title: Study of the PDK1/AKT signaling pathway using selective PDK1 inhibitors, HCS, and enhanced biochemical assays.
abstract: The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP(3)) through interaction with their pleckstrin-homology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-amino-pyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.
authors.affiliation: Department of Oncology, Pfizer, 10578 Science Center Drive, San Diego, CA 92121, USA.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Hofler
authors.forename: Tim
authors.initials: T
authors.lastname: Nichols
authors.forename: Stephan
authors.initials: S
authors.lastname: Grant
authors.forename: Laura
authors.initials: L
authors.lastname: Lingardo
authors.forename: Edward A
authors.initials: EA
authors.lastname: Esposito
authors.forename: Scott
authors.initials: S
authors.lastname: Gridley
authors.forename: Sean T
authors.initials: ST
authors.lastname: Murphy
authors.forename: John C
authors.initials: JC
authors.lastname: Kath
authors.forename: Ciarán N
authors.initials: CN
authors.lastname: Cronin
authors.forename: Michelle
authors.initials: M
authors.lastname: Kraus
authors.forename: Gordon
authors.initials: G
authors.lastname: Alton
authors.forename: Zhi
authors.initials: Z
authors.lastname: Xie
authors.forename: Scott
authors.initials: S
authors.lastname: Sutton
authors.forename: Mike
authors.initials: M
authors.lastname: Gehring
authors.forename: Jacques
authors.initials: J
authors.lastname: Ermolieff
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D000225:Adenine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D057075:Enzyme Assays / Q000379:methods*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011719:Pyrazines / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; C560873:PF 5168899; D047428:Protein Kinase Inhibitors; D011719:Pyrazines; D011994:Recombinant Proteins; D006639:Histidine; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D000225:Adenine