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High-resolution metabolic profiling towards G protein-coupled receptors: Rapid and comprehensive screening of histamine H₄ receptor ligands

Kool, J., Rudebeck, A.F., Fleurbaaij, F., Nijmeijer, S., Falck, D., Smits, R.A., Vischer, H.F., Leurs, R., Niessen, W.M.A.
Journal of chromatography 2012 v.1259 pp. 213-220
G-protein coupled receptors, active ingredients, analytical chemistry, bioassays, cell biology, drugs, fractionation, high performance liquid chromatography, histamine, humans, mass spectrometry, metabolites, metabolomics, pharmacology, screening
In the past years we developed high-resolution screening platforms involving separation of bioactive mixtures and on-line or at-line bioassays for a wide variety of biological targets with parallel mass spectrometric detection and identification. In the current research, we make a major step forward in the development of at-line bioassays by implementation of radioligand receptor binding and functional cellular assays to evaluate bioactvity and selectivity. We demonstrate a new platform for high-resolution metabolic profiling of lead compounds for functional activity and selectivity toward the human histamine H₄ receptor (hH₄R), a member of the large family of membrane-bound G protein-coupled receptors. In this platform analytical chemistry, cell biology and pharmacology are merged. The methodology is based on chromatographic separation of metabolic mixtures by HPLC coupled to high-resolution fractionation onto (multiple) microtiter well plates for complementary assaying. The methodology was used for efficient and rapid metabolic profiling of the drug clozapine and three selective hH₄R lead compounds. With this new platform metabolites with undesired alterations in target selectivity profiles can be readily identified. Moreover, the parallel identification of metabolite structures, with accurate-mass measurements and MS/MS, allowed identification of liable metabolic ‘hotspots’ for further lead optimization and plays a central role in the workflow and in this study. The methodology can be easily adapted for use with other receptor screening formats. The efficient combination of pharmacological assays with analytical techniques by leveraging high-resolution at-line fractionation as a linking technology will allow implementation of comprehensive metabolic profiling in an early phase of the drug discovery process.