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

Mladic M, Scholten DJ, Wijtmans M, Falck D, Leurs R, Niessen WM, Smit MJ, Kool J - Anal Bioanal Chem (2015)

Bottom Line: The method is based on mass spectrometric (MS) identification after liquid chromatographic (LC) separation of metabolic mixtures.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.

View Article: PubMed Central - PubMed

Affiliation: Division of BioAnalytical Chemistry, Amsterdam Institute for Molecules Medicines and Systems, VU University Amsterdam, De Boelelaan 1083, 1081HV, Amsterdam, The Netherlands.

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.

No MeSH data available.


Related in: MedlinePlus

Analysis of a metabolic mixture of VUF11211, a small molecule ligand towards the CXCR3 receptor. Chemical structure of the parent compound with the MS2 fragmentation scheme is inserted in the figure. I and II Reconstructed bioaffinity chromatograms after LC separation and nanofractionation of the metabolic mixture after duplicate injections at two different concentrations corresponding to 20 and 100 μM pre-incubation concentration of the parent compound. III Reconstructed bioaffinity chromatogram after LC separation and nanofractionation of the metabolic mixture after duplicate injections at the concentration corresponding to 50 μM pre-incubation concentration of the parent compound. Radioligand binding assay was performed with 125 pM 125I-CXCL10 radioligand. IV LC-MS traces depicted as extracted ion currents (XICs) of parent compound (black trace) and formed metabolites (color coded notation). Correlation between structure identity and bioaffinity is enabled by the parallel bioaffinity and MS analysis. All bioaffinity chromatograms are scaled equally and shown as an average of a duplicate measurement, where the error bars reflect the variation between the two separate measurements
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Fig7: Analysis of a metabolic mixture of VUF11211, a small molecule ligand towards the CXCR3 receptor. Chemical structure of the parent compound with the MS2 fragmentation scheme is inserted in the figure. I and II Reconstructed bioaffinity chromatograms after LC separation and nanofractionation of the metabolic mixture after duplicate injections at two different concentrations corresponding to 20 and 100 μM pre-incubation concentration of the parent compound. III Reconstructed bioaffinity chromatogram after LC separation and nanofractionation of the metabolic mixture after duplicate injections at the concentration corresponding to 50 μM pre-incubation concentration of the parent compound. Radioligand binding assay was performed with 125 pM 125I-CXCL10 radioligand. IV LC-MS traces depicted as extracted ion currents (XICs) of parent compound (black trace) and formed metabolites (color coded notation). Correlation between structure identity and bioaffinity is enabled by the parallel bioaffinity and MS analysis. All bioaffinity chromatograms are scaled equally and shown as an average of a duplicate measurement, where the error bars reflect the variation between the two separate measurements

Mentions: The validation of the analytical method was proceeded with the metabolic profiling of the two lead compounds NBI-74330 and VUF11211. After in vitro preparation of the metabolic mixtures, the workup procedure involved reconstitution of metabolic mixtures in eluent A after protein precipitation and evaporation. All the measurements have been done in duplicate using optimized method. The metabolic profiles of NBI-74330 and VUF11211 are shown in Figs. 6 and 7, respectively. The bottom part of both figures represents extracted ion chromatograms (XICs) of parent compound and formed metabolites (metabolite number annotation, with corresponding m/z values presented in Tables 1 and 2). The upper part of the figures shows reconstructed bioaffinity chromatograms of two different dilutions of the metabolic mixtures injected. As with the parent compounds, the negative peaks reflect binding to CXCR3 measured in the radioligand binding assay. Correlation between structure identity and bioaffinity is enabled by the parallel bioaffinity and MS analysis. Identification of metabolites is based on shifts in accurate mass of parent and formed metabolites and of their fragments in MS2. Chemical structures of NBI-74330 and VUF11211 showing their fragmentation patterns in MS2 are inserted in Figs. 6 and 7, respectively. Tabulated information on the structure elucidation of the metabolites formed from NBI-74330 and VUF11211 is given in Tables 1 and 2, respectively.Fig. 6


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

Mladic M, Scholten DJ, Wijtmans M, Falck D, Leurs R, Niessen WM, Smit MJ, Kool J - Anal Bioanal Chem (2015)

Analysis of a metabolic mixture of VUF11211, a small molecule ligand towards the CXCR3 receptor. Chemical structure of the parent compound with the MS2 fragmentation scheme is inserted in the figure. I and II Reconstructed bioaffinity chromatograms after LC separation and nanofractionation of the metabolic mixture after duplicate injections at two different concentrations corresponding to 20 and 100 μM pre-incubation concentration of the parent compound. III Reconstructed bioaffinity chromatogram after LC separation and nanofractionation of the metabolic mixture after duplicate injections at the concentration corresponding to 50 μM pre-incubation concentration of the parent compound. Radioligand binding assay was performed with 125 pM 125I-CXCL10 radioligand. IV LC-MS traces depicted as extracted ion currents (XICs) of parent compound (black trace) and formed metabolites (color coded notation). Correlation between structure identity and bioaffinity is enabled by the parallel bioaffinity and MS analysis. All bioaffinity chromatograms are scaled equally and shown as an average of a duplicate measurement, where the error bars reflect the variation between the two separate measurements
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4551560&req=5

Fig7: Analysis of a metabolic mixture of VUF11211, a small molecule ligand towards the CXCR3 receptor. Chemical structure of the parent compound with the MS2 fragmentation scheme is inserted in the figure. I and II Reconstructed bioaffinity chromatograms after LC separation and nanofractionation of the metabolic mixture after duplicate injections at two different concentrations corresponding to 20 and 100 μM pre-incubation concentration of the parent compound. III Reconstructed bioaffinity chromatogram after LC separation and nanofractionation of the metabolic mixture after duplicate injections at the concentration corresponding to 50 μM pre-incubation concentration of the parent compound. Radioligand binding assay was performed with 125 pM 125I-CXCL10 radioligand. IV LC-MS traces depicted as extracted ion currents (XICs) of parent compound (black trace) and formed metabolites (color coded notation). Correlation between structure identity and bioaffinity is enabled by the parallel bioaffinity and MS analysis. All bioaffinity chromatograms are scaled equally and shown as an average of a duplicate measurement, where the error bars reflect the variation between the two separate measurements
Mentions: The validation of the analytical method was proceeded with the metabolic profiling of the two lead compounds NBI-74330 and VUF11211. After in vitro preparation of the metabolic mixtures, the workup procedure involved reconstitution of metabolic mixtures in eluent A after protein precipitation and evaporation. All the measurements have been done in duplicate using optimized method. The metabolic profiles of NBI-74330 and VUF11211 are shown in Figs. 6 and 7, respectively. The bottom part of both figures represents extracted ion chromatograms (XICs) of parent compound and formed metabolites (metabolite number annotation, with corresponding m/z values presented in Tables 1 and 2). The upper part of the figures shows reconstructed bioaffinity chromatograms of two different dilutions of the metabolic mixtures injected. As with the parent compounds, the negative peaks reflect binding to CXCR3 measured in the radioligand binding assay. Correlation between structure identity and bioaffinity is enabled by the parallel bioaffinity and MS analysis. Identification of metabolites is based on shifts in accurate mass of parent and formed metabolites and of their fragments in MS2. Chemical structures of NBI-74330 and VUF11211 showing their fragmentation patterns in MS2 are inserted in Figs. 6 and 7, respectively. Tabulated information on the structure elucidation of the metabolites formed from NBI-74330 and VUF11211 is given in Tables 1 and 2, respectively.Fig. 6

Bottom Line: The method is based on mass spectrometric (MS) identification after liquid chromatographic (LC) separation of metabolic mixtures.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.

View Article: PubMed Central - PubMed

Affiliation: Division of BioAnalytical Chemistry, Amsterdam Institute for Molecules Medicines and Systems, VU University Amsterdam, De Boelelaan 1083, 1081HV, Amsterdam, The Netherlands.

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.

No MeSH data available.


Related in: MedlinePlus