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Rationale: Recently, we demonstrated that the activation of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) signaling facilitates depressive-like behaviors. Additionally, literature findings support the ability of the N/OFQ-NOP system to modulate the hypothalamic-pituitary-adrenal (HPA) axis., Objectives: Considering that dysfunctional HPA axis is strictly related to stress-induced psychopathologies, we aimed to study the role of the HPA axis in the pro-depressant effects of NOP agonists., Methods: Mice were treated prior to stress with the NOP agonist Ro 65-6570, and immobility time in the forced swimming task and corticosterone levels were measured. Additionally, the role of endogenous glucocorticoids and CRF was investigated using the glucocorticoid receptor antagonist mifepristone and the CRF 1 antagonist antalarmin in the mediation of the effects of Ro 65-6570., Results: The NOP agonist in a dose-dependent manner further increased the immobility of mice in the second swimming session compared to vehicle. By contrast, under the same conditions, the administration of the NOP antagonist SB-612111 before stress reduced immobility, while the antidepressant nortriptyline was inactive. Concerning in-serum corticosterone in mice treated with vehicle, nortriptyline, or SB-612111, a significant decrease was observed after re-exposition to stress, but no differences were detected in Ro 65-6570-treated mice. Administration of mifepristone or antalarmin blocked the Ro 65-6570-induced increase in the immobility time in the second swimming session., Conclusions: Present findings suggest that NOP agonists increase vulnerability to depression by hyperactivating the HPA axis and then increasing stress circulating hormones and CRF 1 receptor signaling., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background and Purpose: In animal models of sepsis, increased activation of the Nociceptin/Orphanin FQ (N/OFQ) receptor NOP is associated with mortality and NOP antagonists improved survival. We have explored the role of the N/OFQ-NOP system in freshly isolated volunteer human B- and T-cells incubated with lipopolysaccharide (LPS) and peptidoglycan G (PepG) as a model of in vitro sepsis., Experimental Approach: B- and T-cell NOP expression was measured using the NOP fluorescent probe N/OFQ ATTO594 , N/OFQ content was measured using immunofluorescence, N/OFQ release was tracked using a CHO hNOPGαiq5 biosensor assay and NOP function was measured using transwell migration and cytokine/chemokine release using a 25-plex assay format. Cells were challenged with LPS/PepG., Key Results: CD19-positive B-cells bound N/OFQ ATTO594 ; they also contain N/OFQ. Stimulation with CXCL13/IL-4 increased N/OFQ release. N/OFQ trended to reduced migration to CXCL13/IL-4. Surface NOP expression was unaffected by LPS/PepG, but this treatment increased GM-CSF release in an N/OFQ sensitive manner. CD3-positive T-cells did not bind N/OFQ ATTO594 ; they did contain N/OFQ. Stimulation with CXCL12/IL-6 increased N/OFQ release. When incubated with LPS/PepG, NOP surface expression was induced leading to N/OFQ ATTO594 binding. In LPS/PepG-treated cells, N/OFQ reduced migration to CXCL12/IL-6. LPS/PepG increased GM-CSF release in an N/OFQ sensitive manner., Conclusions and Implications: We suggest both a constitutive and sepsis-inducible N/OFQ-NOP receptor autocrine regulation of B- and T-cell function, respectively. These NOP receptors variably inhibit migration and reduce GM-CSF release. These data provide mechanistic insights to the detrimental role for increased N/OFQ signalling in sepsis and suggest a potential role for NOP antagonists as treatments., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Recently, mixed opioid/NOP agonists came to the spotlight for their favorable functional profiles and promising outcomes in clinical trials as novel analgesics. This study reports on two novel chimeric peptides incorporating the fragment Tyr-c[D-Lys-Phe-Phe]Asp-NH 2 ( RP-170 ), a cyclic peptide with high affinity for µ and κ opioid receptors (or MOP and KOP, respectively), conjugated with the peptide Ac-RYYRIK-NH 2 , a known ligand of the nociceptin/orphanin FQ receptor (NOP), yielding RP-170-RYYRIK-NH 2 ( KW-495 ) and RP-170-Gly 3 -RYYRIK-NH 2 ( KW-496 ). In vitro, the chimeric KW-496 gained affinity for KOP, hence becoming a dual KOP/MOP agonist, while KW-495 behaved as a mixed MOP/NOP agonist with low nM affinity. Hence, KW-495 was selected for further in vivo experiments. Intrathecal administration of this peptide in mice elicited antinociceptive effects in the hot-plate test; this action was sensitive to both the universal opioid receptor antagonist naloxone and the selective NOP antagonist SB-612111. The rotarod test revealed that KW-495 administration did not alter the mice motor coordination performance. Computational studies have been conducted on the two chimeras to investigate the structural determinants at the basis of the experimental activities, including any role of the Gly 3 spacer.

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of an inhibitory G protein coupled receptor named N/OFQ peptide receptor (NOP). Clinical and preclinical findings suggest that the blockade of the NOP signaling induces antidepressant-like effects. Additionally, the blockade of the NOP receptor during inescapable stress exposure prevented the acquisition of the helplessness phenotype, suggesting that NOP antagonists are able to increase stress resilience. BTRX-246040 (aka LY2940094) is a NOP receptor antagonist with high affinity, potency and selectivity for the NOP over classical opioid receptors. BTRX-246040 is under development for the treatment of depression, eating disorders and alcohol abuse and it already entered clinical trials. In the present study, the antidepressant effects of BTRX-246040 were evaluated in mice subjected to the forced swimming test and to the learned helplessness model of depression. Additionally, the ability of BTRX-246040 to prevent the development of the helpless behavior and to modulate adult hippocampal neurogenesis has been investigated. BTRX-246040 (30 mg/kg, i.p.) produced antidepressant-like effects in the forced swimming test and in the learned helplessness model. More interestingly, when given before the stress induction sessions it was able to prevent the development of the helplessness behavior. Under these experimental conditions, BTRX-246040 did not modulate adult hippocampal neurogenesis, neither in naive nor in stressed mice. This study, performed with a clinically viable ligand, further corroborates growing evidence indicating that the blockade of the NOP signaling may provide an innovative strategy for the treatment of stress related psychopathologies., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Opioid receptors are divided into the three classical types: MOP(μ:mu), DOP(δ:delta) and KOP(κ:kappa) that are naloxone-sensitive and an additional naloxone-insensitive nociceptin/orphanin FQ(N/OFQ) peptide receptor(NOP). Studies to determine opioid receptor location and turnover variably rely on; (i) measuring receptor mRNA, (ii) genetically tagging receptors, (iii) labelling receptors with radioligands, (iv) use of antibodies in immunohistochemistry/Western Blotting or (v) measuring receptor function coupled with the use of selective antagonists. All have their drawbacks with significant issues relating to mRNA not necessarily predicting protein, poor antibody selectivity and utility of radiolabels in low expression systems. In this minireview we discuss use of fluorescently labelled opioid receptor ligands. To maintain the pharmacological properties of the corresponding parent ligand fluorescently labelled ligands must take into account fluorophore (brightness and propensity to bleach), linker length and chemistry, and site to which the linker (and hence probe) will be attached. Use of donor and acceptor fluorophores with spectral overlap facilitates use in FRET type assays to determine proximity of ligand or tagged receptor pairs. There is a wide range of probes of agonist and antagonist nature for all four opioid receptor types; caution is needed with agonist probes due to the possibility for internalization. We have produced two novel ATTO based probes; Dermorphin ATTO488 (MOP) and N/OFQ ATTO594 (NOP). These probes label MOP and NOP in a range of preparations and using N/OFQ ATTO594 we demonstrate internalization and ligand-receptor interaction by FRET. Fluorescent opioid probes offer potential methodological advantages over more traditional use of antibodies and radiolabels., (Copyright © 2021 Elsevier Inc. All rights reserved.)

1-cyclohexyl-x-methoxybenzene is a novel psychoactive substance (NPS), first discovered in Europe in 2012 as unknown racemic mixture of its three stereoisomers: ortho, meta and para. Each of these has structural similarities with the analgesic tramadol and the dissociative anesthetic phencyclidine. In light of these structural analogies, and based on the fact that both tramadol and phencyclidine are substances that cause toxic effects in humans, the aim of this study was to investigate the in vitro and in vivo pharmacodynamic profile of these molecules, and to compare them with those caused by tramadol and phencyclidine. In vitro studies demonstrated that tramadol, ortho, meta and para were inactive at mu, kappa and delta opioid receptors. Systemic administration of the three stereoisomers impairs sensorimotor responses, modulates spontaneous motor activity, induces modest analgesia, and alters thermoregulation and cardiorespiratory responses in the mouse in some cases, with a similar profile to that of tramadol and phencyclidine. Naloxone partially prevents only the visual sensorimotor impairments caused by three stereoisomers, without preventing other effects. The present data show that 1-cyclohexyl-x-methoxybenzene derivatives cause pharmaco-toxicological effects by activating both opioid and non-opioid mechanisms and suggest that their use could potentially lead to abuse and bodily harm.

The ability to successfully cope with stress is known as 'resilience', and resilient individuals are less prone to develop psychopathologies. Understanding the neurobiological mechanisms of resilience may be instrumental to improve current therapies and benefit high-risk subjects. This review summarizes the complex interplay that exists between physiological and pathological responses to stressful events and the nociceptin/orphanin FQ (N/OFQ) - N/OFQ receptor (NOP) system, including: the effects of stress in regulating N/OFQ release and NOP expression; the ability of the N/OFQ-NOP system to modulate the hypothalamic-pituitary-adrenal axis; behavioral studies; and evidence in humans correlating this peptidergic system with psychopathologies. Available findings support the view that N/OFQ signaling stimulates the hypothalamic-pituitary-adrenal axis, thus increasing stress circulating hormones and corticotropin-releasing factor signaling. Additionally, activation of the NOP receptor inhibits monoamine transmission, including 5-HT, and this may contribute to maladaptive outcomes of stress. Ultimately, the N/OFQ system seems to have an important role in stress vulnerability, and blockade of NOP signaling may provide an innovative strategy for the treatment of stress related psychopathologies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The nociceptin/orphanin FQ (N/OFQ)/N/OFQ receptor (NOP) system controls different biological functions including pain and cough reflex. Mixed NOP/opioid receptor agonists elicit similar effects to strong opioids but with reduced side effects. In this work, 31 peptides with the general sequence [Tyr/Dmt 1 ,Xaa 5 ]N/OFQ(1-13)-NH 2 were synthesized and pharmacologically characterized for their action at human recombinant NOP/opioid receptors. The best results in terms of NOP versus mu opioid receptor potency were obtained by substituting both Tyr 1 and Thr 5 at the N-terminal portion of N/OFQ(1-13)-NH 2 with the noncanonical amino acid Dmt. [Dmt 1,5 ]N/OFQ(1-13)-NH 2 has been identified as the most potent dual NOP/mu receptor peptide agonist so far described. Experimental data have been complemented by in silico studies to shed light on the molecular mechanisms by which the peptide binds the active form of the mu receptor. Finally, the compound exerted antitussive effects in an in vivo model of cough.

Nociceptin/orphanin FQ (N/OFQ) controls different biological functions via selective stimulation of the N/OFQ peptide (NOP) receptor. The pleiotropic actions of N/OFQ may limit the development of NOP ligands as innovative drugs in different therapeutic areas. The pharmacological concept of functional selectivity (aka biased agonism) might be useful for amplifying beneficial actions and/or counteracting side effects. Thus, molecules with large bias factors toward G protein or β arrestin are required for investigating the translational value of NOP biased modulation. Herein, the biased behavior of a heterogeneous library of NOP-targeting peptide derivatives was evaluated in vitro with the aim to provide possible insights into the structural determinants that govern the selective activation of G protein versus β-arrestin. Our results demonstrate that lipidation of N/OFQ(1-13)-NH 2 is a useful strategy for obtaining G protein biased agonists for the NOP receptor.

Bipolar disorder is a psychiatric pathology characterized by biphasic mood episodes of mania or hypomania and depression. The pharmacotherapy of bipolar disorder has significant adverse effects impairing treatment adherence and patient quality of life. The N/OFQ-NOP receptor system has been widely implicated with mood disorders. Clinical and preclinical findings suggest antidepressants actions for NOP antagonists. More recently, the administration of NOP agonists has shown to promote depressant states. The present study aimed to investigate the effects of non-peptide NOP ligands in methylphenidate-induced manic-like behavior in mice. The NOP agonist Ro 65-6570 (0.01-1 mg/kg, ip), at the higher dose, did not affect spontaneous locomotion per se, but prevented the methylphenidate (10 mg/kg, sc)-induced hyperlocomotion. The NOP partial agonist AT-090 (0.001-0.03 mg/kg, ip) and the NOP antagonist SB-612111 (1-10 mg/kg, ip) did not significantly affect the psychostimulant-induced hyperactivity. Experiments performed with mice lacking the NOP receptor (NOP(-/-)) demonstrated that the treatment with methylphenidate induced similar hyperlocomotion in NOP(-/-) and NOP(+/+) mice. In conclusion, these findings suggest a potential role for NOP agonists in the prevention of manic states, especially by counteracting the hyperactivity symptom of bipolar patients. However, more studies are necessary in order to evaluate these compounds in other features of bipolar disorder., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Rationale: Depression and anxiety frequently co-occur, and this has important clinical implications. Previous studies showed that activation of the nociceptin/orphanin FQ receptor (NOP) elicits anxiolytic effects, while its blockade promotes consistent antidepressant actions. NOP antagonists are effective in reversing footshock-induced depressive-like behaviors, but their effects on stress-induced anxiety are still unclear., Objective: This study aimed to investigate the effects of the NOP antagonist SB-612111 on footshock stress-induced anxiety behaviors., Methods: Male Swiss mice were exposed to inescapable electric footshock stress, and behavioral phenotype was screened based on the ability to escape from footshock (i.e., helpless or non-helpless). Animals were then treated with diazepam (1 mg/kg) and SB-612111 (0.1-10 mg/kg), and their behavior was assessed in the elevated plus-maze (EPM) and open field test., Results: When compared with non-stressed mice, helpless, but not non-helpless, animals displayed significant reductions in the time spent in and entries into open arms in the EPM. Diazepam significantly increased open arms exploration in helpless, non-helpless, and non-stressed mice. However, treatment with the NOP antagonist SB-612111 was inactive in naive mice, while it reversed anxiogenic-related behaviors in helpless mice and increased anxiety states in non-helpless mice. No effects on locomotion were observed., Conclusion: Helpless mice displayed increased anxiety compared to non-stressed and non-helpless animals, thus supporting use of this approach as an animal model to investigate anxiety/depression comorbidity. Additionally, SB-612111 modulated anxiety-like behaviors in male mice depending on individual stress susceptibility. Ultimately, NOP antagonists could be useful for treating anxiety in depressed patients.

The peptide nociceptin/orphanin FQ (N/OFQ) is the natural ligand of the N/OFQ receptor (NOP), which is widely expressed in the central and peripheral nervous system. Selective NOP antagonists are worthy of testing as innovative drugs to treat depression, Parkinson disease, and drug abuse. The aim of this study was to perform a detailed in vitro characterization of BTRX-246040 (also known as LY2940094, [2-[4-[(2-chloro-4,4-difluoro-spiro[5H-thieno[2,3-c]pyran-7,4'-piperidine]-1'-yl)methyl]-3-methyl-pyrazol-1-yl]-3-pyridyl]methanol), a novel NOP antagonist that has been already studied in humans. BTRX-246040 has been tested in vitro in the following assays: calcium mobilization in cells expressing NOP and classic opioid receptors and chimeric G proteins, bioluminescence resonance energy transfer assay measuring NOP interaction with G proteins and β -arrestins, the label-free dynamic mass redistribution assay, and the electrically stimulated mouse vas deferens. BTRX-246040 was systematically compared with the standard NOP antagonist SB-612111. In all assays, BTRX-246040 behaves as a pure and selective antagonist at human recombinant and murine native NOP receptors displaying 3-10-fold higher potency than the standard antagonist SB-612111. BTRX-246040 is an essential pharmacological tool to further investigate the therapeutic potential of NOP antagonists in preclinical and clinical studies. SIGNIFICANCE STATEMENT: NOP antagonists may be innovative antidepressant drugs. In this research, the novel clinically viable NOP antagonist BTRX-246040 has been deeply characterized in vitro in a panel of assays. BTRX-246040 resulted a pure, potent, and selective NOP antagonist., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Morphine, which acts through opioid receptors, is one of the most efficient analgesics for the alleviation of severe pain. However, its usefulness is limited by serious side effects, including analgesic tolerance, constipation, and dependence liability. The growing awareness that multifunctional ligands which simultaneously activate two or more targets may produce a more desirable drug profile than selectively targeted compounds has created an opportunity for a new approach to developing more effective medications. Here, in order to better understand the role of the neurokinin system in opioid-induced antinociception, we report the synthesis, structure-activity relationship, and pharmacological characterization of a series of hybrids combining opioid pharmacophores with either substance P (SP) fragments or neurokinin receptor (NK1) antagonist fragments. On the bases of the in vitro biological activities of the hybrids, two analogs, opioid agonist/NK1 antagonist Tyr-[d-Lys-Phe-Phe-Asp]-Asn-d-Trp-Phe-d-Trp-Leu-Nle-NH 2 ( 2 ) and opioid agonist/NK1 agonist Tyr-[d-Lys-Phe-Phe-Asp]-Gln-Phe-Phe-Gly-Leu-Met-NH 2 ( 4 ), were selected for in vivo tests. In the writhing test, both hybrids showed significant an antinociceptive effect in mice, while neither of them triggered the development of tolerance, nor did they produce constipation. No statistically significant differences in in vivo activity profiles were observed between opioid/NK1 agonist and opioid/NK1 antagonist hybrids.

Background: The peptide nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are implicated in the modulation of emotional states. Previous human and rodent findings support NOP antagonists as antidepressants. However, the role played by the N/OFQ-NOP receptor system in resilience to stress is unclear., Aims: The present study investigated the effects of activation or blockade of NOP receptor signaling before exposure to acute stress., Methods: The behavioral effects of the administration before stress of the NOP agonists Ro 65-6570 (0.01-1 mg/kg) and MCOPPB (0.1-10 mg/kg), and the NOP antagonist SB-612111 (1-10 mg/kg) were assessed in mice exposed to inescapable electric footshock and forced swim as stressors. The behavioral phenotype of mice lacking the NOP receptor (NOP(-/-)) exposed to inescapable electric footshock was also investigated., Results: The activation of NOP receptor signaling with the agonists increased the percentage of mice developing helpless behavior and facilitated immobile posture. In contrast, the blockade of NOP receptor reduced the acquisition of depressive-like phenotypes, and similar resistance to develop helpless behaviors was observed in NOP(-/-) mice. Under the same stressful conditions, the antidepressant nortriptyline (20 mg/kg) did not change the acquisition of helpless behavior and immobile posture., Conclusions: These findings support the view that NOP activation during acute stress facilitates the development of depressive-related behaviors, whereas NOP blockade has a protective outcome. This study showed for first time that NOP antagonists are worthy of investigation as preemptive treatments in patients with severe risk factors for depression.

The general aim of the work was the validation of a new synthetic methodology designed for obtaining bifunctional heterotetrabranched peptide ligands. Applying an easily accessible synthetic route, we provided a small series of heteromultimeric peptide conjugates targeting the nociceptin/orphanin FQ (N/OFQ) peptide receptors (NOP) and mu opioid receptors. Among these, H-PWT1-N/OFQ-[Dmt 1 ]dermorphin demonstrated a similar and high agonist potency at the NOP and mu receptors. The achieved results confirmed the robustness of the approach that is extremely versatile and virtually applicable to different peptide sequences whose pharmacological activity can be combined for generating dual acting multimeric compounds. These innovative pharmacological tools will be extremely helpful for investigating the consequences of the simultaneous activation and/or blockage of different peptidergic receptors.

Cebranopadol is a mixed NOP/opioid receptor agonist currently under development as innovative analgesic. In this study the liability of cebranopadol to produce opioid-type physical dependence has been evaluated in comparison with morphine in wild type mice and in mice knockout for the NOP receptor gene (NOP(-/-)). Mice were treated twice a day for 5 days with increasing doses of cebranopadol or morphine (cumulative doses 10.2 and 255 mg/kg, respectively) and the number of jumping in response to naloxone 10 mg/kg were measured after 2 h from the last injection. In wild type mice naloxone evoked a similar withdrawal jumping behavior in animal pretreated with morphine or cebranopadol. In NOP(-/-) mice morphine treatment produced the same signs of withdrawal as in NOP(+/+) animals, while cebranopadol treatment elicited a stronger withdrawal syndrome in NOP(-/-) than of NOP(+/+) mice. These results demonstrated that the activation of the NOP receptor reduces the liability of cebranopadol to produce opioid-like physical dependence. Thus, the simultaneous activation of NOP and opioid receptors can be an effective pharmacological strategy to counteract physical dependence to opioid drugs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) is a G protein-coupled receptor involved in the regulation of several physiological functions and pathological conditions. Thus, researchers from academia and industry are pursuing NOP to discover and study novel pharmacological entities. In a multidisciplinary effort of pharmacologists, medicinal chemists, and molecular and structural biologists the mechanisms of NOP activation and inhibition have been, at least partially, disentangled. Here, we review the in vitro methodologies employed, which have contributed to our understanding of this target. We hope this chapter guides the reader through the mostly established assay platforms to investigate NOP pharmacology, and gives some hints taking advantage from what has already illuminated the function of other GPCRs. We analyzed the pharmacological results obtained with a large panel of NOP ligands investigated in several assays including receptor binding, stimulation of GTPγS binding, decrease of cAMP levels, calcium flux stimulation via chimeric G proteins, NOP/G protein and NOP/β-arrestin interaction, label-free assays such as dynamic mass redistribution, and bioassays such as the electrically stimulated mouse vas deferens.

Aggressive behaviors can be considered symptoms of bipolar disorder, schizophrenia, post-traumatic stress, intermittent explosive, and personality disorders. Nociceptin/orphanin FQ (N/OFQ) is a peptide acting as endogenous ligand of the NOP receptor. Preclinical and clinical findings suggest the NOP receptor as an innovative target for the treatment of psychopathologies, such as anxiety, depression, and drug abuse. This study investigated the effects of NOP ligands and the behavioral phenotype of mice lacking the NOP receptor in an animal model of aggressiveness, the resident-intruder test. Mood stabilizers, such as valproate, lithium, and carbamazepine reduced aggressive behaviors of resident mice, while diazepam was inactive. In contrast, para-chlorophenylalanine (PCPA), an inhibitor of 5-HT synthesis, increased aggressiveness in mice. Similar to PCPA, the treatment with the NOP agonists Ro 65-6570 and AT-090 also increased aggressive behaviors. The systemic administration of the NOP antagonist SB-612111 did not modify the behavior of resident mice, but it prevented the aggressive behavior of Ro 65-6570. NOP receptor knockout mice did not display any behavioral difference compared to wild-type animals in the resident-intruder test. None of the treatments affected non-agonistic behaviors and spontaneous locomotion. In conclusion, NOP receptor agonists increased aggressiveness, while the pharmacological and genetic blockade of NOP receptor signaling did not modify agonistic behaviors. Ultimately, the aggressive profile of action of NOP agonists should be taken into account in the development of innovative psychiatric drugs targeting the NOP receptor., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Following identification as the endogenous ligand for the NOP receptor, nociceptin/orphanin FQ (N/OFQ) has been shown to control several biological functions including the micturition reflex. N/OFQ elicits a robust inhibitory effect on rat micturition by reducing the excitability of the afferent fibers. After intravesical administration N/OFQ increases urodynamic bladder capacity and volume threshold in overactive bladder patients but not in normal subjects. Moreover daily treatment with intravesical N/OFQ for 10 days significantly reduced urine leakage episodes. Different chemical modifications were combined into the N/OFQ sequence to generate Rec 0438 (aka UFP-112), a peptide NOP full agonist with high potency and selectivity and long-lasting duration of action. Rec 0438 mimicked the robust inhibitory effects of N/OFQ on rat micturition reflex; its action is solely due to NOP receptor stimulation, does not show tolerance liability after 2 weeks of treatment, and can be elicited by intravesical administration. Collectively the evidence summarized and discussed in this chapter strongly suggests that NOP agonists are promising innovative drugs to treat overactive bladder.

The nociceptin/orphanin FQ (N/OFQ)-N/OFQ peptide (NOP) receptor system is widely distributed at both the peripheral and central level where it modulates important biological functions with increasing therapeutic implications. This chapter wants to provide a comprehensive and updated overview focused on the available structure-activity relationship studies on NOP receptor peptide ligands developed through different rational approaches. Punctual modifications and cyclizations of the N/OFQ sequence have been properly combined furnishing potent NOP selective ligands with different pharmacological activities (full and partial agonists, pure antagonists) and enhanced metabolic stability in vivo. The screening of peptide libraries provided a second family of NOP ligands that have been successfully optimized. Moreover, recent findings suggest the possibility to apply different multimerization strategies for the realization of multi-target NOP/opioid receptor ligands or tetrabranched N/OFQ derivatives with extraordinarily prolonged duration of action in vivo. The diverse approaches led to the identification of important pharmacological tools along with drug candidates currently in clinical development such as Rec 0438 (aka UFP-112) for the treatment of overactive bladder and SER 100 (aka ZP120) for the clinical management of systolic hypertension.

The Nociceptin/Orphanin FQ (N/OFQ) peptide NOP receptor is coupled to pertussis toxin (PTX)-sensitive G proteins (Gi/o) whose activation leads to the inhibition of both cAMP production and calcium channel activity, and to the stimulation of potassium currents. The label free dynamic mass redistribution (DMR) approach has been demonstrated useful for investigating the pharmacological profile of G protein-coupled receptors. Herein, we employ DMR technology to systematically characterize the pharmacology of a large panel of NOP receptor ligands. These are of peptide and non-peptide nature and display varying degrees of receptor efficacy, ranging from full agonism to pure antagonism. Using Chinese hamster ovary (CHO) cells expressing the human NOP receptor we provide rank orders of potency for full and partial agonists as well as apparent affinities for selective antagonists. We find the pharmacological profile of NOP receptor ligands to be similar but not identical to values reported in the literature using canonical assays for Gi/o-coupled receptors. Our data demonstrate that holistic label-free DMR detection can be successfully used to investigate the pharmacology of the NOP receptor and to characterize the cellular effects of novel NOP receptor ligands., Competing Interests: The authors declare no conflicts of interest. NTZ is an employee of Astraea Therapeutics. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Several neuropeptidergic systems act as modulators of cognitive performances. Among them, nociceptin, an opioid-like peptide also known as orphanin FQ (N/OFQ), has recently gained attention. Stimulation of its receptor, the N/OFQ opioid receptor (NOP), which is expressed in brain regions involved in emotion, memory and stress response, has inhibitory effects on the acquisition and/or consolidation of spatial and emotional memory in rodents. Recently, N/OFQ was also proposed to be linked to the pathogenesis of Post-Traumatic Stress Disorder in humans. However, until now the effect of the activation of the N/OFQ-NOP system on already consolidated memory, such as during retrieval and reconsolidation phases, has never been explored. In the present study, we investigated the consequences of systemic injection of NOP agonists or i.c.v. injection of the N/OFQ peptide on the retrieval and the reconsolidation of contextual fear memory in mice. We demonstrate that the activation of the N/OFQ system impairs the reconsolidation of context-dependent but not cue-dependent aversive memories. We also show that this amnestic effect is associated with decreased c-Fos expression in the hippocampus and amygdala. Our data thus provide the first evidence that the NOP receptor could be targeted during the reconsolidation process to weaken maladaptive memories. The N/OFQ-NOP system might constitute in the future an interesting pharmacological target for interfering with so-called "pathological memories", in particular those involving maladaptive contextual memories., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A diastereoselective synthesis of the title compound as a single E diastereomer has been efficiently accomplished by assembling the featured pyrano-indole scaffold of the spiro[cyclohexane-dihydropyrano[3,4-b]-indole]-amine framework through an oxa-Pictet-Spengler reaction, promoted by a cheap and green Zeolite catalyst. Basic pharmacological experiments demonstrate that Cebranopadol acts as a mixed nociception/orphanin FQ (NOP) and mu (MOP) opioid receptor agonist useful for treatment of chronic pain.

The peptide nociceptin/orphanin FQ (N/OFQ) and the N/OFQ receptor (NOP) constitute a neuropeptidergic system that modulates various biological functions and is currently targeted for the generation of innovative drugs. In the present study dimeric NOP receptor ligands with spacers of different lengths were generated using both peptide and non-peptide pharmacophores. The novel compounds (12 peptide and 7 nonpeptide ligands) were pharmacologically investigated in a calcium mobilization assay and in the mouse vas deferens bioassay. Both structure- and conformation-activity studies were performed. Results demonstrated that dimerization did not modify the pharmacological activity of both peptide and non-peptide pharmacophores. Moreover, when dimeric compounds were obtained with low potency peptide pharmacophores, dimerization recovered ligand potency. This effect depends on the doubling of the C-terminal address sequence rather than the presence of an additional N-terminal message sequence or modifications of peptide conformation.

The nociceptin (NOP) receptor is a G-protein-coupled receptor whose natural ligand is the NOP/orphanin FQ (N/OFQ) peptide. Evidence from pharmacological studies suggests that the N/OFQ system is implicated in the regulation of several addiction-related phenomena, such as drug intake, withdrawal, and relapse. Here, to further explore the role of NOP system in addiction, we used NOP (-/-) rats to study the motivation for cocaine, heroin, and alcohol self-administration in the absence of N/OFQ function. Conditioned place preference (CPP) and saccharin (0.2% w/v) self-administration were also investigated. Results showed that NOP (-/-) rats self-administer less cocaine (0.25, 0.125, or 0.5 mg/infusion) both under a fixed ratio 1 and a progressive ratio schedule of reinforcement compared with wild-type (Wt) controls. Consistently, cocaine (10 mg/kg, i.p.) was able to induce CPP in Wt but not in NOP (-/-). When NOP (-/-) rats were tested for heroin (20 μg/infusion) and ethanol (10% v/v) self-administration, they showed significantly lower drug intake compared with Wt. Conversely, saccharin self-administration was not affected by NOP deletion, excluding the possibility of nonspecific learning deficits or generalized disruption of reward mechanisms in NOP (-/-) rats. These findings were confirmed with pharmacological experiments using two selective NOP antagonists, SB-612111 and LY2817412. Both drugs attenuated alcohol self-administration in Wt rats but not in NOP (-/-) rats. In conclusion, our results demonstrate that genetic deletion of NOP receptors confers resilience to drug abuse and support a role for NOP receptor antagonism as a potential treatment option for drug addiction.

An innovative chemical strategy named peptide welding technology (PWT) has been developed for the facile synthesis of tetrabranched peptides. [Dmt 1 ]N/OFQ(1-13)-NH 2 acts as a universal agonist for nociceptin/orphanin FQ (N/OFQ) and classical opioid receptors. The present study investigated the pharmacological profile of the PWT derivative of [Dmt 1 ]N/OFQ(1-13)NH 2 (PWT2-[Dmt 1 ]) in several assays in vitro and in vivo after spinal administration in monkeys subjected to the tail withdrawal assay. PWT2-[Dmt 1 ] mimicked the effects of [Dmt 1 ]N/OFQ(1-13)-NH 2 displaying full agonist activity, similar affinity/potency and selectivity at human recombinant N/OFQ (NOP) and opioid receptors in receptor binding, stimulation of [ 35 S]GTPγS binding, calcium mobilization in cells expressing chimeric G proteins, and BRET studies for measuring receptor/G-protein and receptor/β-arrestin 2 interaction. In vivo in monkeys PWT2-[Dmt 1 ] elicited dose-dependent and robust antinociceptive effects being more potent and longer lasting than [Dmt 1 ]N/OFQ(1-13)-NH 2 . The analgesic action of PWT2-[Dmt 1 ] was sensitive to the NOP receptor antagonist J-113397, but not naltrexone. Thus, the present study demonstrated that the tetrabranched derivative of [Dmt 1 ]N/OFQ(1-13)-NH 2 obtained with the PWT technology maintains the in vitro pharmacological profile of the parent peptide but displays higher potency and longer lasting action in vivo., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Nociceptin/Orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ receptor (NOP). In this study novel nonpeptide NOP ligands were characterized in vitro in receptor binding and [ 35 S]GTPγS stimulated binding in membranes of cells expressing human NOP and classical opioid receptors, calcium mobilization assay in cells coexpressing the receptors and chimeric G proteins, bioluminescence resonance energy transfer (BRET) based assay for studying NOP receptor interaction with G protein and arrestin, the electrically stimulated mouse vas deferens and the mouse colon bioassays. The action of the AT compounds were compared with standard NOP agonists (N/OFQ and Ro 65-6570) and the NOP selective antagonist SB-612111. AT compounds displayed high NOP affinity and behaved as NOP agonists in all the functional assays consistently showing the following rank order of potency AT-127≥AT-090≥AT-035>AT-004= AT-001. AT compounds behaved as NOP full agonists in the calcium mobilization and mouse colon assays and as partial agonists in the [ 35 S]GTPγS and BRET assays. Interestingly AT-090 and AT-127, contrary to standard nonpeptide agonists that display G protein biased agonism, behaved as an unbiased agonists. AT-090 and AT-127 displayed higher NOP selectivity than Ro 65-6570 at native mouse receptors. AT-090 and AT-127 might be useful pharmacological tools for investigating the therapeutic potential of NOP partial agonists., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Introduction: Opioid receptors are currently classified as Mu (μ), Delta (δ), Kappa (κ) plus the opioid related nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). Despite compelling evidence for interactions and benefits of targeting more than one receptor type in producing analgesia, clinical ligands are Mu agonists. In this study we have designed a Mu-NOP agonist named DeNo. The Mu agonist component is provided by dermorphin, a peptide isolated from the skin of Phyllomedusa frogs and the NOP component by the endogenous agonist N/OFQ., Methods: We have assessed receptor binding profile of DeNo and compared with dermorphin and N/OFQ. In a series of functional screens we have assessed the ability to (i) increase Ca2+ in cells coexpressing recombinant receptors and a the chimeric protein Gαqi5, (ii) stimulate the binding of GTPγ[35S], (iii) inhibit cAMP formation, (iv) activate MAPKinase, (v) stimulate receptor-G protein and arrestin interaction using BRET, (vi) electrically stimulated guinea pig ileum (gpI) assay and (vii) ability to produce analgesia via the intrathecal route in rats., Results: DeNo bound to Mu (pKi; 9.55) and NOP (pKi; 10.22) and with reasonable selectivity. This translated to increased Ca2+ in Gαqi5 expressing cells (pEC50 Mu 7.17; NOP 9.69), increased binding of GTPγ[35S] (pEC50 Mu 7.70; NOP 9.50) and receptor-G protein interaction in BRET (pEC50 Mu 8.01; NOP 9.02). cAMP formation was inhibited and arrestin was activated (pEC50 Mu 6.36; NOP 8.19). For MAPK DeNo activated p38 and ERK1/2 at Mu but only ERK1/2 at NOP. In the gpI DeNO inhibited electrically-evoked contractions (pEC50 8.63) that was sensitive to both Mu and NOP antagonists. DeNo was antinociceptive in rats., Conclusion: Collectively these data validate the strategy used to create a novel bivalent Mu-NOP peptide agonist by combining dermorphin (Mu) and N/OFQ (NOP). This molecule behaves essentially as the parent compounds in vitro. In the antonocicoeptive assays employed in this study DeNo displays only weak antinociceptive properties.

To investigate whether the endogenous neuropeptide nociceptin/orphanin FQ (N/OFQ) contributes to the death of dopamine neurons in Parkinson's disease, we undertook a genetic and a pharmacological approach using NOP receptor knockout (NOP(-/-)) mice, and the selective and potent small molecule NOP receptor antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111). Stereological unbiased methods were used to estimate the total number of dopamine neurons in the substantia nigra of i) NOP(-/-) mice acutely treated with the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), ii) naïve mice subacutely treated with MPTP, alone or in combination with SB-612111, iii) rats injected with a recombinant adeno-associated viral (AAV) vector overexpressing human mutant p.A53T α-synuclein, treated with vehicle or SB-612111. NOP(-/-) mice showed a 50% greater amount of nigral dopamine neurons spared in response to acute MPTP compared to controls, which was associated with a milder motor impairment. SB-612111, given 4 days after MPTP treatment to mimic the clinical condition, prevented the loss of nigral dopamine neurons and striatal dopaminergic terminals caused by subacute MPTP. SB-612111, administered a week after the AAV injections in a clinically-driven protocol, also increased by 50% both the number of spared nigral dopamine neurons and striatal dopamine terminals, and prevented accompanying motor deficits induced by α-synuclein. We conclude that endogenous N/OFQ contributes to dopamine neuron loss in pathogenic and etiologic models of Parkinson's disease through NOP receptor-mediated mechanisms. NOP receptor antagonists might prove effective as disease-modifying agents in Parkinson's disease, through the rescue of degenerating nigral dopamine neurons and/or the protection of the healthy ones., (Copyright © 2016. Published by Elsevier Inc.)

The NOP receptor (nociceptin/orphanin FQ opioid peptide receptor) is the most recently discovered member of the opioid receptor family and, together with its endogenous ligand, N/OFQ, make up the fourth members of the opioid receptor and opioid peptide family. Because of its more recent discovery, an understanding of the cellular and behavioral actions induced by NOP receptor activation are less well developed than for the other members of the opioid receptor family. All of these factors are important because NOP receptor activation has a clear modulatory role on mu opioid receptor-mediated actions and thereby affects opioid analgesia, tolerance development, and reward. In addition to opioid modulatory actions, NOP receptor activation has important effects on motor function and other physiologic processes. This review discusses how NOP pharmacology intersects, contrasts, and interacts with the mu opioid receptor in terms of tertiary structure and mechanism of receptor activation; location of receptors in the central nervous system; mechanisms of desensitization and downregulation; cellular actions; intracellular signal transduction pathways; and behavioral actions with respect to analgesia, tolerance, dependence, and reward. This is followed by a discussion of the agonists and antagonists that have most contributed to our current knowledge. Because NOP receptors are highly expressed in brain and spinal cord and NOP receptor activation sometimes synergizes with mu receptor-mediated actions and sometimes opposes them, an understanding of NOP receptor pharmacology in the context of these interactions with the opioid receptors will be crucial to the development of novel therapeutics that engage the NOP receptor., (U.S. Government work not protected by U.S. copyright.)

Understanding the mechanism by which ligands affect receptor conformational equilibria is key in accelerating membrane protein structural biology. In the case of G protein-coupled receptors (GPCRs), we currently pursue a brute-force approach for identifying ligands that stabilize receptors and facilitate crystallogenesis. The nociceptin/orphanin FQ peptide receptor (NOP) is a member of the opioid receptor subfamily of GPCRs for which many structurally diverse ligands are available for screening. We observed that antagonist potency is correlated with a ligand's ability to induce receptor stability (Tm) and crystallogenesis. Using this screening strategy, we solved two structures of NOP in complex with top candidate ligands SB-612111 and C-35. Docking studies indicate that while potent, stabilizing antagonists strongly favor a single binding orientation, less potent ligands can adopt multiple binding modes, contributing to their low Tm values. These results suggest a mechanism for ligand-aided crystallogenesis whereby potent antagonists stabilize a single ligand-receptor conformational pair., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Oxaliplatin and paclitaxel are considered central components in the treatment of colorectal and breast cancer, respectively. The development of neuropathy during chronic treatment represents the major dose-limiting side effect that leads to discontinuation or interruption of therapies. The management of neuropathy is a challenge to individuate innovative therapeutic strategies based on new targets and correct routes of administration. We evaluated the hypersensitivity reliever effect of different opioid receptor agonists in rat models of oxaliplatin and paclitaxel-induced neuropathy. Compounds were spinally infused by intrathecal catheter. In oxaliplatin-treated rats, 0.3 nmol morphine induced the reversion of the mechanical hypersensitivity (Paw-pressure test), nociceptin/orphanin FQ (N/OFQ; 0.3-3 nmol) significantly increased the pain threshold without reaching the values of the control animals. The N/OFQ peptide (NOP) receptor full agonist UFP-112 reverted pain threshold alterations at lower dosage (0.1 nmol) vs morphine and N/OFQ, the partial agonist UFP-113 (0.1-1 nmol) was similar to N/OFQ. The higher efficacy of morphine vs N/OFQ was highlighted also in paclitaxel-treated rats. The mechanical hypersensitivity was fully reverted by 0.1 nmol UFP-112 and UFP-113. In conclusion, intrathecal μ opioid peptide (MOP) and NOP receptor agonists relieved chemotherapy-induced neuropathic pain. The synthetic peptides showed valuable potency and efficacy suggesting the NOP system as an exploitable target., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Nociceptin/orphanin FQ is the natural ligand of a Gi-protein coupled receptor named NOP. This peptidergic system is involved in the regulation of mood states and inflammatory responses. The present study aimed to investigate the consequences of blocking NOP signaling in lipopolysaccharide (LPS)-induced sickness and depressive-like behaviors in mice. LPS 0.8mg/kg, ip, significantly induced sickness signs such as weight loss, decrease of water and food intake and depressive-like behavior in the tail suspension test. Nortriptyline (ip, 60min prior the test) reversed the LPS-induced depressive states. The NOP receptor antagonist SB-612111, 30min prior LPS, did not modify LPS-induced sickness signs and depressive-like behavior. However, when injected 24h after LPS, NOP antagonists (UFP-101, icv, and SB-612111, ip) significantly reversed the mood effects of LPS. LPS evoked similar sickness signs and significantly increased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) plasma levels 6h post-injection in wild-type ((NOP(+/+)) and NOP knockout ((NOP(-/-)) mice. However, LPS treatment elicited depressive-like effects in NOP(+/+) but not in NOP(-/-) mice. In conclusion, the pharmacological and genetic blockade of NOP signaling does not affect LPS evoked sickness signs while reversing depressive-like behavior., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Several spiroxatrine derivatives were synthesized and evaluated as potential NOP receptor ligands. Structural modifications of the 1,4-benzodioxane moiety of spiroxatrine have been the focus of this research project. The structure-activity relationships that emerged indicate that the presence of an H-bond donor group (hydroxyl group) is more favorable for NOP activity when it is positioned α with respect to the CH2 linked to the 1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one portion. Moreover, cis diastereoisomers of the hydroxyl derivatives4 and 22 show a moderately higher degree of stereoselectivity than trans isomers. In particular, the spiropiperidine derivative cis-4 has submicromolar agonistic activity, and it will be the reference compound for the design and synthesis of new NOP agonists., (© 2014 John Wiley & Sons A/S.)

The study reports the solid-phase synthesis and biological evaluation of a series of new side chain-to-side chain cyclized opioid peptide analogs of the general structure Tyr-[D-Xaa-Phe-Phe-Asp]NH2, where Xaa = Lys (1), Orn (2), Dab (3), or Dap (4) (Dab = 2,4-diaminobutyric acid, Dap = 2,3-diaminopropionic acid), containing 17- to 14-membered rings. The influence of the ring size on binding to the MOP, DOP and KOP opioid receptors was studied. In general, the reduction of the size of the macrocyclic ring increased the selectivity for the MOP receptor. The cyclopeptide incorporating Xaa = Lys displayed subnanomolar MOP affinity but modest selectivity over the KOP receptor, while the analog with the Orn residue showed increased affinity and selectivity for MOP. The analog with Dab was a weak MOP agonist and did not bind to the other two opioid receptors. Finally, the peptide with Xaa = Dap was completely MOP receptor-selective with subnanomolar affinity. Interestingly, the deletion of one Phe residue from 1 led to the 14-membered Tyr-c[D-Lys-Phe-Asp]NH2 (5), a potent and selective MOP receptor ligand. The in vitro potencies of the new analogs were determined in a calcium mobilization assay performed in Chinese Hamster Ovary (CHO) cells expressing human recombinant opioid receptors and chimeric G proteins. A good correlation between binding and the functional test results was observed. The influence of the ring size, solid support and the N-terminal protecting group on the formation of cyclodimers was studied.

Severe pain occurs in the context of many diseases and conditions and is a leading cause of disability. Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of the N/OFQ peptide (NOP) receptor. This peptidergic system controls pain transmission and in particular spinally administered N/OFQ has robust antinociceptive properties. The aim of this study was to investigate the spinal antinociceptive properties of NOP peptide agonists after acute and subchronic treatment in rats. Doses unable to alter motor coordination were selected. UFP-112 (full NOP agonist) and UFP-113 (partial NOP agonist) were administered intrathecally (i.t.) by spinal catheterization. Acute injection of UFP-112 induced antinociceptive response at lower dosages (0.03-1nmol i.t.) compared to morphine and similar to N/OFQ. UFP-113 was effective in a 0.001-1nmol i.t. dose range. The antinociceptive effects of NOP ligands were no longer evident in rats knockout for the NOP gene, while those of morphine were maintained. The continuous spinal infusion (by osmotic pumps) of 0.1nmol/h UFP-112 and UFP-113 showed antinociceptive action comparable to 1-3nmol/h morphine or N/OFQ. The antinociceptive effect of morphine progressively decreased and was no longer significant after 6 days of treatment. Similar results were obtained with N/OFQ, UFP-112, and UFP-113. The acute i.t. injection of morphine in animals tolerant to N/OFQ and UFP-112 evoked analgesic effects. Neither morphine nor N/OFQ induced antinociceptive effects in morphine- and UFP-113-tolerant rats. In conclusion this study highlights the analgesic efficacy and potency of UFP-112 and UFP-113 underlining the relevance of NOP system in analgesia., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Nociceptin/orphanin FQ (N/OFQ) is a heptadecapeptide acting as the endogenous ligand of the N/OFQ peptide receptor (NOP). N/OFQ(1-13)-NH2 is the shortest N/OFQ sequence maintaining the same potency and efficacy as the natural peptide. Thus N/OFQ(1-13)-NH2 was used as chemical template for investigating the structure activity relationship of threonine in position 5. 28 [X(5)]N/OFQ(1-13)-NH2 derivatives, in which Thr was substituted with natural and unnatural residues, were synthesized and characterized pharmacologically for their effects at the human NOP receptor. Two different functional assays were used: agonist stimulated [(35)S]GTPγS binding in cell membranes and calcium mobilization in whole cells co-expressing chimeric G proteins. All [X(5)]N/OFQ(1-13)-NH2 derivatives behaved as full NOP agonists showing large differences in their potency. There was an excellent correlation between the results obtained in the two assays. The results of this study suggest that: position 5 does not play a pivotal role in receptor activation; the secondary alcoholic function of Thr is not important for receptor binding; side chain size, lipo/hydrophilic balance as well as hydrogen bond capability are also not crucial for receptor binding; an aliphatic amino function positively charged with at least 3 carbon atom distance from the peptide backbone has a huge disrupting effect on receptor binding. In conclusion this study demonstrates that a simple ethyl side chain as in compound 23 is sufficient in N/OFQ position 5 for maintaining bioactivity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The neuropeptide nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of the G-protein-coupled receptor NOP. Cells from the immune system express the precursor preproN/OFQ and the NOP receptor, as well as secrete N/OFQ. The activation of the N/OFQ-NOP pathway can regulate inflammatory and immune responses. Several immune activities, including leukocyte migration, cytokine and chemokine production, and lymphocytes proliferation are influenced by NOP activation. It was demonstrated that cytokines and other stimuli such as Toll-like receptor agonist (e.g., lipopolysaccharide) induce N/OFQ production by cells from innate and adaptive immune response. In this context, N/OFQ could modulate the outcome of inflammatory diseases, such as sepsis and immune-mediated pathologies by mechanisms not clearly elucidated. In fact, clinical studies revealed increased levels of N/OFQ under sepsis, arthritis, and Parkinson's disease. Preclinical and clinical studies pointed to the blockade of NOP receptor signaling as successful strategy for the treatment of inflammatory diseases. This review is focused on experimental and clinical data that suggest the participation of N/OFQ-NOP receptor activation in the modulation of the immune response, highlighting the immunomodulatory potential of NOP antagonists in the inflammatory and immunological disturbances., (© 2015 Elsevier Inc. All rights reserved.)

Branched peptides have been found to be useful in several research fields however their synthesis and purification is complicated. Here we present a novel and facile synthesis of tetra branched derivatives of nociceptin/orphanin FQ (N/OFQ). Three N/OFQ tetra branched derivatives were prepared using novel cores (PWT1, PWT2 and PWT3) containing a maleimido moiety. [Cys(18)]N/OFQ-NH2 was linked to the cores via thiol-Michael reaction characterized by high yield and purity of the desired final product. In the electrically stimulated mouse vas deferens PWT-N/OFQ derivatives mimicked the inhibitory action of the natural sequence showing similar maximal effects and 3 fold higher potencies. The NOP selective antagonist SB-612111 antagonized the effects of N/OFQ and PWT derivatives with similar pKB values (8.02-8.48). In vivo after supraspinal administration PWT2-N/OFQ stimulated food intake in mice mimicking the action of N/OFQ. Compared to the natural peptide PWT2-N/OFQ was 40 fold more potent and elicited larger effects. These findings suggest that the PWT chemical strategy can be successfully applied to biologically active peptides to generate, with unprecedented high purity and yield, tetra branched derivatives displaying an in vitro pharmacological profile similar to that of the natural sequence associated, in vivo, to increased potency and effectiveness., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Morphine and structurally related derivatives are highly effective analgesics, and the mainstay in the medical management of moderate to severe pain. Pharmacological actions of opioid analgesics are primarily mediated through agonism at the µ opioid peptide (MOP) receptor, a G protein-coupled receptor. Position 17 in morphine has been one of the most manipulated sites on the scaffold and intensive research has focused on replacements of the 17-methyl group with other substituents. Structural variations at the N-17 of the morphinan skeleton led to a diversity of molecules appraised as valuable and potential therapeutics and important research probes. Discovery of therapeutically useful morphine-like drugs has also targeted the C-6 hydroxyl group, with oxymorphone as one of the clinically relevant opioid analgesics, where a carbonyl instead of a hydroxyl group is present at position 6. Herein, we describe the effect of N-substituent variation in morphine and oxymorphone on in vitro and in vivo biological properties and the emerging structure-activity relationships. We show that the presence of a N-phenethyl group in position 17 is highly favorable in terms of improved affinity and selectivity at the MOP receptor, potent agonism and antinociceptive efficacy. The N-phenethyl derivatives of morphine and oxymorphone were very potent in stimulating G protein coupling and intracellular calcium release through the MOP receptor. In vivo, they were highly effective against acute thermal nociception in mice with marked increased antinociceptive potency compared to the lead molecules. It was also demonstrated that a carbonyl group at position 6 is preferable to a hydroxyl function in these N-phenethyl derivatives, enhancing MOP receptor affinity and agonist potency in vitro and in vivo. These results expand the understanding of the impact of different moieties at the morphinan nitrogen on ligand-receptor interaction, molecular mode of action and signaling, and may be instrumental to the development of new opioid therapeutics.

Several studies suggest that the N/OFQ (nociceptin/orphanin FQ)-NOP (N/OFQ peptide) receptor pathway is involved in airway physiology. We previously demonstrated a modulation of the endogenous N/OFQ levels in allergen-sensitized mice. Here, we investigated the effects of NOP receptor activation in allergen sensitization using a murine model of allergen-induced airway hyperresponsiveness (AHR). BALB/c mice were intraperitoneally treated with the NOP receptor agonist UFP-112, either during the sensitization phase (30 min before ovalbumin administration) or at the end of sensitization process (15 min before bronchopulmonary reactivity evaluation). At day 21 from the first allergen exposure, bronchopulmonary reactivity and total and differential cell count in bronchoalveolar lavage fluid were evaluated. In a separate set of experiments cell proliferation in lymphocytes, cytokine levels, IgE serum levels, and the effect of UFP-112 on IL-13-induced AHR were evaluated. Pretreatment with UFP-112, during the sensitization phase, caused a significant reduction in allergen-induced AHR and total cell lung infiltration. No effect on allergen-induced AHR was observed when the treatment was performed at the end of sensitization process, on tissues harvested from OVA-sensitized mice and on IL-13-induced AHR. The in vitro proliferative response of lymphocytes was significantly reduced by pretreatment during the sensitization phase with UFP-112. This effect was paralleled by a significant modulation of cytokine secretion in pulmonary tissues and lymphocytes. In conclusion, we demonstrated a role for the NOP receptor and N/OFQ pathway in the AHR induced by allergen, probably through a modulation of the immune response that triggers the development of AHR that involves pro- and anti-inflammatory cytokines.

High throughput calcium mobilization assays are extensively used for pharmacological characterization of GPCR ligands. These approaches, initially developed for G(q)-coupled receptors, can be extended to G(i) coupled GPCRs using chimeric G proteins. Here we used the Gα(qi5) protein to force the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, as well as the classical opioid receptors to signal through the PLC-IP(3)-Ca(2+) pathway in CHO cells. Calcium levels were monitored using the fluorometric imaging plate reader FlexStation II and the Ca(2+) dye Fluo 4 AM. For investigating the pharmacology of the NOP receptor a panel of full and partial agonists and antagonists were assessed, while a small panel of agonists and antagonists was used for evaluating the pharmacological profile of opioid receptors. Some limitations of this assay and differences in the results obtained in comparison with those with G(i) based biochemical assays are described. Overall, the present results confirm that the chimeric G protein strategy is useful for studying the pharmacological activity of G(i) coupled receptor ligands and that the aberrant signaling does not produce any measurable change in the pharmacological profile of the receptor under study. Thus, this G protein strategy is extremely useful for setting up primary screening assays for NOP and classical opioid receptors and likely for other members of the GPCR family.

Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the 'classical' opioid receptors, δ, κ and μ (δ-OR, κ-OR and μ-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR. Although it shares high sequence similarity with classical opioid GPCR subtypes (∼60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands. Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand-receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors κ (ref. 5) and μ (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP-compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands.

Aims: Mucosal mast cells (MMC) are mediators of the stress responses in the gastrointestinal tract. We examined the effect of acute cold-restraint stress and of the neuropeptide nociceptin/orphanin FQ (N/OFQ), implicated in the modulation of stress responses, on MMC density in the rat colon., Main Methods: Stress was induced by restraining the animals into individual cages at 3°C for 3h. N/OFQ and the selective N/OFQ peptide (NOP) receptor antagonist, UFP-101, were infused subcutaneously via Alzet osmotic minipumps. Segments of the distal colon were collected. MMCs were identified immunohistochemically with a monoclonal antibody to rat mast cell protease (RMCP) II and with a rabbit polyclonal antibody to CD117/c-kit receptor., Key Findings: Acute stress caused a decrease in the density of MMCs in the rat colonic mucosa. Short-term peripheral infusion of N/OFQ (0.1 to 10 μg/kg/h for 4h) caused a dose-related reduction of MMC density. Peak reduction occurred after the 4-h infusion of N/OFQ, 1 μg/kg/h. Reduction was maintained after the 52-h infusion period and declined following 7 and 14 days of infusion. The infusion of N/OFQ (1μg/kg/h for 4h) in rats exposed to acute stress caused a decrease in MMC density comparable to that obtained with the single treatments. UFP-101, at the doses of 1 and 10 μg/kg/h, which itself had no significant effect on MMC density, when concurrently infused in stress-exposed rats, abolished the stress-induced decrease of MMC density., Significance: Present results indicate that the peripheral N/OFQ-NOP system is involved in stress-induced reduction of MMC density., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions via selective activation of the N/OFQ peptide receptor (NOP). [(pF)Phe(4) Aib(7) Arg(14) Lys(15) ]N/OFQ-NH(2) (UFP-112) is an NOP receptor ligand designed using a combination of several chemical modifications in the same peptide sequence that increase NOP receptor affinity/potency and/or reduce susceptibility to enzymatic degradation. In the present review article, we summarize data from the literature and present original findings on the in vitro and in vivo pharmacological features of UFP-112. Moreover, important biological actions and possible therapeutic indications of NOP receptor agonists are discussed based on the results obtained with UFP-112 and compared with other peptide and nonpeptide NOP receptor ligands., (© 2010 Blackwell Publishing Ltd.)

Nociceptin/orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ peptide (NOP) receptor. Recently knockout rats for the NOP receptor gene (NOP(-/-)) have been generated; these animals were used in the present study to investigate their emotional (open field, elevated plus maze, and forced swimming test), locomotor (drag and rotarod test), and nociceptive (plantar and formalin test) phenotypes in comparison with their NOP(+/+) littermates. In addition, N/OFQ sensitivity has been assessed in electrically stimulated vas deferens tissues taken from NOP(+/+) and NOP(-/-) rats. In the elevated plus maze and forced swimming tests NOP(-/-) rats showed anxiety- and anti-depressant-like phenotype, respectively. No differences were found in the open field test. NOP(-/-) rats outperformed their NOP(+/+) littermates in two motor behaviour assays. Genetic ablation of the NOP receptor gene produced a statistically significant increase in nociceptive behaviour of the mutant rats in the formalin test. Finally, in the electrically stimulated rat vas deferens taken from NOP(+/+) tissues, N/OFQ inhibited in a concentration-dependent manner the electrically induced twitches while the peptide was inactive in tissues taken from NOP(-/-) animals. These results, in line with previous findings obtained with selective NOP receptor antagonists in mice and rats and with mouse knockout studies, clearly indicate that endogenous N/OFQ-NOP receptor signalling plays an important role in controlling anxiety- and mood-related behaviours, exercise-driven locomotor activity and nociception. These observations are relevant for defining the therapeutic indications (and contraindications) of NOP receptor antagonists., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The pharmacology of G protein-coupled receptors can be influenced by factors such as constitutive receptor activation and Na(+) ions. In this study, we examined the coupling of natively and heterologously expressed nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors with voltage-dependent Ca(2+) channels after exposure to four high-affinity NOP receptor blockers [[Nphe(1)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-101), 1-[1-(cyclooctylmethyl)-1,2,3,6-tetrahydro-5-(hydroxymethyl)-4-pyridinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (Trap-101), 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4'-piperidin-1-yl]propyl}pyrrolidine-2-carboxamide (compound 24), and N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl)benzamide hydrochloride (JTC-801)] in sympathetic neurons. The enhanced tonic inhibition of Ca(2+) currents in the absence of agonists, indicative of constitutively active NOP receptors in transfected neurons, was abolished after pretreatment with pertussis toxin. In control neurons, the four antagonists did not exert any effects when applied alone but significantly blocked the N/OFQ-mediated Ca(2+) current inhibition. Exposure of transfected neurons to UFP-101 resulted in partial agonist effects. In contrast, Trap-101, compound 24, and JTC-801 exerted inverse agonism, as measured by the loss of tonic Ca(2+) current inhibition. In experiments designed to measure the N/OFQ concentration-response relationship under varying Na(+) concentrations, a leftward shift of IC(50) values was observed after Na(+) exposure. Although similar N/OFQ efficacies were measured with all solutions, a significant decrease of Hill coefficient values was obtained with increasing Na(+) concentrations. Examination of the allosteric effects of Na(+) on heterologously overexpressed NOP receptors showed that the tonic Ca(2+) current inhibition was abolished in the presence of the monovalent cation. These results demonstrate that constitutively active NOP receptors exhibit differential blocker pharmacology and allosteric regulation by Na(+). Data are also presented demonstrating that heterologously expressed mu opioid receptors in sympathetic neurons are similarly modulated.

Nociceptin/orphanin FQ (N/OFQ) is an endogenous neuropeptide, which is widely distributed in central and peripheral nervous system. Some N/OFQ sequence unrelated hexapeptides can effectively bind to the N/OFQ peptide (NOP) receptor and they were used as template for structure-activity studies that lead to discovery of the new NOP selective ligands. In the present study, the pharmacological profile of the novel hexapeptide Ac-RYYRIR-ol was investigated using various in vitro assays including receptor binding and G-protein activation in rat brain membranes, mouse and rat vas deferens, guinea pig ileum, mouse colon and Ca(2+) mobilization assay in chinese hamster ovary (CHO) cells co-expressing the human recombinant NOP receptor and the C-terminally modified Galpha(qi5) protein. In rat brain membranes Ac-RYYRIR-ol displaced both [(3)H]nociceptin/OFQ and [(3)H]Ac-RYYRIK-ol with high affinity (pK(i) 9.35 and 8.81, respectively) and stimulated [(35)S]GTPgammaS binding showing however lower maximal effects than N/OFQ (alpha=0.28). The stimulatory effect of Ac-RYYRIR-ol was antagonized by the selective NOP receptor antagonist UFP-101. In the electrically stimulated mouse vas deferens Ac-RYYRIR-ol displayed negligible agonist activity while antagonizing in a competitive manner (pA(2) 7.99) the inhibitory effects of N/OFQ. Similar results were obtained in the rat vas deferens. In the mouse colon Ac-RYYRIR-ol produced concentration dependent contractile effects with similar potency and maximal effects as N/OFQ. Finally, in the Ca(2+) mobilization assay performed with CHO-hNOP-Galpha(qi5) cells Ac-RYYRIR-ol displayed lower potency and maximal effects (alpha=0.87) compared with N/OFQ. In conclusion, the novel NOP receptor selective hexapeptide Ac-RYYRIR-ol has been shown to have fine selectivity, high potency, furthermore agonist and antagonist effects toward the NOP receptors were measured in various assays; this is likely due to its partial agonist pharmacological activity., (Copyright 2009 Elsevier Inc. All rights reserved.)

Twelve derivatives of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) antagonist 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4'-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide (Comp 24) were synthesized and tested in binding experiments performed on CHO(hNOP) cell membranes. Among them, a novel interesting NOP receptor antagonist (compound 35) was identified by blending chemical moieties taken from different NOP receptor ligands. In vitro in various assays, Compound 35 consistently behaved as a pure, highly potent (pA(2) in the range 8.0-9.9), competitive and NOP selective antagonist. However compound 35 was found inactive when challenged against N/OFQ in vivo in the mouse tail withdrawal assay. Thus, the usefulness of the novel NOP ligand compound 35 is limited to in vitro investigations.