Journal: Neuropsychopharmacology

Article Title: Genetic Deletion of the Nociceptin/Orphanin FQ Receptor in the Rat Confers Resilience to the Development of Drug Addiction

doi: 10.1038/npp.2016.171

Figure Lengend Snippet: (a) Effect of the selective NOP receptor antagonist SB-612111 (0.0, 3.0, and 30.0 mg/kg) on 10% ethanol self-administration in NOP (−/− n=8) in black columns and in Wt rats (n=7) shown in white columns. (b) Effect of the selective NOP receptor antagonist LY2817412 (0.0, 3.0, and 10.0 mg/kg) on 10% ethanol self-administration in NOP (−/−) and in Wt rats. Results are expressed as mean±SEM of numbers of rewards (0.1 ml of 10% ethanol) acquired by animals. Differences between Wt and NOP (−/−) rats. ###P<0.001; ##P<0.01. Differences between vehicles and drug-treated groups. **P<0.01 and *P<0.05.

Article Snippet: NOP receptor (−/−) rats were in-licenced from Genoway (Lion, France) and were bred at the University of Camerino.

Techniques:

Journal: Neuropharmacology

Article Title: Pharmacological blockage of NOP receptors decreases ventral tegmental area dopamine neuronal activity through GABA B receptor-mediated mechanism

doi: 10.1016/j.neuropharm.2024.109866

Figure Lengend Snippet: (A-E) The graphs show representative and averaged traces of the spontaneous firing from putative VTA DA neuron before, during, and after LY2817412 (1 μM) slice treatment in (A) absence, and in presence of (B) picrotoxin (100 μM) + CGP55845 (1 μM), (C) picrotoxin (100 μM), (D) CGP55845 (1 μM), or (E) naloxone (10 μM). Scale bar: 1 s. (F) Bar graphs represent the averaged maximal effects of LY2817412 on the firing rate of VTA DA neurons in the same experimental conditions. *** p < 0.001, ### p < 0.001.

Article Snippet: Drugs The NOP receptor antagonist LY2817412 was synthesized and kindly provided by Eli Lilly (IN, USA; [ 66 ]).

Techniques:

Journal: Neuropharmacology

Article Title: Pharmacological blockage of NOP receptors decreases ventral tegmental area dopamine neuronal activity through GABA B receptor-mediated mechanism

doi: 10.1016/j.neuropharm.2024.109866

Figure Lengend Snippet: (A) Representative traces of spontaneous firing activity recorded in cell-attached configuration from a putative VTA DA neuron before (Baseline), during (LY2817412 1 μM), and after (Wash out) LY2817412 slice perfusion. Scale bar: 2 s. (B) The bar graph shows the temporal changes of firing rate (Hz) of the same representative putative VTA DA cell before, during and after LY2817412 slice perfusion. (C) The graph reports the concentration-dependent effect of LY2817412 on putative VTA DA neuronal firing rate. Note that, except for the 10−2 nM concentration, only data from LY2817412-responding cells are reported in this graph.

Article Snippet: Drugs The NOP receptor antagonist LY2817412 was synthesized and kindly provided by Eli Lilly (IN, USA; [ 66 ]).

Techniques: Activity Assay, Concentration Assay

Journal: Neuropharmacology

Article Title: Pharmacological blockage of NOP receptors decreases ventral tegmental area dopamine neuronal activity through GABA B receptor-mediated mechanism

doi: 10.1016/j.neuropharm.2024.109866

Figure Lengend Snippet: (A) Representative sIPSCs traces recorded from putative VTA DA neurons before (Baseline), during LY2817412 (1 μM), and during picrotoxin (100 μM) slice perfusion. Scale bars: 50 pA × 2 s. (B-C) Bar graphs showing the effects of LY2817412 (1 μM) on frequency (D) and amplitude (E) of sIPSCs. (D). Representative mIPSCs traces recorded from putative VTA DA neurons before (vehicle), during TTX (1 μM), TTX + LY2817412 (1 μM), and during TTX + LY2817412 + picrotoxin (100 μM) slice perfusion. Scale bars: 50 pA × 2 s. (E-F) Bar graphs showing the effects of LY2817412 (1 μM) on frequency (D) and amplitude (E) of mIPSCs.

Article Snippet: Drugs The NOP receptor antagonist LY2817412 was synthesized and kindly provided by Eli Lilly (IN, USA; [ 66 ]).

Techniques:

Journal: Science signaling

Article Title: Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists

doi: 10.1126/scisignal.aau8072

Figure Lengend Snippet: (A) Schematic representation of the human nociceptin/orphanin FQ (hNOP) receptor. All potential intracellular phosphate acceptor sites are indicated (gray). Ser346, Ser351 and Thr362/Ser363 were targeted for the generation of phosphosite-specific antibodies and the epitope used for generating a phosphorylation-independent antibody (NOPR) is indicated by a black line. (B) HEK293 cells were transiently transfected with either the wild-type hNOP receptor or its 6S/T-A mutant. After 48 hours, cells were labeled with 200 μCi/ml carrier-free [32P]orthophosphate. Labeled cells were either not treated (–) or treated (+) to 10 μM N/OFQ for 10 min, and whole-cell receptor phosphorylation was determined by SDS-polyacrylamide gel electrophoresis followed by autoradiography. (C) Dot-blot analysis on serial dilutions of peptides P1-P4 to characterize antibody to pThr362/pSer363. (D and E) Characterization of phosphosite-specific antibodies directed against Ser346, Ser351 or Thr362/Ser363 using λ-phosphatase. HEK293 cells stably expressing the HA-tagged hNOP receptor were either not treated (–) or treated (+) with 10 μM N/OFQ for 10 min. Lysates were then either incubated (+) or not (–) with λ-phosphatase and immunoblotted with the phosphosite-specific antibodies to pSer346, pSer351, or pThr362/Ser363. Blots were stripped and reprobed with the phosphorylation-independent antibody to NOPR as a loading control. In all panels, blots are representative from one of three independent experiments. Molecular mass markers (kDA) are indicated, left.

Article Snippet: DNA for human NOP receptor and human NOP receptor mutants were generated via artificial synthesis and cloned into pcDNA3.1 by imaGenes and Eurofins, respectively.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Dot Blot, Stable Transfection, Expressing, Incubation, Control

Journal: Science signaling

Article Title: Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists

doi: 10.1126/scisignal.aau8072

Figure Lengend Snippet: (A to G) Agonist-induced hyperpolarization can be measured by changes in fluorescence intensity of fluorescent oxonol dyes. A reduction of fluorescent signal intensity is indicative of Gβγ protein-mediated GIRK channel activation. AtT20 cells stably expressing the hNOP receptor were first preloaded with the dye. Thereafter, a baseline is measured for 60 seconds before cells were stimulated with vehicle (according to the solvent) or with Ro64–6198 (A), MCOPPB (B), SCH221510 (C), NNC 63–0532 (D), AT-202 (E), BUP (F), cebranopadol (G) or N/OFQ (A to G) at a concentration range of 10−6 to 10−13 M for 180 seconds. Dose-response curves were calculated with OriginPro using sigmoidal non-linear fitting. Vehicle-induced changes in fluorescence signal (background) were subtracted from signals obtained using agonist-containing solutions. Data are mean ± SEM from three independent experiments performed in duplicate. ΔRFU is change in relative fluorescence. (H) Correlation between NOP receptor phosphorylation (pThr362/Ser363) and GIRK channel activation induced by the different ligands, color-coordinated with (A to G). Abscissae: ligand-induced G protein activation (EC50 values). Ordinates: ligand-induced phosphorylation in percentage (normalized to N/OFQ). Solid line, linear regression of the data points; correlation coefficient r = 0.6859.

Article Snippet: DNA for human NOP receptor and human NOP receptor mutants were generated via artificial synthesis and cloned into pcDNA3.1 by imaGenes and Eurofins, respectively.

Techniques: Fluorescence, Activation Assay, Stable Transfection, Expressing, Solvent, Concentration Assay, Phospho-proteomics

Journal: Science signaling

Article Title: Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists

doi: 10.1126/scisignal.aau8072

Figure Lengend Snippet: (A) Sequence of the carboxyl-terminal tail of the hNOP receptor showing all potential phosphate acceptor sites. Serine (S) and threonine (T) residues depicted in gray were exchanged to alanine. (B and C) HEK293 cells stably expressing HA-tagged hNOP, S337/346/351A, T362/S363/T365A, S363A, 6S/T-A, or NOP-10S/T-A were either not treated (–) or treated (+) with 10 μM N/OFQ for 10 min, and lysates were then immunoblotted with the antibodies to pSer346, pSer351, or pThr362/Ser363. Blots were stripped and reprobed with antibodies to NOPR or HA-tag. Blots are representative, (n=3).

Article Snippet: DNA for human NOP receptor and human NOP receptor mutants were generated via artificial synthesis and cloned into pcDNA3.1 by imaGenes and Eurofins, respectively.

Techniques: Sequencing, Stable Transfection, Expressing

Journal: Science signaling

Article Title: Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists

doi: 10.1126/scisignal.aau8072

Figure Lengend Snippet: (A and B) HEK293 cells stably expressing hNOP receptors were preincubated (+) or not (–) with 50 μM naloxone, J-113397, or SB 612111 for 30 min at 37 °C, then treated with vehicle (water; -) or with 10 μM N/OFQ (+) for 10 min at 37 °C. Cell lysates were then immunoblotted with antibodies to pSer346, pSer351, or pThr362/Ser363. Blots were stripped and reprobed for NOPR. Blots are representative, n=3. (C) HEK293 cells stably expressing the NOP receptor were preincubated with antibody to HA-tag and then treated with vehicle (DMSO), 50 μM naloxone, J-113397, or SB 612111 and with or without 10 μM N/OFQ for 60 min at 37 °C. Cells were then fixed and labeled with a peroxidase-conjugated secondary antibody, and receptor internalization was measured by ELISA and quantified as percentage of internalized receptors in agonist-treated cells. Data are means ± SEM from six independent experiments performed in quadruplicate. *P<0.05 vs. N/OFQ by two-way ANOVA with Bonferroni post-test. (D) Cells described and treated as in (C), except treated at 22 °C, were fixed, permeabilized, immunofluorescently stained, and examined using confocal microscopy. Images are representative from one of three independent experiments. Scale bar, 20 μm.

Article Snippet: DNA for human NOP receptor and human NOP receptor mutants were generated via artificial synthesis and cloned into pcDNA3.1 by imaGenes and Eurofins, respectively.

Techniques: Stable Transfection, Expressing, Labeling, Enzyme-linked Immunosorbent Assay, Staining, Confocal Microscopy

Journal: Science signaling

Article Title: Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists

doi: 10.1126/scisignal.aau8072

Figure Lengend Snippet: (A) HEK293 cells stably expressing HA-tagged hNOP receptors were preincubated with HA antibody and then stimulated with 10 μM N/OFQ, Ro64–6198, MCOPPB, SCH221510, NNC 63–0532, AT-202, buprenorphine (BUP), norbuprenorphine (norBUP), cebranopadol or vehicle (according to the solvent) for 60 min at 22 °C. Cells were fixed, permeabilized, immunofluorescently stained, and subsequently examined using confocal microscopy. Images are representative, n=3 independent experiments. Scale bar, 20 μm. (B) NOP receptor-expressing HEK293 cells were treated with the compounds listed in (A) (–, vehicle solvent) at concentrations ranging from 10−9 to 10−5 M for 10 min at 37 °C, and lysates were immunoblotted with antibodies to pSer346, pSer351, or pThr362/Ser363. Blots were stripped and reprobed for NOPR. Blots are representative of n=3–5 experiments. (C) HEK293 cells stably expressing hNOP receptors were preincubated with antibody to HA-tag and treated with vehicle (solvent) or 10 μM N/OFQ, Ro64–6198, MCOPPB, SCH221510, NNC 63–0532, AT-202, BUP, norBUP or cebranopadol for 60 min at 37 °C. Cells were fixed and labeled with a peroxidase-conjugated secondary antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells. Data are means ± SEM from twelve independent experiments performed in quadruplicate. (D) Maximum NOP receptor ligand-induced phosphorylation at Thr362/Ser363 from data in (B). Data are mean ± SEM from three independent experiments. *P<0.05 vs. N/OFQ by two-way ANOVA with Bonferroni post-test. (E) Correlation between NOP receptor phosphorylation and internalization induced by different ligands in HEK293 cells, from data in (A to D). Abscissae: ligand-induced internalization in percentage (normalized to N/OFQ). Ordinates: ligand-induced phosphorylation in percentage (normalized to N/OFQ). Solid line, linear regression of the data points; correlation coefficient r = 0.8581.

Article Snippet: DNA for human NOP receptor and human NOP receptor mutants were generated via artificial synthesis and cloned into pcDNA3.1 by imaGenes and Eurofins, respectively.

Techniques: Stable Transfection, Expressing, Solvent, Staining, Confocal Microscopy, Labeling, Enzyme-linked Immunosorbent Assay, Phospho-proteomics

Journal: Science signaling

Article Title: Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists

doi: 10.1126/scisignal.aau8072

Figure Lengend Snippet: (A) Distribution of NOP receptor in NOP-eGFP knock-in mouse tissue. Anesthetized NOP-eGFP knock-in mice were sacrificed and tissues were removed. The NOP receptor was then immunoprecipitated from homogenates with anti-GFP protein agarose beads, and samples were immunoblotted with the phosphorylation-independent antibody to NOPR or antibody to GFP. Bottom, prolonged ECL detection exposure. Blots are representative from one of three independent experiments. (B) Mass spectrometry coverage of the NOP receptor sequence from mouse brain. The schema represents the secondary structure of the mouse NOP receptor. Filled (blue and black) symbols indicate the protein sequence covered by nanoLC-MS/MS; the phosphorylated residues identified are black. Red circles indicate the trypsin cleavage sites. Pm, plasma membrane; e1-e3, extracellular loops; i1-i4, intracellular loops (C) List of phosphorylated and unphosphorylated NOP receptor peptides identified by nanoLC-MS/MS in the mouse brain. Amino acids belonging to the GFP sequence are in italics. Theo. Mass., theoretical mass (DA); MC, missed cleavage. (D and E) ETD MS/MS spectra of the monophosphorylated peptide 339-EMQVpSDRVR-347 (D; triply charged precursor ion, MH3+, at m/z 400.5128) and 359-pT/pSETVPRPAGSIATMVSK-376 (E; triply charged precursor ion, MH3+, at m/z 637.9797) display series of c- and z-ions indicating that Ser343 (D) and Thr359 or Ser360 (E) are phosphorylated, respectively. Red labels indicate site-determining ions and the corresponding peaks in the spectrum. Blue labels indicate fragment ions that confirm the site localization and exclude another potential site. pS, pT: phosphorylated serine or threonine residues.

Article Snippet: DNA for human NOP receptor and human NOP receptor mutants were generated via artificial synthesis and cloned into pcDNA3.1 by imaGenes and Eurofins, respectively.

Techniques: Knock-In, Immunoprecipitation, Phospho-proteomics, Mass Spectrometry, Sequencing, Tandem Mass Spectroscopy, Clinical Proteomics, Membrane

Journal: Science signaling

Article Title: Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists

doi: 10.1126/scisignal.aau8072

Figure Lengend Snippet: (A)Schematic representation of the human (h) and mouse (m) nociceptin/orphanin FQ receptor. All potential intracellular phosphate acceptor sites are indicated (gray). (B) After i.p. injection of 0.9% NaCl (0) or AT-202 (0.3 to 30 mg/kg) for 30 min, NOP-eGFP knock-in mice were euthanized and brains were removed. NOP receptor was immunoprecipitated with anti-GFP protein agarose beads and immunoblotted with antibodies to pSer346, pSer351, or pThr362/Ser363. Blots were stripped and reprobed for NOPR or GFP. Blots are representative, n=3. (C) As in (B), but mice were treated with SB 612111 or AT-202 singly or pretreated with SB 612111 for 30 min followed by AT-202 (each 30 mg/kg). (D) After intracerebroventricular injection of compound 101 (0.3 to 30 nmol), NOP-eGFP knock-in mice were treated with AT-202 (30 mg/kg for 30 min), euthanized and brains were removed. Homogenates underwent immunoprecipitation with anti-GFP agarose beads, and the resulting samples were immunoblotted for phosphorylated (pThr362/Ser363) NOP receptor. Blots were stripped and reprobed with the GFP antibody as a loading control. Blots are representative, n=3. (E) As in (B), but mice were treated with 0.9% NaCl (–) or 30 mg/kg AT-202, Ro64–6198, NNC 63–0532, SCH221510 or MCOPPB for 30 min. (F and G) Imaging (F) and analysis (G) of NOP receptor internalization in ventral midbrain neurons. Primary cultures from NOPR-eYFP mice were treated for 1 hour with 1 μM of the indicated agonist, followed by live cell spinning disk confocal imaging. Images are representative, and data are means ± SEM of over 40 images from at least two dishes per condition.

Article Snippet: DNA for human NOP receptor and human NOP receptor mutants were generated via artificial synthesis and cloned into pcDNA3.1 by imaGenes and Eurofins, respectively.

Techniques: Injection, Knock-In, Immunoprecipitation, Control, Imaging

Journal: Science signaling

Article Title: Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists

doi: 10.1126/scisignal.aau8072

Figure Lengend Snippet: NOP receptor peptide sequences used for generation of phosphosite-specific antisera. List of peptide sequences used for generating phosphosite-specific antibodies against individual phosphorylated forms of the NOP receptor and a phosphorylation-independent antiserum targeting the NOP receptor at the end of the carboxyl-terminal domain. Endogenous cysteine were exchanged (abu).

Article Snippet: DNA for human NOP receptor and human NOP receptor mutants were generated via artificial synthesis and cloned into pcDNA3.1 by imaGenes and Eurofins, respectively.

Techniques: Phospho-proteomics, Sequencing

Journal:

Article Title: [Nphe 1 ,Arg 14 ,Lys 15 ]Nociceptin-NH 2 , a novel potent and selective antagonist of the nociceptin/orphanin FQ receptor

doi: 10.1038/sj.bjp.0704706

Figure Lengend Snippet: Receptor binding profile of UFP-101 to recombinant DOP, KOP, MOP, and NOP receptors expressed in CHO cells

Article Snippet: We would like to thank Dr F. Marshall and Mrs N. Bevan of Glaxo-Wellcome, Stevenage, Herts U.K. for providing CHO cells expressing the human NOP receptor, Dr D.K.

Techniques: Binding Assay, Recombinant

Journal: Rheumatology (Oxford, England)

Article Title: What is new in pain modification in osteoarthritis?

doi: 10.1093/rheumatology/kex522

Figure Lengend Snippet: Completed randomized, double-blind, placebo-controlled clinical trials for OA pain drugs

Article Snippet: There are several ongoing or recently completed clinical trials with small molecules targeting these molecules for OA pain ( ). table ft1 table-wrap mode="anchored" t5 T able 3 caption a7 Target Drug Trial ID Results GPCRs Bradykinin (BK) B2 receptor Fasitibant {"type":"clinical-trial","attrs":{"text":"NCT02205814","term_id":"NCT02205814"}} NCT02205814 No particular dose different from placebo (WOMAC A) [ 42 ] Delta opioid receptor ADL5859 {"type":"clinical-trial","attrs":{"text":"NCT00979953","term_id":"NCT00979953"}} NCT00979953 No difference from placebo [ 43 ] Delta opioid receptor ADL5747 {"type":"clinical-trial","attrs":{"text":"NCT00979953","term_id":"NCT00979953"}} NCT00979953 No difference from placebo [ 43 ] Kappa opioid receptor CR845 {"type":"clinical-trial","attrs":{"text":"NCT02944448","term_id":"NCT02944448"}} NCT02944448 Positive results for hip OA [ 44 ] Nociceptin/orphanin FQ peptide (NOP) and mu opioid receptors GRT6005 (cebranopadol) {"type":"clinical-trial","attrs":{"text":"NCT01709214","term_id":"NCT01709214"}} NCT01709214 No results; Depomed appears to be continuing development [ 45 ] CB2 {"type":"entrez-nucleotide","attrs":{"text":"GW842166","term_id":"295324833","term_text":"GW842166"}} GW842166 {"type":"clinical-trial","attrs":{"text":"NCT00479427","term_id":"NCT00479427"}} NCT00479427 No results Ion channels Na V 1.7 and other sodium channels TV-45070 {"type":"clinical-trial","attrs":{"text":"NCT02068599","term_id":"NCT02068599"}} NCT02068599 No difference from placebo [ 46 ] Na V 1.8 VX-150 {"type":"clinical-trial","attrs":{"text":"NCT02660424","term_id":"NCT02660424"}} NCT02660424 Positive results [ 47 ] TRPV1 CNTX-4975 {"type":"clinical-trial","attrs":{"text":"NCT02558439","term_id":"NCT02558439"}} NCT02558439 Significant improvement vs placebo in WOMAC A1 scores at week 12 [ 48 ] TRPV1 NEO6860 {"type":"clinical-trial","attrs":{"text":"NCT02712957","term_id":"NCT02712957"}} NCT02712957 Preliminary data suggests an analgesic effect compared with placebo [ 49 ] Open in a separate window GPCRs: G-protein coupled receptors; TRPV1: Transient vanilloid receptor 1.

Techniques: