title: Homogeneous and nonradioactive high-throughput screening platform for the characterization of kinase inhibitors in cell lysates.
abstract: Protein kinases are directly implicated in many human diseases; therefore, kinase inhibitors show great promises as new therapeutic drugs. In an effort to facilitate the screening and the characterization of kinase inhibitors, a novel application of the AlphaScreen technology was developed to monitor JNK activity from (1) purified kinase preparations and (2) endogenous kinase from cell lysates preactivated with different cytokines. The authors confirmed that both adenosine triphosphate (ATP) competitive as well as peptide-based JNK inhibitors were able to block the activity of both recombinant and HepG2 endogenous JNK activity. Using the same luminescence technique adapted for binding studies, the authors characterized peptide inhibitor mechanisms by measuring the binding affinity of the inhibitors for JNK. Because of the versatility of the technology, this cell-based JNK kinase assay could be adapted to other kinases and would represent a powerful tool to evaluate endogenous kinase activity and test a large number of potential inhibitors in a more physiologically relevant environment.
authors.affiliation: Service of Medical Genetics, CHUV, Lausanne, Switzerland.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Guenat
authors.forename: Nathalie
authors.initials: N
authors.lastname: Rouleau
authors.forename: Christelle
authors.initials: C
authors.lastname: Bielmann
authors.forename: Julie
authors.initials: J
authors.lastname: Bedard
authors.forename: Fabienne
authors.initials: F
authors.lastname: Maurer
authors.forename: Nathalie
authors.initials: N
authors.lastname: Allaman-Pillet
authors.forename: Pascal
authors.initials: P
authors.lastname: Nicod
authors.forename: Martina
authors.initials: M
authors.lastname: Bielefeld-Sévigny
authors.forename: Jacques S
authors.initials: JS
authors.lastname: Beckmann
authors.forename: Christophe
authors.initials: C
authors.lastname: Bonny
authors.forename: Roger
authors.initials: R
authors.lastname: Bossé
authors.forename: Raphaël
authors.initials: R
authors.lastname: Roduit
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D011494:Protein Kinases; D048670:MAP Kinase Kinase 4