High throughput screening methods evolution and refinement

by Joshua A Bittker, Nathan T Ross,

High throughput screening methods evolution and refinement High throughput screening remains a key part of early stage drug and tool compound discovery and methods and technologies have seen many fundamental improvements and innovations over the past 20 years This comprehensive book provides a historical survey of the field up to the current state of the art In addition to the specific methods this book also considers cultural and organizational questions that represent opportunities for future success Following thought provoking foreword and introd

Publisher : Royal Society of Chemistry

Author : Joshua A Bittker, Nathan T Ross, Tom Brown, Kira J Weissman, Claes Andersson, Douglas Auld, Peizin Zhu, Sean Ekins, Jaime Cheah, Mark Wigglesworth, Damien Young, Michael Mesleh, Greg Hoffman, Matthew Robers, Christopher Hale, Benjamin Haibe-Kains, Christopher Mader, Richard Eglen, Stephen Hale, Andr

ISBN : 9781782624714

Year : 2016

Language: en

File Size : 12.4 MB

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High Throughput Screening Methods

Evolution and Refinement

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Chemical Biology Series

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Editor-in-Chief:
Tom Brown, University of Oxford, UK

Series Editors:
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Sabine Flitsch, University of Manchester, UK
Nick J. Westwood, University of St Andrews, UK

Editorial Advisor:
Chris L. Dupont, J. Craig Venter Institute, USA

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High Throughput Screening
Methods
Evolution and Refinement

Edited by

Joshua A. Bittker
The Broad Institute of MIT and Harvard, Cambridge, MA, USA
Email: [email protected]

Nathan T. Ross
Novartis Institutes for Biomedical Research, Cambridge, MA, USA
Email: [email protected]

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Chemical Biology No. 1
Print ISBN: 978-1-78262-471-4
PDF eISBN: 978-1-78262-677-0
EPUB eISBN: 978-1-78262-979-5
ISSN: 2055-1975
A catalogue record for this book is available from the British Library
r The Royal Society of Chemistry 2017
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Foreword: Transforming
Medicine by Innovating the
Science of Therapeutics
Human biology is playing an increasingly important role in guiding the early
phase of therapeutic discoveries. For example, analysis of human genetics is
revealing allelic series of risk and protective variants of genes across a range
of diseases. These allelic series demonstrate a dose–response relationship
that relates the activity of a gene to its effect on, for example, the risk of a
disease. Prior to embarking on the discovery of a drug, we can establish
whether perturbing a target, in the context of human physiology, has the
intended effect in terms of safety and efficacy. Biochemical mechanistic
investigations of variant proteins can provide a blueprint for the activities
that drugs should confer on the more common versions of the target proteins in order to be safe and efficacious.
This is an amazing advance with the potential to transform medicine. We
hear about this promise frequently, especially from scientists pioneering
these advances. The promise of these advances has led to powerful terms
such as ‘‘precision medicine’’ being used by President Obama at his State of
the Union speech in 2015, and these terms have even entered the mainstream vernacular of the media and public. However, major hurdles exist to
realize this potential, and these hurdles may not be well appreciated by the
advocates of the human biology/patient based approach to drug discovery.
Without recognizing these hurdles and overcoming them, we are at risk of
this potential revolution in medicine being unfulfilled.
Why this pessimism? For one, the activities suggested by these experiments of nature are challenging and often unfamiliar in the context of the

Chemical Biology No. 1
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Foreword: Transforming Medicine by Innovating the Science of Therapeutics

historical path towards drug discovery. For the most part, to date, drugs that
have these suggested mechanisms of action (MoA) do not exist. In order to
realize the promise of modern biology in medicine, we must innovate in the
science of therapeutics.
Chemistry and chemical biology, as evidenced by this important new book
High Throughput Screening Methods: Evolution and Refinement, are offering
new ways to discover compounds with novel (previously unknown) MoA
(nMoA). Novel methods are being developed to discover specific nMoA
compounds suggested by the human biology, patient based concept. Novel
methods are also being developed to identify vast collections of compounds
where each member has a distinct MoA—known and novel—in anticipation
of their future utility. The range of innovations presented by the authors
is extraordinary and the level of creativity that underlies these advances is
inspiring and makes for exciting reading. The methods described in this
book and their descendants, once integrated into the therapeutics discovery
efforts of laboratories worldwide, offer great promise to bridge the gap
between the knowledge gained from human biology and the thus far elusive
transformative medicines that we hope to derive from them.
In addition to the specific screening methods, this book also hints at
cultural and organizational challenges that represent opportunities for
future success. Foundational capabilities that enable the translation of
insights from human biology to novel therapeutics are in general far easier
to share than compounds, targets and biological insights associated with
specific drug discovery projects. Innovations in the science of therapeutics
can be developed effectively through collaborations involving scientists in
the public and private sectors, especially when the participating not-forprofit and for-profit organizations agree to avoid restrictions in the sharing
of novel innovations. Evidence of this opportunity for interaction and collaboration is seen in the contributions from the authors in this book—the
chapter contributors are from both sectors and cover topics important to
each (for example, the importance of novel asymmetric synthetic chemistry
and small-molecule libraries, integrating new techniques into probe and
drug discovery, and proper annotation and sharing of data, among many
others). The individual chapters also reinforce how overcoming the new
challenges of human biology based precision medicine will need the
foundational capabilities to continue evolving and how researchers will need
to learn from each other as part of this evolution.
Stuart L. Schreiber
Center for the Science of Therapeutics, Broad Institute, Cambridge, USA
Howard Hughes Medical Institute, Department of Chemistry and
Chemical Biology, Harvard University, Cambridge, USA

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Preface
Learning the Future of Technology by Understanding
our Path to the Present
In this collection of reviews from top scientists in academia and industry, we
hope to present the reader with more than simply a survey of the current
state of the field in high throughput screening (HTS) and related technologies. Such reviews, while useful for a brief period of time, are quickly surpassed by changes in the field. This may be due to the discovery and
development of new systems adapted from nature (e.g. gene editing techniques), which provide researchers with powerful new tools that make previously impossible studies relatively routine. Changes may also reflect
simple incremental improvements or more robust commercialization of
existing technologies that make them more readily available to researchers
in a plug and play format (e.g. new fluorophores or luciferase enzyme
systems).
Why, then, assemble such a survey of HTS methods? By examining the
state of the art in relation to how we arrived here and considering what areas
still require improvement, we hope to also encourage our readers to consider
the philosophy underlying technological change. How and why are improvements in technology made? Surely they reflect an underlying demand
from researchers to be able to generate results more quickly and answer
questions more efficiently or more robustly.
Small-molecule activity screening, considered as a specific discipline
practiced initially within the pharmaceutical industry, and now more widely
available to academics and biotechnology firms, could be considered to have
started in the mid-1900s. This involved the routine parallel testing of natural
product extracts and purified compounds, as well as synthetically available
compounds from related industries, in cellular phenotypic assays such as
microbial viability systems or whole animals.1 It became more robust and
Chemical Biology No. 1
High Throughput Screening Methods: Evolution and Refinement
Edited by Joshua A. Bittker and Nathan T. Ross
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Preface

standardized with the shift towards target-based enzymatic assays, large
scale combinatorial synthetic compound collections, and robotic automation in the 1990s, driven in part by genomic studies drastically increasing
the number of putative therapeutic targets.2 This new approach required
significant capital investment, taking it out of reach of all but the largest
companies. However, in the 2000s, through the efforts of government
funding agencies as well as the establishment of contract research organizations offering HTS as a service, a democratization occurred in the field of
small-molecule discovery. This change led both to more routine application
of screening to early biological targets as well as to the development of a
larger variety of assays for measuring biology through alternative and ideally
more relevant methods.
Consider, then, the shifts over the years in the approach to smallmolecule discovery. Why apply automation, previously used in areas such
as industrial manufacturing, to increasing the number of measurements
made? What demands did this increase in throughput have on the related
biology, assays, and number and type of compounds required to feed the
system? What is the most effective use of small-molecule discovery in
academia? Surely these questions are all affected by larger societal and
technological changes, including the information revolution and political
considerations that affect funding decisions. They also relate to the
technologies themselves—each technology is designed to improve some
shortcoming in the existing capabilities, but in turn can lead to its own
problems.
Figure 1 shows the evolution of methods for addressing four key components of HTS: chemical perturbagens, bioassays, data analysis and management, and organizational infrastructure. For example, with the increase
in throughput of cell free enzymatic and cell based reporter assays, more
compounds were required. These were accessed through new methods in
high throughput chemical synthesis, which greatly increased the number of
compounds available but did not always consider the optimal chemical diversity necessary for a range of biological targets.3 Analysis of the desired
properties of compounds changed,4 with many pharmaceutical companies
paring back their collections by 30% or more from their peaks.5 Alternative
approaches, such as encoded libraries and diversity oriented synthesis,
changed the types of libraries available. Lower throughput but arguably
more biologically relevant assays, such as high content imaging and activity
profiling, changed the nature of the information available.2 In turn, this
led to new requirements, as phenotypic assays required systematic methods
for identification of cellular targets. Overall, of course, these different
approaches have different requirements for capital and operational investment, and all scientific research remains a trade-off between available
funding and hoped for return in the understanding of basic biology and the
impact on improvements in human health.
Given these historical and ongoing changes, no one would argue that we
are at the ideal state of HTS discovery now. This book is a snapshot of the

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Preface

Figure 1

ix

The evolution of HTS methods. Each column shows the approximate time
frames when additional techniques, each of which is discussed in this
book, became widely available. The colors of the boxes indicate which key
aspect of HTS the method helps to improve. Some methods involve
contributions to multiple aspects of HTS, indicated by multiple colors
(e.g. encoded libraries are a new source of compound diversity and also
provide a new assay method). By understanding the reasons for developing
each new technology and the current limitations, we can try to understand
what improvements may benefit future HTS and drug discovery. HC: high
content; PPI: protein–protein interaction; uHTS: ultra-high throughput
screening.

state of the field in 2016, as well as a historical survey of how the methods
presented arrived at their current state of the art. Our contributors also
highlight weaknesses and potential solutions to further improve the field.
This, then, is our hope and challenge for our readers: we seek to provide a
means of understanding how and why we have arrived where we are, through
the above-mentioned combination of technological and societal changes.
With that understanding, we hope to illuminate the way forward—what
changes are necessary, what impact will they have, how can they be implemented practically and what future challenges will those changes in turn
bring about?
We cannot predict or understand in a single book written at a single time
all of the technologies yet to come. However, by providing the reader with a
means of understanding how and why technological change occurs, as
illustrated by the evolution and refinement of HTS methods, we hope that
future capabilities can more readily be anticipated, developed, applied and
further improved, to the benefit of researchers and society.
Joshua Bittker and Nathan Ross
Cambridge, MA, USA

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References
´n, Upsala J. Med. Sci., 2014, 119, 162.
1. D. Hughes and A. Karle
2. D. C. Swinney and J. Anthony, Nat. Rev. Drug Discovery, 2011, 10, 507.
3. D. J. Payne, M. N. Gwynn, D. J. Holmes and D. L. Pompliano, Nat. Rev.
Drug Discovery, 2007, 6, 29.
4. M. M. Hann and T. I. Oprea, Curr. Opin. Chem. Biol., 2004, 8, 255.
5. G. A. Bakken, A. S. Bell, M. Boehm, J. R. Everett, R. Gonzalez,
D. Hepworth, J. L. Klug-McLeod, J. Lanfear, J. Loesel, J. Mathias and
T. P. Wood, J. Chem. Inf. Model., 2012, 52, 2937.

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Contents
Chapter 1 HTS Methods: Assay Design and Optimisation
David Murray and Mark Wigglesworth
1.1
1.2

Introduction
HTS at AstraZeneca
1.2.1 Criteria and Acceptance
1.2.2 Robustness/Reliability
1.2.3 Analysing Data to Define
Robustness/Reliability
1.2.4 As Simple to Run as Possible
1.2.5 Assay Validation
1.3 Summary
References
Chapter 2 Considerations Related to Small-molecule Screening
Collections
Damian W. Young
2.1
2.2

Introduction
General Considerations Related to HTS Compound
Collections
2.2.1 Determination of Screening Objectives
2.2.2 Size of HTS Compound Collections
2.2.3 Chemical Diversity in Compound Collections
2.2.4 Quality of Compounds in Screening
Collections

Chemical Biology No. 1
High Throughput Screening Methods: Evolution and Refinement
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2.3

Sources of Compounds in Screening Collections
2.3.1 Natural Products in Screening Collections
2.3.2 Synthetic Drug-like Compounds
2.3.3 Diverse Collections
2.4 Performance of Compounds in Screening
Collections
2.4.1 Background
2.4.2 Performance of Compounds from Different
Sources
2.4.3 Performance Diversity of Compound
Collections
2.4.4 Pan Assay Interference Compounds
2.4.5 Dark Chemical Matter
2.5 Conclusions and Discussion
References
Chapter 3 Combination Screening
Claes R. Andersson, John Moffat and Mats Gustafsson
3.1
3.2

Introduction
Measures of Synergy
3.2.1 Bliss Independence Model of Synergy
3.2.2 Loewe Additivity
3.2.3 Other Measures
3.2.4 Reconciling Measures of Synergy
3.3 Design of Combination Experiments
3.4 Statistical Inference of Combination Effects
3.4.1 The Error Distribution
3.4.2 Bootstrap Intervals
3.4.3 Intervals for Bliss Independence
3.4.4 Intervals for Loewe Additivity Interaction
Index
3.5 Null Hypothesis Significance Testing
3.5.1 Significance Testing of Bliss
3.5.2 Significance Test of Loewe Additivity
3.6 Concluding Remarks
References
Chapter 4 Modern Biophysical Methods for Screening and Drug
Discovery
B. Fulroth, V. K. Kaushik and M. F. Mesleh
4.1

Introduction

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4.2

Physicochemical Properties and High
Concentration Screening
4.2.1 Physicochemical Properties of
Chemical Libraries
4.2.2 High Concentration Screening
4.3 Differential Scanning Fluorimetry
4.4 Surface Plasmon Resonance
4.5 Mass Spectrometry Techniques
4.5.1 Affinity Selection MS
4.5.2 Affinity Chromatography Methods
4.5.3 Protein MS
4.6 NMR Spectroscopy
4.6.1 Protein NMR
4.6.2 Ligand Observed NMR
4.7 Calorimetric Methods
4.7.1 Differential Scanning Calorimetry
4.7.2 Isothermal Titration Calorimetry
4.8 X-Ray Crystallography
4.9 Newer Methods on the Horizon
4.10 Summary and Recommendations
Acknowledgements
References

Chapter 5

Genetic Perturbation Methods, from the ‘Awesome Power’
of Yeast Genetics to the CRISPR Revolution
Gregory R. Hoffman and Dominic Hoepfner
5.1
5.2

Introduction
Genetic Methodologies
5.2.1 Random Mutagenesis
5.2.2 Targeted Genome-wide Deletions
(Homozygous/Heterozygous)
5.2.3 Random Genome-wide Deletions
(Homozygous/Heterozygous)
5.2.4 RNA Interference
5.2.5 CRISPR
5.2.6 Overexpression
5.2.7 Synthetic Biology
5.3 Concluding Remarks
Acknowledgements
References

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Chapter 6 Understanding Luminescence Based Screens
Simona Gokhin and Douglas S. Auld
Why Luminescence? An Introduction to
Bioluminescent and Chemiluminescent Assays
6.1.1 Overview of Common Luminescent
Enzymes Employed in Assays
6.1.2 Chemiluminescence in HTS Assays
6.2 Considerations and Applications of
Bioluminescent Assays
6.2.1 Prevalence of Luciferase Inhibitors in
Compound Libraries
6.2.2 Mechanisms of Luciferase Inhibition
6.2.3 Ligand Based Stabilization of Luciferases:
Impact on RGA Results
6.2.4 Methods to Mitigate Luciferase Inhibitors in
RGAs: Counter-Screens and Orthogonal Assay
Formats
6.2.5 Luciferases as Post-translational Sensors
6.2.6 Use of Luciferases in Biochemical
Applications
6.3 Considerations and Applications of Amplified
Luminescent Proximity Homogenous Assays:
AlphaScreen and AlphaLISA
6.3.1 Example Protocols and Key
Experiments
6.3.2 Interferences with ALPHA
Technology
6.4 Conclusion
References

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6.1

Chapter 7 High Throughput Screening Compatible Methods for
Quantifying Protein Interactions in Living Cells
M. B. Robers, T. Machleidt and K. V. Wood
7.1
7.2

Introduction
Analysis of PPIs in Intact Cells
7.2.1 Two-hybrid Systems
7.2.2 Protein Fragment Complementation
Technologies
7.2.3 FRET for Analysis of PPIs

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7.3

Measuring Target Engagement in Cells
7.3.1 Target Engagement by Protein Stability
Analysis
7.3.2 Real Time, Quantitative Analysis of Target
Engagement via FRET
7.4 Outlook
References
Chapter 8 Approaches to High Content Imaging and Multi-feature
Analysis
C. M. Hale and D. Nojima
8.1
8.2
8.3
8.4

Introduction
Imaging Hardware
Image Analysis
Quality Control of Image Acquisition and
Well Level Data
8.4.1 Quality Control of Image Acquisition
8.4.2 Quality Control of Well Level Data
8.5 Single Cell Analysis
8.6 Analysis of Multiparametric Data
8.6.1 Feature Selection and Dimensional
Reduction
8.6.2 Distance and Similarity
8.7 Machine Learning: Supervised and Unsupervised
Methods
8.7.1 Supervised Learning (Classification)
8.7.2 Unsupervised Learning (Clustering)
8.8 Conclusion
References

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Chapter 9 Pharmacological and Genetic Screening of Molecularly
Characterized Cell Lines
181
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and Benjamin Haibe-Kains
9.1
9.2

Introduction
Cell Lines
9.2.1 Applications in Cancer
9.2.2 Mistaken Identities
9.2.3 Authentication
9.2.4 Molecular Characterizations

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