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Pathway screening using Cell-Based Redistribution^® Assays

Traditional small molecule drug discovery focuses primarily on compounds that modulate cell surface receptors or inhibit the catalytic activity of enzymes.

Redistribution^® assays offer a unique opportunity to screen entire signalling pathways for novel compounds with both traditional (eg. kinase inhibitors) and novel modes of action. The novel modes of action could include molecules that act primarily by modulating protein translocation, as well as modulators of cell surface receptors and intracellular enzymes. Possible intervention points in the PI3K signalling pathway is shown here:

Figure 1. Possible intervention points in the PI3K pathway

Advantages of Redistribution^® based pathway

• Pathway screening addresses both traditional drug targets and previously intractable targets such as transcription factors and structural proteins.
  Since multiple intervention points within disease relevant cell signaling pathways are simultaneously analyzed using a pathway screening approach, proteins such as transcription factors and adaptor/scaffold molecules with no inherent catalytic activity that have historically been difficult to screen are therefore targeted. Since Redistribution^® technology does not rely on the in vitro catalytic activity of the target, it offers an opportunity to direct drug discovery efforts at novel protein classes with unrivalled efficiency.
  
  
• High throughput cell-based imaging assays enable high content screening (HCS).
  Image-based cellular screening has several advantages compared with traditional drug screening approaches. For example, the complex behavior of intracellular signaling molecules can only be studied in living cells, as used in Redistribution^® assays. Second, Redistribution^® technology enables HCS since a degree of drug permeability and toxicity analysis is incorporated, allowing rapid de-selection of unfavorable chemical scaffolds.
  
  
• Compounds that modulate protein translocation may have altered specificity vis-á-vis traditional drugs.
  The domains that regulate intracellular localization of proteins are often distinct from catalytic domains. Therefore, compounds that modulate protein translocation may be more specific than traditional competitive enzyme inhibitors. This is an advantage when targeting multi-protein families such as PKC isoenzymes in which the catalytic domains are very similar.
  
  
• Compounds that modulate protein translocation may belong to novel compound classes.
  Molecules that do not target the same regions of proteins as traditional drugs (such as the ATP-binding site in kinases), or that target novel classes of protein targets, will most likely be built from novel chemical scaffolds. This has obvious implications for both chemistry optimization and intellectual property efforts.

Screening in the PI3K pathway – an example:
BioImage has performed pathway screens covering both the p38 MAPK and the PI3K signalling pathway.

The p38 MAPK pathway was screened using MAPKAP K2 translocation the identified

• Nuclear export inhibitors
• p38 inhibitors as well as
• compound acting upstream in the pathway.

Refer to this publication for details.

We performed a pathway screen in the PI3K pathway using the FKHR Redistribution^® assay. FKHR is a transcription factor that functions as a key regulator of insulin signaling, cell cycle progression and apoptosis downstream of PI3-kinase and Akt.

Compound assay profiles identified in the screen includes:

• PI3K inhibitors
• Akt inhibitors
• FKHR activators

Refer to PI3K pathway project for details.