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 H4 receptor (hH4R) 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 hH4R 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.

Development of Plate Reader and On-Line Microfluidic Screening to Identify Ligands of the 5-Hydroxytryptamine Binding Protein in Venoms

“…The purification of a bioactive from the Pseudonaja affinis snake venom was performed by using a normal bore LC-MS setup with a 10/90% split to MS analysis and fractionation, respectively. The system details are described elsewhere [ 32 ]. In short, the eluents for the LC gradient were pumped with two Shimadzu LC-2AD HLPC pumps at 0.6 mL/min.…”

Trends in the application of high-resolution mass spectrometry for human biomonitoring: An analytical primer to studying the environmental chemical space of the human exposome

“…However, multiplex biological assays were recently combined with MS for the identification of molecules having a biological response and biomarkers of health relevance in an effect-directed approach. For example, gene reporter assays combined with LC-HRMS for determining bioactive estrogenic and anti-estrogenic compounds in environmental water samples (Jonker et al, 2015), cell-based reporter gene assays combined with LC-MS for studying metabolites of the human histamine H4 receptor ligands (Nijmeijer et al, 2012), and a multiplatform approach for protein biomarkers discovery using SOMAscan, a multiplexed aptamer-based technique, and LC-MS (Billing et al, 2016, McArdle et al, 2016). By looking at biological response molecules it can drive future studies on the biological systems affected by exposures (Rappaport, 2012) (Dennis et al, 2016a).…”

Metabolic profiling of ligands for the chemokine receptor CXCR3 by liquid chromatography-mass spectrometry coupled to bioaffinity assessment

“…In this regard, bioactivity is referring to the interaction of such a metabolite with the receptor displayed as, e.g., receptor binding affinity and/or receptor (in)activation. In a parallel study, the histamine H 4 receptor was considered the drug target while metabolite binding to the histamine H 3 receptor was incorporated to assess selectivity loss by off-target binding to the H 3 receptor [ 4 ]. The analytical technology is based on mass spectrometric (MS) identification of individual metabolites after liquid chromatographic (LC) separation of metabolic mixtures.…”

“…The analytical technology is based on mass spectrometric (MS) identification of individual metabolites after liquid chromatographic (LC) separation of metabolic mixtures. The parallel assessment of bioactivity is performed at-line via high-resolution nanofractionation onto high-density microtiter plates followed by performing either receptor binding or functional assays [ 3 , 4 ]. Since the high-resolution nanofractionation collects fractions in the second range, the eventual bioactivities measured from the wells can be reconstructed into bioactivity chromatograms with sufficient resolution to allow accurate correlation of bioactivity to individual metabolites.…”