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 through binding 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
The primary goal of 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.
Importantly, high-throughput screening processes do 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.
Ian Waddell of Charles River Laboratories discusses here what are “the microplate reader requirements of a CRO” for effective hit discovery.
Prof. Christian Heinis from the EPFL in Lausanne makes use of screening in an academic setting to analyse the effectiveness of cyclic peptides as drugs.
In the video below, Mathew Keegan talks about the seamless integration of BMG LABTECH plate readers into Automata´s automation systems.
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 include fluorescence intensity and polarization, FRET, time-resolved fluorescence (e.g.: HTRF®, LANCE®, etc), luminescence (e.g.: NanoBRET), and AlphaScreen®.
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.
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.
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.
PHERAstar FSX: 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 PHERAstar FSX has 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
High-throughput screening applications
Although one of the most targeted groups of receptors in drug screening is G-Protein Coupled Receptors (GPCRs), not every benchtop assay can be converted to an automated HTS, especially when using living cells. AN287: "NanoBRET assay for monitoring of ligand binding to GPCRs in live cells, using the CLARIOstar and the PHERAstar FS" describes how for the first time NanoBRET was successfully used to monitor ligand-GPCRs binding in living cells.
An important issue in HTS is miniaturisation. Samples have to be as small as possible in order to save costs, but still deliver reliable results. The sensitivity of the microplate reader and its ability to reliably measure samples in high-density plate formats is consequently of great importance. In AN229 "Nano high-throughput screening (nHTS) platform - miniaturization of cell-based GPCR and kinase assays", DiscovereX PathHunter reporter cell lines were measured with the PHERAstar FS in both 1536- and 3456-well plates. The reader was able to detect signals coming from as few as 1,000 cells in a 1,536- and 250 cells in a 3,456-well plate. Similarly, AN260: "Miniaturization of a cell-based TNF-α AlphaLISA assay using Echo liquid handler and the PHERAstar FS" shows how an AlphaLISA assay was miniaturized down to 2 µL per sample in a 1536-well plate and detected on the PHERAstar FS.
In drug screening, speed is as important as miniaturization. AN295: "Cellular and biochemical HTRF assays measured in 1536-well microplates" shows how the PHERAstar FSX equipped with the TRF excitation laser, is able to detect HTRF® assays in 1536-well plates “on the fly” in only 36 seconds, still delivering a Z` value > 0.8.
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 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.
Searching for novel kinase inhibitors with the PHERAstar FSX plate reader at BIENTA laboratory in Kyiv, Ukraine. The HTS group screened more than 600,000 compounds in kinase assays using ATP depletion assay, measuring luminescence in a coupled luciferase reaction.
PHERAstar plate readers are routinely used in the laboratory as they provide an accurate signal, high throughput, and sensitivity, as well as stable temperature control required for luminescent kinase assays.