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Homogeneous time-resolved G protein-coupled receptor-ligand binding assay based on fluorescence cross-correlation spectroscopy.
Journal article

Homogeneous time-resolved G protein-coupled receptor-ligand binding assay based on fluorescence cross-correlation spectroscopy.

  • Antoine T Intana Bioscience, DE-82152, Planegg/Martinsried, Germany.
  • Ott D Intana Bioscience, DE-82152, Planegg/Martinsried, Germany.
  • Ebell K Intana Bioscience, DE-82152, Planegg/Martinsried, Germany.
  • Hansen K Intana Bioscience, DE-82152, Planegg/Martinsried, Germany.
  • Henry L Laboratory of Protein Engineering, Swiss Federal Institute of Technology Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
  • Becker F Intana Bioscience, DE-82152, Planegg/Martinsried, Germany.
  • Hannus S Intana Bioscience, DE-82152, Planegg/Martinsried, Germany. Electronic address: stefan.hannus@intana.de.
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  • 2016-03-09
Published in:
  • Analytical biochemistry. - 2016
Affinity determination
Binding kinetics
Fluorescence correlation spectroscopy
Fluorescence cross-correlation spectroscopy
GPCR
Homogeneous time-resolved binding assay
Antibodies, Monoclonal
Binding Sites
Cells, Cultured
Fluorescent Dyes
HEK293 Cells
Humans
Kinetics
Ligands
Peptides
Receptors, G-Protein-Coupled
Small Molecule Libraries
Spectrometry, Fluorescence
Time Factors
English 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.
Language
  • English
Open access status
hybrid
Identifiers
Persistent URL
https://sonar.ch/global/documents/16058
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