Green fluorescent protein-assisted readout for interacting proteins (GRIP) is a universal protein interaction discovery system that can be used to generate truly high throughput screening-compatible cellular assays to be used to screen for inhibitors of protein-protein interactions. The technology uses a "bait and prey" principle based on the distinct translocation behavior of the human cyclic AMP phosphodiesterase 4A4. Here we use the p53-Hdm2 Redistribution assay (Fisher BioImage ApS, Søborg, Denmark) as an example to describe the GRIP technology. The p53-Hdm2 Redistribution assay is a high content imaging assay based on the GRIP technology that is designed to measure the interaction between Hdm2 and the tumor suppressor p53. Hdm2 regulates p53 and inhibits its function by modulating its transcriptional activity and stability. Activation of p53 in tumor cells through inhibition of its physical interaction with Hdm2 is therefore a focus of cancer drug discovery. We have performed a pilot screen by screening 3,165 compounds from a diverse small-molecule library for inhibitors of the p53-Hdm2 interaction by using the p53-Hdm2 Redistribution assay. Here we show that by taking advantage of the translocation behavior of nonbound p53, it is possible to identify true inhibitors of the p53-Hdm2 interaction by extracting high content information from the acquired images.
Redistribution (BioImage) A/S, Søborg, Denmark) is a novel high-throughput screening technology that monitors translocation of specific protein components of intracellular signaling pathways within intact mammalian cells, using green fluorescent protein as a tag. A single Redistribution assay can be used to identify multiple classes of compounds that act at, or upstream of, the level of the protein target used in the primary screening assay. Such compounds may include both conventional and allosteric enzyme inhibitors, as well as protein-protein interaction modulators. We have developed a series of Redistribution assays to discover and characterize compounds that inhibit tumor necrosis factor-alpha biosynthesis via modulation of the p38 mitogen-activated protein kinase (MAPK) pathway. A primary assay was designed to identify low-molecular-weight compounds that inhibit the activation-dependent nuclear export of the p38 kinase substrate MAPK-activated protein kinase 2 (MK2). Hits from the primary screen were categorized, using secondary assays, either as direct inhibitors of MK2 nuclear export, or as inhibitors of the upstream p38 MAPK pathway. Activity profiles are presented for a nuclear export inhibitor, and a compound that structurally and functionally resembles a known p38 kinase inhibitor. These results demonstrate the utility of Redistribution technology as a pathway screening method for the identification of diverse and novel compounds that are active within therapeutically important signaling pathways.
The PI3-kinase/Akt pathway is an important cell survival pathway that is deregulated in the majority of human cancers. Despite the apparent druggability of several kinases in the pathway, no specific catalytic inhibitors have been reported in the literature. The authors describe the development of a fluorometric imaging plate reader (FLIPR)-based Akt1 translocation assay to discover inhibitors of Akt1 activation. Screening of a diverse chemical library of 45,000 compounds resulted in identification of several classes of Akt1 translocation inhibitors. Using a combination of classical in vitro assays and translocation assays directed at different steps of the Akt pathway, the mechanisms of action of 2 selected chemical classes were further defined. Protein translocation assays emerge as powerful tools for hit identification and characterization. (Journal of Biomolecular Screening 2005:20-29)
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