Cytochrome P450 Protocols

320 Pages·2013·5.919 MB·English

by  Douglas S. Auld, Henrike Veith, James J. Cali (auth.), Ian R. Phillips, Elizabeth A. Shephard, Paul R. Ortiz de Montellano (eds.)

Methods in
Molecular Biology 987
Ian R. Phillips
Elizabeth A. Shephard
Paul R. Ortiz de Montellano Editors
Cytochrome
P450
Protocols
Third Edition
M M B ™
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

Cytochrome P450 Protocols
Third Edition
Edited by
Ian R. Phillips
Division of Biosciences, University College London, London, UK;
School of Biological and Chemical Sciences, Queen Mary, University of London, London, UK
Elizabeth A. Shephard
Department of Structural and Molecular Biology, University College London, London, UK
Paul R. Ortiz de Montellano
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA
Editors
Ian R. Phillips Elizabeth A. Shephard
Division of Biosciences Department of Structural and Molecular Biology
University College London University College London
London, UK London, UK
School of Biological and Chemical Sciences
Paul R. Ortiz de Montellano
Queen Mary
Department of Pharmaceutical Chemistry
University of London
University of California
London, UK
San Francisco, CA, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-62703-320-6 978-1-62703-321-3 (eBook)
DOI 10.1007/978-1-62703-321-3
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2013932010
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Preface
Cytochromes P450 (CYPs) comprise a large superfamily of proteins that are of central
importance in the detoxifi cation or activation of a tremendous number of natural and syn-
thetic hydrophobic xenobiotics, including many therapeutic drugs, chemical carcinogens,
and environmental pollutants. Some CYPs catalyze the metabolism of endogenous com-
pounds, particularly the ones involved in signaling. CYPs, therefore, are important in medi-
ating interactions between an organism and its chemical environment and in the regulation
of physiological processes. Many CYPs are inducible by the compounds they metabolize. In
addition, genetic polymorphisms of C YP genes affect expression or activity of the enzymes,
which can result in adverse drug reactions or genetic diseases. Consequently, CYPs are
among the most extensively studied groups of proteins, being investigated by researchers in
fi elds as diverse as biochemistry, molecular biology, pharmacology, toxicology, environmen-
tal biology, and genetics. The wide range of techniques that have been applied to the CYPs
re fl ects the diverse backgrounds of the many researchers active in this fi eld.
Previous editions of C ytochrome P450 Protocols contained collections of key “core”
techniques, most of which are still relevant. The emphasis, however, was on methods for
the investigation of individual CYPs and substrates, mostly in an in vitro context. The cur-
rent edition focuses on high-throughput methods for the simultaneous analysis of multiple
CYPs, substrates, or ligands. Although the emphasis is on CYPs of mammalian origin, it
re fl ects an increasing interest in CYPs of bacterial species. However, most of the methods
described are suitable for the investigation of CYPs from any source. Also included are
chapters on CYP reductase (the redox partner of CYPs) and the fl avin-containing monoox-
ygenases (FMOs), another family of proteins that are important in the metabolism of for-
eign chemicals, and that share several substrates in common with the CYPs.
The chapters of this edition of C ytochrome P450 Protocols , although not formally divided
into sections, are grouped loosely according to topic. Included are high-throughput meth-
ods for identifi cation of substrates, ligands, and inhibitors of CYPs; metabolomic and lipi-
domic approaches for identifi cation of endogenous substrates of CYPs (“de-orphanizing”
CYP substrates); reconstitution systems for the incorporation of modifi ed and novel metal-
loporphyrins into CYPs in vivo or for developing nanoparticle bioreactors for biophysical
and mechanistic studies of CYPs and drug-metabolite profi ling; high-throughput assays for
measuring the activity of CYPs and for identifi cation of their substrates and adducts; meth-
ods for the generation and quantifi cation of novel CYPs and for identifi cation of their
potential substrates; techniques for phenotyping, genotyping, and identifi cation of tran-
scriptional regulatory sequences; a high-throughput method for the generation of libraries
of redox-self-suffi cient CYP biocatalysts; a guide to CYP allele nomenclature; and methods
for the isolation of mouse primary hepatocytes, for the differentiation of a hepatoma cell
line into cells with hepatocyte-like metabolic properties, and for transfection of such cells
with DNA and siRNA constructs to investigate the function and regulation of expression
of CYPs.
Each chapter is written by researchers who have been involved in the development and
application of the particular technique. Protocols are presented in a step-by-step manner,
v
vi Preface
with extensive cross-references to notes that highlight critical steps, potential problems, and
alternative methods. We hope that this format will enable researchers who have no previous
knowledge of the technique to understand the basis of the method and to perform it
successfully.
We are extremely grateful to all the authors who contributed so generously to this
volume and to John Walker, the series editor of M ethods in Molecular Biology , for his advice
and patience.
London, UK Ian R. Phillips
London, UK Elizabeth A. Shephard
San Francisco, CA, USA Paul R. Ortiz de Montellano
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Bioluminescent Assays for Cytochrome P450 Enzymes. . . . . . . . . . . . . . . . . . . . . . 1
Douglas S. Auld, Henrike Veith, and James J. Cali
2 Simultaneous Determination of Multiple CYP Inhibition Constants
using a Cocktail-Probe Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Michael Zientek and Kuresh Youdim
3 High-Throughput Mass Spectrometric Cytochrome
P450 Inhibition Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Kheng B. Lim, Can C. Ozbal, and Daniel B. Kassel
4 The Synthesis, Characterization, and Application of 13C-Methyl
Isocyanide as an NMR Probe of Heme Protein Active Sites. . . . . . . . . . . . . . . . . . . 51
Christopher McCullough, Phani Kumar Pullela, Sang-Choul Im,
Lucy Waskell, and Daniel Sem
5 High-Throughput Fluorescence Assay for
Cytochrome P450 Mechanism-Based Inactivators. . . . . . . . . . . . . . . . . . . . . . . . . . 61
Cesar Kenaan, Haoming Zhang, and Paul F. Hollenberg
6 Identification of Endogenous Substrates of Orphan Cytochrome
P450 Enzymes Through the Use of Untargeted Metabolomics Approaches . . . . . . 71
Qian Cheng and F. Peter Guengerich
7 Genetic and Mass Spectrometric Tools for Elucidating
the Physiological Function(s) of Cytochrome P450 Enzymes
from Mycobacterium tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Hugues Ouellet, Eric D. Chow, Shenheng Guan, Jeffery S. Cox,
Alma L. Burlingame, and Paul R. Ortiz de Montellano
8 An Escherichia coli Expression-Based Approach for Porphyrin
Substitution in Heme Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Michael B. Winter, Joshua J. Woodward, and Michael A. Marletta
9 Expression in E scherichia coli of a Cytochrome P450 Enzyme
with a Cobalt Protoporphyrin IX Prosthetic Group. . . . . . . . . . . . . . . . . . . . . . . . . 107
Wesley E. Straub, Clinton R. Nishida, and Paul R. Ortiz de Montellano
10 Nanodiscs in the Studies of Membrane-Bound Cytochrome
P450 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
A. Luthra, M. Gregory, Y.V. Grinkova, I.G. Denisov, and S.G. Sligar
11 Rapid LC-MS Drug Metabolite Profiling Using Bioreactor Particles. . . . . . . . . . . . 129
Linlin Zhao, Besnik Bajrami, and James F. Rusling
12 Fluorescence-Based Screening of Cytochrome P450 Activities
in Intact Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
M. Teresa Donato and M. José Gómez-Lechón
vii
viii Contents
13 Screening for Cytochrome P450 Reactivity with a Reporter Enzyme. . . . . . . . . . . . 149
Kersten S. Rabe and Christof M. Niemeyer
14 High-Throughput Fluorescence Assay of Cytochrome P450 3A4 . . . . . . . . . . . . . . 157
Qian Cheng and F. Peter Guengerich
15 Targeted Protein Capture for Analysis of Electrophile-Protein Adducts. . . . . . . . . . 163
Rebecca E. Connor, Simona G. Codreanu, Lawrence J. Marnett,
and Daniel C. Liebler
16 DNA Shuffling of Cytochrome P450 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
James B.Y.H. Behrendorff, Wayne A. Johnston,
and Elizabeth M.J. Gillam
17 Measurement of P450 Difference Spectra Using Intact Cells. . . . . . . . . . . . . . . . . . 189
Wayne A. Johnston and Elizabeth M.J. Gillam
18 DNA Shuffling of Cytochromes P450 for Indigoid Pigment Production. . . . . . . . . 205
Nedeljka N. Rosic
19 P450 Oxidoreductase: Genotyping, Expression, Purification of Recombinant
Protein, and Activity Assessments of Wild-Type and Mutant Protein. . . . . . . . . . . . 225
Vishal Agrawal and Walter L. Miller
20 LICRED: A Versatile Drop-In Vector for Rapid Generation
of Redox-Self-Sufficient Cytochromes P450 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Federico Sabbadin, Gideon Grogan, and Neil C. Bruce
21 Update on Allele Nomenclature for Human Cytochromes P450 and the
Human Cytochrome P450 Allele (CYP-Allele) Nomenclature Database . . . . . . . . . 251
Sarah C. Sim and Magnus Ingelman-Sundberg
22 Simultaneous In Vivo Phenotyping of CYP Enzymes . . . . . . . . . . . . . . . . . . . . . . . 261
Sussan Ghassabian and Michael Murray
23 Detection of Regulatory Polymorphisms: High-Throughput Capillary
DNase I Footprinting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Matthew Hancock and Elizabeth A. Shephard
24 Isolation of Mouse Hepatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Mina Edwards, Lyndsey Houseman, Ian R. Phillips,
and Elizabeth A. Shephard
25 Highly Efficient SiRNA and Gene Transfer into Hepatocyte-Like
HepaRG Cells and Primary Human Hepatocytes: New Means
for Drug Metabolism and Toxicity Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Véronique Laurent, Denise Glaise, Tobias Nübel, David Gilot,
Anne Corlu, and Pascal Loyer
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Contributors
VISHAL AGRAWAL • Department of Pediatrics, University of California San Francisco,
San Francisco, CA , USA
DOUGLAS S. AULD • Novartis Institutes for Biomedical Research, Cambridge,
MA, USA
BESNIK BAJRAMI • Department of Chemistry , University of Connecticut, Storrs ,
CT , USA
JAMES B. Y. H. BEHRENDORFF • Australian Institute for Bioengineering and
Nanotechnology, University of Queensland, Brisbane, Australia
NEIL C. BRUCE • Department of Biology, York University, York , UK
ALMA L. BURLINGAME • Department of Pharmaceutical Chemistry ,
University of California, San Francisco, CA , USA
JAMES J. CALI • Promega Corp. , Madison , WI , USA
QIAN CHENG • Department of Biochemistry and Center in Molecular Toxicology,
Vanderbilt University School of Medicine, Nashville, TN, USA
ERIC D. C HOW • Department of Microbiology and Immunology, University
of California, San Francisco, CA , USA
SIMONA G. CODREANU • Department of Biochemistry, Vanderbilt University
School of Medicine, Nashville , TN , USA ; Jim Ayers Institute for Precancer Detection
and Diagnosis, Vanderbilt University School of Medicine, Nashville , TN , USA
REBECCA E. CONNOR • Department of Biochemistry , Vanderbilt University
School of Medicine, Nashville , TN , USA
ANNE CORLU • Inserm UMR991 Foie, Métabolismes et Cancer, Hôpital Pontchaillou
and Université de Rennes 1, Rennes, France
JEFFERY S. COX • Department of Microbiology and Immunology,
University of California, San Francisco, CA , USA
PAUL R. ORTIZ DE MONTELLANO • Department of Pharmaceutical Chemistry ,
University of California, San Francisco, CA , USA
I. G. DENISOV • Department of Biochemistry, University of Illinois, Urbana , IL , USA
M. TERESA DONATO • Departamento de Bioquímica y Biología Molecular, Universidad
de Valencia , Valencia, Spain ; CIBERehd, Fondo de Investigaciones Sanitarias,
Barcelona, Spain
MINA EDWARDS • Institute of Structural and Molecular Biology,
University College London, London , UK
SUSSAN GHASSABIAN • Faculty of Pharmacy, University of Sydney, Sydney, NSW ,
Australia ; Centre for Integrated Preclinical Drug Development,
University of Queensland, Brisbane, Australia
ELIZABETH M. J. GILLAM • School of Chemistry and Molecular Biosciences,
University of Queensland, Brisbane, Australia
DAVID GILOT • CNRS UMR6061, Institut de Génétique et Développement de Rennes,
Université de Rennes 1, Rennes, France
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