Label-Free Technologies For Drug Discovery E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 446

Veröffentlichungsjahr: 2011

Beschreibung

Over the past two decades the benefits of label-free biosensor analysis have begun to make an impact in the market, and systems are beginning to be used as mainstream research tools in many drug discovery laboratories.

Label-Free Technologies For Drug Discovery summarises the latest and emerging developments in label-free detection systems, their underlying technology principles and end-user case studies that reveal the power and limitations of label-free in all areas of drug discovery.

Label-free technologies discussed include SPR, NMR, high-throughput mass spectrometry, resonant waveguide plate-based screening, transmitted-light imaging, isothermal titration calorimetry, optical and impedance cell-based assays and other biophysical methods. The technologies are discussed in relation to their use as screening technologies, high-content technologies, hit finding and hit validation strategies, mode of action and ADME/T, access to difficult target classes, cell-based receptor/ligand interactions particularly orphan receptors, and antibody and small molecule affinity and kinetic analysis.

Label-Free Technologies For Drug Discovery is an essential guide to this emerging class of tools for researchers in drug discovery and development, particularly high-throughput screening and compound profiling teams, medicinal chemists, structural biologists, assay developers, ADME/T specialists, and others interested in biomolecular interaction analysis.

Leseprobe

Contents

Cover

Title Page

Copyright

Preface

List of Contributors

1: The Revolution of Real-Time, Label-Free Biosensor Applications

1.1 Introduction

1.2 SPR Pessimists

1.3 Setting Up Experiments

1.4 Data Processing and Analysis

1.5 The Good News

2: Design and Implementation of Vertically Emitting Distributed Feedback Lasers for Biological Sensing

2.1 Introduction

2.2 DFB Laser Biosensor Design

2.3 Fabrication and Instrumentation

2.4. Experimental Results

2.5 Conclusions

Acknowledgements

3: SPR Screening of Chemical Microarrays for Fragment-Based Discovery

3.1 Introduction

3.2 Key Features of Fragment Screening

3.3 SPR Fragment Screening

3.4 Synthesis of Library Compounds

3.5 Library Design and Array Content

3.6 Chemical Microarray Production

3.7 Surface Plasmon Resonance

3.8 SPR Imaging

3.9 Array Visualization and Analysis

3.10 Follow-up

3.11 Applications: MMP case study

3.12 Other Target Classes

3.13 Conclusion

4: The CellKey® System: A Label-Free Cell-Based Assay Platform for Early Drug Discovery Applications

4.1 Introduction

4.2 Cellular Impedance Technology

4.3 Target Identification and Validation

4.4 Screening and Lead Optimization

4.5 Conclusion

5: Dynamic and Label-Free Cell-Based Assays Using the xCELLigence System

5.1 Introduction

5.2 The xCELLigence System

5.3 Principle of Detection

5.4 Applications

5.5 Functional Assays for G-Protein Coupled Receptors

5.6 Conclusion

6: Selecting the Best HTS Hits to Move Forward: ITC Ligand Binding Characterization Provides Guidance

6.1 Introduction

6.2 Principles of Isothermal Titration Calorimetry (ITC)

6.3 Applications of ITC in Hit Validation

6.4 Applications of ITC in Fragment-Based Drug Discovery

6.5 Applications of ITC in Mechanism of Action Studies

6.6 Applications of ITC in Lead Optimization

6.7 ITC as an Enzyme Activity Monitor

6.8 Conclusion

7: Incorporating Transmitted Light Modalities into High-Content Analysis Assays

7.1 Introduction

7.2 Transmitted Light (Bright Field) Imaging

7.3 Image Analysis of Phase Contrast Images

7.4 Conclusion

8: Nonradioactive Rubidium Efflux Assay Technology for Screening of Ion Channels

8.1 Introduction

8.2 Ion Channels as Drug Targets

8.3 Ion Channel Assays and Screening

8.4 Nonradioactive Rubidium Efflux Assay Based on Atomic Absorption Spectrometry

8.5 A Typical Assay Protocol

8.6 Conclusions

9: Expanding the Scope of HTMS Methods

9.1 Introduction

9.2 Development of the HTMS Method for Underivatized Cystathionine in Biological Samples Spanning IN VIVO Cell Culture, and EX VIVO Assays

9.3 Development of a 2D HTMS Method for Plasma-bound Small Molecules

9.4 Conclusion

10: A Novel Multiplex SPR Array for Rapid Screening and Affinity Determination of Monoclonal Antibodies: The ProteOn XPR36 Label Free System: Kinetic Screening of Monoclonal Antibodies

10.1 Introduction

10.2 Optimized Assay Configuration

10.3 Selection of the Optimal Capture Agent

10.4 Kinetic Analysis of 192 Human Anti-IL-12 Supernatants

10.5 Kinetic Analysis of 243 Human Hemoglobin Supernatants

10.6 Conclusions

11: Biophysics/Label-Free Assays in Hit Discovery and Verification

11.1 Introduction

11.2 Why Biophysics?

11.3 Biophysics / Label-Free Toolbox

11.4 Which Biophysical Measurement at Which Stage of a Drug Discovery Project Flowchart?

11.5 Examples of Higher Throughput Biophysics Applied in Hit and Lead Finding

11.6 Conclusion

11.7 Outlook

12: Harnessing Optical Label-Free on Microtiter Plates for Lead Finding: From Binding to Phenotypes

12.1 Introduction

12.2 Value Proposition and Advantages of Label-Free Methodologies

12.3 Detection Principle of an Optical Label-Free Resonant Grating Sensor

12.4 Biological Applications of Optical Label-Free in Lead Discovery

12.5 Current and Future Challenges

12.6 Conclusion

13: Use of Label-Free Detection Technologies in the Hit-to-Lead Process: Surface Optical Detection of Cellular Processes

13.1 Introduction

13.2 Overview of Label-Free Assay Platforms

13.3 Surface Optical Detection Of Cellular Processes

13.4 Discussion

14: Cellular Screening for 7TM Receptors Using Label-Free Detection

14.1 Introduction

14.2 Results and Discussion

14.3 Conclusions and Perspective

14.4 Materials and Methods

Acknowledgements

15: Novartis Evaluation of the ForteBio Octet RED: A Versatile Instrument for Direct Binding Experiments

15.1 Introduction

15.2 Methods

15.3 Results and Discussion

15.4 Conclusion

16: The Pyramid™ Approach to Fragment-Based Biophysical Screening

16.1 Introduction

16.2 Astex and the Pyramid™ Approach

16.3 Design of Fragment Libraries

16.4 Biophysical Methods in Pyramid™

16.5 Application of Pyramid™ to HSP90

16.6 Summary and Conclusions

Acknowledgements

17: Characterisation of Antibodies Against the Active Conformation of Gαi1 Using the SRU-BIND® Label-Free Detection System

17.1 Introduction

17.2 Materials and Methods

17.3 Results and Discussion

17.4 Conclusions

Acknowledgements

18: SPR-Based Direct Binding Assays in Drug Discovery

18.1 Introduction

18.2 Screening Using SPR-Based Direct Binding Assay

18.3 Lead Selection Using SPR-Based Binding Assay

18.4 Conclusion

Acknowledgements

19: Kinetic Binding Mechanisms: Their Contribution to an Optimal Therapeutic Index

19.1 Introduction

19.2 Why are Binding Mechanisms and Kinetics Important to Drug Action?

19.3 How Can Kinetics Contribute to an Optimal Mechanism?

19.4 Binding Kinetics Differentiate Physiological Responses

19.5 Utilization of Binding Kinetics in Drug Discovery. How to get Maximum Value out of Kinetic Analysis?

19.6 Conclusion

20: ITC: More Than Just Binding Affinities

20.1 Introduction

20.2 Why Should We Care About Enthalpy and Entropy?

20.3 Conclusion

Acknowledgements

Index

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Library of Congress Cataloging-in-Publication Data

Label-free technologies for drug discovery / editors, Matthew Cooper, Lorenz M. Mayr. p. ; cm. Includes bibliographical references and index. ISBN 978-0-470-74683-7 (cloth) 1. Drug development. I. Cooper, M. A. (Matthew A.) II. Mayr, M. Lorenz. [DNLM: 1. Drug Discovery. 2. Biosensing Techniques. 3. Drug Design. QV 744] RM301.25.L33 2011 615′.19–dc22 2010042191

A catalogue record for this book is available from the British Library.

Print ISBN: 9780470746837 ePDF ISBN: 9780470979136 oBook ISBN: 9780470979129 ePub ISBN: 9781119990277

Preface

In the 1980s, surface plasmon resonance (SPR) and related techniques exploiting evanescent waves were first applied to the interrogation of biological and chemical interactions. These techniques allowed us to study the interaction between immobilized receptors and analytes in real time and without labelling of the analyte; leading to the term ‘label-free’. While initially intended as a method of determining affinities, the use of a microfluidic delivery system to the sensor interface allowed kinetics (on and off rates of binding) to be measured. This, in turn, allowed new questions on compound action to be addressed and new compound optimization strategies to be explored. Today it is generally accepted that observed binding rates and binding levels can be interpreted to provide information on the specificity, kinetics and affinity of a drug–receptor interaction that relate to compound mode of action. This builds on the most often quoted maxim used in selecting bioactive compounds; ‘Corpora non agunt nisi fixate’: a drug will not work unless it is bound (Paul Ehrlich; 1854–1915). This now axiomatic statement guided Ehrlich through many scientific discoveries covering haematology, immunology, bacteriology and early chemotherapy. In the drug discovery process, we are now having to consider not just equilibrium-based, static descriptors of drug–receptor interactions (e.g. IC50 and EC50), but also descriptors of the dynamic nature of drug action. For example, similarly structured molecules can bind to a target with similar affinity. However, only one may have a slow enough off rate to effectively block action of an endogenous ligand; only one may bind in an orientation suitable for a catalytic reaction; only one may induce a conformational change in the receptor. At the extreme, two similarly structured molecules may bind with similar affinity, but one may initiate the receptor response (an agonist), whereas the other may block the response (an antagonist). Optimal binding mechanisms can thus define the therapeutic index and the utility of a drug. Label-free techniques can hence help us understand and optimize these parameters, particularly with respect to predictive pharmacodynamics, competition/interaction with endogenous ligands, binding to side effect profiling targets and metabolic enzyme, and many other attributes that lead to differentiation of a drug candidate from competitor compounds.

Since the development of the first commercial label-free biosensors in the late 1980s, their use in research and development has been described in over 5000 scientific publications covering most disciplines found in the pharmaceutical and diagnostic industries. Traditional solution-based thermodynamic techniques, such as isothermal titration calorimetry (ITC), have evolved from cumbersome, labour-intensive techniques to automated systems with much lower requirements for reagents and reduced (200 μl) sample volume. Nonfluorescent (white light) high content cell-based assays have been developed together with automated microscope systems combining rapid auto-focusing, automated stage movement and dedicated analysis software capable of batch processing large numbers of images from 96 and 384 well plates. Mass spectrometry combined with high throughput size exclusion and solid phase extraction methods now allows quantification of free and bound species in minutes. Mass spectrometry is thus now a powerful label-free technique that has transitioned from an analytical quality control tool to a mainstream compound profiling and screening platform. In a similar manner, nuclear magnetic resonance (NMR) has evolved from a method to confirm compound structure to a powerful screening tool for identifying low molecular weight drug ‘fragment’ binders, and even elucidate the specific target binding site of a fragment or lead compound. This approach was pioneered by scientists at Abbott Laboratories, who identified hits from changes in NMR chemical shifts (15N-1H HSQC) and Vertex, who relied on detecting changes in the NMR relaxation properties of the fragments themselves when bound to a protein target.

Finally, in the last five years, the advent of 384- and 1536-well screening systems based on resonant waveguide patterned microtitre plates and electrical impedance 96- and 384-plates has led to an explosion in the application of label-free to GPCR screening. This is highly significant for drug discovery, as at least 800 distinctive human G-protein coupled receptors (GPCRs) are known, with ~350 being estimated to be useful drug targets. Although only ~7% of GPCRs are currently targeted by drugs, this accounts for ~35% of blockbuster pharmaceuticals. Here label-free can really challenge current screening paradigms. It has emerged that different ligands (agonists or antagonists) that bind to the same GPCR, even the same subtype, can display profoundly different biological properties (ligand directed signalling) arising through different regulation of intracellular pathways (e.g. IP3 flux, ERK 1/2 phosphorylation, cAMP activation, Ca2 + release, β-arrestins, etc.). GPCR-mediated pathways, initially thought to be independent, are now known to cross-communicate with other activation paths. For instance, GPCR stimulation can lead to activation of ‘traditional’ tyrosine kinase pathway components such as Raf, MEK and ERK.

Drug candidate screening paradigms typically involve selection of a transfected cell line, over-expressing the target GPCR. This cell line is then used with one, or a variety, of downstream markers of receptor activation, such as Ca2 +, cAMP, inositol phosphate and diacylglyercol flux. Standard assay development can be summarized as: (i) compound selection and synthesis, (ii) cell line selection and (iii) downstream reporter assay selection, all of which lead to a data set predicated by cell line and assay format chosen in the first instance. Although this standard approach has become well-accepted for compound screening and pharmacological characterization, it is fundamentally limited in scope in profiling target-related response outcomes. An ‘agonist’ or ‘antagonist’ may only be so in the specific screen used; a response in a transfected or transduced cell line may not be the same as that found in the disease relevant endogenous cell. In contrast, label-free screening, which can be carried out using parental cell lines, is thought to be indicative of ligand binding induced changes in cell morphology and holistic behaviour. The readout is noninvasive, temporal, cumulative and most importantly, signalling pathway independent. Kinetic responses or ‘fingerprints’ elicited by a compound are mechanistically informative, and profiles for particular G-protein coupling can be determined. Hence, the combination of label-free, pathway independent receptor and whole cell profiling with standard reporter and pathway dependent screening should provide new insight into compound mode of action, in addition to identifying new hits that could be missed by traditional assays.

Label-free continues to grow from a niche technology with a user base comprised of early adopters, towards a mainstream, easy to use (but sometimes not easy to understand) technology. We hope the reader finds this compendium of chapters describing label-free technologies and case studies both useful and thought provoking.

Matt Cooper and Lorenz M. MayrBrisbane, Australia and Basel, Switzerland, July 2010

List of Contributors

Yama A. Abassi ACEA Biosciences Inc., 6779 Mesa Ridge Rd, San Diego, CA 92121, USA

J. Bradley Pfizer Global Research and Development, Sandwich, Kent CT13 9NJ, UK

Tsafrir Bravman Bio-Rad Laboratories, Inc., Gutwirth Park, Technion, Haifa 32000, Israel

Vered Bronner Bio-Rad Laboratories, Inc., Gutwirth Park, Technion, Haifa 32000, Israel

Jason Brown Neurosciences Centre of Excellence in Drug Discovery, GlaxoSmith- Kline, New Frontiers Science Park, Harlow, CM19 5AW, UK

Richard Brown GE Healthcare, MicroCal Products Group, 22 Industrial Drive East, Northampton, MA, USA

Xun Chen FAST (Facility for Automation & Screening Technologies), Merck Research Laboratories, Rahway, NJ, USA

Yen-Wen Chen Molecular Devices, Inc., 1311 Orleans Drive, Sunnyvale, CA 94089, USA

Mike Chin Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA

Bernard K. Choi FAST (Facility for Automation & Screening Technologies), Merck Research Laboratories, Rahway, NJ, USA

Steven S. Choi Micro and Nanotechnology Laboratory, Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, 1406 West Green Street, Urbana, IL 61801 USA

Chun-Wa Chung GlaxoSmithKline, New Frontiers Science Park, Stevenage, Essex CM19 5AW, UK

Brian T. Cunningham Micro and Nanotechnology Laboratory, Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, 1406 West Green Street, Urbana, IL 61801 USA

Mike Doyle Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA

Claude Dufresne FAST (Facility for Automation & Screening Technologies), Merck Research Laboratories, Rahway, NJ, USA

J. Gary Eden Laboratory for Optical Physics and Engineering, Department of Electrical and Computer Engineering University of Illinois at Urbana-Champaign, 1406 West Green Street, Urbana, IL 61801 USA

E. Fairman Pfizer Global Research and Development, Sandwich, Kent CT13 9NJ, UK

Paul Feucht Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA

Ernesto Freire Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA

Debra L. Gallant Molecular Devices, Inc., 1311 Orleans Drive, Sunnyvale, CA 94089, USA

E. Gbekor Pfizer Global Research and Development, Sandwich, Kent CT13 9NJ, UK

Chun Ge Micro and Nanotechnology Laboratory, Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, 1406 West Green Street, Urbana, IL 61801 USA

Neil S. Geoghagen FAST (Facility for Automation & Screening Technologies), Merck Research Laboratories, Rahway, NJ, USA

Robert Graves GE Healthcare Life Sciences, 800 Centennial Avenue, Piscataway, New Jersey 08855-1327, USA

P. Hayter Pfizer Global Research and Development, Sandwich, Kent CT13 9NJ, UK

Tom G. Holt Facility for Automation & Screening Technologies (FAST), Merck Research Laboratories, Rahway, NJ, USA

Walter Huber F.Hoffmann-La RocheAG, Pharma Research Basel, Grenzacherstrasse, 4070 Basel, Switzerland

Kristian K. Jensen FAST (Facility for Automation & Screening Technologies), Merck Research Laboratories, Rahway, NJ, USA

Jeffrey C. Jerman Molecular Discovery Research, 1--3 Burtonhole Lane, London NW7 1AD, UK

Maxine Jonas BioTrove, Inc., Woburn, MA, USA

William A. LaMarr BioTrove, Inc., Woburn, MA, USA

Lukas Leder Novartis Institutes for Biomedical Research, Lichtstrasse 35, CH-4056, Basel, Switzerland

Melanie Leveridge GlaxoSmithKline, New Frontiers Science Park, Stevenage, Essex CM19 5AW, UK

Meng Lu Micro and Nanotechnology Laboratory, Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, 1406 West Green Street, Urbana, IL 61801 USA

Ming-Juan Luo FAST (Facility for Automation & Screening Technologies), Merck Research Laboratories, Rahway, NJ, USA

Qi Luo FAST (Facility for Automation & Screening Technologies), Merck Research Laboratories, Rahway, NJ, USA

Lorraine Malkowitz FAST (Facility for Automation & Screening Technologies), Merck Research Laboratories, Rahway, NJ, USA

Eric Martin Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA

Julio Martin GlaxoSmithKline, Centro de Investigacion Basica, Parque Tecnologico de Madrid, 28760 Tres Cantos, Spain

Ryan P. McGuinness Molecular Devices, Inc., 1311 Orleans Drive, Sunnyvale, CA 94089, USA

Marco Meyerhofer Novartis Institutes for Biomedical Research, Lichtstrasse 35, CH-4056, Basel, Switzerland

David G. Myszka Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City, UT 84132, USA

Oded Nahshol Bio-Rad Laboratories, Inc., Gutwirth Park, Technion, Haifa 32000, Israel

Thomas Neumann Graffinity Pharmaceuticals GmbH, INF 518, 69120 Heidelberg, Germany

Ronan O'Brien GE Healthcare, MicroCal Products Group, 22 Industrial Drive East, Northampton, MA, USA

Johannes Ottl Novartis Institute for BioMedical Research, Centre for Proteomic Chemistry, Forum~1, Novartis Campus, CH-4056, Basel, Switzerland

Can C. Ozbal BioTrove, Inc., Woburn, MA, USA

John M. Proctor Molecular Devices, Inc., 1311 Orleans Drive, Sunnyvale, CA 94089, USA

S. Ramsey Pfizer Global Research and Development, Sandwich, Kent CT13 9NJ, UK

Rebecca L. Rich Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City, UT 84132, USA

Magalie Rocheville Molecular Discovery Research, GlaxoSmithKline, New Frontiers Science Park, Harlow, CM19 5AW, UK

Renate Sekul Graffinity Pharmaceuticals GmbH, INF 518, 69120 Heidelberg, Germany

Kevin Shoemaker Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA

Alexander Sieler Roche Diagnostics GmbH, BP-C1 Nonnenwald 2, 82377 Penzberg, Germany

F. Stuhmeier Pfizer Global Research and Development, Sandwich, Kent CT13 9NJ, UK

David C. Swinney iRND3, Institute for Rare and Neglected Diseases Drug Discovery, 1514 Ridge Road, Belmont, CA 94002, USA

Blisseth Sy Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA

H. Roger Tang Molecular Devices, Inc., 1311 Orleans Drive, Sunnyvale, CA 94089, USA

Georg C. Terstappen Faculty of Pharmacy, University of Siena, Via Fiorentina 1, 53100 Siena, Italy

Trisha A. Tutana Molecular Devices, Inc., 1311 Orleans Drive, Sunnyvale, CA 94089, USA

Clark J. Wagner Laboratory for Optical Physics and Engineering, Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, 1406 West Green Street, Urbana, IL 61801 USA

John Wang Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA

Jun Wang FAST (Facility for Automation & Screening Technologies), Merck Research Laboratories, Rahway, NJ, USA

Xiaobo Wang ACEA Biosciences Inc., 6779 Mesa Ridge Rd, San Diego, CA 92121, USA

Bob Warne Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA

Charles Wartchow FortéBio, Inc., 1360 Willow Road, Suite 201, Menlo Park, CA 94025-1516, USA

Trevor Wattam GlaxoSmithKline, New Frontiers Science Park, Stevenage, Essex CM19 5AW, UK

Manfred Watzele Roche Diagnostics GmbH, BP-C1 Nonnenwald 2, 82377 Penzberg, Germany

Glyn Williams Astex Therapeutics Ltd, 436 Cambridge Science Park, Milton Road, Cambridge CB4 0QA, UK

Donna L. Wilson Molecular Devices, Inc., 1311 Orleans Drive, Sunnyvale, CA 94089, USA

Yusheng Xiong FAST (Facility for Automation & Screening Technologies), Merck Research Laboratories, Rahway, NJ, USA

Xiao Xu ACEA Biosciences Inc., 6779 Mesa Ridge Rd, San Diego, CA 92121, USA

Kelly Yan Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA

Danfeng Yao FortéBio, Inc., 1360 Willow Road, Suite 201, Menlo Park, CA 94025-1516, USA

Jiamin Yu Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA

Isabel Zaror Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA