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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

Nanofractionation and assay optimization. a The optimization of the concentration of the membrane preparation. Fractions were collected in 12 s/well resolution after 100 μL injection and separation of mixture of NBI-74330 and VUF11211 with 20 μM end concentration per compound. Traces I–IV are reconstructed bioaffinity chromatograms from the radioligand binding assay for CXCR3 receptor binding. UV chromatogram trace (V) obtained after injection of 200 μM is given for the correlation with the identity of the compounds. Bioaffinity traces correspond to bioaffinity chromatograms obtained when final concentrations of proteins in the membrane preparation were: 16 μg/well (I), 8 μg/well (II), 4 μg/well (III), and 2 μg/well (IV), respectively. The end concentration of 3H-VUF11211 radioligand was 2 nM. 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. b The optimization of the nanofractionation resolution. Fractions were collected after 100 μL injection and separation of mixture of NBI-74330 and VUF11211 with 20 μM end concentration per compound. Traces I–IV are bioaffinity chromatograms from the radioligand binding assay for CXCR3 receptor binding when four different nanofractionation resolutions were applied. UV chromatogram trace (V) obtained after injection of 200 μM is given for the correlation with the identity of the compounds. Traces I–IV correspond to bioaffinity chromatograms obtained with 24, 12, 6, and 3 s/well nanofractionation resolution, respectively. The end concentration of the membrane preparation was 8 μg/well and [3H]-VUF11211 radioligand concentration was 2 nM. 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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Fig2: Nanofractionation and assay optimization. a The optimization of the concentration of the membrane preparation. Fractions were collected in 12 s/well resolution after 100 μL injection and separation of mixture of NBI-74330 and VUF11211 with 20 μM end concentration per compound. Traces I–IV are reconstructed bioaffinity chromatograms from the radioligand binding assay for CXCR3 receptor binding. UV chromatogram trace (V) obtained after injection of 200 μM is given for the correlation with the identity of the compounds. Bioaffinity traces correspond to bioaffinity chromatograms obtained when final concentrations of proteins in the membrane preparation were: 16 μg/well (I), 8 μg/well (II), 4 μg/well (III), and 2 μg/well (IV), respectively. The end concentration of 3H-VUF11211 radioligand was 2 nM. 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. b The optimization of the nanofractionation resolution. Fractions were collected after 100 μL injection and separation of mixture of NBI-74330 and VUF11211 with 20 μM end concentration per compound. Traces I–IV are bioaffinity chromatograms from the radioligand binding assay for CXCR3 receptor binding when four different nanofractionation resolutions were applied. UV chromatogram trace (V) obtained after injection of 200 μM is given for the correlation with the identity of the compounds. Traces I–IV correspond to bioaffinity chromatograms obtained with 24, 12, 6, and 3 s/well nanofractionation resolution, respectively. The end concentration of the membrane preparation was 8 μg/well and [3H]-VUF11211 radioligand concentration was 2 nM. 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 method was first optimized for the concentration of [3H]-VUF11211 radioligand, the amount of used membranes expressing the CXCR3 receptor, and the nanofractionation resolution after LC separation. The optimal radioligand concentration was selected to be 2 nM per well based on the initial results of the Kd determination that was performed for the radioligand characterization [31]. Subsequently, the method was optimized for the amount of membranes expressing CXCR3 that was used per well of the bioassay microtiter plate. A concentration series of membrane preparations (16, 8, 4, and 2 μg/well) was tested in duplicate, and the results are shown in Fig. 2a (12 s/well nanofractionation resolution). A bioaffinity peak was defined as a decrease in signal more than three times the standard deviation of the average baseline and should consist of more than two data points. The peaks from the bioaffinity traces corresponded to the LC-UV trace (Fig. 2a trace V) and were matched to either VUF11211 or NBI-74330. The identity of compounds was confirmed by using accurate mass measurements obtained with high-resolution MS. The two peaks observed for every superimposed chromatogram in Fig. 2a correspond to the two compounds in the standard mixture and are easily detected in all four bioaffinity traces. For all conditions, the Z′ factor was calculated, which is a statistical measure of effect size taking intra-experimental variability into account. The membrane concentration of 8 μg/well was selected for further experiments as it displayed the highest Z′ factor 0.60, suggesting that it is an excellent assay in terms of baseline separation and intra-assay variability (Fig. 2a).Fig. 2


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)

Nanofractionation and assay optimization. a The optimization of the concentration of the membrane preparation. Fractions were collected in 12 s/well resolution after 100 μL injection and separation of mixture of NBI-74330 and VUF11211 with 20 μM end concentration per compound. Traces I–IV are reconstructed bioaffinity chromatograms from the radioligand binding assay for CXCR3 receptor binding. UV chromatogram trace (V) obtained after injection of 200 μM is given for the correlation with the identity of the compounds. Bioaffinity traces correspond to bioaffinity chromatograms obtained when final concentrations of proteins in the membrane preparation were: 16 μg/well (I), 8 μg/well (II), 4 μg/well (III), and 2 μg/well (IV), respectively. The end concentration of 3H-VUF11211 radioligand was 2 nM. 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. b The optimization of the nanofractionation resolution. Fractions were collected after 100 μL injection and separation of mixture of NBI-74330 and VUF11211 with 20 μM end concentration per compound. Traces I–IV are bioaffinity chromatograms from the radioligand binding assay for CXCR3 receptor binding when four different nanofractionation resolutions were applied. UV chromatogram trace (V) obtained after injection of 200 μM is given for the correlation with the identity of the compounds. Traces I–IV correspond to bioaffinity chromatograms obtained with 24, 12, 6, and 3 s/well nanofractionation resolution, respectively. The end concentration of the membrane preparation was 8 μg/well and [3H]-VUF11211 radioligand concentration was 2 nM. 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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Related In: Results  -  Collection

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Fig2: Nanofractionation and assay optimization. a The optimization of the concentration of the membrane preparation. Fractions were collected in 12 s/well resolution after 100 μL injection and separation of mixture of NBI-74330 and VUF11211 with 20 μM end concentration per compound. Traces I–IV are reconstructed bioaffinity chromatograms from the radioligand binding assay for CXCR3 receptor binding. UV chromatogram trace (V) obtained after injection of 200 μM is given for the correlation with the identity of the compounds. Bioaffinity traces correspond to bioaffinity chromatograms obtained when final concentrations of proteins in the membrane preparation were: 16 μg/well (I), 8 μg/well (II), 4 μg/well (III), and 2 μg/well (IV), respectively. The end concentration of 3H-VUF11211 radioligand was 2 nM. 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. b The optimization of the nanofractionation resolution. Fractions were collected after 100 μL injection and separation of mixture of NBI-74330 and VUF11211 with 20 μM end concentration per compound. Traces I–IV are bioaffinity chromatograms from the radioligand binding assay for CXCR3 receptor binding when four different nanofractionation resolutions were applied. UV chromatogram trace (V) obtained after injection of 200 μM is given for the correlation with the identity of the compounds. Traces I–IV correspond to bioaffinity chromatograms obtained with 24, 12, 6, and 3 s/well nanofractionation resolution, respectively. The end concentration of the membrane preparation was 8 μg/well and [3H]-VUF11211 radioligand concentration was 2 nM. 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 method was first optimized for the concentration of [3H]-VUF11211 radioligand, the amount of used membranes expressing the CXCR3 receptor, and the nanofractionation resolution after LC separation. The optimal radioligand concentration was selected to be 2 nM per well based on the initial results of the Kd determination that was performed for the radioligand characterization [31]. Subsequently, the method was optimized for the amount of membranes expressing CXCR3 that was used per well of the bioassay microtiter plate. A concentration series of membrane preparations (16, 8, 4, and 2 μg/well) was tested in duplicate, and the results are shown in Fig. 2a (12 s/well nanofractionation resolution). A bioaffinity peak was defined as a decrease in signal more than three times the standard deviation of the average baseline and should consist of more than two data points. The peaks from the bioaffinity traces corresponded to the LC-UV trace (Fig. 2a trace V) and were matched to either VUF11211 or NBI-74330. The identity of compounds was confirmed by using accurate mass measurements obtained with high-resolution MS. The two peaks observed for every superimposed chromatogram in Fig. 2a correspond to the two compounds in the standard mixture and are easily detected in all four bioaffinity traces. For all conditions, the Z′ factor was calculated, which is a statistical measure of effect size taking intra-experimental variability into account. The membrane concentration of 8 μg/well was selected for further experiments as it displayed the highest Z′ factor 0.60, suggesting that it is an excellent assay in terms of baseline separation and intra-assay variability (Fig. 2a).Fig. 2

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