There is a great clinical need to develop selective, high affinity kinase inhibitors. While high throughput kinase activity assays have become readily available, easy to use and cost-effective, activity-based assays have significant limitations in terms of both the extent of target coverage and the type of information they can provide about compounds. We have developed a binding assay platform based on Alexa Fluor® 647 conjugated to kinase inhibitor scaffolds that does not require substrate or an activated kinase preparation. 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 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 can be utilized to explore activation-state binding selectivity, such as the case of Imatinib binding preferentially to the non-activated form of Abl. Furthermore, this assay can detect binding of allosteric, non-ATP competitive inhibitors, such as the IKK\#946; inhibitor BMS-3455411 and the allosteric MEK inhibitors, PD98059 and PD0325901. In addition, this format can be used to develop robust assays in cases where activity assay development has proven difficult.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-37.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO