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Evolution of Ultra-High-Throughput Screening

Ultra-high-throughput screening (uHTS) is an automated methodology for conducting hundreds of thousands of biological or chemical screening tests per day using liquid handling devices, sensitive detectors, robotics, data processing, and control software.

by John Buie

Ultra-high-throughput screening (uHTS) is an automated methodology for conducting hundreds of thousands of biological or chemical screening tests per day using liquid handling devices, sensitive detectors, robotics, data processing, and control software. The results of these experiments provide starting points for drug design and for understanding the interaction or role of a particular biochemical process in biology.

The cut-off between high-throughput screening (HTS) and uHTS is somewhat arbitrary. The advent of automated plate-handling and reading instrumentation, and the replacement of radiolabeling assays with luminescence- and fluorescence-based screens, created the opportunity for the several-hundredfold improvement in throughput represented by uHTS. As instrumentation prices have fallen, uHTS is now accessible to small research companies and academic groups.

Recently, in vitro testing has become a viable alternative to in vivo testing for assessing hepatoxicity, a technique that is appealing to manufacturers. The development of quantitative high-throughput screening (qHTS) allows a wide range of toxins to be tested, making it an economical and efficient option for manufacturers.

Here is a summary of some of the main milestones in the history of uHTS from its origins in the late 1980s.

Until the 1980s, the number of compounds that could be screened by a single facility in a week was between 10 and 100.

In 1986, Pfizer was involved in natural products screening by substituting fermentation broths with dimethyl sulfoxide solutions of synthetic compounds, using 96-well plates and reduced assay volumes of 50-100μl. A nominal 30mM source compound concentration provided high μM assay concentrations. Starting at 800 compounds each week, the process reached a steady state of 7200 compounds per week by 1989.

By 1992, technology had advanced enough that thousands of compounds could now be screened by a single facility in a week. By this time, Pfizer was using HTS to produce approximately 40 percent of its ‘hits’ in its Discovery portfolio.

By 1994, tens of thousands of compounds could be screened a week, but 384-well plates were still extremely rare.

The 1994 International Forum on Advances in Screening Technologies and Data Management saw the first mention of the term ‘Ultra-High-Throughput Screening’ in a presentation by Harry Stylli entitled, ‘An Integrated Approach to High-Throughput Screening’.

By 1996, uHTS was considered a realistic goal, and 384-well plates were being used in proof-of-principle applications. Around this time, thousands of compounds could be screened a day.

In 1996, Evotek, in collaboration with Novartis and SmithKlineBeecham, developed a high-throughput screening system, EVOscreen®, which was eventually launched in 2000.

The Evotek EVOscreen

In 1997, LJL Biosystems, Inc. raised the bar in the number of compounds that could be screened in a single day with its first high-throughput screening system, ANALYST. This instrument was capable of throughputs of approximately 70,000 assays per day, exceeding traditional throughput averages by up to five to ten times.

By 1998, tens of thousands of compounds could be screened per day, and genomic targets were a reality. By this time, 384-well plates were widely used, and 1536- well plates were being used in proof-of-principle testing.

In 2000, Aurora Biosciences completed development of an ultra-high throughput screening system for Merck. This platform combined compound management, plate replication, assay preparation, hit identification, selection and re-tests of the hits, fluorescence detection, and data analysis into one fully-integrated and automated system that enabled the performance of miniaturized assays with a ten-fold increase in efficiency over conventional highthroughput screening methods.

In 2001, Molecular Devices launched the FLIPR High-throughput Screening System. This was followed in 2004 by the FLIPR Tetra Ultra High-throughput Screening System for identifying early leads against GPCR and ion channel receptors. Because the company recognized that assay needs vary during the different stages of the drug discovery screening process, the FLIPR Tetra System was designed to be user-configurable and available with either a fluorescence or luminescence camera.

In 2005, high-throughput screening reached new levels of efficiency when RTS Life Science began selling the Symphony ultra-high-throughput screening system developed in conjunction with Novartis to European customers. The Symphony system was conceived as a multi-lane robot system combining local compound storage, reformatting, assay plate creation and screening and was designed for use in laboratories needing to screen in excess of 1,000,000 compounds per day.

The FLIPR Tetra

In 2006, Molecular Devices made high-throughput screening available to a greater number of laboratories when it launched a benchtop high-speed confocal imaging system that streamlined high-throughput imaging and hit selection in a fully-integrated environment. This system was intended to offer higher performance high-throughput screening at a lower price than existing instruments and was specifically intended to compete with similar products offered by GE Healthcare and Evotec Technologies.

The RF360 High Resolution System

In 2007, PerkinElmer responded to the increasing importance of flash luminescence in primary and secondary screening of drug targets by releasing the first dedicated flash luminescence uHTS instrument, the 1536-Head LumiLux Cellular Screening Platform. This instrument enabled 1536 simultaneous “inject and read” flash luminescence assays, allowing laboratories to produce over 100,000 data points per day.

The 1536-Head LumiLux Cellular Screening Platform

In 2008, Sanofi-aventis launched the dScreen Consortium, a research initiative conducted with RainDance Technologies and Louis Pasteur University, Strasbourg, France, to develop the next generation of high-throughput screening for drug discovery applications.

In 2009, BioTrove announced the launch of the RapidFire 300 system for high-throughput screening of in vitro ADME assays, enabling researchers to perform a wide range of assays with 24-hour, unattended operation. The RapidFire system streamlined drug discovery workflow, significantly decreasing the processing time compared to conventional MS-based technologies and helping to eliminate bottlenecks in drug discovery while providing accurate results for data-driven decision making.

The AU5800 Automated Chemistry System

In 2010, BIOCIUS Life Sciences and Agilent Technologies developed the RF360 High Resolution System for the high-throughput screening of in vitro ADME assays. This system combined time-of-flight mass spectrometry with the high sample processing speed of RapidFire technology for efficient in vitro ADME analysis by eliminating a notorious bottleneck in drug discovery.

Also in 2010, PharmaDiagnostics launched the So- PRano assay kit for in-house assay development enabling users to develop assays for a range of applications, including fragment screening, focused high throughput screening, antibody discovery, and hit confirmation.

In 2011, Beckman Coulter, Inc. released the AU5800 Automated Chemistry System designed to improve processing time compared with existing systems and meet the demands of high- and ultrahigh- volume clinical laboratories. This system was capable of up to 2,000 photometric chemistry tests per hour for the single module, and up to 8,000 photometric tests per hour with a four-unit configuration.

Also in 2011, High Throughput Genomics launched the M3 series of multiplex mini-microarrays based on the qNPA ArrayPlate that improved the precision of data generated from samples by increasing automation. This technology allowed hundreds to thousands of samples to be analyzed per day.

Future of ultra-high-throughput screening

Ultra-high-throughput screening is a new field which is evolving at an astonishing rate. The advances made over the past 20 years have revolutionized drug screening and development, and this pace of change is only likely to increase over the coming years.

In the future, uHTS is likely to move to even higher density formats, with no limit on the number of wells that can theoretically be analyzed simultaneously. Chip-based screening systems are likely to become increasingly important, as well as micro-channel flow systems, and plateless systems.