Screening
of the target), HCS assays are enabling a more focused readout on the target of interest (eg pro- tein phosphorylation) in addition to the monitor- ing of the compounds’ effect on the whole cell physiology (eg toxicity or morphological changes). Third, the use of multiplexing and sophisticated image analysis in HCS offers more information from the hits than traditional screens. For instance, multiple nodes of a cellular pathway can be measured already at the stage of primary screening (eg protein translocation trig- gered by a phosphorylation event), enhancing the content and quality of the derived hitlist. Fourth, the use of multi-parametric image and data analysis can reduce the rate of false positive hits lowering the need to perform counter screens to sort out compounds having unspecific effects. Image visualisation tools can support quality control to reduce the false positives as well as to identify assay artifacts that could lead to false negatives (eg absence of staining).
Figure 1 The automated plate
preparation platform dedicated to immuno-staining protocols. A Catalyst 5 robot (Thermo Scientific) is integrated with 3 BNX1536 (Bionex), 2 Cytomat 5 (Thermo Scientific) for incubation at 37˚C or 4˚C as well as with a Teleshake (Thermo Scientific). The system is operated by the Momentum software
folds were identified as hits. While this study represents only one HCS and RGA assay format the outcome might change when comparing other pathways or assay setups.
Why enable HCS in a high-throughput format?
There are mainly four reasons for high-through- put high-content screens. The first and most obvious reason is to fill a gap. Many targets are not suited to be screened with biochemical or conventional cellular assays. HCS is expanding the field by enabling screens that used to be impossible with a decent throughput. Examples of these assays are the quantitative analysis of protein aggregation and granularity, as well as relocation events or morphological changes. The second reason is that in contrast to classical cel- lular assays (eg a reporter gene which provides an indirect readout that can be far downstream
20
How are high-throughput HCI assays performed? We started implementing HCS at the Lead Finding Platform of NIBR in Basel in May 2005. The first high-throughput HCS was carried out only recently in 2010. With our high-content instrumentation we support assays for multiple disease areas of NIBR, eg oncology, respiratory diseases, immunology, cardiovascular and infec- tious diseases. Depending on the readout type, the sensitivity and the statistical quality of the assay and taking into account the time and cost con- straints, the team decides if the imaging technolo- gy can be used or if a conventional cell-based assay is better suited. The HCS assay formats and readouts are variable including nuclear transloca- tion, protein phosphorylation, receptor internali- sation, intracellular trafficking of proteins and virus infection. Not all of these assays are amenable to being tested with a million com- pounds due to technical or biological constraints such as plate format, incubation times, or cell line stability; therefore these parameters need to be assessed project specifically.
HTS process
Generic processes for plate preparation, image acquisition and data handling need to be imple- mented to efficiently perform HCI in high through- put. The imaging time is generally the bottleneck compared to the plate preparation time which is faster in most cases. In order to reach the maxi- mum throughput, flexibility in the use of the
Drug Discovery World Winter 2011/12
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