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

Francesco Ansideri; Andreas Lange; Ahmed El-Gokha; Frank M Boeckler; Pierre Koch

doi:10.1016/j.ab.2016.02.018

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

Anal Biochem. 2016 Jun 15:503:28-40. doi: 10.1016/j.ab.2016.02.018. Epub 2016 Mar 5.

Authors

Francesco Ansideri¹, Andreas Lange², Ahmed El-Gokha³, Frank M Boeckler², Pierre Koch⁴

Affiliations

¹ Institute of Pharmaceutical Sciences, Department of Medicinal and Pharmaceutical Chemistry, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany.
² Institute of Pharmaceutical Sciences, Molecular Design and Pharmaceutical Biophysics, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany.
³ 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.
⁴ Institute of Pharmaceutical Sciences, Department of Medicinal and Pharmaceutical Chemistry, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany. Electronic address: pierre.koch@uni-tuebingen.de.

PMID: 26954235
DOI: 10.1016/j.ab.2016.02.018

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.

Keywords: Binding assay; Fluorescence polarization; c-Jun N-terminal kinase 3; p38α MAP kinase.

MeSH terms

Binding Sites / drug effects
Dose-Response Relationship, Drug
Fluorescence Polarization*
Fluorescent Dyes / chemical synthesis
Fluorescent Dyes / chemistry*
Humans
Imidazoles / chemical synthesis
Imidazoles / chemistry*
Mitogen-Activated Protein Kinase 10 / antagonists & inhibitors*
Mitogen-Activated Protein Kinase 10 / chemistry
Mitogen-Activated Protein Kinase 10 / metabolism
Mitogen-Activated Protein Kinase 14 / antagonists & inhibitors*
Mitogen-Activated Protein Kinase 14 / chemistry
Mitogen-Activated Protein Kinase 14 / metabolism
Molecular Structure
Protein Kinase Inhibitors / chemistry
Protein Kinase Inhibitors / pharmacology*
Structure-Activity Relationship

Substances

Fluorescent Dyes
Imidazoles
Protein Kinase Inhibitors
Mitogen-Activated Protein Kinase 10
Mitogen-Activated Protein Kinase 14