title: Fluorescence polarization-based assays for detecting compounds binding to inactive c-Jun N-terminal kinase 3 and p38α mitogen-activated protein kinase.
abstract: Two fluorescein-labeled pyridinylimidazoles were synthesized and evaluated as probes for the binding affinity determination of potential kinase inhibitors to the c-Jun N-terminal kinase 3 (JNK3) and p38α mitogen-activated protein kinase (MAPK). Fluorescence polarization (FP)-based competition binding assays were developed for both enzymes using 1-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)-3-(4-((4-(4-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yl)amino)phenyl)thiourea (5) as an FP probe (JNK3: Kd = 3.0 nM; p38α MAPK: Kd = 5.7 nM). The validation of the assays with known inhibitors of JNK3 and p38α MAPK revealed that both FP assays correlate very well with inhibition data received by the activity assays. This, in addition to the viability of both FP-based binding assays for the high-throughput screening procedure, makes the assays suitable as inexpensive prescreening protocols for JNK3 and p38α MAPK inhibitors.
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Medicinal and Pharmaceutical Chemistry, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany.
authors.forename: Francesco
authors.initials: F
authors.lastname: Ansideri
authors.affiliation: Institute of Pharmaceutical Sciences, Molecular Design and Pharmaceutical Biophysics, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Lange
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Medicinal and Pharmaceutical Chemistry, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany; Department of Chemistry, Faculty of Science, Menofia University, Menofia, Egypt.
authors.forename: Ahmed
authors.initials: A
authors.lastname: El-Gokha
authors.affiliation: Institute of Pharmaceutical Sciences, Molecular Design and Pharmaceutical Biophysics, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany.
authors.forename: Frank M
authors.initials: FM
authors.lastname: Boeckler
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Medicinal and Pharmaceutical Chemistry, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany. Electronic address: pierre.koch@uni-tuebingen.de.
authors.forename: Pierre
authors.initials: P
authors.lastname: Koch
mesh_terms: D001665:Binding Sites / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D007093:Imidazoles / Q000138:chemical synthesis / Q000737:chemistry*; D048057:Mitogen-Activated Protein Kinase 10 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D048308:Mitogen-Activated Protein Kinase 14 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D005456:Fluorescent Dyes; D007093:Imidazoles; D047428:Protein Kinase Inhibitors; D048057:Mitogen-Activated Protein Kinase 10; D048308:Mitogen-Activated Protein Kinase 14
keywords: Binding assay; Fluorescence polarization; c-Jun N-terminal kinase 3; p38α MAP kinase