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Background Stress-induced analgesia (SIA) is an evolutionarily conserved phenomenon during stress. Neuropeptide S (NPS), orexins, substance P, glutamate and endocannabinoids are known to be involved in stress and/or SIA, however their causal links remain unclear. Here, we reveal an unprecedented sequential cascade involving these mediators in the lateral hypothalamus (LH) and ventrolateral periaqueductal gray (vlPAG) using a restraint stress-induced SIA model. Methods Male C57BL/6 mice of 8–12 week-old were subjected to intra-cerebroventricular (i.c.v.) and/or intra-vlPAG (i.pag.) microinjection of NPS, orexin-A or substance P alone or in combination with selective antagonists of NPS receptors (NPSRs), OX1 receptors (OX1Rs), NK1 receptors (NK1Rs), mGlu5 receptors (mGlu5Rs) and CB1 receptors (CB1Rs), respectively. Antinociceptive effects of these mediators were evaluated via the hot-plate test. SIA in mice was induced by a 30-min restraint stress. NPS levels in the LH and substance P levels in vlPAG homogenates were compared in restrained and unrestrained mice. Results NPS (i.c.v., but not i.pag.) induced antinociception. This effect was prevented by i.c.v. blockade of NPSRs. Substance P (i.pag.) and orexin-A (i.pag.) also induced antinociception. Substance P (i.pag.)-induced antinociception was prevented by i.pag. Blockade of NK1Rs, mGlu5Rs or CB1Rs. Orexin-A (i.pag.)-induced antinociception has been shown previously to be prevented by i.pag. blockade of OX1Rs or CB1Rs, and here was prevented by NK1R or mGlu5R antagonist (i.pag.). NPS (i.c.v.)-induced antinociception was prevented by i.pag. blockade of OX1Rs, NK1Rs, mGlu5Rs or CB1Rs. SIA has been previously shown to be prevented by i.pag. blockade of OX1Rs or CB1Rs. Here, we found that SIA was also prevented by i.c.v. blockade of NPSRs or i.pag. blockade of NK1Rs or mGlu5Rs. Restrained mice had higher levels of NPS in the LH and substance P in the vlPAG than unrestrained mice. Conclusions These results suggest that, during stress, NPS is released and activates LH orexin neurons via NPSRs, releasing orexins in the vlPAG. Orexins then activate OX1Rs on substance P-containing neurons in the vlPAG to release substance P that subsequently. Activates NK1Rs on glutamatergic neurons to release glutamate. Glutamate then activates perisynaptic mGlu5Rs to initiate the endocannabinoid retrograde inhibition of GABAergic transmission in the vlPAG, leading to analgesia.

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.

Evaluation of stimulus salience is critical for any higher organism, as it allows for prioritizing of vital information, preparation of responses, and formation of valuable memory. The paraventricular nucleus of the thalamus (PVT) has recently been identified as an integrator of stimulus salience but the neurochemical basis and afferent input regarding salience signaling have remained elusive. Here we report that neuropeptide S (NPS) signaling in the PVT is necessary for stimulus salience encoding, including aversive, neutral and reinforcing sensory input. Taking advantage of a striking deficit of both NPS receptor (NPSR1) and NPS precursor knockout mice in fear extinction or novel object memory formation, we demonstrate that intra-PVT injections of NPS can rescue the phenotype in NPS precursor knockout mice by increasing the salience of otherwise low-intensity stimuli, while intra-PVT injections of NPSR1 antagonist in wild type mice partially replicates the knockout phenotype. The PVT appears to provide stimulus salience encoding in a dose- and NPS-dependent manner. PVT NPSR1 neurons recruit the nucleus accumbens shell and structures in the prefrontal cortex and amygdala, which were previously linked to the brain salience network. Overall, these results demonstrate that stimulus salience encoding is critically associated with NPS activity in the PVT.

Background Cebranopadol, a mixed nociceptin/opioid receptor full agonist, can effectively relieve pain in rodents and humans. However, it is unclear to what degree different opioid receptor subtypes contribute to its antinociception and whether cebranopadol lacks acute opioid-associated side effects in primates. The authors hypothesized that coactivation of nociceptin receptors and μ receptors produces analgesia with reduced side effects in nonhuman primates. Methods The antinociceptive, reinforcing, respiratory-depressant, and pruritic effects of cebranopadol in adult rhesus monkeys (n = 22) were compared with μ receptor agonists fentanyl and morphine using assays, including acute thermal nociception, IV drug self-administration, telemetric measurement of respiratory function, and itch-scratching responses. Results Subcutaneous cebranopadol (ED50, 2.9 [95% CI, 1.8 to 4.6] μg/kg) potently produced antinociception compared to fentanyl (15.8 [14.6 to 17.1] μg/kg). Pretreatment with antagonists selective for nociceptin and μ receptors, but not δ and κ receptor antagonists, caused rightward shifts of the antinociceptive dose–response curve of cebranopadol with dose ratios of 2 and 9, respectively. Cebranopadol produced reinforcing effects comparable to fentanyl, but with decreased reinforcing strength, i.e., cebranopadol (mean ± SD, 7 ± 3 injections) versus fentanyl (12 ± 3 injections) determined by a progressive-ratio schedule of reinforcement. Unlike fentanyl (8 ± 2 breaths/min), systemic cebranopadol at higher doses did not decrease the respiratory rate (17 ± 2 breaths/min). Intrathecal cebranopadol (1 μg) exerted full antinociception with minimal scratching responses (231 ± 137 scratches) in contrast to intrathecal morphine (30 μg; 3,009 ± 1,474 scratches). Conclusions In nonhuman primates, the μ receptor mainly contributed to cebranopadol-induced antinociception. Similar to nociceptin/μ receptor partial agonists, cebranopadol displayed reduced side effects, such as a lack of respiratory depression and pruritus. Although cebranopadol showed reduced reinforcing strength, its detectable reinforcing effects and strength warrant caution, which is critical for the development and clinical use of cebranopadol. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

Fentanyl derivatives (FENS) belongs to the class of Novel Synthetic Opioids that emerged in the illegal drug market of New Psychoactive Substances (NPS). These substances have been implicated in many cases of intoxication and death with overdose worldwide. Therefore, the aim of this study is to investigate the pharmaco-dynamic profiles of three fentanyl (FENT) analogues: Acrylfentanyl (ACRYLF), Ocfentanyl (OCF) and Furanylfentanyl (FUF). In vitro, we measured FENS opioid receptor efficacy, potency, and selectivity in calcium mobilization studies performed in cells coexpressing opioid receptors and chimeric G proteins and their capability to promote the interaction of the mu receptor with G protein and β-arrestin 2 in bioluminescence resonance energy transfer (BRET) studies. In vivo, we investigated the acute effects of the systemic administration of ACRYLF, OCF and FUF (0.01-15 mg/kg i.p.) on mechanical and thermal analgesia, motor impairment, grip strength and cardiorespiratory changes in CD-1 male mice. Opioid receptor specificity was investigated in vivo using naloxone (NLX; 6 mg/kg i.p) pre-treatment. In vitro, the three FENS were able to activate the mu opioid receptor in a concentration dependent manner with following rank order potency: FUF FENT=OCF ACRYLF. All compounds were able to elicit maximal effects similar to that of dermorphin, with the exception of FUF which displayed lower maximal effects thus behaving as a partial agonist. In the BRET G-protein assay, all compounds behaved as partial agonists for the β-arrestin 2 pathway in comparison with dermorphin, whereas FUF did not promote β-arrestin 2 recruitment, behaving as an antagonist. In vivo, all the compounds increased mechanical and thermal analgesia with following rank order potency ACRYLF = FENT FUF OCF and impaired motor and cardiorespiratory parameters. Among the substances tested, FUF showed lower potency for cardiorespiratory and motor effects. These findings reveal the risks associated with the use of FENS and the importance of studying the pharmaco-dynamic properties of these drugs to better understand possible therapeutic interventions in the case of toxicity.

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-[Dmt1]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.

In insects, tyramine receptor 1 (TAR1) has been shown to control several physiological functions, including olfaction. We investigated the molecular and functional profile of the Halyomorpha halys type 1 tyramine receptor gene (HhTAR1) and its role in olfactory functions of this pest. Molecular and pharmacological analyses confirmed that the HhTAR1 gene codes for a true TAR1. RT-qPCR analysis revealed that HhTAR1 is expressed mostly in adult brain and antennae as well as in early development stages (eggs, 1st and 2nd instar nymphs). In particular, among the antennomeres that compose a typical H. halys antenna, HhTAR1 was more expressed in flagellomeres. Scanning electron microscopy investigation revealed the type and distribution of sensilla on adult H. halys antennae: both flagellomeres appear rich in trichoid and grooved sensilla, known to be associated with olfactory functions. Through an RNAi approach, topically delivered HhTAR1 dsRNA induced a 50% downregulation in gene expression after 24 h in H. halys 2nd instar nymphs. An innovative behavioural assay revealed that HhTAR1 RNAi-silenced 2nd instar nymphs were less susceptible to the alarm pheromone component (E)-2 decenal as compared with controls. These results provide critical information concerning the role of TAR1 in olfaction regulation, especially alarm pheromone reception, in H. halys. Furthermore, considering the emerging role of TAR1 as target of biopesticides, this work opens the way for further investigation on innovative methods for controlling H. halys.

Neurons containing neuropeptide S (NPS) and orexins are activated during stress. Previously, we reported that orexins released during stress, via orexin OX1 receptors (OX1 Rs), contribute to the reinstatement of cocaine seeking through endocannabinoid/CB1 receptor (CB1 R)-mediated dopaminergic disinhibition in the ventral tegmental area (VTA). Here, we further demonstrated that NPS released during stress is an up-stream activator of this orexin-endocannabinoid cascade in the VTA, leading to the reinstatement of cocaine seeking. Mice were trained to acquire cocaine conditioned place preference (CPP) by context-pairing cocaine injections followed by the extinction training with context-pairing saline injections. Interestingly, the extinguished cocaine CPP in mice was significantly reinstated by intracerebroventricular injection (i.c.v.) of NPS (1 nmol) in a manner prevented by intraperitoneal injection (i.p.) of SHA68 (50 mg/kg), an NPS receptor antagonist. This NPS-induced cocaine reinstatement was prevented by either i.p. or intra-VTA microinjection (i.vta.) of SB-334867 (15 mg/kg, i.p. or 15 nmol, i.vta.) and AM 251 (1.1 mg/kg, i.p. or 30 nmol, i.vta.), antagonists of OX1 Rs and CB1 Rs, respectively. Besides, NPS (1 nmol, i.c.v.) increased the number of c-Fos-containing orexin neurons in the lateral hypothalamus (LH) and increased orexin-A level in the VTA. The latter effect was blocked by SHA68. Furthermore, a 30-min restraint stress in mice reinstated extinguished cocaine CPP and was prevented by SHA68. These results suggest that NPS is released upon stress and subsequently activates LH orexin neurons to release orexins in the VTA. The released orexins then reinstate extinguished cocaine CPP via an OX1 R- and endocannabinoid-CB1 R-mediated signaling in the VTA.

The role of stress in the etiology of depression has been largely reported. In this line, exogenous glucocorticoids are employed to mimic the influence of stress on the development of depression. The N/OFQ-NOP receptor system has been implicated in the modulation of stress and emotional behaviors. In fact, the blockade of NOP receptors induces antidepressant effects and increases resilience to acute stress. This study investigated the effects of the NOP receptor blockade on dexamethasone-treated mice exposed to acute and prolonged swimming stress. Swiss and NOP(+/+) and NOP(−/−) mice were treated with dexamethasone, and the protective effects of the NOP antagonist SB-612111 (10 mg/kg, ip) or imipramine (20 mg/kg, ip) were investigated in three swimming sessions. The re-exposure to swim stress increased immobility time in Swiss and NOP(+/+), but not in NOP(−/−) mice. Acute and repeated dexamethasone administration induced a further increase in the immobility time, and facilitated body weight loss in Swiss mice. Single administration of SB-612111, but not imipramine, prevented swimming stress- and dexamethasone-induced increase in the immobility time. Repeated administrations of SB-612111 prevented the deleterious effects of 5 days of dexamethasone treatment. Imipramine also partially prevented the effects of repeated glucocorticoid administration on the immobility time, but did not affect the body weight loss. NOP(−/−) mice were more resistant than NOP(+/+) mice to inescapable swimming stress, but not dexamethasone-induced increase in the immobility time and body weight loss. In conclusion, the blockade of the NOP receptor facilitates an active stress copying response and attenuates body weight loss due to repeated stress.

In insects, the tyramine receptor 1 (TAR1) has been shown to control several physiological functions, including olfaction. We investigated the molecular and functional profile of the Halyomorpha halys type 1 tyramine receptor gene (HhTAR1) and its role in olfactory functions of this pest. Molecular and pharmacological analyses confirmed that the HhTAR1 gene codes for a true TAR1. The RT-qPCR analysis revealed that HhTAR1 is expressed mostly in adult brain and antennae as well as in early development stages (eggs, 1st and 2nd instar nymphs). In particular, among the antennomeres that compose a typical H. halys antenna, HhTAR1 was more expressed in flagellomeres. Scanning electron microscopy (SEM) investigation revealed the type and distribution of sensilla on adult H. halys antennae: both flagellomeres appear rich in trichoid and grooved sensilla, known to be associated with olfactory functions. Through a RNAi approach, topically delivered HhTAR1 dsRNA induced a 50 % gene downregulation after 24 h in H. halys 2nd instar nymphs. An innovative behavioral assay revealed that HhTAR1 RNAi-silenced 2nd instar nymphs were less susceptible to the alarm pheromone component (E)-2 decenal as compared to control. These results provide critical information concerning the TAR1 role in olfaction regulation, especially alarm pheromone reception, in H. halys. Furthermore, considering the emerging role of TAR1 as target of biopesticides, this work paves the way for further investigation on innovative methods for controlling H. halys.

Opioids such as morphine—acting at the mu opioid receptor—are the mainstay for treatment of moderate to severe pain and have good efficacy in these indications. However, these drugs produce a plethora of unwanted adverse effects including respiratory depression, constipation, immune suppression and with prolonged treatment, tolerance, dependence and abuse liability. Studies in β-arrestin 2 gene knockout (βarr2(−/−)) animals indicate that morphine analgesia is potentiated while side effects are reduced, suggesting that drugs biased away from arrestin may manifest with a reduced-side-effect profile. However, there is controversy in this area with improvement of morphine-induced constipation and reduced respiratory effects in βarr2(−/−) mice. Moreover, studies performed with mice genetically engineered with G-protein-biased mu receptors suggested increased sensitivity of these animals to both analgesic actions and side effects of opioid drugs. Several new molecules have been identified as mu receptor G-protein-biased agonists, including oliceridine (TRV130), PZM21 and SR–17018. These compounds have provided preclinical data with apparent support for bias toward G proteins and the genetic premise of effective and safer analgesics. There are clinical data for oliceridine that have been very recently approved for short term intravenous use in hospitals and other controlled settings. While these data are compelling and provide a potential new pathway-based target for drug discovery, a simpler explanation for the behavior of these biased agonists revolves around differences in intrinsic activity. A highly detailed study comparing oliceridine, PZM21 and SR–17018 (among others) in a range of assays showed that these molecules behave as partial agonists. Moreover, there was a correlation between their therapeutic indices and their efficacies, but not their bias factors. If there is amplification of G-protein, but not arrestin pathways, then agonists with reduced efficacy would show high levels of activity at G-protein and low or absent activity at arrestin; offering analgesia with reduced side effects or ‘apparent bias’. Overall, the current data suggests—and we support—caution in ascribing biased agonism to reduced-side-effect profiles for mu-agonist analgesics.

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.

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.

A small library of 3-thia-7,9-diazabicyclo[3.3.1]nonanes was synthesized and their opioid receptors affinity and selectivity evaluated. Among these novel sulfur-bridged compounds, the (E) 9-[3′-(3-chlorophenyl)-but-2′-en-1′-yl]-7-propionyl-3-thia-7,9-diazabicyclo[3.3.1]nonane 2i emerged as the derivative with the highest μ receptor affinity (Ki = 85 nM) and selectivity (Ki μ/δ = 58.8, Ki μ/κ > 117.6). The antinociceptive activity of 2i was also evaluated in acute thermal pain. Docking studies disclosed the specific pattern of interactions of these derivatives.

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)-NH2 is a useful strategy for obtaining G protein biased agonists for the NOP receptor.

Two similar proteolytic cascades lead to release of bradykinin and lysbradykinin in tissues; these peptides act as autocoids, the first favouring blood clotting and endothelial protection, the second by initiating and maintaining tissue reactions to noxious stimuli. Kinin biological actions are mediated by two different receptor types, the constitutive B2 which promotes initial reactions and the inducible B1 which may later maintain mechanisms of defence and repair. Keywords: kallikreins; kinins; B1 and B2 receptors

Background and purpose: The nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is a member of the opioid receptor family and is involved in a number of physiological responses, pain and immune regulation as examples. In this study, we conjugated a red fluorophore-ATTO594 to the peptide ligand N/OFQ (N/OFQATTO594 ) for the NOP receptor and explored NOP receptor function at high (in recombinant systems) and low (on immune cells) expression. Experimental approach: We assessed N/OFQATTO594 receptor binding, selectivity and functional activity in recombinant (CHO) cell lines. Live cell N/OFQATTO594 binding was measured in (i) HEK cells expressing NOP and NOPGFP receptors, (ii) CHO cells expressing the hNOPGαqi5 chimera (to force coupling to measurable Ca2+ responses) and (iii) freshly isolated human polymorphonuclear cells (PMN). Key results: N/OFQATTO594 bound to NOP receptor with nM affinity and high selectivity. N/OFQATTO594 activated NOP receptor by reducing cAMP formation and increasing Ca2+ levels in CHOhNOPGαqi5 cells. N/OFQATTO594 was also able to visualize NOP receptors at low expression levels on PMN cells. In NOP-GFP-tagged receptors, N/OFQATTO594 was used in a FRET protocol where GFP emission activated ATTO, visualizing ligand-receptor interaction. When the NOPGFP receptor is activated by N/OFQATTO594 , movement of ligand and receptor from the cell surface to the cytosol can be measured. Conclusions and implications: In the absence of validated NOP receptor antibodies and issues surrounding the use of radiolabels (especially in low expression systems), these data indicate the utility of N/OFQATTO594 to study a wide range of N/OFQ-driven cellular responses.

Morphine and related drugs, which are the most effective analgesics for the relief of severe pain, act through activating opioid receptors. The endogenous ligands of these receptors are opioid peptides which cannot be used as antinociceptive agents due to their low bioactivity and stability in biological fluids. The major goal of opioid research is to understand the mechanism of action of opioid receptor agonists in order to improve therapeutic utility of opioids. Analgesic effects of morphine are mediated mostly through activation of the mu opioid receptor. However, in the search for safer and more effective drug candidates, analogs with mixed opioid receptor profile gained a lot of interest. Recently, the concept of biased agonists able to differentially activate GPCR downstream pathways, became a new approach in the design of novel drug candidates. It is hypothesized that compounds promoting G-protein signaling may produce analgesia while β-arrestin recruitment may be responsible for opioid side effects. In this report we showed that replacement of the tyrosine residue in the mu-selective ligand Tyr-c[d-Lys-Phe-Asp]NH2 with 2',6'-dimethyltyrosine (Dmt) produced a cyclopeptide Dmt-c[d-Lys-Phe-Asp]NH2 with mu/delta opioid receptor agonist profile. This analog showed improved antinociception in the hot-plate test, probably due to the simultaneous activation of mu and delta receptors but also significantly inhibited the gastrointestinal transit. Using the bioluminescence resonance energy transfer (BRET) assay it was shown that this analog was a mu receptor agonist biased toward β-arrestin. β-Arrestin-dependent signaling is most likely responsible for the observed inhibition of gastrointestinal motility exerted by the novel cyclopeptide. This work was supported by the Polpharma Scientific Foundation grant (11/XIII/2014 to KG) and grants from the Medical University of Lodz (No 503/1-156-02/503-11-002 and 502-03/1-156-02/502-14-301) and from the University of Ferrara (FAR grant to G.C.). KG is recipients of the Polish L’Oréal UNESCO Awards for Women in Science.

Neuropeptide S (NPS) is an endogenous neuropeptide controlling anxiolysis, wakefulness, and analgesia. NPS containing neurons exist near to the locus coeruleus (LC) involved in the descending anti-nociceptive system. NPS interacts with central noradrenergic neurons; thus brain noradrenergic signaling may be involved in NPS-induced analgesia. We tested NPS analgesia in noradrenergic neuron-lesioned rats using a selective LC noradrenergic neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). A total 66 male Sprague–Dawley rats weighing 350–450 g were used. Analgesic effects of NPS were evaluated using hot-plate and tail-flick test with or without DSP-4. The animal allocated into 3 groups; hot-plate with NPS alone intracerebroventricular (icv) (0.0, 1.0, 3.3, and 10.0 nmol), tail-flick NPS alone icv (0.0 and 10.0 nmol), and hot-plate with NPS and DSP-4 (0 or 50 mg/kg ip). In hot-plate with NPS and DSP-4 group, noradrenaline content in the cerebral cortex, pons, hypothalamus, were measured. NPS 10 nmol icv prolonged hot plate (%MPE) but not tail flick latency at 30 and 40 min after administration. DSP-4 50 mg/kg decreased noradrenaline content in the all 3 regions. The NA depletion inhibited NPS analgesic effect in the hot plate test but not tail flick test. There was a significant correlation between hot plate latency (percentage of maximum possible effect: %MPE) with NPS 10 nmol and NA content in the cerebral cortex (p = 0.017, r 2 = 0.346) which noradrenergic innervation arisen mainly from the LC. No other regions had the correlation. NPS analgesia interacts with LC noradrenergic neuronal activity.

Nociceptin/orphanin FQ (N/OFQ) modulates several biological functions, including pain transmission via selective activation of a specific receptor named NOP. The aim of this study was the investigation of the antinociceptive properties of NOP agonists and their interaction with opioids in the trigeminal territory. The orofacial formalin (OFF) test in mice was used to investigate the antinociceptive potential associated to the activation of NOP and opioid receptors. Mice subjected to OFF test displayed the typical biphasic nociceptive response and sensitivity to opioid and NSAID drugs. Mice knockout for the NOP gene displayed a robust pronociceptive phenotype. The NOP selective agonist Ro 65-6570 (0.1-1mgkg-1) and morphine (0.1-10mgkg-1) elicited dose dependent antinociceptive effects in the OFF with the alkaloid showing larger effects; the isobologram analysis of their actions demonstrated an additive type of interaction. The mixed NOP/opioid receptor agonist cebranopadol elicited potent (0.01-0.1mgkg-1) and robust antinociceptive effects. In the investigated dose range, all drugs did not modify the motor performance of the mice in the rotarod test. Collectively the results of this study demonstrated that selective NOP agonists and particularly mixed NOP/opioid agonists are worthy of development as innovative drugs to treat painful conditions of the trigeminal territory.

MT-45 is a synthetic opioid that was developed in the 1970s as an analgesic compound. However, in recent years MT-45 has been associated with multiple deaths in Europe and has been included in the class of novel psychoactive substances known as novel synthetic opioids (NSOs). Little is known about the pharmaco-toxicological effects of MT-45. Therefore, we used a dynamic mass redistribution (DMR) assay to investigate the pharmacodynamic profile of this NSO in vitro compared with morphine. We then used in vivo studies to investigate the effect of the acute systemic administration of MT-45 (0.01–15 mg/kg i.p.) on motor and sensorimotor (visual, acoustic and tactile) responses, mechanical and thermal analgesia, muscle strength and body temperature in CD-1 male mice. Higher doses of MT-45 (6–30 mg/kg i.p.) were used to investigate cardiorespiratory changes (heart rate, respiratory rate, SpO2 saturation and pulse distention). All effects of MT-45 were compared with those of morphine. In vitro DMR assay results demonstrated that at human recombinant opioid receptors MT-45 behaves as a potent selective mu agonist with a slightly higher efficacy than morphine. In vivo results showed that MT-45 progressively induces tail elevation at the lowest dose tested (0.01 mg/kg), increased mechanical and thermal antinociception (starting from 1 to 6 mg/kg), decreased visual sensorimotor responses (starting from 3 to 6 mg/kg) and reduced tactile responses, modulated motor performance and induced muscle rigidity at higher doses (15 mg/kg). In addition, at higher doses (15–30 mg/kg) MT-45 impaired the cardiorespiratory functions. All effects were prevented by the administration of the opioid receptor antagonist naloxone. These findings reveal the risks associated with the ingestion of opioids and the importance of studying these drugs and undertaking more clinical studies of the current molecules to better understand possible therapeutic interventions in the case of toxicity.

This study proposes a biochemical and molecular model for the interaction between the Drosophila suzukii type 1 tyramine receptor (DsTAR1) and monoterpenes. A preliminary molecular and functional characterization of DsTAR1 cDNA revealed that a 1.8 kb long ORF codes for a 600 amino acid polypeptide featuring seven transmembrane domains, as expected for a GPCR. A stable HEK 293 cell line expressing DsTAR1 was tested for responsiveness to tyramine (TA) and octopamine (OA). In intracellular calcium mobilization studies, TA led to a concentration-dependent increase in [Ca2+]i (pEC50 ~ 6.40), completely abolished by pre-incubation with the antagonist yohimbine 1 μM. Besides, in dynamic mass redistribution (DMR) studies, TA evoked a positive DMR signal in a concentration-dependent manner (pEC50 ~ 6.80). The recombinant cell line was then used to test three monoterpenes (thymol, carvacrol and α-terpineol) as putative ligands for DsTAR1. The terpenoids showed no agonist effects in both DMR and calcium mobilization assays, but they increased the potency of the endogenous ligand, TA, acting as positive allosteric modulators. Moreover, expression analysis on adults D. suzukii, exposed for 24, 72 or 120 h to a sublethal concentration of the three monoterpenes, showed a downregulation of DsTAR1. This evidence has led to hypothesize that the downregulation of DsTAR1 might be a compensatory mechanism in response to the positive allosteric modulation of the receptor induced by monoterpenes. Therefore, these findings might be useful for the development of a new generation of biopesticides against Drosophila suzukii, targeting TAR1.

Opioid and opioid-like receptors are activated by a variety of endogenous peptides including [Met]enkephalin (met), [Leu]enkephalin (leu), β-endorphin (β-end), α-neodynorphin, dynorphin A (dynA), dynorphin B (dynB), big dynorphin (Big dyn), nociceptin/orphanin FQ (N/OFQ); endomorphin-1 and endomorphin-2 are also potential endogenous peptides. The Greek letter nomenclature for the opioid receptors, μ, δ and κ, is well established, and NC-IUPHAR considers this nomenclature appropriate, along with the symbols spelled out (mu, delta, and kappa), and the acronyms, MOP, DOP, and KOP. [116, 96, 88]. The human N/OFQ receptor, NOP, is considered 'opioid-related' rather than opioid because, while it exhibits a high degree of structural homology with the conventional opioid receptors [282], it displays a distinct pharmacology. Currently there are numerous clinically used drugs, such as morphine and many other opioid analgesics, as well as antagonists such as naloxone, however only for the μ receptor.

Neuropeptide S (NPS) is the endogenous ligand of a G-protein coupled receptor named NPS receptor. The NPS system controls several biological functions, including anxiety, wakefulness, locomotor activity, food intake, and pain transmission. A growing body of evidence supports facilitatory effects for NPS over dopaminergic neurotransmission. The present study was aimed to investigate the role of dopamine receptors signaling in the antinociceptive effects of NPS in the mouse formalin test. The following dopamine receptor antagonists were employed: SCH 23390 (selective dopamine D

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.

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.

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.

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.

Neuropeptide S (NPS) is the endogenous ligand of a G-protein coupled receptor named NPS receptor. The NPS system controls several biological functions, including anxiety, wakefulness, locomotor activity, food intake, and pain transmission. A growing body of evidence supports facilitatory effects for NPS over dopaminergic neurotransmission. The present study was aimed to investigate the role of dopamine receptors signaling in the antinociceptive effects of NPS in the mouse formalin test. The following dopamine receptor antagonists were employed: SCH 23390 (selective dopamine D1 antagonist, 0.05 mg/kg, ip), haloperidol (non-selective dopamine D2-like receptor antagonist; 0.03 mg/kg, ip), and sulpiride (selective dopamine D2-like receptor antagonist; 25 mg/kg, ip). Mice were pretreated with dopamine antagonists before the supraspinal administration of NPS (0.1 nmol, icv). Morphine (5 mg/kg, sc) and indomethacin (10 mg/kg, ip) were used as positive controls to set up the experimental conditions. Morphine-induced antinociceptive effects were observed during phases 1 and 2 of the test, while indomethacin was only active at phase 2. Central NPS significantly reduced formalin-induced nociception during both phases. The systemic administration of SCH 23390 slightly blocked the effects of NPS only during phase 2. Haloperidol prevented NPS-induced antinociceptive effects. Similar to haloperidol, sulpiride also counteracted the antinociceptive effects of NPS in both phases of the formalin test. In conclusion, the present findings suggest that the analgesic effects of NPS are linked with dopaminergic neurotransmission mainly through dopamine D2-like receptor signaling.

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&ndash, 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-NH2 (2) and opioid agonist/NK1 agonist Tyr-[d-Lys-Phe-Phe-Asp]-Gln-Phe-Phe-Gly-Leu-Met-NH2 (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.

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 pivotal role of the stereocenter at the N-substituent of the 6,7-benzomorphan scaffold was investigated combining synthetic and pharmacological approaches. 2R- and 2S-diastereoisomers of the multitarget MOR/DOR antinociceptive ligand LP2 (1) were synthesized and their pharmacological profile was evaluated in in vitro and vivo assays. From our results, 2S-LP2 (5) showed an improved pharmacological profile in comparison to LP2 (1) and 2R-LP2 (4). 2S-LP2 (5) elicited an antinociceptive effect with a 1.5- and 3-times higher potency than LP2 (1) and R-antipode (4), respectively. In vivo effect of 2S-LP2 (5) was consistent with the improved MOR/DOR efficacy profile assessed by radioligand binding assay, to evaluate the opioid receptor affinity, and BRET assay, to evaluate the capability to promote receptor/G-protein and receptor/β-arrestin 2 interaction. 2S-LP2 (5) was able to activate, with different efficacy, G-protein pathway over β-arrestin 2, behaving as biased agonist at MOR and mainly at DOR. Considering the therapeutic potential of both multitarget MOR/DOR agonism and functional selectivity over G-protein, the 2S-LP2 (5) biased multitarget MOR/DOR agonist could provide a safer treatment opportunity.