Three Mechanistically Distinct Kinase Assays Compared: Measurement of Intrinsic ATPase Activity Identified the Most Comprehensive Set of ITK Inhibitors

Abstract

Numerous assay methods have been developed to identify small-molecule effectors of protein kinases, but no single method can be applied to all isolated kinases. The authors developed a set of 3 high-throughput screening (HTS)-compatible biochemical assays that can measure 3 mechanistically distinct properties of a kinase active site, with the goal that at least 1 of the 3 would be applicable to any kinase selected as a target for drug discovery efforts. Two assays measure catalytically active enzyme: A dissociation-enhanced lanthanide fluoroimmuno assay (DELFIA) uses an antibody to quantitate the generation of phosphorylated substrate; a second assay uses luciferase to measure the consumption of adenosine triphosphate (ATP) during either phosphoryl-transfer to a peptide substrate or to water (intrinsic ATPase activity). A third assay, which is not dependent on a catalytically active enzyme, measures the competition for binding to kinase between an inhibitor and a fluorescent ATP binding site probe. To evaluate the suitability of these assays for drug discovery, the authors compared their ability to identify inhibitors of a nonreceptor protein tyrosine kinase from the Tec family, interleukin-2-inducible T cell kinase (ITK). The 3 assays agreed on 57% of the combined confirmed hit set identified from screening a 10,208-compound library enriched with known kinase inhibitors and molecules that were structurally similar. Among the 3 assays, the one measuring intrinsic ATPase activity produced the largest number of unique hits, the fewest unique misses, and the most comprehensive hit set, missing only 2.7% of the confirmed inhibitors identified by the other 2 assays combined. Based on these data, all 3 assay formats are viable for screening and together provide greater options for assay design depending on the targeted kinase. (Journal of Biomolecular

Measuring and interpreting the selectivity of protein kinase inhibitors

“…Decisions on whether to use a binding assay or measure phosphorylation, whether to use colorimetric, fluorescent, radiometric or luminescence endpoints and what format to use are all important. It has been noted that different assay formats can give different results, so one approach is to carry out a number of assay types in parallel to look for consistent patterns [78,107,128].…”

Development and Applications of a Broad-Coverage, TR-FRET-Based Kinase Binding Assay Platform

“…When the same assay was reconfigured in the TR-FRET format presented here, the observed IC 50 for staurosporine was reduced to 3 nM (data not shown). 37 The ability to use a sub-K d concentration of kinase, as well as a concentration of tracer that is close to the K d for the kinase⋅tracer interaction, is particularly advantageous when using tracers that have relatively low affinity for the kinase (>100 nM K d ) and is a distinguishing feature of the TR-FRET format that we have presented when compared to binding assays performed in FLT, 5 FP, 2 or EFC 7 formats. Although the convenience of using an epitope tag for the purification or visualization of a recombinant kinase is well established, such tags could potentially alter the activity of the kinase being studied.…”

“…Common requirements for all activity-based assays include a suitable substrate for phosphorylation, sufficient catalytic activity to generate an adequate amount of product for detection, and a direct or indirect means of detecting the phosphorylation event. There are exceptions to these requirements, such as the use of nonactivated kinases in "cascade" types of assays 1 or the use of water as a nonspecific phosphoacceptor substrate, 2 but these exceptions often come with their own caveats such as complications from nonlinear product formation or slower reaction rates. In contrast, the requirements for an assay to directly measure the affinity of a compound for a kinase are much less demanding.…”

Differential Sensitivity to Itk Kinase Signals for T Helper 2 Cytokine Production and Chemokine-Mediated Migration

“…Our data suggest that by altering signals coming from this kinase one can manipulate these T cell responses. A number of inhibitors have recently been developed that target the kinase activity of Itk, which completely inhibit T cell responses (17)(18)(19)(20)(21), but can potentially lead to immunosuppression. Our work in this study suggests that manipulation of the dose of these inhibitors to selectively alter T cell migration while maintaining normal systemic T cell responses may provide researchers with a better and more effective strategy to treat allergic asthma.…”

“…Because mice lacking Itk are resistant to developing allergic asthma (6,7), a number of inhibitors have recently been developed that target the kinase activity of Itk (17)(18)(19)(20)(21). However, the role of kinase activity of Itk in the induction of allergic asthma is still unknown.…”