ORL1 Research Articles

Nociceptin/orphanin FQ opioid receptor (NOP) selective ligand MCOPPB links anxiolytic and senolytic effects.

Accumulation of senescent cells may drive age-associated alterations and pathologies. Senolytics are promising therapeutics that can preferentially eliminate senescent cells. Here, we performed a high-throughput automatized screening (HTS) of the commercial LOPAC®Pfizer library on aphidicolin-induced senescent human fibroblasts, to identify novel senolytics. We discovered the nociceptin receptor FQ opioid receptor (NOP) selective ligand 1-[1-(1-methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole (MCOPPB, a compound previously studied as potential anxiolytic) as the best scoring hit. The ability of MCOPPB to eliminate senescent cells in in vitro models was further tested in mice and in C. elegans. MCOPPB reduced the senescence cell burden in peripheral tissues but not in the central nervous system. Mice and worms exposed to MCOPPB also exhibited locomotion and lipid storage changes. Mechanistically, MCOPPB treatment activated transcriptional networks involved in the immune responses to external stressors, implicating Toll-like receptors (TLRs). Our study uncovers MCOPPB as a NOP ligand that, apart from anxiolytic effects, also shows tissue-specific senolytic effects.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11357-021-00487-y.

Pharmacological and metabolic characterization of the novel synthetic opioid brorphine and its detection in routine casework

After their first emergence in 2009, Novel synthetic opioids (NSO) have become an emerging class of New Psychoactive Substances (NPS) on the market for these new drugs. So far, 67 NSO have been reported to the Early Warning system of the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). It is presumed that NSO mainly target the four known opioid receptors, i.e. the μ-opioid (MOR), the δ-opioid (DOR), the κ-opioid (KOR) and nociceptin receptors and that their consumption can result in serious adverse effects such as massive respiratory depression or death. In the present study we investigated the in vivo and in vitro metabolism of brorphine, a NSO that was first identified on the NPS market in August 2019 in the United States, using both a pooled human liver microsome assay and real forensic case samples. For the detection of metabolites LC-HR-MS/MS was used and quantification of brorphine was performed using an LC-MS/MS method. Additionally, we pharmacologically characterized brorphine regarding its activation of the MOR and KOR via G protein recruitment using the [35S]-GTPγS assay. In forensic urine samples, 14 distinct metabolites were identified, whereas in blood only four metabolites could be found. The pooled human liver microsome assay generated six distinct in vitro phase I metabolites. The most prominent in vivo metabolite was formed by N-oxydation, whereas the main in vitro metabolite was formed by hydroxylation. The pharmacological characterization at the MOR and KOR revealed brorphine to be a potent MOR agonist and a weak, partial KOR agonist in the [35S]-GTPγS assay.

This Month in Anesthesiology

This Month in Anesthesiology

Maternal deprivation by early weaning causesthe nociceptinto alter the palatability and cookie consumption

The deprivation of maternal care during the initial development period, as well as interruption of early breastfeeding, promotes changes in eating behavior, however, the underlying neuromodulatory mechanisms have not been fully understood. Studies demonstrate theimportance of the nociceptin receptor (NOP) in modulating the immediate hedonic impact and the hyperphagic effect associated with nociceptin/orphanin FQ (N/OFQ or nociceptin) overexpression. In the current study, experiments have beenperformedto understand the impact of early weaningon the control of hedonic eating behavior and the NOP system's modulatory performance on the resulting phenotype of this injury. Early weaning promotespreference and increasespalatable food consumption, compared to the control group. The NOP receptor antagonist (SB 612111) isable to normalize the palatable food consumption in animals of theearlyweanedgroup, in addition to reducing this consumption in the control group. These results are concordant with the hypothesis that changein hedonic eating behavior, resulting from early weaning are influenced by the NOP system,which, in turn, enhances its potential as a therapeutic target for obesity.

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

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

Structure-Based SAR in the Design of Selective or Bifunctional Nociceptin (NOP) Receptor Agonists.

The nociceptin opioid receptor (NOP), the fourth member of the opioid receptor family, and its endogenous peptide ligand, nociceptin or orphanin FQ (N/OFQ), play a vital role in several central nervous system pathways regulating pain, reward, feeding, anxiety, motor control and learning/memory. Both selective NOP agonists as well as bifunctional agonists at the NOP and mu opioid receptor (MOP) have potential therapeutic applications in CNS disorders related to these processes. Using Surflex-Dock protocols, we conducted a computational structure-activity study of four scaffold classes of NOP ligands with varying NOP-MOP selectivity. By docking these compounds into the orthosteric binding sites within an active-state NOP homology model, and an active-state MOP crystal structure, the goal of this study was to use a structure-based drug design approach to modulate NOP affinity and NOP vs. MOP selectivity. We first docked four parent compounds (no side chain) to determine their binding interactions within the NOP and MOP binding pockets. Various polar sidechains were added to the heterocyclic A-pharmacophore to modulate NOP ligand affinity. The substitutions mainly contained a 1-2 carbon chain with a polar substituent such as an amine, alcohol, sulfamide, or guanidine. The SAR analysis is focused on the impact of structural changes in the sidechain, such as chain length, hydrogen bonding capability, and basic vs neutral functional groups on binding affinity and selectivity at both NOP and MOP receptors. This study highlights structural modifications that can be leveraged to rationally design both selective NOP and bifunctional NOP-MOP agonists with different ratios of functional efficacy.

The role of nociceptin in opioid regulation of brain functions

This review discusses our current knowledge on the nociceptin/orphanin (N/OFQ) system regarding its role in regulation of brain functions. Nociceptin receptor (NOPr) was identified in 1994 [Bunzow et al., 1994; Mollereau et al., 1994]. In 1995 a 17 amino acid endogenous peptide was found to be the high-affinity ligand for the NOPr [Reinscheid et al., 1995]. N/OFQ has a broad spectrum of activity and can act as on opioid-like as well as an anti-opioid peptide. Considering high level of N/OFQ and NOPr mRNA expression in the limbic brain regions, the N/OFQ/NOP system is suggested to be involved in regulation of emotions, resward, pain sensitivity, stress responsibility, sexual behavior, aggression, drug abuse and addiction. However it is still not well understood whether an increased vulnerability to drugs of abuse may be associated with dysregulation of N/OFQ/NOP system. Current review further highlights a need for further research on N/OFQ/NOP system as it could have clinical utility for substance abuse, depression, and anxiety pharmacotherapy.

Potential of Nociceptin/Orphanin FQ Peptide Analogs for Drug Development.

Nociceptin receptor (NOP) belongs to the family of opioid receptors but was discovered and characterized much later than the so called classical opioid receptors, μ, δ and κ (or MOP, DOP and KOP, resp.). Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of this receptor and it controls numerous important functions in the central nervous system and in the periphery, so its analogs may be developed as innovative drugs for the treatment of a variety of conditions and pathological states. Availability of potent and selective ligands with high affinity to NOP receptor is essential to fully understand the role of NOP-N/OFQ system in the body, which in turn may lead to designing novel therapeutics. Here, we have focused on reviewing the structure of potent peptide-based agonists, antagonists, biased analogs and bivalent ligands that target NOP receptor.

A method for identifying G protein-coupled receptor dimers and their interfaces

The G protein-coupled receptor (GPCR) dimer interface plays an important role in the formation and stabilization of the dimer. Therefore, identifying the potential receptor-receptor interface is an important part of studying GPCRs. Various strategies have been employed to study the GPCR dimer interface and explore its functional significance, but experimental methods lack robustness and calculations are laborious. Herein, we report a combined optimized experimental and calculation approach for identifying and structurally characterizing GPCR dimer interfaces, and constructing atomic resolution models. Using a transmembrane domain (TM) peptide containing a human immunodeficiency virus trans-acting transcriptional activator (HIV-TAT) protein transduction motif, matrix-assisted laser desorption tandem time-of-flight mass spectrometry (MALDITOF-MS), and bioluminescence resonance energy transfer (BRET), we successfully identified Apelin receptor (APJ)/Nociceptin receptor 1 (ORL1) and APJ/Vasopressin receptor 2 (V2R) heterodimer interfaces, and two key sites mediating dimerization. This method can identify dimer interfaces of GPCR homodimers and heterodimers.

Alternative Splicing of Opioid Receptor Genes Shows a Conserved Pattern for 6TM Receptor Variants

The opioid receptor (OPR) family comprises the mu-, delta-, and kappa-opioid, and nociceptin receptors that belong to the superfamily of 7-transmembrane spanning G protein-coupled receptors (GPCRs). The mu-opioid receptor is the main target for clinically used opioid analgesics, and its biology has been extensively studied. The N-terminally truncated 6TM receptors isoform produced through alternative splicing of the OPRM1 gene displays unique signaling and analgesic properties, but it is unclear if other OPRs have the same ability. In this study, we have built a comprehensive map of alternative splicing events that produce 6TM receptor variants in all the OPRs and demonstrated their evolutionary conservation. We then obtained evidence for their translation through ribosomal footprint analysis. We discovered that N-terminally truncated 6TM GPCRs are rare in the human genome and OPRs are overrepresented in this group. Finally, we also observed a significant enrichment of 6TM GPCR genes among genes associated with pain, psychiatric disorders, and addiction. Understanding the biology of 6TM receptors and leveraging this knowledge for drug development should pave the way for novel therapies.

Serum Activity Against G Protein-Coupled Receptors and Severity of Orthostatic Symptoms in Postural Orthostatic Tachycardia Syndrome.

BackgroundPostural orthostatic tachycardia syndrome (POTS) is characterized by excessive heart rate increase on standing and orthostatic intolerance. Previous data indicate autoimmune involvement. We studied serum activity against G protein–coupled receptors in relation to symptoms in patients with POTS and controls using a commercial cell‐based assay.Methods and ResultsForty‐eight patients with POTS (aged 28.6±10.5 years; 44 women) and 25 healthy individuals (aged 30.7±8.6 years; 21 women) were included. The 10‐item Orthostatic Hypotension Questionnaire (OHQ) was completed by 33 patients with POTS and all controls. Human embryonic kidney 293 cells overexpressing one G protein–coupled receptor: adrenergic α1 receptor, adrenergic β2 receptor, cholinergic muscarinic type 2 receptor, and opioid receptor‐like 1 were treated with sera from all patients. Receptor response was analyzed using a β‐arrestin–linked transcription factor driving transgenic β‐lactamase transcription by fluorescence resonance energy transfer method. Receiver operating characteristic curves were constructed. G protein–coupled receptor activation was related to OHQ indices in linear regression models. Sera from patients with POTS activated all 4 receptors to a higher degree compared with controls (P<0.01 for all). The area under the curve was 0.88 (0.80–0.97, P<0.001) combining all 4 receptors. Adrenergic α1 receptor activation associated with OHQ composite score (β=0.77 OHQ points per SD of activity, P=0.009) and with reduced tolerability for prolonged standing (P=0.037) and walking for short (P=0.042) or long (P=0.001) periods. All 4 receptors were associated with vision problems (P<0.05 for all).ConclusionsOur results indicate the presence of circulating proteins activating adrenergic, muscarinic, and nociceptin receptors in patients with POTS. Serum‐mediated activation of these receptors has high predictive value for POTS. Activation of adrenergic α1 receptor is associated with orthostatic symptoms severity in patients with POTS.

Pain, Motivation, Migraine, and the Microbiome: New Frontiers for Opioid Systems and Disease.

For decades the broad role of opioids in addiction, neuropsychiatric disorders, and pain states has been somewhat well established. However, in recent years, with the rise of technological advances, not only is the existing dogma being challenged, but we are identifying new disease areas in which opioids play a critical role. This review highlights four new areas of exploration in the opioid field. The most recent addition to the opioid family, the nociceptin receptor system, shows promise as the missing link in understanding the neurocircuitry of motivation. It is well known that activation of the kappa opioid receptor system modulates negative affect and dysphoria, but recent studies now implicate the kappa opioid system in the modulation of negative affect associated with pain. Opioids are critical in pain management; however, the often-forgotten delta opioid receptor system has been identified as a novel therapeutic target for headache disorders and migraine. Lastly, changes to the gut microbiome have been shown to directly contribute to many of the symptoms of chronic opioid use and opioid related behaviors. This review summarizes the findings from each of these areas with an emphasis on identifying new therapeutic targets. SIGNIFICANCE STATEMENT: The focus of this minireview is to highlight new disease areas or new aspects of disease in which opioids have been implicated; this includes pain, motivation, migraine, and the microbiome. In some cases, this has resulted in the pursuit of a novel therapeutic target and resultant clinical trial. We believe this is very timely and will be a refreshing take on reading about opioids and disease.

The Rewarding and Anxiolytic Properties of Ethanol within the Central Nucleus of the Amygdala: Mediated by Genetic Background and Nociceptin.

In humans, alcohol is consumed for its rewarding and anxiolytic effects. The central nucleus of the amygdala (CeA) is considered a neuronal nexus that regulates fear, anxiety, and drug self-administration. Manipulations of the CeA alter ethanol (EtOH) consumption under numerous EtOH self-administration models. The experiments determined whether EtOH is reinforcing/anxiolytic within the CeA, whether selective breeding for high alcohol consumption alters the rewarding properties of EtOH in the CeA, and whether the reinforcing/anxiolytic effects of EtOH in the CeA are mediated by the neuropeptides corticotropin-releasing factor (CRF) and nociceptin. The reinforcing properties of EtOH were determined by having male Wistar and Taconic alcohol-preferring (tP) rats self-administer EtOH directly into the CeA. The expression of anxiety-like behaviors was assessed through multiple behavioral models (social interaction, acoustic startle, and open field). Coadministration of EtOH and a CRF1 antagonist (NBI35965) or nociceptin on self-administration into the CeA and anxiety-like behaviors was determined. EtOH was self-administered directly into the lateral CeA, and tP rats self-administered a lower concentration of EtOH than Wistar rats. EtOH microinjected into the lateral CeA reduced the expression of anxiety-like behaviors, indicating an anxiolytic effect. Coadministration of NBI35965 failed to alter the rewarding/anxiolytic properties of EtOH in the CeA. In contrast, coadministration of the nociceptin enhanced both EtOH reward and anxiolysis in the CeA. Overall, the data indicate that the lateral CeA is a key anatomic location that mediates the rewarding and anxiolytic effects of EtOH, and local nociceptin receptors, but not local CRF1 receptors, are involved in these behaviors. SIGNIFICANCE STATEMENT: Alcohol is consumed for the stimulatory, rewarding, and anxiolytic properties of the drug of abuse. The current data are the first to establish that alcohol is reinforcing and anxiolytic within the lateral central nucleus of the amygdala (CeA) and that the nociceptin system regulates these effects of alcohol within the CeA.

Opioid Receptor Regulation of Neuronal Voltage-Gated Calcium Channels.

Neuronal voltage-gated calcium channels play a pivotal role in the conversion of electrical signals into calcium entry into nerve endings that is required for the release of neurotransmitters. They are under the control of a number of cellular signaling pathways that serve to fine tune synaptic activities, including G-protein coupled receptors (GPCRs) and the opioid system. Besides modulating channel activity via activation of second messengers, GPCRs also physically associate with calcium channels to regulate their function and expression at the plasma membrane. In this mini review, we discuss the mechanisms by which calcium channels are regulated by classical opioid and nociceptin receptors. We highlight the importance of this regulation in the control of neuronal functions and their implication in the development of disease conditions. Finally, we present recent literature concerning the use of novel μ-opioid receptor/nociceptin receptor modulators and discuss their use as potential drug candidates for the treatment of pain.

Imaging Nociceptin Receptor Expression in First Episode Psychosis (FEP) Using Positron Emission Tomography With the Novel Ligand [11C] NOP-1A

Imaging Nociceptin Receptor Expression in First Episode Psychosis (FEP) Using Positron Emission Tomography With the Novel Ligand [11C] NOP-1A

Functional nociceptin receptors are upregulated on human umbilical vein endothelial cells during experimental in vitro sepsis

Functional nociceptin receptors are upregulated on human umbilical vein endothelial cells during experimental in vitro sepsis

The nociceptin/orphanin FQ receptor system as a target to alleviate cancer-induced bone pain in rats: Model validation and pharmacological evaluation.

Background and PurposeCancer‐induced bone pain remains inadequately controlled, and current standard of care analgesics is accompanied by several side effects. Nociceptin/orphanin FQ peptide (NOP) receptor agonists have demonstrated broad analgesic properties in rodent neuropathic and inflammatory pain models. Here, we investigate the analgesic potential of NOP receptor activation in a rodent cancer‐induced bone pain model.Experimental ApproachModel validation by intratibial inoculation in male Sprague Dawley rats was performed with varying MRMT‐1/Luc2 cell quantities (0.5–1.5 × 106·ml−1) and a behavioural battery (>14 days post‐surgery) including evoked and non‐evoked readouts: paw pressure test, cold plate, von Frey, open field, and weight distribution. Anti‐allodynic potential of the endogenous NOP receptor ligand nociceptin (i.t.) and NOP receptor agonist Ro65‐6570 ( i.p.) was tested using von Frey filaments, followed by a combination experiment with Ro65‐6570 and the NOP receptor antagonist J‐113397 (i.p.). Plasma cytokine levels and NOP receptor gene expression in dorsal root ganglion (DRG, L4‐L6) and bone marrow were examined.Key ResultsInoculation with 1.5 × 106·ml−1 of MRMT‐1/Luc2 cells resulted in a robust and progressive pain‐related phenotype. Nociceptin and Ro65‐6570 treatment inhibited cancer‐induced mechanical allodynia. J‐113397 selectively antagonized the effect of Ro65‐6570. MRMT‐1/Luc2‐bearing animals demonstrated elevated plasma cytokine levels of IL‐4, IL‐5, IL‐6 and IL‐10 plus unaltered NOP‐r gene expression in DRG and reduced expression in bone marrow.Conclusion and ImplicationsNociceptin and Ro65‐6570 selectively and dose‐dependently reversed cancer‐induced bone pain‐like behaviour. The NOP receptor system may be a potential target for cancer‐induced bone pain treatment.LINKED ARTICLESThis article is part of a themed issue on The molecular pharmacology of bone and cancer‐elated bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc

Discovery and Structure–Activity Relationships of Nociceptin Receptor Partial Agonists That Afford Symptom Ablation in Parkinson’s Disease Models

A novel series of C(3)-substituted piperdinylindoles were developed as nociceptin opioid receptor (NOP) partial agonists to explore a pharmacological hypothesis that NOP partial agonists would afford a dual pharmacological action of attenuating Parkinson's disease (PD) motor symptoms and development of levodopa-induced dyskinesias. SAR around the C-3 substituents investigated effects on NOP binding, intrinsic activity, and selectivity and showed that while the C(3)-substituted indoles are selective, high affinity NOP ligands, the steric, polar, and cationic nature of the C-3 substituents affected intrinsic activity to afford partial agonists with a range of efficacies. Compounds 4, 5, and 9 with agonist efficacies between 25% and 35% significantly attenuated motor deficits in the 6-OHDA-hemilesioned rat model of PD. Further, unlike NOP antagonists, which appear to worsen dyskinesia expression, these NOP partial agonists did not attenuate or worsen dyskinesia expression. The NOP partial agonists and their SAR reported here may be useful to develop nondopaminergic treatments for PD.

Cebranopadol: A First-in-Class Nociceptin Receptor Agonist for Managing Chronic Pain.

Cebranopadol: A First-in-Class Nociceptin Receptor Agonist for Managing Chronic Pain.

Cytoprotective effect of non-opioid leu-enkephalin analogue in primary culture of pulmonary fibroblasts in oxidative stress

Aim. Analysis of the cytoprotective effect of non-opioid leu-enkephalin analogue (Phe-D-Ala-Gly-Phe-Leu-Arg) in the primary culture of pulmonary fibroblasts in conditions of oxidative stress.&#x0D; Methods. Pulmonary fibroblasts were incubated with the peptide of non-opioid leu-enkephalin analogue (Phe-D-Ala-Gly-Phe-Leu-Arg) in the concentration 0.1 μM for 6 hours. To simulate oxidative stress, 60 μM H2O2 was added to the culture medium for 2 hours. Experimental series included (1) «control»; (2) «non-opiate leu-enkephalin analogue» (the peptide was added to the culture medium 44 hours after the final passage); (3) «oxidative stress» (H2O2 was added to the culture medium 48 hours after the final passage); (4) «non-opiate leu-enkephalin analogue + oxidative stress» (the peptide and H2O2 were added to the culture medium 44 and 48 hours respectively after the final passage). In order to evaluate the generation of superoxide anion by pulmonary fibroblasts, the method of lucigenin-dependent chemiluminescence was used. Computer morphometry of the nucleo-nucleolar apparatus of fibroblasts stained with silver nitrate by the AgNOR method was used to assess the cell state: the area of fibroblast nuclei, the total nucleoli area in the nucleus, and the number of nucleoli in the nucleus were measured. These parameters correlate with the activity of anabolic processes in the cells.&#x0D; Results. The effect of H2O2 on the primary culture of pulmonary fibroblasts caused an increase of superoxide anion generation by the fibroblasts, reduction of fibroblast nuclei size, decrease of nucleoli amount and size. Pre-incubation of pulmonary fibroblasts with a non-opioid leu-enkephalin analogue reduced the H2O2-induced generation of superoxide anion, corrected changes in the nucleo-nucleolar apparatus of fibroblasts caused by oxidative stress. In our previous studies, similar effect in the same model was shown for non-selective μ/δ-opioid receptor agonist peptide sedatin (dermorphin analogue). The mechanism of cytoprotective effect of non-opioid leu-enkephalin analogue may include the affinity of this peptide to nociceptin receptors (NOR receptors) that requires further studies.&#x0D; Conclusion. The results of the study indicate a direct cytoprotective effect of the peptide Phe-D-Ala-Gly-Phe-Leu-Arg (non-opioid leu-enkephalin analogue) in oxidative stress.