Fig. 4. Activity of opioid modulators and development of LIH383 as ACKR3-selective agonist.

a Agonist (green color scale) and antagonist (red color scale) activity towards ACKR3 of opioid modulators (10 µM, 1 µM, and 100 nM) commonly used for research purposes or in clinic and comparison with the opioid receptors MOR, DOR, KOR, and NOP monitored in a β-arrestin-1 recruitment assay. Antagonist activity was measured following addition of BAM22 (50 nM), met-enkephalin (70 nM), dynorphin A (50 nM), nociceptin (70 nM) and CXCL12 (4 nM) on MOR, DOR, KOR, NOP, and ACKR3, respectively (b, e) Agonist activity of LIH383 (FGGFMRRK) towards human (b) and mouse (e) ACKR3 and comparison with other endogenous ACKR3 chemokine ligands, and a control peptide (MRRKFGGF), consisting of the eight amino acids building LIH383 in a different arrangement. c, d LIH383 selectivity evaluated by comparison of β-arrestin-1 recruitment to ACKR3 and the opioid receptors MOR, DOR, KOR and NOP (c) or all the other chemokine receptors known to recruit β-arrestin (3 µM) (d). f Selective binding of fluorescently labeled LIH383 (LIH383-Cy5) to ACKR3-expressing cells. All assays were performed in U87 cells. Results represent the mean (a) or mean ± S.E.M (b–f) of three to five independent experiments (n = 3–5) except for LIH383 on hACKR3 (n = 9). All fitted values are available in Supplementary Table 6. For statistical analysis, one-way ANOVA with Bonferroni multiple comparison test was used. ****p < 0.0001. Source data and statistical analysis parameters are provided as Source Data file.