Mapping the N/OFQ(1−13)-NH2 Address Domain to Identify G Protein-Biased NOP Receptor Agonists
Giulio Meneguzzo, Erika Morrone, Daniele Scarpa, Alessandra Rizzo, Erika Marzola, Antonella Ciancetta, Davide Illuminati, Valentina Albanese, Tiziano De Ventura, Delia Preti, Girolamo Caló, Davide Malfacini, Salvatore Pacifico, Remo GuerriniAbstract
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.