title: Targeting the gatekeeper residue in phosphoinositide 3-kinases.
abstract: A single residue in the ATP binding pocket of protein kinases-termed the gatekeeper-has been shown to control sensitivity to a wide range of small molecule inhibitors (Chem. Biol.2004, 11, 691; Chem. Biol.1999, 6, 671). Kinases that possess a small side chain at this position (Thr, Ala, or Gly) are readily targeted by structurally diverse classes of inhibitors, whereas kinases that possess a larger residue at this position are broadly resistant. Recently, lipid kinases of the phosphoinositide 3-kinase (PI3-K) family have become the focus of intense research interest as potential drug targets (Chem. Biol.2003, 10, 207; Curr. Opin. Pharmacol.2003, 3, 426). In this study, we identify the residue that corresponds structurally to the gatekeeper in PI3-Ks, and explore its importance in controlling enzyme activity and small molecule sensitivity. Isoleucine 848 of p110alpha was mutated to alanine and glycine, but the mutated kinase was found to have severely impaired enzymatic activity. A structural bioinformatic comparison of this kinase with its yeast orthologs identified second site mutations that rescued the enzymatic activity of the I848A kinase. To probe the dimensions of the gatekeeper pocket, a focused panel of analogs of the PI3-K inhibitor LY294002 was synthesized and its activity against gatekeeper mutated and wild-type p110alpha was assessed.
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143-2280, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Alaimo
authors.forename: Zachary A
authors.initials: ZA
authors.lastname: Knight
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D019556:COS Cells; D002522:Chlorocebus aethiops; D002867:Chromones / Q000138:chemical synthesis / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007532:Isoleucine / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D009025:Morpholines / Q000138:chemical synthesis / Q000494:pharmacology; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship
chemical_list: D002867:Chromones; D007532:Isoleucine; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one