title: A high-throughput microfluidic assay for SH2 domain-containing inositol 5-phosphatase 2.
abstract: 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.
authors.affiliation: Amgen Inc., Thousand Oaks, CA 91320-1799, USA.
authors.forename: Todd
authors.initials: T
authors.lastname: Rowe
authors.forename: Clarence
authors.initials: C
authors.lastname: Hale
authors.forename: Aileen
authors.initials: A
authors.lastname: Zhou
authors.forename: Robert J M
authors.initials: RJ
authors.lastname: Kurzeja
authors.forename: Arisha
authors.initials: A
authors.lastname: Ali
authors.forename: Anthony
authors.initials: A
authors.lastname: Menjares
authors.forename: Minghan
authors.initials: M
authors.lastname: Wang
authors.forename: John D
authors.initials: JD
authors.lastname: McCarter
mesh_terms: D002855:Chromatography, Thin Layer; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D046210:Microfluidic Analytical Techniques; D051059:PTEN Phosphohydrolase / Q000032:analysis / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000032:analysis / Q000235:genetics; D018129:Phosphatidylinositol Phosphates / Q000032:analysis / Q000235:genetics; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis; D015398:Signal Transduction; D014162:Transfection
chemical_list: D004121:Dimethyl Sulfoxide; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010744:Phosphoric Monoester Hydrolases; D011994:Recombinant Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C098706:INPPL1 protein, human