Automation and validation of the Transfluor~(TM) technology: a universal screening assay for G protein-coupled receptors

摘要

G protein-coupled receptors (GPCRs) are historically the richest targets for drug discovery, accounting for nearly 60% of prescription drugs. The ligands and functions of only 200 out of possibly 1000 GPCRs are known. Screening methods that directly and accurately measure GPCR activation and inhibition are required to identify ligands for orphan receptors and cultivate superior drugs for known GPCRs. Norak Biosciences utilizes the redistribution of a fluorescently-labeled protein, arrestin, as a novel screen for monitoring GPCR activation. In contrast to the present methods of analyzing GPCR function, the power of the Transfluor~(TM) technology is in its simplicity, large signal to noise ratio, and applicability to all GPCRs (both known and orphan). Here, we demonstrate that the Transfluor~(TM) technology can be automated and quantitated on high throughput image analysis systems. Cells transfected with an arrestin-green fluorescent protein conjugate (arrestin-GFP) and the neurokinin-1 (NK-1) GPCR were seeded on 96-well plates. Activation of the NK-1 receptor with Substance P induced translocation of arrestin-GFP from the cytosol to the receptor. Image quantitation of the arrestin-GFP translocation was used to generate dose dependent curves. These results reveal that the Transfluor~(TM) technology combined with an image analysis system forms a universal platform capable of measuring ligand-receptor interactions for all GPCRs.