title: Baculovirus production of fully-active phosphoinositide 3-kinase alpha as a p85alpha-p110alpha fusion for X-ray crystallographic analysis with ATP competitive enzyme inhibitors.
abstract: Phosphoinositide 3-kinases have been targeted for therapeutic research because they are key components of a cell signaling cascade controlling proliferation, growth, and survival. Direct activation of the PI3Kalpha pathway contributes to the development and progression of solid tumors in breast, endometrial, colon, ovarian, and gastric cancers. In the context of a drug discovery effort, the availability of a robust crystallographic system is a means to understand the subtle differences between ATP competitive inhibitor interactions with the active site and their selectivity against other PI3Kinase enzymes. To generate a suitable recombinant design for this purpose, a p85alpha-p110alpha fusion system was developed which enabled the expression and purification of a stoichiometrically homogeneous, constitutively active enzyme for structure determination with potent ATP competitive inhibitors (Raha et al., in preparation) [56]. This approach has yielded preparations with activity and inhibition characteristics comparable to those of the full-length PI3Kalpha from which X-ray diffracting crystals were grown with inhibitors bound in the active site.
authors.affiliation: Biological Reagents and Assay Development Department, Molecular Discovery Research, GlaxoSmithKline, Upper Providence, 1250 South Collegeville Rd., Collegeville, PA 19426, USA. Robert_H_Sinnamon@gsk.com
authors.forename: Robert H
authors.initials: RH
authors.lastname: Sinnamon
authors.forename: Patrick
authors.initials: P
authors.lastname: McDevitt
authors.forename: Beth L
authors.initials: BL
authors.lastname: Pietrak
authors.forename: Vaughan R
authors.initials: VR
authors.lastname: Leydon
authors.forename: Yu
authors.initials: Y
authors.lastname: Xue
authors.forename: Ruth
authors.initials: R
authors.lastname: Lehr
authors.forename: Hongwei
authors.initials: H
authors.lastname: Qi
authors.forename: Matthew
authors.initials: M
authors.lastname: Burns
authors.forename: Patricia
authors.initials: P
authors.lastname: Elkins
authors.forename: Paris
authors.initials: P
authors.lastname: Ward
authors.forename: Giorgia
authors.initials: G
authors.lastname: Vincentini
authors.forename: Donald
authors.initials: D
authors.lastname: Fisher
authors.forename: Maggie
authors.initials: M
authors.lastname: Grimes
authors.forename: Martin
authors.initials: M
authors.lastname: Brandt
authors.forename: Kurt R
authors.initials: KR
authors.lastname: Auger
authors.forename: Thau
authors.initials: T
authors.lastname: Ho
authors.forename: Kyung
authors.initials: K
authors.lastname: Johanson
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Jones
authors.forename: Benjamin
authors.initials: B
authors.lastname: Schwartz
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Sweitzer
authors.forename: Robert B
authors.initials: RB
authors.lastname: Kirkpatrick
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019677:Artificial Gene Fusion; D016367:Baculoviridae / Q000378:metabolism; D001665:Binding Sites; D002478:Cells, Cultured; D058541:Class II Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000235:genetics; D018360:Crystallography, X-Ray; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D008958:Models, Molecular; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D058541:Class II Phosphatidylinositol 3-Kinases; D058543:Class Ia Phosphatidylinositol 3-Kinase