title: Development of a fluorescence polarization bead-based coupled assay to target different activity/conformation states of a protein kinase.
abstract: Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states. Hence, alternative assay formats that can identify compounds targeting the inactive form of a protein kinase are desirable. The authors describe the development and optimization of an Immobilized Metal Assay for Phosphochemicals (IMAP)-based couple d assay using PDK1 and inactive Akt-2 enzymes. PDK1 phosphorylates Akt-2 at Thr 309 in the catalytic domain, leading to enzymatic activation. Activation of Akt by PDK1 is measured by quantitating the phosphorylation of Akt-specific substrate peptide using the IMAP assay format. This IMAP-coupled assay has been formatted in a 384-well microplate format with a Z' of 0.73 suitable for high-throughput screening. This assay was evaluated by screening the biologically active sample set LOPAC trade mark and validated with the protein kinase C inhibitor staurosporine. The IC(50) value generated was comparable to the value obtained by the radioactive (33)P-gamma-ATP flashplate transfer assay. This coupled assay has the potential to identify compounds that target the inactive form of Akt and prevent its activation by PDK1, in addition to finding inhibitors of PDK1 and activated Akt enzymes.
authors.affiliation: Department of Tumor Biology, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.
authors.forename: Zhuomei
authors.initials: Z
authors.lastname: Lu
authors.forename: Zhizhang
authors.initials: Z
authors.lastname: Yin
authors.forename: Linda
authors.initials: L
authors.lastname: James
authors.forename: Rosalinda
authors.initials: R
authors.lastname: Syto
authors.forename: Jill M
authors.initials: JM
authors.lastname: Stafford
authors.forename: Sandra
authors.initials: S
authors.lastname: Koseoglu
authors.forename: Todd
authors.initials: T
authors.lastname: Mayhood
authors.forename: Joseph
authors.initials: J
authors.lastname: Myers
authors.forename: William
authors.initials: W
authors.lastname: Windsor
authors.forename: Paul
authors.initials: P
authors.lastname: Kirschmeier
authors.forename: Ahmed A
authors.initials: AA
authors.lastname: Samatar
authors.forename: Bruce
authors.initials: B
authors.lastname: Malcolm
authors.forename: Tammy C
authors.initials: TC
authors.lastname: Turek-Etienne
authors.forename: C Chandra
authors.initials: CC
authors.lastname: Kumar
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D000255:Adenosine Triphosphate / Q000378:metabolism; D016231:Fluorescence Polarization Immunoassay / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D050356:Lipid Metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D064413:3-Phosphoinositide-Dependent Protein Kinases; C494918:AKT1 protein, human; C494921:AKT2 protein, human; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt