Using Bioluminescent Kinase Profiling Strips to Identify Kinase Inhibitor Selectivity and Promiscuity.
The advancement of a kinase inhibitor throughout drug discovery and development is predicated upon its selectivity towards the target of interest. Thus, profiling the compound against a broad panel of kinases is important for providing a better understanding of its activity and for obviating any off-target activities that can result in undesirable consequences. To assess the selectivity and potency of an inhibitor against multiple kinases, it is desirable to use a universal assay that can monitor the activity of all classes of kinases regardless of the nature of their substrates. The luminescent ADP-Glo kinase assay is a universal platform that measures kinase activity by quantifying the amount of the common kinase reaction product ADP. Here we present a method using standardized kinase profiling systems for inhibitor profiling studies based on ADP detection by luminescence. The kinase profiling systems are sets of kinases organized by family, presented in multi-tube strips containing eight enzymes, each with corresponding substrate strips, and standardized for optimal kinase activity. We show that using the kinase profiling strips we could quickly and easily generate multiple selectivity profiles using small or large kinase panels, and identify compound promiscuity within the kinome.
Promega Corporation, Research and Development department, 2800 Woods Hollow Road, Madison, WI, 53711, USA. Hicham.zegzouti@promega.com.
Hicham
H
Zegzouti
Promega Corporation, Research and Development department, 2800 Woods Hollow Road, Madison, WI, 53711, USA.
Jacquelyn
J
Hennek
Promega Corporation, Research and Development department, 2800 Woods Hollow Road, Madison, WI, 53711, USA. said.goueli@promega.com.|University of Wisconsin School of Medicine and Public Health, Madison, WI, USA. said.goueli@promega.com.
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Goueli
D000244:Adenosine Diphosphate / Q000032:analysis; D055808:Drug Discovery / Q000379:methods; D001330:Electronic Data Processing; D006801:Humans; D007202:Indicators and Reagents; D049409:Luciferases, Firefly / Q000378:metabolism; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D011934:Reagent Strips; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013379:Substrate Specificity
D007202:Indicators and Reagents; D047428:Protein Kinase Inhibitors; D011934:Reagent Strips; D000244:Adenosine Diphosphate; D049409:Luciferases, Firefly; D020794:Receptor Protein-Tyrosine Kinases
ADP detection; Bioluminescence; Kinase assay; Kinase profiling; Selectivity profiles