title: Comparative analysis of various in vitro COT kinase assay formats and their applications in inhibitor identification and characterization.
abstract: Cancer osaka thyroid (COT) is a member of the mitogen-activated protein kinase kinase kinase family of enzymes and plays a pivotal role in tumor necrosis factor-alpha production in macrophages. Consequently, COT is considered to be a promising target for antiinflammatory drug discovery. We describe here the development of in vitro COT assays in several formats and the advantages and disadvantages of each. A cascade assay requires very small amounts of enzyme and can provide a useful tool for high-throughput screening, but it is not desirable for compound mechanistic studies due to complicated kinetics. Direct assays are superior to cascade assays and are suitable for both compound screening and mechanistic studies. Among the direct assays, the homogeneous time-resolved fluorescence (HTRF) format is preferred over the radiometric format due to the robustness, throughput, and ease of use of the HTRF format. When the physiological protein substrate MEK1 (MAP/Erk kinase 1) was used to determine inhibitor potencies, false positives were observed due to compound interference by binding to MEK1. Using a MEK1 peptide substrate, these false positives were eliminated. In addition, we describe a simple method to study the ATP competitiveness of compounds. The knowledge gained through our studies with COT, and the methods described for our assays and compound mechanistic studies, can be readily applied to other kinase targets.
authors.affiliation: Department of Molecular Pharmacology, Abbott Bioresearch Center, Worcester, MA 01605, USA. yong.jia@abbott.com
authors.forename: Yong
authors.initials: Y
authors.lastname: Jia
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Quinn
authors.forename: Anca
authors.initials: A
authors.lastname: Clabbers
authors.forename: Robert
authors.initials: R
authors.lastname: Talanian
authors.forename: Yajun
authors.initials: Y
authors.lastname: Xu
authors.forename: Neil
authors.initials: N
authors.lastname: Wishart
authors.forename: Hamish
authors.initials: H
authors.lastname: Allen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors*; D020935:MAP Kinase Signaling System; D010761:Phosphorus Radioisotopes; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors*
chemical_list: D010761:Phosphorus Radioisotopes; D011518:Proto-Oncogene Proteins; D000255:Adenosine Triphosphate; D020930:MAP Kinase Kinase Kinases; C069937:MAP3K8 protein, human; D048369:MAP Kinase Kinase 1