title: EphB4 cellular kinase activity assayed using an enzymatic protein interaction system.
abstract: Receptor tyrosine kinases (RTKs) are important players in various cellular processes, including proliferation, migration, metabolism, and neuronal development. EphB4 RTK is essential for the development of a functional arterial-venous network in embryonic and adult neoangiogenesis. To develop novel inhibitors of EphB4 that might have applications in severe diseases like cancer and retinopathies, assays need to be in place that resemble, in a most physiological fashion, the activation and downstream function of the kinase. In addition, such assays need to be amenable to high-throughput screening to serve efficiently the modern drug discovery processes in the pharmaceutical industry. The authors have developed an enzyme fragment complementation assay that measures the interaction of a downstream docking protein to the activated and phosphorylated full-length EphB4 kinase in cells. The assay is specific, robust, and amenable to miniaturization and high-throughput screening. It covers most steps in the activation process of EphB4, including ligand binding, autophosphorylation, and docking of a downstream interactor. This assay format can be transferred to other RTKs and adds an important cell-based kinase assay option to researchers in the field.
authors.affiliation: DiscoveRx Corporation, Fremont, California 94538, USA. twehrman@discoverx.com
authors.forename: Tom
authors.initials: T
authors.lastname: Wehrman
authors.forename: Mimi
authors.initials: M
authors.lastname: Nguyen
authors.forename: Wei
authors.initials: W
authors.lastname: Feng
authors.forename: Benjamin
authors.initials: B
authors.lastname: Bader
mesh_terms: D055808:Drug Discovery / Q000379:methods*; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D036224:Receptor, EphB4 / Q000032:analysis* / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D036224:Receptor, EphB4