title: Development of a Novel Phosphorylated AMPK Protection Assay for High-Throughput Screening Using TR-FRET Assay.
abstract: AMP-activated protein kinase (AMPK), a conserved heterotrimeric kinase, serves as an energy sensor maintaining energy balance at both cellular and whole-body levels and plays multiple beneficial roles in carbohydrate and lipid metabolism, which makes AMPK an attractive target for diabetes and other metabolic disorders. To date, establishment of the physiologically relevant biochemical assay for AMPK has not been reported. Here we developed a phosphorylated AMPK protection assay based on a time-resolved fluorescence resonance energy transfer (TR-FRET) assay, using the protein phosphatase 2A (PP2A) to dephosphorylate AMPK. The partially dephosphorylated AMPK by PP2A had lower activity than phosphorylated AMPK. This specific TR-FRET assay for AMPK was optimized in the 384-well format and produced similar EC(50) values for AMPK activators AMP and A769662 and a similar IC(50) value for AMPK inhibitor compound C, as previously reported. Under the optimized conditions, the assay Z' factor calculated over 160 data points has an optimal value greater than 0.5, which is suitable for high-throughput screening. In conclusion, this phosphorylated AMPK protection assay we developed is very robust, sensitive, and simple to perform and may be useful as a high-throughput assay for identifying AMPK activators with the ability of preventing activated AMPK against dephosphorylation by phosphatase in the physiological conditions.
authors.affiliation: Jiangsu Key Laboratory of Drug Screening, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, P.R. China.
authors.forename: Yazhou
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Jiangsu Key Laboratory of Drug Screening, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, P.R. China.
authors.forename: Yunjie
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Jiangsu Key Laboratory of Drug Screening, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, P.R. China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Drug Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, P.R. China.
authors.forename: Jia
authors.initials: J
authors.lastname: Li
authors.affiliation: Jiangsu Key Laboratory of Drug Screening, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, P.R. China.
authors.forename: Hong
authors.initials: H
authors.lastname: Liao
authors.affiliation: Jiangsu Key Laboratory of Drug Screening, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, P.R. China.
authors.forename: Luyong
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Jiangsu Key Laboratory of Drug Screening, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, P.R. China Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China Pharmaceutical University, Nanjing, P.R. China Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, DC, USA tpang@cpu.edu.cn lyzhang@cpu.edu.cn liaohong56@hotmail.com.
authors.forename: Tao
authors.initials: T
authors.lastname: Pang
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays; D006801:Humans; D010766:Phosphorylation; D054648:Protein Phosphatase 2 / Q000378:metabolism
chemical_list: D055372:AMP-Activated Protein Kinases; D054648:Protein Phosphatase 2
keywords: AMPK; AMPK activator; TR-FRET; high-throughput screening; phosphatase PP2A