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

¹ Boehringer Ingelheim Pharmaceuticals, Inc., Department of Medicinal Chemistry, Ridgefield, CT.
² Boehringer Ingelheim Pharmaceuticals, Inc., Department of Medicinal Chemistry, Ridgefield, CT.; Aerie Pharmaceuticals, Inc., Research Triangle Park, NC.
³ Boehringer Ingelheim Pharmaceuticals, Inc., Department of Cardiovascular Diseases, Ridgefield, CT.
⁴ Boehringer Ingelheim Pharmaceuticals, Inc., Department of Biologics/Biomolecular Sciences, Ridgefield, CT.
⁵ Boehringer Ingelheim Pharmaceuticals, Inc., Department of Translational Science, Ridgefield, CT.

^* To whom correspondence should be addressed. E-mail: mkashem{at}rdg.boehringer-ingelheim.com.

	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.

Key Words: kinase assay, ATPase activity, fluorescence polarization/anisotropy, DELFIA, luminescence, luciferase

First published on December 8, 2006, doi:10.1177/1087057106296047

Journal of Biomolecular Screening 2007;12:70.

A more recent version of this article appeared on February 1, 2007


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