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Development of a Fluorescence Polarization Bead-Based Coupled Assay to Target Different Activity/Conformation States of a Protein KinaseDepartment of Tumor Biology, Schering-Plough Research Institute, Kenilworth, New Jersey
Department of Tumor Biology, Schering-Plough Research Institute, Kenilworth, New Jersey
Department of Tumor Biology, Schering-Plough Research Institute, Kenilworth, New Jersey
Department of Structural Chemistry, Schering-Plough Research Institute, Kenilworth, New Jersey
Department of New Lead Discovery, Schering-Plough Research Institute, Kenilworth, New Jersey
Department of Tumor Biology, Schering-Plough Research Institute, Kenilworth, New Jersey
Department of Structural Chemistry, Schering-Plough Research Institute, Kenilworth, New Jersey
Department of Structural Chemistry, Schering-Plough Research Institute, Kenilworth, New Jersey
Department of Structural Chemistry, Schering-Plough Research Institute, Kenilworth, New Jersey
Department of Tumor Biology, Schering-Plough Research Institute, Kenilworth, New Jersey
Department of Tumor Biology, Schering-Plough Research Institute, Kenilworth, New Jersey
Department of Antiviral Research, Schering-Plough Research Institute, Kenilworth, New Jersey
Department of New Lead Discovery, Schering-Plough Research Institute, Kenilworth, New Jersey
Department of Tumor Biology, Schering-Plough Research Institute, Kenilworth, New Jersey
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 (IMAPTM)-based coupleTMd 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 LOPACTM and validated with the protein kinase C inhibitor staurosporine. The IC50 value generated was comparable to the value obtained by the radioactive 33P-
Key Words: protein kinase inhibitors • IMAP • high-throughput screening • Akt
Journal of Biomolecular Screening, Vol. 9, No. 4,
309-321 (2004) |
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-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.