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...