Application of BRET to monitor ligand binding to GPCRs

Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a BRET approach to monitor ligand binding to G protein–coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a bioluminescent protein (NanoLuc) that can be readily expressed on the N terminus of GPCRs

Development of fluorescent probes that target serotonin 5-HT2B receptors

Some 5-HT2B fluorescent probes were obtained by tagging 1-(2,5-dimethoxy-4-iodophenyl)-propan-2-amine (DOI) with a subset of fluorescent amines. Some of the resulting fluorescent ligands showed excellent affinity and selectivity profiles at the 5-HT2B receptors (e.g. 12b), while retain the agonistic functional behaviour of the model ligand (DOI). The study highlighted the most salient features of the structure-activity relationship in this series and these were substantiated by a molecular modelling study based on a receptor-driven docking model constructed on the basis of the crystal structure of the human 5-HT2B receptor. One of the fluorescent ligands developed in this work, compound 12i, specifically labelled CHO-K1 cells expressing 5-HT2B receptors and not parental CHO-K1 cells in a concentration-dependent manner. 12i enables imaging and quantification of specific 5-HT2B receptor labelling in live cells by automated fluorescence microscopy as well as quantification by measurements of fluorescence intensity using a fluorescence plate reader.This research was carried out within the framework of the Cost Action GLISTEN and financially supported by the Spanish Government (grant numbers SAF2009-13609-C04-03 and GPC2014/003 (PS09/63) to E.S. and SAF2014-57138-C2-1-R to M.C. and M.I.L.). Authors also thanks financial support from Consellería de Cultura, Educación e Ordenación Universitaria of the Galician Government: (grant: GPC2014/03), Centro Singular de Investigación de Galicia accreditation 2016-2019 (ED431G/09). J. S. acknowledges financial support from Instituto de Salud Carlos III FEDER (CP12/03139 and PI15/00460)