Fluorescent ligands targeting the intracellular allosteric binding site of the chemokine receptor CCR2

Fluorescently labeled ligands are versatile molecular tools to study G protein-coupled receptors (GPCRs) and can be used for a range of different applications, including bioluminescence resonance energy transfer (BRET) assays. Here, we report the structure-based development of fluorescent ligands targeting the intracellular allosteric binding site (IABS) of the CC chemo-kine receptor 2 (CCR2), a class A GPCR that has been pursued as a drug target in oncology and inflammation. Starting from previously reported intracellular CCR2 antagonists, several tetramethylrhodamine (TAMRA) labeled CCR2 ligands were designed, synthesized and tested for their suitability as fluorescent reporters to probe binding to the IABS of CCR2. By means of these studies, we developed 14 as a fluorescent CCR2 ligand enabling cell-free as well as cellular NanoBRET-based binding studies in a non-isotopic and high-throughput manner. Further, we show that 14 can be used as a tool for fragment-based screening approaches as well. Thus, our small molecule-based fluorescent CCR2 ligand 14 represents a promising tool for future studies of CCR2 pharmacology.