title: An ultrasensitive high-throughput electrochemiluminescence immunoassay for the Cdc42-associated protein tyrosine kinase ACK1.
abstract: Several drugs inhibiting protein kinases have been launched successfully, demonstrating the attractiveness of protein kinases as therapeutic targets. Functional genomics research within both academia and industry has led to the identification of many more kinases as potential drug targets. Although a number of well-known formats are used for measuring protein kinase activity, some less well-characterized protein kinases identified through functional genomics present particular challenges for existing assay formats when there is limited knowledge of the endogenous substrates or activation mechanisms for these novel kinase targets. This is especially the case when a very sensitive assay is required to differentiate often highly potent inhibitors developed by late-stage medicinal chemistry programs. ACK1 is a non-receptor tyrosine kinase that has been shown to be involved in tumorigenesis and metastasis. Here we describe the development of an extremely sensitive high-throughput assay for ACK1 capable of detecting 240 fmol per well of the kinase reaction product employing a BV-tag-based electrochemiluminescence assay. This assay is universally applicable to protein tyrosine kinases using a BV-tag-labeled monoclonal antibody against phosphotyrosine. Furthermore, this assay can be extended to the evaluation of Ser/Thr kinases in those cases where an antibody recognizing the phospho-product is available.
authors.affiliation: Chemistry Research and Discovery, Amgen, South San Francisco, CA 94080, USA. josh.xiao@amgen.com
authors.forename: Shou-Hua
authors.initials: SH
authors.lastname: Xiao
authors.forename: Ellyn
authors.initials: E
authors.lastname: Farrelly
authors.forename: John
authors.initials: J
authors.lastname: Anzola
authors.forename: Daniel
authors.initials: D
authors.lastname: Crawford
authors.forename: Xianyun
authors.initials: X
authors.lastname: Jiao
authors.forename: Jinqian
authors.initials: J
authors.lastname: Liu
authors.forename: Merrill
authors.initials: M
authors.lastname: Ayres
authors.forename: Shyun
authors.initials: S
authors.lastname: Li
authors.forename: Linda
authors.initials: L
authors.lastname: Huang
authors.forename: Rajiv
authors.initials: R
authors.lastname: Sharma
authors.forename: Frank
authors.initials: F
authors.lastname: Kayser
authors.forename: Holger
authors.initials: H
authors.lastname: Wesche
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Young
mesh_terms: D000911:Antibodies, Monoclonal; D004563:Electrochemistry / Q000379:methods; D007700:Kinetics; D008163:Luminescent Measurements / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors; D011743:Pyrimidines / Q000494:pharmacology; D017975:Ruthenium Compounds / Q000737:chemistry; D012680:Sensitivity and Specificity
chemical_list: C412373:AG 1879; D000911:Antibodies, Monoclonal; D011743:Pyrimidines; D017975:Ruthenium Compounds; D011505:Protein-Tyrosine Kinases; C403262:TNK2 protein, human