Development and Characterization of a Potent Free Fatty Acid Receptor 1 (FFA1) Fluorescent Tracer

The free fatty acid
receptor 1 (FFA1/GPR40) is a potential target
for treatment of type 2 diabetes. Although several potent agonists
have been described, there remains a strong need for suitable tracers
to interrogate ligand binding to this receptor. We address this by
exploring fluorophore-tethering to known potent FFA1 agonists. This
led to the development of 4, a high affinity FFA1 tracer
with favorable and polarity-dependent fluorescent properties. A close
to ideal overlap between the emission spectrum of the NanoLuciferase
receptor tag and the excitation spectrum of 4 enabled
the establishment of a homogeneous BRET-based binding assay suitable
for both detailed kinetic studies and high throughput competition
binding studies. Using 4 as a tracer demonstrated that
the compound acts fully competitively with selected synthetic agonists
but not with lauric acid and allowed for the characterization of binding
affinities of a diverse selection of known FFA1 agonists, indicating
that 4 will be a valuable tool for future studies at
FFA1.