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- Title
Identification of Allosteric PIF-Pocket Ligands for PDK1 using NMR-Based Fragment Screening and <sup>1</sup>H-<sup>15</sup>N TROSY Experiments.
- Authors
Stockman, Brian J.; Kothe, Michael; Kohls, Darcy; Weibley, Laura; Connolly, Brendan J.; Sheils, Alissa L.; Qing Cao; Cheng, Alan C.; Lily Yang; Kamath, Ajith V.; Yuan-Hua Ding; Charlton, Maura E.
- Abstract
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.
- Publication
Chemical Biology & Drug Design, 2009, Vol 73, Issue 2, p179
- ISSN
1747-0277
- Publication type
Academic Journal
- DOI
10.1111/j.1747-0285.2008.00768.x