At-line coupling of LC-MS to bioaffinity and selectivity assessment for metabolic profiling of ligands towards chemokine receptors CXCR1 and CXCR2

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Abstract

This study describes an analytical method for bioaffinity and selectivity assessment of CXCR2 antagonists and their metabolites. The method is based on liquid chromatographic separation (LC) of metabolic mixtures followed by parallel mass spectrometry (MS) identification and bioaffinity determination. The bioaffinity is assessed using radioligand binding assays in 96-well plates after at-line nanofractionation. The described method was optimized for chemokines and low-molecular weight CXCR2 ligands. The limits of detection (LODs; injected amounts) for MK-7123, a high affinity binder to both CXCR1 and CXCR2 receptors belonging to the diaminocyclobutendione chemical class, were 40pmol in CXCR1 binding and 8pmol in CXCR2 binding. For CXCL8, the LOD was 5pmol in both binding assays. A control compound was always taken along with each bioassay plate as triplicate dose-response curve. For MK-7123, the calculated IC50 values were 314±59nM (CXCR1 binding) and 38±11nM (CXCR2 binding). For CXCL8, the IC50 values were 6.9±1.4nM (CXCR1 binding) and 2.7±1.3nM (CXCR2 binding). After optimization, the method was applied to the analysis of metabolic mixtures of eight LMW CXCR2 antagonists generated by incubation with pig liver microsomes. Moreover, metabolic profiling of the MK-7123 compound was described using the developed method. Three bioactive metabolites were found, two of which were (partially) identified. This method is suitable for bioaffinity and selectivity assessment of mixtures targeting the CXCR2. In contrary to conventional LC-MS based metabolic profiling studies done at the early lead discovery stage, additional qualitative bioactivity information of drug metabolites is obtained with the method described.

Original languageEnglish
Pages (from-to)42-53
Number of pages12
JournalJournal of Chromatography B
Volume1002
DOIs
Publication statusPublished - 2015

Fingerprint

Chemokine Receptors
Metabolites
Mass spectrometry
Mass Spectrometry
Ligands
Assays
Interleukin-8A Receptors
Liquids
Interleukin-8B Receptors
Bioassay
Bioactivity
Chemokines
Liver
Binders
Inhibitory Concentration 50
Molecular weight
Radioligand Assay
Pharmaceutical Preparations
Liver Microsomes
2-hydroxy-N,N-dimethyl-3-(2-((1-(5-methylfuran-2-yl)propyl)amino)-3,4-dioxocyclobut-1-enylamino)benzamide

Keywords

  • Chromatography, Liquid
  • HEK293 Cells
  • Humans
  • Ligands
  • Limit of Detection
  • Mass Spectrometry
  • Receptors, Interleukin-8A
  • Receptors, Interleukin-8B
  • Journal Article
  • Research Support, Non-U.S. Gov't
  • Validation Studies

Cite this

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title = "At-line coupling of LC-MS to bioaffinity and selectivity assessment for metabolic profiling of ligands towards chemokine receptors CXCR1 and CXCR2",
abstract = "This study describes an analytical method for bioaffinity and selectivity assessment of CXCR2 antagonists and their metabolites. The method is based on liquid chromatographic separation (LC) of metabolic mixtures followed by parallel mass spectrometry (MS) identification and bioaffinity determination. The bioaffinity is assessed using radioligand binding assays in 96-well plates after at-line nanofractionation. The described method was optimized for chemokines and low-molecular weight CXCR2 ligands. The limits of detection (LODs; injected amounts) for MK-7123, a high affinity binder to both CXCR1 and CXCR2 receptors belonging to the diaminocyclobutendione chemical class, were 40pmol in CXCR1 binding and 8pmol in CXCR2 binding. For CXCL8, the LOD was 5pmol in both binding assays. A control compound was always taken along with each bioassay plate as triplicate dose-response curve. For MK-7123, the calculated IC50 values were 314±59nM (CXCR1 binding) and 38±11nM (CXCR2 binding). For CXCL8, the IC50 values were 6.9±1.4nM (CXCR1 binding) and 2.7±1.3nM (CXCR2 binding). After optimization, the method was applied to the analysis of metabolic mixtures of eight LMW CXCR2 antagonists generated by incubation with pig liver microsomes. Moreover, metabolic profiling of the MK-7123 compound was described using the developed method. Three bioactive metabolites were found, two of which were (partially) identified. This method is suitable for bioaffinity and selectivity assessment of mixtures targeting the CXCR2. In contrary to conventional LC-MS based metabolic profiling studies done at the early lead discovery stage, additional qualitative bioactivity information of drug metabolites is obtained with the method described.",
keywords = "Chromatography, Liquid, HEK293 Cells, Humans, Ligands, Limit of Detection, Mass Spectrometry, Receptors, Interleukin-8A, Receptors, Interleukin-8B, Journal Article, Research Support, Non-U.S. Gov't, Validation Studies",
author = "Marija Mladic and Scholten, {Danny J} and Niessen, {Wilfried M A} and Somsen, {Govert W} and Smit, {Martine J} and Jeroen Kool",
note = "Copyright {\circledC} 2015 Elsevier B.V. All rights reserved.",
year = "2015",
doi = "10.1016/j.jchromb.2015.08.004",
language = "English",
volume = "1002",
pages = "42--53",
journal = "Journal of Chromatography B",
issn = "1570-0232",
publisher = "Elsevier",

}

TY - JOUR

T1 - At-line coupling of LC-MS to bioaffinity and selectivity assessment for metabolic profiling of ligands towards chemokine receptors CXCR1 and CXCR2

AU - Mladic, Marija

AU - Scholten, Danny J

AU - Niessen, Wilfried M A

AU - Somsen, Govert W

AU - Smit, Martine J

AU - Kool, Jeroen

N1 - Copyright © 2015 Elsevier B.V. All rights reserved.

PY - 2015

Y1 - 2015

N2 - This study describes an analytical method for bioaffinity and selectivity assessment of CXCR2 antagonists and their metabolites. The method is based on liquid chromatographic separation (LC) of metabolic mixtures followed by parallel mass spectrometry (MS) identification and bioaffinity determination. The bioaffinity is assessed using radioligand binding assays in 96-well plates after at-line nanofractionation. The described method was optimized for chemokines and low-molecular weight CXCR2 ligands. The limits of detection (LODs; injected amounts) for MK-7123, a high affinity binder to both CXCR1 and CXCR2 receptors belonging to the diaminocyclobutendione chemical class, were 40pmol in CXCR1 binding and 8pmol in CXCR2 binding. For CXCL8, the LOD was 5pmol in both binding assays. A control compound was always taken along with each bioassay plate as triplicate dose-response curve. For MK-7123, the calculated IC50 values were 314±59nM (CXCR1 binding) and 38±11nM (CXCR2 binding). For CXCL8, the IC50 values were 6.9±1.4nM (CXCR1 binding) and 2.7±1.3nM (CXCR2 binding). After optimization, the method was applied to the analysis of metabolic mixtures of eight LMW CXCR2 antagonists generated by incubation with pig liver microsomes. Moreover, metabolic profiling of the MK-7123 compound was described using the developed method. Three bioactive metabolites were found, two of which were (partially) identified. This method is suitable for bioaffinity and selectivity assessment of mixtures targeting the CXCR2. In contrary to conventional LC-MS based metabolic profiling studies done at the early lead discovery stage, additional qualitative bioactivity information of drug metabolites is obtained with the method described.

AB - This study describes an analytical method for bioaffinity and selectivity assessment of CXCR2 antagonists and their metabolites. The method is based on liquid chromatographic separation (LC) of metabolic mixtures followed by parallel mass spectrometry (MS) identification and bioaffinity determination. The bioaffinity is assessed using radioligand binding assays in 96-well plates after at-line nanofractionation. The described method was optimized for chemokines and low-molecular weight CXCR2 ligands. The limits of detection (LODs; injected amounts) for MK-7123, a high affinity binder to both CXCR1 and CXCR2 receptors belonging to the diaminocyclobutendione chemical class, were 40pmol in CXCR1 binding and 8pmol in CXCR2 binding. For CXCL8, the LOD was 5pmol in both binding assays. A control compound was always taken along with each bioassay plate as triplicate dose-response curve. For MK-7123, the calculated IC50 values were 314±59nM (CXCR1 binding) and 38±11nM (CXCR2 binding). For CXCL8, the IC50 values were 6.9±1.4nM (CXCR1 binding) and 2.7±1.3nM (CXCR2 binding). After optimization, the method was applied to the analysis of metabolic mixtures of eight LMW CXCR2 antagonists generated by incubation with pig liver microsomes. Moreover, metabolic profiling of the MK-7123 compound was described using the developed method. Three bioactive metabolites were found, two of which were (partially) identified. This method is suitable for bioaffinity and selectivity assessment of mixtures targeting the CXCR2. In contrary to conventional LC-MS based metabolic profiling studies done at the early lead discovery stage, additional qualitative bioactivity information of drug metabolites is obtained with the method described.

KW - Chromatography, Liquid

KW - HEK293 Cells

KW - Humans

KW - Ligands

KW - Limit of Detection

KW - Mass Spectrometry

KW - Receptors, Interleukin-8A

KW - Receptors, Interleukin-8B

KW - Journal Article

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

KW - Validation Studies

U2 - 10.1016/j.jchromb.2015.08.004

DO - 10.1016/j.jchromb.2015.08.004

M3 - Article

VL - 1002

SP - 42

EP - 53

JO - Journal of Chromatography B

JF - Journal of Chromatography B

SN - 1570-0232

ER -