Fluorescent Ligand Enables Target Engagement Studies for the Intracellular Allosteric Binding Site of the Chemokine Receptor CXCR2

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 and fluorescence microscopy. Herein, we report the structure-based development of a fluorescent ligand targeting the intracellular allosteric binding site (IABS) of the CXC chemokine receptor 2 (CXCR2), a class A GPCR that has been pursued as a drug target in oncology and inflammation. Starting from the co-crystallized intracellular CXCR2 antagonist 00767013 (1), a tetramethylrhodamine (TAMRA)-labeled CXCR2 ligand was designed, synthesized, and tested for its suitability as fluorescent reporter to probe binding to the IABS of CXCR2. By means of these studies, we developed Mz438 (9) as a high affinity fluorescent CXCR2 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a non-isotopic and high-throughput manner. Further, we show that 9 can be used as a tool to visualize intracellular target engagement for CXCR2 via fluorescence microscopy. Thus, our small molecule-based fluorescent CXCR2 ligand 9 represents a promising tool for future studies of CXCR2 pharmacology.