Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and β-Arrestins 2

Abstract

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP). Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or β-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes) allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/β-arrestin 2 interactions (evaluated in whole cells). In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gβ1 or NOP/β-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified.

Nociceptin/Orphanin FQ Receptor Structure, Signaling, Ligands, Functions, and Interactions with Opioid Systems

“…In addition, although the effects of 100 nmol N/OFQ lasted for 2.5 hours (Ko et al, 2006), those elicited by 1 nmol PWT2-N/OFQ were still statistically significant 24 hours after the spinal injection . These recent findings demonstrated that that the PWT can be successfully applied to the peptide sequence of N/OFQ to generate tetrabranched derivatives characterized by a Calo' et al, 2000b), b (Malfacini et al, 2015), c (McDonald et al, 2003b), d ), e (Calo' et al, 2002b, f (Trapella et al, 2006), g (Bigoni et al, 2000), h , i (Fischetti et al, 2009b). Underscored data indicate pA 2 values obtained from Schild plots.…”

“…The sodium binding pocket collapses upon receptor activation, thus suggesting that presence of sodium may stabilize the receptor in an inactive conformation . Very recently using a BRET-based assay to investigate NOP receptor/G-protein interactions, it was demonstrated that GDP was not able to significantly inhibit the baseline BRET ratio (Malfacini et al, 2015). However, in membranes expressing the other opioid receptors, under similar experimental conditions, GDP can suppress the baseline BRET ratio, indicating a reduction in spontaneous receptor/G-protein interactions, with maximal effects 4-5 times greater at delta than mu receptors (Vezzi et al, 2013).…”

“…How the NOP receptor engages these various arrestins and whether agonists of varying efficacies and potencies can induce differential rates of internalization and divergent arrestin2/3 recruitment remains an active area of study. Recent evidence suggests that compounds acting as partial agonists with respect to NOP/G-protein signaling behave as antagonists with little to no activity in NOP/arrestin coupling (Chang et al, 2015b;Malfacini et al, 2015). In fact, NOP receptors indeed functionally recruit both arrestin2 and arrestin3, yet may recruit arrestin3 in a more efficacious manner (Chang et al, 2015b).…”

“…35 S]GTPgS binding, and a BRET-based assay measuring NOP/G-protein interaction (Malfacini et al, 2015). These ligands were also tested in calcium mobilization studies performed in whole cells coexpressing the NOP receptor and the chimeric G-protein Ga qi5 ) and in bioassay experiments performed in N/OFQ-sensitive isolated tissues such as the electrically stimulated mouse (Berzetei-Gurske et al, 1996;Calo' et al, 1996) and rat vas deferens.…”

“…The investigation into NOP receptor biased signaling is in its infacy. Initial studies have demonstrated that partial agonists for G-protein NOP Receptor Biology and Function coupling are very poor in b-arrestin-mediated signal transduction (Chang et al, 2015b;Malfacini et al, 2015). New techniques in resonance energy transfer and genetic tools (i.e., conditional arrestin knockout mice) will help characterize new ligands to better understand how NOP receptor biased signaling translates to their in vivo actions.…”