title: Discovery of PDK1 kinase inhibitors with a novel mechanism of action by ultrahigh throughput screening.
abstract: The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, K(m) values were determined as 5.6 mum for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1x and 10x K(m) concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1.
authors.affiliation: Merck Research Laboratories, Boston, Massachusetts 02115, USA. ebobkova@burnham.org
authors.forename: Ekaterina V
authors.initials: EV
authors.lastname: Bobkova
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Weber
authors.forename: Zangwei
authors.initials: Z
authors.lastname: Xu
authors.forename: Yan-Ling
authors.initials: YL
authors.lastname: Zhang
authors.forename: Joon
authors.initials: J
authors.lastname: Jung
authors.forename: Peter
authors.initials: P
authors.lastname: Blume-Jensen
authors.forename: Alan
authors.initials: A
authors.lastname: Northrup
authors.forename: Priya
authors.initials: P
authors.lastname: Kunapuli
authors.forename: Jannik N
authors.initials: JN
authors.lastname: Andersen
authors.forename: Ilona
authors.initials: I
authors.lastname: Kariv
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D001703:Biophysics / Q000379:methods; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D015398:Signal Transduction
chemical_list: D004791:Enzyme Inhibitors; C000631429:PDK1 protein, human; D010455:Peptides; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases
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