Identification of allosteric PIF-pocket ligands for PDK1 using NMR-based fragment screening and 1H-15N TROSY experiments
| Autor: | Yuan-Hua Ding, Brendan Connolly, Alissa L. Sheils, Michael Kothe, Alan C. Cheng, Ajith V. Kamath, Darcy Kohls, Maura E. Charlton, Laura Weibley, Lily Yang, Qing Cao, Brian J. Stockman |
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| Rok vydání: | 2009 |
| Předmět: |
animal structures
Magnetic Resonance Spectroscopy Nitrogen Protein Conformation Allosteric regulation Fluorine-19 NMR Protein Serine-Threonine Kinases Ligands Biochemistry 3-Phosphoinositide-Dependent Protein Kinases Catalytic Domain Drug Discovery Humans Computer Simulation PI3K/AKT/mTOR pathway Pharmacology Kinase Chemistry Organic Chemistry Nuclear magnetic resonance spectroscopy Protein Structure Tertiary Docking (molecular) Molecular Medicine Phosphorylation Signalling cascades Allosteric Site Hydrogen |
| Zdroj: | Chemical biologydrug design. 73(2) |
| ISSN: | 1747-0285 |
| Popis: | Aberrant activation of the phosphoinositide 3-kinase pathway because of genetic mutations of essential signalling proteins has been associated with human diseases including cancer and diabetes. The pivotal role of 3-phosphoinositide-dependent kinase-1 in the PI3K signalling cascade has made it an attractive target for therapeutic intervention. The N-terminal lobe of the 3-phosphoinositide-dependent kinase-1 catalytic domain contains a docking site which recognizes the non-catalytic C-terminal hydrophobic motifs of certain substrate kinases. The binding of substrate in this so-called PDK1 Interacting Fragment pocket allows interaction with 3-phosphoinositide-dependent kinase-1 and enhanced phosphorylation of downstream kinases. NMR spectroscopy was used to a screen 3-phosphoinositide-dependent kinase-1 domain construct against a library of chemically diverse fragments in order to identify small, ligand-efficient fragments that might interact at either the ATP site or the allosteric PDK1 Interacting Fragment pocket. While majority of the fragment hits were determined to be ATP-site binders, several fragments appeared to interact with the PDK1 Interacting Fragment pocket. Ligand-induced changes in 1H-15N TROSY spectra acquired using uniformly 15N-enriched PDK1 provided evidence to distinguish ATP-site from PDK1 Interacting Fragment-site binding. Caliper assay data and 19F NMR assay data on the PDK1 Interacting Fragment pocket fragments and structurally related compounds identified them as potential allosteric activators of PDK1 function. |
| Databáze: | OpenAIRE |
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