Development of an online p38α mitogen-activated protein kinase binding assay and integration of LC-HR-MS.
A high-resolution screening method was developed for the p38α mitogen-activated protein kinase to detect and identify small-molecule binders. Its central role in inflammatory diseases makes this enzyme a very important drug target. The setup integrates separation by high-performance liquid chromatography with two parallel detection techniques. High-resolution mass spectrometry gives structural information to identify small molecules while an online enzyme binding detection method provides data on p38α binding. The separation step allows the individual assessment of compounds in a mixture and links affinity and structure information via the retention time. Enzyme binding detection was achieved with a competitive binding assay based on fluorescence enhancement which has a simple principle, is inexpensive, and is easy to interpret. The concentrations of p38α and the fluorescence tracer SK&F86002 were optimized as well as incubation temperature, formic acid content of the LC eluents, and the material of the incubation tubing. The latter notably improved the screening of highly lipophilic compounds. For optimization and validation purposes, the known kinase inhibitors BIRB796, TAK715, and MAPKI1 were used among others. The result is a high-quality assay with Z' factors around 0.8, which is suitable for semi-quantitative affinity measurements and applicable to various binding modes. Furthermore, the integrated approach gives affinity data on individual compounds instead of averaged ones for mixtures.
Falck
David
D
LACDR-Division of Biomolecular Analysis, Department of Chemistry and Pharmaceutical Sciences, VU University Amsterdam, 1081 HV Amsterdam, The Netherlands.
de Vlieger
Jon S B
JS
Niessen
Wilfried M A
WM
Kool
Jeroen
J
Honing
Maarten
M
Giera
Martin
M
Irth
Hubertus
H
D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D048308:Mitogen-Activated Protein Kinase 14 / Q000737:chemistry*; D011485:Protein Binding
D048308:Mitogen-Activated Protein Kinase 14