Time for change
13 Jun 2007 by Evoluted New Media
The spiraling cost of developing drugs is due, in part, to time spent assessing compounds that subsequently fail. High-content screening is an established technology that has the potential to deliver higher quality candidates and change the course of drug discovery
The spiraling cost of developing drugs is due, in part, to time spent assessing compounds that subsequently fail. High-content screening is an established technology that has the potential to deliver higher quality candidates and change the course of drug discovery
FOR over a decade, pharmaceutical companies have routinely used High Throughput Screening (HTS) assays to identify compounds acting on drug targets in an effort to find the next blockbuster drug. HTS assesses compound libraries as a first step to determine the effects on targets of interest, generally using homogenous, non-cellular assay methodologies. Traditionally, primary screening was performed using techniques such as biochemical screens, in which unravelling the complex biochemical pathways that constitute and underlie the cell machinery has involved the study of isolated proteins in homogeneous in vitro assays. Similarly, pathway resolution was determined by reconstituting proteins with appropriate substrates and applied known co-factors. These methods investigate protein activity in a well controlled manner, but their overall relevance to protein function in the whole cell environment has been questioned. This move towards to primary drug screening has not delivered the anticipated number of successful drugs to the market because this (non-cell based) minimalist approach to screening results in many hit compounds failing further down the drug discovery process. A new process was needed, which has led to screening of smaller targeted compound libraries plus a renewed focus on target validation in a whole cell environment to provide more information, including the evaluation of multiple biochemical and morphological parameters for each compound tested. Thus, the now established technology of High Content Screening (HCS) was born.
HCS can provide researchers with the comprehensive level of biological and chemical information needed to reduce late-stage failures and it is maturing rapidly, but is still not fully applied within the pharmaceutical industry as researchers continue to define its value. It offers whole cell analysis at various stages of the drug discovery process - from target identification through to lead optimisation - while retaining the ability to screen large numbers of compounds. HCS allows the generation of secondary screening information (for example, determining if the compound is toxic). Potentially, HCS also provides greatly improved data quality, as it monitors multiple cellular events in response to the effects of a drug at much earlier stages of drug discovery than were previously possible. However, the current challenge is to make HCS compatible with the requirements for primary screening. Today's instrumentation is moving towards screening similar numbers of compounds to those used in HTS. However, one of the key issues is how high content cell based assays developed for target identification and validation can meet the differing requirements for high throughput screening campaigns. HTS relies on automated technologies delivering rapid, yes/no readouts at low cost. To deliver meaningful results, HCS assays must not only meet these criteria, but also be rigorous enough to cope with extensive libraries of poorly characterised compounds of unknown biological behaviour.
High content cell based assays principally measure fluorescence spectroscopy at the level of individual cells. Three distinct technological solutions have been used in this arena: laser-scanning fluorescence microplate cytometry, fluorescence microscopy and flow cytometry. Although based on quite different technologies, each analyses multicolour fluorescence in fixed- and/or live-cells by detecting cellular events using fluorescently-tagged markers, enabling researchers to screen for multiple cellular readouts on a per cell basis within a single experiment.
Laboratories, however, are now beginning to realise that high content instrumentation used to understand the underlying biology for target identification and validation, does not necessarily meet the very different requirements of HCS (Table 2). The aim of HCS is to profile compound libraries in a fast and economical manner to generate hits against new therapeutic targets. High levels of information are not necessarily required for simple hit/no-hit answers in a primary screen, as often 99.9% of compounds in a standard HTS assay will have no effect. To comply with these requirements, HCS assays need to be robust (with Z’ typically greater than 0.5), have fixed endpoints to permit batch processing of large numbers of plates, and preferably offer whole well analysis to account for intra-well variation (Figure 1). In addition, the number of cells required should be low, and data file sizes kept small to cope with the increased throughput and the lack of requirement for re-analysis.
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| Figure 1: High content analysis of protein kinase activation (ERK1/2) using an Acumen eX3 microplate cytometer. Panel A (left) shows a well view from a partially active well. Panel B (right) shows the same well with the cells classified by the Acumen eX3: green cells are active, red are inactive |
These requirements are met by multiple laser-scanning microplate cytometers, which can analyse the whole well, and process data directly to rapidly detect and quantify all fluorescent objects within each well. They also offer
| Cell adhesion Neurite outgrowth Micronucleus formation Protein translocation Cell cycle analysis Cell differentiation Adipogenesis Reporter gene analysis Cell viability Cell spreading Cell proliferation | Apoptosis Microtubule formation Receptor trafficking Protein kinase activation Mitotic index Proteasome activity Oligosaccharide synthesis Cell migration Membrane potential Gap junction formation P-glycoprotein activity |
| Table 1: Examples of High Content Screens |
exceptionally fast read times and their use of thresholding algorithms to identify objects results in small data files – ideal for primary screening. Although some fluorescence microscopy systems have the speed to run large primary screens they are best suited for validating hits that are derived from High Content cell based assays. This is due to the large file sizes generated (up to terabytes), which require expensive storage and retrieval solutions and their analysis of small areas of each well. Flow cytometry offers the user high sensitivity assays with great multiplexing capability. However, this technology does not lend itself for use within a screening environment due to its relatively low throughput and the difficulty of automating assay protocols. A requisite for HTS-compatible high content assays to be useful in a screening environment is their efficient transfer from the development to the screening laboratory. The limitations associated with fluorescent microscopy and flow cytometry mean that while they can perform low throughput screening they are best suited for validating hits, while laser scanning microplate cytometry stands out as the technology best suited for HCS.
| High Content Primary Screening | High Content Hit Validation |
| • compound capacity is in the 10,000 - 100,000s • requires yes/no answer - only interested in hits • readout is fixed endpoint for signal stability and ease of automation • ideally whole well analysis to account for possible variations in well • small data files needed to cope with increased throughput | • compound capacity is in the 100-1,000s • requires multi-parametric data from each cell, often from multiple fluorescent colours • readout may be kinetic or single endpoint assay performed on live or fixed cells • high resolution analysis of sub-population of cells in each well • large data files produced to store all information for detailed analysis |
| Table 2: Comparison of the key features of high content analysis and high content screening |
HCS can improve the quality of hits identified from primary screens, by the ability to evaluate multiple readouts. It is possible to evaluate a compound against a specific target and simultaneously determine the compound’s cytotoxicity. Effects such as these are undesirable and as such having this information enables compounds to be discounted at this early stage. The potential for reducing false positives makes secondary screening and “hit to lead” more efficient and cheaper. In an industry preoccupied with the quest for the next blockbuster drug, the advantages and benefits of HCS-enabled lead discovery cannot be ignored.
HCS has a bright future and an important - possibly pivotal - role to play in primary drug screening, therapeutic drug evaluation and delivery of novel therapeutic agents.
By Paul Wylie and Andrew Goulter of TTP LabTech, Hertfordshire, UK
