Development and Characterization of a Fluorescent Tracer for the Free Fatty Acid Receptor 2 (FFA2/GPR43)

J Med Chem. 2017 Jul 13;60(13):5638-5645. doi: 10.1021/acs.jmedchem.7b00338. Epub 2017 Jun 16.

Abstract

The free fatty acid receptor 2 (FFA2/GPR43) is considered a potential target for treatment of metabolic and inflammatory diseases. Here we describe the development of the first fluorescent tracer for FFA2 intended as a tool for assessment of thermodynamic and kinetic binding parameters of unlabeled ligands. Starting with a known azetidine FFA2 antagonist, we used a carboxylic acid moiety known not to be critical for receptor interaction as attachment point for a nitrobenzoxadiazole (NBD) fluorophore. This led to the development of 4 (TUG-1609), a fluorescent tracer for FFA2 with favorable spectroscopic properties and high affinity, as determined by bioluminescence resonance energy transfer (BRET)-based saturation and kinetic binding experiments, as well as a high specific to nonspecific BRET binding signal. A BRET-based competition binding assay with 4 was also established and used to determine binding constants and kinetics of unlabeled ligands.

MeSH terms

  • Azetidines / chemistry
  • Azetidines / metabolism
  • Binding Sites
  • Binding, Competitive
  • Cell Line
  • Drug Evaluation, Preclinical
  • Fluorescent Dyes / chemistry*
  • Fluorescent Dyes / metabolism
  • Humans
  • Ligands
  • Oxadiazoles / chemistry*
  • Oxadiazoles / metabolism
  • Receptors, Cell Surface / analysis*
  • Receptors, Cell Surface / antagonists & inhibitors
  • Receptors, Cell Surface / metabolism
  • Spectrometry, Fluorescence

Substances

  • Azetidines
  • FFA2R protein, human
  • Fluorescent Dyes
  • Ligands
  • Oxadiazoles
  • Receptors, Cell Surface
  • azetidine