Quantitative imaging of tyrosine kinase-drug interactions in cells.

Abstract

Kinases play a crucial role in regulating cellular signaling cascades, making them therapeutic targets for several human diseases. In human cancers, mis-regulation and mutations of kinases such as EGFR (epidermal growth factor receptor) have been found to drive malignant transformation. Due to the conserved structural elements of protein kinases, the majority of kinase inhibitors available have a tendency to inhibit multiple targets. The biological impact of this promiscuity is insufficiently defined and the prevalence of cellular compensatory mechanisms additionally varies the clinical responses to drug treatment. In order to understand the relationship between selectivity and efficacy, prior to clinical trials, it is essential to characterize how inhibitors interact with the kinome within a cellular context. Monitoring inhibitor-target interactions generally involves in vitro assaying with purified proteins or protein domains, which compromises the native integrity of the kinases. Cellbased assays either gain outcomes from bulk populations that average out cell variance or phenotypic assays that lack molecular resolution. To obtain information on drug interactions on a single cell level, we have developed a method to measure the direct binding of kinase inhibitors to their targets in situ and in vivo. Kinase inhibitors are chemically tagged with fluorophores that serve as acceptors to genetically tagged donor fluorophores on the enzyme and the interaction is measured using FRET-FLIM. With epidermal growth factor receptor (EGFR) and irreversible EGFR inhibitors as the model system, this approach has been applied to image inhibitor-kinase interactions in live and fixed cells. Using this method, a small panel of tyrosine kinase targets, and labeled inhibitors, we were able to investigate the cross-specificity within the panel. Additionally it was found that the specificity of inhibitors for specific kinase conformations enables the distinction between EGFR in the active and inactive conformation by the inhibitor-probes.