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 a 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.