scholarly journals Impact of a Red-Shifted Dye Label for High Throughput Fluorescence Polarization Assays of G Protein-Coupled Receptors

2000 ◽  
Vol 5 (5) ◽  
pp. 329-334 ◽  
Author(s):  
Peter Banks ◽  
Mylene Gosselin ◽  
Linda Prystay
Keyword(s):  
G Protein ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
False Negative ◽  
G Protein Coupled Receptors ◽  
Negative Results ◽  
False Negative Results ◽  
Assay Precision ◽  
G Protein Coupled

High throughput screening fluorescence polarization assays using G protein-coupled receptors (GPCRs) as targets have been compared using fluorescein and BODIPY TMR-labeled peptides. The red-shifted BODIPY TMR dye exhibits improved assay performance relative to fluorescein due to improvement in both ligand affinity to the GPCRs and assay precision brought about by the higher intensity probe. Furthermore, the red-shifted dye demonstrates an insensitivity to the effects of the highly colored compound tartrazine, which can produce false-negative results for assays conducted with fluorescein as a label.

scholarly journals A Homogeneous Enzyme Fragment Complementation-Based β-Arrestin Translocation Assay for High-Throughput Screening of G-Protein-Coupled Receptors

2008 ◽  
Vol 13 (8) ◽  
pp. 737-747 ◽  
Author(s):  
Xiaoning Zhao ◽  
Adrie Jones ◽  
Keith R. Olson ◽  
Kun Peng ◽  
Tom Wehrman ◽  
...  
Keyword(s):  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Somatostatin Receptor ◽  
Gene Families ◽  
G Protein Coupled Receptors ◽  
Functional Assay ◽  
Gpcr Activation ◽  
Fragment Complementation ◽  
G Protein Coupled

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between β-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (~4 kDa), optimized α fragment peptide (termed ProLink™) derived from β-galactosidase, and β-arrestin is fused to an N-terminal deletion mutant of β-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the β-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active β-galactosidase enzyme, and thus GPCR activation can be determined by quantifying β-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a Gαi-coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified. ( Journal of Biomolecular Screening 2008:737-747)

scholarly journals Fluorescence Polarization Assays for High Throughput Screening of G Protein-Coupled Receptors

2000 ◽  
Vol 5 (3) ◽  
pp. 159-167 ◽  
Author(s):  
Peter Banks ◽  
Mylene Gosselin ◽  
Linda Prystay
Keyword(s):  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Rank Order ◽  
Drug Binding ◽  
G Protein Coupled Receptors ◽  
Microtiter Plates ◽  
High Throughput Drug Screening ◽  
G Protein Coupled ◽  
Speed Of Analysis

Fluorescence polarization assays in 384-well microtiter plates have been demonstrated. The performance is suitable for high throughput drug screening applications with respect to speed of analysis, displaceable signal, precision, and sensitivity to various reagents. Rank order of potency was maintained relative to ['251]-ligand filtration assays, and the effects of the highly colored compounds, tartrazine and Chicago Sky Blue, were insignificant on the polarization signal up to a concentration of 1 tiM. These attributes suggest that accurate assessment of drug binding can be obtained.

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