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Fluorescent G protein-coupled receptors (GPCRs)

By: Mike Earley, Market Segment Manager, Sigma Life Science, Biofiles Vol. 8, No. 4

G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins and the targets for almost half of the clinical drugs in the market today. Advances in X-ray crystallography and other biophysical techniques have led to many exciting findings related to receptor structure and conformational shifting. Traditional GPCR membrane preparations expressing recombinant or endogenous receptors like the adenosine transporter (Cat. No. A8352) provide a useful platform for initial screening studies and ligand identification. Radiolabeled ligand binding assays using GPCR membrane preparations allow researchers to compare novel ligands to gold standard compounds of interest, such as NBTI (Cat. No. N2255), a potent adenosine uptake inhibitor, and offer a consistent and affordable platform for high throughput screening. However, these techniques remove the target receptor from its cellular context.

A study illustrating successful GPCR screening within live cells was recently published by Nottingham University researchers.1 They identified a putative adenosine A3 receptor antagonist as a result of a high-content primary screen using live cells with native levels of receptor and an innovative fluorescent receptor ligand. CA200645 (Cat. No. SML0157) is a fluorescent adenosine A3 receptor antagonist derived from Xanthine Amine Congener. This molecule maintains its fluorescent properties while bound, allowing visualization through fluorescent confocal microscopy. Upon development of a competition binding assay utilizing CA200645, a high-content fragment-based screen for adenosine receptor binders was performed. Briefly, cells were incubated with unlabeled compounds, washed, and then incubated with CA200645. Fluorescence intensity was measured with a confocal imaging plate reader to assay for molecules that would displace the CA200645. Use of this system to screen a 248 compound fragment library resulted in 38 hits that were validated by a secondary screen.

The researchers synthesized structural analogs of the most potent hit and successfully identified a compound with a higher binding affinity that could distinguish between adenosine A1 and adenosine A3 receptor subtypes. This novel screening assay, incorporating CA200645 as its central reagent, was shown to be a powerful approach for studying GPCRs in live cells, preserving the physiological context of the target receptors.




  1. Stoddart, LA et al., Fragment Screening at Adenosine-A3 Receptors in Living Cells Using a Fluorescent-Based Binding Assay. Chemistry & Biology, 19:1105-1115 (2012).


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