In vitro pharmacological characterization of a novel unbiased NOP receptor-selective nonpeptide agonist AT-403.

Nociceptin/orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ receptor (NOP), a member of the opioid receptor family. We recently identified a new high affinity and highly selective NOP agonist AT-403. In this study, we characterized the functional profile of AT-403 and compared it to other known nonpeptide NOP agonists Ro 65-6570, Ro 2q, SCH-221510, MCOPPB, AT-202 and SCH-486757, using the following assays: GTPγ[ S] stimulated binding, calcium mobilization assay in cells-expressing human NOP or classical opioid receptors and chimeric G proteins, bioluminescence resonance energy transfer (BRET) based assay for studying NOP receptor interaction with G protein and arrestin, and the electrically stimulated mouse vas deferens bioassay. All compounds behaved as NOP full agonists consistently showing the following rank order of potency MCOPPB > AT-403 > Ro 65-6570 = Ro 2q > SCH-221510 >  AT-202 > SCH-486757. AT-403 and MCOPPB displayed the highest NOP selectivity both at human and murine receptors. Interestingly, while all the other nonpeptide NOP agonists displayed bias toward G protein-mediated signaling in the BRET assay, AT-403, similar to the natural ligand N/OFQ, behaved as an unbiased agonist, activating G-protein-mediated function as well as arrestin recruitment. AT-403 may be a useful nonpeptide tool compound to study the pharmacology of NOP activation in disease states.