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

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.