title: Biosensor-based approach to the identification of protein kinase ligands with dual-site modes of action.
abstract: The authors have used a surface plasmon resonance (SPR)-based biosensor approach to identify and characterize compounds with a unique binding mode to protein kinases. Biacore was used to characterize hits from an enzymatic high-throughput screen of the Tec family tyrosine kinase, IL2-inducible T cell kinase (ITK). Complex binding kinetics was observed for some compounds, which led to identification of compounds that bound simultaneously at both the adenosine triphosphate (ATP) binding site and a second, allosteric site on ITK. The presence of the second binding site was confirmed by X-ray crystallography. The second site is located in the N-terminal lobe of the protein kinase catalytic domain, adjacent to but distinct from the ATP site. To enable rapid optimization of binding properties, a competition-based Biacore assay has been developed to successfully identify second site noncompetitive binders that have been confirmed by X-ray crystallographic studies. The authors have found that SPR technology is a key method for rapid identification of compounds with dual-site modes of action.
authors: Structural Biology and Biophysics, Pfizer Global Research and Development, Sandwich, Kent, UK. i.navratilova@dundee.ac.uk
authors: Iva
authors: I
authors: Navratilova
authors: Graeme
authors: G
authors: Macdonald
authors: Colin
authors: C
authors: Robinson
authors: Samantha
authors: S
authors: Hughes
authors: John
authors: J
authors: Mathias
authors: Chris
authors: C
authors: Phillips
authors: Andrew
authors: A
authors: Cook
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry*; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D008024:Ligands; D047428:Protein Kinase Inhibitors; C089848:Tec protein-tyrosine kinase; D011505:Protein-Tyrosine Kinases; C080813:emt protein-tyrosine kinase