Development of a Profiling Strategy for Metabolic Mixtures by Combining Chromatography and Mass Spectrometry with Cell-Based GPCR Signaling

作者：Nijmeijer Saskia; Vischer Henry F; Rudebeck Anders F; Fleurbaaij Frank; Falck David; Leurs Rob; Niessen Wilfried M A; Kool Jeroen^*

来源：Journal of Biomolecular Screening, 2012, 17(10): 1329-1338.

DOI：10.1177/1087057112451922

• histamine H-4 receptor
• ligand metabolites
• in-line screening
• signal transduction
• GPCR
• high-resolution fractionation
• miniaturization

In this study, we developed an in-line methodology that combines analytical with pharmacological techniques to characterize metabolites of human histamine H-4 receptor (hH(4)R) ligands. Liquid chromatographic separation of metabolic mixtures is coupled to high-resolution fractionation into 96- or 384-well plates and directly followed by a cell-based reporter gene assay to measure receptor signaling. The complete methodology was designed, optimized, validated, and ultimately miniaturized into a high-density well plate format. Finally, the methodology was demonstrated in a metabolic profiling setting for three hH(4)R lead compounds and the drug clozapine. This new methodology comprises integrated analytical separations, mass spectrometry, and a cell-based signal transduction-driven reporter gene assay that enables the implementation of comprehensive metabolic profiling earlier in the drug discovery process.

• 出版日期2012-12