Comprehensive transduceromic profiling of NOP receptor ligands at different Gα subunits

Background and Purpose: The nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) is an opioid receptor family member with distinct pharmacology and broad therapeutic potential. NOP receptors regulate functions such as nociception, stress reactivity, reward, mood, autonomic tone, immunity and sleep/wake cycle. Biased signalling has been proposed to enhance efficacy and minimise adverse effects at many G protein-coupled receptors (GPCRs), yet the transducer coupling preferences of NOP remain poorly defined. Experimental Approach: We applied the (TRansdUcer PATHways) BRET-based G protein profiling platform (TRUPATH) biosensor suite to resolve NOP receptor coupling. Human NOP receptor was expressed in HEK293 cells and challenged with 20 chemically diverse ligands, including the endogenous neuropeptide, as well as peptide and non-peptide agonists. We compared TRUPATH data with those obtained in validated assays, that is, calcium mobilisation (via Gαqi5), NOP–G protein and NOP–β-Arrestin 2 interaction. Key Results: N/OFQ activated six inhibitory G proteins (Gi1, Gi2, Gi3, GoA, GoB, Gz) with high potency (pEC50 9.08–10.49) but modest signal amplitude (15–30% of basal). Ligands UFP-113 and Comp 26 showed the most pronounced deviations across G protein isoforms. Conclusions and Implications: This study provides a comprehensive fingerprint of NOP receptor pharmacology at the single-transducer level. The lack of marked G protein bias at the NOP receptor, more pronounced than for the classical opioid receptors, may reflect a combination of intrinsic receptor properties and assay limitations. These findings underscore the need for innovative chemotypes and complementary experimental strategies to fully resolve potential transducer bias at the NOP receptor.