title: Development of a profiling strategy for metabolic mixtures by combining chromatography and mass spectrometry with cell-based GPCR signaling.
abstract: 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.
authors: Medicinal Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, VU University, Amsterdam, The Netherlands.
authors: Saskia
authors: S
authors: Nijmeijer
authors: Henry F
authors: HF
authors: Vischer
authors: Anders F
authors: AF
authors: Rudebeck
authors: Frank
authors: F
authors: Fleurbaaij
authors: David
authors: D
authors: Falck
authors: Rob
authors: R
authors: Leurs
authors: Wilfried M A
authors: WM
authors: Niessen
authors: Jeroen
authors: J
authors: Kool
mesh_terms: D002460:Cell Line; D002853:Chromatography, Liquid; D000242:Cyclic AMP / Q000378:metabolism; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry; D055442:Metabolome; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D011968:Receptors, Histamine / Q000378:metabolism; D000074040:Receptors, Histamine H4; D020218:Response Elements; D015398:Signal Transduction
chemical_list: C418607:HRH4 protein, human; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D011968:Receptors, Histamine; D000074040:Receptors, Histamine H4; D000242:Cyclic AMP