Fluorescence polarization-based assays for detecting compounds binding to inactive c-Jun N-terminal kinase 3 and p38α mitogen-activated protein kinase

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 p38 alpha 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-(44(4-(4-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yeamino) phenyl)thiourea (5) as an FP probe (JNK3: K-d = 3.0 nM; p38 alpha MAPK: K-d = 5.7 nM). The validation of the assays with known inhibitors of JNK3 and p38 alpha 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 p38 alpha MAPK inhibitors. (C) 2016 Elsevier Inc. All rights reserved.