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.
Highlights
In cancer, oncogenic transformations are frequently associated with increased activity of protein-serine/threonine kinases, many of which are key signaling molecules in the phosphoinositide 3-kinase and mitogen-activated protein kinase (MAPK)2 pathways
The serine/threonine protein kinases can be divided into two major families: the MAPK family, the members of which respond to the mitogen-activated G-protein RAS, and the extended AGC family of kinases [1]
One is located in the kinase activation loop, whereas the other is located C-terminal to the catalytic domain, referred to as the hydrophobic motif (HM) or PDK1-interacting fragment (PIF) [9, 10]
Summary
The serine/threonine protein kinases can be divided into two major families: the MAPK family, the members of which respond to the mitogen-activated G-protein RAS, and the extended AGC family of kinases [1] The latter comprises over 40 distinct human members including candidate oncology drug targets AKT, p70S6K, p90RSK, PRK3, protein kinase C, and PDK1. Upon T-loop phosphorylation, the phosphorylated HM of the substrate kinase is released from the PIF pocket of PDK1, forming an intramolecular interaction with its own HM-binding pocket This fully stabilizes the active enzyme conformation, resulting in phosphorylation of downstream effector molecules and downstream signal transduction [13]. We describe the enzymatic properties of the full-length PDK1, screening assays, and uHTS campaign and report on the discovery of alkaloids as PIF pocket-specific ligands
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