Sept 11, 2003 | AS THE PHARMACEUTICAL industry battles the escalating cost and time of drug discovery and development, there is a growing need to increase confidence in "validated" targets, improve lead selection, and reduce late-stage attrition. One technology making an impact in target and lead selection is high-content cellular screening (HCS).
By tracking cellular activity with multiple fluorescent reporter systems, combined with high-resolution imaging and high-throughput image analysis, researchers can observe multiple intracellular events in individual cells. HCS enables functional analysis of target and pathway modulation in living cells, making it ideal for target and pathway validation, primary screening, and lead optimization. While the potential of HCS has been evident for years, the technology is just beginning to approach the capabilities necessary for large-scale adoption in drug discovery.
"The new-generation cellular assays are more complex and demanding," explains Dietrich Ruehlmann, development integration scientist at Amersham Biosciences. "But their true value is the ability to multiplex analysis of various targets within a single cell, offering researchers a closer look at living systems in a high-throughput manner."
Under the microscope: UV-irradiated skin fibroblasts show mitochondrial damage; mitochondria (green), cytoskeletal proteins (red), and nuclei (blue) are visualized using fluorescent dyes.
The key segments of the HCS market include powerful imaging technologies; robust and easy-to-use cellular assays and biomolecular labeling methods; and automated image analysis and data storage.
Development of high-resolution, high-throughput imaging systems capable of multi-color fluorescence detection in fixed and live cells provides a key enabling technology for HCS. Examples include Cellomics' ArrayScan system and KineticScan workstation; Amersham's IN Cell Analyzer 1000 and 3000; Acumen Bioscience's Explorer system; CompuCyte's iCyte imaging cytometer and LSC laser scanning cytometer; Atto Bioscience's Pathway HT kinetic cell imaging system; Universal Imaging's (subsidiary of Molecular Devices) Discovery-1 system; and Q3DM's EIDAQ 100 High-Throughput Microscopy (HTM) system.
Green fluorescent protein (GFP) and other labeling methods provide easy-to-use approaches to monitoring intracellular events. Fluorescent labeling offers real-time analysis with high sensitivity and good dynamic range, minimal interference with normal cellular function, ability to multiplex analysis of multiple targets in a single experiment, and relative abundance of commercial reagents.
The final piece of the puzzle is image analysis and data storage, which has to keep up with the increased throughput of new imaging technologies. High-throughput HCS systems can generate as many as 40,000 images per day; these images need to be analyzed and stored for future query. Considering that image files can take up orders of magnitude more space than numerical data, companies must make difficult choices about storing terabytes of image data.
"As good as the technology is, it's still in the early stages," warns Ralph Garippa, group leader of cell-based high-throughput screening (HTS) and robotics at Roche Discovery Technologies. "The equipment, image algorithms, and processing speed still need to improve. The probes and reagents should be developed beyond GFP and CyDye fluors to allow more live-cell analysis. Lower prices could also make the technology more affordable for early introduction into the field."
Spanning Drug Discovery
"The advantage of HCS lies in its ability to monitor the cell's dynamic state — where proteins are being activated, deactivated, or translocated within a cell," Garippa explains. "Tagging these proteins and tracking them by microscopy opens a new avenue for screening."
HCS was first adopted in secondary/tertiary drug screening and lead optimization. The high-content cellular information on lead specificity, bioavailability, and ADME-Tox allows researchers to prioritize leads with more confidence and reduce late-stage attrition.
The second area of drug discovery adopting HCS is target and pathway validation. Tracking the effects of target/pathway modulation on cellular processes and phenotype — combined with tools from functional genomics, RNA interference, and chemical genomics — provides a deeper understanding of molecular pathways and enables better target prioritization.
HCS technologies are also approaching the throughput, automation, and robustness necessary to handle primary screening. HCS in whole living cells allows researchers to observe the effects of compound-target interaction, determine toxicity and specificity of compounds, and identify cell-to-cell variability in drug response. It also allows researchers to screen targets that are intractable using conventional in vitro assays. Availability of high-content information in primary screening promises to increase confidence in hits and reduce the need for secondary screens.
"While just a few years ago, HCS was a 'good idea,' now you are actually seeing the real applications," Garippa says. He attributes successful technology adoption to a changing mindset, improvements in instrumentation and processing speed, and a better selection of commercially available reagents.
Availability of high-content cellular information at early stages of drug discovery promises to improve the quality of targets, hits, and leads; reduce late-stage attrition; and shorten time and cost of development. According to Amersham's Ruehlmann, "the market and the technology are evolving in synchrony."
Julia Boguslavsky is the conference director for Cambridge Healthtech Institute. She can be reached at firstname.lastname@example.org.
PHOTO COURTESY MOLECULAR PROBES INC.