G Protein-Coupled Receptor Screening Assays: Methods and Protocols

301 Pages·2015·10.417 MB·English

by  Duarte Miguel F. Prazeres, Sofia Aires M. Martins (eds.)

Methods in
Molecular Biology 1272
Duarte Miguel F. Prazeres
Sofi a Aires M. Martins Editors
G Protein-
Coupled Receptor
Screening Assays
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

G Protein-Coupled Receptor
Screening Assays
Methods and Protocols
Edited by
Duarte Miguel F. Prazeres
IBB – Institute for Biotechnology and Bioengineering, Lisbon, Portugal
and
Department of Bioengineering, Instituto Superior Técnico,
Universidade de Lisboa, Lisbon, Portugal
Sofia Aires M. Martins
IBB – Institute for Biotechnology and Bioengineering, Lisbon, Portugal
Editors
Duarte Miguel F. Prazeres Sofi a Aires M . Martins
IBB – Institute for Biotechnology IBB – Institute for Biotechnology
and Bioengineering and Bioengineering
Lisbon, Portugal Lisbon, Portugal
Department of Bioengineering
Instituto Superior Técnico
Universidade de Lisboa
Lisbon, Portugal
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-4939-2335-9 ISBN 978-1-4939-2336-6 (eBook)
DOI 10.1007/978-1-4939-2336-6
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2014958478
© Springer Science+Business Media New York 2 015
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this
legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifi cally for
the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work.
Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the
Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions
for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution
under the respective Copyright Law.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
While the advice and information in this book are believed to be true and accurate at the date of publication, neither
the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be
made. The publisher makes no warranty, express or implied, with respect to the material contained herein.
Printed on acid-free paper
Humana Press is a brand of Springer
Springer is part of Springer Science+Business Media (www.springer.com)
Prefa ce
G Protein-Coupled Receptors (GPCRs) are one of the largest known families of membrane
proteins, with around 1,000 GPCR-like sequences identifi ed in the human genome. These
highly specialized proteins play a key role in signal transduction, converting changes in
extracellular information into changes in intracellular functions. The diversity of ligands
that interact with GPCRs is vast and includes neurotransmitters, metals, odorants, taste
ligands, biogenic amines, fatty acids, amino acids, peptides, proteins, steroids, and light.
The involvement of GPCRs in many physiological or disease-related processes has made
them one of the favorite targets for researchers in academia and in the pharmaceutical
industry. This signifi cance is emphasized by the fact that one third of the drugs currently
available in the market interact in some way with a GPCR. Still, the fact that those drugs
target only ~30 members of the family and that ligands and structure are unknown for
around 100 “orphan” GPCRs makes it clear that their pharmacological potential remains
largely untapped.
In the past 20 years, considerable efforts have been directed towards the development
of GPCR screening assays in order to disclose GPCR acting compounds, elucidate signaling
mechanisms, or evaluate compound’s effi cacy. The majority of the approaches target par-
ticular steps in the GPCR signaling cascade ranging from the ligand binding event to func-
tional, cell-based assays where partners from both signaling and regulation mechanisms are
screened. Furthermore, the combination of genetically engineered cells expressing a large
variety of GPCRs with automatic fl uid handling and read-outs has originated high-throughput
(HTS) or high-content (HCS) platforms that can screen and assay millions of compounds
in a parallel fashion. As these technologies are solidly introduced in drug discovery pro-
grams, new and exciting fi ndings regarding GPCR signaling are being disclosed. It is now
well recognized that GPCRs can signal independently from their associated G proteins, that
in living cell the GPCR signaling is a product of a complex network of positive and negative
feedbacks from multiple receptors and that some ligands stabilize different receptor confor-
mations in such a way that different signaling pathways can be favored in detriment to oth-
ers. As a result, new screening assays, where reconstituted cell lines are being replaced with
more realistic cellular systems such as tissue or animal models and biosensor technology for
noninvasive “in vivo” cell testing, are being implemented.
These new opportunities along with the widely recognized need for better and safer
pharmaceutical drugs constitute the main motivation for editing this book. Acknowledging
the principle that no screening assays are ideal, the book “GPCR Screening Assays” intends
to provide the reader, both the beginner and the experienced researcher, with an updated
overview of not only the established but also the innovative technologies that promise to
advance GPCR drug research. The book targets all those involved in the discovery of
GPCR-active drugs, whether they come from academia or industry, but also the novice
who is being introduced to the subject.
The book is organized into two major sections: (1) Introduction and (2) GPCR
screening assays. The topics presented and discussed in the introductory chapter of the fi rst
v
vi Preface
section provide the necessary foundations for the understanding of GPCR action and the
rationale behind the design of the available screening assays. In Part II, detailed protocols
are provided for different screening approaches. The individual chapters were selected and
laid down to provide a transversal overview of the different levels of GPCR signaling that
are addressable in the different screening strategies and present practical examples of how
current assay technologies are contributing to new paradigms in GPCR drug research.
Lisbon, Portugal D uarte Miguel F. Prazeres
Sofia Aires M. Martins
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
PART I INTRODUCTION
1 G protein-Coupled Receptors: An Overview of Signaling
Mechanisms and Screening Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Duarte M iguel F . P razeres and Sofia A ires M . M artins
PART II GPCR SCREENING ASSAYS
2 Time-Resolved FRET Strategy to Screen GPCR Ligand Library . . . . . . . . . . . 2 3
Nadia Oueslati , Candide Hounsou , Abderazak Belhocine ,
Thieric R odriguez , Elodie Dupuis , J urriaan M . Z wier , Eric T rinquet ,
Jean-Philippe P in , and Thierry Durroux
3 Homogeneous Fluorescence Anisotropy-Based Assay
for Characterization of Ligand Binding Dynamics to GPCRs
in Budded Baculoviruses: The Case of Cy3B-NDP-α-MSH Binding
to MC Receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4
Santa Veiksina , S ergei Kopanchuk , Olga Mazina , Reet L ink ,
Anne Lille , and Ago R inken
4 C onstruction of Recombinant HEK293 Cell Lines
for the Expression of the Neurotensin Receptor NTSR1 . . . . . . . . . . . . . . . . . 51
Su X iao , J oseph Shiloach , and Reinhard G risshammer
5 c AMP Assay for GPCR Ligand Characterization:
Application of BacMam Expression System . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 5
Olga M azina , Anni A llikalt , A nnika H einloo ,
Reet R einart-Okugbeni , S ergei Kopanchuk , and Ago Rinken
6 C a2+ Mobilization Assays in GPCR Drug Discovery. . . . . . . . . . . . . . . . . . . . . 79
Grzegorz Woszczek and E lisabeth F uerst
7 U sing Constitutive Activity to Define Appropriate High-T hroughput
Screening Assays for Orphan G Protein- Coupled Receptors. . . . . . . . . . . . . . . 91
Tony N go , J ames L . J. Coleman , and Nicola J . Smith
8 Monitoring G Protein-Coupled Receptor Activation
Using the Protein Fragment Complementation Technique Split TEV . . . . . . . 107
Michael C. W ehr , S abrina G alinski , and Moritz J . Rossner
vii
viii Contents
9 Quantifying GPCR Internalization: A Focus on the Kisspeptin Receptor. . . . . 1 19
Macarena Pampillo and Andy V . B abwah
10 G PCR Oligomerization Analysis by Means of BRET and dFRAP. . . . . . . . . . . 133
Francisco C iruela and V íctor F ernández-Dueñas
11 U se of ImageJ to Recover Information from Individual Cells
in a G Protein-Coupled Receptor Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
João R . C. T rabuco , S ofia Aires M . M artins ,
and Duarte M iguel F . P razeres
12 Methods to Immobilize GPCR on the Surface of SPR Sensors . . . . . . . . . . . . 1 73
Laura M artínez-Muñoz , R ubén B arroso , A nabel G uedán Paredes ,
Mario M ellado , and José M iguel R odríguez-Frade
13 O lfactory Receptor Screening Assay Using Nanovesicle-I mmobilized
Carbon Nanotube Transistor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Jong Hyun Lim , Juhun P ark , S eunghun H ong , and Tai H yun Park
14 Label-Free Biosensor Assays in GPCR Screening. . . . . . . . . . . . . . . . . . . . . . . 1 99
Manuel Grundmann and Evi K ostenis
15 M ultidimensional GPCR Profiling and Screening
Using Impedance-Based Label-Free and Real-Time Assay . . . . . . . . . . . . . . . . 2 15
Ning K e , K hanh Nguyen , J effery I relan , and Yama A. Abassi
16 L abel-Free Functional Selectivity Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 27
Ann M. F errie , Vasiliy G oral , C haoming W ang , and Ye F ang
17 Measurement of Surface-Mediated Ca2+ Transients
on the Single-Cell Level in a Microfluidic Lab-on-a-Chip Environment. . . . . . 247
Michael K irschbaum , Magnus S . Jaeger , and Claus Duschl
18 Cell-Based Assays and Animal Models for GPCR Drug Screening . . . . . . . . . . 2 57
Hideo T akakura , Mitsuru H attori , Miho Tanaka , and Takeaki Ozawa
19 Computer-Aided Design of GPCR Ligands. . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Hugo Gutiérrez-de-Terán , H enrik K eränen , Jhonny Azuaje ,
David R odríguez , Johan Åqvist , and Eddy Sotelo
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 93
Contributors
YAMA A. A BASSI • ACEA Biosciences , San Diego, C A , U SA
ANNI A LLIKALT • Institute of Chemistry , U niversity of Tartu , T artu , E stonia ;
Competence Centre on Health Technologies , T artu, E stonia
JOHAN ÅQVIST • Department of Cell and Molecular Biology , U ppsala University,
Biomedical Center , U ppsala , S weden
JHONNY AZUAJE • Center for Research in Biological Chemistry and Molecular Materials,
University of Santiago de Compostela , A. Coruña, S pain
ANDY V. BABWAH • Children’s Health Research Institute, Victoria Research Laboratories ,
London , O N, C anada ; L awson Health Research Institute , L ondon , ON, C anada ;
Department of Obstetrics and Gynecology , T he University of Western Ontario , London ,
ON , C anada ; Department of Physiology and Pharmacology, The University of Western
Ontario , L ondon , ON, Canada
RUBÉN BARROSO • Department of Immunology and Oncology , C entro Nacional de
Biotecnología (CNB/CSIC) , M adrid, Spain
ABDERAZAK BELHOCINE • Institut de Génomique Fonctionnelle, CNRS, UMR 5203 ,
Montpellier, F rance ; INSERM , M ontpellier, France ; U niversité Montpellier , M ontpellier,
France
FRANCISCO CIRUELA • Unitat de Farmacologia, Departament Patologia i Terapèutica
Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona , Barcelona,
Spain
JAMES L. J. COLEMAN • Molecular Cardiology Division, Victor Chang Cardiac Research
Institute , D arlinghurst, NSW , A ustralia ; St. Vincent’s Clinical School, U niversity of
New South Wales , Sydney, N SW , Australia
ELODIE DUPUIS • Cisbio Bioassays , C odolet , F rance
THIERRY D URROUX • Institut de Génomique Fonctionnelle, CNRS, UMR 5203 ,
Montpellier, F rance ; INSERM , Montpellier, France ; U niversité Montpellier , M ontpellier,
France
CLAUS DUSCHL • Fraunhofer Institute for Cell Therapy and Immunology IZI Branch
Bioanalytics and Bioprocesses , Potsdam, G ermany
YE FANG • Science and Technology Division, Biochemical Technologies, C orning
Incorporated , C orning, NY , U SA
VÍCTOR FERNÁNDEZ-DUEÑAS • Unitat de Farmacologia, Departament Patologia i
Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona ,
Barcelona, S pain
ANN M. F ERRIE • Science and Technology Division, Biochemical Technologies, C orning
Incorporated , C orning, NY , U SA
ELISABETH FUERST • Division of Asthma, Allergy and Lung Biology, King’s College
London , L ondon , U K ; MRC & Asthma UK Centre in Allergic Mechanisms of Asthma ,
London, U K
ix