Kinase activity assays are cost-effective and widely used to drive early drug discovery and lead identification.  However, activity-based assays have limitations both in terms of the extent of target coverage and the type of information they can provide about test compounds.  We have developed a competitive TR-FRET binding assay platform based on Alexa Fluor� 647 conjugated to kinase inhibitor scaffolds for characterization of protein kinase inhibitors.  This platform has advantages over traditional activity-based assays by eliminating the need for substrate or an activated kinase preparation, the ability to measure binding events in real-time, and overall simplicity of the assay.  Binding of the conjugate to a kinase is detected by addition of a europium-labeled anti-tag antibody, which binds specifically to the kinase.  Binding of both the tracer and antibody to a kinase results in a high degree of FRET, whereas displacement of the tracer with a kinase inhibitor results in a loss of FRET.  This assay has been validated for more than 200 kinases using both Type I and Type II inhibitors.  The ability of this assay format to detect �allosteric� kinase inhibitors has been examined and, although these compounds have been described as non-competitive or un-competitive with respect to ATP, the clear majority are detected in this assay format, indicating that the binding sites are either close to the ATP site or that they elicit a conformation change impacting the ATP site.  Thus, in addition to measuring Type I and Type II kinase inhibitors, this assay format is able to detect and provide mechanistic insight into many allosteric kinase inhibitors.

Characterization of both allosteric and ATP-competitive kinase inhibitors with TR-FRET binding assays

239th National Meeting (2010)

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