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 K m), 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.
Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA, 92121, USA. yjia@gnf.org.
Yong
Y
Jia
Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA, 92121, USA.
Mari
M
Manuia
Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA, 92121, USA.
Jose
J
Juarez
D066246:ErbB Receptors / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D020128:Inhibitory Concentration 50; D007700:Kinetics; D009154:Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors
D007202:Indicators and Reagents; D047428:Protein Kinase Inhibitors; C512478:EGFR protein, human; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases
Assay development; HTRF; Inhibitor characterization; Kinase; MoA studies; TR-FRET