High Throughput Screening Methods: Evolution and Refinement: Volume 1 (Chemical Biology) - Hardcover

Hardcover

ISBN 10: 1782624716 ISBN 13: 9781782624714

Verlag: Royal Society of Chemistry, 2016

Inhaltsangabe

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 introduction from Professor Stuart Schreiber and the editors, chapters from leading experts across academia and industry cover initial considerations for screening, methods appropriate for different goals in small molecule discovery, newer technologies that provide alternative approaches to traditional miniaturization procedures, and practical aspects such as cost and resourcing. Within the context of their historical development, authors explain common pitfalls and their solutions.
This book will serve as both a practical reference and a thoughtful guide to the philosophy underlying technological change in such a fast-moving area for postgraduates and researchers in academia and industry, particularly in the areas of chemical biology, pharmacology, structural biology and assay development.

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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.
Chapters from leading experts across academia and industry cover initial considerations for screening, methods appropriate for different goals in small molecule discovery, newer technologies that provide alternative approaches to traditional miniaturization procedures, and practical aspects such as cost and resourcing. Within the context of their historical development, authors explain common pitfalls and their solutions.
This book will serve as both a practical reference and a thoughtful guide to the philosophy underlying technological change in such a fast-moving area for postgraduates and researchers in academia and industry, particularly in the areas of chemical biology, pharmacology, structural biology and assay development.

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High Throughput Screening Methods

Evolution and Refinement

By Joshua A. Bittker, Nathan T. Ross

The Royal Society of Chemistry

Copyright © 2017 The Royal Society of Chemistry
All rights reserved.

ISBN: 978-1-78262-471-4

Contents

Chapter 1 HTS Methods: Assay Design and Optimisation David Murray and Mark Wigglesworth, 1, 
Chapter 2 Considerations Related to Small-molecule Screening Collections Damian W. Young, 16, 
Chapter 3 Combination Screening Claes R. Andersson, John Moffat and Mats Gustafsson, 37, 
Chapter 4 Modern Biophysical Methods for Screening and Drug Discovery B. Fulroth, V. K. Kaushik and M. F. Mesleh, 58, 
Chapter 5 Genetic Perturbation Methods, from the 'Awesome Power' of Yeast Genetics to the CRISPR Revolution Gregory R. Hoffman and Dominic Hoepfner, 87, 
Chapter 6 Understanding Luminescence Based Screens Simona Gokhin and Douglas S. Auld, 117, 
Chapter 7 High Throughput Screening Compatible Methods for Quantifying Protein Interactions in Living Cells M. B. Robers, T. Machleidt and K. V. Wood, 143, 
Chapter 8 Approaches to High Content Imaging and Multi-feature Analysis C. M. Hale and D. Nojima, 162, 
Chapter 9 Pharmacological and Genetic Screening of Molecularly Characterized Cell Lines Zhaleh Safikhani, Heather Selby, Azin Sayad, Christos Hatzis and Benjamin Haibe-Kains, 181, 
Chapter 10 Multidimensional Profile Based Screening: Understanding Biology through Cellular Response Signatures Christopher C. Mader, Aravind Subramanian and Joshua Bittker, 214, 
Chapter 11 3D Cell Culture and Dish Based Organogenesis: Optimizing In vitro Cellular Physiology David H. Randle, Ye Fang and Richard M. Eglen, 239, 
Chapter 12 Small-molecule-mediated Targeted Protein Degradation for Drug Discovery Rohan E. J. Beckwith, 252, 
Chapter 13 Phenotypic Screens with Model Organisms Peixin Zhu, Gerald J. Sun and Brant K. Peterson, 275, 
Chapter 14 Encoded Compound Libraries to Accelerate Small-molecule Therapeutic Discovery Stephen P. Hale, 303, 
Chapter 15 Research Data Management Shuba Gopal and Andrea de Souza, 324, 
Chapter 16 Small-molecule Bioactivity Databases Sean Ekins, Alex M. Clark, Christopher Southan, Barry A. Bunin and Antony J. Williams, 344, 
Chapter 17 "So You Want to Run a High-throughput Screen: Do You Know How Much That Costs?"; Costs of High Throughput Screens and How to Fund Them Jaime H. Cheah and Joshua A. Bittker, 372, 
Subject Index, 390, 

CHAPTER 1

HTS Methods: Assay Design and Optimisation

DAVID MURRAY AND MARK WIGGLESWORTH

Discovery Sciences, Global HTS Centre, AstraZeneca, Alderley Park, SK10 4TG, UK

1.1 Introduction

High throughput screening (HTS) remains the key methodology for finding hit and lead compounds within the pharmaceutical industry and also, recently, in the academic drug discovery community. HTS has changed significantly in AstraZeneca over the last 15–20 years with a massive expansion of the number of compounds available to screen, increasing industrialisation and automation of the process to cope with larger numbers of compounds, and more recently, the running of screens from external collaborators via open innovation initiatives. It has also become evident over the last 5 or so years (at least in AstraZeneca) that a more nuanced approach to HTS is required, where a large repertoire of assays is needed that spans from very high throughput "industrial" biochemical assays for targets such as kinases to highly complex cell based phenotypic assays on hard to source cells such as primary cells, genetically engineered human cell lines and induced pluripotent stem cells. We are also using a wider range of detection methods, from standard plate reader assays through to technologies such as flow cytometry, imaging and high throughput mass spectrometry. This presents very significant challenges in designing and developing complex cell and biochemical assays for the assay development teams, and huge challenges to the HTS group to run hundreds of thousands to millions of compounds through these assays.

There are perhaps two core models of how to run HTS in drug discovery. The simplest and arguably most efficient model is to limit the repertoire of assays to very few detection technologies, and if the assay cannot run in this mode it will not be run. This allows both operational and cost efficiencies, and increases the productivity of a limited team. However, this model can also limit the impact of HTS on drug discovery by narrowing the targets that undergo HTS. Within AstraZeneca we run a model of HTS where we will try and run complex biochemical or cell based assay as high throughput screens. This promises to find a wider range of hits against a wider range of targets but it does require very sophisticated and costly automation platforms and considerable effort is needed to develop assays robust enough to screen large compound libraries. This requires staff with a wide range of experience and expertise. We need people with experience in running large scale assays and the management of the logistics of such assays. We also require experts in automation, informatics and statistics plus more specialised technologies such as flow cytometry and mass spectrometry. We also need to mirror some, but not all, of this expertise in the assay development teams. This makes staffing of such an HTS department more difficult, and with a need for more specialisation, departments can become less flexible.

In this chapter we will discuss how this approach to HTS has been developed within AstraZeneca and pay particular attention to how the optimisation and validation of the wide spectrum of assays that we have to deal with in our group is done. We will discuss how we accept assays into HTS from our assay development groups and describe how these assays are validated and optimised for use as an HTS screen.

1.3 HTS at AstraZeneca

Within AstraZeneca we have a single global HTS centre that provides high throughput screening for all AstraZeneca disease areas as well as our collaborators who have taken advantage of the various open innovation initiatives that AstraZeneca has launched. The HTS centre sits in an organisation within AstraZeneca called Discovery Sciences. Discovery Sciences supplies a large set of scientific and technical services to AstraZeneca, allowing for consolidation of the expertise and infrastructure to supply these vital components of the drug discovery value chain. This results in the HTS group interacting widely across the business as well as outside of it. In terms of reagent supply and assay development of high throughput screens, this is carried out by a separate group within Discovery Sciences called Reagents and Assay Development (RAD). Although introducing a handover step into the HTS process, this again allows the consolidation of expertise and infrastructure to both save cost and increase quality. However, the handover does present challenges to both the assay development and HTS groups, who must make sure that all assays required for HTS are of the quality that is required to support the costly undertaking of a screen. This organisational structure has led to a considered process of defining the criteria for an acceptable screen, accepting the screen and validating the screening assay to ensure that it is indeed suitable for an HTS campaign without incurring a large bureaucratic burden. Although some have questioned the need for these criteria, it is our experience that the standards defined within them are vital to facilitate the transfer and deployment of successful screening assays. Without this foundation we have found that standards inevitably slip and different practices spring up within and across groups, leading to issues with assays of varying quality being prepared for HTS.

1.2.1 Criteria and Acceptance

HTS is both costly to set up, with a high initial capital outlay, and a demanding process to maintain and run, yet it remains a good return on investment by being the most productive hit finding strategy we employ. To be able to screen millions of compounds and get a set of reliable data is difficult. Equally, HTS is the main method for finding novel chemistry for projects within AstraZeneca and beyond, and it is critical for keeping the pipeline of drug discovery projects filled with high quality chemical equity. Within HTS we have developed a set of criteria that will result in assays that are fit for the task of finding chemical leads.

However, it is worth reiterating that these are not hard and fast rules. What is important is that these guide the scientists to have a conversation regarding what risks are acceptable, where the problems lie and how they can be overcome. It would be our advice to anyone looking at these criteria to assess the quality of assays as early as possible as this will minimise the possibility of re-work later in assay development. These "mini" validation experiments combined with the recommended statistics can really help to define why and how an assay needs to be modified to become a good HTS assay.

The overriding aim is the development of robust assays. In many respects HTS is an anomaly in drug discovery in that the vast majority of data are generated by taking a single concentration of a compound and testing it just once in an attempt to see if it is active against a biological target. Of course there is an element of replicate testing in large HTS collections as there are clusters of compounds of similar structures, but an HTS assay needs to be sensitive enough to detect relatively weak compounds and robust enough that the false positive and negative rates are low. Much focus is on false negatives, as of course we do not like to think we have missed something. However, managing false positives is, arguably, a greater challenge and can lead to compounds being missed as teams try to separate true hits from many hundreds or thousands of false hits. Additionally, the assay has to be of a form that can actually be run on the automation platforms we have or be run on manual workstations at a throughput that allows the assay to complete in the time frame required to allow the flow of projects through a portfolio of assays. An assay also has to be reliable in that if it is run twice it will find the majority of active compounds both times, confirming that the hits are not due to random events. It quickly becomes apparent that there are some key criteria that an HTS assay has to fulfil to maximise its utility in finding hit compounds:

• Robustness

• Reliability

• As simple to run as possible

• Affordable

• Relevant

In this discussion we will focus on the first three bullet points in explaining how we have generated a set of criteria to help design good HTS assays. Affordable is a given in many respects in that an assay has to fit within a budget. We do run assays with quite a range of different costs but there always has to be a balance between the cost and maximising both how easy the assay is to run and the ability to find hit compounds. Relevant is a key criterion and may seem obvious but is worth stating. The assay has to be relevant to the biological or disease process that we are wishing to disrupt or stimulate. Anything other than this wastes the investment in the screen. Robustness and reliability in many respects overlap, and in fact, robustness should lead to reliability.

1.2.2 Robustness/Reliability

Within HTS at AstraZeneca robustness of an assay is key. In our experience, a lack of robustness is the key reason we will struggle with an assay or in extreme circumstances stop the assay running. Determining robustness is a large topic with many differing opinions. We will discuss what works for us and it should be noted that in many of these topics another criterion we use is to keep things simple and understandable for the scientists doing the screening (and indeed the assay developers) whilst having a fit for purpose set of criteria. In Figure 1.1 we give the criteria that we use when setting out to develop an HTS assay. These are an attempt to generate robust and reliable assays that will pass assay validation. They are derived from our experience across AstraZeneca and other Pharma companies in HTS over the last 15 years and are there to guide the user to make informed decisions rather than being a simplistic check list. They are by no means an exhaustive list but are what we consider to be key. Below we look at some of these criteria one by one.

Z'-factor is a widely used parameter to help determine the robustness of an assay and its use for single shot screening. It is simple to understand and is popular across assay development and screening groups due to its proven utility. We use the robust Z-factor to determine how sensitive the assay will be in finding hits and as a measure of the robustness of the assay from the performance of the control wells. We do not use Z-factor routinely (although it is calculated in our data analysis package) as we screen focussed libraries of compounds, which can be an issue because the very high hit rates result in a compound activity distribution that does not define the true central reference for the assay, leading to an artificially low Z-factor. Additionally, sticking to the Z'-factor gives consistency across the assay development and HTS groups when comparing data. The reason why we have adopted the robust Z'-factor {where standard deviation is replaced by robust standard deviation [median absolute deviation (MAD)×1.483] and mean by median in the equation derived by Zhang et al.} is to remove the influence of outliers on the Z-factor and to remove the need for human intervention, which can result in people chasing a target Z'-factor value with subjective removal of "outlier" data. Although Zhang et al.3 state that assays can be used with a Z/Z'-factor as low as 0 to give a yes/no answer for an HTS primary screen, our experience has shown us that assays need to have a robust Z'-factor of at least [is greater than or equal to] 0.5 to perform robustly and reliably. We of course remain pragmatic and will take assays with a lower Z'-factor when the target is very high value and there is no alternative assay and/or nothing more can be done to improve an assay. In these circumstances we will look at other approaches to improve robustness such as replication in the assay, which almost certainly will reduce the number of compounds screened, or perform a quantitative HTS (qHTS) where concentration responses are run as a primary screen, again on a significantly reduced number of compounds.

A signal to background ratio (S: B) of >3 is used to ensure robustness and our experience again shows us that a relatively poor robust Z'-factor and a small S: B most likely will result in a poor assay unsuitable for HTS. This may again seem obvious but there is pressure from project teams to run assays, after all not running an assay guarantees not finding hits, and without a clear set of criteria clear decisions are harder to achieve. It is important that assay developers do not try and configure assay parameters solely to ensure the measurement of very high potency compounds to the detriment of a good S : B, especially as HTS most likely will not find such high potency compounds, and even if there were such compounds, an accurate measurement of potency at the HTS stage is not important; detection of active compounds is what we need.

Measuring the percentage coefficient of variation (%CV) across whole plates ensures that the dispensers and readers are functioning correctly, and if they are available, running known pharmacological standards as concentration responses gives confidence that the assay will find active chemistry in a screen, displays the same rank order of potency expected and can reliably estimate the potency of compounds. This in itself does not test the reliability of the primary single shot assay but is the foundation of a reliable assay. It is also important during screening to give confidence that assay sensitivity remains acceptable throughout an extended screening run.

Our assay development groups also run what we call a mini-validation set to test the reliability of the assay in detecting hit compounds. The mini-validation set is 1408 compounds from our main validation set (see Section 1.1.2.5) on both 1536 and 384 plates. Although it does not always contain hit compounds against all targets it is a useful set to run in that it does not take much effort, does not use too many precious reagents and will quickly flag issues such as high hit rates or poor reproducibility. The full validation set could of course be used at this stage but as we move to more complex screens with expensive and sometimes hard to resource reagents it is usually prudent to use the mini-validation set to preserve these reagents. With these data we can determine some simple parameters and assess the data to determine whether the assay is suitable for hit finding and can be moved to the HTS group for full validation and subsequent transfer. In Table 1.1 we show how the mini-validation data are used to determine, in this case, the screening concentration to be used. In the case of this epi-genetic target, we expect a low real hit rate and a high artefact hit rate and the mini-validation data nicely show how we can determine key parameters for the screen at an early stage in the assay transfer. Additionally, we can use the output of the mini-validation exercise to determine the efficacy of any downstream assay to successfully remove false hits and allow the identification of true hits.

It is important that both the HTS and assay development groups use similar (ideally the same) equipment to remove any issues that can arise during assay transfer with different equipment that perform differently. We have experienced transfers taking longer than necessary when we have used different equipment across the groups and this leads to whitespace (a term we use to describe downtime) in the project whilst the differences are investigated and corrected. The criteria we apply to the mini-validation assessment are essentially the same as for a full validation and are listed in Figure 1.1. The hit rate and reproducibility are key at this stage. A high hit rate can be particularly problematic and we try to ensure that the full screening cascade is in place prior to transfer so that we can test the ability of the assays to remove false hits and confirm real hits. We also have a small library of problematic compound classes such as redox compounds, aggregators and thiol-reactives to probe the sensitivity of a target and its assay to such compounds, which are commonly referred to as pan-assay interference compounds (PAINS). This is a key step in checking the robustness of an assay and also helps us to understand what assays are needed to remove a high hit rate associated with a class or classes of PAINS. This early information allows us to test the validity of the screening cascade. Having this view early on is key to ensure that we can successfully transfer an assay, validate and run the HTS and successfully prosecute its output.

(Continues...)

Excerpted from High Throughput Screening Methods by Joshua A. Bittker, Nathan T. Ross. Copyright © 2017 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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Details zur Ausgabe

• Verlag: Royal Society of Chemistry
• Erscheinungsdatum: 2016
• ISBN 10: 1782624716
• ISBN 13: 9781782624714
• Einband: Tapa dura
• Sprache: Englisch
• Auflage: 1
• Anzahl der Seiten: 396
• Herausgeber: Bittker Joshua A, Ross Nathan T