A tool for running millions of biological or chemical tests in a short time is high-throughput screening. It is mainly used in drug discovery processes to identify biologically relevant compounds. Following you will learn what high throughput screening is, what is required to run high-throughput tests and which assays are primarily used.
Dr Tobias Pusterla
What is high-throughput screening?
High-throughput screening (HTS)is a drug discovery process that allows automated testing of large numbers of chemical and/or biological compounds for a specific biological target, for example throughbinding assays. High-throughput screening methods are extensively used in the pharmaceutical industry, leveraging robotics and automation to quickly test the biological or biochemical activity of a large number of molecules, usually drugs. They accelerate target analysis, as large-scale compound libraries can quickly be screened in a cost-effective way. HTS is a useful tool for assessing for instance pharmacological targets, pharmacologically profiling agonists, and antagonists for receptors (such as GPCRs) and enzymes.
High-throughput screening in drug discovery
Theprimary goalof HTS is to identify through compound library screenings, candidates that affect the target in the desired way, so-called “hits” or “leads”. This is usually achieved employing liquid handling devices, robotics, plate readers as detectors, and dedicated software for instrumentation control and data processing.Fig. 1: PHERAstar FSX plate reader integrated in a Labcyte Access lab workstation at Cancer Research UK, Manchester Institute.Importantly,high-throughput screening processesdo not usually identify drugs, as several properties that are critical to the development of a new drug cannot be assessed by compound library screening. For instance, HTS cannot evaluate toxicity and bioavailability. The primary role of high-throughput screening assays is instead to identify “leads” and provide suggestions for their optimization. The results of HTS assays provide hence the starting point for further steps in the drug discovery pipeline like drug design, and for understanding the interaction or role of a particular biochemical process.
Consequently, high-throughput screening should be seen as a fast scan of biological processes by which compounds with a poor or no effect can be rapidly excluded from the analysis pipeline.
High-throughput screening services: pharma, CROs, and universities
In recent times, HTS was largely enabled by the modern advances in robotics, liquid handling, plate reader detection as well as high-speed computers. Nevertheless, to run effectively, high-throughput screening still requires a highly specialized and expensive screening facility that not every lab can afford. As an alternative to setting up one´s own facility, institutions with limited budgets usually access HTS services provided by third-party providers such as Contract Research Organisations (CRO) or, mainly in an academic environment, core facilities.
In the video below, Mathew Keegan talks about the seamless integration of BMG LABTECH plate readers into Automata´s automation systems.
The high-throughput screening process: basic steps
From a technological point of view, the major points in HTS are miniaturization, automation, and assay readout. Generally, high-throughput screening consists of the following four steps:
Samples are usually of cellular or biochemical nature, depending on the assay to be run. High-throughput screening necessitates that samples are prepared in an arrayed format. The key platform or sample carrier used is therefore themicroplate. Typical formats include 384-, 1536-, or 3456-well plates. The nature of the sample and of the detection assay may affect the choice of the microplate format and its colour.
Screening facilities usually keep their compound library collections stored in so-called “stock plates”. Stock plates are not directly used in experiments. Instead, when needed, compounds from a stock plate are “copied” to an assay plate through a pipetting station.
Establishment of a method suitable for automation
Automationis an important element in HTS as the conversion of a benchtop to an automated high-throughput screening assay enforces specific constraints affecting the practical assay design. Ideally, a HTS assay is performed in a single well, with a low amount of reagents (miniaturization), and minimal or no further manipulation than injection of the sample/compound to be tested. Accordingly, the choice of the optimal detection mode and assay has to be subordinated to automation issues. For more information on the identification of false positives in screening attempts please seeAN 359: Identification of false positives in a HTRF® screen for small molecule inhibitors of PD-1/PD-L1.
Establishing a stringent assay and an effective quality control method are major issues when setting up an automated screen. The clearer the distinction between negative and positive controls, the higher the possibility to obtain high-quality data with a neglectable number of false-negative but especially false-positive results.
In his testimonial, Building a bridge between organoid research and drug discovery, Elad Katz from Navigate Precision Biology discusses the establishment of an organoid platform in 384-well format that can be used for drug screening purposes and how the PHERAstar FSX has been instrumental to developing accurate tests.
Configuration of a robotic workstation
The scope of a robotic platform is to autonomously manage multiple plates simultaneously, significantly speeding up data acquisition. Robotic platforms for high-throughput screenings range from simple automated liquid handling machines to multidimensional workstations performing multiple functions. This is usually achieved with the support of one or more mechanical arms. Typically, a robotic system manages microplates from station to station for several steps such as reagent addition, mixing, incubation, and detection.
Acquisition and handling of data
In high-throughput screenings data acquisition is usually performed by an optical measurement, quantifying the amount of light “produced” by the sample. Different readouts such as fluorescent or luminescent detection, colourimetry, or light scatter (turbidity) are available. Common detection modes includefluorescence intensityandpolarization, FRET,time-resolved fluorescence(e.g.: HTRF®, LANCE®, etc),luminescence(e.g.:NanoBRET), andAlphaScreen®.Fig. 2: HTRF ratios obtained for the IP-One assay with a CCD-based HTS Device.
Fig. 3: HTRF® ratios obtained for the IP-One assay with the PHERAstar FS.
Depending on the biological question to be answered, data quality as well as cost-effectiveness, different light-based detection readouts may be chosen. Specialized instrumentation, like multi-mode microplate readers, can sequentially perform different experiments or apply different detection protocols on the wells. The output thereof is a grid of numeric values.
Fig. 5: 4 PHERAstar FSX plate readers integrated in a HighRes Biosolutions modular robotic workstation at AstraZeneca UK.
To better understand what are the microplate reader requirements of screening facilities, read here Mark Wigglesworth´s full interview. Mark is Director of High-Throughput Screening at AstraZeneca in the UK and has integrated a PHERAstar FSX in AstraZeneca's screening facility.
„We found that the improvements in the new PHERAstar FSX outperformed our historic experiences and the other readers in our trial. The reader has fitted well into our assay development and screening groups, where it has been used frequently for a variety of technologies.“
Mark Wiggelsworth, AstraZeneca, UK
A HTS-dedicated plate reader can measure hundreds of plates in a single day, generating a considerable amount of data points. Consequently, data management is a critical point in automated high-throughput screening, given the large number of compounds tested, the variety of chemical libraries, and the necessity to correlate and compare results from different screening campaigns. Dedicated data analysis and management platforms (e.g.: Genedata) intertwine data originated from different screening campaigns with compound structures, as well as performed assays to each other, facilitating the extraction of detailed information from different perspectives. In addition, applying artificial intelligence to drug discovery can provide a more rapid and cost‑effective route to discovering candidates. Martin Redhead elaborates on this topic in the customer interview: Searching for optimised drugs: combining AI with human expertise.
A good example of how the whole high-throughput screening process woks, from assay development to target identification, is discussed in the scientific talk here below.
PHERAstarFSX: the dedicated high-throughput screening plate reader
Compound library screenings have to be usually measured as fast as possible, in very small volumes (a few µL), and produce robust results. Besides the ease of use and flexibility to perform different assays, this requires very sensitive and precise instrumentation. Watch Céline Legros from Servier, France, commenting on her experience with the PHERAstar platform.
Through its past generations, the PHERAstarFSXhas been looked at as the reference reader for the field. The following features make it the gold standard for high-throughput screening:
Highest sensitivity on the market for fluorescence intensity and polarization
Measurement in 384-, 1536- as well as 3456-well plate formats
Simultaneous Dual Emission detection for fast and robust detection of fluorescence polarization assays, BRET, FRET, and TR-FRET as well as AlphaScreen®assays
Dedicated AlphaScreen®/AlphaLISA®excitation laser
High-frequency TRF laser allows efficient measurement of 1536-well plates on the fly (1 flash) in just 36 seconds
All plates in a batch or a screening campaign are measured under the same conditions thanks to the AAS system
Fig. 6: Signal to Blank (S/B), Z´value and read times for a Histone-BRD4 interaction assay measured on the PHERAstar FSX. The use of the TRF laser reduces read times, increases S/B, while keeping a very high Z´value.
In drug screening, speed is as important as miniaturization. AN295: "Cellular and biochemical HTRF assays measured in 1536-well microplates" shows how the PHERAstarFSXequipped with the TRF excitation laser, is able to detectHTRF®assays in 1536-well plates “on the fly” in only 36 seconds, still delivering a Z` value > 0.8.
You can learn more about Z prime (Z’) values and their uses in high throughput screening in the BMG LABTECH blog Z prime value (Z’).
Customer success stories
PHERAstar plate readers are routinely used in HTS facilities as they provide an accurate signal, sensitivity, and high throughput capacity.
The Pivot Park Screening Centre in Oss, the Netherlands, relies on the PHERAstar FSX for effective hit discovery. In our customer success story “Improving hit discovery efficiency at Europe’s leading screening centre”, their CTO, Steven van Helden discusses innovation in drug discovery and the future of screening technology.
In this testimonial, Yanlong Li, Ph.D. tells us how the PHERAstar helped him to cut reagent usage by half.
In the post „Choosing the best microplate reader“, Mark Wigglesworth, Director High Throughput Screening at AstraZeneca UK discusses the microplate reader requirements needed to keep up with AZ´s screening necessities and why AZ chose the PHERAstar FSX.
In her testimonial, Tackling rare and neglected cancers with advanced screening, Charlotte Walsh-Tripp from Cancer Research Horizons UK discusses working on cancer targets that are often neglected by other research initiatives and which role the PHERAstar plays in her work.
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Mark Wiggelsworth, AstraZeneca, UK 
