A bead-based activity screen for small-molecule inhibitors of signal transduction in chronic myelogenous leukemia...

Chronic myelogenous leukemia is characterized by the presence of the chimeric BCR-ABL gene, which is expressed as the constitutively active Bcr-Abl kinase. Although kinase activity is directly responsible for the clinical phenotype, current diagnostic and prognostic methods focus on a genetic classification system in which molecularly distinct subcategories are used to predict patient responses to small-molecule inhibitors of the Bcr-Abl kinase. Point mutations in the kinase domain are a central factor regulating inhibitor resistance; however, compensatory signaling caused by the activation of unrelated kinases can influence inhibitor efficacy. Kinase activity profiling can be used as a complementary approach to genetic screening and allows direct screening of small-molecule inhibitors. We developed a quantitative assay to monitor tyrosine kinase activities and inhibitor sensitivities in a model of chronic myelogenous leukemia using peptide reporters covalently immobilized on Luminex beads. Kinase activity is quantified by nonlinear regression from well-specific internal standard curves. Using optimized synthetic substrates and peptides derived from native substrates as probes, we measured kinase inhibition in cell lysates by the signal transduction inhibitors imatinib and dasatinib. Taking advantage of a convenient 96-well plate format, this assay also allows a straightforward and quantitative analysis of the differential effects of ATP and inhibitors on kinase activity. This method for analyzing a focused signaling network benefits from rigorous statistical analysis and short processing times, thereby offering a powerful tool for drug discovery and clinical testing.