Fig. 1. Opioid-peptide library screening on ACKR3 and hit activity comparison.

a The ability of 58 compounds, including natural opioid peptides from the four opioid families, variants thereof and small molecule opioid receptor modulators, to induce β-arrestin-2 recruitment to ACKR3, CXCR4 and CXCR3 in U87 cells at a concentration of 5 µM. For full peptide names and sequences, see Supplementary Table 1. Chemokine positive controls (Ctrls) were used at a concentration of 300 nM. Results are expressed as fold change over vehicle-treated cells and presented as mean of two technical replicates for ACKR3 and CXCR4. A single measurement was performed for CXCR3. b–f Comparison of potency and efficacy of ACKR3-activating and other representative opioid peptides in inducing β-arrestin-1 recruitment to the opioid receptors MOR (b), DOR (c), KOR (d), NOP (e), and ACKR3 (f) in U87 cells. Results are expressed as percentage of indicated agonist response. The corresponding EC₅₀ and Emax values are summarized in Table 1. g Binding competition of ACKR3-activating and other representative opioid peptides with Alexa Fluor 647-labeled CXCL12 (5 nM) on U87-ACKR3 cells determined by flow cytometry. Results from b-g are presented as mean ± S.E.M of three or four independent experiments (n = 3 or 4). Peptides from the enkephalin, dynorphin, nociceptin and endorphin families are depicted in blue, green, orange and gray scale, respectively. h Schematic representation of the interactions of the four opioid receptors and chemokine receptors with their respective ligands. The newly identified opioid peptide pairings with ACKR3 are shown, highlighting the cross-family selectivity of ACKR3. Source data are provided as Source Data file.