High content screening has several benefits over other drug lead screening methods but has yet to reach its full potential, argues Dr Stephen Hammond of Scottish Biomedical
Around 20 years ago high throughput screening (HTS) of chemical libraries of drug-like small molecules against single biochemical targets in microtitre (96-, 384- or 1536-well) format replaced the time worn route where teams of medicinal chemists toiled synthesising hundreds of analogues of known drugs or natural products in the hope of finding better or safer compounds. In the 1990s, the availability of chemical libraries consisting of hundreds of thousands of compounds coupled with improvements in miniaturisation, automation, fluid handling robotics, endpoint detection technologies and data handling software allowed the Industry to screen hundreds of thousands of compounds against multiple targets each month.
Impressive though this sounds, the largest of such libraries could only contain a small subset of all chemical possibilities. The estimated total number of chemical structures possible ranges from 1018 to 10200, with a consensus around 1060 compounds that could actually be synthesised1 but to date chemists have only made around 5 x 107 of these. It is likely than only a small fraction of the total "chemical space" will ever be available for screening by the Industry.
Many studies have demonstrated that the productivity of any chemical library in producing drug leads depends more upon the quality of the assay used and the informational content the assay generates rather than the total number of compounds screened.
Analysis of the human genome suggests it contains around 35,000 proteins around 10-20% of which are likely "drugable" targets (current estimates suggest that to date between one to two thousand of these have been the subject of HTS.2
Thanks to automation, robotics, miniaturisation, data handling and more sophisticated assay techniques, modern HTS systems now can comfortably screen 100,000 to 200,000 compounds a day against multiple targets. Some of the larger pharmaceutical companies are able to ratchet that up to 500,000 to 1,000,000 compounds on dozens of targets per day. Disappointingly, while HTS has led to the discovery a large number of potent and specific inhibitors of their respective targets, it has so far failed to deliver sufficient new drug leads to reverse the year on year decline in new drug approvals.3
Over the past five years the Industry has turned to the use of High Content Screening (HCS) to improve its chances of finding innovative therapies. HCS is not a new concept but is a natural extension of existing technologies. In HCS the endpoint in expressed in a single cell or a population of cells rather than isolated target in solution. In general HCS have three requirements, a cellular component, instrumentation to conduct and monitor the assay, and informatics to capture manage and analyse the data coming from the assay.
HCS can provide detailed information on sub-cellular temporal and molecular events and can allow the discrimination and analysis of different cell populations. HCS often uses automated fluorescence techniques that allow high-resolution multicolour fluorescence imaging of individual or multiple targets or pathways within living cells Because HCS makes multi-parameter measurements it generates much more information per microtitre well on the pharmacological potential of each compound under test. As a result HCS requires the application of sophisticated bioinformatics tools to interpret with the sheer volume and complexity of the information produced.
HCS are widely promoted as the way to overcome present hurdles in drug discovery and to "fast fail" flawed candidates. HCS allows walk away automation of certain targets not addressable by HTS. In HCS the conformation and activity of the drug target, as well as the read out that monitors the drug candidate, are presented in a cellular context that better represents the natural physiological state that in biochemical assays. There is no need to purify target. Since HCS collects multiple pieces of useful information per micro-plate well it may detect subtle or graded response missed by HTS that use single point measurement.
By their very nature cell based assays select against compounds that are cytotoxic or that are membrane impermeable. Additionally HCS can be used to identify cell sub-populations and sub cellular compartments and gives valuable insights into the mode of action of drug candidates. HCS can capture a greater variety of possible drug-target interactions e.g., inhibitors, activators, allosteric modulators, altered target conformation or interactions and, unlike HTS, can discriminate between agonists and antagonists. All in all HCS generates more relevant, valid leads than HTS presenting a saving in both time and cost.
However the successful application of HCS to drug discovery does require a change in the philosophy and skill set assay development and screening teams.
Fundamental to HCS is the engineering and growth of cell lines. This is time-consuming and when coupled to the limitations in current HCS equipment means significantly lower throughputs than with conventional HTS. To some extent we can compensate by using integrated solution focussed libraries (the product of in silico screening) rather than the using the larger chemical diversity libraries and by the introduction of second-generation instrumentation.
Increased acceptance of HCS would be facilitated by new technology that shortens the assay development time in particular improved technologies for cloning, transfection and establishing stable reporter cell lines. Better expression vectors have been introduced and advances have been made in the automation of colony selection (picking), automated cell counting and confluence measurements miniaturisation and automation of cell dispensing. These have come together to facilitate the production of consistently reproducible reporter cell lines. The advent of improved more flexible instrumentation (particularly imaging equipment) with faster performance coupled with the use of multiplexed assays that permit simultaneous screen of pathways or multiple targets are yielding significant gains in daily HCS throughputs
The complexity of the data produced by HCS is not generally compatible with current HTS IT packages. HCS requires detailed strategy on image format, retention and the choice of algorithms to handle the data produced. Often HCS produces large amounts of data needing bespoke bioinformatics packages to render it meaningful. There is obvious value in designing screening systems to extract the maximum information in the simplest way.
HCS of large compound libraries requires expensive and complex reading and data analysis systems. One key problem that remains is the management of DMSO sensitivity/compound toxicity over the longer incubations required for HCS.
Acceptance of HCS is growing in the Industry. It will never completely supplant HTS but certainly will run along side it. At Scottish Biomedical at least 50% of all drug discovery assays requested by pharma and biotech clients are now cell-based and we have consistently observed that HCS allows the earlier identification of higher quality leads and the delivery of novel therapeutic agents for our clients.