Three mechanistically distinct kinase assays compared: Measurement of intrinsic ATPase activity identified the most comprehensive set of ITK inhibitors.
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.
Kashem
Mohammed A
MA
Boehringer Ingelheim Pharmaceuticals, Inc., Department of Medicinal Chemistry, Ridgefield, CT 06877-0368, USA. mkashem@rdg.boehringer-ingelheim.com
Nelson
Richard M
RM
Yingling
Jeffrey D
JD
Pullen
Steven S
SS
Prokopowicz
Anthony S
AS
Jones
Jessi Wildeson
JW
Wolak
John P
JP
Rogers
George R
GR
Morelock
Maurice M
MM
Snow
Roger J
RJ
Homon
Carol Ann
CA
Jakes
Scott
S
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D001681:Biological Assay / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D047428:Protein Kinase Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*
D005456:Fluorescent Dyes; D047428:Protein Kinase Inhibitors; D011505:Protein-Tyrosine Kinases; C080813:emt protein-tyrosine kinase; D000251:Adenosine Triphosphatases