SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) is a potential drug target for the treatment of type 2 diabetes. This enzyme serves as a negative regulator of insulin-mediated signal transduction by catalyzing the dephosphorylation of the second messenger lipid molecule phosphatidylinositol 3,4,5-triphosphate. Traditionally, assays for phosphoinositide phosphatases such as SHIP2 have relied on radiolabeled phosphatidylinositol-containing lipid membranes and chromatographic separation of labeled phospholipid substrate from product by thin-layer chromatography. We have expressed and purified catalytically active phosphatase domain constructs of SHIP2 from Escherichia coli and developed a sensitive and antibody- or binding protein-independent assay for SHIP2 amenable to high-throughput screening of phosphoinositide phosphatases or phosphoinositide kinases. This microfluidic assay, with Z' values approximately 0.8, is based upon the difference in mobility within an electric field between a fluorophore-labeled phosphatidylinositol 3,4,5-triphosphate substrate and the corresponding 3,4-bisphosphate product. High-throughput screening of a 91,060-member compound library in 384-well format resulted in the identification of SHIP2 inhibitors.
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