Cell-Based Assays: Advancing Discovery

The majority of screening assays in either drug discovery target validation or lead compound identification/optimization now utilize cell-based technologies. Many of these assays use miniaturized fluid addition protocols, accompanied by highly sensitive detection techniques and automated liquid handling instruments. One implicit assumption is that in order to achieve an optimal understanding of the physiology of the biological target, as well as its pharmacological interaction with novel compounds, understanding the response in a correct physiological (ie, cellular) context is critical.

Although immortalized cell lines are widely used in cell-based screening, they are not without drawbacks. Historically, classical cellbased assays have used phenotypes more for their ease-of-use and compatibility with screening instrumentation, rather than for their optimal physiological relevance. However, since cell-based assays frequently utilize cell phenotypes that are different from those in native human physiology, one may question the clinical relevance of the validated target, the lead compound identified, or both. Consequently, a growing number of discovery programs are using primary or stem cells in High-Throughput Screening (HTS) and High-Content Screening (HCS) protocols. This suggests that the use of primary cells, HTS and HCS are rapidly converging as enabling techniques in drug discovery.

Cell-Based Assays Drug Discovery

Common approaches in primary screening use assays in which recombinant immortalized cells express a discrete molecular target. The activity of compounds at this target is then assayed using functional responses that are detected and quantified via sophisticated automated liquid handling and detection systems. Recombinant immortalized cells are used to screen large libraries of small molecules. Potential “hits” identified are then “validated” through multiple counter-screens to identify compound potency and specificity.

Via sub-structural searching techniques, libraries can be used to optimize lead compounds for “druggable” properties including solubility and pharmacokinetic characteristics, and potential cellular toxicities. Through the iterative re-screening of focused libraries, the quantification of interactions at molecular targets can result in potential candidates for later in vivo and clinical testing. The approach has met with some success in identifying lead compounds for further development. This is despite the occurrences of disparities between compound effects on drug targets recombinantly expressed and in vivo efficacy.

Compromises in Using Immortalized Cells

As noted, there are advantages to using immortalized cell lines in drug discovery. First, near-unlimited amounts of transfected cells can be cultured in reproducible batches, providing a consistent, homogenous vehicle for HTS campaigns. Second, they provide a null background for target expression and assay response measurement. The stable expression of drug targets can be achieved for physiological relevance. Additional reporter proteins can be transfected into these cells by using established cloning and expression techniques, for monitoring compound/receptor interactions via gene expression readouts.

However, emerging data indicates limitations in screening compounds using these systems, particularly in regard to the physiological relevance of data generated with respect to human physiology. Indeed the genetic and therefore molecular phenotype of cultured immortalized cells can significantly differ from that of native cells in vivo. This raises the question of which functional response one should measure in a HTS assay ie, which response is most relevant in identifying clinically relevant leads.

Primary, Stem Cells in HTS

There are differences between the physiological environment when using recombinant cell lines and those found in natural tissue. This understanding has led to an increased interest in using primary mammalian cells for HTS – in which the endogenous target is assumed to be expressed in a setting far closer to that found in the human disease, and at levels that resemble those found endogenously. The presumption is that new potential drugs characterized using primary cells will act more predictably, and in line with disease state interaction, than those characterized by immortalized cells, according to Eglen, Comb Chem & HTS, 2008.

One barrier to the widespread adoption of primary mammalian cells in HTS is their lack of abundance and homogeneity. This is partially offset by the use of highly sensitive assay techniques and miniaturized detection systems, enabling the use of far fewer cells per assay data point. However, the growing availability of embryonic stem cells (ESCs) may provide cells that can be grown in abundance (in a similar manner to immortalized cells), yet which retain

key phenotypic characteristics of natural cells. Furthermore, ESCs can be differentiated into distinct cell types, corresponding to required organs and tissue, according to McNeish, Curr Op Pharmacol 2007. Surprisingly, few primary cells or ESCs are currently used in primary screening, instead being relegated to secondary screening campaigns or lead optimization studies.

Possibilities for ESCs in HTS

The lack of cell availability restricts the use of primary cells in HTS. However, this may be overcome with the use of pluripotent ESCs, which can be grown in an almost unlimited fashion. These cells can be differentiated into specific cell types including neurons, hepatocytes or myocytes, and protein reporters can be recombinantly engineered and inserted. Transfected ESCs can be selected, expanded, and then using specific growth factors, induced to differentiate into populations enriched for a selective lineage (eg, neurons, myocytes, and hepatocytes).

According to Pouton, CW and Haynes, JW Nature, 2007, although human ESCs provide considerable advantages in screening, their disadvantages are that they do not propagate and divide, while being difficult to maintain and expand. An alternative is to use human induced pluripotent stem cells, which can be obtained from cord blood, bone marrow and several other tissue.

The advantages of ESCs include the use of previously unavailable cell types and the ability to study cellular regeneration and differentiation. Small molecules have historically been recognized to reproducibly impact the differentiation of stem or progenitor cells. The increased expertise in human stem cell cultures to facilitate HTS has allowed the identification of new chemical series that serve to direct cellular renewal, regeneration, expansion and differentiation, particularly when used with adult pluripotent stem cells (iPS cells).

High Throughput Imaging

Primary human cells are used in several therapeutic areas, and such biologically relevant cell assays are becoming increasingly recognized as robust and amenable screening tools for HTS. Cellular imaging is emerging as an important tool that integrates biological complexity into drug discovery. Current imaging systems allow the high-resolution analysis of single cells, high throughput and kinetic studies on live cells, and are linked to efficient data storage systems via user-friendly image analysis programs.

The key feature of modern cellular imaging systems in drug discovery is to provide a multidimensional aspect for each experiment performed, allowing the measurement of multiple parameters, which in turn enables the analysis of cellular responses against different stimuli, according to Yarrow, Comb Chem & HTS, 2008. ESCs permit the development of predictive screening assays that can deliver higher-quality leads. The emergence of high content imaging instruments coupled with plate handling equipment also allows high throughput assays to be carried out successfully by imaging target cells within heterogeneous cultures.

One area that is benefiting from screening via automated confocal imaging systems is the ability to undertake a cellular phenotypic approach to drug discovery. The use of HCS techniques to permit phenotypic profiling of compounds based on changes in cellular activity looks likely to grow in drug discovery. However, this will require the development of techniques to analyze large image datasets, as well as precise correlations of phenotypic changes and compound mechanism of action, according to Young, Nature Chem Biol, 2008.

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Looking Ahead

The use of cell-based assays in all phases of drug discovery, and notably in HTS, has accelerated in the last five years. In many screening campaigns, the cell phenotype has been subservient to assay technology, instrument, or liquid handling systems in the laboratory. Consequently, heterologous expressions in immortalized cell lines of G protein-coupled receptors (GPCR), ion channels or kinase targets, as well as their ancillary signaling partners, provide the mainstay of most cell-based screening assays. Clearly, such cells are poor substitutes for cells reflecting human diseases.

Furthermore, current cell-based Absorption, Distribution, Metabolism, and Excretion/ Toxicity (ADME/Tox) assays, now moving into HTS, are relatively poor predictors of the human response due to the nature of the cells being used in the studies. Because of these limitations in standard HTS programs, the growing interest in the use of primary cells and ultimately stem cells in drug discovery is increasingly justified.

The use of primary cells in secondary screening assays is also increasing apace, particularly in studies where confocal imaging is used, as well as in target validation studies in conjunction with gene silencing techniques. The convergence of high throughput confocal imaging, primary cells and stem cells are likely to collectively allow the earlier and more effective identification of novel clinical candidates.

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