title: Characterization of PI3K class IA isoforms with regulatory subunit p55alpha using a scintillation proximity assay.
abstract: Differential activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway has been linked to cancer. Activation occurs through gene amplification and activating mutations. High-frequency mutations in the gene encoding the p110alpha catalytic subunit of PI3K (PIK3CA) have been observed in a variety of tumors including colon, brain, breast, ovarian, and gastric. Inhibition of PI3K kinase activity may provide a specific way to treat multiple types of human cancer. A scintillation proximity assay (SPA) was developed to detect phosphatidylinositol 3-kinase catalytic activity. Using this assay format, steady-state kinetic parameters were compared for the PI3K class IA enzymes p110alpha, p110beta, and p110delta, each coexpressed with the regulatory subunit p85alpha or splice variant p55alpha. Inhibition by the natural product wortmannin and LY294002 was detected with potencies consistent with alternate assay formats. Other biochemical assay formats have been described for phosphoinositide 3-kinases but each has its unique limitations. The simple, inexpensive, sensitive high-throughput nature of the SPA format has advanced our knowledge of isoform-specific enzymology and will facilitate the discovery of novel PI3K inhibitors.
authors.affiliation: Department of Enzymology and Mechanistic Pharmacology, Oncology CEDD, GlaxoSmithKline, Collegeville, PA 19426, USA. glenn.s.van.aller@gsk.com
authors.forename: Glenn S
authors.initials: GS
authors.lastname: Van Aller
authors.forename: Jeff D
authors.initials: JD
authors.lastname: Carson
authors.forename: Christine
authors.initials: C
authors.lastname: Fernandes
authors.forename: Ruth
authors.initials: R
authors.lastname: Lehr
authors.forename: Robert H
authors.initials: RH
authors.lastname: Sinnamon
authors.forename: Robert B
authors.initials: RB
authors.lastname: Kirkpatrick
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Tummino
authors.forename: Lusong
authors.initials: L
authors.lastname: Luo
mesh_terms: D055162:Biocatalysis / Q000187:drug effects; D001688:Biological Products / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D008863:Microspheres; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012588:Scintillation Counting / Q000379:methods*; D017186:Titrimetry
chemical_list: D001688:Biological Products; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D021122:Protein Subunits