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.游离脂肪酸受体2（free fatty acid receptor 2, FFA2/GPR43）被认为是治疗代谢性与炎症性疾病的潜在靶点。本研究首次开发了针对FFA2的首款荧光示踪剂，该工具可用于评估非标记配体的热力学与动力学结合参数。研究以一款已知的氮杂环丁烷类FFA2拮抗剂为起始化合物，选取一个被证实不影响受体相互作用的羧酸基团作为连接位点，偶联硝基苯并恶二唑（NBD）荧光团，最终得到化合物4（TUG-1609）。经基于生物发光共振能量转移（BRET）的饱和结合与动力学结合实验验证，该示踪剂具备优异的光谱学特性与高亲和力，且其BRET结合信号具有极高的特异性结合与非特异性结合比值。此外，本研究还建立了以化合物4为探针的BRET竞争结合测定法，用于测定非标记配体的结合常数与动力学参数。