Three mechanistically distinct kinase assays compared: Measurement of intrinsic ATPase activity identified the most comprehensive set of ITK inhibitors

• Published in: Journal of biomolecular screening Vol. 12; no. 1; pp. 70 - 83
• Main Authors: Kashem, Mohammed A, Nelson, Richard M, Yingling, Jeffrey D, Pullen, Steven S, Prokopowicz, 3rd, Anthony S, Jones, Jessi Wildeson, Wolak, John P, Rogers, George R, Morelock, Maurice M, Snow, Roger J, Homon, Carol Ann, Jakes, Scott
• Format: Journal Article
• Language: English
• Published: United States 01.02.2007
• Subjects: Adenosine Triphosphatases - metabolism; Binding Sites - drug effects; Biological Assay - methods; Fluorescent Dyes - chemistry; Humans; Kinetics; Protein Kinase Inhibitors - analysis; Protein Kinase Inhibitors - pharmacology; Protein-Tyrosine Kinases - antagonists & inhibitors
• ISSN: 1087-0571; 2472-5552
• DOI: 10.1177/1087057106296047

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.