Biosensor-Based Approach to the Identification of Protein Kinase Ligands with Dual-Site Modes of Action

Iva Navratilova, Graeme Macdonald, Colin Robinson, Samantha Hughes, John Mathias, Chris Phillips, Andrew Cook

    Research output: Contribution to journalArticlepeer-review

    22 Citations (Scopus)


    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.

    Original languageEnglish
    Pages (from-to)183-193
    Number of pages11
    JournalJournal of Biomolecular Screening
    Issue number2
    Publication statusPublished - Feb 2012


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