HTRF Kinase Assay Development and Methods in Inhibitor Characterization
Due to their important roles in cellular signaling and their dysfunctions being linked to diseases, kinases have become a class of proteins being actively pursued as potential drug targets. Biochemical assays for kinases have been developed in various formats to facilitate inhibitor screening and selectivity profiling. Here, we focus on one such technology: homogeneous time-resolved fluorescence (HTRF). In this chapter, we describe the methods of developing an HTRF kinase assay using mutant EGFR enzyme as an example. We show how to determine the kinetic parameter of the enzyme (ATP Km), as well as how to study the inhibitor mechanism of action (MoA) exemplified by inhibitors of different MoAs. All methods described here can be readily applied to other kinases with minor modifications.
Key wordsHTRF TR-FRET Kinase Assay development MoA studies Inhibitor characterization
- 8.Fabbro D, Garcia-Echeverria C (2002) Targeting protein kinases in cancer therapy. Curr Opin Drug Discov Dev 5:701–712Google Scholar
- 9.Myers MR, He W, Hulme C (1997) Inhibitors of tyrosine kinases involved in inflammation and autoimmune disease. Curr Pharm Des 3:473–502Google Scholar