Traumatic Brain Injury Induces Nociceptin/Orphanin FQ and Nociceptin Opioid Peptide Receptor Expression within 24 Hours.

Traumatic brain injury (TBI) is a major cause of mortality and disability around the world, for which no treatment has been found. Nociceptin/Orphanin FQ (N/OFQ) and the nociceptin opioid peptide (NOP) receptor are rapidly increased in response to fluid percussion, stab injury, and controlled cortical impact (CCI) TBI. TBI-induced upregulation of N/OFQ contributes to cerebrovascular impairment, increased excitotoxicity, and neurobehavioral deficits. Our objective was to identify changes in N/OFQ and NOP receptor peptide, protein, and mRNA relative to the expression of injury markers and extracellular regulated kinase (ERK) 24 h following mild (mTBI) and moderate TBI (ModTBI) in wildtype (WT) and NOP receptor-knockout (KO) rats. N/OFQ was quantified by radioimmunoassay, mRNA expression was assessed using real-time PCR and protein levels were determined by immunoblot analysis. This study revealed increased N/OFQ mRNA and peptide levels in the CSF and ipsilateral tissue of WT, but not KO, rats 24 h post-TBI; NOP receptor mRNA increased after ModTBI. Cofilin-1 activation increased in the brain tissue of WT but not KO rats, ERK activation increased in all rats following ModTBI; no changes in injury marker levels were noted in brain tissue at this time. In conclusion, this study elucidates transcriptional and translational changes in the N/OFQ-NOP receptor system relative to TBI-induced neurological deficits and initiation of signaling cascades that support the investigation of the NOP receptor as a therapeutic target for TBI.

Acute single non-sedative doses of NOP receptor agonists affect acquisition of object location memory but repeated high doses do not induce long-lasting deficits.

The Nociceptin/Orphanin FQ (N/OFQ) system has been shown to modulate various aspects of long-term memory. It is therefore important to study the effects on memory impairment by nociceptin receptor (NOP) agonists under preclinical development. In the present study, we investigated the effect of systemic injection of two small molecule selective NOP agonists, AT-202 and AT-524, in the object location memory task in male and female mice. Since high doses of NOP agonists have been shown to induce sedation, we first determined the sedative doses for the two compounds and found them to be higher in female than in male mice. We then observed that sub-sedative doses of NOP agonists administered before learning, induced memory impairment during a test session performed 24 h later. Again, female mice were less sensitive to the amnesic effects than males. On the contrary, in male mice, NOP agonists did not produce amnesia when they were injected after learning, suggesting that they do not affect the consolidation of object location memory. Finally, repeated administration of high doses of NOP agonists over 7 days did not impair long-term spatial memory. Together, our data show for the first time that NOP receptor agonists impair the acquisition of object location memory with sex-dependent potency but do not affect memory consolidation, and that repeated stimulation of the receptor does not compromise long-term episodic-like spatial memory.

Discovery and structure-activity relationships (SAR) of a novel class of 2-substituted N-piperidinyl indole-based nociceptin opioid receptor ligands.

The development of SAR around substituted N-piperidinyl indole-based nociceptin opioid receptor (NOP) ligands led to the discovery of a novel series of 2-substituted N-piperidinyl indoles that provide both selective NOP full agonists and bifunctional NOP full agonists-µ opioid (MOP) receptor partial agonists. 2-substituted N-piperidinyl indoles have improved potency at the NOP receptor and are NOP full agonists, compared to our previously reported 3-substituted N-piperidinyl indoles that are selective NOP partial agonists. SAR in this series of 2-substituted N-piperidinyl indoles shows that 2-substitution versus 3-substitution on the indole moiety affects their intrinsic activity and opioid receptor selectivity. Molecular docking of these 2-substituted N-piperidinyl indoles in an active-state NOP homology model and MOP receptor structures provides a rationale for the differences observed in the binding, functional profiles and selectivity of 2-substituted versus 3-substituted N-piperidinyl indoles.

Spotlight on Nociceptin/Orphanin FQ Receptor in the Treatment of Pain.

In our society today, pain has become a main source of strain on most individuals. It is crucial to develop novel treatments against pain while focusing on decreasing their adverse effects. Throughout the extent of development for new pain therapies, the nociceptin/orphanin FQ receptor (NOP receptor) has appeared to be an encouraging focal point. Concentrating on NOP receptor to treat chronic pain with limited range of unwanted effects serves as a suitable alternative to prototypical opioid morphine that could potentially lead to life-threatening effects caused by respiratory depression in overdose, as well as generate abuse and addiction. In addition to these harmful effects, the uprising opioid epidemic is responsible for becoming one of the most disastrous public health issues in the US. In this article, the contributing molecular and cellular structure in controlling the cellular trafficking of NOP receptor and studies that support the role of NOP receptor and its ligands in pain management are reviewed.

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.

Nociceptin/orphanin FQ peptide receptor mediates inhibition of N-type calcium currents in vestibular afferent neurons of the rat.

The vestibular system is modulated by various neuromodulators including opioid peptides. The current study was conducted to determine whether activation of nociceptin/orphanin FQ peptide (NOP) receptors modulates voltage-gated calcium currents and action potential discharge of rat vestibular afferent neurons. We performed whole cell patch-clamp recordings on cultured vestibular afferent neurons from P7-P10 Long-Evans rats. Application of nociceptin/orphanin FQ (N/OFQ), a 17-amino acid neuropeptide that is the endogenous ligand for NOP receptor, inhibits the high-voltage activated (HVA) component of the calcium current in a concentration-dependent manner with a half inhibitory concentration of 26 nM. Said inhibitory action on the calcium current is voltage-dependent, which was made clear by the fact that it was reverted in 80% by a depolarizing prepulse. Furthermore, the effect of N/OFQ was blocked by application of the specific NOP-antagonist UFP101, by preincubation with G-protein blocker pertussis toxin, and by coapplication of the specific N-type calcium-current blocker ω-conotoxin-MVIIA. N/OFQ application causes an increase in the duration and maximum rate of repolarization of action potentials. It also decreases repetitive discharge and discharge elicited by sinusoidal stimulation. These results show that in vestibular afferents, NOP receptor activation inhibits N-type calcium current by activating G proteins, mostly through the Gßγ subunit. This suggests that NOP activation produces a presynaptic modulation of primary vestibular afferent neurons' output into the vestibular nuclei, thus taking part in the integration and gain setting of vestibular information in second-order vestibular nucleus neurons.NEW & NOTEWORTHY Our results show that in primary vestibular afferent neurons, activation of the nociceptin/orphanin FQ peptide receptor inhibits the N-type calcium current by a mechanism mediated by G proteins. We propose that calcium current inhibition modulates neurotransmitter release from vestibular afferents, producing a presynaptic modulation of vestibular input to vestibular nuclei, thus contributing to gain control in the vestibular afferent input.

Nociceptin impairs acquisition of novel object recognition memory in perirhinal cortex.

Nociceptin/Orphanin FQ (N/OFQ) plays an important role in the regulation of spatial, fear and recognition memories. N/OFQ receptors are highly distributed in the perirhinal cortex, which is a key brain area involved in modulating novel object recognition (NOR) memory. However, the role of N/OFQ in NOR memory in the perirhinal cortex was still unknown. Moreover, the effects of N/OFQ on different stages of NOR memory were still unclear. In NOR task, we found that pre-training intracerebroventricular (icv) injection of N/OFQ (0.3 and 1 nmol) impaired long-term memory in a dose-dependent manner. However, icv infusion of N/OFQ immediately after training did not affect NOR memory consolidation even at a high dose of 3 nmol. Pre-test icv injection of N/OFQ (1 nmol) also did not influence NOR memory retrieval. These data indicate that N/OFQ negatively modulates long-term NOR memory during the acquisition phase. Furthermore, the amnesia effect of N/OFQ (1 nmol, icv) could be antagonist by pre-treatment with the selective N/OFQ receptor antagonist [Nphe1]N/OFQ(1-13)NH2 (10 nmol, icv), indicating pharmacological specificity. Then, we found that pre-training infusion of N/OFQ (0.1 and 0.3 nmol/side) into the bilateral perirhinal cortex impaired long-term NOR memory, suggesting the perirhinal cortex is a critical brain structure in mediating the amnesic effect of N/OFQ in NOR task. In conclusion, our data, for the first time, indicate that N/OFQ in the perirhinal cortex impairs NOR memory acquisition through the NOP receptors.

NOP Receptor Antagonists Decrease Alcohol Drinking in the Dark in C57BL/6J Mice.

BACKGROUND: The nociceptin/orphanin FQ opioid peptide (NOP) receptor and its endogenous ligand N/OFQ have been implicated in the regulation of drug and alcohol use disorders (AUD). In particular, evidence demonstrated that NOP receptor activation blocks reinforcing and motivating effects of alcohol across a range of behavioral measures, including alcohol intake, conditioned place preference, and vulnerability to relapse. METHODS: Here, we show the effects of pharmacological activation and inhibition of NOP receptors on binge-like alcohol consumption, as measured by the "drinking in the dark" (DID) model in C57BL/6J mice. RESULTS: We found that 2 potent and selective NOP agonists AT-202 (0, 0.3, 1, 3 mg/kg) and AT-312 (0, 0.3, 1 mg/kg) did not affect binge alcohol drinking at doses that do not affect locomotor activity. AT-202 also failed to alter DID behavior when administered to mice previously exposed to chronic alcohol treatment with an alcohol-containing liquid diet. Conversely, treatment with either the high affinity NOP receptor antagonist SB-612111 (0, 3, 10, 30 mg/kg) or the selective antagonist LY2817412 (0, 3, 10, 30 mg/kg) decreased binge drinking. SB-612111 was effective at all doses examined, and LY2817412 was effective at 30 mg/kg. Consistently, NOP receptor knockout mice consumed less alcohol compared to wild type. SB-612111 reduced DID and increased sucrose consumption at doses that do not appear to affect locomotor activity. However, the high dose of SB-612111 (30 mg/kg) reduced alcohol intake but failed to inhibit preference in a 2-bottle choice DID model that can assess moderate alcohol intake. CONCLUSIONS: The present results suggest that NOP receptor inhibition rather than activation may represent a valuable approach for treatment of AUD characterized by excessive alcohol consumption such as binge drinking.

NOP Receptor Signaling Cascades.

The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor is a G protein-coupled receptor with wide distribution throughout the peripheral and central nervous system. Similar to other opioid receptors, NOP receptors couple to intracellular second messengers and regulatory proteins to affect biological systems. In this chapter, we review the current literature for NOP signaling cascades including their role as classic GPCRs, the investigation of their kinase and arrestin signaling pathways, and the importance of examining biased signaling to critically evaluate the therapeutic potential of novel NOP agonists.

Nociceptin/Orphanin FQ and Urinary Bladder.

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.

Pharmacological Assays for Investigating the NOP Receptor.

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/ß-arrestin interaction, label-free assays such as dynamic mass redistribution, and bioassays such as the electrically stimulated mouse vas deferens.

NOP-Related Mechanisms in Substance Use Disorders.

Nociceptin/orphanin FQ (N/OFQ) is a 17 amino acid peptide that was deorphanized in 1995 and has been widely studied since. The role of the N/OFQ system in drug abuse has attracted researchers' attention since its initial discovery. The first two scientific papers describing the effect of intracranial injection of N/OFQ appeared 20 years ago and reported efficacy of the peptide in attenuating alcohol intake, whereas heroin self-administration was insensitive. Since then more than 100 scientific articles investigating the role of the N/OFQ and N/OFQ receptor (NOP) system in drug abuse have been published. The present article provides an historical overview of the advances in the field with focus on three major elements. First, the most robust data supportive of the efficacy of NOP agonists in treating drug abuse come from studies in the field of alcohol research, followed by psychostimulant and opioid research. In contrast, activation of NOP appears to facilitate nicotine consumption. Second, emerging data challenge the assumption that activation of NOP is the most appropriate strategy to attenuate consumption of substances of abuse. This assumption is based first on the observation that animals carrying an overexpression of NOP system components are more prone to consume substances of abuse, whereas NOP knockout rats are less motivated to self-administer heroin, alcohol, and cocaine. Third, administration of NOP antagonists also reduces alcohol consumption. In addition, NOP blockade reduces nicotine self-administration. Hypothetical mechanisms explaining this apparent paradox are discussed. Finally, we focus on the possibility that co-activation of NOP and mu opioid (MOP) receptors is an alternative strategy, readily testable in the clinic, to reduce the consumption of psychostimulants, opiates, and, possibly, alcohol.

NOP-Related Mechanisms in Pain and Analgesia.

Since the discovery of the NOP receptor and N/OFQ as the endogenous ligand, evidence has appeared demonstrating the involvement of this receptor system in pain. This was not surprising for members of the opioid receptor and peptide families, particularly since both the receptor and N/OFQ are highly expressed in brain regions involved in pain, spinal cord, and dorsal root ganglia. What has been surprising is the complicated picture that has emerged from 25 years of research. The original finding that N/OFQ decreased tail flick and hotplate latency, when administered i.c.v., led to the hypothesis that NOP receptor antagonists could have analgesic activity without abuse liability. However, as data accumulated, it became clear that not only the potency but the activity per se was different when N/OFQ or small molecule NOP agonists were administered in the brain versus the spinal cord and it also depended upon the pain assay used. When administered systemically, NOP receptor agonists are generally ineffective in attenuating heat pain but are antinociceptive in an acute inflammatory pain model. Most antagonists administered systemically have no antinociceptive activity of their own, even though selective peptide NOP antagonists have potent antinociceptive activity when administered i.c.v. Chronic pain models provide different results as well, as small molecule NOP receptor agonists have potent anti-allodynic and anti-hyperalgesic activity after systemic administration. A considerable number of electrophysiological and anatomical experiments, in particular with NOP-eGFP mice, have been conducted in an attempt to explain the complicated profile resulting from NOP receptor modulation, to examine receptor plasticity, and to elucidate mechanisms by which selective NOP agonists, bifunctional NOP/mu agonists, or NOP receptor antagonists modulate acute and chronic pain.

N/OFQ-NOP System in Food Intake.

While lifestyle modifications should be the first-line actions in preventing and treating obesity and eating disorders, pharmacotherapy also provides a necessary tool for the management of these diseases.However, given the limitations of current anti-obesity drugs, innovative treatments that improve efficacy and safety are needed.Since the discovery that the activation of the Nociceptin/Orphanin (N/OFQ) FQ peptide (NOP) receptor by N/OFQ induces an increase of food intake in laboratory animals, and the finding that this effect can be blocked by NOP antagonists, many NOP agonists and antagonists have been synthesized and tested in vitro and in vivo for their potential regulation of feeding behavior. Promising results seem to suggest that the N/OFQergic system may be a potential therapeutic target for the neural control of feeding behavior and related pathologies, especially in binge-like eating behavior.

Therapeutic Approaches for NOP Receptor Antagonists in Neurobehavioral Disorders: Clinical Studies in Major Depressive Disorder and Alcohol Use Disorder with BTRX-246040 (LY2940094).

Conventional antidepressants increase the efflux of biogenic amine neurotransmitters (the monoamine hypothesis of depression) in the central nervous system (CNS) and are the principle drugs used to treat major depressive disorder (MDD). However, the lack of efficacy in some patients, the slow onset of action, and the side effect profiles of existing antidepressants necessitate the exploration of additional treatment options. The discovery of the nociceptin/orphanin FQ peptide NOP receptor (N/OFQ-NOP receptor) system and its characterization in preclinical biological and pharmacological stress-related conditions supports the potential antidepressant and anti-stress properties of a NOP receptor antagonist for the treatment of neurobehavioral disorders. BTRX-246040 (formerly LY2940094) was designed to test this hypothesis in the clinic. A small clinical proof of concept study demonstrated efficacy of BTRX-246040 in MDD patients. In this study, BTRX-246040 (40 mg, p.o.) significantly reduced negative bias as assessed by the facial recognition test within 1 week of treatment and decreased depression symptoms after 8 weeks. BTRX-246040 also reduced depression symptoms in a second trial with heavy alcohol drinkers. Given the comorbidity of MDD and alcohol use disorder, a compound with such effects in patients could be a valuable addition to the medications available. A proof of concept study showed efficacy of BTRX-246040 in reducing heavy drinking and increasing the probability of abstinence in individuals diagnosed with alcohol dependence. In addition, plasma levels of gamma-glutamyl transferase were decreased by BTRX-246040 compared to placebo control implying improvement in liver function. Collectively, the clinical data reviewed within this chapter suggest that BTRX-264040 functions to normalize dysfunction in reward circuits. The overall efficacy and safety of this compound with a novel mechanism of action are encouraging of further clinical development. BTRX-246040 is currently under development for MDD by BlackThorn Therapeutics.

Regulation of the Genes Encoding the ppN/OFQ and NOP Receptor.

Over the years, the ability of N/OFQ-NOP receptor system in modulating several physiological functions, including the release of neurotransmitters, anxiety-like behavior responses, modulation of the reward circuitry, inflammatory signaling, nociception, and motor function, has been examined in several brain regions and at spinal level. This chapter collects information related to the genes encoding the ppN/OFQ and NOP receptor, their regulation, and relative transcriptional control mechanisms. Furthermore, genetic manipulations, polymorphisms, and epigenetic alterations associated with different pathological conditions are discussed. The evidence here collected indicates that the study of ppN/OFQ and NOP receptor gene expression may offer novel opportunities in the field of personalized therapies and highlights this system as a good "druggable target" for different pathological conditions.

N/OFQ-NOP System in Peripheral and Central Immunomodulation.

Classical opioids (µ: mu, MOP; δ: delta, DOP and κ: kappa, KOP) variably affect immune function; they are immune depressants and there is good clinical evidence in the periphery. In addition, there is evidence for a central role in the control of a number of neuropathologies, e.g., neuropathic pain. Nociceptin/Orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor, NOP; peripheral and central activation can modulate immune function. In the periphery, NOP activation generally depresses immune function, but unlike classical opioids this is in part driven by NOP located on circulating immune cells. Peripheral activation has important implications in pathologies like asthma and sepsis. NOP is expressed on central neurones and glia where activation can modulate glial function. Microglia, as resident central 'macrophages', increase/infiltrate in pain and following trauma; these changes can be reduced by N/OFQ. Moreover, the interaction with other glial cell types such as the ubiquitous astrocytes and their known cross talk with microglia open a wealth of possibilities for central immunomodulation. At the whole animal level, clinical ligands with wide central and peripheral distribution have the potential to modulate immune function, and defining the precise nature of that interaction is important in mitigating or even harnessing the adverse effect profile of these important drugs.

A novel orvinol analog, BU08028, as a safe opioid analgesic without abuse liability in primates.

Despite the critical need, no previous research has substantiated safe opioid analgesics without abuse liability in primates. Recent advances in medicinal chemistry have led to the development of ligands with mixed mu opioid peptide (MOP)/nociceptin-orphanin FQ peptide (NOP) receptor agonist activity to achieve this objective. BU08028 is a novel orvinol analog that displays a similar binding profile to buprenorphine with improved affinity and efficacy at NOP receptors. The aim of this preclinical study was to establish the functional profile of BU08028 in monkeys using clinically used MOP receptor agonists for side-by-side comparisons in various well-honed behavioral and physiological assays. Systemic BU08028 (0.001-0.01 mg/kg) produced potent long-lasting (i.e., >24 h) antinociceptive and antiallodynic effects, which were blocked by MOP or NOP receptor antagonists. More importantly, the reinforcing strength of BU08028 was significantly lower than that of cocaine, remifentanil, or buprenorphine in monkeys responding under a progressive-ratio schedule of drug self-administration. Unlike MOP receptor agonists, BU08028 at antinociceptive doses and ∼10- to 30-fold higher doses did not cause respiratory depression or cardiovascular adverse events as measured by telemetry devices. After repeated administration, the monkeys developed acute physical dependence on morphine, as manifested by precipitated withdrawal signs, such as increased respiratory rate, heart rate, and blood pressure. In contrast, monkeys did not show physical dependence on BU08028. These in vivo findings in primates not only document the efficacy and tolerability profile of bifunctional MOP/NOP receptor agonists, but also provide a means of translating such ligands into therapies as safe and potentially abuse-free opioid analgesics.

"In silico" study of the binding of two novel antagonists to the nociceptin receptor.

Antagonists of the nociceptin receptor (NOP) are raising interest for their possible clinical use as antidepressant drugs. Recently, the structure of NOP in complex with some piperidine-based antagonists has been revealed by X-ray crystallography. In this study, a multi-flexible docking (MF-docking) procedure, i.e. docking to multiple receptor conformations extracted by preliminary molecular dynamics trajectories, together with hybrid quantum mechanics/molecular mechanics (QM/MM) simulations have been carried out to provide the binding mode of two novel NOP antagonists, one of them selective (BTRX-246040, formerly named LY-2940094) and one non selective (AT-076), i.e. able to inactivate NOP as well as the classical µ- k- and δ-opioid receptors (MOP KOP and DOP). According to our results, the pivotal role of residue D1303,32 (upper indexes are Ballesteros-Weinstein notations) is analogous to that enlighten by the already known X-ray structures of opioid receptors: binding of the molecules are predicted to require a slight readjustment of the hydrophobic pocket (residues Y1313,33, M1343,36, I2195,43, Q2806,52 and V2836,55) in the orthosteric site of NOP, accommodating either the pyridine-pyrazole (BTRX-246040) or the isoquinoline (AT-076) moiety of the ligand, in turn allowing the protonated piperidine nitrogen to maximize interaction (salt-bridge) with residue D1303,32 of the NOP, and the aromatic head to be sandwiched in optimal π-stacking between Y1313,33 and M1343,36. The QM/MM optimization after the MF-docking procedure has provided the more likely conformations for the binding to the NOP receptor of BTRX-246040 and AT-076, based on different pharmacophores and exhibiting different selectivity profiles. While the high selectivity for NOP of BTRX-246040 can be explained by interactions with NOP specific residues, the lack of selectivity of AT-076 could be associated to its ability to penetrate into the deep hydrophobic pocket of NOP, while retaining a conformation very similar to the one assumed by the antagonist JDTic into the K-opioid receptor. The proposed binding geometries fit better the binding pocket environment providing clues for experimental studies aimed to design selective or multifunctional opioid drugs.

Knock-In Mice with NOP-eGFP Receptors Identify Receptor Cellular and Regional Localization.

The nociceptin/orphanin FQ (NOP) receptor, the fourth member of the opioid receptor family, is involved in many processes common to the opioid receptors including pain and drug abuse. To better characterize receptor location and trafficking, knock-in mice were created by inserting the gene encoding enhanced green fluorescent protein (eGFP) into the NOP receptor gene (Oprl1) and producing mice expressing a functional NOP-eGFP C-terminal fusion in place of the native NOP receptor. The NOP-eGFP receptor was present in brain of homozygous knock-in animals in concentrations somewhat higher than in wild-type mice and was functional when tested for stimulation of [(35)S]GTPγS binding in vitro and in patch-clamp electrophysiology in dorsal root ganglia (DRG) neurons and hippocampal slices. Inhibition of morphine analgesia was equivalent when tested in knock-in and wild-type mice. Imaging revealed detailed neuroanatomy in brain, spinal cord, and DRG and was generally consistent with in vitro autoradiographic imaging of receptor location. Multicolor immunohistochemistry identified cells coexpressing various spinal cord and DRG cellular markers, as well as coexpression with µ-opioid receptors in DRG and brain regions. Both in tissue slices and primary cultures, the NOP-eGFP receptors appear throughout the cell body and in processes. These knock-in mice have NOP receptors that function both in vitro and in vivo and appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function. SIGNIFICANCE STATEMENT: The NOP receptor, the fourth member of the opioid receptor family, is involved in pain, drug abuse, and a number of other CNS processes. The regional and cellular distribution has been difficult to determine due to lack of validated antibodies for immunohistochemical analysis. To provide a new tool for the investigation of receptor localization, we have produced knock-in mice with a fluorescent-tagged NOP receptor in place of the native NOP receptor. These knock-in mice have NOP receptors that function both in vitro and in vivo and have provided a detailed characterization of NOP receptors in brain, spinal cord, and DRG neurons. They appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function.