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

Richard M. Nelson

Boehringer Ingelheim Pharmaceuticals, Inc., Department of Medicinal Chemistry, Ridgefield, CT

Jeffrey D. Yingling

Boehringer Ingelheim Pharmaceuticals, Inc., Department of Medicinal Chemistry, Ridgefield, CT; Aerie Pharmaceuticals, Inc., Research Triangle Park, NC

Steven S. Pullen

Boehringer Ingelheim Pharmaceuticals, Inc., Department of Cardiovascular Diseases, Ridgefield, CT

Anthony S. Prokopowicz, III

Boehringer Ingelheim Pharmaceuticals, Inc., Department of Medicinal Chemistry, Ridgefield, CT

Jessi Wildeson Jones

Boehringer Ingelheim Pharmaceuticals, Inc., Department of Biologics/Biomolecular Sciences, Ridgefield, CT

John P. Wolak

George R. Rogers

Boehringer Ingelheim Pharmaceuticals, Inc., Department of Medicinal Chemistry, Ridgefield, CT

Maurice M. Morelock

Boehringer Ingelheim Pharmaceuticals, Inc., Department of Translational Science, Ridgefield, CT

Roger J. Snow

Carol Ann Homon

Boehringer Ingelheim Pharmaceuticals, Inc., Department of Medicinal Chemistry, Ridgefield, CT

Scott Jakes

Boehringer Ingelheim Pharmaceuticals, Inc., Department of Cardiovascular Diseases, Ridgefield, CT

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

This version was published on February 1, 2007

Journal of Biomolecular Screening, Vol. 12, No. 1, 70-83 (2007)
DOI: 10.1177/1087057106296047


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