High Throughput Fluorescence Polarization: A Homogeneous Alternative to Radioligand Binding for Cell Surface Receptors

Abstract

High throughput fluorescence polarization (FP) assays are described that offer a nonradioactive, homogeneous, and low-cost alternative to radioligand binding assays for cell surface receptors (G protein-coupled receptors and ligand-gated ion channels). FP assays were shown to work across a range of both peptide (vasopressin V1a and delta-opioid) and nonpeptide (beta1-adrenoceptor, 5-hydroxytryptamine3) receptors. Structure-activity relationships were investigated at beta1-receptors and were found to be consistent with radioligand binding assays. FP was shown to tolerate up to 5% DMSO with no loss in sensitivity or signal window. From a random set of 1,280 compounds, 1.9% were found to significantly interfere with FP measurement. If fluorescent or quenching compounds were eliminated (3% of all compounds), less than 0.4% of compounds were found to interfere with FP measurement. Assays could be run in 384-well plates with little loss of signal window or sensitivity compared to 96-well plate assays. New advances in FP measurement have therefore enabled FP to offer a high throughput alternative to radioligand binding for cell surface receptors.

A Flow-Through Fluorescence Polarization Detection System for Measuring GPCR-Mediated Modulation of cAMP Production

“…Nowadays, several rapid and relatively easily performed homogeneous FP plate reader assays for drug discovery in general (e.g., for kinases, phosphatases, proteases, and nuclear receptors 23 ) and for GPCRs specifically 13,24 have been reported. For the readout of functional responses of signaling molecules such as cAMP with FP plate reader assays, usually expensive cAMP kits based on antibodies are used; moreover, specially equipped FP plate readers are needed.…”

“…This strategy renders the FP assay formats less prone to false positives mediated by interferences. The here described flow-through FP detection technology developed for proof-of-principle reasons was successfully validated and could be used for the transformation of other FP-based plate reader assay formats 4,24,27,[30][31][32] into flow-through FP assay formats when FP plate readers are not available. Moreover, the flow-through FP detector allows the use of FP bioassays in flowthrough postcolumn detection to identify individual bioactive compounds in mixtures.…”

Identification of 2-oxohistidine Interacting Proteins Using E. coli Proteome Chips

“…On the other hand, fluorescence polarization assay is a homogeneous binding detection method to mimic the interaction between two compounds in the cellular solution environment (55)(56)(57)(58)(59). We used fluorescence polarization assays to investigate the binding between fluorescent peptide probes and the 10 identified interacting proteins.…”

Miniaturization of Fluorescence Polarization Receptor-Binding Assays Using CyDye-Labeled Ligands

“…In addition, these BODIPY-FL and fluorescein-labeled ligands have been used in the development of FP receptor assays. 4 Accordingly, the relative affinities, based on IC 50 , of the Cy5-and Cy3B-labeled nonpeptide and peptide ligands used in this study were determined by radioactive, competitive binding assays using the appropriate receptor source and radiolabeled ligand. Table 1 shows the IC 50 values generated for Cy5-and Cy3B-telenzepine compared with those for unlabeled telenzepine and telenzepine amine congener.…”