Design, Synthesis, and Application of Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the CXC Chemokine Receptor 2

The inhibition of CXC chemokine receptor 2 (CXCR2), akey inflammatorymediator, is a potential strategy in the treatment of several pulmonarydiseases and cancers. The complexity of endogenous chemokine interactionwith the orthosteric binding site has led to the development of CXCR2negative allosteric modulators (NAMs) targeting an intracellular pocketnear the G protein binding site. Our understanding of NAM bindingand mode of action has been limited by the availability of suitabletracer ligands for competition studies, allowing direct ligand bindingmeasurements. Here, we report the rational design, synthesis, andpharmacological evaluation of a series of fluorescent NAMs, basedon navarixin (2), which display high affinity and preferentialbinding for CXCR2 over CXCR1. We demonstrate their application influorescence imaging and NanoBRET binding assays, in whole cells ormembranes, capable of kinetic and equilibrium analysis of NAM binding,providing a platform to screen for alternative chemophores targetingthese receptors.