Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists

Agonists of the nociceptin/orphanin FQ opioid peptide (NOP) receptor, a member of the opioid receptor family, are under active investigation as novel analgesics, but their modes of signaling are less well characterized than those of other members of the opioid receptor family. Therefore, we investigated whether different NOP receptor ligands showed differential signaling or functional selectivity at the NOP receptor. Using newly developed phosphosite-specific antibodies to the NOP receptor, we found that agonist-induced NOP receptor phosphorylation occurred primarily at four carboxyl-terminal serine (Ser) and threonine (Thr) residues, namely, Ser346, Ser351, Thr362, and Ser363, and proceeded with a temporal hierarchy, with Ser346 as the first site of phosphorylation. G protein–coupled receptor kinases 2 and 3 (GRK2/3) cooperated during agonist-induced phosphorylation, which, in turn, facilitated NOP receptor desensitization and internalization. A comparison of structurally distinct NOP receptor agonists revealed dissociation in functional efficacies between G protein–dependent signaling and receptor phosphorylation. Furthermore, in NOP-eGFP and NOP-eYFP mice, NOP receptor agonists induced multisite phosphorylation and internalization in a dose-dependent and agonist-selective manner that could be blocked by specific antagonists. Our study provides new tools to study ligand-activated NOP receptor signaling in vitro and in vivo. Differential agonist-selective NOP receptor phosphorylation by chemically diverse NOP receptor agonists suggests that differential signaling by NOP receptor agonists may play a role in NOP receptor ligand pharmacology.

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Functional Selectivity Does Not Predict Antinociceptive/Locomotor Impairing Potencies of NOP Receptor Agonists

Frontiers in Neuroscience ◽

10.3389/fnins.2021.657153 ◽

2021 ◽

Vol 15 ◽

Author(s):

Joaquim Azevedo Neto ◽

Chiara Ruzza ◽

Chiara Sturaro ◽

Davide Malfacini ◽

Salvatore Pacifico ◽

...

Keyword(s):

G Protein ◽

Knockout Mice ◽

Motor Impairment ◽

Therapeutic Index ◽

Label Free ◽

Nop Receptor ◽

Orphanin Fq ◽

Biased Agonism

Nociceptin/orphanin FQ controls several functions, including pain transmission, via stimulation of the N/OFQ peptide (NOP) receptor. Here we tested the hypothesis that NOP biased agonism may be instrumental for identifying innovative analgesics. In vitro experiments were performed with the dynamic mass redistribution label free assay and the NOP non-peptide agonists Ro 65-6570, AT-403 and MCOPPB. In vivo studies were performed in wild type and β-arrestin 2 knockout mice using the formalin, rotarod and locomotor activity tests. In vitro all compounds mimicked the effects of N/OFQ behaving as potent NOP full agonists. In vivo Ro 65-6570 demonstrated a slightly higher therapeutic index (antinociceptive vs. motor impairment effects) in knockout mice. However, all NOP agonists displayed very similar therapeutic index in normal mice despite significant differences in G protein biased agonism. In conclusion the different ability of inducing G protein vs. β-arrestin 2 recruitment of a NOP agonist cannot be applied to predict its antinociceptive vs. motor impairment properties.

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6,5-Fused Ring, C2-Salvinorin Ester, Dual Kappa and Mu Opioid Receptor Agonists as Analgesics Devoid of Anxiogenic Effects

10.26434/chemrxiv.14204834.v1 ◽

2021 ◽

Author(s):

Nicholas S. Akins ◽

Nisha Mishra ◽

Hannah M. Harris ◽

Narendar Dudhipala ◽

Seong Jong Kim ◽

...

Keyword(s):

Opioid Receptor ◽

Opioid Receptors ◽

Analgesic Activity ◽

Mu Opioid Receptor ◽

Kappa Opioid Receptor ◽

Mu Opioid ◽

Receptor Agonists ◽

Opioid Receptor Agonists

Analgesia is commonly mediated through the mu or kappa opioid receptor agonism. Unfortunately, selective mu or kappa receptor agonists often cause harmful side effects. Recently, ligands exhibiting dual agonism to the opioid receptors, such as to mu and kappa, or to mu and delta, have been suggested to temper undesirable adverse effects while retaining analgesic activity. Herein we report an introduction of various 6,5-fused rings to C2 of the salvinorin scaffold via an ester linker. In vitro studies showed that some of these compounds have dual agonism on kappa and mu opioid receptors, while some have triple agonism on kappa, mu, and delta. In vivo studies on the lead dual kappa and mu opioid receptor agonist, compound 10, showed that it produced analgesic activity while avoiding anxiogenic effects in murine models, thus providing further strong evidence for the therapeutic advantages of dual opioid receptor agonists over selective opioid receptor agonists.

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Molecular Modeling of µ Opioid Receptor Ligands with Various Functional Properties: PZM21, SR-17018, Morphine, and Fentanyl—Simulated Interaction Patterns Confronted with Experimental Data

Molecules ◽

10.3390/molecules25204636 ◽

2020 ◽

Vol 25 (20) ◽

pp. 4636 ◽

Cited By ~ 2

Author(s):

Sabina Podlewska ◽

Ryszard Bugno ◽

Lucja Kudla ◽

Andrzej J. Bojarski ◽

Ryszard Przewlocki

Keyword(s):

Molecular Modeling ◽

G Protein ◽

Opioid Receptor ◽

Pearson Correlation ◽

Interaction Patterns ◽

Receptor Ligands ◽

Opioid Receptor Ligands ◽

Dynamics Simulations

Molecular modeling approaches are an indispensable part of the drug design process. They not only support the process of searching for new ligands of a given receptor, but they also play an important role in explaining particular activity pathways of a compound. In this study, a comprehensive molecular modeling protocol was developed to explain the observed activity profiles of selected µ opioid receptor agents: two G protein-biased µ opioid receptor agonists (PZM21 and SR-17018), unbiased morphine, and the β-arrestin-2-biased agonist, fentanyl. The study involved docking and molecular dynamics simulations carried out for three crystal structures of the target at a microsecond scale, followed by the statistical analysis of ligand–protein contacts. The interaction frequency between the modeled compounds and the subsequent residues of a protein during the simulation was also correlated with the output of in vitro and in vivo tests, resulting in the set of amino acids with the highest Pearson correlation coefficient values. Such indicated positions may serve as a guide for designing new G protein-biased ligands of the µ opioid receptor.

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Opioid receptor agonists in the rabbit colon: Comparison of in vivo and in vitro studies

Life Sciences ◽

10.1016/0024-3205(84)90176-0 ◽

1984 ◽

Vol 35 (16) ◽

pp. 1653-1658 ◽

Cited By ~ 11

Author(s):

M. Pairet ◽

Y. Ruckebusch

Keyword(s):

Opioid Receptor ◽

In Vitro Studies ◽

Receptor Agonists ◽

Rabbit Colon ◽

Opioid Receptor Agonists

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NOP Receptor Ligands as Potential Agents for Inflammatory and Autoimmune Diseases

Journal of Amino Acids ◽

10.4061/2011/836569 ◽

2011 ◽

Vol 2011 ◽

pp. 1-10 ◽

Cited By ~ 15

Author(s):

Elaine C. Gavioli ◽

Pedro R. T. Romão

Keyword(s):

Immune Cells ◽

Inflammatory Diseases ◽

Nop Receptor ◽

Orphanin Fq ◽

Experimental Conditions ◽

Amino Acid Peptide ◽

Cellular And Humoral Immunity ◽

Preclinical And Clinical Studies

Nociceptin/orphanin FQ (N/OFQ) is a seventeen-amino acid peptide that is the endogenous ligand of a G-protein-coupled receptor (NOP). Various immune cells express the precursor protein and secrete N/OFQ as well as display binding sites for this peptide. The functional capacity of NOP receptor was demonstrated in vitro and in vivo studies by the ability of N/OFQ to induce chemotaxis of immune cells, to regulate the expression of cytokines and other inflammatory mediators, and to control cellular and humoral immunity. In this context, N/OFQ could modulate the outcome of some inflammatory diseases, such as sepsis and autoimmune pathologies by mechanisms not clearly elucidated yet. In fact, human body fluid revealed increased levels of N/OFQ under sepsis, arthritis, and Parkinson's diagnose. Preclinical studies pointed to the blockade of NOP receptor signaling as successful in treating these experimental conditions. Further preclinical and clinical studies are required to investigate the potential of NOP ligands in treating inflammatory diseases.

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β-Arrestin-Selective G Protein-Coupled Receptor Agonists Engender Unique Biological Efficacy in Vivo

Molecular Endocrinology ◽

10.1210/me.2012-1091 ◽

2013 ◽

Vol 27 (2) ◽

pp. 296-314 ◽

Cited By ~ 48

Author(s):

Diane Gesty-Palmer ◽

Ling Yuan ◽

Bronwen Martin ◽

William H. Wood ◽

Mi-Hye Lee ◽

...

Keyword(s):

Bone Formation ◽

G Protein ◽

Wild Type ◽

G Protein Coupled Receptor ◽

Calvarial Bone ◽

Matrix Mineralization ◽

Receptor Agonists ◽

G Protein Coupled

Abstract Biased G protein-coupled receptor agonists are orthosteric ligands that possess pathway-selective efficacy, activating or inhibiting only a subset of the signaling repertoire of their cognate receptors. In vitro, d-Trp12,Tyr34-bPTH(7–34) [bPTH(7–34)], a biased agonist for the type 1 PTH receptor, antagonizes receptor-G protein coupling but activates arrestin-dependent signaling. In vivo, both bPTH(7–34) and the conventional agonist hPTH(1–34) stimulate anabolic bone formation. To understand how two PTH receptor ligands with markedly different in vitro efficacy could elicit similar in vivo responses, we analyzed transcriptional profiles from calvarial bone of mice treated for 8 wk with vehicle, bPTH(7–34) or hPTH(1–34). Treatment of wild-type mice with bPTH(7–34) primarily affected pathways that promote expansion of the osteoblast pool, notably cell cycle regulation, cell survival, and migration. These responses were absent in β-arrestin2-null mice, identifying them as downstream targets of β-arrestin2-mediated signaling. In contrast, hPTH(1–34) primarily affected pathways classically associated with enhanced bone formation, including collagen synthesis and matrix mineralization. hPTH(1–34) actions were less dependent on β-arrestin2, as might be expected of a ligand capable of G protein activation. In vitro, bPTH(7–34) slowed the rate of preosteoblast proliferation, enhanced osteoblast survival when exposed to an apoptotic stimulus, and stimulated cell migration in wild-type, but not β-arrestin2-null, calvarial osteoblasts. These results suggest that bPTH(7–34) and hPTH(1–34) affect bone mass in vivo through predominantly separate genomic mechanisms created by largely distinct receptor-signaling networks and demonstrate that functional selectivity can be exploited to change the quality of G protein-coupled receptor efficacy.

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In Vitro Analyses of Spinach-Derived Opioid Peptides, Rubiscolins: Receptor Selectivity and Intracellular Activities through G Protein- and β-Arrestin-Mediated Pathways

Molecules ◽

10.3390/molecules26196079 ◽

2021 ◽

Vol 26 (19) ◽

pp. 6079

Author(s):

Yusuke Karasawa ◽

Kanako Miyano ◽

Hideaki Fujii ◽

Takaaki Mizuguchi ◽

Yui Kuroda ◽

...

Keyword(s):

Side Effects ◽

G Protein ◽

Opioid Receptor ◽

Opioid Receptors ◽

Opioid Peptides ◽

Opioid Analgesics ◽

Receptor Agonists ◽

Δ Opioid Receptor ◽

Opioid Receptor Agonists

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the β-arrestin-mediated pathway, which leads to unfavorable side effects. Hence, G-protein-biased opioid agonists are preferable as opioid analgesics. Rubiscolins, the spinach-derived naturally occurring opioid peptides, are selective δ opioid receptor agonists, and their p.o. administration exhibits antinociceptive effects. Although the potency and effect of rubiscolins as G-protein-biased molecules are partially confirmed, their in vitro profiles remain unclear. We, therefore, evaluated the properties of rubiscolins, in detail, through several analyses, including the CellKeyTM assay, cADDis® cAMP assay, and PathHunter® β-arrestin recruitment assay, using cells stably expressing µ, δ, κ, or µ/δ heteromer opioid receptors. In the CellKeyTM assay, rubiscolins showed selective agonistic effects for δ opioid receptor and little agonistic or antagonistic effects for µ and κ opioid receptors. Furthermore, rubiscolins were found to be G-protein-biased δ opioid receptor agonists based on the results obtained in cADDis® cAMP and PathHunter® β-arrestin recruitment assays. Finally, we found, for the first time, that they are also partially agonistic for the µ/δ dimers. In conclusion, rubiscolins could serve as attractive seeds, as δ opioid receptor-specific agonists, for the development of novel opioid analgesics with reduced side effects.

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Nociceptin/Orphanin FQ Peptide Receptor-Related Ligands as Novel Analgesics

Current Topics in Medicinal Chemistry ◽

10.2174/1568026620666200508082615 ◽

2020 ◽

Vol 20 (31) ◽

pp. 2878-2888 ◽

Cited By ~ 2

Author(s):

Norikazu Kiguchi ◽

Huiping Ding ◽

Shiroh Kishioka ◽

Mei-Chuan Ko

Keyword(s):

Opioid Receptor ◽

Receptor Agonist ◽

Physical Dependence ◽

Opioid Peptide ◽

Receptor Activation ◽

Abuse Liability ◽

Similar Distribution ◽

Nop Receptor ◽

Orphanin Fq ◽

Receptor Agonists

Despite similar distribution patterns and intracellular events observed in the nociceptin/ orphanin FQ peptide (NOP) receptor and other opioid receptors, NOP receptor activation displays unique pharmacological profiles. Several researchers have identified a variety of peptide and nonpeptide ligands to determine the functional roles of NOP receptor activation and observed that NOP receptor- related ligands exhibit pain modality-dependent pain processing. Importantly, NOP receptor activation results in anti-nociception and anti-hypersensitivity at the spinal and supraspinal levels regardless of the experimental settings in non-human primates (NHPs). Given that the NOP receptor agonists synergistically enhance mu-opioid peptide (MOP) receptor agonist-induced anti-nociception, it has been hypothesized that dual NOP and MOP receptor agonists may display promising functional properties as analgesics. Accumulating evidence indicates that the mixed NOP/opioid receptor agonists demonstrate favorable functional profiles. In NHP studies, bifunctional NOP/MOP partial agonists (e.g., AT-121, BU08028, and BU10038) exerted potent anti-nociception via NOP and MOP receptor activation; however, dose-limiting adverse effects associated with the MOP receptor activation, including respiratory depression, itch sensation, physical dependence, and abuse liability, were not observed. Moreover, a mixed NOP/opioid receptor agonist, cebranopadol, presented promising outcomes in clinical trials as a novel analgesic. Collectively, the dual agonistic actions on NOP and MOP receptors, with appropriate binding affinities and efficacies, may be a viable strategy to develop innovative and safe analgesics.

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