title: A direct ELISA assay for quantitative determination of the inhibitory potency of small molecules inhibitors for JNK3.
abstract: The c-jun N-terminal kinase 3 (JNK3) is a promising drug target for the treatment of neurological disorders. Here we report a direct ELISA including the optimization of a nonradioactive immunosorbent JNK3 activity assay to determine inhibitory potency of small-molecule inhibitors. Based on our previous JNK3 assay and our recently optimized p38α mitogen activated protein kinase (MAPK) protocol for monitoring the phosphorylation of activating-transcription factor 2 (ATF-2), we present a rapid and straightforward alternative to conventional radioactive and indirect ELISA kinase assays. To validate the assay with the optimized assay conditions we used reference compounds and achieved well comparable IC(50) results to published data. The use of a linked monoclonal antibody increased the specificity and the sensitivity of the assay, reducing the required antibody concentration by approximately 100-fold. The novel protocol is an accurate, easy-to-handle and robust screening assay for JNK3 and the assay performance was reduced from 7.5 to 3h.
authors.affiliation: Department of Pharmaceutical and Medicinal Chemistry, Eberhard Karls University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
authors.forename: Márcia
authors.initials: M
authors.lastname: Goettert
authors.forename: Sabine
authors.initials: S
authors.lastname: Luik
authors.forename: Ralph
authors.initials: R
authors.lastname: Graeser
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Laufer
mesh_terms: D051698:Activating Transcription Factor 2 / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000465:Algorithms; D000911:Antibodies, Monoclonal; D045163:Antibodies, Phospho-Specific; D055808:Drug Discovery / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology; D017248:Enzyme Multiplied Immunoassay Technique; D006863:Hydrogen-Ion Concentration; D048057:Mitogen-Activated Protein Kinase 10 / Q000037:antagonists & inhibitors*; D009994:Osmolar Concentration; D010766:Phosphorylation / Q000187:drug effects; D013997:Time Factors
chemical_list: D051698:Activating Transcription Factor 2; D000911:Antibodies, Monoclonal; D045163:Antibodies, Phospho-Specific; D004791:Enzyme Inhibitors; D000255:Adenosine Triphosphate; D048057:Mitogen-Activated Protein Kinase 10