Abstract

G protein-coupled receptors (GPCRs) have been described to form hetero-oligomers. The importance of these complexes in physiology and pathology is considered crucial, and heterodimers represent promising new targets to discover innovative therapeutics. However, there is a lack of binding assays to allow the evaluation of ligand affinity for GPCR hetero-oligomers. Using dopamine receptors and more specifically the D<sub>1</sub> and D<sub>3</sub> receptors as GPCR models, we developed a new time-resolved FRET (TR-FRET) based assay to determine ligand affinity for the D<sub>1</sub>/D<sub>3</sub> heteromer. Based on the high-resolution structure of the dopamine D<sub>3</sub> receptor (D<sub>3</sub>R), six fluorescent probes derived from a known D<sub>3</sub>R partial agonist (BP 897) were designed, synthesized and evaluated as high affinity and selective ligands for the D<sub>3</sub>/D<sub>2</sub> receptors, and for other dopamine receptor subtypes. The highest affinity ligand <b>21</b> was then employed in the development of the D<sub>1</sub>/D<sub>3</sub> heteromer assay. The TR-FRET was monitored between a fluorescent tag donor carried by the D<sub>1</sub> receptor (D<sub>1</sub>R) and a fluorescent acceptor D<sub>3</sub>R ligand <b>21</b>. The newly reported assay, easy to implement on other G protein-coupled receptors, constitutes an attractive strategy to screen for heteromer ligands

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Last time updated on 12/02/2018

This paper was published in FigShare.

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