Nociceptin Receptor Agonist Research Articles

SmartHomeCage: a platform for automation of rodent behavior monitoring at homecages

We have developed a SmartHomeCage system that allow for insertion of normal rodent cages to monitor animal's behaviors in its homecage with bedding, food and water for days. Each apparatus is comprised of multiple sensors and up to 16 apparatuses can be simultaneously operated by one PC. Parameters measured include sleep/wake states, locomotion (traveling distance and speed), rearing, movement patterns and rotations. For validating the system, we showed that cocaine (30 mg/kg, ip) produced an increase in activity including distance traveled and rearing. The nociceptin receptor agonist SR 14150 (10 mg/kg, ip) that was previously shown to induce hypolocomotion produced a decrease in activity. Using a dark box inserted in the normal cage, we were able to assess the anxiolytic effect of diazepam (3mg/kg, i.p.), as indicated by an increased latency of first entry to the dark box and increased the transition number between light and dark compartments. In pilot studies through collaboration with universities, we showed this system is sensitive enough to quantify mouse behavioral changes following experimental spinal injury or middle cerebral artery occlusion. Sleep/wake state automated classification using the SmartHomeCage will be further validated by comparing with simultaneous EEG recordings. This higher throughput behavioral monitoring system for behavioral phenotype of transgenic and gene knockout mice are currently underway.

Synthesis and Pharmacological Evaluation of 1,2-Dihydrospiro[isoquinoline-4(3H),4′-piperidin]-3-ones as Nociceptin Receptor Agonists

Some synthesized 1,2-dihydrospiro[isoquinoline-4(3 H),4'-piperidin]-3-ones were evaluated as ligands for nociceptin receptor (NOP receptor). Their affinity was established by binding studies, and efficacy was investigated by GTP binding experiments. Selectivity toward DOP, KOP, and MOP receptors was assessed, and structural requirements affecting affinity and selectivity were remarked. Most notably, compound 6d displayed nanomolar NOP receptor affinity and showed more than 800-fold selectivity. The new structures exerted full or partial agonistic activity.

Endogenous Nociceptin (Orphanin FQ) Suppresses Basal Hedonic State and Acute Reward Responses to Methamphetamine and Ethanol, but Facilitates Chronic Responses

The opioid peptide nociceptin (orphanin FQ) suppresses drug reward, drug self-administration, and impedes some of the processes believed to underlie the transition to addiction. As virtually all previous studies have used administration of nociceptin receptor agonists to evaluate the role of nociceptin on addiction-like behavior, the current study used a pharmacological (nociceptin receptor antagonist) and genetic (nociceptin receptor knockout mice) approach to elucidate the role of endogenous nociceptin. The nociceptin receptor antagonist UFP-101 induced a modest place preference, and enhanced the conditioned place preference induced by methamphetamine. In agreement with this, nociceptin receptor knockout mice had slightly enhanced methamphetamine and ethanol conditioned place preferences compared to wild-type mice. This effect did not appear to depend on differences in learning ability, as nociceptin receptor knockout mice had slightly weaker-conditioned place aversions to lithium chloride, the kappa-opioid receptor agonist, U50488H, and the general opiate antagonist, naloxone. The development of behavioral sensitization to methamphetamine was lower in nociceptin receptor knockout mice, and attenuated by UFP-101 administration to wild-type mice. Additionally, ethanol consumption and preference in a two-bottle choice test was lower in nociceptin receptor knockout mice, though ethanol-stimulated locomotion was stronger. Whereas the rewarding effect of methamphetamine and ethanol following chronic treatment, as measured by place conditioning, strengthened in wild-type mice, this effect was absent in nociceptin receptor knockout mice. These results suggest that endogenous N/OFQ suppresses basal and drug-stimulated increases in hedonic state, and plays either a permissive or facilitatory role in the development of addiction.

Small-molecule agonists and antagonists of the opioid receptor-like receptor (ORL1, NOP): Ligand-based analysis of structural factors influencing intrinsic activity at NOP

The recently discovered fourth member of the opioid receptor family, the nociceptin receptor (NOP) and its endogenous ligand, the heptadecapeptide nociceptin, are involved in several central nervous system pathways, such as nociception, reward, tolerance, and feeding. The discovery of small-molecule ligands for NOP is being actively pursued for several therapeutic applications. This review presents a brief overview of the several recently reported NOP ligands, classified as NOP agonists and antagonists, with an emphasis on the analysis of the structural features that may be important for modulating the agonist/antagonist profile (intrinsic activity) of these ligands. Structure-activity relationships in our own series of dihydroindolinone-based NOP ligands and those of the various reported ligands indicate that the lipophilic substituent on the common basic nitrogen present in all NOP ligands plays a role in determining the agonist/antagonist profile of the NOP ligand. This analysis provides a basis for the rational drug design of NOP ligands of desired intrinsic activity and provides a framework for developing pharmacophore models for high affinity binding and intrinsic activity at the NOP receptor. Since NOP agonists and antagonists both have therapeutic value, rational approaches for obtaining both within a high-affinity binding class of compounds are very useful for designing potent and selective NOP ligands with the desired profile of intrinsic efficacy.

Assessment of the activity of a novel nociceptin/orphanin FQ analogue at recombinant human nociceptin/orphanin FQ receptors expressed in Chinese hamster ovary cells

The neuropeptide nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the nociceptin receptor (NOP). In an attempt to identify high potency NOP agonists for use in the brain we have compared the activity of a novel N/OFQ analogue [Phe 1Ψ(CH 2-O)Gly 2]N/OFQ(1-13)NH 2 ([F/G-O]) with the existing [Phe 1Ψ(CH 2-NH)Gly 2]N/OFQ(1-13)NH 2 ([F/G]). Both peptides are modified between the first two N-terminal amino acids and are further compared with the agonist template N/OFQ(1-13)NH 2 in [ 3H]N/OFQ binding, GTPγ[ 35S] binding and cAMP inhibition studies using Chinese hamster ovary cells expressing the recombinant human NOP. All peptides displaced [ 3H]N/OFQ, stimulated GTPγ[ 35S] binding and inhibited cAMP formation. In [ 3H]N/OFQ binding and GTPγ[ 35S] binding the rank order affinity and potency was N/OFQ(1-13)NH 2>[F/G-O]>[F/G]. In GTPγ[ 35S] binding [F/G] was a clear partial agonist with intrinsic activity ( E max stimulation factor, mean±SEM, n=4) of 7.75±1.02 compared with N/OFQ(1-13)NH 2 of 11.13±1.76. The efficacy of [F/G-O] (10.17±1.88) approached that of the full agonist N/OFQ(1-13)NH 2. Downstream, at the level of cAMP formation, all peptides were full agonists with the following rank order potency: N/OFQ(1-13)NH 2>[F/G-O]=[F/G]. The enhanced potency and intrinsic activity of the novel [F/G-O] modification makes this an interesting peptide for further in vivo analysis.

Characterisation of the non–peptide nociceptin receptor agonist, Ro64-6198 in Chinese hamster ovary cells expressing recombinant human nociceptin receptors

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the opioid receptor-like receptor or nociceptin receptor (NOP). We have compared a novel non–peptide NOP agonist Ro64-6198 with N/OFQ in a series of GTPγ 35S binding and inhibition of forskolin stimulated cAMP formation assays. GTPγ 35S binding assays were performed in membranes prepared from Chinese hamster ovary cells expressing the recombinant human NOP (CHO hNOP). cAMP inhibition studies were performed in whole CHO hNOP cells. Both Ro64-6198 and N/OFQ stimulated GTPγ 35S binding with pEC 50 values (95%CL) of 7.61 (0.18) and 8.58 (0.21) respectively. Both Ro64-6198 and N/OFQ inhibited cAMP formation with pEC 50 values of 8.45 (0.09) and 9.28 (0.28) respectively. In each assay Ro64-6198 and N/OFQ were full agonists. Ro64-6198 stimulation of GTPγ 35S binding and inhibition of cAMP formation was competitively antagonised by the NOP antagonists [Nphe 1]NC(1–13)NH 2 (10μM), J-113397 (100nM) and III-BTD (1μM) with pK B values of 7.04 (0.34) and 6.29 (0.10), 8.65 (0.34) and 7.90 (0.30) and 7.59 (0.22) and 7.60 (0.22) respectively. Despite the slightly reduced potency of Ro64-6198 compared with N/OFQ, by virtue of high selectivity and relative metabolic stability this molecule will be of considerable use in studies of the actions of the NOP.

(8-Naphthalen-1-ylmethyl-4-oxo-1-phenyl-1,3,8-triaza-spiro[4. 5]dec-3-yl)-acetic acid methyl ester (NNC 63-0532) is a novel potent nociceptin receptor agonist.

Spiroxatrine was identified as a moderately potent (K:(i)=118 nM) but non-selective agonist at the human nociceptin/orphanin FQ receptor, ORL1. This compound was subject to chemical modification and one of the resulting compounds, (8-naphthalen-1-ylmethyl-4-oxo-1-phenyl-1,3,8-triaza-s piro[4. 5]dec-3-yl)-acetic acid methyl ester (NNC 63-0532) was shown to have high affinity for ORL1 (K:(i)=7.3 nM). NNC 63-0532 showed only moderate affinity for the following receptors (K:(i) values in parentheses): mu-opioid (140 nM), kappa-opioid (405 nM), dopamine D(2S) (209 nM), dopamine D(3) (133 nM) and dopamine D(4.4) (107 nM) out of 75 different receptors, ion-channels and transporters. In functional assays, NNC 63-0532 was shown to be an agonist at ORL1 (EC(50)=305 nM), a much weaker agonist at the mu-opioid receptor (EC(50)>10 microM) and an antagonist or weak partial agonist at dopamine D(2S) (IC(50)=2830 nM). Thus, NNC 63-0532 is a novel non-peptide agonist with approximately 12 fold selectivity for ORL1 and may be useful for exploring the physiological roles of this receptor owing to its brain-penetrating properties.

Opioid activity profiles indicate similarities between the nociceptin/orphanin FQ and opioid receptors

Nociceptin (orphanin FQ) is the recently discovered peptide agonist for the orphan receptor opioid receptor-like 1 (ORL1). Despite the high sequence homology between ORL1 and the opioid receptors, most opioids lack affinity for the nociceptin receptor. The affinity and functional profile of opioids possessing activity at the nociceptin receptor was determined using [ 3H]nociceptin and nociceptin-stimulated [ 35S]GTPγS binding. The μ-opioid receptor-selective agonist lofentanil potently and competitively displaced [ 3H]nociceptin at rat brain receptors (IC 50 62 nM). Lofentanil exhibited full agonism for enhancement of [ 35S]GTPγS binding to human recombinant ORL1 receptors (EC 50 50 nM). The related piperidines ohmefentanyl and sufentanil and the nonselective opioid receptor agonist etorphine were less potent nociceptin receptor agonists. The κ 1+κ 3-opioid receptor agonist/μ-opioid receptor antagonist naloxone benzoylhydrazone was a pure antagonist at both rat brain and human ORL1 receptors. The nonselective opioid receptor partial agonist buprenorphine and the nonselective opioid receptor antagonist (−)-quadazocine exhibited pure antagonism at rat brain receptors, but displayed partial agonism at human ORL1 receptors. Thus, opioids displaying full agonism at the nociceptin receptor are also opioid receptor agonists, whereas opioids that are antagonists or partial agonists at the nociceptin receptor show antagonism or partial agonism at opioid receptors. In addition, the stereospecificity required at opioid receptors appears to be retained at the nociceptin receptor, since (+)-quadazocine is inactive at both receptors. These findings illustrate the structural and functional homology of the opioid recognition site on these two receptor classes and suggest that opioids may provide leads for the design of nonpeptide nociceptin receptor agonists and antagonists lacking affinity for the classical opioid receptors.

Nociceptin receptor activation inhibits tachykinergic non adrenergic non cholinergic contraction of guinea pig isolated bronchus

we studied the action of nociceptin (NC) on the atropine-resistant contractions of the guinea pig isolated bronchus evoked by the electrical field stimulation (EFS), an effect that is mediated by the activation of excitatory non adrenergic-non cholinergic (eNANC) nerves and the subsequent release of tachykinins. The functional site by which NC acts in this preparation was investigated using few different NC receptor agonists and the newly discovered NC receptor antagonist, [Phe 1ψ(CH 2-NH)Gly 2]NC(1–13)NH 2 ([ F G ]NC(1–13)NH 2). NC inhibited in a concentration dependent manner (pEC 50 7.14; E max − 87 ± 3% of control values) EPS induced contractions. NC effect was mimicked by the NC analogues, NCNH 2 and NC(1–13)NH 2, but not by NC(1–9)NH 2 NC (1 μM) did not affect the contractile effects of exogenously applied neurokinin A (1 μM). [ F G ]NC(1–13)NH 2 (10 μM) completely prevented the inhibition induced by NC (1 μM), whereas naloxone (1 μM) was found inactive. Both naloxone and ([ F G ]NC(1–13)NH 2 were per se inactive on basal resting tone as well as on the electrically induced contractions. The present findings show that NC inhibits the atropine-resistant EFS-induced contraction in the guinea pig bronchus by inhibiting eNANC nerves, and suggest the presence of NC receptors, distinct from opioid receptors, on the nerves of the guinea pig bronchus.