title: Homogeneous time-resolved G protein-coupled receptor-ligand binding assay based on fluorescence cross-correlation spectroscopy.
abstract: G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs.
authors.affiliation: Intana Bioscience, DE-82152, Planegg/Martinsried, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Antoine
authors.affiliation: Intana Bioscience, DE-82152, Planegg/Martinsried, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Ott
authors.affiliation: Intana Bioscience, DE-82152, Planegg/Martinsried, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Ebell
authors.affiliation: Intana Bioscience, DE-82152, Planegg/Martinsried, Germany.
authors.forename: Kerrin
authors.initials: K
authors.lastname: Hansen
authors.affiliation: Laboratory of Protein Engineering, Swiss Federal Institute of Technology Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
authors.forename: Luc
authors.initials: L
authors.lastname: Henry
authors.affiliation: Intana Bioscience, DE-82152, Planegg/Martinsried, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Becker
authors.affiliation: Intana Bioscience, DE-82152, Planegg/Martinsried, Germany. Electronic address: stefan.hannus@intana.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Hannus
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors* / Q000737:chemistry; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D008024:Ligands; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D054852:Small Molecule Libraries
keywords: Affinity determination; Binding kinetics; Fluorescence correlation spectroscopy; Fluorescence cross-correlation spectroscopy; GPCR; Homogeneous time-resolved binding assay