Differential In Vitro Pharmacological Profiles of Structurally Diverse Nociceptin Receptor Agonists in Activating G Protein and Beta-Arrestin Signaling at the Human Nociceptin Opioid Receptor

Abstract

Agonists at the nociceptin opioid peptide receptor (NOP) are under investigation as therapeutics for nonaddicting analgesia, opioid use disorder, Parkinson's disease, and other indications. NOP full and partial agonists have both been of interest, particularly since NOP partial agonists show a reduced propensity for behavioral disruption than NOP full agonists. Here, we investigated the in vitro pharmacological properties of chemically diverse NOP receptor agonists in assays measuring functional activation of the NOP receptor such as guanosine 5'-O-[gamma-thio]triphosphate (GTPγS) binding, cAMP inhibition, G protein-coupled inwardly rectifying potassium (GIRK) channel activation, phosphorylation, β-arrestin recruitment and receptor internalization. When normalized to the efficacy of the natural agonist nociceptin/orphanin FQ (N/OFQ), we found that different functional assays that measure intrinsic activity produce inconsistent levels of agonist efficacy, particularly for ligands that were partial agonists. Agonist efficacy obtained in the GTPγS assay tended to be lower than that in the cAMP and GIRK assays. These structurally diverse NOP agonists also showed differential receptor phosphorylation profiles at the phosphosites we examined and induced varying levels of receptor internalization. Interestingly, although the rank order for β-arrestin recruitment by these NOP agonists was consistent with their ability to induce receptor internalization, their phosphorylation signatures at the time point we investigated were not indicative of the levels of β-arrestin recruitment or internalization induced by these agonists. It is possible that other phosphorylation sites, yet to be identified, drive the recruitment of NOP receptor ensembles and subsequent receptor trafficking by some nonpeptide NOP agonists. These findings potentially help understand NOP agonist pharmacology in the context of ligand-activated receptor trafficking. SIGNIFICANCE STATEMENT: Chemically diverse agonist ligands at the nociceptin opioid receptor G protein-coupled receptor showed differential efficacy for activating downstream events after receptor binding, in a suite of functional assays measuring guanosine 5'-O-[gamma-thio]triphosphate binding, cAMP inhibition, G protein-coupled inwardly rectifying protein channel activation, β-arrestin recruitment, receptor internalization and receptor phosphorylation. These analyses provide a context for understanding nociceptin opioid peptide receptor (NOP) agonist pharmacology driven by ligand-induced differential NOP receptor signaling.

Fig. 1.

Chemical structures of NOP agonists investigated.

Fig. 3.

Phosphorylation and internalization induced by AT compounds at human NOP receptor. (A) HEK293 cells expressing human NOP receptors were treated with 10 μM of N/OFQ, AT-004, AT-200, AT-403, or vehicle (−) or with 1 nM to 10 μM of AT-090, AT-312, and AT-390, at 37°C for 10 minutes, and cell lysates were immunoblotted with antibodies to phosphorylated (p) S346, pS351, or pT362/S363. Blots were stripped and reprobed for human NOP receptor to ensure equal loading. All blots were representative of 3–5 experiments. (B) HEK293 cells stably expressing HA-tagged human NOP receptors were preincubated with HA antibody and then stimulated with 10 μM of N/OFQ, AT-004, AT-200, AT-312, or solvent vehicle at 22°C for 60 minutes. Cells were then fixed, permeabilized, stained immunofluorescently, and examined subsequently using confocal microscopy. All images were representative of three independent experiments. (C) HEK293 cells stably expressing human NOP receptors were preincubated with antibody to HA-tag and treated with 10 μM of N/OFQ, AT-390, AT-090, or AT-403 at 37°C for 60 minutes. Then, the cells were fixed and labeled with a peroxidase-conjugated secondary antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in the cells treated with agonists. Data represent means ± S.D. from 12 independent experiments performed in quadruplicate. The P values were comparative with N/OFQ and were obtained using two sample Student’s t tests assuming unequal variances. *P < 0.05 versus N/OFQ.

Fig. 2.

Maximal stimulation of G protein signaling of human NOP receptor by NOP agonists, measured in GTPγS, cAMP and GIRK assays, and normalized to N/OFQ (100%). Each value represents the average value and S.D. obtained from three experiments done on different days, with triplicates in each experiment.

Fig. 4.

Stimulation of β-arrestin recruitment at human NOP receptor induced by AT-004, AT-090, AT-200, AT-312, AT-390, and AT-403, normalized to N/OFQ (100%). On day 1, HTLA cells were plated at 10 × 10⁶ cells in 150-mm cell culture dishes. On day 2, the cells were transfected with NOP receptor plasmids using the Fugene 6 HD transfection reagent. On day 3, transfected cells were harvested by trypsinization and then transferred into white flat-bottom 96-well tissue culture plates. On day 4, the cells were treated with corresponding AT compounds or N/OFQ control overnight at 37°C for at least 18 hours. On day 5, the luciferase reading in each well was detected by using a Bright Glo Luciferase Reporter Assay on a plate reader. Percentage of β-arrestin recruitment was normalized to that of N/OFQ (at 1 μM). Each experiment was repeated at least 3 times, with each drug concentration in quintuplicate wells to obtain the average value ± S.D. The potency (EC₅₀ nM) of each NOP agonist for β-arrestin recruitment is labeled in the graph for each agonist.

Fig. 5.

Graphical side-by-side representation of the levels of NOP receptor internalization and β-arrestin recruitment by NOP agonists.