期刊
ANALYTICAL BIOCHEMISTRY
卷 502, 期 -, 页码 24-35出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2016.02.017
关键词
GPCR; Homogeneous time-resolved binding assay; Fluorescence correlation spectroscopy; Fluorescence cross-correlation spectroscopy; Affinity determination; Binding kinetics
资金
- German Federal Ministry of Education and Research (BMBF) [031A239A]
- European Union [289278]
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. (C) 2016 The Authors. Published by Elsevier Inc.
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