G Protein-Coupled Receptors
Redistribution assay technology relies on the imaging and quantification of intracellular protein translocation. For development of Redistribution assays, BioImage (www.bioimage.com) uses Aequorea victoria GFP and other fluorescent proteins fused to translocating targets of interest.
G protein-coupled receptors (GPCRs) account for some 50 percent of all current drug targets, says Arne Heydorn, scientist at BioImage, whereas marketed drugs only target about 30 percent of the GPCRs in the genome. We developed Redistribution cell-based assays for use in both primary screening and lead optimization of compounds targeting GPCRs.
Redistribution assays are quantitative assays that can be run in standard microtiter plates. The assay response is most commonly measured by capturing an image from an assay well, and the response is quantified by performing image analysis.
Redistribution assays are independent of the imaging platform and have been shown to give consistent results on a number of instruments on the market, including: IN Cell 3000 (GE Healthcare), Opera (Evotec Technologies), ArrayScan (Cellomics), Pathway HT (BD Biosciences), Discovery1 (Molecular Devices), and Icyte (CompuCyte).
Multiplexing refers to technology that enables the simultaneous measurement of multiple parameters in one test compound well. Redistribution assays can be multiplexed by either expressing two targets in the same cell line labeled with different colors, or by mixing different assay cell lines directly in the microtiter plate well.
These assays allow monitoring of GPCR desensitization, i.e., receptor internalization and recycling/degradation, by quantifying the intracellular translocation of GPCR-GFP fusion constructs.
A second class of assays is designed to report functional activity for Gq-, Gi-, and Gs-coupled GPCRs, by monitoring activation of downstream signaling pathways, such as nuclear factor of activated T cells (NFAT) or protein kinase A (PKA). For these assays, the GPCR of interest is transfected into a cell line expressing a fusion construct of GFP with the appropriate downstream target. Recent studies have shown that the effects of agonists and antagonists on receptor desensitization are variable, and that receptor desensitization plays a key role in drug behavior in vivo.
What we do is focus on the biology; we dont do the algorithms, Heydorn adds.