Mechanistic insights into ligand selectivity in μ- and δ-opioid receptors beyond the “message–address” conceptual framework

Ligand selectivity between μ -opioid receptors ( μ -OR) and δ -opioid receptors ( δ -OR) is key for improving opioid analgesics. While the “message–address” hypothesis has been central to explaining this selectivity, we present evidence for an additional mechanism. Chimeric receptor and mutagenesis studies identify residue 2.63 in the orthosteric pocket as a key determinant of morphine's μ / δ -OR selectivity, while EM-1's selectivity involves a combination of residues at positions 2.63 and 3.29, the N-terminus, and extracellular loop 3 (ECL3). Approaches like voltage-clamp fluorometry, engineered zinc-bridge and microscale thermophoresis show that EM-1's Y 1 P 2 W 3 F 4 sequence confers over 1500-fold μ / δ -OR selectivity, driven by steric hindrance and a β -turn structure. β -Endorphin, with a more flexible sequence, binds non-selectively to both receptors. Morphine's rigid isoquinoline scaffold and broader geometry restrict it to binding through the wide ECL2-transmembrane (TM) 5 cleft, explaining its modest μ / δ -OR selectivity. These findings reveal that μ / δ -OR selectivity is driven by both “message–address” interactions and receptor–specific binding pathways, advancing opioid drug design. μ / δ -OR selectivity is dictated by a combination of canonical “message–address” interactions and receptor-specific binding pathways, advancing our understanding of endogenous peptide sequence evolution and informing the rational design of selective opioid therapeutics.