Mapping the N/OFQ(1−13)-NH2 Address Domain to Identify G Protein-Biased NOP Receptor Agonists

The nociceptin/orphanin FQ peptide receptor (NOP) couples to G proteins and β-arrestins, but how the peptide address domain influences transducer preference remains unclear. We synthesized ninety N/OFQ(1−13)-NH2 analogues with acylated lysine-based or related non-natural residues at positions 5−13 and evaluated NOP−G protein and NOP−β-arrestin 2 interactions in BRET assays. Substitutions at positions 5−7 most affected signaling. Position 7 was the main hotspot for G protein bias, while bulky benzoyl- and cyclohexanoyl-lysine substitutions at position 5 also produced significant bias. Position 8 was poorly tolerant to modification, whereas positions 9−13 generally retained agonist activity and balanced signaling. No reproducible β-arrestin 2-biased profile was identified. Molecular dynamics and allosteric network analyses of [Lys(CyCO)5]N/OFQ(1−13)-NH2 suggested that engagement of a hydrophobic pocket at the TM3−TM5/ECL2 interface may contribute to its profile. These findings provide a position-dependent map for designing NOP peptide agonists with altered transducer preference.