Chemical & Processing
Oil & Gas
Pharma Biotech
Infrastructure & Design

Advanced Cell Screening Technologies Accelerate Drug Discovery Process
- Mattias Karlsson, Chief Technology Officer, Cellectricon
- Johan Pihl, Product Manager, Cellaxess Platforms Cellectricon

In this article, the authors review the latest developments in phenotypic assay technologies employed in modern drug discovery.

The majority of the new “first-inclass” drugs approved in the past 10 years were developed from candidates generated through phenotypic screening, despite the fact that the focus in the industry has been on target-based approaches. With this becoming more apparent, information-rich phenotypic screening strategies have gained strong attention.

Despite extensive efforts, drug discovery, particularly in the field of CNS and pain, has seen a number of late-stage failures in recent years. Arguably this is a consequence partially spanning from the lack of predictive in-vitro models. In order to better envisage target engagement and effects of candidate compounds it is important to use relevant cell and tissue models, taking into account the complexity of interactions in both health and disease, as early as possible. Traditionally, cell-based screening in drug discovery has mainly been carried out using recombinant cell lines coupled with homogenous downstream assays, principally due to cost and convenience. Furthermore, reductionist approaches such as targetbased screening have been widely employed for a variety of reasons, with limited success.

Recent observations indicate that phenotypic screening has been more productive in generating “first-in-class” drugs, while an overemphasis on individual targets has been suggested as one of the reasons for the lack of productivity in drug discovery1. Using phenotypic approaches may lead to the identification of molecules that modify a disease by acting on previously undescribed target(s) or by acting simultaneously on multiple targets.

Consequently, there is a large emphasis in the pharmaceutical industry to develop screening strategies with capability to identify phenotypic effects in highly relevant biological models. In particular, there is a strong focus on developing invitro disease models from either native tissue9,10 or stem cell-derived cultures2. There is also a need for enabling assay hightechnologies4 that can support screening of this type of sophisticated biology that in general is less amenable to traditional high-throughput methodologies. Ideally, these strategies should be employed early in a project’s lifecycle to add real value to the decision of whether to pursue or eliminate a compound, ultimately translating to considerable cost savings in the drug development business.

Biological Relevance, Primary Cells and IPSC-Derived Disease Models
Because of availability and convenience, immortalised cell lines have traditionally been used in place of more biologically relevant cell models in drug discovery. However, few cell lines, if any, can serve as accurate in-vitro models. Although derived from primary cells, these lines typically consist of immortalised or cancerous cells that divide continuously, suggesting their pattern of gene and protein expression is significantly different from terminally differentiated and functioning cells. In terms of cellular composition and complexity, dissociated primary cultures more closely resemble in-vivo biology than immortalised cell lines. Accordingly, these cellular systems allow evaluation of features and processes that are hard to assess in overexpressed cell lines. Examples include cell morphology and polarisation, energy metabolism, signal transduction, toxicity response and neurotransmitter release. When using native cells or tissue is not practical or possible, iPSC-derived cell models have risen as a highly attractive model for use in drug discovery2. Although lack of validation is still an issue for many of the models5, they hold great promise for a variety of disease models in drug discovery6.

Technologies for Phenotypic Screening
In addition to cell models and systems providing biological relevance, a number of phenotypic and information-rich screening technologies have emerged that enable accurate interrogation of the more biologically relevant cell models. By far, the most established route today is the shift from homogenous and/or plate reader-based readout assays to various types of high-content based ones. While mostly used as an endpoint assay, highcontent methods allow for detailed analysis of a wide range of cellular phenotypes. Furthermore, the data sets generated by high-content screening allows for refinement of the analysis in a way that is not possible with a single-parametric, endpoint readout.

The available high-content screening instrumentation and analysis methods have evolved immensely since the advent of high-content screening in the 1990s, and there is now instrumentation available ranging from bench-top systems with lampbased wide field illumination to confocal systems with laser-based illumination. A plethora of analysis software options exist to allow the analysis of virtually any cellular phenotype or process, ranging from cell cycle and proliferation, intracellular trafficking and translocation, infection, differentiation, gap junction formation and more8-10. However, despite the versatility of the current high-content screening platforms, in many cases they lack the ability to apply physiologically relevant stimulus, and this is particularly true in the case of excitable cells.

A recent technology combining physiological relevance with an informationrich assay readout is Cellaxess Elektra discovery platform, which enables high throughput in-situ electric field stimulation of adherent cell cultures and tissues in a microplate format. Simultaneous dynamic image-based fluorescence readouts can be employed to monitor the response of the cell culture to electric field exposure in real time. This offers a unique methodology to identify novel modulators of excitable cells for a multitude of diseases in the field of CNS/pain, such as Alzheimer’s disease, Schizophrenia, Parkinson’s and neuropathic pain.

The potential assays include stimulation of native or iPSC-derived cells using specific Electric Field Stimulation (EFS) protocols to characterise the effects of compounds on excitability parameters. One specific example is the pain peripheral sensitisation assay, where primary Dorsal Root Ganglion (DRG) neurons are cultured in microplates and supplemented with nerve growth factor to mimic peripheral sensitisation. The Cellaxess Elektra platform is used to excite the sensory neurons, and the resulting dynamic response assists in the identification of small molecules that have the potential to treat chronic pain11.

On a similar note, Hempel et al has described the development of a highthroughput assay technology for performing assays of synaptic function in primary neurons7. The system was designed to study synaptic vesicle cycling assays in parallel with high sensitivity, precision, uniformity and reproducibility. By screening libraries of pharmacologically defined compounds on rat forebrain cultures, researchers used this system to identify novel effects of compounds on specific aspects of presynaptic function. As a system for unbiased compound and genomic screening, this technology has significant applications for basic neuroscience research, as well as for the discovery of novel, mechanism-based treatments for central nervous system disorders using a phenotypic approach.

The development of more biologically relevant cell systems and enabling technology platforms has provided a new toolbox of cell-based assays in drug discovery. Taking into account the realisation that target-based and/or reductionist screening techniques may not provide the high quality candidates needed to drive the development of new drugs has led to a significant shift in screening towards these innovative new technologies, and cell-based phenotypic screens are regularly being employed alongside traditional target-based screening4.

Interestingly, phenotypic screens may also allow rescreening of previously rejected compounds tested against specific targets, perhaps revealing a new and unexpected function.

In time, phenotypic screening technologies are sure to improve the early stages of the drug discovery process - in the form of more and higher quality drug candidates, while also leading towards an overall more efficient drug discovery process in the longer term.

1. Swinney, D. C. & Anthony, J. How were new medicines discovered? Nature Reviews Drug Discovery 10, 507-519 (2011).
2. Chambers SM et al. Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nature Biotechnology 30, 715-720 (2012).
3. Elliott, N. T. & Yuan, F. A review of threedimensional in vitro tissue models for drug discovery and transport studies. Journal of Pharmaceutical Sciences 100, 59-74 (2011).
4. Kotz J. Phenotypic screening, take two. SciBX 5, doi:10.1038/scibx.2012.380 (2012).
5. Saha, K. & Jaenisch, R. Technical Challenges in Using Human Induced Pluripotent Stem Cells to Model Disease. Cell Stem Cell 5, 584-595 (2009).
6. Devine MJ et al. Parkinson's disease induced pluripotent stem cells with triplication of the a-synuclein locus. Nature Communications 23, 440-449 (2011).
7. Hempel, C. M. et al. A System for Performing High Throughput Assays of Synaptic Function. PLoS ONE 6, e25999 EP - (2011).
8. Bickle M. The beautiful cell: high-content screening in drug discovery. Analytical and Bioanalytical Chemistry 398, 219-226 (2010).
9. Lie M, Grover M & Whitlon DS. Accelerated neurite growth from spiral ganglion neurons exposed to the Rho kinase inhibitor H-1152. Neuroscience 169, 855-862 (2010).
10. Blackmore MG et al. High content screening of cortical neurons identifies novel regulators of axon growth. Molecular and Cellular Neuroscience 44, 43-54 (2010).
11. Karila P. in 6th Annual Targeting Pain with Novel Therapeutics (Cambridge Healthtech Institute, 2013).