After having shown that our fluorescent CCR2 ligand 14 is a suitable tool to study binding of high to low affinity binders to the IABS of CCR2 in a cell-free assay setup, we were curious to see whether we could also use this approach in a live cell environment. In general, the assessment of the interactions between a drug and its protein target in a physiologically relevant cellular environment is a critical step in preclinical drug discovery. This step, also referred to as cellular target engagement, is highly important for successfully delivering compounds with the desired biological and ultimately clinical effects, as the on-target activity of small molecules can be affected significantly when transitioning from a cell-free to a cellular environment. A loss of activity in a cellular environment can be attributed to various factors, for example low cell permeability, compound efflux, or offtarget protein binding. For our cell-based assay setup, we used live HEK293T cells transiently expressing a Nluctagged CCR2 with no linker between the C-terminus of CCR2 and the Nluc-tag (hereafter referred to as CCR2_Nluc), as this construct resulted in a larger and more robust assay window compared to CCR2_GSSG_Nluc under cellular conditions (Figure ). With this cell-based assay setup, a Kd value of 114.3 ± 22.0 nM for 14 was determined (Figure ), demonstrating that 14 is able to pass the cell membrane and bind to CCR2 on the intracellular side of the receptor. In a cell-based displacement assay, we detected low nanomolar Ki values for literature-known intracellular CCR2 antagonists SD-24 (1) (Ki = 8.50 ± 2.49 nM) and CCR2-RA (4) (Ki = 4.20 ± 1.39 nM), see Figure . These results are consistent with the affinities detected in the cell-free setup (Fig. ) as well as with the rank order determined in a cellular assay system (EC50 = 236 nM for SD-24 (1) and EC50 = 93 nM for CCR2-RA-[R] (4-[R]) in a cellular IP turnover activation assay). For 27 and 44, we detected a cellular CCR2 Ki value of 4.40 ± 0.96 nM and 55.5 ± 7.6 nM, respectively. Overall, results from live-cell NanoBRET and membrane-based experiments were in good agreement with each other. Thus, our fluorescent CCR2 ligand 14 is a valuable tool to study binding to the IABS of CCR2 in a cellular environment. Besides providing mere binding affinity, our cell-based assay allows initial insights into pharmacokinetic properties, especially membrane permeability. This is of utmost importance as the IABS represents an intracellular target site and ligands targeting this site should feature appropriate pharmacokinetics, especially membrane permeability, to be considered as suitable candidates for further development.