@article{MTMT:31655073, title = {Impact of T161, Y318 and S363 alanine mutations on regulation of the human delta-opioid receptor (hDOPr) induced by peptidic and alkaloid agonists}, url = {https://m2.mtmt.hu/api/publication/31655073}, author = {Camacho, Elise and Marie, Nicolas and Dupas, Quentin and Martel, Caroline and Nowoczyn, Marie and Elie, Nicolas and Rochais, Christophe and Tóth, Géza and Allouche, Stephane}, doi = {10.1016/j.neuropharm.2020.108286}, journal-iso = {NEUROPHARMACOLOGY}, journal = {NEUROPHARMACOLOGY}, volume = {179}, unique-id = {31655073}, issn = {0028-3908}, abstract = {Previously, we showed a differential regulation of the human delta-opioid receptor (hDOPr) by etorphine and [D-Pen(2), D-Pen(5)] enkephalin (DPDPE). To understand the molecular basis of such differences, we introduced 3 alanine mutations at the residues T161. Y318 and S363. Both wild type (WT) and hDOPr mutants were expressed in HEK cells containing endogenous arrestins or CFP-tagged arrestin 3, then desensitization, internalization, recycling and phosphorylation were studied. In a context of endogenous arrestin expression, a major difference in DOPr desensitization was observed between agonists that was modified with the T161A mutation upon etorphine and with the S363A substitution upon DPDPE exposure. While both agonists induced a major receptor internalization, T161A and S363A impaired DOPr sequestration only for etorphine. However, similar level of S363 phosphorylation was measured between agonists. When CFP-tagged arrestin 3 was over-expressed, a similar profile of desensitization was measured for both agonists. In this context, all the 3 alanine mutations decreased etorphine-induced receptor desensitization. Using FRET, we showed similar interactions between WT hDOPr and arrestin 3 under DPDPE and etorphine stimulation which were delayed by both the Y318A and the S363A substitutions for etorphine. Finally, hDOPr recycling was qualitatively evaluated by microscopy and showed neither arrestin 3/hDOPr colocalization nor major impact of alanine mutations except for the S363A which impaired internalization and recycling for etorphine. The T161, Y318 and S363 residues of hDOPr could underlie the differential regulation promoted by DPDPE and etorphine.}, year = {2020}, eissn = {1873-7064} } @article{MTMT:3213626, title = {Characterization of [3H] oxymorphone binding sites in mouse brain: Quantitative autoradiography in opioid receptor knockout mice}, url = {https://m2.mtmt.hu/api/publication/3213626}, author = {Yoo, JH and Borsodi, Anna and Tóth, Géza and Benyhe, Sándor and Gáspár, Róbert and Matifas, A and Kieffer, BL and Metaxas, A and Kitchen, I and Bailey, A}, doi = {10.1016/j.neulet.2017.02.002}, journal-iso = {NEUROSCI LETT}, journal = {NEUROSCIENCE LETTERS}, volume = {643}, unique-id = {3213626}, issn = {0304-3940}, abstract = {Oxymorphone, one of oxycodone's metabolic products, is a potent opioid receptor agonist which is thought to contribute to the analgesic effect of its parent compound and may have high potential abuse liability. Nonetheless, the in vivo pharmacological binding profile of this drug is still unclear. This study uses mice lacking mu (MOP), kappa (KOP) or delta (DOP) opioid receptors as well as mice lacking all three opioid receptors to provide full characterisation of oxymorphone binding sites in the brain. Saturation binding studies using [H-3]oxymorphone revealed high affinity binding sites in mouse brain displaying Kd of 1.7 nM and Bmax of 147 fmol/mg. Furthermore, we performed quantitative autoradiography binding studies using [H-3]oxymorphone in mouse brain. The distribution of [H-3]oxymorphone binding sites was found to be similar to the selective MOP agonist [H-3]DAMGO in the mouse brain. [H-3]Oxymorphone binding was completely abolished across the majority of the brain regions in mice lacking MOP as well as in mice lacking all three opioid receptors. DOP and KOP knockout mice retained [H-3]oxymorphone binding sites suggesting oxymorphone may not target DOP or KOP. These results confirm that the MOP, and not the DOP or the KOP is the main high affinity binding target for oxymorphone. (C) 2017 Elsevier B.V. All rights reserved.}, keywords = {GENE; ACTIVATION; AUTORADIOGRAPHY; MOUSE; MU; Dimerization; Knockout; WITHDRAWAL; ABUSE; Extended-release; oxycodone; Opioid; BACK-PAIN; DOPAMINE TRANSPORTER BINDING; [H-3]Oxymorphone}, year = {2017}, eissn = {1872-7972}, pages = {16-21}, orcid-numbers = {Gáspár, Róbert/0000-0002-1571-7579} } @article{MTMT:3157890, title = {A NOVEL NON-OPIOID BINDING SITE FOR ENDOMORPHIN-1}, url = {https://m2.mtmt.hu/api/publication/3157890}, author = {Lengyel, Imre and Tóth, Fanni and Biyashev, Daureen and Szatmári, Ildikó and Monory, K and Tömböly, Csaba and Tóth, Géza and Benyhe, Sándor and Borsodi, Anna}, journal-iso = {J PHYSIOL PHARMACOL}, journal = {JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY}, volume = {67}, unique-id = {3157890}, issn = {0867-5910}, abstract = {Endomorphins are natural amidated opioid tetrapeptides with the following structure: Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2). Endomorphins interact selectively with the mu-opioid or MOP receptors and exhibit nanomolar or sub-nanomolar receptor binding affinities, therefore they suggested to be endogenous agonists for the mu-pioid receptors. Endomorphins mediate a number of characteristic opioid effects, such as antinociception, however there are several physiological functions in which endomorphins appear to act in a fashion that does not involve binding to and activation of the mu-opioid receptor. Our recent data indicate that a radiolabelled [H-3]endomorphin-1 with a specific radioactivity of 2.35 TBq/mmol -prepared by catalytic dehalogenation of the diiodinated peptide precursor in the presence of tritium gas-is able to bind to a second, naloxone insensitive recognition site in rat brain membranes. Binding heterogeneity, i.e., the presence of higher (K-d = 0.4 nM /B-max = 120 fmol/mg protein) and lower (K-d = 8.2 nM /B-max,, = 432 fmol/mg protein) affinity binding components is observed both in saturation binding experiments followed by Schatchard analysis, and in equilibrium competition binding studies. The signs of receptor multiplicity, e.g., curvilinear Schatchard plots or biphasic displacement curves are seen only if the nonspecific binding is measured in the presence of excess unlabeled endomorphin-1 and not in the presence of excess unlabeled naloxone. The second, lower affinity non-opioid binding site is not recognized by heterocyclic opioid alkaloid ligands, neither agonists such as morphine, nor antagonists such as naloxone. On the contrary, endomorphin-1 is displaced from its lower affinity, higher capacity binding site by several natural neuropeptides, including methionine-enkephalin-Arg-Phe, nociceptin-orphanin FQ, angiotensin and FMRF-amide. This naloxone-insensitive, consequently non-opioid binding site seems to be present in nervous tissues carrying low density or no mu-opioid receptors, such as rodent cerebellum, or brain of mu-opioid receptor deficient (MOPr-/-) transgenic or 'knock-out' (K.O.) mice. The newly described non-opioid binding component is not coupled to regulatory G-proteins, nor does it affect adenylyl cyclase enzyme activity. Taken together endomorphin-1 carries opioid and, in addition to non-opioid functions that needs to be taken into account when various effects of endomorphin-1 are evaluated in physiological or pathologic conditions.}, keywords = {CELLS; RAT-BRAIN; ADENYLYL-CYCLASE; AGONIST; RADIOLIGAND BINDING; MORPHINE; OPIATE RECEPTOR; DELTA; Rat brain; MU-OPIOID-RECEPTOR; KNOCK-OUT MICE; Tritiated endomorphin-1; Naloxone insensitive site; AFFINITY STATES; mu-opioid peptide receptor; G-protein activation}, year = {2016}, eissn = {1899-1505}, pages = {605-616}, orcid-numbers = {Szatmári, Ildikó/0000-0003-1040-145X} } @article{MTMT:3093931, title = {Synthesis and binding characteristics of [H-3]neuromedin N, a NTS2 receptor ligand}, url = {https://m2.mtmt.hu/api/publication/3093931}, author = {Tóth, Fanni and Mallareddy, Jayapelreddy and Tourwe, D and Lipkowski, AW and Bujalska-Zadrozny, M and Benyhe, Sándor and Ballet, S and Tóth, Géza and Kleczkowska, P}, doi = {10.1016/j.npep.2015.12.004}, journal-iso = {NEUROPEPTIDES}, journal = {NEUROPEPTIDES}, volume = {57}, unique-id = {3093931}, issn = {0143-4179}, abstract = {Neurotensin (NT) and its analog neuromedin N (NN) are formed by the processing of a common precursor in mammalian brain tissue and intestines. The biological effects mediated by NT and NN (e.g. analgesia, hypothermia) result from the interaction with G protein-coupled receptors. The goal of this study consisted of the synthesis and radiolabeling of NN, as well as the determination of the binding characteristics of [H-3]NN and G protein activation by the cold ligand. In homologous displacement studies a weak affinity was determined for NN, with IC50 values of 454 nM in rat brain and 425 nM in rat spinal cord membranes. In saturation binding experiments the Kd value proved to be 264.8 +/- 30.18 nM, while the B-max value corresponded to 3.8 02 pmol/mg protein in rat brain membranes. The specific binding of [H-3]NN was saturable, interacting with a single set of homogenous binding sites. In sodium sensitivity experiments, a very weak inhibitory effect of Na+ ions was observed on the binding of resulting in an IC50 of 150.6 inM. In [S-35]GTP gamma S binding experiments the E-max value was 112.3 +/- 1.4% in rat brain and 112.9 +/- 2.4% in rat spinal cord membranes and EC50 values of 0.7 nM and 0.79 nM were determined, respectively. NN showed moderate agonist activities in stimulating G proteins. The stimulatory effect of NN could be maximally inhibited via use of the NTS2 receptor antagonist levocabastine, but not by the opioid receptor specific antagonist naloxone, nor by the NTS1 antagonist SR48692. These observations allow us to conclude that [H-3]NN labels NTS2 receptors in rat brain membranes. (C) 2015 Elsevier Ltd. All rights reserved.}, keywords = {SITES; INACTIVATION; ANTAGONIST; RAT-BRAIN; CLONING; SENSITIVITY; POTENTIATION; aspartate residue; NEUROMEDIN-N; NEUROTENSIN-LIKE PEPTIDE; NTS2; Receptor binding assays; Neuromedin N; rat}, year = {2016}, eissn = {1532-2785}, pages = {15-20} } @article{MTMT:2915571, title = {Transfer of opiorphin through a blood-brain barrier culture model}, url = {https://m2.mtmt.hu/api/publication/2915571}, author = {Bocsik, Alexandra and Darula, Zsuzsanna and Tóth, Géza and Deli, Mária Anna and Wollemann, Mária}, doi = {10.1016/j.arcmed.2015.06.009}, journal-iso = {ARCH MED RES}, journal = {ARCHIVES OF MEDICAL RESEARCH}, volume = {46}, unique-id = {2915571}, issn = {0188-4409}, year = {2015}, eissn = {1873-5487}, pages = {502-506}, orcid-numbers = {Deli, Mária Anna/0000-0001-6084-6524} } @article{MTMT:2882389, title = {Synthesis and Pharmacological Evaluation of [H-3]HS665, a Novel, Highly Selective Radioligand for the Kappa Opioid Receptor}, url = {https://m2.mtmt.hu/api/publication/2882389}, author = {Guerrieri, E and Mallareddy, Jayapelreddy and Tóth, Géza and Schmidhammer, H and Spetea, M}, doi = {10.1021/cn5002792}, journal-iso = {ACS CHEM NEUROSCI}, journal = {ACS CHEMICAL NEUROSCIENCE}, volume = {6}, unique-id = {2882389}, issn = {1948-7193}, abstract = {Herein we report the radiolabeling and pharmacological investigation of a novel radioligand, the N-cyclobutylmethyl substituted diphenethylamine [H-3]HS665, designed to bind selectively to the kappa opioid peptide (KOP) receptor, a target of therapeutic interest for the treatment of a variety of human disorders (i.e., pain, affective disorders, drug addiction, and psychotic disorders). HS665 was prepared in tritium-labeled form by a dehalotritiated method resulting in a specific activity of 30.65 Ci/mmol. Radioligand binding studies were performed to establish binding properties of [H-3]HS665 to the recombinant human KOP receptor in membranes from Chinese hamster ovary cells stably expressing human KOP receptors (CHOhKOP) and to the native neuronal KOP receptor in guinea pig brain membranes. Binding of [H-3]HS665 was specific and saturable in both tissue preparations. A single population of high affinity binding sites was labeled by [H-3]HS665 in membranes from CHOhKOP cells and guinea pig brain with similar equilibrium dissociation constants, K-d, 0.45 and 0.64 nM, respectively. Average receptor density of [H-3]HS665 recognition sites were 5564 and 154 fmol/mg protein in CHOhKOP cells and guinea pig brain, respectively. This study shows that the new radioligand distinguishes and labels KOP receptors specifically in neuronal and cellular systems expressing KOP receptors, making this molecule a valuable tool in probing structural and functional mechanisms governing ligand-KOP receptor interactions in both a recombinant and native in vitro setting.}, year = {2015}, eissn = {1948-7193}, pages = {456-463} } @article{MTMT:2844779, title = {In vitro pharmacological evaluation of the radiolabeled C-terminal substance P analogue Lys-Phe-Phe-Gly-Leu-Met-NH2: Does a specific binding site exist?}, url = {https://m2.mtmt.hu/api/publication/2844779}, author = {Tomczyszyn, A and Csibrány, Balázs and Keresztes, Attila and Mallareddy, Jayapelreddy and Dyniewicz, J and Misicka, A and Tóth, Géza and Lipkowski, AW}, doi = {10.5114/fn.2014.47839}, journal-iso = {FOLIA NEUROPATHOL}, journal = {FOLIA NEUROPATHOLOGICA}, volume = {52}, unique-id = {2844779}, issn = {1641-4640}, abstract = {In the present paper, we report the synthesis, radiolabeling and comprehensive pharmacological evaluation of a C-terminally truncated tachykinin derivative, H-3-KFFGLM-NH2. The C-terminal fragments of endogenous tachykinins are pharmacophores responsible for interaction with the tachykinin receptors NK1, NK2 and NK3. The N-terminal fragments are responsible for modulation of receptor selectivity and interactions with other receptor systems. To evaluate and separate the function of an NK-pharmacophore from the activity of its parent neurokinin, KFFGLM-NH2 was synthesized in both tritiated and unlabeled forms. It has been proposed that the obtained NK-binding profiles of specific reference ligands and KFFGLM-NH2 differentiate monomeric and dimeric forms of NK receptors. We hypothesize that dimers of NK receptors could be specific receptor(s) for C-terminal fragments of all neurokinins as well as their C-terminal fragments, including H-KFFGLM-NH2. Dissociation of dimers into monomers opens access to additional allosteric binding sites. Fully elongated undecapeptide substance P interacts with both the "tachykinin pocket" and the "allosteric pocket" on the monomeric NK1 receptor, resulting in high and selective activation. However C-terminal hexapeptide fragment analogues, recognizing only the "tachykinin pocket", may have less specific interactions with all tachykinin receptors in both monomeric and dimeric forms.}, year = {2014}, eissn = {1509-572X}, pages = {383-393} } @article{MTMT:2774715, title = {Direct high-performance liquid chromatographic enantioseparation of secondary amino acids on Cinchona alkaloid-based chiral zwitterionic stationary phases. Unusual temperature behavior}, url = {https://m2.mtmt.hu/api/publication/2774715}, author = {Ilisz, István and Gecse, Zsanett and Pataj, Zoltán and Fülöp, Ferenc and Tóth, Géza and Lindner, W and Péter, Antal}, doi = {10.1016/j.chroma.2014.06.087}, journal-iso = {J CHROMATOGR A}, journal = {JOURNAL OF CHROMATOGRAPHY A}, volume = {1363}, unique-id = {2774715}, issn = {0021-9673}, abstract = {Two chiral stationary phases containing a quinine- or a quinidine-based zwitterionic ion-exchanger as chiral selector were applied for the enantioseparation of 27 unusual cyclic secondary alpha-amino acids. The effects of the nature and concentration of the bulk solvent, the acid and base additives, the structures of the analytes and temperature on the enantioresolution were investigated. To study the effects of temperature and to obtain thermodynamic parameters, experiments were carried out at constant mobile phase compositions in the temperature range -5 to 55 degrees C. The thermodynamic parameters indicated that in most cases the separations were enthalpy-driven, but some entropy-driven separations were also observed. The sequence of elution of the enantiomers was determined in most cases. (C) 2014 Elsevier B.V. All rights reserved.}, year = {2014}, eissn = {1873-3778}, pages = {169-177}, orcid-numbers = {Ilisz, István/0000-0001-8282-457X; Fülöp, Ferenc/0000-0003-1066-5287} } @article{MTMT:2731289, title = {Effects of synthetic analogues of human opiorphin on rat brain opioid receptors.}, url = {https://m2.mtmt.hu/api/publication/2731289}, author = {Benyhe, Z and Tóth, Géza and Wollemann, Mária and Borsodi, Anna and Helyes, Zsuzsanna and Rougeot, C and Benyhe, Sándor}, journal-iso = {J PHYSIOL PHARMACOL}, journal = {JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY}, volume = {65}, unique-id = {2731289}, issn = {0867-5910}, abstract = {Human opiorphin (Gln-Arg-Phe-Ser-Arg; QRFSR-peptide) is a physiological inhibitor of enkephalin-inactivating peptidases. We previously demonstrated that opiorphin can substitute for the classic mixture of peptidase inhibitors and greatly improves the specific binding and affinity of the enkephalin-related peptide [(3)H]MERF (Tyr-Gly-Gly-Phe-Met-Arg-Phe; YGGFMRF) for rat brain opioid receptors. To extend the metabolic stability of opiorphin in human plasma two functional derivatives were designed, i.e., Cys-[(CH2)6]-QRF-[Ser-O-octanoyl]-R peptide (monomeric CC6-opiorphin) and its cystine-dipeptide (dimeric CC6-opiorphin) derivative. We found that, in homologous competition experiments, the affinity of [(3)H]MERF for rat brain opioid receptors was significantly increased in the presence of monomeric and dimeric CC6-opiorphin, compared to control-Tris buffer. In addition ten times lower concentrations (5 muM) than those required for native opiorphin (50 muM) were sufficient. In heterologous competition experiments, using unlabeled dynorphin(1-10), affinity increases were also observed: increases in binding were similar with either monomeric or dimeric CC6-opiorphin. Surprisingly, these opiorphin analogues displayed weak competitive effects on [(3)H]MERF binding to rat brain opioid receptors in the absence of unlabeled MERF, effects never observed for the native opiorphin. In conclusion, CC6-opiorphin compounds are certainly more potent than the native opiorphin in increasing the binding and the affinity of homologous and heterologous competition, but the binding enhancement occurs only at temperatures much higher than 0 degrees C, specifically at 24 degrees C.}, year = {2014}, eissn = {1899-1505}, pages = {525-530} } @article{MTMT:2597721, title = {Knockout subtraction autoradiography: A novel ex vivo method to detect heteromers finds sparse KOP receptor/DOP receptor heterodimerization in the brain}, url = {https://m2.mtmt.hu/api/publication/2597721}, author = {Yoo, JH and Bailey, A and Borsodi, Anna and Tóth, Géza and Matifas, A and Kieffer, BL and Kitchen, I}, doi = {10.1016/j.ejphar.2014.03.007}, journal-iso = {EUR J PHARMACOL}, journal = {EUROPEAN JOURNAL OF PHARMACOLOGY}, volume = {731}, unique-id = {2597721}, issn = {0014-2999}, abstract = {Several methodological approaches suggest that receptor heteromers exist in cell systems, but their presence in physiological tissue is widely contentious. We describe a novel method to determine if heterodimers exist in brain tissue sections using autoradiographic binding comparisons from single and double gene knockout mice, where tissues either have a full receptor complement and can form heterodimers, or are incapable of making heterodimers. We have tested this model, which we have named Knockout Subtraction Autoradiography, to determine if heterodimerisation of the kappa (KOP) and delta opioid (DOP) receptors occurs, as evidence from binding studies in cell systems suggest they are present in the brain. Using labeling of putative KOP receptor/DOP receptor heterodimers with either [H-3]bremazocine or with [H-3]naltrindole, two ligands which were used to provide evidence suggesting that these opioid receptor subtypes heterodimerize, we have applied a subtraction equation model based on the principle that receptor gene double knockout of either MOP receptor/KOP receptor (DOP receptor expression only) or MOP receptor/DOP receptor (KOP receptor expression only) produces tissue incapable of making the KOP receptor/DOP receptor heterodimer. We have shown in most brain regions that the labeling fits a simple additive model of monomer labeling, but that in a few brain regions opioid receptor heterodimerization does occur. The data does not support the conclusion that KOP receptor/DOP receptor heterodimerisation is widespread in the central nervous system, but does indicate that this novel methodology can detect heterodimerisation, when ligands with distinct binding affinities for monomer and heterodimer forms exist. (C) 2014 Elsevier B.V. All rights reserved.}, year = {2014}, eissn = {1879-0712}, pages = {1-7} }