Characterization of irreversible kinase inhibitors by directly detecting covalent bond formation: A tool for dissecting kinase drug resistance

Targeting protein kinases in cancer therapy with irreversible small-molecule inhibitors is moving to the forefront of kinase-inhibitor research and is thought to be an effective means of overcoming mutation-associated drug resistance in epidermal growth factor receptor kinase (EGFR). We generated a detection technique that allows direct measurements of covalent bond formation without relying on kinase activity, thereby allowing the straightforward investigation of the influence of steric clashes on covalent inhibitors in different resistant kinase mutants. The obtained results are discussed together with structural biology and biochemical studies of catalytic activity in both wild-type and gatekeeper mutated kinase variants to draw conclusions about the impact of steric hindrance and increased catalytic activity in drug-resistant kinase variants. Dissecting the mechanisms of kinase drug resistance: We describe a straightforward assay system, which allowed real-time detection of irreversible kinase inhibition without requiring ATP or time-dependent IC50 measurements. This assay system provided an effective tool for dissecting drug-resistance mechanisms resulting from point mutations at the gatekeeper position. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.