In order to evaluate the binding affinities of the synthesized fluorescent CCR2 ligands, we developed a NanoBRET-based binding assay (Figure and S2A-G). Therefore, we fused CCR2 at its intracellular C-terminus to a small and bright luciferase variant (nanoluciferase, Nluc). Successful surface expression of CCR2-Nluc fusion proteins was confirmed by ELISA directed against an N-terminal HA-tag (Figure ). In saturation binding experiments using membranes from HEK293T cells transiently expressing Nluc-tagged CCR2 with a GSSG linker between the C-terminus of CCR2 and the Nluc-tag (hereafter referred to as CCR2_GSSG_Nluc), 14 showed the highest binding affinity among the tested ligands, with a Kd value of 266 ± 28 nM (Table , Figure and). The obtained affinity data indicates that our approach for the design of fluorescently labeled intracellular CCR2 ligands was most successful, when the biaryl sulfonamide-based pharmacophore of 2 was used as a CCR2 binding unit. The difference in affinity between 12 and 14 might be rationalized by additional polar interactions of the triazole moiety with polar amino acids at the entrance of the intracellular allosteric binding pocket of CCR2 (see Figure ). Fluorescent ligands based on the biaryl sulfonamide-based pharmacophore of 1 (10, 13, 15, 16) showed reduced affinities, especially when the ether and benzamide substituents were placed in ortho position to each other (15-16). Low affinities were also detected for our pyrrolone-based fluorescent ligands (8-9). Kinetic binding studies with 14 revealed a very fast association of the ligand-receptor-complex by exhibiting a rate constant of kon = 6.00 ± 0.39 •10 6 M -1 min -1 (Figure ). With a rate constant of koff = 0.101 ± 0.005 min -1 and a residence time of tr = 9.93 ± 0.43 min, the dissociation of the ligandreceptor-complex was detected to be fast as well. This is in contrast to a recently reported fluorescent ligand targeting the IABS of the related chemokine receptor CCR9, which showed a much longer residence time (tr = 90 min). The difference between the resulting kinetic Kd value of 16.8 ± 0.1 nM and the equilibrium Kd value of 266 ± 28 nM can be rationalized by the very fast association of ligand-receptorcomplex, which mainly takes place in the first seconds of coincubation and can therefore only be roughly estimated by the applied assay setup.