title: A homogeneous and nonisotopic assay for phosphatidylinositol 4-kinases.
abstract: Phosphatidylinositol 4-kinases (PI 4-kinases) catalyze the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate (PtdIns4P). The four known mammalian PI 4-kinases, PI4KA, PI4KB, PI4K2A, and PI4K2B have roles in intracellular lipid and protein trafficking. PI4KA and PI4KB also assist in the replication of several positive-sense RNA viruses. The identification of selective inhibitors of these kinases would be facilitated by assays suitable for high-throughput screening. We describe a homogeneous and nonisotopic assay for PI 4-kinase activity based on the bioluminescent detection of the ADP produced by kinase reactions. We have evaluated this assay with known nonselective inhibitors of PI 4-kinases and show that it performs similar to radiometric assay formats previously described in the literature. In addition, this assay generates Z-factor values of >0.7 for PI4KA in a 384-well format, demonstrating its suitability for high-throughput screening applications.
authors.affiliation: Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48105, USA. andrewwt@med.umich.edu
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Tai
authors.forename: Naveen
authors.initials: N
authors.lastname: Bojjireddy
authors.forename: Tamas
authors.initials: T
authors.lastname: Balla
mesh_terms: D019870:1-Phosphatidylinositol 4-Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000241:Adenosine / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000032:analysis; D000255:Adenosine Triphosphate / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D057075:Enzyme Assays / Q000379:methods*; D057166:High-Throughput Screening Assays; D006801:Humans; D007553:Isotope Labeling; D007700:Kinetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011874:Radiometry; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D047428:Protein Kinase Inhibitors; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D019870:1-Phosphatidylinositol 4-Kinase; D000241:Adenosine; D000077191:Wortmannin; D004121:Dimethyl Sulfoxide