title: Development of high-throughput TR-FRET and AlphaScreen assays for identification of potent inhibitors of PDK1.
abstract: The PI3K/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a Ser/Thr protein kinase, which catalyzes the phosphorylation of a conserved residue in the activation loop of a number of AGC kinases, including proto-oncogenes Akt, p70S6K, and RSK kinases. To find new small-molecule inhibitors of this important regulator kinase, the authors have developed PDK1-specific high-throughput enzymatic assays in time-resolved fluorescence resonance energy transfer (TR-FRET) and AlphaScreen formats, monitoring phosphorylation of a biotinylated peptide substrate derived from the activation loop of Akt. Development of homogeneous assays enabled screening of a focused kinase library of approximately 21,500 compounds in 1536-well TR-FRET format in duplicate. Upon validation of hits in an alternative 384-well AlphaScreen assay, several classes of structurally diverse PDK1 inhibitors, including tetracyclics, tricyclics, azaindoles, indazoles, and indenylpyrazoles, were identified, thus confirming the utility and sensitivity of the developed assays. Further testing in PC3 prostate cancer cells confirmed that representatives of the tetracyclic series showed intracellular modulation of the PDK1 activity, as evident from decreased phosphorylation levels of AKT, RSK, and S6-ribosomal protein.
authors.affiliation: Merck Research Laboratories, Boston, Massachusetts 02115, USA.
authors.forename: Zangwei
authors.initials: Z
authors.lastname: Xu
authors.forename: Kumiko
authors.initials: K
authors.lastname: Nagashima
authors.forename: Dongyu
authors.initials: D
authors.lastname: Sun
authors.forename: Thomas
authors.initials: T
authors.lastname: Rush
authors.forename: Alan
authors.initials: A
authors.lastname: Northrup
authors.forename: Jannik N
authors.initials: JN
authors.lastname: Andersen
authors.forename: Ilona
authors.initials: I
authors.lastname: Kariv
authors.forename: Ekaterina V
authors.initials: EV
authors.lastname: Bobkova
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D000255:Adenosine Triphosphate / Q000378:metabolism; D019921:Biotinylation / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D010455:Peptides / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects; D013997:Time Factors
chemical_list: D010455:Peptides; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt