Dataset Information

A Targeted in Vivo SILAC Approach for Quantification of Drug Metabolism Enzymes: Regulation by the Constitutive Androstane Receptor

ABSTRACT: The modulation of drug metabolism enzyme (DME) expression by therapeutic agents is a central mechanism of drug–drug interaction and should be assessed as early as possible in preclinical drug development. Direct measurement of DME levels is typically achieved by Western blotting, qPCR, or microarray, but these techniques have their limitations; antibody cross-reactivity among highly homologous subfamilies creates ambiguity, while discordance between mRNA and protein expression undermines observations. The aim of this study was to design a simple targeted workflow by combining in vivo SILAC and label-free proteomics approaches for quantification of DMEs in mouse liver, facilitating a rapid and comprehensive evaluation of metabolic potential at the protein level. A total of 197 peptides, representing 51 Phase I and Phase II DMEs, were quantified by LC-MS/MS using targeted high resolution single ion monitoring (tHR/SIM) with a defined mass-to-charge and retention time window for each peptide. In a constitutive androstane receptor (Car) activated mouse model, comparison of tHR/SIM-in vivo SILAC with Western blotting for analysis of the expression of cytochromes P450 was favorable, with agreement in fold-change values between methods. The tHR/SIM-in vivo SILAC approach therefore permits the robust analysis of multiple DME in a single protein sample, with clear utility for the assessment of the drug–drug interaction potential of candidate therapeutic compounds.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Hepatocyte, Liver

SUBMITTER: Kenneth MacLeod

LAB HEAD: Jeffrey Tze-Jen Huang

PROVIDER: PXD000675 | Pride | 2014-02-14

REPOSITORIES: Pride

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Publications

A targeted in vivo SILAC approach for quantification of drug metabolism enzymes: regulation by the constitutive androstane receptor.

Macleod A Kenneth AK Zang Tuo T Riches Zoe Z Henderson Colin J CJ Wolf C Roland CR Huang Jeffrey T-J JT

Journal of proteome research 20131212 2

The modulation of drug metabolism enzyme (DME) expression by therapeutic agents is a central mechanism of drug-drug interaction and should be assessed as early as possible in preclinical drug development. Direct measurement of DME levels is typically achieved by Western blotting, qPCR, or microarray, but these techniques have their limitations; antibody cross-reactivity among highly homologous subfamilies creates ambiguity, while discordance between mRNA and protein expression undermines observa ...[more]

PMID: 24303842

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Project description:Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics and drug-drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically-labelled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labelled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II and 8 control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11 and 3a13, carboxylesterase (Ces) 2a and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of import to xenobiotic metabolism, with improved utility for the study of drug pharmacokinetics, pharmacodynamics, and of chemically-treated and genetically-modified mouse models.

Dataset Information

A Targeted in Vivo SILAC Approach for Quantification of Drug Metabolism Enzymes: Regulation by the Constitutive Androstane Receptor

Publications

A targeted in vivo SILAC approach for quantification of drug metabolism enzymes: regulation by the constitutive androstane receptor.

Similar Datasets

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