Cell Based Technologies

In contrast to enzyme assays, cell-based assays present the target in a more physiological milieu. With enzyme assays, it may be difficult to purify and express active kinases and phosphatases in their full-length forms and they may require the use of fusion proteins with kinase activity domains. Cell-based technologies, on the other hand, present the opportunity to express the targets with regulatory domains included. Furthermore, cell-based assays usually detect only cell-permeable inhibitors and have the potential to identify more unusual mechanisms, as described earlier.

However, cell-based technologies present the potential for artifacts resulting from "off-target" effects of compounds. They also tend to be more complicated with respect to automation and reagent addition steps, often requiring a lysis, a plate-to-plate transfer step, and several washes. Cellular assays also tend to be more difficult and demanding in obtaining statistical quality for HTS, related to the accurate and reproducible production of cell batches. One usually needs to use downstream assay methods such as enzyme assays to validate the mechanism of action and guide structure-based lead optimization.

The principles for cell-based assays are based on signal transduction. The assay technologies used can, in one way or another, detect intracellular changes in the phosphorylation states of downstream targets of the kinase or phosphatase of interest (Figure 1.2). Alternatively, they can detect some consequences of kinase or phosphatase inhibition or activation such as cell growth or differentiation or specific gene expression. For this purpose, specialized anti-phosphopeptide site antibodies or kinase-regulated reporter gene systems must be made often. These projects represent a challenge because the antibodies must be of suitable affinity and specificity and because the cell line must

Reporter Gene Assays

Luciferase constructs changes in native protein expression

Morphological changes high-throughput microscopy

Cell Survival

Alamar Blue

Cell-titerGlo (ATP detection)

Neuroplasticity, neurogenesis, cell survival and resilience

FIGURE 1.2 (See color insert following page 114.) Cell-based assay concepts for kinase and phosphatase HTS.

Autophosphorylation

ELISA, AlphaScreen TR-FRET InCell Western

Phosphoprotein detection

ELISA AlphaScreen TR-FRET InCell Western

Nuclear translocation high-throughput microscopy

Reporter Gene Assays

Luciferase constructs changes in native protein expression

Morphological changes high-throughput microscopy

Cell Survival

Alamar Blue

Cell-titerGlo (ATP detection)

Neuroplasticity, neurogenesis, cell survival and resilience

FIGURE 1.2 (See color insert following page 114.) Cell-based assay concepts for kinase and phosphatase HTS.

have high enough expression levels of analytes to be detectable with the desired readout technology. In the following sections, we will review some of the basic cellular techniques for kinase and phosphatase HTS.

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