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

Marija Mladic, Danny J Scholten, M. Wijtmans, David Falck, Rob Leurs, Wilfried M A Niessen, Martine J Smit, Jeroen Kool

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Chemokine receptors belong to the class of G protein-coupled receptors and are important in the host defense against infections and inflammation. However, aberrant chemokine signaling is linked to different disorders such as cancer, central nervous system and immune disorders, and viral infections [Scholten DJ et al. (2012) Br J Pharmacol 165(6):1617-1643]. Modulating the chemokine receptor function provides new ways of targeting specific diseases. Therefore, discovery and development of drugs targeting chemokine receptors have received considerable attention from the pharmaceutical industry in the past decade. Along with that, the determination of bioactivities of individual metabolites derived from lead compounds towards chemokine receptors is crucial for drug selectivity, pharmacodynamics, and potential toxicity issues. Therefore, advanced analytical methodologies are in high demand. This study is aimed at the optimization of a new analytical method for metabolic profiling with parallel bioaffinity assessment of CXCR3 ligands of the azaquinazolinone and piperazinyl-piperidine class and their metabolites. The method is based on mass spectrometric (MS) identification after liquid chromatographic (LC) separation of metabolic mixtures. The bioaffinity assessment is performed "at-line" via high-resolution nanofractionation onto 96-well plates allowing direct integration of radioligand binding assays. This new method enables identification of metabolites from lead compounds with associated estimation of their individual bioaffinity. Moreover, the identification of the metabolite structures via accurate mass measurements and MS(2) allows the identification of liable metabolic "hotspots" for further lead optimization. The efficient combination of chemokine receptor ligand binding assays with analytical techniques, involving nanofractionation as linking technology, allows implementation of comprehensive metabolic profiling in an early phase of the drug discovery process.

Original languageEnglish
Pages (from-to)7067-81
Number of pages15
JournalAnalytical and Bioanalytical Chemistry
Volume407
Issue number23
DOIs
Publication statusPublished - Sep 2015

Fingerprint

Chemokine Receptors
Liquid chromatography
Liquid Chromatography
Mass spectrometry
Mass Spectrometry
Metabolites
Ligands
Lead compounds
Assays
Autoimmune Diseases of the Nervous System
Pharmacodynamics
Radioligand Assay
Central Nervous System Diseases
Drug Industry
Neurology
Virus Diseases
Drug Discovery
Drug Delivery Systems
G-Protein-Coupled Receptors
Bioactivity

Keywords

  • Chemokines
  • Chromatography, High Pressure Liquid
  • HEK293 Cells
  • Humans
  • Protein Interaction Mapping
  • Receptors, CXCR3
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Spectrometry, Mass, Electrospray Ionization
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

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title = "Metabolic profiling of ligands for the chemokine receptor CXCR3 by liquid chromatography-mass spectrometry coupled to bioaffinity assessment",
abstract = "Chemokine receptors belong to the class of G protein-coupled receptors and are important in the host defense against infections and inflammation. However, aberrant chemokine signaling is linked to different disorders such as cancer, central nervous system and immune disorders, and viral infections [Scholten DJ et al. (2012) Br J Pharmacol 165(6):1617-1643]. Modulating the chemokine receptor function provides new ways of targeting specific diseases. Therefore, discovery and development of drugs targeting chemokine receptors have received considerable attention from the pharmaceutical industry in the past decade. Along with that, the determination of bioactivities of individual metabolites derived from lead compounds towards chemokine receptors is crucial for drug selectivity, pharmacodynamics, and potential toxicity issues. Therefore, advanced analytical methodologies are in high demand. This study is aimed at the optimization of a new analytical method for metabolic profiling with parallel bioaffinity assessment of CXCR3 ligands of the azaquinazolinone and piperazinyl-piperidine class and their metabolites. The method is based on mass spectrometric (MS) identification after liquid chromatographic (LC) separation of metabolic mixtures. The bioaffinity assessment is performed {"}at-line{"} via high-resolution nanofractionation onto 96-well plates allowing direct integration of radioligand binding assays. This new method enables identification of metabolites from lead compounds with associated estimation of their individual bioaffinity. Moreover, the identification of the metabolite structures via accurate mass measurements and MS(2) allows the identification of liable metabolic {"}hotspots{"} for further lead optimization. The efficient combination of chemokine receptor ligand binding assays with analytical techniques, involving nanofractionation as linking technology, allows implementation of comprehensive metabolic profiling in an early phase of the drug discovery process.",
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author = "Marija Mladic and Scholten, {Danny J} and M. Wijtmans and David Falck and Rob Leurs and Niessen, {Wilfried M A} and Smit, {Martine J} and Jeroen Kool",
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month = "9",
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language = "English",
volume = "407",
pages = "7067--81",
journal = "Analytical and Bioanalytical Chemistry",
issn = "1618-2642",
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Metabolic profiling of ligands for the chemokine receptor CXCR3 by liquid chromatography-mass spectrometry coupled to bioaffinity assessment. / Mladic, Marija; Scholten, Danny J; Wijtmans, M.; Falck, David; Leurs, Rob; Niessen, Wilfried M A; Smit, Martine J; Kool, Jeroen.

In: Analytical and Bioanalytical Chemistry, Vol. 407, No. 23, 09.2015, p. 7067-81.

Research output: Contribution to JournalArticleAcademicpeer-review

TY - JOUR

T1 - Metabolic profiling of ligands for the chemokine receptor CXCR3 by liquid chromatography-mass spectrometry coupled to bioaffinity assessment

AU - Mladic, Marija

AU - Scholten, Danny J

AU - Wijtmans, M.

AU - Falck, David

AU - Leurs, Rob

AU - Niessen, Wilfried M A

AU - Smit, Martine J

AU - Kool, Jeroen

PY - 2015/9

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AB - Chemokine receptors belong to the class of G protein-coupled receptors and are important in the host defense against infections and inflammation. However, aberrant chemokine signaling is linked to different disorders such as cancer, central nervous system and immune disorders, and viral infections [Scholten DJ et al. (2012) Br J Pharmacol 165(6):1617-1643]. Modulating the chemokine receptor function provides new ways of targeting specific diseases. Therefore, discovery and development of drugs targeting chemokine receptors have received considerable attention from the pharmaceutical industry in the past decade. Along with that, the determination of bioactivities of individual metabolites derived from lead compounds towards chemokine receptors is crucial for drug selectivity, pharmacodynamics, and potential toxicity issues. Therefore, advanced analytical methodologies are in high demand. This study is aimed at the optimization of a new analytical method for metabolic profiling with parallel bioaffinity assessment of CXCR3 ligands of the azaquinazolinone and piperazinyl-piperidine class and their metabolites. The method is based on mass spectrometric (MS) identification after liquid chromatographic (LC) separation of metabolic mixtures. The bioaffinity assessment is performed "at-line" via high-resolution nanofractionation onto 96-well plates allowing direct integration of radioligand binding assays. This new method enables identification of metabolites from lead compounds with associated estimation of their individual bioaffinity. Moreover, the identification of the metabolite structures via accurate mass measurements and MS(2) allows the identification of liable metabolic "hotspots" for further lead optimization. The efficient combination of chemokine receptor ligand binding assays with analytical techniques, involving nanofractionation as linking technology, allows implementation of comprehensive metabolic profiling in an early phase of the drug discovery process.

KW - Chemokines

KW - Chromatography, High Pressure Liquid

KW - HEK293 Cells

KW - Humans

KW - Protein Interaction Mapping

KW - Receptors, CXCR3

KW - Reproducibility of Results

KW - Sensitivity and Specificity

KW - Spectrometry, Mass, Electrospray Ionization

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1007/s00216-015-8867-z

DO - 10.1007/s00216-015-8867-z

M3 - Article

VL - 407

SP - 7067

EP - 7081

JO - Analytical and Bioanalytical Chemistry

JF - Analytical and Bioanalytical Chemistry

SN - 1618-2642

IS - 23

ER -