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February 01, 2009 (Vol. 29, No. 3)

Simplification of Kinase Binding Assays

TR-FRET Tools Broaden the Potential of Drug Screening

  • Multiple Classes of Kinase Inhibitors

    Click Image To Enlarge +
    Figure 4. Preferential binding of the Type II inhibitor Gleevec® to dephosphorylated Abl.

    The first FDA-approved kinase inhibitor, Gleevec, was discovered in cell-based assays and later shown (through biochemical assays) to bind preferentially to a non-phosphorylated (nonactivated) form of Abl. The LanthaScreen Eu Kinase Binding Assay has been used to demonstrate tight binding of Gleevec to dephosphorylated Abl and weaker binding to active (phosphorylated) Abl (Figure 4); Gleevec-bound phosphatase-treated Abl has nearly 40-fold higher affinity than untreated Abl (the affinity of the control compound staurosporine was similar for the two forms).

    These experiments demonstrate the flexibility of this binding assay for analyzing kinase inhibitors—it is possible to modify the phosphorylation state of a kinase by adding a phosphatase, and, in many cases, another kinase, without the need to repurify the target kinase prior to analysis. This is in contrast to activity assays, for which contaminating phosphatase and kinase activities would be much more likely to interfere with analysis.

  • Assay the Right Kinase

    A challenge often faced when developing kinase activity assays, especially for less-studied targets and those with low specific activity, is ensuring that the measured activity is due to the target of interest and not to low levels of contaminating kinases from host cells.

    This is particularly true when using generic substrates and when specific control inhibitors are unavailable. This complication is removed in the LanthaScreen Eu Kinase Binding Assay, as the signal is derived only from an epitope-tagged kinase and is not affected by low levels of any contaminating kinase or phosphatase activities.

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