bind to both PPARgamma and PPARalpha. Using this FP binding assay, we have identified a large number of PPARalpha/gamma dual activators. A similar assay platform may be easily adapted to other members of the nuclear hormone receptor family.
authors.affiliation: Drug Discovery Division, Pharmaceutical Research Institute, Bristol-Myers Squibb, Princeton, NJ 08543, USA. ramakrishna.seethala@bms.com <ramakrishna.seethala@bms.com>
authors.forename: Ramakrishna
authors.initials: R
authors.lastname: Seethala
authors.forename: Rajasree
authors.initials: R
authors.lastname: Golla
authors.forename: Zhengping
authors.initials: Z
authors.lastname: Ma
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.forename: Kevin
authors.initials: K
authors.lastname: O'Malley
authors.forename: Jonathan
authors.initials: J
authors.lastname: Lippy
authors.forename: Lin
authors.initials: L
authors.lastname: Cheng
authors.forename: Kasim
authors.initials: K
authors.lastname: Mookhtiar
authors.forename: Dennis
authors.initials: D
authors.lastname: Farrelly
authors.forename: Litao
authors.initials: L
authors.lastname: Zhang
authors.forename: Narayanan
authors.initials: N
authors.lastname: Hariharan
authors.forename: Peter T W
authors.initials: PT
authors.lastname: Cheng
mesh_terms: D002850:Chromatography, Gel; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D015394:Molecular Structure; D047493:PPAR alpha / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D047495:PPAR gamma / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D015203:Reproducibility of Results
chemical_list: D008024:Ligands; D047493:PPAR alpha; D047495:PPAR gamma; D004121:Dimethyl Sulfoxide