Development of a Novel Phosphorylated AMPK Protection Assay for High-Throughput Screening Using TR-FRET Assay
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 EC50 values for AMPK activators AMP and A769662 and a similar IC50 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.