title: Development of an ELISA for High-Throughput Screening of Inhibitors of Cdk5-Mediated PPARγ Phosphorylation.
abstract: The peroxisome proliferator-activated receptor gamma (PPARγ) is the target for the thiazolidinedione class of potent insulin-sensitizing drugs, which includes rosiglitazone and pioglitazone. However, their usage has been restricted due to severe side effects. Recent data have shown that specifically inhibiting the cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation of PPARγ at Ser273 may lead to novel insulin sensitizers with fewer side effects. Here we describe a novel enzyme-linked immunosorbent assay (ELISA) in the 384-well format, which enables screening for PPARγ ligands that inhibit phosphorylation at Ser273 by Cdk5. The assay is robust with a Z-factor  > 0.6, demonstrating its suitability for high-throughput screening. We demonstrate the suitability of this assay for profiling of published PPARγ ligands and identification of novel compounds that prevent the Cdk5-mediated phosphorylation of PPARγ at Ser273 in a 622 compound pilot study. Our assay enables the discovery and development of novel therapeutic agents for use in type-2 diabetes. Furthermore, our results in combination with structural analysis of reported PPARγ ligand binding domain X-ray structures give a molecular rationale for the Cdk5-mediated phosphorylation of PPARγ at Ser273.
authors.affiliation: 1 Discovery Sciences, AstraZeneca R&D , Gothenburg, Sweden .
authors.forename: Nina
authors.initials: N
authors.lastname: Prokoph
authors.affiliation: 1 Discovery Sciences, AstraZeneca R&D , Gothenburg, Sweden .
authors.forename: Mats
authors.initials: M
authors.lastname: Ormö
authors.affiliation: 2 Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, AstraZeneca R&D , Gothenburg, Sweden .
authors.forename: Gavin
authors.initials: G
authors.lastname: O'Mahony
authors.affiliation: 2 Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, AstraZeneca R&D , Gothenburg, Sweden .
authors.forename: Anders
authors.initials: A
authors.lastname: Hogner
authors.affiliation: 1 Discovery Sciences, AstraZeneca R&D , Gothenburg, Sweden .
authors.forename: Jane
authors.initials: J
authors.lastname: McPheat
authors.affiliation: 1 Discovery Sciences, AstraZeneca R&D , Gothenburg, Sweden .
authors.forename: Ulla
authors.initials: U
authors.lastname: Karlsson
authors.affiliation: 1 Discovery Sciences, AstraZeneca R&D , Gothenburg, Sweden .
authors.forename: Lovisa
authors.initials: L
authors.lastname: Holmberg Schiavone
authors.affiliation: 1 Discovery Sciences, AstraZeneca R&D , Gothenburg, Sweden .
authors.forename: Jianming
authors.initials: J
authors.lastname: Liu
mesh_terms: D051360:Cyclin-Dependent Kinase 5 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D047495:PPAR gamma / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D047495:PPAR gamma; D047428:Protein Kinase Inhibitors; D051360:Cyclin-Dependent Kinase 5; C495905:CDK5 protein, human