title: Kinetic analysis of PI3K reactions with fluorescent PIP2 derivatives.
abstract: Phosphatidylinositol 3-kinase (PI3K) signaling plays important roles in cell differentiation, proliferation, and migration. Increased mutations and expression levels of PI3K are hallmarks for the development of certain cancers. Pharmacological targeting of PI3K activity has also been actively pursued as a novel cancer therapeutic. Consequently, measurement of PI3K activity in different cell types or patient samples holds the promise as being a novel diagnostic tool. However, the direct measurement of cellular PI3K activity has been a challenging task. We report here the characterization of two fluorescent PIP(2) derivatives as reporters for PI3K enzymatic activity. The reporters are efficiently separated from their corresponding PI3K enzymatic products through either thin layer chromatography (TLC) or capillary electrophoresis (CE), and can be detected with high sensitivity by fluorescence. The biophysical and kinetic properties of the two probes are measured, and their suitability to characterize PI3K inhibitors is explored. Both probes show similar capacity as PI3K substrates for inhibitor characterization, yet also possess distinct properties that may suggest their different applications. These characterizations have laid the groundwork to systematically measure cellular PI3K activity, and have the potential to generate molecular fingerprints for diagnostic and therapeutic applications.
authors.affiliation: Division of Medicinal Chemistry and Natural Products, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Weigang
authors.initials: W
authors.lastname: Huang
authors.forename: Dechen
authors.initials: D
authors.lastname: Jiang
authors.forename: Xiaoyang
authors.initials: X
authors.lastname: Wang
authors.forename: Kelong
authors.initials: K
authors.lastname: Wang
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Sims
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
authors.forename: Qisheng
authors.initials: Q
authors.lastname: Zhang
mesh_terms: D002855:Chromatography, Thin Layer / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012680:Sensitivity and Specificity
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D019269:Phosphatidylinositol 4,5-Diphosphate; D000081082:Phosphoinositide-3 Kinase Inhibitors; D058539:Phosphatidylinositol 3-Kinase