Endomorphin-2: a biased agonist at the μ-opioid receptor.

PubMed

Rivero, Guadalupe; Llorente, Javier; McPherson, Jamie; Cooke, Alex; Mundell, Stuart J; McArdle, Craig A; Rosethorne, Elizabeth M; Charlton, Steven J; Krasel, Cornelius; Bailey, Christopher P; Henderson, Graeme; Kelly, Eamonn

2012-08-01

Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the μ-opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K(+) current in a concentration-dependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K(+) current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins.

Facilitatory effects of selective agonists for tachykinin receptors on cholinergic neurotransmission: evidence for species differences.

PubMed Central

Belvisi, M. G.; Patacchini, R.; Barnes, P. J.; Maggi, C. A.

1994-01-01

1. Exogenous tachykinins modulate cholinergic neurotransmission in rabbit and guinea-pig airways. We have investigated the effect of selective tachykinin receptor agonists and antagonists on cholinergic neurotransmission evoked by electrical field stimulation (EFS) of bronchial rings in rabbit, guinea-pig and human airways in vitro to assess which type of tachykinin receptor is mediating this facilitatory effect. 2. Bronchial rings were set up for isometric tension recording. Contractile responses to EFS (60 V, 0.4 ms, 2 Hz for 10 s every min) and exogenous acetylcholine (ACh) were obtained and the effects of selective tachykinin agonists and antagonists were investigated. 3. In rabbit bronchi the endogenous tachykinins, substance P (SP) and neurokinin A (NKA) (10 nM) potentiated cholinergic responses to EFS (by 287.6 +/- 121%, P < 0.01 and 181.4 +/- 56.5%, P < 0.001 respectively). 4. The NK1 receptor selective agonist, [Sar9]SP sulphone (10 nM) evoked a maximal facilitatory action on cholinergic responses of 334.9 +/- 63% (P < 0.01) (pD2 = 8.5 +/- 0.06) an effect which was blocked by the selective NK1-receptor antagonist, CP 96,345 (100 nM) (P < 0.05) but not by the NK2 receptor antagonist, MEN 10,376 (100 nM). The NK2 receptor selective agonist, [beta Ala8]NKA(4-10) (10 nM), produced a maximum enhancement of 278 +/- 83.5% (P < 0.01) (pD2 = 8.7 +/- 0.1) an effect which was blocked by MEN 10,376 (100 nM) (P < 0.05) and not by CP 96,345. [MePhe7]NKB, an NK3 receptor selective agonist was without effect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7516799

Inhibition by spinal mu- and delta-opioid agonists of afferent-evoked substance P release.

PubMed

Kondo, Ichiro; Marvizon, Juan Carlos G; Song, Bingbing; Salgado, Frances; Codeluppi, Simone; Hua, Xiao-Ying; Yaksh, Tony L

2005-04-06

Opioid mu- and delta-receptors are present on the central terminals of primary afferents, where they are thought to inhibit neurotransmitter release. This mechanism may mediate analgesia produced by spinal opiates; however, when they used neurokinin 1 receptor (NK1R) internalization as an indicator of substance P release, Trafton et al. (1999) noted that this evoked internalization was altered only modestly by morphine delivered intrathecally at spinal cord segment S1-S2. We reexamined this issue by studying the effect of opiates on NK1R internalization in spinal cord slices and in vivo. In slices, NK1R internalization evoked by dorsal root stimulation at C-fiber intensity was abolished by the mu agonist [D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO) (1 microM) and decreased by the delta agonist [D-Phe2,5]-enkephalin (DPDPE) (1 microM). In vivo, hindpaw compression induced NK1R internalization in ipsilateral laminas I-II. This evoked internalization was significantly reduced by morphine (60 nmol), DAMGO (1 nmol), and DPDPE (100 nmol), but not by the kappa agonist trans-(1S,2S)-3,4-dichloro-N-mathyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride (200 nmol), delivered at spinal cord segment L2 using intrathecal catheters. These doses of the mu and delta agonists were equi-analgesic as measured by a thermal escape test. Lower doses neither produced analgesia nor inhibited NK1R internalization. In contrast, morphine delivered by percutaneous injections at S1-S2 had only a modest effect on thermal escape, even at higher doses. Morphine decreased NK1R internalization after systemic delivery, but at a dose greater than that necessary to produce equivalent analgesia. All effects were reversed by naloxone. These results indicate that lumbar opiates inhibit noxious stimuli-induced neurotransmitter release from primary afferents at doses that are confirmed behaviorally as analgesic.

Beta-methyl substitution of cyclohexylalanine in Dmt-Tic-Cha-Phe peptides results in highly potent delta opioid antagonists.

PubMed

Tóth, Géza; Ioja, Eniko; Tömböly, Csaba; Ballet, Steven; Tourwé, Dirk; Péter, Antal; Martinek, Tamás; Chung, Nga N; Schiller, Peter W; Benyhe, Sándor; Borsodi, Anna

2007-01-25

The opioid peptide TIPP (H-Tyr-Tic-Phe-Phe-OH, Tic:1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was substituted with Dmt (2',6'-dimethyltyrosine) and a new unnatural amino acid, beta-MeCha (beta-methyl-cyclohexylalanine). This double substitution led to a new series of opioid peptides displaying subnanomolar delta antagonist activity and mu agonist or antagonist properties depending on the configuration of the beta-MeCha residue. The most promising analog, H-Dmt-Tic-(2S,3S)-beta-MeCha-Phe-OH was a very selective delta antagonist both in the mouse vas deferens (MVD) assay (Ke = 0.241 +/- 0.05 nM) and in radioligand binding assay (K i delta = 0.48 +/- 0.05 nM, K i mu/K i delta = 2800). The epimeric peptide H-Dmt-Tic-(2S,3R)-beta-MeCha-Phe-OH and the corresponding peptide amide turned out to be mixed partial mu agonist/delta antagonists in the guinea pig ileum and MVD assays. Our results constitute further examples of the influence of Dmt and beta-methyl substitution as well as C-terminal amidation on the potency, selectivity, and signal transduction properties of TIPP related peptides. Some of these compounds represent valuable pharmacological tools for opioid research.

Structure-Activity Relationship Studies on a Macrocyclic Agouti-Related Protein (AGRP) Scaffold Reveal Agouti Signaling Protein (ASP) Residue Substitutions Maintain Melanocortin-4 Receptor Antagonist Potency and Result in Inverse Agonist Pharmacology at the Melanocortin-5 Receptor.

PubMed

Ericson, Mark D; Freeman, Katie T; Schnell, Sathya M; Fleming, Katlyn A; Haskell-Luevano, Carrie

2017-10-12

The melanocortin system consists of five reported receptors, agonists from the proopiomelanocortin gene transcript, and two antagonists, agouti-signaling protein (ASP) and agouti-related protein (AGRP). For both ASP and AGRP, the hypothesized Arg-Phe-Phe pharmacophores are on exposed β-hairpin loops. In this study, the Asn and Ala positions of a reported AGRP macrocyclic scaffold (c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro]) were explored with 14-compound and 8-compound libraries, respectively, to generate more potent, selective melanocortin receptor antagonists. Substituting diaminopropionic acid (Dap), DDap, and His at the Asn position yielded potent MC4R ligands, while replacing Ala with Ser maintained MC4R potency. Since these substitutions correlate to ASP loop residues, an additional Phe to Ala substitution was synthesized and observed to maintain MC4R potency. Seventeen compounds also possessed inverse agonist activity at the MC5R, the first report of this pharmacology. These findings are useful in developing molecular probes to study negative energy balance conditions and unidentified functions of the MC5R.

Adenosine-A1 Receptor Agonist Induced Hyperalgesic Priming Type II

PubMed Central

Araldi, Dioneia; Ferrari, Luiz F.; Levine, Jon D.

2016-01-01

We have recently shown that repeated exposure of the peripheral terminal of the primary afferent nociceptor to the mu-opioid receptor (MOR) agonist DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt) induces a model of the transition to chronic pain that we have termed Type II hyperalgesic priming. Similar to Type I hyperalgesic priming, there is a markedly prolonged response to subsequent administration of proalgesic cytokines, prototypically prostaglandin E2 (PGE2). However, Type II hyperalgesic priming differs from Type I in being rapidly induced, protein kinase A (PKA), rather than PKCε dependent, not reversed by a protein translation inhibitor, occurring in female as well as in male rats, and isolectin B4-negative neuron dependent. We report that as with the repeated injection of a MOR agonist, the repeated administration of an agonist at the A1-adenosine receptor, also a Gi-protein coupled receptor, N6-Cyclopentyladenosine (CPA), also produces priming similar to DAMGO-induced Type II hyperalgesic priming. In this study we demonstrate that priming induced by repeated exposure to this A1-adenosine receptor agonist shares the same mechanisms as MOR-agonist induced priming. However, the prolongation of PGE2 hyperalgesia induced by repeated administration of CPA depends on G-protein αi subunit activation, differently from DAMGO-induced Type II priming, in which it depends on the β/γ subunit. These data implicate a novel form of Gi-protein signaling pathway in the Type II hyperalgesic priming induced by repeated administration of an agonist at A1-adenosine receptor to the peripheral terminal of the nociceptor. PMID:26588695

N-terminal fatty acylated His-dPhe-Arg-Trp-NH(2) tetrapeptides: influence of fatty acid chain length on potency and selectivity at the mouse melanocortin receptors and human melanocytes.

PubMed

Todorovic, Aleksandar; Holder, Jerry Ryan; Bauzo, Rayna M; Scott, Joseph Walker; Kavanagh, Renny; Abdel-Malek, Zalfa; Haskell-Luevano, Carrie

2005-05-05

The melanocortin system is involved in the regulation of a diverse number of physiologically important pathways including pigmentation, feeding behavior, weight and energy homeostasis, inflammation, and sexual function. All the endogenous melanocortin agonist ligands possess the conserved His-Phe-Arg-Trp tetrapeptide sequence that is postulated to be important for melanocortin receptor molecular recognition and stimulation. Previous studies by our laboratory resulted in the discovery that increasing alkyl chain length at the N-terminal "capping" region of the His-dPhe-Arg-Trp-NH(2) tetrapeptide resulted in a 100-fold increased melanocortin receptor agonist potency. This study was undertaken to systematically evaluate the pharmacological effects of increasing N-capping alkyl chain length of the CH(3)(CH(2))(n)CO-His-dPhe-Arg-Trp-NH(2) (n = 6-16) tetrapeptide template. Twelve analogues were synthesized and pharmacologically characterized at the mouse melanocortin receptors MC1R and MC3R-MC5R and human melanocytes known to express the MC1R. These peptides demonstrated melanocortin receptor selectivity profiles different from those of previously published tetrapeptides. The most notable results of enhanced ligand potency (20- to 200-fold) and receptor selectivity were observed at the MC1R. Tetrapeptides that possessed greater than nine alkyl groups were superior to alpha-MSH in terms of the stimulation of human melanocyte tyrosinase activity. Additionally, the n-pentadecanoyl derivative had a residual effect on tyrosinase activity that existed for at least 4 days after the peptide was removed from the human melanocyte culture medium. These data demonstrate the utility, potency, and residual effect of melanocortin tetrapeptides by adding N-terminal fatty acid moieties.

Optical resolution of phenylthiohydantoin-amino acids by capillary electrophoresis and identification of the phenylthiohydantoin-D-amino acid residue of [D-Ala2]-methionine enkephalin.

PubMed

Kurosu, Y; Murayama, K; Shindo, N; Shisa, Y; Ishioka, N

1996-11-01

This is an initial report to propose a protein sequence analysis system with DL differentiation using capillary electrophoresis (CE). This system consists of a protein sequencer and a CE system. After fractionation of phenyl-thiohydantoin (PTH)-amino acids using a protein sequencer, optical resolution for each PTH-amino acid is performed by CE using some chiral selectors such as digitonin, beta-escin and others. As a model peptide, [D-Ala2]-methionine enkephalin (L-Tyr-D-Ala-Gly-L-Phe-L-Met), was used and the sequence with DL differentiation was determined, with the exception of the fourth amino acid, L-Phe, using our proposed system.

In vitro and in vivo activity of cyclopeptide Dmt-c[d-Lys-Phe-Asp]NH2, a mu opioid receptor agonist biased toward β-arrestin.

PubMed

Gach-Janczak, Katarzyna; Piekielna-Ciesielska, Justyna; Adamska-Bartłomiejczyk, Anna; Wtorek, Karol; Ferrari, Federica; Calo', Girolamo; Szymaszkiewicz, Agata; Piasecka-Zelga, Joanna; Janecka, Anna

2018-07-01

Morphine and related drugs, which are the most effective analgesics for the relief of severe pain, act through activating opioid receptors. The endogenous ligands of these receptors are opioid peptides which cannot be used as antinociceptive agents due to their low bioactivity and stability in biological fluids. The major goal of opioid research is to understand the mechanism of action of opioid receptor agonists in order to improve therapeutic utility of opioids. Analgesic effects of morphine are mediated mostly through activation of the mu opioid receptor. However, in the search for safer and more effective drug candidates, analogs with mixed opioid receptor profile gained a lot of interest. Recently, the concept of biased agonists able to differentially activate GPCR downstream pathways, became a new approach in the design of novel drug candidates. It is hypothesized that compounds promoting G-protein signaling may produce analgesia while β-arrestin recruitment may be responsible for opioid side effects. In this report we showed that replacement of the tyrosine residue in the mu-selective ligand Tyr-c[d-Lys-Phe-Asp]NH 2 with 2',6'-dimethyltyrosine (Dmt) produced a cyclopeptide Dmt-c[d-Lys-Phe-Asp]NH 2 with mu/delta opioid receptor agonist profile. This analog showed improved antinociception in the hot-plate test, probably due to the simultaneous activation of mu and delta receptors but also significantly inhibited the gastrointestinal transit. Using the bioluminescence resonance energy transfer (BRET) assay it was shown that this analog was a mu receptor agonist biased toward β-arrestin. β-Arrestin-dependent signaling is most likely responsible for the observed inhibition of gastrointestinal motility exerted by the novel cyclopeptide. Copyright © 2018. Published by Elsevier Inc.

Engineering an ATP-dependent D-Ala:D-Ala ligase for synthesizing amino acid amides from amino acids.

PubMed

Miki, Yuta; Okazaki, Seiji; Asano, Yasuhisa

2017-05-01

We successfully engineered a new enzyme that catalyzes the formation of D-Ala amide (D-AlaNH 2 ) from D-Ala by modifying ATP-dependent D-Ala:D-Ala ligase (EC 6.3.2.4) from Thermus thermophilus, which catalyzes the formation of D-Ala-D-Ala from two molecules of D-Ala. The new enzyme was created by the replacement of the Ser293 residue with acidic amino acids, as it was speculated to bind to the second D-Ala of D-Ala-D-Ala. In addition, a replacement of the position with Glu performed better than that with Asp with regards to specificity for D-AlaNH 2 production. The S293E variant, which was selected as the best enzyme for D-AlaNH 2 production, exhibited an optimal activity at pH 9.0 and 40 °C for D-AlaNH 2 production. The apparent K m values of this variant for D-Ala and NH 3 were 7.35 mM and 1.58 M, respectively. The S293E variant could catalyze the synthesis of 9.3 and 35.7 mM of D-AlaNH 2 from 10 and 50 mM D-Ala and 3 M NH 4 Cl with conversion yields of 93 and 71.4 %, respectively. This is the first report showing the enzymatic formation of amino acid amides from amino acids.

Fast Modulation of μ-Opioid Receptor (MOR) Recycling Is Mediated by Receptor Agonists*

PubMed Central

Roman-Vendrell, Cristina; Yu, Y. Joy; Yudowski, Guillermo Ariel

2012-01-01

The μ-opioid receptor (MOR) is a member of the G protein-coupled receptor family and the main target of endogenous opioid neuropeptides and morphine. Upon activation by ligands, MORs are rapidly internalized via clathrin-coated pits in heterologous cells and dissociated striatal neurons. After initial endocytosis, resensitized receptors recycle back to the cell surface by vesicular delivery for subsequent cycles of activation. MOR trafficking has been linked to opioid tolerance after acute exposure to agonist, but it is also involved in the resensitization process. Several studies describe the regulation and mechanism of MOR endocytosis, but little is known about the recycling of resensitized receptors to the cell surface. To study this process, we induced internalization of MOR with [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) and morphine and imaged in real time single vesicles recycling receptors to the cell surface. We determined single vesicle recycling kinetics and the number of receptors contained in them. Then we demonstrated that rapid vesicular delivery of recycling MORs to the cell surface was mediated by the actin-microtubule cytoskeleton. Recycling was also dependent on Rab4, Rab11, and the Ca2+-sensitive motor protein myosin Vb. Finally, we showed that recycling is acutely modulated by the presence of agonists and the levels of cAMP. Our work identifies a novel trafficking mechanism that increases the number of cell surface MORs during acute agonist exposure, effectively reducing the development of opioid tolerance. PMID:22378794

The novel endomorphin degradation blockers Tyr-Pro-DClPhe-Phe-NH (EMDB-1) and Tyr-Pro-Ala-NH (EMDB-2) prolong endomorphin-2 action in rat ileum in vitro.

PubMed

Fichna, Jakub; Perlikowska, Renata; Gach, Katarzyna; do-Rego, Jean-Claude; Cravezic, Aurore; Janecka, Anna; Storr, Martin A

2010-07-01

The endogenous opioid system is involved in the control of gastrointestinal (GI) motility. The potential use of endogenous MOR ligands, endomorphins (EMs), as therapeutics is limited because of their rapid enzymatic degradation and short duration of action. Targeting enzymatic degradation is an approach to prolong EM activity. In the present study, we characterized the effects of novel blockers of EM degradation in GI tissue preparation in vitro. The effects of actinonin, diprotin A (DIP) and the novel peptide EM degradation blockers Tyr-Pro-DClPhe-Phe-NH(2) (EMDB-1), Tyr-Pro-Ala-NH(2) (EMDB-2) and Tyr-Pro-Ala-OH (EMDB-3) on EM-2-mediated inhibition of electrically induced cholinergic twitch contractions were compared in rat ileum in vitro using an organ bath. EMDB-1 and EMDB-2 significantly prolonged the inhibitory effect of EM-2 on smooth muscle contractility in rat ileum. EMDB-2 extended the EM-2 action for up to 60 min compared to 10 min in controls and was more potent than the conventional peptidase inhibitor DIP. EMDB-1 and EMDB-2 are potent EM degradation blockers, which prolong the inhibitory effects of EM-2 on smooth muscle contractility in rat ileum. These novel compounds may be of future use when targeting the endogenous opioid system in the treatment of GI motility disorders such as diarrhea.

Potent and selective oxytocin receptor agonists without disulfide bridges.

PubMed

Adachi, Yusuke; Sakimura, Katsuya; Shimizu, Yuji; Nakayama, Masaharu; Terao, Yasuko; Yano, Takahiko; Asami, Taiji

2017-06-01

Oxytocin (OT) is a neuropeptide involved in a wide variety of physiological actions, both peripherally and centrally. Many human studies have revealed the potential of OT to treat autism spectrum disorders and schizophrenia. OT interacts with the OT receptor (OTR) as well as vasopressin 1a and 1b receptors (V 1a R, V 1b R) as an agonist, and agonistic activity for V 1a R and V 1b R may have a negative impact on the therapeutic effects of OTR agonism in the CNS. An OTR-selective agonistic peptide, FE 202767, in which the structural differences from OT are a sulfide bond instead of a disulfide bond, and N-alkylglycine replacement for Pro at position 7, was reported. However, the effects of amino acid substitutions in OT have not been comprehensively investigated to compare OTR, V 1a R, and V 1b R activities. This led us to obtain a new OTR-selective analog by comprehensive amino acid substitutions of OT and replacement of the disulfide bond. A systematic amino acid scanning (Ala, Leu, Phe, Ser, Glu, or Arg) of desamino OT (dOT) at positions 2, 3, 4, 5, 7, and 8 revealed the tolerability for the substitution at positions 7 and 8. Further detailed study showed that trans-4-hydroxyproline (trans-Hyp) at position 7 and γ-methylleucine [Leu(Me)] at position 8 were markedly effective for improving receptor selectivity without decreasing the potency at the OTR. Subsequently, a combination of these amino acid substitutions with the replacement of the disulfide bond of dOT analogs with a sulfide bond (carba analog) or an amide bond (lactam analog) yielded several promising analogs, including carba-1-[trans-Hyp 7 ,Leu(Me) 8 ]dOT (14) with a higher potency (7.2pM) at OTR than that of OT and marked selectivity (>10,000-fold) over V 1a R and V 1b R. Hence, we investigated comprehensive modification of OT and obtained new OT analogs that exhibited high potency at OTR with marked selectivity. These OTR-selective agonists could be useful to investigate OTR-mediated effects on

Intravitreally-administered dopamine D2-like (and D4), but not D1-like, receptor agonists reduce form-deprivation myopia in tree shrews.

PubMed

Ward, Alexander H; Siegwart, John T; Frost, Michael R; Norton, Thomas T

2017-01-01

We examined the effect of intravitreal injections of D1-like and D2-like dopamine receptor agonists and antagonists and D4 receptor drugs on form-deprivation myopia (FDM) in tree shrews, mammals closely related to primates. In eleven groups (n = 7 per group), we measured the amount of FDM produced by monocular form deprivation (FD) over an 11-day treatment period. The untreated fellow eye served as a control. Animals also received daily 5 µL intravitreal injections in the FD eye. The reference group received 0.85% NaCl vehicle. Four groups received a higher, or lower, dose of a D1-like receptor agonist (SKF38393) or antagonist (SCH23390). Four groups received a higher, or lower, dose of a D2-like receptor agonist (quinpirole) or antagonist (spiperone). Two groups received the D4 receptor agonist (PD168077) or antagonist (PD168568). Refractions were measured daily; axial component dimensions were measured on day 1 (before treatment) and day 12. We found that in groups receiving the D1-like receptor agonist or antagonist, the development of FDM and altered ocular component dimensions did not differ from the NaCl group. Groups receiving the D2-like receptor agonist or antagonist at the higher dose developed significantly less FDM and had shorter vitreous chambers than the NaCl group. The D4 receptor agonist, but not the antagonist, was nearly as effective as the D2-like agonist in reducing FDM. Thus, using intravitreally-administered agents, we did not find evidence supporting a role for the D1-like receptor pathway in reducing FDM in tree shrews. The reduction of FDM by the dopamine D2-like agonist supported a role for the D2-like receptor pathway in the control of FDM. The reduction of FDM by the D4 receptor agonist, but not the D4 antagonist, suggests an important role for activation of the dopamine D4 receptor in the control of axial elongation and refractive development.

Intravitreally-administered dopamine D2-like (and D4), but not D1-like, receptor agonists reduce form-deprivation myopia in tree shrews

PubMed Central

Ward, Alexander H.; Siegwart, John T.; Frost, Michael R.; Norton, Thomas T.

2017-01-01

We examined the effect of intravitreal injections of D1-like and D2-like dopamine receptor agonists and antagonists and D4 receptor drugs on form-deprivation myopia (FDM) in tree shrews, mammals closely related to primates. In eleven groups (n = 7 per group), we measured the amount of FDM produced by monocular form deprivation (FD) over an 11-day treatment period. The untreated fellow eye served as a control. Animals also received daily 5 μL intravitreal injections in the FD eye. The reference group received 0.85% NaCl vehicle. Four groups received a higher, or lower, dose of a D1-like receptor agonist (SKF38393) or antagonist (SCH23390). Four groups received a higher, or lower, dose of a D2-like receptor agonist (quinpirole) or antagonist (spiperone). Two groups received the D4 receptor agonist (PD168077) or antagonist (PD168568). Refractions were measured daily; axial component dimensions were measured on day 1 (before treatment) and day 12. We found that in groups receiving the D1-like receptor agonist or antagonist, the development of FDM and altered ocular component dimensions did not differ from the NaCl group. Groups receiving the D2-like receptor agonist or antagonist at the higher dose developed significantly less FDM and had shorter vitreous chambers than the NaCl group. The D4 receptor agonist, but not the antagonist, was nearly as effective as the D2-like agonist in reducing FDM. Thus, using intravitreally-administered agents, we did not find evidence supporting a role for the D1-like receptor pathway in reducing FDM in tree shrews. The reduction of FDM by the dopamine D2-like agonist supported a role for the D2-like receptor pathway in the control of FDM. The reduction of FDM by the D4 receptor agonist, but not the D4 antagonist, suggests an important role for activation of the dopamine D4 receptor in the control of axial elongation and refractive development. PMID:28304244

Characterization of Gly-D-Phe, Gly-L-Leu, and D-Phe as affinity ligands to thermolysin.

PubMed

Yasukawa, Kiyoshi; Kusano, Masayuki; Nakamura, Koji; Inouye, Kuniyo

2006-04-01

In this study, glycyl-D-phenylalanine (Gly-D-Phe), glycyl-L-leucine (Gly-L-Leu), and D-phenylalanine (D-Phe) were characterized for their abilities as affinity ligands to thermolysin. Each of the ligands was immobilized to the resin. The optimum pH for adsorption of thermolysin is 5.0-6.0 for each of the ligands. By the affinity column chromatography in which 2mg thermolysin was applied onto 4 ml volume of the resins at pH 5.5, the adsorption ratios based on casein hydrolysis activity were 100% for each of the ligands. However, the adsorption ratios of the resins containing Gly-L-Leu and D-Phe, unlike that of Gly-D-Phe, were progressively decreased with increasing the amounts of thermolysin applied to the column. Measurement of adsorption isotherms showed that the association constant to thermolysin at pH 5.5 of the resins containing Gly-D-Phe was (3.3+/-0.8)x10(5)M(-1), while those of Gly-L-Leu and D-Phe were approximately ten times less. This result is coincident with the observations of performances in affinity column chromatography. On the other hand, maximum thermolysin binding capacities were almost the same among the resins examined. These results indicate that Gly-D-Phe is more suitable than Gly-L-Leu and D-Phe as an affinity ligand for purification of thermolysin.

Identification of putative agouti-related protein(87-132)-melanocortin-4 receptor interactions by homology molecular modeling and validation using chimeric peptide ligands.

PubMed

Wilczynski, Andrzej; Wang, Xiang S; Joseph, Christine G; Xiang, Zhimin; Bauzo, Rayna M; Scott, Joseph W; Sorensen, Nicholas B; Shaw, Amanda M; Millard, William J; Richards, Nigel G; Haskell-Luevano, Carrie

2004-04-22

Agouti-related protein (AGRP) is one of only two naturally known antagonists of G-protein-coupled receptors (GPCRs) identified to date. Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these melanocortin receptors. Insight into putative interactions between the antagonist AGRP amino acids with the melanocortin-4 receptor (MC4R) may be important for the design of unique ligands for the treatment of obesity related diseases and is currently lacking in the literature. A three-dimensional homology molecular model of the mouse MC4 receptor complex with the hAGRP(87-132) ligand docked into the receptor has been developed to identify putative antagonist ligand-receptor interactions. Key putative AGRP-MC4R interactions include the Arg111 of hAGRP(87-132) interacting in a negatively charged pocket located in a cavity formed by transmembrane spanning (TM) helices 1, 2, 3, and 7, capped by the acidic first extracellular loop (EL1) and specifically with the conserved melanocortin receptor residues mMC4R Glu92 (TM2), mMC4R Asp114 (TM3), and mMC4R Asp118 (TM3). Additionally, Phe112 and Phe113 of hAGRP(87-132) putatively interact with an aromatic hydrophobic pocket formed by the mMC4 receptor residues Phe176 (TM4), Phe193 (TM5), Phe253 (TM6), and Phe254 (TM6). To validate the AGRP-mMC4R model complex presented herein from a ligand perspective, we generated nine chimeric peptide ligands based on a modified antagonist template of the hAGRP(109-118) (Tyr-c[Asp-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2)). In these chimeric ligands, the antagonist AGRP Arg-Phe-Phe residues were replaced by the melanocortin agonist His/D-Phe-Arg-Trp amino acids. These peptides resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs). The most notable results include the identification of a novel subnanomolar melanocortin peptide

Tachykinin receptors in the guinea-pig renal pelvis: activation by exogenous and endogenous tachykinins.

PubMed Central

Maggi, C. A.; Patacchini, R.; Eglezos, A.; Quartara, L.; Giuliani, S.; Giachetti, A.

1992-01-01

1. The contractile response to substance P, neurokinin A, selective agonists for the NK1, NK2 and NK3 tachykinin receptors and the activity of receptor-selective antagonists has been investigated in circular muscle strips of the guinea-pig isolated renal pelvis in the presence of indomethacin (3 microM). 2. Neurokinin A was the most potent agonist tested, being about 32 times more potent than substance P. The action of both substance P and neurokinin A was enhanced by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The selective NK2 receptor agonist [beta Ala8] neurokinin A (4-10), was slightly less potent and effective than neurokinin A itself. The selective NK1 receptor agonist [Sar9] substance P sulphone was effective at low (nM) concentrations but its maximal effect did not exceed 30% of maximal response to substance P or neurokinin A. The NK3-selective agonist [MePhe7] neurokinin B was effective only at high (microM) concentrations. 3. The pseudopeptide derivative of neurokinin A(4-10), MDL 28,564, displayed a clear-cut agonist character, although it was less potent than neurokinin A. 4. The responses to roughly equieffective (25-35% of maximal response) concentrations of [beta Ala8] neurokinin A (4-10), MDL 28,564 and [MePhe7] neurokinin B were antagonized to a similar extent by MEN 10,376 (3 microM), a selective NK2 tachykinin receptor antagonist, while the response to [Sar9] substance P sulphone was unchanged. 5. The response to [Sar9] substance P sulphone was inhibited by the NK1 receptor-selective antagonist, GR 82,334 (3 microM) while the response to [beta Ala8] neurokinin A (4-10) was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1384907

A Macrocyclic Agouti-Related Protein/[Nle4,DPhe7]α-Melanocyte Stimulating Hormone Chimeric Scaffold Produces Subnanomolar Melanocortin Receptor Ligands.

PubMed

Ericson, Mark D; Freeman, Katie T; Schnell, Sathya M; Haskell-Luevano, Carrie

2017-01-26

The melanocortin system consists of five receptor subtypes, endogenous agonists, and naturally occurring antagonists. These receptors and ligands have been implicated in numerous biological pathways including processes linked to obesity and food intake. Herein, a truncation structure-activity relationship study of chimeric agouti-related protein (AGRP)/[Nle4,DPhe7]α-melanocyte stimulating hormone (NDP-MSH) ligands is reported. The tetrapeptide His-DPhe-Arg-Trp or tripeptide DPhe-Arg-Trp replaced the Arg-Phe-Phe sequence in the AGRP active loop derivative c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro], where Xxx was the native Asn of AGRP or a diaminopropionic (Dap) acid residue previously shown to increase antagonist potency at the mMC4R. The Phe, Ala, and Dap/Asn residues were successively removed to generate a 14-member library that was assayed for agonist activity at the mouse MC1R, MC3R, MC4R, and MC5R. Two compounds possessed nanomolar agonist potency at the mMC4R, c[Pro-His-DPhe-Arg-Trp-Asn-Ala-Phe-DPro] and c[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro], and may be further developed to generate novel melanocortin probes and ligands for understanding and treating obesity.

(D-Phe/sup 12/)bombesin analogues: a new class of bombesin receptor antagonists

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heinz-Erian, P.; Coy, D.H.; Tamura, M.

1987-03-01

Previous attempts to develop analogues of bombesin that function as specific receptor antagonists have been unsuccessful. Alteration of the histidine in luteinizing hormone releasing factor has resulted in analogues that function as competitive antagonists. In the present study the authors have used a similar strategy and altered the histidine in bombesin. (D-Phe/sup 12/)bombesin, (D-Phe/sup 12/,Leu/sup 14/)bombesin, and (Try/sup 4/, D-)je/sup 12/) bombesin did not stimulate amylase release from guinea pig pancreatic acini when present alone, but each analog inhibited bombesin-stimulated secretion. For each analog, detectable inhibition occurred at 1 ..mu..M and half-maximal inhibition at 4 ..mu..M. Each analog inhibited amylasemore » release by bombesin and other agonists that stimulate secretion by interacting with bombesin receptors. The analogues of bombesin did not alter stimulation by substance P or other agonists that interact with other receptors. The inhibition of the action of bombesin was competitive with Schild plots having slopes of 1.0. Each analog also inhibited binding of /sup 125/I-labeled (Try/sup 4/) bombesin but not /sup 125/I-labeled substance P. These results demonstrate that (D-Phe/sup 12/) analogues of bombesin function as bombesin receptor antagonists and are the only bombesin receptor antagonists that interact only with the bombesin receptor. Because of their specificity, these analogues may prove useful for defining the role of bombesin in various physiological or pathological processes.« less

Bifunctional [2’,6’-Dimethyl-l-tyrosine1]Endomorphin-2 Analogues Substituted at Position 3 with Alkylated Phenylalanine Derivatives Yield Potent Mixed μ-Agonist/δ-Antagonist and Dual μ-/δ-Agonist Opioid Ligands

PubMed Central

Li, Tingyou; Shiotani, Kimitaka; Miyazaki, Anna; Tsuda, Yuko; Ambo, Akihiro; Sasaki, Yusuke; Jinsmaa, Yunden; Marczak, Ewa; Bryant, Sharon D.; Lazarus, Lawrence H.; Okada, Yoshio

2009-01-01

Endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) and [Dmt1]EM-2 (Dmt = 2’,6’-dimethyl-l-tyrosine) analogues were synthesized containing alkylated Phe3 derivatives, 2’-monomethyl (2, 2’), 3’,5’- and 2’,6’-dimethyl (3, 3’, and 4’, respectively), 2’,4’,6’-trimethyl (6, 6’), 2’-ethyl-6’-methyl (7, 7’) and 2’-isopropyl-6’-methyl (8, 8’) groups or Dmt (5, 5’). They had the following characteristics: (i) [Xaa3]EM-2 analogues improved μ- and δ-opioid receptor affinities, the latter were inconsequential (Kiδ= 491–3,451 nM); (ii) [Dmt1,Xaa3]EM-2 analogues enhanced μ- and δ-opioid receptor affinities (Kiμ = 0.069–0.32 nM; Kiδ = 1.83–99.8 nM) and lacked interaction with κ-opioid receptors, and (iii) elevated μ-bioactivity (IC50 = 0.12–14.4 nM) and abolished δ-agonism (IC50 > 10 µM; 2’, 3’, 4’, 5’, 6’); however, 4’ and 6’ exhibited mixed μ-agonism/δ-antagonism (4’: IC50μ = 0.12, pA2 = 8.15; 6’: IC50μ = 0.21 nM, pA2 = 9.05), and 7’ was a dual μ-/δ -agonist (IC50μ = 0.17 nM; IC50δ = 0.51 nM). Alteration of EM-2 activity by Dmt1 and alkylated Phe3 residues retained μ-receptor bioactivity and formed dual μ-/δ -agonists and mixed μ-agonists/δ-antagonists. PMID:17497839

Analogues of luteinizing hormone-releasing hormone containing cytotoxic groups.

PubMed

Janáky, T; Juhász, A; Bajusz, S; Csernus, V; Srkalovic, G; Bokser, L; Milovanovic, S; Redding, T W; Rékási, Z; Nagy, A

1992-02-01

In an attempt to produce better cytotoxic analogues, chemotherapeutic antineoplastic radicals including an alkylating nitrogen mustard derivative of D-phenylalanine (D-melphalan), reactive cyclopropane, anthraquinone derivatives [2-(hydroxymethyl)anthraquinone and the anticancer antibiotic doxorubicin], and an antimetabolite (methotrexate) were coupled to suitably modified agonists and antagonists of luteinizing hormone-releasing hormone (LH-RH). Analogues with D-lysine6 and D-ornithine6 or N epsilon-(2,3-diaminopropionyl)-D-lysine and N delta-(2,3-diaminopropionyl)-D-ornithine were used as carriers for one or two cytotoxic moieties. The enhanced biological activities produced by the incorporation of D amino acids into position 6 of the agonistic analogues were further increased by the attachment of hydrophobic cytotoxic groups, resulting in compounds with 10-50 times higher activity than LH-RH. Most of the monosubstituted agonistic analogues showed high affinities for the membrane receptors of human breast cancer cells, while the receptor binding affinities of peptides containing two cytotoxic side chains were lower. Antagonistic carriers [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Trp3,Arg5,D-Lys6,D-Ala10] LH-RH [where Nal(2) is 3-(2-naphthyl)alanine], [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Trp3,Arg5,N epsilon-(2,3-diaminopropionyl)-D-Lys6,D-Ala10]LH-RH, and their D-Pal(3)3 homologs [Pal(3) is 3-(3-pyridyl)alanine] as well as [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Pal(3)3,Tyr5,N epsilon-(2,3-diamino-propionyl)-D-Lys6,D-Ala10]LH-RH were linked to cytotoxic compounds. The hybrid molecules inhibited ovulation in rats at doses of 10 micrograms and suppressed LH release in vitro. The receptor binding of cytotoxic analogues was decreased compared to the precursor peptides, although analogues with 2-(hydroxymethyl)anthraquinone hemiglutarate had high affinities. All of the cytotoxic analogues tested inhibited [3H]thymidine incorporation into DNA in cultures of human breast and prostate cancer cell lines

Analogues of luteinizing hormone-releasing hormone containing cytotoxic groups.

PubMed Central

Janáky, T; Juhász, A; Bajusz, S; Csernus, V; Srkalovic, G; Bokser, L; Milovanovic, S; Redding, T W; Rékási, Z; Nagy, A

1992-01-01

In an attempt to produce better cytotoxic analogues, chemotherapeutic antineoplastic radicals including an alkylating nitrogen mustard derivative of D-phenylalanine (D-melphalan), reactive cyclopropane, anthraquinone derivatives [2-(hydroxymethyl)anthraquinone and the anticancer antibiotic doxorubicin], and an antimetabolite (methotrexate) were coupled to suitably modified agonists and antagonists of luteinizing hormone-releasing hormone (LH-RH). Analogues with D-lysine6 and D-ornithine6 or N epsilon-(2,3-diaminopropionyl)-D-lysine and N delta-(2,3-diaminopropionyl)-D-ornithine were used as carriers for one or two cytotoxic moieties. The enhanced biological activities produced by the incorporation of D amino acids into position 6 of the agonistic analogues were further increased by the attachment of hydrophobic cytotoxic groups, resulting in compounds with 10-50 times higher activity than LH-RH. Most of the monosubstituted agonistic analogues showed high affinities for the membrane receptors of human breast cancer cells, while the receptor binding affinities of peptides containing two cytotoxic side chains were lower. Antagonistic carriers [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Trp3,Arg5,D-Lys6,D-Ala10] LH-RH [where Nal(2) is 3-(2-naphthyl)alanine], [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Trp3,Arg5,N epsilon-(2,3-diaminopropionyl)-D-Lys6,D-Ala10]LH-RH, and their D-Pal(3)3 homologs [Pal(3) is 3-(3-pyridyl)alanine] as well as [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Pal(3)3,Tyr5,N epsilon-(2,3-diamino-propionyl)-D-Lys6,D-Ala10]LH-RH were linked to cytotoxic compounds. The hybrid molecules inhibited ovulation in rats at doses of 10 micrograms and suppressed LH release in vitro. The receptor binding of cytotoxic analogues was decreased compared to the precursor peptides, although analogues with 2-(hydroxymethyl)anthraquinone hemiglutarate had high affinities. All of the cytotoxic analogues tested inhibited [3H]thymidine incorporation into DNA in cultures of human breast and prostate cancer cell lines

Bacterial resistance to vancomycin: overproduction, purification, and characterization of VanC2 from Enterococcus casseliflavus as a D-Ala-D-Ser ligase.

PubMed

Park, I S; Lin, C H; Walsh, C T

1997-09-16

The VanC phenotype for clinical resistance of enterococci to vancomycin is exhibited by Enterococcus gallinarum and Enterococcus casseliflavus. Based on the detection of the cell precursor UDP-N-acetylmuramic acid pentapeptide intermediate terminating in D-Ala-D-Ser instead of D-Ala-D-Ala, it has been predicted that the VanC ligase would be a D-Ala-D-Ser rather than a D-Ala-D-Ala ligase. Overproduction of the E. casseliflavus ATCC 25788 vanC2 gene in Escherichia coli and its purification to homogeneity allowed demonstration of ATP-dependent D-Ala-D-Ser ligase activity. The kcat/Km2 (Km2 = Km for D-Ser or C-terminal D-Ala) ratio for D-Ala-D-Ser/D-Ala-D-Ala dipeptide formation is 270/0.69 for a 400-fold selection against D-Ala in the C-terminal position. VanC2 also has substantial D-Ala-D-Asn ligase activity (kcat/Km2 = 74 mM-1min-1).

(-)-N-[(11)C]propyl-norapomorphine: a positron-labeled dopamine agonist for PET imaging of D(2) receptors.

PubMed

Hwang, D R; Kegeles, L S; Laruelle, M

2000-08-01

Imaging neuroreceptors with radiolabeled agonists might provide valuable information on the in vivo agonist affinity states of receptors of interest. We report here the radiosynthesis, biodistribution in rodents, and imaging studies in baboons of [(11)C]-labeled (-)-N-propyl-norapomorphine [(-)-NPA]. (-)-[(11)C]NPA was prepared by reacting norapomorphine with [(11)C]propionyl chloride and a lithium aluminum hydride reduction. [(11)C]Propionyl chloride was prepared by reacting [(11)C]CO(2) with ethylmagnesium bromide, followed by reacting with phthaloyl chloride. The radiochemical yield of (-)-[(11)C]NPA was 2.5% at end of synthesis (EOS), and the synthesis time was 60 min. The specific activity was 1700+/-1900 mCi/micromol ( N=7; ranged 110-5200 mCi/micromol at EOS). Rodent biodistribution studies showed high uptake of [(11)C](-)-NPA in D(2) receptor-rich areas, and the striatum/cerebellum ratios were 1.7, 3.4, and 4.4 at 5 min, 30 min, and 60 min postinjection, respectively. Pretreating the animals with haloperidol (1 mg/kg) decreased the striatum/cerebellum ratio at 30 min postinjection to 1.3. (-)-[(11)C]NPA was also evaluated via baboon positron emission tomography (PET) studies. Under control conditions ( N=4), rapid uptake of the tracer was observed and the striatum/cerebellum ratio reached 2.86+/-0.15 at 45 min postinjection. Following haloperidol pretreatment (0.2 mg/kg IV), the striatum/cerebellum ratio was 1.29 at 45 min postinjection. The result demonstrated the existence of specific binding of this new tracer to the D(2) receptor. To our knowledge, the current finding of a striatum/cerebellum ratio of 2.8 in baboon was the highest reported with a radiolabeled D(2) agonist. (-)-[(11)C]NPA is a promising new D(2) agonist PET tracer for probing D(2) receptors in vivo using PET.

Dopamine D2-receptor activation elicits akinesia, rigidity, catalepsy, and tremor in mice expressing hypersensitive α4 nicotinic receptors via a cholinergic-dependent mechanism

PubMed Central

Zhao-Shea, Rubing; Cohen, Bruce N.; Just, Herwig; McClure-Begley, Tristan; Whiteaker, Paul; Grady, Sharon R.; Salminen, Outi; Gardner, Paul D.; Lester, Henry A.; Tapper, Andrew R.

2010-01-01

Recent studies suggest that high-affinity neuronal nicotinic acetylcholine receptors (nAChRs) containing α4 and β2 subunits (α4β2*) functionally interact with G-protein-coupled dopamine (DA) D2 receptors in basal ganglia. We hypothesized that if a functional interaction between these receptors exists, then mice expressing an M2 point mutation (Leu9′Ala) rendering α4 nAChRs hypersensitive to ACh may exhibit altered sensitivity to a D2-receptor agonist. When challenged with the D2R agonist, quinpirole (0.5–10 mg/kg), Leu9′Ala mice, but not wild-type (WT) littermates, developed severe, reversible motor impairment characterized by rigidity, catalepsy, akinesia, and tremor. While striatal DA tissue content, baseline release, and quinpirole-induced DA depletion did not differ between Leu9′Ala and WT mice, quinpirole dramatically increased activity of cholinergic striatal interneurons only in mutant animals, as measured by increased c-Fos expression in choline acetyltransferase (ChAT)-positive interneurons. Highlighting the importance of the cholinergic system in this mouse model, inhibiting the effects of ACh by blocking muscarinic receptors, or by selectively activating hypersensitive nAChRs with nicotine, rescued motor symptoms. This novel mouse model mimics the imbalance between striatal DA/ACh function associated with severe motor impairment in disorders such as Parkinson’s disease, and the data suggest that a D2R–α4*-nAChR functional interaction regulates cholinergic interneuron activity.—Zhao-Shea, R., Cohen, B. N., Just, H., McClure-Begley, T., Whiteaker, P., Grady, S. R., Salminen, O., Gardner, P. D., Lester, H. A., Tapper, A. R. Dopamine D2-receptor activation elicits akinesia, rigidity, catalepsy, and tremor in mice expressing hypersensitive α4 nicotinic receptors via a cholinergic-dependent mechanism. PMID:19720621

Phe317 Is Essential for Rubber Oxygenase RoxA Activity

PubMed Central

Birke, Jakob; Hambsch, Nadja; Schmitt, Georg; Altenbuchner, Josef

2012-01-01

RoxA is an extracellular c-type diheme cytochrome secreted by Xanthomonas sp. strain 35Y during growth on rubber. RoxA cleaves poly(cis-1,4-isoprene) to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). Analysis of the RoxA structure revealed that Phe317 is located in close proximity (≈5 Å) to the N-terminal heme that presumably represents the active site. To find evidence of whether Phe317 is important for catalysis, we changed it to tyrosine, tryptophan, leucine, histidine, or alanine. All five RoxA muteins were expressed after integration of the respective gene into the chromosome of a Xanthomonas sp. ΔroxA strain. Residual clearing zone formation on opaque latex agar was found for Xanthomonas sp. strains expressing the Phe317Leu, Phe317Ala, or Phe317His variant (wild type > Leu > Ala > His). Strains in which Phe317 was changed to tyrosine or tryptophan were inactive. Phe317Ala and Phe312Leu RoxA muteins were purified, and polyisoprene cleavage activities were reduced to ≈3% and 10%, respectively. UV-visible spectroscopy of RoxA muteins confirmed that both heme groups were present in an oxidized form, but spectral responses to the addition of low-molecular-weight (inhibitory) ligand molecules such as imidazole and pyridine were different from those of wild-type RoxA. Our results show that residue 317 is involved in interaction with substrates. This is the first report on structure-function analysis of a polyisoprene-cleaving enzyme and on the identification of an amino acid that is essential for polyisoprene cleavage activity. PMID:22941074

Introduction of antigenic determining 2,4-dinitrophenyl residues into 4-thiouridine, N3-(3-L-amino-3-carboxypropyl) uridine and tRNA-Phe from E. coli.

PubMed Central

Seela, F; Hansske, F; Watanabe, K; Cramer, F

1977-01-01

The introduction of antigenic determining 2,4-dinitrophenyl residues into the rare ribonucleosides 4-thiouridine (1a), and N3-(3-L-amino-3-carboxypropyl) uridine (2) as well as into tRNA-Phe from E. coli has been investigated. Alkylation of 1a with omega-bromo-2,4-dinitroacetophenone (3b) gives S-(2,4-dinitrophenacyl)-4-thiouridine (5A). Applying the reaction to the 5'-monophosphate of 1a, 5b is formed, but this product decomposes at pH 7. However, acylation of 2 with 2,4-dinitrobenzoic acid N-hydroxysuccinimide ester (4b) leads to N3-[3-carboxy-3-L-(2,4-dinitrobenzamido)propyl]uridine (6) which is stable in aqueous solution. The latter reaction was used for the introduction of an antigenic determining 2,4-dinitrophenyl residue into tRNA-Phe from E. coli. The modified tRNA-Phe was isolated and by degradation of the molecule with RNase T2 and alkaline phosphatase the nucleoside derivative 6 was obtained and found to be identical with the synthetic product. PMID:68463

Cooperative regulation of anxiety and panic-related defensive behaviors in the rat periaqueductal grey matter by 5-HT1A and μ-receptors.

PubMed

Roncon, Camila M; Biesdorf, Carla; Coimbra, Norberto C; Audi, Elisabeth A; Zangrossi, Hélio; Graeff, Frederico G

2013-12-01

Previous results with the elevated T-maze (ETM) test indicate that the antipanic action of serotonin (5-HT) in the dorsal periaqueductal grey (dPAG) depends on the activation endogenous opioid peptides. The aim of the present work was to investigate the interaction between opioid- and serotonin-mediated neurotransmission in the modulation of defensive responses in rats submitted to the ETM. The obtained results showed that intra-dPAG administration of morphine significantly increased escape latency, a panicolytic-like effect that was blocked by pre-treatment with intra-dPAG injection of either naloxone or the 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1 piperazinyl] ethyl] -N- 2- pyridinyl-ciclohexanecarboxamide maleate (WAY-100635). In addition, previous administration of naloxone antagonized both the anti-escape and the anti-avoidance (anxiolytic-like) effect of the 5-HT1A agonist (±)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), but did not affect the anti-escape effect of the 5-HT2A agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI). Moreover, the combination of sub-effective doses of locally administered 5-HT and morphine significantly impaired ETM escape performance. Finally, the µ-antagonist D-PHE-CYS-TYR-D-TRP-ORN-THR-PEN (CTOP) blocked the anti-avoidance as well as the anti-escape effect of 8-OHDPAT, and the association of sub-effective doses of the µ-opioid receptor agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin acetate salt (DAMGO) and of 8-OHDPAT had anti-escape and anti-avoidance effects in the ETM. These results suggest a synergic interaction between the 5-HT1A and the µ-opioid receptor at post-synaptic level on neurons of the dPAG that regulate proximal defense, theoretically related to panic attacks.

Regulation of His-dTrp-Ala-Trp-dPhe-Lys-NH2 (GHRP-6)-induced GH secretion in the rat.

PubMed

Mallo, F; Alvarez, C V; Benitez, L; Burguera, B; Coya, R; Casanueva, F F; Dieguez, C

1993-01-01

His-dTrp-Ala-Trp-dPhe,Lys-NH2(GHRP-6) is a synthetic compound that releases GH in a dose-response and specific manner in several species and that may well be related to an endogenous compound of similar structure. The aim of this study was to investigate the in vivo GH responses to GHRP-6 in pentobarbital anesthetized rats. Specifically and in order to avoid the influence of endogenous GHRH and somatostatin secretion we studied the GH responses to GHRP-6 in animals with surgical ablation of the hypothalamus, confirmed by histological assessment, as well as in hypophysectomyzed-transplanted rats bearing two hypophyses under the renal capsule. Since it has been previously reported that rats pretreated with GHRH (10 micrograms/kg i.p. every 12 h for 15 days) rather than saline-treated rats have greater GH responses to acutely administered GHRH, we compared the self-potentiating effect of chronic GH pretreatment with GHRP-6 (10 micrograms/kg i.p. every 12 h). Furthermore we also studied the influence of estrogens, glucocorticoids, free fatty acids (FFA) and bombesin on somatotroph responsiveness to GHRP-6 in intact rats. We found a greater GH response to GHRP-6 in rats that underwent a surgical ablation of the hypothalamus 36 h prior to the test than in sham-operated rats. A direct stimulatory effect of GHRP-6 on in vivo GH secretion was demonstrated by a clear GH response to GHRP-6 in hypophysectomyzed-transplanted rats. In addition, we found a similar response whether the animals were pretreated with GHRH or GHRP-6 over the previous 2 weeks. Finally, we found that both estrogen- and testosterone-treated rats have greater GH responses to GHRP-6 than untreated rats. On the other hand, chronic dexamethasone administration, acute elevation of circulating FFA levels and bombesin administration markedly inhibited GH responses to GHRP-6. In contrast to the effects exerted on GH responses to GHRP-6 estrogen administration led to a decrease in GH responses to GHRH while

Biphalin analogs containing β(3)-homo-amino acids at the 4,4' positions: Synthesis and opioid activity profiles.

PubMed

Frączak, Oliwia; Lasota, Anika; Kosson, Piotr; Leśniak, Anna; Muchowska, Adriana; Lipkowski, Andrzej W; Olma, Aleksandra

2015-04-01

Biphalin, a synthetic opioid octapeptide with a palindromic sequence has high analgesic activity. Biphalin displays a strong affinity for μ and δ-opioid receptors, and a significant to κ-receptor. The paper reports the synthesis of novel analogs of biphalin containing β(3)-homo-amino acid residues at the 4,4' positions and a hydrazine or 1,2-phenylenediamine linker. The potency and selectivity of the peptides were evaluated by a competitive receptor-binding assay in rat brain homogenate using [(3)H]DAMGO (a μ ligand) and [(3)H]DELT (a δ ligand). Analogs with β(3)-h-p-NO2Phe in positions 4 and 4' are the most active compounds. Selectivity depends on the degree of freedom between the two pharmacophore moieties. Analogs with a hydrazine linker show noticeable binding selectivity to μ receptors (IC50(μ)=0.72nM; IC50(δ)=4.66nM), while the peptides with a 1,2-phenylenediamine linker show slight δ selectivity (IC50(μ)=10.97nM; IC50(δ)=1.99nM). Tyr-d-Ala-Gly-β(3)-h-p-NO2PheNHNH-β(3)-h-p-NO2Phe (1) and (Tyr-d-Ala-Gly-β(3)-h-p-NO2PheNH)2 (2) produced greater antinociceptive effect compared to morphine after i.t. administration. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparison of the functional potencies of ropinirole and other dopamine receptor agonists at human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells

PubMed Central

Coldwell, Martyn C; Boyfield, Izzy; Brown, Tony; Hagan, Jim J; Middlemiss, Derek N

1999-01-01

The aim of the present study was to characterize functional responses to ropinirole, its major metabolites in man (SKF-104557 (4-[2-(propylamino)ethyl]-2-(3H) indolone), SKF-97930 (4-carboxy-2-(3H) indolone)) and other dopamine receptor agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) receptors separately expressed in Chinese hamster ovary cells using microphysiometry.All the receptor agonists tested (ropinirole, SKF-104557, SKF-97930, bromocriptine, lisuride, pergolide, pramipexole, talipexole, dopamine) increased extracellular acidification rate in Chinese hamster ovary clones expressing the human D2, D3 or D4 receptor. The pEC50s of ropinirole at hD2, hD3 and hD4 receptors were 7.4, 8.4 and 6.8, respectively. Ropinirole is therefore at least 10 fold selective for the human dopamine D3 receptor over the other D2 receptor family members.At the hD2 and hD3 dopamine receptors all the compounds tested were full agonists as compared to quinpirole. Talipexole and the ropinirole metabolite, SKF-104557, were partial agonists at the hD4 receptor.Bromocriptine and lisuride had a slow onset of agonist action which precluded determination of EC50s.The rank order of agonist potencies was dissimilar to the rank order of radioligand binding affinities at each of the dopamine receptor subtypes. Functional selectivities of the dopamine receptor agonists, as measured in the microphysiometer, were less than radioligand binding selectivities.The results show that ropinirole is a full agonist at human D2, D3 and D4 dopamine receptors. SKF-104557 the major human metabolite of ropinirole, had similar radioligand binding affinities to, but lower functional potencies than, the parent compound. PMID:10455328

Opiate agonist-induced re-distribution of Wntless, a mu-opioid receptor interacting protein, in rat striatal neurons.

PubMed

Reyes, B A S; Vakharia, K; Ferraro, T N; Levenson, R; Berrettini, W H; Van Bockstaele, E J

2012-01-01

Wntless (WLS), a mu-opioid receptor (MOR) interacting protein, mediates Wnt protein secretion that is critical for neuronal development. We investigated whether MOR agonists induce re-distribution of WLS within rat striatal neurons. Adult male rats received either saline, morphine or [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO) directly into the lateral ventricles. Following thirty minutes, brains were extracted and tissue sections were processed for immunogold silver detection of WLS. In saline-treated rats, WLS was distributed along the plasma membrane and within the cytoplasmic compartment of striatal dendrites as previously described. The ratio of cytoplasmic to total dendritic WLS labeling was 0.70±0.03 in saline-treated striatal tissue. Morphine treatment decreased this ratio to 0.48±0.03 indicating a shift of WLS from the intracellular compartment to the plasma membrane. However, following DAMGO treatment, the ratio was 0.85±0.05 indicating a greater distribution of WLS intracellularly. The difference in the re-distribution of the WLS following different agonist exposure may be related to DAMGO's well known ability to induce internalization of MOR in contrast to morphine, which is less effective in producing receptor internalization. Furthermore, these data are consistent with our hypothesis that MOR agonists promote dimerization of WLS and MOR, thereby preventing WLS from mediating Wnt secretion. In summary, our findings indicate differential agonist-induced trafficking of WLS in striatal neurons following distinct agonist exposure. Adaptations in WLS trafficking may represent a novel pharmacological target in the treatment of opiate addiction and/or pain. Copyright © 2011 Elsevier Inc. All rights reserved.

The delta-selective opioid peptide dermenkephalin and the mu-selective hybrid peptide dermenkephalin-[1-4]-dermophin-[5-7] display strikingly different conformations despite identical tetrapeptide N-termini. A quantitative 2-D NMR and molecular modeling analysis.

PubMed

Riand, J; Baron, D; Nicolas, P; Benajiba, A; Teng, Y; Naim, M

1999-12-01

The selective recognition of the aminoterminal binding pharmacophore Tyr-D-Xaa-Phe of the opioid heptapeptide dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (DRM)1, and of dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (DREK), by the mu-opioid receptor and delta-opioid receptor, respectively, depends upon the constitution / conformation of the C-terminal tripeptide. The hybrid peptide DREK-[1-4]-DRM-[5-7] is very potent at, and exquisitely selective for the mu-opioid receptor, and differs only from dermenkephalin by its C-terminal tripeptide. Comparison of the structural features of DREK-[1-4]-DRM-[5-7] and dermenkephalin by nmr analysis and molecular modeling revealed striking differences, as well in the trans (Tyr5 - Pro6) isomer (population 75%) than in the cis isomer.. Whereas the folded C-terminal tail of dermenkephalin influenced the tertiary structure of the N-terminal tetrapeptide and placed the Tyr1 and Phe3 aromatic rings in definite orientations that are best suited for the delta-receptor, there were only weak contacts, as shown by NOE data, between the aminoterminal and carboxyterminal parts of the hybrid peptide. This promoted increased flexibility of the whole backbone and relaxed orientations for the side-chains of Tyr1 and Phe3 that are compatible with the mu-receptor but unsuitable for the delta-receptor. The steric hindrance introduced by Pro6 in DREK-[1-4]-DRM-[5-7], plus the absence of large hydrophobic side-chains in positions 5 and 6 may prevent close contacts between the N-terminal and C-terminal domains and reorientation of the main pharmacophoric elements Tyr1 and Phe3.

Synthesis and reactivity of bis(tetramethylcyclopentadienyl) yttrium metallocenes including the reduction of Me(3)SiN(3) to [(Me(3)Si)(2)N](-) with [(C(5)Me(4)H)(2)Y(THF)](2)(mu-eta(2):eta(2)-N(2)).

PubMed

Lorenz, Sara E; Schmiege, Benjamin M; Lee, David S; Ziller, Joseph W; Evans, William J

2010-07-19

The metallocene precursors needed to provide the tetramethylcyclopentadienyl yttrium complexes (C(5)Me(4)H)(3)Y, [(C(5)Me(4)H)(2)Y(THF)](2)(mu-eta(2):eta(2)-N(2)), and [(C(5)Me(4)H)(2)Y(mu-H)](2) for reactivity studies have been synthesized and fully characterized, and their reaction chemistry has led to an unexpected conversion of an azide to an amide. (C(5)Me(4)H)(2)Y(mu-Cl)(2)K(THF)(x), 1, synthesized from YCl(3) and KC(5)Me(4)H reacts with allylmagnesium chloride to make (C(5)Me(4)H)(2)Y(eta(3)-C(3)H(5)), 2, which is converted to [(C(5)Me(4)H)(2)Y][(mu-Ph)(2)BPh(2)], 3, with [Et(3)NH][BPh(4)]. Complex 3 reacts with KC(5)Me(4)H to form (C(5)Me(4)H)(3)Y, 4. The reduced dinitrogen complex, [(C(5)Me(4)H)(2)Y(THF)](2)(mu-eta(2):eta(2)-N(2)), 5, can be synthesized from either [(C(5)Me(4)H)(2)Y](2)[(mu-Ph)(2)BPh(2)], 3, or (C(5)Me(4)H)(3)Y, 4, with potassium graphite under a dinitrogen atmosphere. The (15)N labeled analogue, [(C(5)Me(4)H)(2)Y(THF)](2)(mu-eta(2):eta(2)-(15)N(2)), 5-(15)N, has also been prepared, and the (15)N NMR data have been compared to previously characterized reduced dinitrogen complexes. Complex 2 reacts with H(2) to form the corresponding hydride, [(C(5)Me(4)H)(2)Y(mu-H)](2), 6. Complex 5 displays similar reactivity to that of the analogous [(C(5)Me(4)H)(2)Ln(THF)](2)(mu-eta(2):eta(2)-N(2)) complexes (Ln = La, Lu), with substrates such as phenazine, anthracene, and CO(2). In addition, 5 reduces Me(3)SiN(3) to form (C(5)Me(4)H)(2)Y[N(SiMe(3))(2)], 7.

Comparisons of the potential utility of LHRH agonists and antagonists for fertility control.

PubMed

Vickery, B H

1985-11-01

Prospects for the use of LHRH analogs for human fertility control have been reviewed with particular reference to two highly potent representatives. Nafarelin acetate, the LHRH agonist, has a potency about 200 X that of LHRH and is consistently effective in suppressing gonadal function in females through a desensitization of LHRH receptors in the pituitary. Such agents show promise as ovulation inhibitors for women although concern has been expressed over the dangers of unopposed estrogen or alternatively hypoestrogenemia. Although early studies indicated luteolysis in women and interceptive action in baboons it is now clear that the LHRH agonists will not be useful clinically to terminate pregnancy. Wide species differences in the male response to LHRH agonists exist. Unfortunately azoospermia has not been achieved in men. The LHRH antagonists, typified by [N-Ac-D-Nal(2)1, D-pCl-Phe2, D-Trp3, D-hArg(Et2)6, D-Ala10]LHRH, require high doses to competitively inhibit responses to endogenous LHRH. Their advantages include a rapid induction of the hypogonadal state with apparently little species or sexual variation in response. Based on animal studies, preferable utility of the antagonists would lie in male contraception and pregnancy interception.

μ-Opioid Receptor Trafficking on Inhibitory Synapses in the Rat Brainstem

PubMed Central

Browning, Kirsteen N.; Kalyuzhny, Alexander E.; Travagli, R. Alberto

2011-01-01

Whole-cell recordings were made from identified gastric-projecting rat dorsal motor nucleus of the vagus (DMV) neurons. The amplitude of evoked IPSCs (eIPSCs) was unaffected by perfusion with met-enkephalin (ME) or by μ-, δ-, or κ-opioid receptor selective agonists, namely d-Ala2-N-Me-Phe4-Glycol5-enkephalin (DAMGO), cyclic [d-Pen2-d-Pen5]-enkephalin, or trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolytinil)-cyclohexyl]-benzeneacetamide methane sulfonate (U50,488), respectively. Brief incubation with the adenylate cyclase activator forskolin or the nonhydrolysable cAMP analog 8-bromo-cAMP, thyrotropin releasing hormone, or cholecystokinin revealed the ability of ME and DAMGO to inhibit IPSC amplitude; this inhibition was prevented by pretreatment with the μ-opioid receptor (MOR1) selective antagonist d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2. Conversely, incubation with the adenylate cyclase inhibitor dideoxyadenosine, with the protein kinase A (PKA) inhibitor N-[2-(p-Bromocinnamyl-amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89), or with the Golgi-disturbing agent brefeldin A, blocked the ability of forskolin to facilitate the inhibitory actions of ME. Immunocytochemical experiments revealed that under control conditions, MOR1 immunoreactivity (MOR1-IR) was colocalized with glutamic acid decarboxylase (GAD)-IR in profiles apposing DMV neurons only after stimulation of the cAMP–PKA pathway. Pretreatment with H89 or brefeldin A or incubation at 4°C prevented the forskolin-mediated insertion of MOR1 on GAD-IR-positive profiles. These results suggest that the cAMP–PKA pathway regulates trafficking of μ-opioid receptors into the cell surface of GABAergic nerve terminals. By consequence, the inhibitory actions of opioid peptides in the dorsal vagal complex may depend on the state of activation of brainstem vagal circuits. PMID:15317860

Novel selective agonists and antagonists confirm neurokinin NK1 receptors in guinea-pig vas deferens.

PubMed Central

Hall, J. M.; Morton, I. K.

1991-01-01

1. This study investigated the recognition characteristics of neurokinin receptors mediating potentiation of the contractile response to field stimulation in the guinea-pig vas deferens. 2. A predominant NK1 receptor population is strongly suggested by the relative activities of the common naturally-occurring tachykinin agonists, which fall within less than one order of magnitude. This conclusion is supported by the relative activities of the synthetic NK1 selective agonists substance P methyl ester, [Glp6,L-Pro9]-SP(6-11) and delta-aminovaleryl-[L-Pro9,N-MeLeu10]- SP(7-11) (GR73632) which were 0.78, 9.3 and 120 as active as substance P, respectively. Furthermore, the NK2 selective agonist [Lys3, Gly8,-R-gamma-lactam-Leu9]-NKA(3-10) (GR64349) was active only at the highest concentrations tested (greater than 10 microM), and the NK3 selective agonist, succ-[Asp6,N-MePhe8]-SP(6-11) (senktide) was essentially inactive (10 nM-32 microM). 3. [D-Arg1,D-Pro2,D-Trp7,9,Leu11]-SP(1-11) antagonized responses to neurokinin A, neurokinin B, physalaemin, eledoisin, [Glp6,D-Pro9]-SP(6-11), GR73632 and GR64349 (apparent pKB s 5.6-6.2), but was less potent in antagonizing responses to substance P, substance P methyl ester and [Glp6,L-Pro9]-SP(6-11) (apparent pKB s less than or equal to 5.0-5.0). 4. In contrast, the recently developed NK1-selective receptor antagonist [D-Pro9[Spiro-gamma-lactam]Leu10,Trp11]-SP(1-11) (GR71251) did not produce agonist-dependent pKB estimates. Schild plot analysis indicated a competitive interaction with a single receptor population where the antagonist had an estimated overall pKB of 7.58 +/- 0.13 for the four agonists of differing subtype selectivity tested (GR73632, GR64349, substance P methyl ester and neurokinin B).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1707714

Structure-activity relationships of the melanocortin tetrapeptide Ac-His-DPhe-Arg-Trp-NH(2) at the mouse melanocortin receptors: part 2 modifications at the Phe position.

PubMed

Holder, Jerry Ryan; Bauzo, Rayna M; Xiang, Zhimin; Haskell-Luevano, Carrie

2002-07-04

The melanocortin pathway is an important participant in skin pigmentation, steroidogenesis, obesity, energy homeostasis and exocrine gland function. The centrally located melanocortin-3 and melanocortin-4 receptors (MC3R, MC4R) are involved in the metabolic and food intake aspects of energy homeostasis and are stimulated by melanocortin agonists such as alpha-melanocyte stimulation hormone (alpha-MSH). The melanocortin agonists contain the putative message sequence "His-Phe-Arg-Trp," and it has been well-documented that inversion of chirality of the Phe to DPhe results in a dramatic increase in melanocortin receptor potency. Herein, we report a tetrapeptide library, based upon the template Ac-His-DPhe-Arg-Trp-NH(2), consisting of 26 members that have been modified at the DPhe(7) position (alpha-MSH numbering) and pharmacologically characterized for agonist and antagonist activity at the mouse melanocortin receptors MC1R, MC3R, MC4R, and MC5R. The most notable results of this study include the identification of the tetrapeptide Ac-His-(pI)DPhe-Arg-Trp-NH(2) that is a full nanomolar agonist at the mMC1 and mMC5 receptors, a mMC3R partial agonist with potent antagonist activity (pA(2) = 7.25, K(i) = 56 nM) and, but unexpectedly, is a potent agonist at the mMC4R (EC(50) = 25 nM). This ligand possesses novel melanocortin receptor pharmacology, as compared to previously reported peptides, and is potentially useful for in vivo studies to differentiate MC3R vs MC4R physiological roles in animal models, such as primates, where "knockout" animals are not viable options. The DNal(2') substitution for DPhe resulted in a mMC3R partial agonist with antagonist activity (pA(2) = 6.5, K(i) = 295 nM) and a mMC4R (pA(2) = 7.8, K(i) = 17 nM) antagonist possessing 60- and 425-fold decreased potency, respectively, as compared with SHU9119 at these receptors. Examination of this DNal(2')-containing tetrapeptide at the F254S and F259S mutant mMC4Rs resulted in agonist activity of this m

Synthesis, structure, luminescence, and magnetic properties of a single-ion magnet "mer"-[tris(N-[(imidazol-4-yl)-methylidene]-DL-phenylalaninato)terbium(III) and related "fac"-DL-alaninato derivative.

PubMed

Yamauchi, Suguru; Fujinami, Takeshi; Matsumoto, Naohide; Mochida, Naotaka; Ishida, Takayuki; Sunatsuki, Yukinari; Watanabe, Masayuki; Tsuchimoto, Masanobu; Coletti, Cecilia; Re, Nazzareno

2014-06-16

Two Tb(III) complexes with the same N6O3 donor atoms but different coordination geometries, "fac"-[Tb(III)(HL(DL-ala))3]·7H2O (1) and "mer"-[Tb(III)(HL(DL-phe))3]·7H2O (2), were synthesized, where H2L(DL-ala) and H2L(DL-phe) are N-[(imidazol-4-yl)methylidene]-DL-alanine and -DL-phenylalanine, respectively. Each Tb(III) ion is coordinated by three electronically mononegative NNO tridentate ligands to form a coordination geometry of a tricapped trigonal prism. Compound 1 consists of enantiomers "fac"-[Tb(III)(HL(D-ala))3] and "fac"-[Tb(III)(HL(L-ala))3], while 2 consists of "mer"-[Tb(III)(HL(D-phe))2(HL(L-phe))] and "mer"-[Tb(III)(HL(D-phe))(HL(L-phe))2]. Magnetic data were analyzed by a spin Hamiltonian including the crystal field effect on the Tb(III) ion (4f(8), J = 6, S = 3, L = 3, gJ = 3/2, (7)F6). The Stark splitting of the ground state (7)F6 was evaluated from magnetic analysis, and the energy diagram pattern indicated easy-plane and easy-axis (Ising type) magnetic anisotropies for 1 and 2, respectively. Highly efficient luminescences with Φ = 0.50 and 0.61 for 1 and 2, respectively, were observed, and the luminescence fine structure due to the (5)D4 → (7)F6 transition is in good accordance with the energy diagram determined from magnetic analysis. The energy diagram of 1 shows an approximate single-well potential curve, whereas that of 2 shows a double- or quadruple-well potential within the (7)F6 multiplets. Complex 2 displayed an onset of the out-of-phase signal in alternating current (ac) susceptibility at a direct current bias field of 1000 Oe on cooling down to 1.9 K. A slight frequency dependence was recorded around 2 K. On the other hand, 1 did not show any meaningful out-of-phase ac susceptibility. Pulsed-field magnetizations of 1 and 2 were measured below 1.6 K, and only 2 exhibited magnetic hysteresis. This finding agrees well with the energy diagram pattern from crystal field calculation on 1 and 2. DFT calculation allowed us to estimate the

D2-Thr92Ala, thyroid hormone levels and biochemical hypothyroidism in preeclampsia.

PubMed

Procopciuc, Lucia Maria; Caracostea, Gabriela; Hazi, Georgeta; Nemeti, Georgiana; Stamatian, Florin

2017-02-01

To identify if there is a relationship between the deiodinase D2-Thr92Ala genetic variant, thyroid hormone levels and biochemical hypothyroidism in preeclampsia. We genotyped 125 women with preeclampsia and 131 normal pregnant women using PCR-RFLP. Serum thyroid hormone levels were determined using ELISA. Our study showed higher TSH and FT4 levels and lower FT3 levels in women with preeclampsia compared to normal pregnant women, with statistical significance for women with mild and severe preeclampsia. The risk to develop pregnancy-induced hypertension (PIH), mild or severe preeclampsia was increased in carriers of at least one D2-Ala92 allele. TSH and FT4 levels were significantly higher and FT3 levels were significantly lower in preeclamptic women with severe preeclampsia if they carried the D2-Ala92 allele compared to non-carriers. Pregnant women with PIH and mild preeclampsia, carriers of at least one D2-Ala92 allele, delivered at lower gestational age neonates with a lower birth weight compared to non-carriers, but the results were statistically significant only in severe preeclampsia. The D2-Thr92Ala genetic variant is associated with the severity and the obstetric outcome of preeclampsia, and it also influences thyroid hormone levels. The study demonstrates non-thyroidal biochemical hypothyroidism - as a result of deiodination effects due to D2 genotypes.

Both substance P agonists and antagonists inhibit ion conductance through nicotinic acetylcholine receptors on PC12 cells.

PubMed

Eardley, D; McGee, R

1985-08-07

Substance P stimulates substance P receptors but also inhibits ion conductance through nicotinic acetylcholine receptors. Substance P analogs, classified as agonists or antagonists based on their actions on smooth muscle, were tested to determine if they also could act at nicotinic receptors on the pheochromocytoma, PC12. All of the analogs tested, [D-Pro2, D-Trp7,9]SP, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]SP, [pGlu5, MePhe8, Sar9]SP-(5-11), and [D-Pro4, D-Trp7,9,10]SP-(4-11), inhibited agonist-induced uptake of 86Rb+ through the nicotinic receptors at concentrations quite similar to those required for action at substance P receptors on smooth muscle. Thus, the chemical modifications in the analogs do not substantially alter their ability to inhibit nicotinic receptors.

Development of Spexin-based Human Galanin Receptor Type II-Specific Agonists with Increased Stability in Serum and Anxiolytic Effect in Mice.

PubMed

Reyes-Alcaraz, Arfaxad; Lee, Yoo-Na; Son, Gi Hoon; Kim, Nam Hoon; Kim, Dong-Kyu; Yun, Seongsik; Kim, Dong-Hoon; Hwang, Jong-Ik; Seong, Jae Young

2016-02-24

The novel neuropeptide spexin (SPX) was discovered to activate galanin receptor 2 (GALR2) and 3 (GALR3) but not galanin receptor 1 (GALR1). Although GALR2 is known to display a function, particularly in anxiety, depression, and appetite regulation, the further determination of its function would benefit from a more stable and selective agonist that acts only at GALR2. In the present study, we developed a GALR2-specific agonist with increased stability in serum. As galanin (GAL) showed a low affinity to GALR3, the residues in SPX were replaced with those in GAL, revealing that particular mutations such as Gln5 → Asn, Met7 → Ala, Lys11 → Phe, and Ala13 → Pro significantly decreased potencies toward GALR3 but not toward GALR2. Quadruple (Qu) mutation of these residues still retained potency to GALR2 but totally abolished the potency to both GALR3 and GALR1. The first amino acid modifications or D-Asn1 substitution significantly increased the stability when they are incubated in 100% fetal bovine serum. Intracerebroventricular administration of the mutant peptide with D-Asn1 and quadruple substitution (dN1-Qu) exhibited an anxiolytic effect in mice. Taken together, the GALR2-specific agonist with increased stability can greatly help delineation of GALR2-mediated functions and be very useful for treatments of anxiety disorder.

Peptide and small molecules rescue the functional activity and agonist potency of dysfunctional human melanocortin-4 receptor polymorphisms.

PubMed

Xiang, Zhimin; Pogozheva, Irina D; Sorenson, Nicholas B; Wilczynski, Andrzej M; Holder, Jerry Ryan; Litherland, Sally A; Millard, William J; Mosberg, Henry I; Haskell-Luevano, Carrie

2007-07-17

The melanocortin pathway, specifically the melanocortin-4 receptor and the cognate endogenous agonist and antagonist ligands, have been strongly implicated in the regulation of energy homeostasis and satiety. Genetic studies of morbidly obese human patients and normal weight control patients have resulted in the discovery of over 70 human melanocortin-4 receptor (MC4R) polymorphisms observed as both heterozygous and homozygous forms. A number of laboratories have been studying these hMC4R polymorphisms attempting to understand the molecular mechanism(s) that might explain the obese human phenotype. Herein, we have studied 13 polymorphic hMC4Rs that have been identified to possess statistically significant decreased endogenous agonist potency with synthetic peptides and small molecules attempting to identify ligands that can pharmacologically rescue the hMC4R polymorphic agonist response. The ligands examined in this study include NDP-MSH, MTII, Ac-His-DPhe-Arg-Trp-NH2 (JRH887-9), Ac-Anc-DPhe-Arg-Trp-NH2 (amino-2-naphtylcarboxylic acid, Anc, JRH420-12), Ac-His-(pI)DPhe-Arg-Trp-NH2 (JRH322-18), chimeric AGRP-melanocortin based ligands (Tyr-c[Cys-His-DPhe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2, AMW3-130 and Ac-mini-(His-DPhe-Arg-Trp)-hAGRP-NH2, AMW3-106), and the small molecules JB25 and THIQ. The hMC4R polymorphisms included in this study are S58C, N97D, I102S, L106P, S127L, T150I, R165Q, R165W, L250Q, G252S, C271Y, Y287Stop, and I301T. These studies resulted in the NDP-MSH, MTII, AMW3-130, THIQ, and AMW3-106 ligands possessing nanomolar to subnanomolar agonist potency at the hMC4R polymorphisms examined in this study. Thus, these ligands could generically rescue the potency and stimulatory response of the abnormally functioning hMC4Rs studied and may provide tools to further clarify the molecular mechanism(s) involving these receptor modifications.

Micro-opioid receptor agonist diminishes POMC gene expression and anorexia by central insulin in neonatal chicks.

PubMed

Shiraishi, Jun-Ichi; Yanagita, Kouchi; Fujita, Masanori; Bungo, Takashi

2008-07-18

Pro-opiomelanocortin (POMC) neurons in the hypothalamus are direct targets of peripheral satiety signals, such as leptin and insulin in mammals. The stimulation of these signals activates hypothalamic POMC neurons and elevates POMC-derived melanocortin peptides that inhibit food intake in mammals. On the other hand, it has been recognized that beta-endorphin, a post-translational processing of POMC, acts in an autoreceptor manner to the micro-opioid receptor (MOR) on POMC neurons, diminishing POMC neuronal activity in mammals. Recently, we found that central insulin functions as an anorexic peptide in chicks. Thus, the present study was done to elucidate whether beta-endorphin affects the activation of POMC neurons by insulin in neonatal chicks. Consequently, quantitative real-time PCR analysis shows that intracerebroventricular (ICV) injection of insulin with beta-endorphin significantly decreases brain POMC mRNA expression when compared with insulin alone. In addition, co-injection of MOR agonist (beta-endorphin or [d-Ala2, N-MePhe4, Gly5-ol]-enkephalin (DAMGO)) significantly attenuates insulin-induced hypophagia in chicks. These data suggest that beta-endorphin regulates the activity of the central melanocortin system, and its activation may provide an inhibitory feedback mechanism in the brain of neonatal chicks.

Novel Analogues of (R)-5-(Methylamino)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (Sumanirole) Provide Clues to Dopamine D2/D3 Receptor Agonist Selectivity

PubMed Central

2016-01-01

Novel 1-, 5-, and 8-substituted analogues of sumanirole (1), a dopamine D2/D3 receptor (D2R/D3R) agonist, were synthesized. Binding affinities at both D2R and D3R were higher when determined in competition with the agonist radioligand [3H]7-hydroxy-N,N-dipropyl-2-aminotetralin (7-OH-DPAT) than with the antagonist radioligand [3H]N-methylspiperone. Although 1 was confirmed as a D2R-preferential agonist, its selectivity in binding and functional studies was lower than previously reported. All analogues were determined to be D2R/D3R agonists in both GoBRET and mitogenesis functional assays. Loss of efficacy was detected for the N-1-substituted analogues at D3R. In contrast, the N-5-alkyl-substituted analogues, and notably the n-butyl-arylamides (22b and 22c), all showed improved affinity at D2R over 1 with neither a loss of efficacy nor an increase in selectivity. Computational modeling provided a structural basis for the D2R selectivity of 1, illustrating how subtle differences in the highly homologous orthosteric binding site (OBS) differentially affect D2R/D3R affinity and functional efficacy. PMID:27035329

Capillary permeability induced by intravenous neurokinins. Receptor characterization and mechanism of action.

PubMed

Jacques, L; Couture, R; Drapeau, G; Regoli, D

1989-08-01

The effects on plasma extravasation of three increasing doses from 6.5 pmol to 650 nmol/kg of substance P (SP), SP fragments, neurokinin A (NKA), neurokinin B (NKB) and selective agonists for neurokinin receptors were assessed in three cutaneous tissues (skin of hind paws, dorsal skin and ears) by intravenous (i.v.) administration in the pentobarbitone anaesthetized rat. Dose-dependent increases in plasma extravasation were observed with the following rank orders of potency (SP greater than NKA greater than NKB) for neurokinins and (SP greater than [p-Glu6]SP(6-11) greater than SP(4-11) greater than [p-Glu5]SP(5-11) greater than SP(7-11] for C-terminal SP fragments. The metabolically stable SP analogue [p-Glu5, MePhe8, Sar9]SP(5-11) was slightly more potent than [p-Glu5]SP(5-11). The N-terminal fragments SP(1-4), SP(1-7) and SP(1-9) were inactive up to 650 nmol/kg. The NK-1 receptor selective agonists [Sar9, Met(O2)11]SP and [beta-Ala4, Sar9, Met (O2)11]SP(4-11) were more potent than the NK-2 [( Nle10]NKA(4-10] and NK-3 [( MePhe7]NKB and [beta-Asp4, MePhe7]NKB(4-10] receptor selective agonists. Plasma extravasation induced by SP (6.5 nmol/kg) was unchanged in the presence of atropine, methysergide, diphenhydramine or during the i.v. and intra-arterial (i.a.) infusion of D-Arg0[Hyp3.D-Phe7]BK, an antagonist of bradykinin. Plasma extravasation induced by SP and [Sar9, Met(O2)11]SP was significantly reduced by indomethacin while that induced by NKA, NKB, [beta-Ala4, Sar9, Met(O2)11]SP(4-11), SP(4-11) and [p-Glu6]SP(6-11) was unaffected by the cyclooxygenase inhibitor. Compound 48/80 (0.75 mg/kg), histamine (10 mg/kg) and 5-HT (10 mg/kg) caused an increase in plasma extravasation, only the effect of compound 48/80 was abolished by indomethacin. Pretreatment with compound 48/80 prevented its own action on plasma extravasation and significantly reduced that induced by 6.5 nmol/kg of SP. These results rule out the involvement of acetylcholine (muscarinic receptors), 5-HT

Novel selective human melanocortin-3 receptor ligands: Use of the 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) scaffold

PubMed Central

Ballet, Steven; Mayorov, Alexander V.; Cai, Minying; Tymecka, Dagmara; Chandler, Kevin B.; Palmer, Erin S.; Van Rompaey, Karolien; Misicka, Aleksandra; Tourwé, Dirk; Hruby, Victor J.

2008-01-01

In search of new selective antagonists and/or agonists for the human melanocortin receptor subtypes hMC1R to hMC5R to elucidate the specific biological roles of each GPCR, we modified the structures of the superagonist MT-II (Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]-NH2) and the hMC3R/hMC4R antagonist SHU9119 (Ac-Nle-c[Asp-His-D-Nal(2′)-Arg-Trp-Lys]-NH2) by replacing the His-D-Phe and His-D-Nal(2′) fragments in MT-II and SHU9119, respectively, with Aba-Xxx (4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one-Xxx) dipeptidomimetics (Xxx = D-Phe/pCl-D-Phe/D-Nal(2′)). Employment of the Aba mimetic yielded novel selective high affinity hMC3R and hMC3R/hMC5R antagonists. PMID:17314042

Antinociceptive potency of a fluorinated cyclopeptide Dmt-c[D-Lys-Phe-p-CF3-Phe-Asp]NH2.

PubMed

Piekielna-Ciesielska, Justyna; Mollica, Adriano; Pieretti, Stefano; Fichna, Jakub; Szymaszkiewicz, Agata; Zielińska, Marta; Kordek, Radzisław; Janecka, Anna

2018-12-01

Opioid peptides and opiate drugs such as morphine, mediate their analgesic effects, but also undesired side effects, mostly through activation of the mu opioid receptor. However, delta- and kappa-opioid receptors can also contribute to the analgesic effects of opioids. Recent findings showed that simultaneous activation of multiple opioid receptors may result in additional analgesia with fewer side effects. Here, we evaluated the pharmacological profile of our formerly developed mixed mu/kappa-opioid receptor ligands, Dmt-c[D-Lys-Phe-Phe-Asp]NH 2 (C-36) and Dmt-c[D-Lys-Phe-p-CF 3 -Phe-Asp]NH 2 (F-81). The ability of these peptides to cross the blood-brain barrier was tested in the parallel artificial membrane permeability (PAMPA) assay. On the basis of the hot-plate test in mice after central and peripheral administration, analog F-81 was selected for the anti-nociceptive and anti-inflammatory activity assessment after peripheral administration.

Identification of Dmt-D-Lys-Phe-Phe-OH as a highly antinociceptive tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20.

PubMed

Kleczkowska, Patrycja; Bojnik, Engin; Leśniak, Anna; Kosson, Piotr; Van den Eynde, Isabelle; Ballet, Steven; Benyhe, Sandor; Tourwé, Dirk; Lipkowski, Andrzej W

2013-01-01

Recently, we presented a novel compound (PK20, Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH) that targets single entity opioid and neurotensin pharmacophores. This endomorphin-2-like opioid peptide was introduced as a highly active analgesic because it elicited a strong dose- and time-dependent antinociceptive response when administered centrally and peripherally. Its pain-relieving activity was observed as rapidly as 5 min after drug injection. Such promising results led us to perform further studies, such as determining the resistance to enzymatic degradation, which resulted in obtaining a very stable opioid pharmacore PK20 metabolite. The synthesis of PK20 and its N-terminal tetrapeptide fragment has been accomplished using solid phase peptide chemistry. The biological stability of peptides has been measured in human serum and analyzed by HPLC/MS. Peptides were pharmacologically characterized in in vitro MOP and DOP receptor binding as well as [(35)S]GTPγS receptor binding assays. Antinociceptive properties of compounds were measured by in vivo assays in C57Bl6 mice after intravenous or intrathecal applications. Dmt-D-Lys-Phe-Phe-OH (PK20M), an N-terminal tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20, is characterized by a long duration of action, as demonstrated by a preserved, long-lasting analgesic effect even 2 h post-injection (average % MPE = 69.33). In rat brain membranes, PK20M efficiently displaced both the MOP and DOP receptor selective radioprobes [(3)H]DAMGO and [(3)H]DIDI (pKi of 9.52 and 7.86, respectively) and potently stimulated [(35)S]GTPγS binding, proving full agonism at both receptor types. In the [(35)S]GTPγS assay, which measured the agonist-mediated G protein activation, PK20M together with PK20 and Met-enkephalin were potent stimulators of the regulatory G proteins. The relative affinities of PK20M for the μ and δ receptor subtypes revealed μ-receptor selectivity. The novel MOP receptor selective metabolite

Crystal and NMR Structures of a Peptidomimetic β-Turn That Provides Facile Synthesis of 13-Membered Cyclic Tetrapeptides.

PubMed

Cameron, Alan J; Squire, Christopher J; Edwards, Patrick J B; Harjes, Elena; Sarojini, Vijayalekshmi

2017-12-14

Herein we report the unique conformations adopted by linear and cyclic tetrapeptides (CTPs) containing 2-aminobenzoic acid (2-Abz) in solution and as single crystals. The crystal structure of the linear tetrapeptide H 2 N-d-Leu-d-Phe-2-Abz-d-Ala-COOH (1) reveals a novel planar peptidomimetic β-turn stabilized by three hydrogen bonds and is in agreement with its NMR structure in solution. While CTPs are often synthetically inaccessible or cyclize in poor yield, both 1 and its N-Me-d-Phe analogue (2) adopt pseudo-cyclic frameworks enabling near quantitative conversion to the corresponding CTPs 3 and 4. The crystal structure of the N-methylated peptide (4) is the first reported for a CTP containing 2-Abz and reveals a distinctly planar 13-membered ring, which is also evident in solution. The N-methylation of d-Phe results in a peptide bond inversion compared to the conformation of 3 in solution. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The rabbit iris sphincter contains NK1 and NK3 but not NK2 receptors: a study with selective agonists and antagonists.

PubMed

Wang, Z Y; Håkanson, R

1993-04-08

Tachykinin analogues, claimed to be selective NK1, NK2 and NK3 receptor agonists, contracted the isolated rabbit iris sphincter muscle in a concentration-dependent manner. The contractions were not modified by the enkephalinase inhibitor thiorphan and the angiotensin-converting enzyme inhibitor captopril (10(-5) M of each). The pD2 values for (Sar9,Met(O2)11)SP (NK1 receptor agonist), (Nle10)NKA(4-10) (NK2 receptor agonist) and (MePhe7)NKB (NK3 receptor agonist) were 8.3, 6.1 and 8.2, respectively. (Sar9,Met(O2)11)SP was the most efficacious of the three agonists. The results are compatible with the presence of NK1 and NK3 receptors. The low pD2 value for the NK2 agonist may reflect a lack of NK2 receptors and interaction of the NK2 agonist with NK1 receptors. The contraction caused by the NK1 receptor agonist was inhibited competitively by the highly selective NK1 receptor antagonist (+/-) CP-96,345; the pA2 value was 5.5. Also the contraction caused by the NK2 receptor agonist was inhibited competitively by (+/-) CP-96,345 with a pA2 value of 5.7, supporting the view that the two agonists (Sar9,Met(O2)11)SP and (Nle10)NKA(4-10) interact with the same receptor. The selective NK2 receptor antagonist actinomycin D did not affect the contraction caused by the NK2 receptor agonist. We conclude that the rabbit iris sphincter muscle contains NK1 and probably NK3 receptors. We obtained no evidence for the presence of NK2 receptors.

Thermally activated persistent photoconductivity & donor binding energy in high mobility AlAs QWs

NASA Astrophysics Data System (ADS)

Dasgupta, S.; Knaak, C.; Fontcuberta, A.; Bichler, M.; Abstreiter, G.; Grayson, M.

2008-03-01

In AlAs, valley index is important quantum number which can help understand interactions. However, important parameters for growth such as donor binding energy and Si δ-doping efficiency were unknown and AlAs quantum wells (QWs) typically did not conduct in dark. We grew series of (001) and (110) oriented double-sided doped n-type AlAs QWs and deduced Si donor binding energy δ in Al0.45Ga0.55 As and doping efficiency η. They work in dark possibly because dilute charge traps in substrate are screened by backside doping. From dark saturation density for doping series we deduced δdk=65.2 meV [1]. Our studies show thermally activated PPC where sample is illuminated at 4 K and returned to dark without appreciable density increase. As temperature is increased to 30 K, density doubles, indicating shallow binding energy δPIA=0 meV post-illumination anneal (PIA). Doping efficiency after illumination for (001) facet was found to be η=n2D/nSi=35% and for (110) η=17%. With this understanding, we designed (001) AlAs QW with PIA density n=2.4 x 10^11 cm-2 and mobility μ=4.3 x 10^5 cm^2/Vs(330 mK), improvement of almost an order of magnitude over published results. [1] Dasgupta, et al. APL (2007)

Structure-activity relationship studies and pharmacological characterization of N5-heteroarylalkyl-substituted-2-(2-furanyl)thiazolo[5,4-d]pyrimidine-5,7-diamine-based derivatives as inverse agonists at human A2A adenosine receptor.

PubMed

Varano, Flavia; Catarzi, Daniela; Vincenzi, Fabrizio; Falsini, Matteo; Pasquini, Silvia; Borea, Pier Andrea; Colotta, Vittoria; Varani, Katia

2018-06-09

This paper describes the synthesis and characterization of N 5 -(hetero)arylalkyl-substituted-thiazolo [5,4-d]pyrimidine-5,7-diamine derivatives (4-19) as novel human (h) A 2A adenosine receptor (AR) inverse agonists. Competition binding and cyclic AMP assays indicate that the examined compounds behave as hA 2A AR inverse agonists showing binding affinity values in the nanomolar or subnanomolar range. Notably, compounds 4, 5, 6 and 11 showed two affinity values for the hA 2A ARs with the highest (KH) falling in the femtomolar range and the lowest (KL) of the nanomolar order. In addition, in cyclic AMP assays, compounds 4, 5, 6 and 11 exhibited potency (IC 50 ) values in the picomolar range. This study has confirmed that 2-(2-furanyl)thiazolo [5,4-d]pyrimidine-5,7-diamine-based derivatives represent a unique new class of hA 2A AR inverse agonists. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Characterization of opioid receptors that modulate nociceptive neurotransmission in the trigeminocervical complex

PubMed Central

Storer, R J; Akerman, S; Goadsby, P J

2003-01-01

Opioid agonists have been used for many years to treat all forms of headache, including migraine. We sought to characterize opioid receptors involved in craniovascular nociceptive pathways by in vivo microiontophoresis of μ-receptor agonists and antagonists onto neurons in the trigeminocervical complex of the cat. Cats were anaesthetized with α-chloralose 60 mg kg−1, i.p. and 20 mg kg−1, i.v. supplements after induction and surgical preparation using halothane. Units were identified in the trigeminocervical complex responding to supramaximal electrical stimulation of the superior sagittal sinus, and extracellular recordings of activity made. Seven- or nine-barrelled glass micropipettes incorporating tungsten recording electrodes in their centre barrels were used for microiontophoresis of test substances onto cell bodies. Superior sagittal sinus (SSS)-linked cells whose firing was evoked by microiontophoretic application of L-glutamate (n=8 cells) were reversibly inhibited by microiontophoresis of H2N-Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) (n=12), a selective μ-receptor agonist, in a dose dependent manner, but not by control ejection of sodium or chloride ions from a barrel containing saline. The inhibition by DAMGO of SSS-linked neurons activated with L-glutamate could be antagonized by microiontophoresis of selective μ-receptor antagonists D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) or D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), or both, in all cells tested (n=4 and 6, respectively). Local iontophoresis of DAMGO during stimulation of the superior sagittal sinus resulted in a reduction in SSS-evoked activity. This effect was substantially reversed 10 min after cessation of iontophoresis. The effect of DAMGO was markedly inhibited by co-iontophoresis of CTAP. Thus, we found that μ-receptors modulate nociceptive input to the trigeminocervical complex. Characterizing the sub-types of opioid receptors that influence trigeminovascular nociceptive

Molecular modeling of the human vasopressin V2 receptor/agonist complex

NASA Astrophysics Data System (ADS)

Czaplewski, Cezary; Kaźmierkiewicz, Rajmund; Ciarkowski, Jerzy

1998-05-01

The V2 vasopressin renal receptor (V2R), which controls antidiuresis in mammals, is a member of the large family of heptahelical transmembrane (7TM) G protein-coupled receptors (GPCRs). Using the automated GPCR modeling facility available via Internet (http://expasy.hcuge.ch/swissmod/SWISS-MODEL.html) for construction of the 7TM domain in accord with the bovine rhodopsin (RD) footprint, and the SYBYL software for addition of the intra- and extracellular domains, the human V2R was modeled. The structure was further refined and its conformational variability tested by the use of a version of the Constrained Simulated Annealing (CSA) protocol developed in this laboratory. An inspection of the resulting structure reveals that the V2R (likewise any GPCR modeled this way) is much thicker and accordingly forms a more spacious TM cavity than most of the hitherto modeled GPCR constructs do, typically based on the structure of bacteriorhodopsin (BRD). Moreover, in this model the 7TM helices are arranged differently than they are in any BRD-based model. Thus, the topology and geometry of the TM cavity, potentially capable of receiving ligands, is in this model quite different than it is in the earlier models. In the subsequent step, two ligands, the native [arginine8]vasopressin (AVP) and the selective agonist [d-arginine8]vasopressin (DAVP) were inserted, each in two topologically non-equivalent ways, into the TM cavity and the resulting structures were equilibrated and their conformational variabilities tested using CSA as above. The best docking was selected and justified upon consideration of ligand-receptor interactions and structure-activity data. Finally, the amino acid residues were indicated, mainly in TM helices 3-7, as potentially important in both AVP and DAVP docking. Among those Cys112, Val115-Lys116, Gln119, Met123 in helix 3; Glu174 in helix 4; Val206, Ala210, Val213-Phe214 in helix 5; Trp284, Phe287-Phe288, Gln291 in helix 6; and Phe307, Leu310, Ala314 and

Effects of receptor-selective neurokinin agonists and a neurokinin antagonist on the electrical activity of spinal cord neurones in culture.

PubMed

Wienrich, M; Reuss, K; Harting, J

1989-11-01

1. Rat spinal cord neurones grown in tissue culture were used to examine the electrophysiological effects of the neurokin in (NK)-selective agonists (pGlu6, Pro9) substance P(6-11) (septide; NK1, 10(-6)M) and (pGlu5, MePhe8, MeGly9)SP(1-7) (DiMe-C7; NK3, 10(-6)M). In addition, the effect of the neurokinin antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11)SP (10(-5)M) on the neurokinin-evoked responses was investigated. 2. Neurokinin-evoked responses consisted of an increase in neuronal activity with or without long-lasting (mean: 50s) depolarizations of the membrane potential of up to 25mV. The latter also occurred in the presence of tetrodotoxin (10(-7)M) (direct response). 3. In a number of spinal cord neurones (n = 17) only septide induced a membrane depolarization while DiMe-C7 elicited no response. On the other hand, in 2 neurones a response was exclusively evoked by DiMe-C7. 4. The neurokinin antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11)SP had no effect of its own but blocked the septide- and DiMe-C7-induced depolarizations. It had no effect on the glutamate (10(-5)M)-evoked depolarization. 5. It is concluded that by the use of neurokinin receptor-selective agonists, subpopulations of spinal cord neurones in primary dissociated cell culture can be differentiated which express the NK1 or the NK3 receptor. Cells expressing only the NK1 receptor outnumber those expressing only the NK3 receptor subtype. Both receptors can be blocked by the neurokinin antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11)SP.

Controlled self-assembling structures of ferrocene-dipeptide conjugates composed of Ala-Pro-NHCH2CH2SH chain.

PubMed

Moriuchi, Toshiyuki; Nishiyama, Taiki; Tayano, Yoshiki; Hirao, Toshikazu

2017-12-01

Bioorganometallic ferrocene-dipeptide conjugates with the Ala-Pro-cysteamine chain, Fc-L-Ala-L-Pro-NHCH 2 CH 2 SH (2) and Fc-L-Ala-D-Pro-NHCH 2 CH 2 SH (4) (Fc=ferrocenoyl), were prepared by the reduction of the ferrocene-dipeptide conjugates, Fc-L-Ala-L-Pro-cystamine-L-Pro-L-Ala-Fc (1) or Fc-L-Ala-D-Pro-cystamine-D-Pro-L-Ala-Fc (3), respectively. Control of the self-assembling structures of the ferrocene-dipeptide conjugates was demonstrated by changing the chirality of the amino acid. The molecular structure of 2 composed of the L-Ala-L-Pro-NHCH 2 CH 2 SH chain confirmed the formation of intramolecular hydrogen bond of N-H⋯N pattern between the NH of cysteamine moiety and the nitrogen of Pro moiety. Furthermore, intermolecular hydrogen bonds between NH (Ala) and CO (Pro of another molecule) and between NH (cysteamine) and CO (the ferrocenoyl moiety of another molecule) were formed, wherein each molecule is connected to four neighboring molecules by continuous intermolecular hydrogen bonds to form the hydrogen-bonded molecular assembling structure. On the contrary, the left-handed helical assembly through an intermolecular hydrogen-bonding network of 15-membered intermolecularly hydrogen-bonded ring between NH (Ala) and CO (the ferrocenoyl moiety of another molecule) and between NH (the cysteamine moiety of another molecule) and CO (Ala) was observed in the crystal packing of 4 composed of the L-Ala-D-Pro-NHCH 2 CH 2 SH chain. Copyright © 2017 Elsevier Inc. All rights reserved.

New antagonists of LHRH. II. Inhibition and potentiation of LHRH by closely related analogues.

PubMed

Bajusz, S; Csernus, V J; Janaky, T; Bokser, L; Fekete, M; Schally, A V

1988-12-01

Modifications of the previously described LHRH antagonists, [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and the corresponding D-Hci6 analogue, have been made to alter the hydrophobicity of the N-terminal acetyl-tripeptide portion. Substitution of D-Trp3 with the less hydrophobic D-Pal(3) had only marginal effects on the antagonistic activities and receptor binding potencies of the D-Cit/D-Hci6 analogues, but it appeared to further improve the toxicity lowering effect of D-Cit/D-Hci6 substitution. Antagonists containing D-Pal(3)3 and D-Cit/D-Hci6 residues, i.e. [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH (SB-75) and [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Hci6, D-Ala10]LHRH (SB-88), were completely free of the toxic effects, such as cyanosis and respiratory depression leading to death, which have been observed in rats with the D-Trp3, D-Arg6 antagonist and related antagonists. Replacement of the N-acetyl group with the hydrophilic carbamoyl group caused a slight decrease in antagonistic activities, particularly in vitro. Introduction of urethane type acyl group such as methoxycarbonyl (Moc) or t-butoxycarbonyl (Boc) led to analogues that showed LHRH-potentiating effect. The increase in potency induced by these analogues, e.g. [Moc-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and [Boc-D-Phe1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH, was 170-260% and persisted for more than 2 h when studied in a superfused rat pituitary system.

Free-Operant Avoidance Behavior by Rats after Reinforcer Revaluation Using Opioid Agonists and d-Amphetamine

PubMed Central

Urcelay, Gonzalo; Mar, Adam; Dickinson, Anthony; Robbins, Trevor

2014-01-01

The associative processes that support free-operant instrumental avoidance behavior are still unknown. We used a revaluation procedure to determine whether the performance of an avoidance response is sensitive to the current value of the aversive, negative reinforcer. Rats were trained on an unsignaled, free-operant lever press avoidance paradigm in which each response avoided or escaped shock and produced a 5 s feedback stimulus. The revaluation procedure consisted of noncontingent presentations of the shock in the absence of the lever either paired or unpaired with systemic morphine and in a different cohort with systemic d-amphetamine. Rats were then tested drug free during an extinction test. In both the d-amphetamine and morphine groups, pairing of the drug and shock decreased subsequent avoidance responding during the extinction test, suggesting that avoidance behavior was sensitive to the current incentive value of the aversive negative reinforcer. Experiment 2 used central infusions of D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO), a mu-opioid receptor agonist, in the periacqueductal gray and nucleus accumbens shell to revalue the shock. Infusions of DAMGO in both regions replicated the effects seen with systemic morphine. These results are the first to demonstrate the impact of revaluation of an aversive reinforcer on avoidance behavior using pharmacological agents, thereby providing potential therapeutic targets for the treatment of avoidance behavior symptomatic of anxiety disorders. PMID:24790199

Pharmacological characterization of 30 human melanocortin-4 receptor polymorphisms with the endogenous proopiomelanocortin-derived agonists, synthetic agonists, and the endogenous agouti-related protein antagonist.

PubMed

Xiang, Zhimin; Proneth, Bettina; Dirain, Marvin L; Litherland, Sally A; Haskell-Luevano, Carrie

2010-06-08

The melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic biomarker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and nonobese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [alpha-, beta-, and gamma(2)-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87-132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-dPhe-Arg-Trp-NH(2) (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4R's and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface

The Unique α4(+)/(−)α4 Agonist Binding Site in (α4)3(β2)2 Subtype Nicotinic Acetylcholine Receptors Permits Differential Agonist Desensitization Pharmacology versus the (α4)2(β2)3 Subtype

PubMed Central

Eaton, J. Brek; Lucero, Linda M.; Stratton, Harrison; Chang, Yongchang; Cooper, John F.; Lindstrom, Jon M.; Lukas, Ronald J.

2014-01-01

Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (α4)2(β2)3) or low-sensitivity (LS) (α4)3(β2)2) isoforms of human α4β2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using 86Rb+ efflux in a stably transfected SH-EP1-hα4β2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS α4β2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced α4β2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only α4(+)/(−)α4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using β2-subunit–specific [125I]mAb295 labeling. However, maximum function is approximately five times greater on a “per-receptor” basis for unmodified LSP versus HSP α4β2-nAChRs. Thus, recruitment of the α4(+)/(−)α4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of α4(+)/(−)β2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two α4β2-nAChR isoforms, and demonstrate that HS versus LS α4β2-nAChR activity can be selectively manipulated using pharmacological approaches. Since α4β2 nAChRs are the predominant neuronal subtype, these discoveries likely

Enhancement of blood-tumor barrier permeability by Sar-[D-Phe8]des-Arg9BK, a metabolically resistant bradykinin B1 agonist, in a rat C6 glioma model

PubMed Central

Cardoso, Ronie Cleverson; Lobão-Soares, Bruno; Bianchin, Marino Muxfeldt; Carlotti, Carlos Gilberto; Walz, Roger; Alvarez-Silva, Márcio; Trentin, Andréa Gonçalves; Nicolau, Mauro

2004-01-01

Background While it is well known that bradykinin B2 agonists increase plasma protein extravasation (PPE) in brain tumors, the bradykinin B1 agonists tested thus far are unable to produce this effect. Here we examine the effect of the selective B1 agonist bradykinin (BK) Sar-[D-Phe8]des-Arg9BK (SAR), a compound resistant to enzymatic degradation with prolonged activity on PPE in the blood circulation in the C6 rat glioma model. Results SAR administration significantly enhanced PPE in C6 rat brain glioma compared to saline or BK (p < 0.01). Pre-administration of the bradykinin B1 antagonist [Leu8]-des-Arg (100 nmol/Kg) blocked the SAR-induced PPE in the tumor area. Conclusions Our data suggest that the B1 receptor modulates PPE in the blood tumor barrier of C6 glioma. A possible role for the use of SAR in the chemotherapy of gliomas deserves further study. PMID:15458573

Substitution of the Lys Linker with the β-Ala Linker Dramatically Decreased the Renal Uptake of 99mTc-Labeled Arg-X-Asp-Conjugated and X-Ala-Asp-Conjugated α-Melanocyte Stimulating Hormone Peptides

PubMed Central

2015-01-01

The purpose of this study was to examine whether the substitution of the Lys linker with the β-Ala could reduce the renal uptake of 99mTc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-β-Ala-(Arg11)CCMSH (1) {c[Arg-Ser-Asp-dTyr-Asp]-β-Ala-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RTD-β-Ala-(Arg11)CCMSH (2), RVD-β-Ala-(Arg11)CCMSH (3), RAD-β-Ala-(Arg11)CCMSH (4), NAD-β-Ala-(Arg11)CCMSH (5), and EAD-β-Ala-(Arg11)CCMSH (6) peptides were synthesized and evaluated for their melanocortin 1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of their 99mTc-conjugates were determined in B16/F1 melanoma-bearing C57 mice. The substitution of the Lys linker with β-Ala linker dramatically reduced the renal uptake of all six 99mTc-peptides. 99mTc-4 exhibited the highest melanoma uptake (15.66 ± 6.19% ID/g) and the lowest kidney uptake (20.18 ± 3.86% ID/g) among these 99mTc-peptides at 2 h postinjection. The B16/F1 melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT)/CT using 99mTc-4 as an imaging probe. PMID:25290883

Substitution of the Lys linker with the β-Ala linker dramatically decreased the renal uptake of 99mTc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone peptides.

PubMed

Flook, Adam M; Yang, Jianquan; Miao, Yubin

2014-11-13

The purpose of this study was to examine whether the substitution of the Lys linker with the β-Ala could reduce the renal uptake of (99m)Tc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-β-Ala-(Arg(11))CCMSH (1) {c[Arg-Ser-Asp-dTyr-Asp]-β-Ala-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RTD-β-Ala-(Arg(11))CCMSH (2), RVD-β-Ala-(Arg(11))CCMSH (3), RAD-β-Ala-(Arg(11))CCMSH (4), NAD-β-Ala-(Arg(11))CCMSH (5), and EAD-β-Ala-(Arg(11))CCMSH (6) peptides were synthesized and evaluated for their melanocortin 1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of their (99m)Tc-conjugates were determined in B16/F1 melanoma-bearing C57 mice. The substitution of the Lys linker with β-Ala linker dramatically reduced the renal uptake of all six (99m)Tc-peptides. (99m)Tc-4 exhibited the highest melanoma uptake (15.66 ± 6.19% ID/g) and the lowest kidney uptake (20.18 ± 3.86% ID/g) among these (99m)Tc-peptides at 2 h postinjection. The B16/F1 melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT)/CT using (99m)Tc-4 as an imaging probe.

Studies on the vascular permeability induced by intrathecal substance P and bradykinin in the rat.

PubMed

Jacques, L; Couture, R

1990-08-02

The effects of substance P (SP), SP fragments, neurokinin A (NKA), neurokinin B (NKB) and selective agonists for neurokinin receptors were assessed on cutaneous vascular permeability after intrathecal (i.t.) administration in rats. Dose-dependent increases in plasma extravasation were observed with the following rank orders of potency ([p-Glu6]SP-(6-11) greater than SP greater than or equal to SP-(4-11) greater than [p-Glu5,MePhe8,Sar9]SP-(5-11) = [p-Glu5]SP-(5-11) greater than SP-(7-11) and SP greater than NKA greater than NKB). The N-terminal fragments SP-(1-4), SP-(1-7) and SP-(1-9) were inactive up to 65 nmol. The NK-1 receptor selective agonists [( beta-Ala4,Sar9,Met(O2)11]SP-(4-11) and [Pro9,Met(O2)11]SP) were more potent than the NK-2 ([Nle10]NKA-(4-10] and NK-3 ([beta-Asp4,MePhe7]NKB-(4-10) and [MePhe7]NKB) receptor-selective agonists. Plasma extravasation was also increased by i.t. bradykinin (BK, 8.1 nmol) while the fragment BK-(1-8), a potent B1-receptor-selective agonist, produced only a slight effect at 81 nmol. When BK was given after prior i.t. administration of 6.1 nmol of [Thi5.8,D-Phe7]BK, an antagonist of BK at the B2-receptor, the increase in vascular permeability was significantly attenuated. The analogue [Leu8]BK-(1-8) (10.3 nmol), an antagonist of BK at the B1-receptor, failed to modify the BK-induced plasma extravasation. Plasma extravasation induced by SP (6.5 nmol) and BK (8.1 nmol) was abolished in cervically vagotomized rats, and significantly reduced in both spinal rats and in capsaicin-treated animals. Conversely, bilateral adrenalectomy (48 h earlier) and intercollicular decerebration (30 min earlier) had no major effect on the response elicited either by SP or BK. The response to SP remained unaffected by methysergide and hexamethonium but was significantly reduced by methylnitrate atropine and diphenhydramine. Indomethacin significantly enhanced the plasma extravasation induced by SP. These results suggest that SP and BK may play a

Novel sst2-selective somatostatin agonists. Three-dimensional consensus structure by NMR

PubMed Central

Grace, Christy Rani R.; Erchegyi, Judit; Koerber, Steven C.; Reubi, Jean Claude; Rivier, Jean; Riek, Roland

2008-01-01

The three-dimensional NMR structures of six octapeptide agonist analogues of somatostatin (SRIF) in the free form are described. These analogues, with the basic sequence H-DPhe/Phe2-c[Cys3-Xxx7-DTrp8-Lys9-Thr10-Cys14]-Thr-NH2 (the numbering refers to the position in native SRIF), with Xxx7 being Ala/Aph, exhibit potent and highly selective binding to human SRIF type 2 (sst2) receptors. The backbone of these sst2-selective analogues have the usual type-II’ β-turn reported in the literature for sst2/3/5-subtype-selective analogues. Correlating biological results and NMR studies led to the identification of the side chains of DPhe2, DTrp8 and Lys9 as the necessary components of the sst2 pharmacophore. This is the first study to show that the aromatic ring at position 7 (Phe7) is not critical for sst2 binding and that it plays an important role in sst3 and sst5 binding. This pharmacophore is therefore different from that proposed by others for sst2/3/5 analogues. PMID:16854054

Accumulation of polyubiquitylated proteins in response to Ala-Ala-Phe-chloromethylketone is independent of the inhibition of Tripeptidyl peptidase II.

PubMed

Villasevil, Eugenia M; Guil, Sara; López-Ferreras, Lorena; Sánchez, Carlos; Del Val, Margarita; Antón, Luis C

2010-09-01

In the present study we have addressed the issue of proteasome independent cytosolic protein degradation. Tripeptidyl peptidase II (TPPII) has been suggested to compensate for a reduced proteasome activity, partly based on evidence using the inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk). Here we show that AAF-cmk induces the formation of polyubiquitin-containing accumulations in osteosarcoma and Burkitt's lymphoma cell lines. These accumulations meet many of the landmarks of the aggresomes that form after proteasome inhibition. Using a combination of experiments with chemical inhibitors and interference of gene expression, we show that TPPII inhibition is not responsible for these accumulations. Our evidence suggests that the relevant target(s) is/are in the ubiquitin-proteasome pathway, most likely upstream the proteasome. We obtained evidence supporting this model by inhibition of Hsp90, which also acts upstream the proteasome. Although our data suggest that Hsp90 is not a target of AAF-cmk, its inhibition resulted in accumulations similar to those obtained with AAF-cmk. Therefore, our results question the proposed role for TPPII as a prominent alternative to the proteasome in cellular proteolysis. Copyright 2010 Elsevier B.V. All rights reserved.

Tyrocidine A Analogues Bearing the Planar d-Phe-2-Abz Turn Motif: How Conformation Impacts Bioactivity.

PubMed

Cameron, Alan J; Edwards, Patrick J B; Harjes, Elena; Sarojini, Vijayalekshmi

2017-12-14

The d-Phe-Pro β-turn of the cyclic β-hairpin antimicrobial decapeptide tyrocidine A, (Tyrc A) was substituted with the d-Phe-2-aminobenzoic acid (2-Abz) motif in a synthetic analogue (1). The NMR structure of 1 demonstrated that compound 1 retained the β-hairpin structure of Tyrc A with additional planarity, resulting in approximately 30-fold reduced hemolysis than Tyrc A. Although antibacterial activity was partially compromised, a single Gln to Lys substitution (2) restored activity equivalent to Tyrc A against S. aureus, enhanced activity against two Gram negative strains and maintained the reduced hemeloysis of 1. Analysis by transmission electron microscopy (TEM) suggested a membrane lytic mechanism of action for these peptides. Compound 2 also exhibits nanomolar antifungal activity in synergy with amphotericin B. The d-Phe-2-Abz turn may serve as a tool for the synthesis of structurally predictable β-hairpin libraries. Unlike traditional β-turn motifs such as d-Pro-Gly, both the 2-Abz and d-Phe rings may be further functionalized.

Structure of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser of Aβ-peptide with phospholipase A2 from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution.

PubMed

Mirza, Zeenat; Pillai, Vikram Gopalakrishna; Zhong, Wei-Zhu

2014-03-10

Alzheimer's disease (AD) is one of the most significant social and health burdens of the present century. Plaques formed by extracellular deposits of amyloid β (Aβ) are the prime player of AD's neuropathology. Studies have implicated the varied role of phospholipase A2 (PLA2) in brain where it contributes to neuronal growth and inflammatory response. Overall contour and chemical nature of the substrate-binding channel in the low molecular weight PLA2s are similar. This study involves the reductionist fragment-based approach to understand the structure adopted by N-terminal fragment of Alzheimer's Aβ peptide in its complex with PLA2. In the current communication, we report the structure determined by X-ray crystallography of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser (DAEFRHDS) of Aβ-peptide with a Group I PLA2 purified from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution (Protein Data Bank (PDB) Code: 3JQ5). This is probably the first attempt to structurally establish interaction between amyloid-β peptide fragment and hydrophobic substrate binding site of PLA2 involving H bond and van der Waals interactions. We speculate that higher affinity between Aβ and PLA2 has the therapeutic potential of decreasing the Aβ-Aβ interaction, thereby reducing the amyloid aggregation and plaque formation in AD.

Potent agonists of growth hormone-releasing hormone. Part I.

PubMed

Zarandi, M; Serfozo, P; Zsigo, J; Bokser, L; Janaky, T; Olsen, D B; Bajusz, S; Schally, A V

1992-03-01

Analogs of the 29 amino acid sequence of growth hormone-releasing hormone (GH-RH) with agmatine (Agm) in position 29 have been synthesized by the solid phase method, purified, and tested in vitro and in vivo. The majority of the analogs contained desaminotyrosine (Dat) in position 1, but a few of them had Tyr1, or N-MeTyr1. Some peptides contained one or more additional L- or D-amino acid substitutions in positions 2, 12, 15, 21, 27, and/or 28. Compared to the natural sequence of GH-RH(1-29)NH2, [Dat1,Ala15]GH-RH(1-28)Agm (MZ-3-191) and [D-Ala2,Ala15]GH-RH(1-28)Agm (MZ-3-201) were 8.2 and 7.1 times more potent in vitro, respectively. These two peptides contained Met27. Their Nle27 analogs, [Dat1,Ala15,Nle27]GH-RH(1-28)Agm(MZ-2-51), prepared previously (9), and [D-Ala2,Ala15,Nle28]GH-RH(1-28)Agm(MZ-3-195) showed relative in vitro potencies of 10.5 and 2.4, respectively. These data indicate that replacement of Met27 by Nle27 enhanced the GH-releasing activity of the analog when the molecule contained Dat1-Ala2 residues at the N-terminus, but peptides containing Tyr1-D-Ala2 in addition to Nle27 showed decreased potencies. Replacement of Ser28 with Asp in multi-substituted analogs of GH-RH(1-28)Agm resulted in a decrease in in vitro potencies compared to the parent compound. Thus, the Ser28-containing MZ-2-51, and [Dat1,Ala15,D-Lys21,Nle27]GH-RH(1-28)Agm, its Asp28 homolog (MZ-3-149), possessed relative activities of 10.5 and 5.6, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Tachykinin receptors in the circular muscle of the guinea-pig ileum.

PubMed Central

Maggi, C. A.; Patacchini, R.; Giachetti, A.; Meli, A.

1990-01-01

1. We have studied the mechanical response of circular strips of the guinea-pig ileum to tachykinins and characterized the receptors involved by means of receptor-selective agonists. 2. The strips responded to both substance P (SP) and neurokinin A (NKA), as well as to [Pro9]-SP sulphone (selective NK1-receptor agonist), [beta Ala8]-NKA(4-10) (selective NK2-receptor agonist) and [MePhe7]-neurokinin B (selective NK3-receptor agonist). The ED50s of the various peptides (calculated as the concentration of agonist which produced 50% of the response to 10 microM carbachol) were similar, in the range of 40-200 nM, i.e. no clearcut rank order of potency was evident. 3. The response to a submaximal (10 nM) concentration of SP or NKA was unaffected in the presence of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 4. The response to the NK1-agonist was totally atropine-resistant, but was reduced (about 30% inhibition) by tetrodotoxin. The response to the NK3-receptor agonist was halved by atropine and abolished by tetrodotoxin. The response to the NK2-agonist was unaffected by either atropine or tetrodotoxin. 5. The response to the selective NK2-agonist was unchanged after desensitization of NK1- or NK3-receptors. 6. The response to the NK2-selective agonist was strongly inhibited by [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10) (MEN 10,207) a selective NK2-receptor antagonist which did not modify the response to the NK1-selective agonist. 7. Our findings indicate that all the three known types of tachykinin receptors mediate the contractile response of the circular muscle of the guinea-pig ileum to peptides of this family.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1707710

Desleucyl-Oritavancin with a Damaged d-Ala-d-Ala Binding Site Inhibits the Transpeptidation Step of Cell-Wall Biosynthesis in Whole Cells of Staphylococcus aureus.

PubMed

Kim, Sung Joon; Singh, Manmilan; Sharif, Shasad; Schaefer, Jacob

2017-03-14

We have used solid-state nuclear magnetic resonance to characterize the exact nature of the dual mode of action of oritavancin in preventing cell-wall assembly in Staphylococcus aureus. Measurements performed on whole cells labeled selectively in vivo have established that des-N-methylleucyl-N-4-(4-fluorophenyl)benzyl-chloroeremomycin, an Edman degradation product of [ 19 F]oritavancin, which has a damaged d-Ala-d-Ala binding aglycon, is a potent inhibitor of the transpeptidase activity of cell-wall biosynthesis. The desleucyl drug binds to partially cross-linked peptidoglycan by a cleft formed between the drug aglycon and its biphenyl hydrophobic side chain. This type of binding site is present in other oritavancin-like glycopeptides, which suggests that for these drugs a similar transpeptidase inhibition occurs.

Rotigotine is a potent agonist at dopamine D1 receptors as well as at dopamine D2 and D3 receptors.

PubMed

Wood, Martyn; Dubois, Vanessa; Scheller, Dieter; Gillard, Michel

2015-02-01

Rotigotine acts as a dopamine receptor agonist with high affinity for the dopamine D2, D3, D4 and D5 receptors but with a low affinity for the dopamine D1 receptor. We have investigated this further in radioligand binding and functional studies and compared the profile of rotigotine with that of other drugs used in the treatment of Parkinson's disease (PD). The binding of rotigotine to human dopamine D1, D2, D3, D4 and D5 receptors was determined in radioligand binding studies using [(3)H]rotigotine and compared with that of standard antagonist radioligands. Functional interactions of rotigotine with human dopamine receptors was also determined. [(3)H]rotigotine can be used as an agonist radioligand to label all dopamine receptor subtypes and this can be important to derive agonist affinity estimates. Rotigotine maintains this high affinity in functional studies at all dopamine receptors especially D1, D2 and D3 receptors and, to a lesser extent, D4 and D5 receptors. Rotigotine, like apomorphine but unlike ropinirole and pramipexole, was a potent agonist at all dopamine receptors. Rotigotine is a high-potency agonist at human dopamine D1, D2 and D3 receptors with a lower potency at D4 and D5 receptors. These studies differentiate rotigotine from conventional dopamine D2 agonists, used in the treatment of PD, such as ropinirole and pramipexole which lack activity at the D1 and D5 receptors, but resembles that of apomorphine which has greater efficacy in PD than other dopamine agonists but has suboptimal pharmacokinetic properties. © 2014 The British Pharmacological Society.

Rotigotine is a potent agonist at dopamine D1 receptors as well as at dopamine D2 and D3 receptors

PubMed Central

Wood, Martyn; Dubois, Vanessa; Scheller, Dieter; Gillard, Michel

2015-01-01

Background and Purpose Rotigotine acts as a dopamine receptor agonist with high affinity for the dopamine D2, D3, D4 and D5 receptors but with a low affinity for the dopamine D1 receptor. We have investigated this further in radioligand binding and functional studies and compared the profile of rotigotine with that of other drugs used in the treatment of Parkinson's disease (PD). Experimental Approach The binding of rotigotine to human dopamine D1, D2, D3, D4 and D5 receptors was determined in radioligand binding studies using [3H]rotigotine and compared with that of standard antagonist radioligands. Functional interactions of rotigotine with human dopamine receptors was also determined. Key Results [3H]rotigotine can be used as an agonist radioligand to label all dopamine receptor subtypes and this can be important to derive agonist affinity estimates. Rotigotine maintains this high affinity in functional studies at all dopamine receptors especially D1, D2 and D3 receptors and, to a lesser extent, D4 and D5 receptors. Rotigotine, like apomorphine but unlike ropinirole and pramipexole, was a potent agonist at all dopamine receptors. Conclusions and Implications Rotigotine is a high-potency agonist at human dopamine D1, D2 and D3 receptors with a lower potency at D4 and D5 receptors. These studies differentiate rotigotine from conventional dopamine D2 agonists, used in the treatment of PD, such as ropinirole and pramipexole which lack activity at the D1 and D5 receptors, but resembles that of apomorphine which has greater efficacy in PD than other dopamine agonists but has suboptimal pharmacokinetic properties. PMID:25339241

Novel kinin B1 receptor agonists with improved pharmacological profiles.

PubMed

Côté, Jérôme; Savard, Martin; Bovenzi, Veronica; Bélanger, Simon; Morin, Josée; Neugebauer, Witold; Larouche, Annie; Dubuc, Céléna; Gobeil, Fernand

2009-04-01

There is some evidence to suggest that inducible kinin B1 receptors (B1R) may play beneficial and protecting roles in cardiovascular-related pathologies such as hypertension, diabetes, and ischemic organ diseases. Peptide B1R agonists bearing optimized pharmacological features (high potency, selectivity and stability toward proteolysis) hold promise as valuable therapeutic agents in the treatment of these diseases. In the present study, we used solid-phase methodology to synthesize a series of novel peptide analogues based on the sequence of Sar[dPhe(8)]desArg(9)-bradykinin, a relatively stable peptide agonist with moderate affinity for the human B1R. We evaluated the pharmacological properties of these peptides using (1) in vitro competitive binding experiments on recombinant human B1R and B2R (for index of selectivity determination) in transiently transfected human embryonic kidney 293 cells (HEK-293T cells), (2) ex vivo vasomotor assays on isolated human umbilical veins expressing endogenous human B1R, and (3) in vivo blood pressure tests using anesthetized lipopolysaccharide-immunostimulated rabbits. Key chemical modifications at the N-terminus, the positions 3 and 5 on Sar[dPhe(8)]desArg(9)-bradykinin led to potent analogues. For example, peptides 18 (SarLys[Hyp(3),Cha(5), dPhe(8)]desArg(9)-bradykinin) and 20 (SarLys[Hyp(3),Igl(5), dPhe(8)]desArg(9)-bradykinin) outperformed the parental molecule in terms of affinity, functional potency and duration of action in vitro and in vivo. These selective agonists should be valuable in future animal and human studies to investigate the potential benefits of B1R activation.

Redoubling the ring size of an endomorphin-2 analog transforms a centrally acting mu-opioid receptor agonist into a pure peripheral analgesic.

PubMed

Piekielna, Justyna; De Marco, Rossella; Gentilucci, Luca; Cerlesi, Maria Camilla; Calo', Girolamo; Tömböly, Csaba; Artali, Roberto; Janecka, Anna

2016-05-01

The study reports the synthesis and biological evaluation of two opioid analogs, a monomer and a dimer, obtained as products of the solid-phase, side-chain to side-chain cyclization of the pentapeptide Tyr-d-Lys-Phe-Phe-AspNH2 . The binding affinities to the mu, delta, and kappa opioid receptors, as well as results obtained in a calcium mobilization functional assay are reported. Tyr-[d-Lys-Phe-Phe-Asp]2 -NH2 1 was a potent and selective full agonist of mu with sub-nanomolar affinity, while the dimer (Tyr-[d-Lys-Phe-Phe-Asp]2 -NH2 )2 2 showed a significant mixed mu/kappa affinity, acting as an agonist at the mu. Molecular docking computations were utilized to explain the ability of the dimeric cyclopeptide 2 to interact with the receptor. Interestingly, in spite of the increased ring size, the higher flexibility allowed 2 to fold and fit into the mu receptor binding pocket. Both cyclopeptides were shown to elicit strong antinociceptive activity after intraventricular injection but only cyclomonomer 1 was able to cross the blood-brain barrier. However, the cyclodimer 2 displayed a potent peripheral antinociceptive activity in a mouse model of visceral inflammatory pain. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 309-317, 2016. © 2016 Wiley Periodicals, Inc.

Receptor binding properties and antinociceptive effects of chimeric peptides consisting of a micro-opioid receptor agonist and an ORL1 receptor antagonist.

PubMed

Kawano, Susumu; Ito, Risa; Nishiyama, Miharu; Kubo, Mai; Matsushima, Tomoko; Minamisawa, Motoko; Ambo, Akihiro; Sasaki, Yusuke

2007-07-01

Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the micro-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) or its analog YRFB (Tyr-D-Arg-Phe-betaAla-NH(2)) linked to the ORL1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)). All chimeric peptides were found to possess high receptor binding affinities for both micro-opioid and ORL1 receptors in mouse brain membranes although their binding affinities for both receptors in spinal membranes were significantly lower. Among them, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH(2) connected by a long spacer, had the highest binding affinity towards both receptors. In the tail-flick test following intrathecal (i.t.) administration to mice, all chimeric peptides showed potent and dose-dependent antinociceptive activities with an ED(50) of 1.34-4.51 (pmol/mouse), nearly comparable to dermorphin alone (ED(50); 1.08 pmol/mouse). In contrast to their micro-opioid receptor binding profiles, intracerebroventricular (i.c.v.) administration of the chimeric peptides resulted in much less potent antinociceptive activity (ED(50) 5.55-100< pmol/mouse) than when administered i.t. (ED(50): 1.34-4.51 pmol/mouse). These results suggest the involvement of nociceptin-like agonistic effects of the Ac-RYYRIK pharmacophore in the peptides, and the regulation of mu-opioid receptor-mediated antinociception in brain. The present chimeric peptides may be useful as pharmacological tools for studies on micro-opioid receptor/ORL1 receptor heterodimers.

Emergence of the pre-Bötzinger respiratory rhythm generator in the mouse embryo.

PubMed

Thoby-Brisson, Muriel; Trinh, Jean-Baptiste; Champagnat, Jean; Fortin, Gilles

2005-04-27

To obtain insights into the emergence of rhythmogenic circuits supporting respiration, we monitored spontaneous activities in isolated brainstem and medullary transverse slice preparations of mouse embryos, combining electrophysiological and calcium imaging techniques. At embryonic day 15 (E15), in a restricted region ventral to the nucleus ambiguus, we observed the onset of a sustained high-frequency (HF) respiratory-like activity in addition to a preexisting low-frequency activity having a distinct initiation site, spatial extension, and susceptibility to gap junction blockers. At the time of its onset, the HF generator starts to express the neurokinin 1 receptor, is connected bilaterally, requires active AMPA/kainate glutamatergic synapses, and is modulated by substance P and the mu-opioid agonist D-Ala2-N-Me-Phe4-Glycol5-enkephalin. We conclude that a rhythm generator sharing the properties of the neonatal pre-Bötzinger complex becomes active during E15 in mice.

In vitro pharmacology of aripiprazole, its metabolite and experimental dopamine partial agonists at human dopamine D2 and D3 receptors.

PubMed

Tadori, Yoshihiro; Forbes, Robert A; McQuade, Robert D; Kikuchi, Tetsuro

2011-10-15

Aripiprazole is the first dopamine D(2)/D(3) receptor partial agonist successfully developed and ultimately approved for treatment of a broad spectrum of psychiatric and neurological disorders. Aripiprazole's dopamine D(2) and serotonin 5-HT(1A) receptor partial agonist activities have been postulated to confer clinical efficacy without marked sedation, and a relatively favorable overall side-effect profile. Using aripiprazole's unique profile as a benchmark for new dopamine partial agonist development may facilitate discovery of new antipsychotics. We conducted an in vitro comparative analysis between aripiprazole, and its human metabolite OPC-14857 (7-(4-[4-(2,3-dichlorophenyl)-1-piperazinyl)butoxy)-2(1H)-quinolinone)); RGH-188 (trans-1-[4-[2-[4-(2,3-dichlorophenyl)piperazine-1-yl]ethyl]cyclohexyl]-3,3-dimethylurea), and its metabolite didesmethyl-RGH-188 (DDM-RGH-188); as well as bifeprunox, sarizotan, N-desmethylclozapine (NDMC; clozapine metabolite), and SDZ 208-912 (N-[(8α)-2-chloro-6-methylergolin-8-yl]-2,2-dimethylpropanamide). In vitro pharmacological assessment included inhibition of forskolin-stimulated cAMP accumulation and the reversal of dopamine-induced inhibition in clonal Chinese hamster ovary cell lines expressing D(2S), D(2L), D(3) Ser-9 and D(3) Gly-9 for human dopamine receptors. All test compounds behaved as dopamine D(2)/D(3) receptor partial agonists. Aripiprazole's intrinsic activity at dopamine D(2S) and D(2L) receptors was similar to that of OPC-14857 and RGH-188; lower than that of dopamine and bifeprunox; and higher than that of DDM-RGH-188, SDZ 208-912, sarizotan, and NDMC. Aripiprazole's intrinsic activity at dopamine D(3) Ser-9 and D(3) Gly-9 receptors was similar to that of OPC-14857 and sarizotan; lower than that of dopamine, bifeprunox, RGH-188 and DDM-RGH-188; and higher than that of SDZ 208-912 and NDMC. A consolidated assessment of these findings may help defining the most appropriate magnitude of intrinsic activity at

Role of dopamine D4 receptors in copulatory behavior: Studies with selective D4 agonists and antagonists in male rats.

PubMed

Sanna, Fabrizio; Contini, Andrea; Melis, Maria Rosaria; Argiolas, Antonio

2015-10-01

Dopamine influences the anticipatory and consummatory phases of sexual behavior, by acting on receptors of the D2 family (D2, D3 and D4) and in particular of the D2 subtype, although evidence for a role of D4 receptors in erectile function and copulatory behavior is also available. In order to clarify such a role of D4 receptors, the effect of selective D4 receptor agonists and antagonists on copulatory behavior of sexually potent male rats in classic copulation tests with a receptive female, was compared with that of apomorphine and haloperidol, a classic dopamine receptor agonist and antagonist, respectively. PD-168,077 (0.05-0.2mg/kg) and ABT-724 (0.01-0.04mg/kg), two selective D4 receptor agonists, given subcutaneously, improved dose-dependently copulatory behavior as shown by the decrease of mount frequency and post ejaculatory interval induced by PD-168,077, and of mount frequency, ejaculation latency, post ejaculatory and inter intromission intervals induced by ABT-724, and by the increase of ejaculation frequency and copulatory efficacy induced by both drugs. Conversely, L-745,870 (1-5mg/kg), a selective D4 receptor antagonist, given intraperitoneally, impaired dose-dependently copulatory behavior, as shown by the increase in intromission and ejaculation latencies, mount frequency, post ejaculatory interval and the decrease in ejaculation frequency and copulatory efficacy induced by this drug. L-745,870 (5mg/kg) administered before PD-168,077 (0.2mg/kg) or ABT-724 (0.04mg/kg), also abolished completely the facilitatory effects of both PD-168,077 and ABT-724 on sexual behavior. These results confirm the involvement of D4 receptors in specific aspects of male rat copulatory behavior that overlap only partially with those influenced by apomorphine and haloperidol. Copyright © 2015 Elsevier Inc. All rights reserved.

d-Ala-d-Ser VanN-Type Transferable Vancomycin Resistance in Enterococcus faecium▿

PubMed Central

Lebreton, François; Depardieu, Florence; Bourdon, Nancy; Fines-Guyon, Marguerite; Berger, Pierre; Camiade, Sabine; Leclercq, Roland; Courvalin, Patrice; Cattoir, Vincent

2011-01-01

Enterococcus faecium UCN71, isolated from a blood culture, was resistant to low levels of vancomycin (MIC, 16 μg/ml) but susceptible to teicoplanin (MIC, 0.5 μg/ml). No amplification was observed with primers specific for the previously described glycopeptide resistance ligase genes, but a PCR product corresponding to a gene called vanN was obtained using degenerate primers and was sequenced. The deduced VanN protein was related (65% identity) to the d-alanine:d-serine VanL ligase. The organization of the vanN gene cluster, determined using degenerate primers and by thermal asymmetric interlaced (TAIL)-PCR, was similar to that of the vanC operons. A single promoter upstream from the resistance operon was identified by rapid amplification of cDNA ends (RACE)-PCR. The presence of peptidoglycan precursors ending in d-serine and d,d-peptidase activities in the absence of vancomycin indicated constitutive expression of the resistance operon. VanN-type resistance was transferable by conjugation to E. faecium. This is the first report of transferable d-Ala-d-Ser-type resistance in E. faecium. PMID:21807981

Effect of [D-Phe6] bombesin (6-13) methylester, a bombesin receptor antagonist, towards bombesin-induced contractions in the guinea-pig and rat isolated urinary bladder.

PubMed

Maggi, C A; Coy, D H; Giuliani, S

1992-08-01

1. The effect of [D-Phe6] bombesin (6-13) methylester (OMe), a newly developed potent antagonist of bombesin receptors, has been investigated against bombesin-induced contractions of the guinea-pig and rat isolated urinary bladder. 2. Bombesin (0.1 nM-10 microM) produced a concentration-dependent contraction of the guinea-pig isolated bladder which approached the same maximum response as KCl (80 mM). The response to bombesin was antagonized in a competitive manner (rightward shift of the concentration-response curve without depression of the maximal response) by [D-Phe6] bombesin (6-13) OMe (0.3-10 microM). Degree of antagonism was concentration-dependent between 0.3 and 3 microM (dose ratios = 2.4, 9 and 39 in the presence of 0.3, 1, 3 microM of the antagonist). However, a larger concentration (10 microM) of the antagonist was not more effective (dose ratio = 36) than 3 microM. 3. Neither the action of bombesin nor the activity of the antagonist was influenced by peptidase inhibitors (bestatin, captopril and thiorphan 3 microM each) or by atropine, indomethacin, chlorpheniramine and desensitization of P2x purinoceptors by alpha, beta methylene ATP. 4. The bombesin antagonist was ineffective against contraction of the guinea-pig urinary bladder produced by the NK-1 tachykinin receptor-selective agonist, [Sar9] substance P sulphone. The action of the NK-1 receptor agonist was antagonized by L 668, 169 (3 microM), a cyclic peptide tachykinin antagonist. L 668, 169 had no effect toward bombesin-induced contraction. 5. The bombesin antagonist (1-10 microM) had no effect against the non-adrenergic non-cholinergic response of the guinea-pig isolated urinary bladder to electrical field stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate specificity of platypus venom L-to-D-peptide isomerase.

PubMed

Bansal, Paramjit S; Torres, Allan M; Crossett, Ben; Wong, Karen K Y; Koh, Jennifer M S; Geraghty, Dominic P; Vandenberg, Jamie I; Kuchel, Philip W

2008-04-04

The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.

Compositions of supersaturated solutions for enhanced growth of {alpha}-NiSO{sub 4} . 6H{sub 2}O, Me{sub 2}Ni(SO{sub 4}){sub 2} . 6H{sub 2}O, MeH{sub 2}PO{sub 4} [Me = Li, Na, K, Rb, Cs, NH{sub 4}], and K(H{sub x}D{sub 1-x}){sub 2}PO{sub 4} single crystals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soboleva, L. V., E-mail: afkonst@ns.crys.ras.ru

2008-05-15

The possibility of determining the optimal compositions and temperatures of supersaturated solutions for enhanced growth of single crystals of congruently and incongruently dissolving solid phases from the solubility diagrams of ternary systems is shown, and this approach is justified. The NiSO{sub 4}-H{sub 2}SO{sub 4}-H{sub 2}O, Me{sub 2}SO{sub 4}-NiSO{sub 4}-H{sub 2}O, and Me{sub 2}O-P{sub 2}O{sub 5}-H{sub 2}O(D{sub 2}O) systems have been used to determine the optimal compositions and temperatures of supersaturated solutions for growth of {alpha}-NiSO{sub 4} . 6H{sub 2}O, Me{sub 2}Ni(SO{sub 4}){sub 2} . 6H{sub 2}O, MeH{sub 2}PO{sub 4} [Me = Li, Na, K, Rb, Cs, NH{sub 4}], and Kmore » (H{sub x} D{sub 1-x}){sub 2}PO{sub 4} (D is deuterium) single crystals.« less

Characterization of kappa 1 and kappa 2 opioid binding sites in frog (Rana esculenta) brain membrane preparation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benyhe, S.; Varga, E.; Hepp, J.

1990-09-01

The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappa 1 is the dominant receptor subtype, frog brain contains mainly the kappa 2 subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition experiments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazidomorphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa 2 ligands (Met5)enkephalin-Arg6-Phe7 and etorphine also show high affinities to the frog brain.more » Kappa 1 binding sites measured in the presence of 5 microM/D-Ala2-Leu5/enkephalin represent 25-30% of (3H)ethylketocyclazocine binding in frog brain membranes. The kappa 2 subtype in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and /D-Ala2-(Me)Phe4-Gly5-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain.« less

First measurement of the 2.4 MeV and 2.9 MeV 6He three-cluster resonant states via the 3H(4He, pα)2n four-body reaction

NASA Astrophysics Data System (ADS)

Mandaglio, Giuseppe; Povoroznyk, Orest; Gorpinich, Olga K.; Jachmenjov, Olexiy O.; Anastasi, Antonio; Curciarello, Francesca; de Leo, Veronica; Mokhnach, Hanna V.; Ponkratenko, Oleg; Roznyuk, Yuri; Fazio, Giovanni; Giardina, Giorgio

2014-06-01

Two new low-lying 6He levels at excitation energies of about 2.4 MeV and 2.9 MeV were observed in the experimental investigation of the p-α coincidence spectra obtained by the 3H(4He, pα)2n four-body reaction at E4He beam energy of 27.2 MeV. The relevant E* peak energy and Γ energy width spectroscopic parameters for such 6He* excited states decaying into the α+n+n channel were obtained by analyzing the bidimensional (Ep, Eα) energy spectra. The present new result of two low-lying 6He* excited states above the 4He+2n threshold energy of 0.974 MeV is important for the investigation of the nuclear structure of neutron-rich light nuclei and also as a basic test for theoretical models in the study of the three-cluster resonance feature of 6He.

4,2':6',4"- and 3,2':6',3"-Terpyridines: The Conflict between Well-Defined Vectorial Properties and Serendipity in the Assembly of 1D-, 2D- and 3D-Architectures.

PubMed

Klein, Y Maximilian; Prescimone, Alessandro; Constable, Edwin C; Housecroft, Catherine E

2017-06-30

A comparative investigation of the coordination assemblies formed between Co(NCS)₂ and two monotopic 4,2':6',4''-terpyridine (4,2':6',4"-tpy) ligands or two related ditopic ligands is reported. Crystals were grown by layering MeOH solutions of Co(NCS)₂ over a CHCl₃ or 1,2-C₆H₄Cl₂ solution of the respective ligand at room temperature. With 4'-(2-methylpyrimidin-5-yl)-4,2':6',4"-terpyridine ( 6 ), the 1D-coordination polymer {[Co₂(NCS)₄(MeOH)₄( 6 )₂]∙2MeOH∙8H₂O} n assembles with 6 coordinating only through the outer N-donors of the 4,2':6',4"-tpy unit; coordination by the MeOH solvent blocks two cobalt coordination sites preventing propagation in a higher-dimensional network. A combination of Co(NCS)₂ and 1-(4,2':6',4"-terpyridin-4'-yl)ferrocene ( 7 ) leads to {[Co(NCS)₂( 7 )₂]∙4CHCl₃} n which contains a (4,4) net; the 2D-sheets associate through π-stacking interactions between ferrocenyl and pyridyl units. A 3D-framework is achieved through use of the ditopic ligand 1,4-bis( n propoxy)-2,5-bis(4,2':6',4"-terpyridin-4'-yl)benzene ( 8 ) which acts as a 4-connecting node in {[Co(NCS)₂( 8 )₂] . 2C₆H₄Cl₂} n ; the combination of metal and ligand planar 4-connecting nodes results in a {6⁵.8} cds net. For a comparison with the coordinating abilities of the previously reported 1,4-bis( n octoxy)-2,5-bis(4,2':6',4"-terpyridin-4'-yl)benzene ( 3 ), a more flexible analogue 9 was prepared. {[Co(NCS)₂( 9 )]∙2CHCl₃} n contains a (4,4) net defined by both metal and ligand planar 4-connecting nodes. The n octoxy tails of 9 protrude from each side of the (4,4) net and thread through adjacent sheets; the arene-attached n octoxy chains associate through a combination of van der Waals and C-H...π interactions.

Behavioural and biochemical responses following activation of midbrain dopamine pathways by receptor selective neurokinin agonists.

PubMed

Elliott, P J; Mason, G S; Stephens-Smith, M; Hagan, R M

1991-06-01

Preferential activation of mesolimbic and nigro-striatal dopamine (DA) pathways by receptor-selective and peptidase-resistant neurokinin (NK) agonists is reported. The DA cell body region of the mesolimbic pathway appears to be activated by NK agonists selective for NK-1 and NK-3 receptors whereas the DA cell bodies in the substantia nigra are under an excitatory NK-2 receptor-mediated influence. Stimulation of the mesolimbic DA pathway by NK-1 (Ava[L-Pro9,N-Me-Leu10]SP (7-11) [GR73632]) or NK-3 (Senktide) agonists increase locomotor activity. Additional studies showed that this elevated motor response observed after intra-VTA infusion of GR73632 was accompanied by a corresponding increase in DA turnover in the terminal fields of this pathway. Similarly, unilateral activation of the nigro-striatal DA pathway by NK-2 selective agonists (Ava (D-Pro9) SP (7-11) [GR51667] or [Lys3,Gly8,R-Lac-Leu9]NKA (3-10) [GR64349]) elicit contralateral rotational activity and an increase in DA turnover in the ipsilateral striatum. The rotational response was attenuated by prior administration of an NK-2 antagonist (cyclo (Gln, Trp, Phe, Gly, Leu, Met)] L-659877]) into the nigra. Peripheral injection of haloperidol, a DA antagonist, also blocked the NK-2 agonist induced rotations.

Discovery of N-(4-aryl-5-aryloxy-thiazol-2-yl)-amides as potent RORγt inverse agonists.

PubMed

Wang, Yonghui; Yang, Ting; Liu, Qian; Ma, Yingli; Yang, Liuqing; Zhou, Ling; Xiang, Zhijun; Cheng, Ziqiang; Lu, Sijie; Orband-Miller, Lisa A; Zhang, Wei; Wu, Qianqian; Zhang, Kathleen; Li, Yi; Xiang, Jia-Ning; Elliott, John D; Leung, Stewart; Ren, Feng; Lin, Xichen

2015-09-01

A novel series of N-(4-aryl-5-aryloxy-thiazol-2-yl)-amides as RORγt inverse agonists was discovered. Binding mode analysis of a RORγt partial agonist (2c) revealed by co-crystal structure in RORγt LBD suggests that the inverse agonists do not directly interfere with the interaction between H12 and the RORγt LBD. Detailed SAR exploration led to identification of potent RORγt inverse agonists such as 3m with a pIC50 of 8.0. Selected compounds in the series showed reasonable activity in Th17 cell differentiation assay as well as low intrinsic clearance in mouse liver microsomes. Copyright © 2015 Elsevier Ltd. All rights reserved.

IL1R2, CCR2, and CXCR4 May Form Heteroreceptor Complexes with NMDAR and D2R: Relevance for Schizophrenia

PubMed Central

Borroto-Escuela, Dasiel O.; Tarakanov, Alexander O.; Bechter, Karl; Fuxe, Kjell

2017-01-01

The mild neuroinflammation hypothesis of schizophrenia was introduced by Bechter in 2001. It has been hypothesized that a hypofunction of glutamatergic signaling via N-methyl-D-aspartate receptors (NMDARs) and hyperactivation of dopamine D2 receptors play a role in schizophrenia. The triplet puzzle theory states that sets of triplet amino acid homologies guide two different receptors toward each other and contributes to the formation of a receptor heteromer. It is, therefore, proposed that putative NMDAR-C-C chemokine receptor type 2 (CCR2), NMDAR-C-X-C chemokine receptor type 4 (CXCR4), and NMDAR- interleukin 1 receptor type II (IL1R2) heteromers can be formed in the neuronal networks in mild neuroinflammation due to demonstration of Gly-Leu-Leu (GLL), Val-Ser-Thr (VST), and/or Ser-Val-Ser (SVS) amino acid homologies between these receptor protomers. This molecular process may underlie the ability to produce symptoms of schizophrenia in mild neuroinflammation. In this state, volume transmission (VT) is increased involving increased extracellular vesicle-mediated VT from microglia and astroglia. These vesicles may contain CCR2, CXCR4, and/or IL1R2 as well as their ligands and upon internalization by endocytic pathways into neurons can form heteroreceptor complexes with NMDAR in the plasma membrane with pathological allosteric receptor–receptor interactions involving increased internalization and reduced NMDAR signaling. The triplet puzzle theory also suggests the formation of putative D2R-CCR2, D2R-CXCR4, and D2R-IL1R2 heteromers in mild neuroinflammation in view of their demonstrated sets of Leu-Tyr-Ser (LYS), Leu-Pro-Phe (LPF), and/or Ser-Leu-Ala (SLA) triplet homologies. These D2R heteroreceptor complexes may also contribute to schizophrenia-like symptoms in mild neuroinflammation by enhancing D2R protomer function. PMID:28261115

Identification of novel thiazolo[5,4-d]pyrimidine derivatives as human A1 and A2A adenosine receptor antagonists/inverse agonists.

PubMed

Varano, Flavia; Catarzi, Daniela; Falsini, Matteo; Vincenzi, Fabrizio; Pasquini, Silvia; Varani, Katia; Colotta, Vittoria

2018-07-23

In this study a new set of thiazolo[5,4-d]pyrimidine derivatives was synthesized. These derivatives bear different substituents at positions 2 and 5 of the thiazolopyrimidine core while maintaining a free amino group at position-7. The new compounds were tested for their affinity and potency at human (h) A 1 , A 2A , A 2B and A 3 adenosine receptors expressed in CHO cells. The results reveal that the higher affinity of these new set of thiazolopyrimidines is toward the hA 1 and hA 2A adenosine receptors subtypes and is tuned by the substitution pattern at both the 2 and 5 positions of the thiazolopyrimidine nucleus. Functional studies evidenced that the compounds behaved as dual A 1 /A 2A antagonists/inverse agonists. Compound 3, bearing a 5-((2-methoxyphenyl) methylamino) group and a phenyl moiety at position 2, displayed the highest affinity (hA 1 K i  = 10.2 nM; hA 2A K i  = 4.72 nM) and behaved as a potent A 1 /A 2A antagonist/inverse agonist (hA 1 IC 50  = 13.4 nM; hA 2A IC 50  = 5.34 nM). Copyright © 2018 Elsevier Ltd. All rights reserved.

Discovery of Mixed Pharmacology Melanocortin-3 Agonists and Melanocortin-4 Receptor Tetrapeptide Antagonist Compounds (TACOs) Based on the Sequence Ac-Xaa1-Arg-(pI)DPhe-Xaa4-NH2.

PubMed

Doering, Skye R; Freeman, Katie T; Schnell, Sathya M; Haslach, Erica M; Dirain, Marvin; Debevec, Ginamarie; Geer, Phaedra; Santos, Radleigh G; Giulianotti, Marc A; Pinilla, Clemencia; Appel, Jon R; Speth, Robert C; Houghten, Richard A; Haskell-Luevano, Carrie

2017-05-25

The centrally expressed melanocortin-3 and -4 receptors (MC3R/MC4R) have been studied as possible targets for weight management therapies, with a preponderance of studies focusing on the MC4R. Herein, a novel tetrapeptide scaffold [Ac-Xaa 1 -Arg-(pI)DPhe-Xaa 4 -NH 2 ] is reported. The scaffold was derived from results obtained from a MC3R mixture-based positional scanning campaign. From these results, a set of 48 tetrapeptides were designed and pharmacologically characterized at the mouse melanocortin-1, -3, -4, and -5 receptors. This resulted in the serendipitous discovery of nine compounds that were MC3R agonists (EC 50 < 1000 nM) and MC4R antagonists (5.7 < pA 2 < 7.8). The three most potent MC3R agonists, 18 [Ac-Arg-Arg-(pI)DPhe-Tic-NH 2 ], 1 [Ac-His-Arg-(pI)DPhe-Tic-NH 2 ], and 41 [Ac-Arg-Arg-(pI)DPhe-DNal(2')-NH 2 ] were more potent (EC 50 < 73 nM) than the melanocortin tetrapeptide Ac-His-DPhe-Arg-Trp-NH 2 . This template contains a sequentially reversed "Arg-(pI)DPhe" motif with respect to the classical "Phe-Arg" melanocortin signaling motif, which results in pharmacology that is first-in-class for the central melanocortin receptors.

β2-Agonist Induced cAMP Is Decreased in Asthmatic Airway Smooth Muscle Due to Increased PDE4D

PubMed Central

Trian, Thomas; Burgess, Janette K.; Niimi, Kyoko; Moir, Lyn M.; Ge, Qi; Berger, Patrick; Liggett, Stephen B.; Black, Judith L.; Oliver, Brian G.

2011-01-01

Background and Objective Asthma is associated with airway narrowing in response to bronchoconstricting stimuli and increased airway smooth muscle (ASM) mass. In addition, some studies have suggested impaired β-agonist induced ASM relaxation in asthmatics, but the mechanism is not known. Objective To characterize the potential defect in β-agonist induced cAMP in ASM derived from asthmatic in comparison to non-asthmatic subjects and to investigate its mechanism. Methods We examined β2-adrenergic (β2AR) receptor expression and basal β-agonist and forskolin (direct activator of adenylyl cyclase) stimulated cAMP production in asthmatic cultured ASM (n = 15) and non-asthmatic ASM (n = 22). Based on these results, PDE activity, PDE4D expression and cell proliferation were determined. Results In the presence of IBMX, a pan PDE inhibitor, asthmatic ASM had ∼50% lower cAMP production in response to isoproterenol, albuterol, formoterol, and forskolin compared to non-asthmatic ASM. However when PDE4 was specifically inhibited, cAMP production by the agonists and forskolin was normalized in asthmatic ASM. We then measured the amount and activity of PDE4, and found ∼2-fold greater expression and activity in asthmatic ASM compared to non-asthmatic ASM. Furthermore, inhibition of PDE4 reduced asthmatic ASM proliferation but not that of non-asthmatic ASM. Conclusion Decreased β-agonist induced cAMP in ASM from asthmatics results from enhanced degradation due to increased PDE4D expression. Clinical manifestations of this dysregulation would be suboptimal β-agonist-mediated bronchodilation and possibly reduced control over increasing ASM mass. These phenotypes appear to be “hard-wired” into ASM from asthmatics, as they do not require an inflammatory environment in culture to be observed. PMID:21611147

Highly potent analogues of luteinizing hormone-releasing hormone containing D-phenylalanine nitrogen mustard in position 6.

PubMed Central

Bajusz, S; Janaky, T; Csernus, V J; Bokser, L; Fekete, M; Srkalovic, G; Redding, T W; Schally, A V

1989-01-01

The nitrogen mustard derivatives of 4-phenylbutyric acid and L-phenylalanine, called chlorambucil (Chl) and melphalan (Mel), respectively, have been incorporated into several peptide hormones, including luteinizing hormone-releasing hormone (LH-RH). The alkylating analogues of LH-RH were prepared by linking Chl, as an N-acyl moiety, to the complete amino acid sequence of agonistic and antagonistic analogues. These compounds, in particular the antagonistic analogues, showed much lower potency than their congeners carrying other acyl groups. To obtain highly potent alkylating analogues of LH-RH, the D enantiomer of Mel was incorporated into position 6 of the native hormone and some of its antagonistic analogues. Of the peptides prepared, [D-Mel6]LH-RH (SB-05) and [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,Arg5,D-Mel6,D-Ala10++ +]LH-RH [SB-86, where Nal(2) is 3-(2-naphthyl)alanine and Pal(3) is 3-(3-pyridyl)alanine] possessed the expected high agonistic and antagonistic activities, respectively, and also showed high affinities for the membrane receptors of rat pituitary cells, human breast cancer cells, human prostate cancer cells, and rat Dunning R-3327 prostate tumor cells. These two analogues exerted cytotoxic effects on human and rat mammary cancer cells in vitro. Thus these two D-Mel6 analogues seem to be particularly suitable for the study of how alkylating analogues of LH-RH could interfere with intracellular events in certain cancer cells. PMID:2548207

Highly potent analogues of luteinizing hormone-releasing hormone containing D-phenylalanine nitrogen mustard in position 6.

PubMed

Bajusz, S; Janaky, T; Csernus, V J; Bokser, L; Fekete, M; Srkalovic, G; Redding, T W; Schally, A V

1989-08-01

The nitrogen mustard derivatives of 4-phenylbutyric acid and L-phenylalanine, called chlorambucil (Chl) and melphalan (Mel), respectively, have been incorporated into several peptide hormones, including luteinizing hormone-releasing hormone (LH-RH). The alkylating analogues of LH-RH were prepared by linking Chl, as an N-acyl moiety, to the complete amino acid sequence of agonistic and antagonistic analogues. These compounds, in particular the antagonistic analogues, showed much lower potency than their congeners carrying other acyl groups. To obtain highly potent alkylating analogues of LH-RH, the D enantiomer of Mel was incorporated into position 6 of the native hormone and some of its antagonistic analogues. Of the peptides prepared, [D-Mel6]LH-RH (SB-05) and [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,Arg5,D-Mel6,D-Ala10++ +]LH-RH [SB-86, where Nal(2) is 3-(2-naphthyl)alanine and Pal(3) is 3-(3-pyridyl)alanine] possessed the expected high agonistic and antagonistic activities, respectively, and also showed high affinities for the membrane receptors of rat pituitary cells, human breast cancer cells, human prostate cancer cells, and rat Dunning R-3327 prostate tumor cells. These two analogues exerted cytotoxic effects on human and rat mammary cancer cells in vitro. Thus these two D-Mel6 analogues seem to be particularly suitable for the study of how alkylating analogues of LH-RH could interfere with intracellular events in certain cancer cells.

Potent and selective agonists of alpha-melanotropin (alphaMSH) action at human melanocortin receptor 5; linear analogs of alpha-melanotropin.

PubMed

Bednarek, Maria A; MacNeil, Tanya; Tang, Rui; Fong, Tung M; Cabello, M Angeles; Maroto, Marta; Teran, Ana

2007-05-01

Alpha-melanotropin, Ac-Ser(1)-Tyr-Ser-Met-Glu-His(6)-Phe(7)-Arg(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2)(1), is a non-selective endogenous agonist for the melanocortin receptor 5; the receptor present in various peripheral tissues and in the brain, cortex and cerebellum. Most of the synthetic analogs of alphaMSH, including a broadly used and more potent the NDP-alphaMSH peptide, Ac-Ser(1)-Tyr-Ser-Nle(4)-Glu-His(6)-D-Phe(7)-Arg(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2), are also not particularly selective for MC5R. To elucidate physiological functions of the melanocortin receptor 5 in rodents and humans, the receptor subtype selective research tools are needed. We report herein syntheses and pharmacological evaluation in vitro of several analogs of NDP-alphaMSH which are highly potent and specific agonists for the human MC5R. The new linear peptides, of structures and solubility properties similar to those of the endogenous ligand alphaMSH, are exemplified by compound 7, Ac-Ser(1)-Tyr-Ser-Met-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2) (Oic: octahydroindole-2-COOH, 4,4'-Bip: 4,4'-biphenylalanine, Pip: pipecolic acid), shortly NODBP-alphaMSH, which has an IC(50)=0.74 nM (binding assay) and EC(50)=0.41 (cAMP production assay) at hMC5R nM and greater than 3500-fold selectivity with respect to the melanocortin receptors 1b, 3 and 4. A shorter peptide derived from NODBP-alphaMSH: Ac-Nle-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8)-Trp(9) -NH(2) (17) was measured to be an agonist only 10-fold less potent at hMC5R than the full length parent peptide. In the structure of this smaller analog, the Nle-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8) segment was found to be critical for high agonist potency, while the C-terminal Trp(9) residue was shown to be required for high hMC5R selectivity versus hMC1b,3,4R.

Structure-activity relationship of linear peptide Bu-His6-DPhe7-Arg8-Trp9-Gly10-NH2 at the human melanocortin-1 and -4 receptors: DPhe7 and Trp9 substitution.

PubMed

Danho, Waleed; Swistok, Joseph; Cheung, Adrian Wai-Hing; Kurylko, Grazyna; Franco, Lucia; Chu, Xin-Jie; Chen, Li; Yagaloff, Keith

2003-02-24

A series of pentapeptides, based on hMC4R pentapeptide agonist (Bu-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2)), was prepared in which either DPhe(7) or Trp(9) residue was systematically substituted. A number of interesting DPhe surrogates (D-Thi, D-3-CF(3)Phe, D-2-Nal and D-3,4-diClPhe) as well as Trp surrogates (2-Nal and Bta) were identified in this study.

A critical period in the supraspinal control of pain: opioid-dependent changes in brainstem rostroventral medulla function in preadolescence

PubMed Central

Hathway, Gareth J.; Vega-Avelaira, David; Fitzgerald, Maria

2012-01-01

We have previously shown that the balance of electrically evoked descending brainstem control of spinal nociceptive reflexes undergoes a switch from excitation to inhibition in preadolescent rats. Here we show that the same developmental switch occurs when μ-opioid receptor agonists are microinjected into the rostroventral medulla (RVM). Microinjections of the μ-opioid receptor agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) into the RVM of lightly anaesthetised adult rats produced a dose-dependent decrease in mechanical nociceptive hindlimb reflex electromyographic activity. However, in preadolescent (postnatal day 21 [P21]) rats, the same doses of DAMGO produced reflex facilitation. RVM microinjection of δ-opioid receptor or GABAA receptor agonists, on the other hand, caused reflex depression at both ages. The μ-opioid receptor-mediated descending facilitation is tonically active in naive preadolescent rats, as microinjection of the μ-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) into the RVM at this age decreases spinal nociceptive reflexes while having no effect in adults. To test whether tonic opioid central activity is required for the preadolescent switch in RVM descending control, naloxone hydrochloride was delivered continuously from subcutaneous osmotic mini-pumps for 7-day periods, at various postnatal stages. Blockade of tonic opioidergic activity from P21 to P28, but not at earlier or later ages, prevented the normal development of descending RVM inhibitory control of spinal nociceptive reflexes. Enhancing opioidergic activity with chronic morphine over P7 to P14 accelerated this development. These results show that descending facilitation of spinal nociception in young animals is mediated by μ-opioid receptor pathways in the RVM. Furthermore, the developmental transition from RVM descending facilitation to inhibition of pain is determined by activity in central opioid networks at a critical period of

Tachykinin receptors in the circular muscle of the guinea-pig ileum.

PubMed

Maggi, C A; Patacchini, R; Giachetti, A; Meli, A

1990-12-01

1. We have studied the mechanical response of circular strips of the guinea-pig ileum to tachykinins and characterized the receptors involved by means of receptor-selective agonists. 2. The strips responded to both substance P (SP) and neurokinin A (NKA), as well as to [Pro9]-SP sulphone (selective NK1-receptor agonist), [beta Ala8]-NKA(4-10) (selective NK2-receptor agonist) and [MePhe7]-neurokinin B (selective NK3-receptor agonist). The ED50s of the various peptides (calculated as the concentration of agonist which produced 50% of the response to 10 microM carbachol) were similar, in the range of 40-200 nM, i.e. no clearcut rank order of potency was evident. 3. The response to a submaximal (10 nM) concentration of SP or NKA was unaffected in the presence of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 4. The response to the NK1-agonist was totally atropine-resistant, but was reduced (about 30% inhibition) by tetrodotoxin. The response to the NK3-receptor agonist was halved by atropine and abolished by tetrodotoxin. The response to the NK2-agonist was unaffected by either atropine or tetrodotoxin. 5. The response to the selective NK2-agonist was unchanged after desensitization of NK1- or NK3-receptors. 6. The response to the NK2-selective agonist was strongly inhibited by [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10) (MEN 10,207) a selective NK2-receptor antagonist which did not modify the response to the NK1-selective agonist. 7. Our findings indicate that all the three known types of tachykinin receptors mediate the contractile response of the circular muscle of the guinea-pig ileum to peptides of this family. The response to activation of NK3-receptors is totally neurogenic and partially mediated by endogenous acetylcholine, the response to activation of NK1-receptors is partly neurogenic and largely myogenic and the response to activation of NK2-receptors is totally myogenic.

Tachykinin receptors in the guinea-pig isolated bronchi.

PubMed

Maggi, C A; Patacchini, R; Quartara, L; Rovero, P; Santicioli, P

1991-05-17

The aim of the study was to assess which tachykinin receptors mediate the contractile response in the guinea-pig isolated bronchi. Experiments with natural tachykinins and receptor-selective tachykinin agonists were performed in the absence or presence of peptidase inhibitors and in bronchi pretreated with phenoxybenzamine. Both NK-1 (substance P, substance P methylester and septide) and NK-2 (neurokinin A, [beta-Ala8]neurokinin A-(4-10) and MDL 28,564) receptor agonists produced concentration-dependent contraction. NK-3 agonists (senktide and [MePhe7]neurokinin B) were active only at high concentrations. Phenoxybenzamine pretreatment reduced the maximal response to NK-1 agonists and produced a rightward shift of the curve to NK-2 agonists, without depression of the maximum. Five tachykinin antagonists selective for the NK-1 (L 668,169) or the NK-2 (MEN 10,207, MEN 10,376, L 659,877 and R 396) receptor were tested against substance P methylester and [beta-Ala8]neurokinin A-(4-10). The results indicated that these receptor-selective antagonists maintain their characteristic even when tested in a multireceptor assay such as the guinea-pig bronchus. The rank order of potency of NK-2 antagonists against [beta-Ala8]neurokinin A-(4-10) was MEN 10,207 = MEN 10,376 greater than L 659,877 much greater than R 396. This pattern, with the observation of the full agonist activity of MDL 28,564, indicates that in addition to NK-1 receptors, NK-2 receptors also are present in the guinea-pig bronchi and belong to the same subtype (NK-2A) as present in the rabbit pulmonary artery.

Differential agonist and inverse agonist profile of antipsychotics at D2L receptors coupled to GIRK potassium channels.

PubMed

Heusler, Peter; Newman-Tancredi, Adrian; Castro-Fernandez, Annabelle; Cussac, Didier

2007-03-01

The D(2) dopaminergic receptor represents a major target of antipsychotic drugs. Using the coupling of the human D(2long) (hD(2L)) receptor to G protein-coupled inward rectifier potassium (GIRK) channels in Xenopus laevis oocytes, we examined the activity of antipsychotic agents of different classes - typical, atypical, and a "new generation" of compounds, exhibiting a preferential D(2) and 5-HT(1A) receptor profile. When the hD(2L) receptor was coexpressed with GIRK channels, a series of reference compounds exhibited full agonist (dopamine, and quinpirole), partial agonist (apomorphine, (-)3-PPP, and (+)-UH232) or inverse agonist (raclopride, and L741626) properties. Sarizotan exhibited only very weak partial agonist action. At higher levels of receptor cRNA injected per oocyte, both partial agonist activity and inverse agonist properties were generally more pronounced. The inverse agonist action of L741626 was reversed by interaction with sarizotan, thus confirming the constitutive activity of wild-type hD(2L) receptors in the oocyte expression system. When antipsychotic agents were tested for their actions at the hD(2L) receptor, typical (haloperidol) as well as atypical (nemonapride, ziprasidone, and clozapine) compounds acted as inverse agonists. In contrast, among D(2)/5-HT(1A) antipsychotics, only SLV313 and F15063 behaved as inverse agonists, whilst the other members of this group (bifeprunox, SSR181507 and the recently marketed antipsychotic, aripiprazole) exhibited partial agonist properties. Thus, the X. laevis oocyte expression system highlights markedly different activity of antipsychotics at the hD(2L) receptor. These differential properties may translate to distinct therapeutic potential of these compounds.

5-Amino-6-chloro-N-[(1-isobutylpiperidin-4-yl)methyl]-2-methylimidazo[1,2-alpha]pyridine-8-carboxamide (CJ-033,466), a novel and selective 5-hydroxytryptamine4 receptor partial agonist: pharmacological profile in vitro and gastroprokinetic effect in conscious dogs.

PubMed

Mikami, Tadayoshi; Ochi, Yasuo; Suzuki, Keiko; Saito, Toshiyuki; Sugie, Yutaka; Sakakibara, Minoru

2008-04-01

5-Hydroxytryptamine (5-HT) receptors and dopamine(2) (D(2)) receptor modulate gastrointestinal motility. Gastroprokinetic agents that act on several 5-HT receptor subtypes and/or D(2) receptors are used clinically. Although the 5-HT(4) receptor is known to mediate the gastroprokinetic effects of these agents, the absence of highly selective 5-HT(4) receptor agonists has made it difficult to confirm the physiological consequences of selective 5-HT(4) receptor stimulation. In this study, we report the in vitro pharmacological profiles and the in vivo gastroprokinetic effects of 5-amino-6-chloro-N-[(1-isobutylpiperidin-4-yl)methyl]-2-methylimidazo[1,2-alpha]pyridine-8-carboxamide (CJ-033,466), a novel, potent, and selective 5-HT(4) partial agonist. Compared with preceding 5-HT(4) agonists such as cisapride, mosapride, and tegaserod, CJ-033,466 had a superior in vitro profile, with nanomolar agonistic activities for the 5-HT(4) receptor and 1000-fold greater selectivity for the 5-HT(4) receptor over other 5-HT and D(2) receptors. In vivo studies in conscious dogs showed that CJ-033,466 dose-dependently stimulated gastric antral motility in both the fasted and postprandial states at the same dose range and that it was 30 times more potent than cisapride. Furthermore, CJ-033,466 accelerated the gastric emptying rate in a gastroparesis dog model at the minimally effective dose established in the gastric motility study. In conclusion, CJ-033,466 is a potent and highly selective 5-HT(4) agonist that stimulates physiologically coordinated gastric motility, and it has no activity on other 5-HT receptor subtypes and D(2) receptors. Therefore, CJ-033,466 could be used to treat gastroparesis, providing better gastroprokinetics and reduced side effects mediated by the other receptors.

F15063, a compound with D2/D3 antagonist, 5-HT1A agonist and D4 partial agonist properties: (III) Activity in models of cognition and negative symptoms

PubMed Central

Depoortère, R; Auclair, A L; Bardin, L; Slot, L Bruins; Kleven, M S; Colpaert, F; Vacher, B; Newman-Tancredi, A

2007-01-01

Background and purpose: The D2/D3 receptor antagonist, D4 receptor partial agonist, and high efficacy 5-HT1A receptor agonist F15063 was shown to be highly efficacious and potent in rodent models of activity against positive symptoms of schizophrenia. However F15063 induced neither catalepsy nor the ‘serotonin syndrome'. Here, we evaluated its profile in rat models predictive of efficacy against negative symptoms/cognitive deficits of schizophrenia. Experimental approach: F15063, given i.p., was assessed in models of behavioural deficits induced by interference with the NMDA/glutamatergic (phencyclidine: PCP) or cholinergic (scopolamine) systems. Key results: Through 5-HT1A activation, F15063 partially alleviated (MED: 0.04 mg kg−1) PCP-induced social interaction deficit between two adult rats, without effect by itself, underlining its potential to combat negative symptoms. At doses above 0.16 mg kg−1, F15063 reduced interaction by itself. F15063 (0.16 mg kg−1) selectively re-established PCP-impaired ‘cognitive flexibility' in a reversal learning task, suggesting potential against adaptability deficits. F15063 (0.04–0.63 mg kg−1) also reversed scopolamine-induced amnesia in a juvenile-adult rat social recognition test, indicative of a pro-cholinergic influence. Activity in this latter test is consistent with its D4 partial agonism, as it was blocked by the D4 antagonist L745,870. Finally, F15063 up to 40 mg kg−1 did not disrupt basal prepulse inhibition of startle reflex in rats, a marker of sensorimotor gating. Conclusions and implications: The balance of D2/D3, D4 and 5-HT1A receptor interactions of F15063 yields a promising profile of activity in models of cognitive deficits and negative symptoms of schizophrenia. PMID:17375085

Does dinitrogen hydrogenation follow different mechanisms for [(eta5-C5Me4H)2Zr]2(mu2,eta2,eta2-N2) and {[PhP(CH2SiMe2NSiMe2CH2)PPh]Zr}2(mu2,eta2,eta2-N2) complexes? A computational study.

PubMed

Bobadova-Parvanova, Petia; Wang, Qingfang; Quinonero-Santiago, David; Morokuma, Keiji; Musaev, Djamaladdin G

2006-09-06

The mechanisms of dinitrogen hydrogenation by two different complexes--[(eta(5)-C(5)Me(4)H)(2)Zr](2)(mu(2),eta(2),eta(2)-N(2)), synthesized by Chirik and co-workers [Nature 2004, 427, 527], and {[P(2)N(2)]Zr}(2)(mu(2),eta(2),eta(2)-N(2)), where P(2)N(2) = PhP(CH(2)SiMe(2)NSiMe(2)CH(2))(2)PPh, synthesized by Fryzuk and co-workers [Science 1997, 275, 1445]--are compared with density functional theory calculations. The former complex is experimentally known to be capable of adding more than one H(2) molecule to the side-on coordinated N(2) molecule, while the latter does not add more than one H(2). We have shown that the observed difference in the reactivity of these dizirconium complexes is caused by the fact that the former ligand environment is more rigid than the latter. As a result, the addition of the first H(2) molecule leads to two different products: a non-H-bridged intermediate for the Chirik-type complex and a H-bridged intermediate for the Fryzuk-type complex. The non-H-bridged intermediate requires a smaller energy barrier for the second H(2) addition than the H-bridged intermediate. We have also examined the effect of different numbers of methyl substituents in [(eta(5)-C(5)Me(n)H(5)(-)(n))(2)Zr](2)(mu(2),eta(2),eta(2)-N(2)) for n = 0, 4, and 5 (n = 5 is hypothetical) and [(eta(5)-C(5)H(2)-1,2,4-Me(3))(eta(5)-C(5)Me(5))(2)Zr](2)(mu(2),eta(2),eta(2)-N(2)) and have shown that all complexes of this type would follow a similar H(2) addition mechanism. We have also performed an extensive analysis on the factors (side-on coordination of N(2) to two Zr centers, availability of the frontier orbitals with appropriate symmetry, and inflexibility of the catalyst ligand environment) that are required for successful hydrogenation of the coordinated dinitrogen.

Radiosynthesis and in vitro evaluation of 2-(N-alkyl-N-1'-11C-propyl)amino-5-hydroxytetralin analogs as high affinity agonists for dopamine D-2 receptors.

PubMed

Shi, B; Narayanan, T K; Yang, Z Y; Christian, B T; Mukherjee, J

1999-10-01

We have developed radiotracers based on agonists that may potentially allow the in vivo assessment of the high affinity (HA) state of the dopamine D-2 receptors. The population of HA state, which is likely the functional state of the receptor, may be altered in certain diseases. We carried out radiosyntheses and evaluated the binding affinities, lipophilicity, and in vitro autoradiographic binding characteristics of three dopamine D-2 receptor agonists: (+/-)-2-(N,N-dipropyl)amino-5-hydroxytetralin (5-OH-DPAT), (+/-)-2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (PPHT), and (+/-)-2-(N-cyclohexylethyl-N-propyl)amino-5-hydroxytetralin (ZYY-339). In 3H-spiperone assays using rat striata, ZYY-339 exhibited subnanomolar affinity for D-2 receptor sites (IC50 = 0.010 nM), PPHT was somewhat weaker (IC50 = 0.65 nM), and 5-OH-DPAT exhibited the weakest affinity (IC50 = 2.5 nM) of the three compounds. Radiosynthesis of these derivatives, 2-(N-propyl-N-1'-11C-propyl)amino-5-hydroxytetralin (11C-5-OH-DPAT), 2-(N-phenethyl-N-1'-11C-propyl)amino-5-hydroxytetralin (11C-PPHT), and 2-(N-cyclohexylethyl-N-1'-11C-propyl)amino-5-hydroxytetralin (11C-ZYY-339) was achieved by first synthesizing 11C-1-propionyl chloride and subsequent coupling with the appropriate secondary amine precursor to form the respective amide, which was then reduced to provide the desired tertiary amine products. The final products were obtained by reverse-phase high performance liquid chromatography (HPLC) purification in radiochemical yields of 5-10% after 60-75 min from the end of 11CO2 trapping and with specific activities in the range of 250-1,000 Ci/mmol. In vitro autoradiographs in rat brain slices with 11C-5-OH-DPAT, 11C-PPHT, and 11C-ZYY-339 revealed selective binding of the three radiotracers to the dopamine D-2 receptors in the striata.

Tachykinins mediate contraction of the human lower esophageal sphincter in vitro via activation of NK2 receptors.

PubMed

Huber, O; Bertrand, C; Bunnett, N W; Pellegrini, C A; Nadel, J A; Nakazato, P; Debas, H T; Geppetti, P

1993-08-03

The contractile response to natural tachykinins and selective peptide agonists for tachykinin receptors was studied in strips of circular smooth muscle of human lower esophageal sphincter in vitro. The effects of phosphoramidon, which inhibits neutral endopeptidase (EC.3.4.24.11) and of the non-peptide compounds, SR 48968 and CP-96,345, which selectively block NK1 and NK2 receptors, respectively, were also investigated. Substance P, neurokinin A and neurokinin B produced a concentration-dependent contractile response. The rank order of potency was neurokinin A > neurokinin B > substance P. Phosphoramidon (1 microM) potentiated the response to substance P without changing the order of potency of natural tachykinins. The NK2-selective agonist, ([ beta Ala8]neurokinin A-(4-10)), produced a concentration-dependent contraction. The NK1 ([Sar9,Met(O2)11]substance P, 1 microM) and NK3 ([MePhe7]neurokinin B, 1 microM) selective agonists, however, did not exert any contractile effect. The selective NK2 antagonist, SR 48968, potently inhibited in a concentration-dependent (10 nM-1 microM) manner the response to neurokinin A, without affecting the response to carbachol. The selective NK1 antagonist, CP-96,345 (1 microM), did not affect the response to neurokinin A. These results indicate that tachykinins contract the circular muscle of human lower esophageal sphincter, and that this effect is mediated by NK2 receptor stimulation. Moreover, a phosphoramidon-sensitive mechanism plays a role in the regulation of the response to substance P.

A periplasmic D-alanyl-D-alanine dipeptidase in the gram-negative bacterium Salmonella enterica.

PubMed

Hilbert, F; García-del Portillo, F; Groisman, E A

1999-04-01

The VanX protein is a D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase essential for resistance to the glycopeptide antibiotic vancomycin. While this enzymatic activity has been typically associated with vancomycin- and teicoplainin-resistant enterococci, we now report the identification of a D-Ala-D-Ala dipeptidase in the gram-negative species Salmonella enterica. The Salmonella enzyme is only 36% identical to VanX but exhibits a similar substrate specificity: it hydrolyzes D-Ala-D-Ala, DL-Ala-DL-Phe, and D-Ala-Gly but not the tripeptides D-Ala-D-Ala-D-Ala and DL-Ala-DL-Lys-Gly or the dipeptides L-Ala-L-Ala, N-acetyl-D-Ala-D-Ala, and L-Leu-Pro. The Salmonella dipeptidase gene, designated pcgL, appears to have been acquired by horizontal gene transfer because pcgL-hybridizing sequences were not detected in related bacterial species and the G+C content of the pcgL-containing region (41%) is much lower than the overall G+C content of the Salmonella chromosome (52%). In contrast to wild-type Salmonella, a pcgL mutant was unable to use D-Ala-D-Ala as a sole carbon source. The pcgL gene conferred D-Ala-D-Ala dipeptidase activity upon Escherichia coli K-12 but did not allow growth on D-Ala-D-Ala. The PcgL protein localizes to the periplasmic space of Salmonella, suggesting that this dipeptidase participates in peptidoglycan metabolism.

Differential α4(+)/(−)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms*

PubMed Central

Lucero, Linda M.; Weltzin, Maegan M.; Eaton, J. Brek; Cooper, John F.; Lindstrom, Jon M.; Lukas, Ronald J.; Whiteaker, Paul

2016-01-01

Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(−)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(−)α4 site with lower agonist affinity than the α4(+)/(−)β2 sites. However, the relative roles of the conserved α4(+)/(−)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (−)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with 125I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(−)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(−)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect. PMID:26644472

The delta-opioid receptor agonist SNC80 [(+)-4-[alpha(R)-alpha-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-(3-methoxybenzyl)-N,N-diethylbenzamide] synergistically enhances the locomotor-activating effects of some psychomotor stimulants, but not direct dopamine agonists, in rats.

PubMed

Jutkiewicz, Emily M; Baladi, Michelle G; Folk, John E; Rice, Kenner C; Woods, James H

2008-02-01

The nonpeptidic delta-opioid agonist SNC80 [(+)-4-[alpha(R)-alpha-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-(3-methoxybenzyl)-N,N-diethylbenzamide] produces many stimulant-like behavioral effects in rodents and monkeys, such as locomotor stimulation, generalization to cocaine in discrimination procedures, and antiparkinsonian effects. Tolerance to the locomotor-stimulating effects of SNC80 develops after a single administration of SNC80 in rats; it is not known whether cross-tolerance develops to the effects of other stimulant compounds. In the initial studies to determine whether SNC80 produced cross-tolerance to other stimulant compounds, it was discovered that amphetamine-stimulated locomotor activity was greatly enhanced in SNC80-pretreated rats. This study evaluated acute cross-tolerance between delta-opioid agonists and other locomotor-stimulating drugs. Locomotor activity was measured in male Sprague-Dawley rats implanted with radiotransmitters, and activity levels were recorded in the home cage environment. Three-hour SNC80 pretreatment produced tolerance to further delta-opioid receptor stimulation but also augmented greatly amphetamine-stimulated locomotor activity in a dose-dependent manner. Pretreatments with other delta-opioid agonists, (+)BW373U86 [(+)-4-[alpha(R)-alpha-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide] and oxymorphindole (17-methyl-6,7-dehydro-4,5-epoxy-3,14-dihydroxy-6,7,2',3'-indolomorphinan), also modified amphetamine-induced activity levels. SNC80 pretreatment enhanced the stimulatory effects of the dopamine/norepinephrine transporter ligands cocaine and nomifensine (1,2,3,4-tetrahydro-2-methyl-4-phenyl-8-isoquinolinanmine maleate salt), but not the direct dopamine receptor agonists SKF81297 [R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide] and quinpirole [trans-(-)-(4alphaR)-4,4a, 5,6,7,8,8a,9-octahydro-5-propyl-1H-pyrazolo[3,4-g] quinoline

Modified melanocortin tetrapeptide Ac-His-dPhe-Arg-Trp-NH at the arginine side chain with ureas and thioureas.

PubMed

Joseph, C G; Sorensen, N B; Wood, M S; Xiang, Z; Moore, M C; Haskell-Luevano, C

2005-11-01

The Ac-His-dPhe-Arg-Trp-NH2 tetrapeptide is a nonselective melanocortin agonist and replacement of Arg in the tetrapeptide with acidic, basic or neutral amino acids results in reduced potency at the melanocortin receptor (MCR) isoforms (MC1R and MC3-5R). To determine the importance of the positive charge and the guanidine moiety for melanocortin activity, a series of urea- and thiourea-substituted tetrapeptides were designed. Replacement of Arg with Lys or ornithine reduced agonist activity at the mouse mMC1 and mMC3-5 receptors, thus supporting the hypothesis that the guanidine moiety is important for receptor potency, particularly at the MC3-5 receptors. The Arg side chain-modified tetrapeptides examined in this study include substituted phenyl, naphthyl, and aliphatic urea and thiourea residues using a Lys side-chain template. These ligands elicit full-agonist pharmacology at the mouse MCRs examined in this study.

Design and Synthesis of 2-Alkylpyrimidine-4,6-diol and 6-Alkylpyridine-2,4-diol as Potent GPR84 Agonists.

PubMed

Liu, Yang; Zhang, Qing; Chen, Lin-Hai; Yang, Hui; Lu, Wei; Xie, Xin; Nan, Fa-Jun

2016-06-09

A series of alkylpyrimidine-4,6-diol derivatives were designed and synthesized as novel GRP84 agonists based on a high-throughput screening (HTS) hit 1. 6-Nonylpyridine-2,4-diol was identified as the most potent agonist of GPR84 reported so far, with an EC50 of 0.189 nM. These novel GPR84 agonists will provide valuable tools for the study of the physiological functions of GPR84.

Design and Synthesis of 2-Alkylpyrimidine-4,6-diol and 6-Alkylpyridine-2,4-diol as Potent GPR84 Agonists

PubMed Central

2016-01-01

A series of alkylpyrimidine-4,6-diol derivatives were designed and synthesized as novel GRP84 agonists based on a high-throughput screening (HTS) hit 1. 6-Nonylpyridine-2,4-diol was identified as the most potent agonist of GPR84 reported so far, with an EC50 of 0.189 nM. These novel GPR84 agonists will provide valuable tools for the study of the physiological functions of GPR84. PMID:27326330

Highly potent analogues of luteinizing hormone-releasing hormone containing D-phenylalanine nitrogen mustard in position 6

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bajusz, S.; Janaky, T.; Csernus, V.J.

The nitrogen mustard derivatives of 4-phenylbutyric acid and L-phenylalanine, called chlorambucil (Chl) and melphalan (Mel), respectively, have been incorporated into several peptide hormones, including luteinizing hormone-releasing hormone (LH-RH). The alkylating analogues of LH-RH were prepared by linking Chl, as an N-acyl moiety, to the complete amino acid sequence of agonistic and antagonistic analogues. These compounds, in particular the antagonistic analogues, showed much lower potency than their congeners carrying other acyl groups. To obtain highly potent alkylating analogues of LH-RH, the D enantiomer of Mel was incorporated into position 6 of the native hormone and some of its antagonistic analogues. Ofmore » the peptides prepared, (D-Mel{sup 6})LH-RH (SB-05) and (Ac-D-Nal(2){sup 1},D-Phe(pCl){sup 2},D-Pal(3){sup 3},Arg{sup 5},D-Mel{sup 6},D-Ala{sup 10})LH-RH (SB-86, where Nal(2) is 3-(2-naphthyl)alanine and Pal(3) is 3-(3-pyridyl)alanine) possessed the expected high agonistic and antagonistic activities, respectively, and also showed high affinities for the membrane receptors of rat pituitary cells, human breast cancer cells, human prostate cancer cells, and rat Dunning R-3327 prostate tumor cells. These two analogues exerted cytotoxic effects on human and rat mammary cancer cells in vitro. Thus these two D-Mel{sup 6} analogues seem to be particularly suitable for the study of how alkylating analogues of LH-RH could interfere with intracellular events in certain cancer cells.« less

Working memory span capacity improved by a D2 but not D1 receptor family agonist.

PubMed

Tarantino, Isadore S; Sharp, Richard F; Geyer, Mark A; Meves, Jessica M; Young, Jared W

2011-06-01

Patients with schizophrenia exhibit poor working memory (WM). Although several subcomponents of WM can be measured, evidence suggests the primary subcomponent affected in schizophrenia is span capacity (WMC). Indeed, the NIMH-funded MATRICS initiative recommended assaying the WMC when assessing the efficacy of a putative therapeutic for FDA approval. Although dopamine D1 receptor agonists improve delay-dependent memory in animals, evidence for improvements in WMC due to dopamine D1 receptor activation is limited. In contrast, the dopamine D2-family agonist bromocriptine improves WMC in humans. The radial arm maze (RAM) can be used to assess WMC, although complications due to ceiling effects or strategy confounds have limited its use. We describe a 12-arm RAM protocol designed to assess whether the dopamine D1-family agonist SKF 38393 (0, 1, 3, and 10 mg/kg) or bromocriptine (0, 1, 3, and 10 mg/kg) could improve WMC in C57BL/6N mice (n=12) in cross-over designs. WMC increased and strategy usage decreased with training. The dopamine D1 agonist SKF 38393 had no effect on WMC or long-term memory. Bromocriptine decreased WMC errors, without affecting long-term memory, consistent with human studies. These data confirm that WMC can be measured in mice and reveal drug effects that are consistent with reported effects in humans. Future research is warranted to identify the subtype of the D2-family of receptors responsible for the observed improvement in WMC. Finally, this RAM procedure may prove useful in developing animal models of deficient WMC to further assess putative treatments for the cognitive deficits in schizophrenia. Copyright © 2011 Elsevier B.V. All rights reserved.

Solvent selection and optimization of α-chymotrypsin-catalyzed synthesis of N-Ac-Phe-Tyr-NH2 using mixture design and response surface methodology.

PubMed

Hu, Shih-Hao; Kuo, Chia-Hung; Chang, Chieh-Ming J; Liu, Yung-Chuan; Chiang, Wen-Dee; Shieh, Chwen-Jen

2012-01-01

A peptide, N-Ac-Phe-Tyr-NH(2) , with angiotensin I-converting enzyme (ACE) inhibitor activity was synthesized by an α-chymotrypsin-catalyzed condensation reaction of N-acetyl phenylalanine ethyl ester (N-Ac-Phe-OEt) and tyrosinamide (Tyr-NH(2) ). Three kinds of solvents: a Tris-HCl buffer (80 mM, pH 9.0), dimethylsulfoxide (DMSO), and acetonitrile were employed in this study. The optimum reaction solvent component was determined by simplex centroid mixture design. The synthesis efficiency was enhanced in an organic-aqueous solvent (Tris-HCl buffer: DMSO: acetonitrile = 2:1:1) in which 73.55% of the yield of N-Ac-Phe-Tyr-NH(2) could be achieved. Furthermore, the effect of reaction parameters on the yield was evaluated by response surface methodology (RSM) using a central composite rotatable design (CCRD). Based on a ridge max analysis, the optimum condition for this peptide synthesis included a reaction time of 7.4 min, a reaction temperature of 28.1°C, an enzyme activity of 98.9 U, and a substrate molar ratio (Phe:Tyr) of 1:2.8. The predicted and the actual (experimental) yields were 87.6 and 85.5%, respectively. The experimental design and RSM performed well in the optimization of synthesis of N-Ac-Phe-Tyr-NH(2) , so it is expected to be an effective method for obtaining a good yield of enzymatic peptide. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

Neurokinin receptors in the rabbit iris sphincter characterised by novel agonist ligands.

PubMed

Hall, J M; Mitchell, D; Morton, I K

1991-06-18

We have used novel selective agonist ligands to examine neurokinin receptors mediating the contractile response to tachykinins in the rabbit iris sphincter preparation in vitro. The selective NK-1 receptor agonist delta-amino valeryl-[L-Pro9,N-Me Leu10]SP-(7-11) (GR73632) and the NK-3 receptor-selective agonist succ-[Asp6,N-Me-Phe8] SP-(6-11) (senktide) were both very active (concentration range 0.032 pM-10 nM and 0.1 pM-32 nM respectively), and were 933 and 16.6 times more potent than substance P, respectively, in contracting the iris. In contrast, the NK-2 selective agonist [Lys3,Gly8-R-gamma-lactam,Leu9]NKA-(3-10) (GR64349) was active only at the highest concentrations tested (3.2 nM-32 microM), and had 0.054 the activity of substance P. The presence of several peptidase inhibitors was without effect on the concentration-response relationship to substance P, GR73632, GR64349 or senktide. Tachykinins differed in their offset kinetics. Responses to GR73632, GR64349 and senktide were rapid in offset (times to reach half maximal responses were 1.5, 1.1 and 5.1 min, respectively), whereas responses to substance P were very much more prolonged in duration (time to reach half maximal response was 35.3 min). These results suggest the presence of both NK-1 and NK-3 receptors mediating contraction of the rabbit iris sphincter preparation. In addition, differences in response offset kinetics seem not to be due to differences in peptide metabolism, and suggest a property of substance P not shared by the other tachykinins used in this study.

cDNAs encoding [D-Ala2]deltorphin precursors from skin of Phyllomedusa bicolor also contain genetic information for three dermorphin-related opioid peptides.

PubMed

Richter, K; Egger, R; Negri, L; Corsi, R; Severini, C; Kreil, G

1990-06-01

We present the structure of four precursors for [D-Ala2]deltorphins I and II as deduced from cDNAs cloned from skin of the frog Phyllomedusa bicolor. These contain the genetic information for one copy of [D-Ala2]deltorphin II and zero, one, or three copies of [D-Ala2]deltorphin I. In each case, the D-alanine of the end product is encoded by a normal GCG codon for L-alanine. In addition, the existence of three peptides related to dermorphin was predicted from the amino acid sequence of the precursors. These peptides were synthesized with a D-alanine in position 2 and their pharmacological properties were tested. Two of them, [Lys7]dermorphin-OH and [Trp4,Asn7]dermorphin-OH, were found to have roughly the same affinity and selectivity for mu-type opioid receptors as dermorphin.

Selective melanocortin MC4 receptor agonists reverse haemorrhagic shock and prevent multiple organ damage

PubMed Central

Giuliani, D; Mioni, C; Bazzani, C; Zaffe, D; Botticelli, A R; Capolongo, S; Sabba, A; Galantucci, M; Iannone, A; Grieco, P; Novellino, E; Colombo, G; Tomasi, A; Catania, A; Guarini, S

2007-01-01

Background and purpose: In circulatory shock, melanocortins have life-saving effects likely to be mediated by MC4 receptors. To gain direct insight into the role of melanocortin MC4 receptors in haemorrhagic shock, we investigated the effects of two novel selective MC4 receptor agonists. Experimental approach: Severe haemorrhagic shock was produced in rats under general anaesthesia. Rats were then treated with either the non-selective agonist [Nle4, D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH) or with the selective MC4 agonists RO27-3225 and PG-931. Cardiovascular and respiratory functions were continuously monitored for 2 h; survival rate was recorded up to 24 h. Free radicals in blood were measured using electron spin resonance spectrometry; tissue damage was evaluated histologically 25 min or 24 h after treatment. Key results: All shocked rats treated with saline died within 30-35 min. Treatment with NDP-α-MSH, RO27-3225 and PG-931 produced a dose-dependent (13-108 nmol kg-1 i.v.) restoration of cardiovascular and respiratory functions, and improved survival. The three melanocortin agonists also markedly reduced circulating free radicals relative to saline-treated shocked rats. All these effects were prevented by i.p. pretreatment with the selective MC4 receptor antagonist HS024. Moreover, treatment with RO27-3225 prevented morphological and immunocytochemical changes in heart, lung, liver, and kidney, at both early (25 min) and late (24 h) intervals. Conclusions and Implications: Stimulation of MC4 receptors reversed haemorrhagic shock, reduced multiple organ damage and improved survival. Our findings suggest that selective MC4 receptor agonists could have a protective role against multiple organ failure following circulatory shock. PMID:17245369

Metabolism of the tryptamine-derived new psychoactive substances 5-MeO-2-Me-DALT, 5-MeO-2-Me-ALCHT, and 5-MeO-2-Me-DIPT and their detectability in urine studied by GC-MS, LC-MSn , and LC-HR-MS/MS.

PubMed

Caspar, Achim T; Gaab, Jonas B; Michely, Julian A; Brandt, Simon D; Meyer, Markus R; Maurer, Hans H

2018-01-01

Many N,N-dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5-methoxy-2-methyl-N,N-diallyltryptamine (5-MeO-2-Me-DALT), 5-methoxy-2-methyl-N-allyl-N-cyclohexyltryptamine (5-MeO-2-Me-ALCHT), and 5-methoxy-2-methyl-N,N-diisopropyltryptamine (5-MeO-2-Me-DIPT) using gas chromatography-mass spectrometry (GC-MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC-MS n ), and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS). For metabolism studies, urine was collected over a 24 h period after administration of the compounds to male Wistar rats at 20 mg/kg body weight (BW). Phase I and II metabolites were identified after urine precipitation with acetonitrile by LC-HR-MS/MS. 5-MeO-2-Me-DALT (24 phase I and 12 phase II metabolites), 5-MeO-2-Me-ALCHT (24 phase I and 14 phase II metabolites), and 5-MeO-2-Me-DIPT (20 phase I and 11 phase II metabolites) were mainly metabolized by O-demethylation, hydroxylation, N-dealkylation, and combinations of them as well as by glucuronidation and sulfation of phase I metabolites. Incubations with mixtures of pooled human liver microsomes and cytosols (pHLM and pHLC) confirmed that the main metabolic reactions in humans and rats might be identical. Furthermore, initial CYP activity screenings revealed that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were involved in hydroxylation, CYP2C19 and CYP2D6 in O-demethylation, and CYP2C19, CYP2D6, and CYP3A4 in N-dealkylation. For SUSAs, GC-MS, LC-MS n , and LC-HR-MS/MS were applied to rat urine samples after 1 or 0.1 mg/kg BW doses, respectively. In contrast to the GC-MS SUSA, both LC-MS SUSAs were able to detect an intake of 5-MeO-2-Me-ALCHT and 5-MeO-2-Me-DIPT via their metabolites following 1 mg/kg BW administrations and 5-MeO-2-Me-DALT following

Self-assembled decanuclear Na(I)2Mn(II)4Mn(III)4 complexes: from discrete clusters to 1-D and 2-D structures, with the Mn(II)4Mn(III)4 unit displaying a large spin ground state and probable SMM behaviour.

PubMed

Langley, Stuart K; Chilton, Nicholas F; Moubaraki, Boujemaa; Murray, Keith S

2011-12-07

The synthesis, magnetic characterization and X-ray crystal structures are reported for five new manganese compounds, [Mn(III)(teaH(2))(sal)]·(1/2)H(2)O (1), [Na(I)(2)Mn(II)(4)Mn(III)(4)(teaH)(6)(sal)(4)(N(3))(2)(MeOH)(4)]·6MeOH (2), [Na(I)(2)Mn(II)(4)Mn(III)(4)(teaH)(6)(sal)(4)(N(3))(2)(MeOH)(2)](n)·7MeOH (3), [Na(I)(2)Mn(II)(4)Mn(III)(4)(teaH)(6)(sal)(4)(N(3))(2)(MeOH)(2)](n)·2MeOH·Et(2)O (4) and [K(I)(2)Mn(II)(4)Mn(III)(4)(teaH)(6)(sal)(4)(N(3))(2)(H(2)O)(2)](n)·5MeOH (5). Complex 1 is a mononuclear compound, formed via the reaction of Mn(NO(3))(2)·4H(2)O, triethanolamine (teaH(3)) and salicylic acid (salH(2)) in a basic methanolic solution. Compound 2 is a mixed-valent hetero-metallic cluster made up of a Mn(8)Na(2) decanuclear core and is formed via the reaction of sodium azide (NaN(3)) with 1. Compounds 3-5 are isolated as 1- or 2-D coordination polymers, each containing the decanuclear Mn(8)M(2) (M = Na(+) or K(+)) core building block as the repeating unit. Compound 3 is isolated when 1 is reacted with NaN(3) over a very short reaction time and forms a 1-D coordination polymer. Each unit displays inter-cluster bridges via the O-atoms of teaH(2-) ligands bonding to the sodium ions of an adjacent cluster. Increasing the reaction time appears to drive the formation of 4 which forms 2-D polymeric sheets and is a packing polymorph of 3. The addition of KMnO(4) and NaN(3) to 1 resulted in compound 5, which also forms a 1-D coordination polymer of the decanuclear core unit. The 1-D chains are now linked via inter-cluster potassium and salicylate bridges. Solid state DC susceptibility measurements were performed on compounds 1-5. The data for 1 are as expected for an S = 2 Mn(III) ion, with the isothermal M vs. H data being fitted by matrix diagonalization methods to give values of g and the axial (D) and rhombic (E) zero field splitting parameters of 2.02, -2.70 cm(-1) and 0.36 cm(-1) respectively. The data for 2-5, each with an identical Mn(II)(4)Mn(III)(4

Assembly of bicyclic or monocyclic clusters from [(η5-C5Me5)2Mo2(μ3-S)4(CuMeCN)2]2+ with tetraphosphine or N,P mixed ligands: syntheses, structures and enhanced third-order NLO performances.

PubMed

Ren, Zhi-Gang; Sun, Sha; Dai, Min; Wang, Hui-Fang; Lü, Chun-Ning; Lang, Jian-Ping; Sun, Zhen-Rong

2011-09-07

Reactions of the preformed cluster [(η(5)-C(5)Me(5))(2)Mo(2)(μ(3)-S)(4)Cu(2)(MeCN)(2)](ClO(4))(2) (1) with two tetraphosphine ligands, 1,4-N,N,N',N'-tetra(diphenylphosphanylmethyl)benzene diamine (dpppda) and N,N,N',N'-tetra(diphenylphosphanylmethyl)ethylene diamine (dppeda), produced two bicyclic clusters {[(η(5)-C(5)Me(5))(2)Mo(2)(μ(3)-S)(4)Cu(2)](2)(L)}(ClO(4))(4) (3: L = dpppda; 4: L = dppeda). Analogous reactions of 1 or [(η(5)-C(5)Me(5))(2)Mo(2)(μ(3)-S)(4)Cu(2)(MeCN)(2)](PF(6))(2) (2) with two N,P mixed ligands, N,N-bi(diphenylphosphanylmethyl)-2-aminopyridine (bdppmapy) and N-diphenylphosphanylmethyl-4-aminopyridine (dppmapy), afforded two monocyclic clusters {[(η(5)-C(5)Me(5))(2)Mo(2)(μ(3)-S)(4)Cu(2)](2)(L)(2)}X(4) (5: L = bdppmapy, X = ClO(4); 6: L = dppmapy, X = PF(6)). Compounds 3-6 were fully characterized by elemental analysis, IR spectra, UV-vis spectra, (1)H and (31)P{(1)H} NMR spectra, ESI-MS and single-crystal X-ray crystallography. In the tetracations of 3-6, two cubane-like [Mo(2)(μ(3)-S)(4)Cu(2)] cores are linked either by one dpppda or dppeda bridge to form a bicyclic structure or by a pair of bdppmapy or dppmapy bridges to afford a monocyclic structure. The third-order nonlinear optical (NLO) properties of 1 and 3-6 in MeCN were also investigated by femtosecond degenerate four-wave mixing (DFWM) technique with a 50 fs pulse width at 800 nm. Compounds 3-6 exhibited enhanced third-order NLO performances relative to that of 1. This journal is © The Royal Society of Chemistry 2011

cDNAs encoding [D-Ala2]deltorphin precursors from skin of Phyllomedusa bicolor also contain genetic information for three dermorphin-related opioid peptides.

PubMed Central

Richter, K; Egger, R; Negri, L; Corsi, R; Severini, C; Kreil, G

1990-01-01

We present the structure of four precursors for [D-Ala2]deltorphins I and II as deduced from cDNAs cloned from skin of the frog Phyllomedusa bicolor. These contain the genetic information for one copy of [D-Ala2]deltorphin II and zero, one, or three copies of [D-Ala2]deltorphin I. In each case, the D-alanine of the end product is encoded by a normal GCG codon for L-alanine. In addition, the existence of three peptides related to dermorphin was predicted from the amino acid sequence of the precursors. These peptides were synthesized with a D-alanine in position 2 and their pharmacological properties were tested. Two of them, [Lys7]dermorphin-OH and [Trp4,Asn7]dermorphin-OH, were found to have roughly the same affinity and selectivity for mu-type opioid receptors as dermorphin. PMID:2352951

Pharmacological characterization of extracellular acidification rate responses in human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells

PubMed Central

Coldwell, M C; Boyfield, I; Brown, A M; Stemp, G; Middlemiss, D N

1999-01-01

This study characterized pharmacologically the functional responses to agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) zreceptors separately expressed in cloned cells using the cytosensor microphysiometer. Dopaminergic receptor agonists caused increases in extracellular acidification rate in adherent Chinese hamster ovary (CHO) clones expressing hD2, hD3 or hD4 receptors. Acidification rate responses to agonists in other cell lines expressing these receptors were smaller than those in adherent CHO cells. The time courses and maximum increases in acidification rate of the agonist responses in adherent CHO cells were different between the three dopamine receptor clones. Responses were blocked by pretreatment of cells with pertussis toxin or amiloride analogues. Most agonists had full intrinsic activity at each of the dopamine receptor subtypes, as compared to quinpirole, however both enantiomers of UH-232 and (−)3-PPP were partial agonists in this assay system. The functional potency of full agonists at each of the three receptors expressed in CHO cells was either higher than, or similar to, the apparent inhibition constants (Ki) determined in [125I]-iodosulpride competition binding studies. Functional selectivities of the agonists were less than radioligand binding selectivities. The rank orders of agonist potencies and selectivities were similar, but not identical, to the rank orders of radioligand binding affinities and selectivities. The dopamine receptor antagonists, iodosulpride and clozapine, had no effect on basal acidification rates but inhibited acidification responses in CHO cells to quinpirole in an apparently competitive manner. Antagonist potencies closely matched their radioligand binding affinities in these cells. PMID:10455259

Effects of Serotonergic and Opioidergic Drugs on Escape Behaviors and Social Status of Male Crickets

NASA Astrophysics Data System (ADS)

Dyakonova, V. E.; Schürmann, F.-W.; Sakharov, D. A.

We examined the effects of selective serotonin depletion and opioid ligands on social rank and related escape behavior of the cricket Gryllus bimaculatus. Establishment of social rank in a pair of males affected their escape reactions. Losers showed a lower and dominants a higher percentage of jumps in response to tactile cercal stimulation than before a fight. The serotonin-depleting drug α-methyltryptophan (AMTP) caused an activation of the escape reactivity in socially naive crickets. AMTP-treated animals also showed a lower ability to become dominants. With an initial 51.6+/-3.6% of wins in the AMTP group, the percentage decreased to 26+/-1.6% on day 5 after injection. The opiate receptor antagonist naloxone affected fight and escape similarly as AMTP. In contrast to naloxone, the opioid agonist [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin decreased escape responsiveness to cercal stimulation in naive and subordinate crickets. We suggest that serotonergic and opioid systems are involved in the dominance induced depression of escape behavior.

Design, synthesis and evaluation of a potent substrate analog inhibitor identified by scanning Ala/Phe mutagenesis, mimicking substrate co-evolution, against multidrug-resistant HIV-1 protease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yedidi, Ravikiran S.; Muhuhi, Joseck M.; Liu, Zhigang

Highlights: •Inhibitors against MDR HIV-1 protease were designed, synthesized and evaluated. •Lead peptide (6a) showed potent inhibition (IC{sub 50}: 4.4 nM) of MDR HIV-1 protease. •(6a) Showed favorable binding isotherms against NL4-3 and MDR proteases. •(6a) Induced perturbations in the {sup 15}N-HSQC spectrum of MDR HIV-1 protease. •Molecular modeling suggested that (6a) may induce total flap closure inMDR protease. -- Abstract: Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: (1TW7)), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded activemore » site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC{sub 50}: 4.4 nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6aagainst both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of {sup 15}N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV.« less

Potentiation of kinin analogues by ramiprilat is exclusively related to their degradation.

PubMed

Dendorfer, A; Reibetamann, S; Wolfrum, S; Raasch, W; Dominiak, P

2001-07-01

The potentiation of kinin actions represents a cardioprotective property of ACE inhibitors. Although a clear contribution to this effect is related to the inhibition of bradykinin (BK) breakdown, the high efficacy of potentiation and the ability of ACE inhibitors to provoke a B(2)-receptor-mediated response even after receptor desensitization has also triggered hypotheses concerning additional mechanisms of kinin potentiation. The application of kinin analogues with enhanced metabolic stability for the demonstration of degradation-independent mechanisms of potentiation, however, has yielded inconsistent results. Therefore, the relation between the susceptibility of B(2)-agonists to ACE and the potentiation of their actions by ACE inhibitors was investigated with the use of minimally modified kinin derivatives that varied in their degree of ACE resistance. The B(2)-agonists BK, D-Arg-[Hyp(3)]-BK, [Hyp,(3) Tyr(Me)(8)]-BK, [DeltaPhe(5)]-BK, [D-NMF(7)]-BK, and [Phe(8)psi(CH(2)-NH)Arg(9)]-BK were tested for degradation by purified rabbit ACE and for their potency in contracting the endothelium-denuded rabbit jugular vein in the absence and presence of ramiprilat. Purified ACE degraded D-Arg-[Hyp(3)]-BK and [Hyp,(3) Tyr(Me)(8)]-BK at 81% and 71% of BK degradation activity, respectively, whereas other peptides were highly ([DeltaPhe(5)]-BK) or completely ([D-NMF(7)]-BK, [Phe(8)psi(CH(2)-NH)Arg(9)]-BK) resistant. The EC(50) of BK-induced venoconstriction (1.15+/-0.2 nmol/L) was reduced by a factor of 5.7 in the presence of ramiprilat. Likewise, D-Arg-[Hyp(3)]-BK and [Hyp,(3) Tyr(Me)(8)]-BK were both significantly potentiated by a factor of 4.4, whereas the activities of the other agonists were not affected. Ramiprilat exerted no influence on the maximum contraction induced by any of the agonists. It is concluded that the potentiation of kinin analogues during ACE inhibition correlates quantitatively with the susceptibility of each substance to degradation by ACE. As such

Antagonists of substance P. Further modifications of substance P antagonists obtained by replacing either positions 7, 9 or 7, 8 and 11 of SP with D-amino acid residues.

PubMed

Dutta, A S; Gormley, J J; Graham, A S; Briggs, I; Growcott, J W; Jamieson, A

1986-07-01

Antagonists of SP and the C-terminal (6-11)-hexapeptide have been obtained by multiple D-amino acid substitutions in various positions of SP and by protecting the N alpha-Arg1 and N epsilon Lys3 amino groups with benzyloxycarbonyl groups. On the guinea pig ileum a number of these antagonized both SP and the hexapeptide. Except [N alpha-Z-Arg1,D-Pro2,N epsilon-Z-Lys3,Asn5,Arg6,D-Phe7,D-Trp9]-SP-OMe (4) and the corresponding amide 7, which were more potent antagonists of SP than the hexapeptide, all the others, e.g., [N alpha-Z-Arg1,D-Pro2,4,N epsilon-Z-Lys3,D-Phe7,8,Sar9,D-Met11]-SP-OMe (9), [N alpha-Z-Arg1,D-Pro2,4,N epsilon-Z-Lys3,D-Phe7,8,Sar9,MeLeu10,D-Met11]-SP -OMe (11), were more potent antagonists of the hexapeptide. On the rat spinal cord preparation, most of the antagonists were only active against the hexapeptide. A few antagonized SP, but these also reduced carbachol or both carbachol and glutamate responses. Two of the antagonists, [D-Pro2,Asn5,Lys6,D-Phe7,D-Trp9]-SP-OMe (2) and [Boc-D-Pro4,D-Phe7,8,Sar9,D-Met11]-SP(4-11)-OMe (10), were inactive on the ileum but still antagonized the hexapeptide on the spinal cord. The smallest peptides to antagonize SP and the hexapeptide were two heptapeptides, 6 and 21, [Z-Asn5,Arg6,D-Phe7,8,Gly9 psi (CH2S)D-Leu10,D-Met11]-SP(5-11)-OMe (21) being more potent than 6. None of the antagonists showed significant analgesic activity without side effects. Some of the antagonists were shown to release histamine from isolated rat peritoneal cells.

Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses

PubMed Central

Ferreira, Joana S; Papouin, Thomas; Ladépêche, Laurent; Yao, Andrea; Langlais, Valentin C; Bouchet, Delphine; Dulong, Jérôme; Mothet, Jean-Pierre; Sacchi, Silvia; Pollegioni, Loredano; Paoletti, Pierre; Oliet, Stéphane Henri Richard; Groc, Laurent

2017-01-01

The subunit composition of synaptic NMDA receptors (NMDAR), such as the relative content of GluN2A- and GluN2B-containing receptors, greatly influences the glutamate synaptic transmission. Receptor co-agonists, glycine and D-serine, have intriguingly emerged as potential regulators of the receptor trafficking in addition to their requirement for its activation. Using a combination of single-molecule imaging, biochemistry and electrophysiology, we show that glycine and D-serine relative availability at rat hippocampal glutamatergic synapses regulate the trafficking and synaptic content of NMDAR subtypes. Acute manipulations of co-agonist levels, both ex vivo and in vitro, unveil that D-serine alter the membrane dynamics and content of GluN2B-NMDAR, but not GluN2A-NMDAR, at synapses through a process requiring PDZ binding scaffold partners. In addition, using FRET-based FLIM approach, we demonstrate that D-serine rapidly induces a conformational change of the GluN1 subunit intracellular C-terminus domain. Together our data fuels the view that the extracellular microenvironment regulates synaptic NMDAR signaling. DOI: http://dx.doi.org/10.7554/eLife.25492.001 PMID:28598327

In situ reaction mechanism studies on the Ti(NMe{sub 2}){sub 2}(O{sup i}Pr){sub 2}-D{sub 2}O and Ti(O{sup i}Pr){sub 3}[MeC(N{sup i}Pr){sub 2}]-D{sub 2}O atomic layer deposition processes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomczak, Yoann, E-mail: yoann.tomczak@helsinki.fi; Knapas, Kjell; Leskelä, Markku

2014-01-15

Reaction mechanisms in the Ti(NMe{sub 2}){sub 2}(O{sup i}Pr){sub 2}-D{sub 2}O and Ti(O{sup i}Pr){sub 3}[MeC(N{sup i}Pr){sub 2}] [also written Ti(O{sup i}Pr){sub 3}(N{sup i}Pr-Me-amd)]-D{sub 2}O atomic layer deposition processes were studied in situ with quartz crystal microbalance (QCM) and quadrupole mass spectrometry (QMS) at 275 °C. For the Ti(NMe{sub 2}){sub 2}(O{sup i}Pr){sub 2}-D{sub 2}O process, both QCM and QMS results indicated adsorption of the Ti(NMe{sub 2}){sub 2}(O{sup i}Pr){sub 2} molecule through an exchange of at least one of its –NMe{sub 2} ligands with surface hydroxyl groups. Regarding the Ti(O{sup i}Pr){sub 3}(N{sup i}Pr-Me-amd)-D{sub 2}O process, a mismatch between the QCM and QMS results revealedmore » more complex reactions: the decomposition of the [MeC(N{sup i}Pr){sub 2}] [also written (N{sup i}Pr-Me-amd)] ligand is suggested by the shape of the QCM data and the intensity of the QMS signals belonging to fragments of the [MeC(N{sup i}Pr){sub 2}] [also written (N{sup i}Pr-Me-amd)] ligand. A simple calculation model associating the growth rate per cycle of a crystalline film and the surface area taken by the ligands remaining after saturation was also used to support the decomposition of the [MeC(N{sup i}Pr){sub 2}] [also written (N{sup i}Pr-Me-amd)] ligand. The observed high growth rate is incompatible with the whole [MeC(N{sup i}Pr){sub 2}] (also written [N{sup i}Pr-Me-amd)] ligand remaining on the surface.« less

A new anti-MRSA antibiotic complex, WAP-8294A II. Structure characterization of minor components by ESI LCMS and MS/MS.

PubMed

Kato, Azusa; Hirata, Haruhisa; Ohashi, Yoshitami; Fujii, Kiyonaga; Mori, Kenji; Harada, Ken-ichi

2011-05-01

The anti-MRSA antibiotic, WAP-8294A, was isolated from the fermentation broth of Lysobacter sp. The major component, WAP-8294A2, is composed of 1 mol of Gly, L-Leu, L-Glu, D-Asn, D-Trp, D-threo-β-hydroxyasparagine, N-Me-D-Phe and N-Me-L-Val, and 2 mol of L-Ser, D-Orn and D-3-hydroxy-7-Me-octanoic acid. The structure of the WAP-8294A2 was mainly determined as a cyclic depsipeptide by 2D NMR experiments. However, it was difficult to use the NMR experiment to determine the minor components, A1, A4 and Ax13, isolated in small amounts. In the present study, ESI MS/MS was applied to the structure elucidation of these minor components. The structures of these minor components were determined on the basis of the fragmentation pattern of the product ions of WAP-8294A2 in the ESI MS/MS. As a result, it was confirmed that A1 and A4 had the same amino acid sequence as A2, while A1 and A4 had the 3-OH-octanoic acid and 3-OH-8-Me-nonanoic acid, respectively, in the place of the 3-OH-7-Me-octanoic acid in A2. In the structure of Ax13, it was found that Gly of A2 was changed to β-Ala of Ax13. © 2011 Japan Antibiotics Research Association All rights reserved

Conformational analysis of α-helical polypeptide included L-proline residue by high-resolution solid-state NMR measurement and quantum chemical calculation

NASA Astrophysics Data System (ADS)

Souma, Hiroyuki; Shoji, Akira; Kurosu, Hiromichi

2008-10-01

We challenged the problem about the stabilization mechanism of an α-helix formation for polypeptides containing L-proline (Pro) residue. We computed the optimized structure of α-helical poly( L-alanine) molecules including a Pro residue, H-(Ala) 8-Pro-(Ala) 9-OH, based on the molecular orbital calculation with density functional theory, B3LYP/6-31G(d) and the 13C and 15N chemical shift values based on the GIAO-CHF method with B3LYP/6-311G(d,p), respectively. It was found that two kinds of optimized structures, 'Bent structure' and 'Included α-helix structure', were preferred structures in H-(Ala) 8-Pro-(Ala) 9-OH. In addition, based on the precise 13C and 15N chemical shift data of the simple model, we successfully analyzed the secondary structure of well-defined synthetic polypeptide H-(Phe-Leu-Ala) 3-Phe C-Pro-Ala N-(Phe-Leu-Ala) 2-OH (FLA-11P), the secondary structure of which was proven to the 'Included α-helix structure'.

Truncation studies of alpha-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity.

PubMed

Haskell-Luevano, C; Sawyer, T K; Hendrata, S; North, C; Panahinia, L; Stum, M; Staples, D J; Castrucci, A M; Hadley, M F; Hruby, V J

1996-01-01

Truncation studies of alpha-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity: PEPTIDES 17(6) 995-1002, 1996.-Systematic analysis of fragment derivatives of the superpotent alpha-MSH analogue. Ac-Ser.Tyr-Ser-Nle4-Glu- His-DPhe7-Arg-Trp-Gly-Lys-Pro-Val-NH2(NDP-MSH), led to the discovery of tripeptide agonists possessing prolonged bioactivity in the frog skin assay. Of particular significance to this discovery was Ac-DPhe-Arg-DTrp-NH2, which was the most potent tripeptide in this series exhibiting sustained melanotropic activity. Different pharmacophore models appear to exist that are dependent on the substructure and stereochemistry of the MSH(6-9) "active site." The tripeptides Ac-DPhe-Arg-Trp-NH2, Ac-DPhe-Arg-DTrp-NH2, and Ac-DPhe-DArg-Trp-NH2 stereo-chemical combinations require only Phe7-Xaa8-Trp9, whereas Ac-DPhe-DArg-DTrp-NH2, Ac-Phe-Arg-DTrp-NH2, and Ac-Phe-Arg-Trp-NH2 additionally require His4 for minimal biological activity. Ac-DPhe-Arg-DTrp-NH2 represents a novel prototype lead for the development of MSH-based peptidomimetic agonists.

Mass Spectrometry Identification of N-Chlorinated Dipeptides in Drinking Water.

PubMed

Huang, Guang; Jiang, Ping; Li, Xing-Fang

2017-04-04

We report the identification of N-chlorinated dipeptides as chlorination products in drinking water using complementary high-resolution quadrupole time-of-flight (QTOF) and quadrupole ion-trap mass spectrometry techniques. First, three model dipeptides, tyrosylglycine (Tyr-Gly), tyrosylalanine (Tyr-Ala), and phenylalanylglycine (Phe-Gly), reacted with sodium hypochlorite, and these reaction solutions were analyzed by QTOF. N-Cl-Tyr-Gly, N,N-di-Cl-Tyr-Gly, N-Cl-Phe-Gly, N,N-di-Cl-Phe-Gly, N-Cl-Tyr-Ala, and N,N-di-Cl-Tyr-Ala were identified as the major products based on accurate masses, 35 Cl/ 37 Cl isotopic patterns, and MS/MS spectra. These identified N-chlorinated dipeptides were synthesized and found to be stable in water over 10 days except N,N-di-Cl-Phe-Gly. To enable sensitive detection of N-chlorinated dipeptides in authentic water, we developed a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method with multiple reaction monitoring (MRM) mode. N-Cl-Tyr-Gly, N,N-di-Cl-Tyr-Gly, N-Cl-Phe-Gly, N-Cl-Tyr-Ala, and N,N-di-Cl-Tyr-Ala along with their corresponding dipeptides were detected in authentic tap water samples. The dipeptides were clearly detected in the raw water, but the N-chlorinated dipeptides were at background levels. These results suggest that the N-chlorinated dipeptides are produced by chlorination. This study has identified N-chlorinated dipeptides as new disinfection byproducts in drinking water. The strategy developed in this study can be used to identify chlorination products of other peptides in drinking water.

Off-target effect of the Epac agonist 8-pCPT-2'-O-Me-cAMP on P2Y12 receptors in blood platelets.

PubMed

Herfindal, Lars; Nygaard, Gyrid; Kopperud, Reidun; Krakstad, Camilla; Døskeland, Stein Ove; Selheim, Frode

2013-08-09

The primary target of the cAMP analogue 8-pCPT-2'-O-Me-cAMP is exchange protein directly activated by cAMP (Epac). Here we tested potential off-target effects of the Epac activator on blood platelet activation signalling. We found that the Epac analogue 8-pCPT-2'-O-Me-cAMP inhibits agonist-induced-GPCR-stimulated, but not collagen-stimulated, P-selectin surface expression on Epac1 deficient platelets. In human platelets, 8-pCPT-2'-O-Me-cAMP inhibited P-selectin expression elicited by the PKC activator PMA. This effect was abolished in the presence of the extracellular ADP scavenger system CP/CPK. In silico modelling of 8-pCPT-2'O-Me-cAMP binding into the purinergic platelet receptor P2Y12 revealed that the analogue docks similar to the P2Y12 antagonist 2MeSAMP. The 8-pCPT-2'-O-Me-cAMP analogue per se, did not provoke Rap 1 (Rap 1-GTP) activation or phosphorylation on the vasodilator-stimulated phosphoprotein (VASP) at Ser-157. In addition, the protein kinase A (PKA) antagonists Rp-cAMPS and Rp-8-Br-cAMPS failed to block the inhibitory effect of 8-pCPT-2'-O-Me-cAMP on thrombin- and TRAP-induced Rap 1 activation, thus suggesting that PKA is not involved. We conclude that the 8-pCPT-2'-O-Me-cAMP analogue is able to inhibit agonist-induced-GPCR-stimulated P-selectin independent from Epac1; the off-target effect of the analogue appears to be mediated by antagonistic P2Y12 receptor binding. This has implications when using cAMP analogues on specialised system involving such receptors. We found, however that the Epac agonist 8-Br-2'-O-Me-cAMP did not affect platelet activation at similar concentrations. Copyright © 2013 Elsevier Inc. All rights reserved.

Neonatal (+)-methamphetamine exposure in rats alters adult locomotor responses to dopamine D1 and D2 agonists and to a glutamate NMDA receptor antagonist, but not to serotonin agonists

PubMed Central

Graham, Devon L.; Amos-Kroohs, Robyn M.; Braun, Amanda A.; Grace, Curtis E.; Schaefer, Tori L.; Skelton, Matthew R.; Williams, Michael T.; Vorhees, Charles V.

2015-01-01

Neonatal exposure to (+)-methamphetamine (Meth) results in long-term behavioural abnormalities but its developmental mechanisms are unknown. In a series of experiments, rats were treated from post-natal days (PD) 11–20 (stage that approximates human development from the second to third trimester) with Meth or saline and assessed using locomotor activity as the readout following pharmacological challenge doses with dopamine, serotonin and glutamate agonists or antagonists during adulthood. Exposure to Meth early in life resulted in an exaggerated adult locomotor hyperactivity response to the dopamine D1 agonist SKF-82958 at multiple doses, a high dose only under-response activating effect of the D2 agonist quinpirole, and an exaggerated under-response to the activating effect of the N-methyl-D-aspartic acid (NMDA) receptor antagonist, MK-801. No change in locomotor response was seen following challenge with the 5-HT releaser p-chloroamphetamine or the 5-HT2/3 receptor agonist, quipazine. These are the first data to show that PD 11-20 Meth exposure induces long-lasting alterations to dopamine D1, D2 and glutamate NMDA receptor function and may suggest how developmental Meth exposure leads to many of its long-term adverse effects. PMID:22391043

Gas chromatographic determination and mechanism of formation of D-amino acids occurring in fermented and roasted cocoa beans, cocoa powder, chocolate and cocoa shell.

PubMed

Pätzold, R; Brückner, H

2006-07-01

Fermented cocoa beans of various countries of origin (Ivory Coast, Ghana, Sulawesi), cocoa beans roasted under defined conditions (100-150 degrees C; 30-120 min), low and high fat cocoa powder, various brands of chocolate, and cocoa shells were analyzed for their contents of free L-and D-amino acids. Amino acids were isolated from defatted products using a cation exchanger and converted into volatile N(O)-pentafluoropropionyl amino acid 2-propyl esters which were analyzed by enantioselective gas chromatography mass spectrometry on a Chirasil-L-Val capillary column. Besides common protein L-amino acids low amounts of D-amino acids were detected in fermented cocoa beans. Quantities of D-amino acids increased on heating. On roasting cocoa beans of the Forastero type from the Ivory Coast at 150 degrees C for 2 h, relative quantities of D-amino acids approached 17.0% D-Ala, 11.7% D-Ile, 11.1% D-Asx (Asp + Asn), 7.9% D-Tyr, 5.8% D-Ser, 4.8% D-Leu, 4.3% D-Phe, 37.0% D-Pro, and 1.2% D-Val. In cocoa powder and chocolate relative quantities amounted to 14.5% D-Ala, 10.6% D-Tyr, 9.8% D-Phe, 8.1% L-Asx, and 7.2% D-Ile. Lower quantities of other D-amino acids were also detected. In order to corroborate our hypothesis that D-amino acids are generated from Amadori compounds (fructose amino acids) formed in the course of the Maillard reaction, fructose-L-phenylalanine and fructose-D-phenylalanine were synthesized and heated at 200 degrees C for 5-60 min. Already after 5 min release of 11.7% D-Phe and 11.8% L-Phe in the free form could be analyzed. Based on the data a racemization mechanism is presented founded on the intermediate and reversible formation of an amino acid carbanion in the Amadori compounds.

Step-by-step thermal transformations of a new porous coordination polymer [(H{sub 2}O){sub 5}CuBa(Me{sub 2}mal){sub 2}]{sub n} (Me{sub 2}mal{sup 2-}=dimethylmalonate): Thermal degradation to barium cuprate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zauzolkova, Natalya, E-mail: zauzolkova@igic.ras.ru; Dobrokhotova, Zhanna; Lermontov, Anatoly

The reactions of CuSO{sub 4}{center_dot}5H{sub 2}O, dimethylmalonic acid and Ba(OH){sub 2}{center_dot}H{sub 2}O (Cu: H{sub 2}Me{sub 2}mal: Ba=1: 2: 2) in aqueous and aqueous-ethanol solutions (H{sub 2}O: EtOH=1: 1) resulted in formation of 3D-porous coordination polymers [(H{sub 2}O){sub 3}({mu}-H{sub 2}O){sub 2}CuBa({mu}{sub 3}-Me{sub 2}mal)(Me{sub 2}mal)]{sub n} (1) and [({mu}-H{sub 2}O)CuBa({mu}{sub 3}-Me{sub 2}mal)({mu}{sub 4}-Me{sub 2}mal)]{sub n} (2), respectively. It has been shown that compound 2 was an intermediate in the thermal degradation of compound 1. Thorough studies of solid-state thermolysis of 1 and 2 allowed to detect formation of coordination polymer [CuBa({mu}{sub 4}-Me{sub 2}mal)({mu}{sub 5}-Me{sub 2}mal)]{sub n} (3), structure of which was determinedmore » by X-ray powder diffraction. It has been found that the channels in polymer 3 were accessible for guest molecules (MeOH). Theoretical estimation of methanol diffusion barrier was carried out. Complete solid-phase thermolysis of 1 and 2 leads to a mixture of BaCuO{sub 2}, BaCO{sub 3}, and CuO. Special conditions for obtaining of a crystalline phase of pure cubic BaCuO{sub 2} were determined. - Graphical abstract: Step-by-step transformation of new coordination polymer [(H{sub 2}O){sub 5}CuBa(Me{sub 2}mal){sub 2}]{sub n} to [(H{sub 2}O)CuBa(Me{sub 2}mal){sub 2}]{sub n} and [CuBa(Me{sub 2}mal){sub 2}]{sub n} were performed. Dehydration of initial compound leads to structural changes of 12-membered ring fragment. All compounds have porous structure. The final product of thermal decomposition is crystalline phase of individual cubic BaCuO{sub 2}. Highlights: Black-Right-Pointing-Pointer New 3D-polymers [(H{sub 2}O){sub 5}CuBa(Me{sub 2}mal){sub 2}]{sub n} and [(H{sub 2}O)CuBa(Me{sub 2}mal){sub 2}]{sub n} were synthesized. Black-Right-Pointing-Pointer Thermal analysis showed step-by-step transformations of [(H{sub 2}O){sub 5}CuBa(Me{sub 2}mal){sub 2}]{sub n}. Black

Subchronic intermittent caffeine administration to unilaterally 6-hydroxydopamine-lesioned rats sensitizes turning behaviour in response to dopamine D(1) but not D(2) receptor agonists.

PubMed

Cauli, Omar; Pinna, Annalisa; Morelli, Micaela

2005-12-01

The effects of caffeine, an antagonist of adenosine A(1) and A(2A) receptors, are significantly influenced by modifications in dopamine transmission. Administration of caffeine to unilaterally 6-hydroxydopamine-lesioned rats induces ipsilateral turning behaviour in rats never exposed to a dopamine receptor agonist, whereas contralateral turning is elicited if rats are repeatedly primed with a dopamine receptor agonist. In this study, rats unilaterally lesioned with 6-hydroxydopamine and subchronically treated with an intermittent administration of caffeine (15 mg/kg) or vehicle, were administered, 3 days after discontinuations of the treatment, with the dopamine D(1) receptor agonist 1-phenyl 1,2,3,4,5-tetrahydro(1H)-3-benzazepine-7,8-diolhydrochloride (SKF 38393), the D(2)/D(3) receptor agonist quinpirole, the D(2) receptor agonist R(-)-propylnorapomorphine or the dopamine precursor L-3,4-dihydroxyphenyl-alanine. Administration of SKF 38393 (1.5 mg/kg) or L-3,4-dihydroxyphenyl-alanine (6 mg/kg), but not quinpirole (0.15 mg/kg) or R(-)-propylnorapomorphine (0.01 mg/kg), induced a significantly higher contralateral turning behaviour in rats subchronically treated with caffeine than in vehicle-pretreated rats. The results show that repeated intermittent caffeine exposure enhances the motor stimulant effects elicited by dopamine agonists by a preferential sensitization of dopamine D(1) receptors.

Discovery of 4-anilino-N-methylthieno[3,2-d]pyrimidines and 4-anilino-N-methylthieno[2,3-d]pyrimidines as potent apoptosis inducers.

PubMed

Kemnitzer, William; Sirisoma, Nilantha; May, Chris; Tseng, Ben; Drewe, John; Cai, Sui Xiong

2009-07-01

We report the discovery of N-((benzo[d][1,3]dioxol-5-yl)methyl)-6-phenylthieno[3,2-d]pyrimidin-4-amine (2a) as an apoptosis inducer using our proprietary cell- and caspase-based ASAP HTS assay, and SAR study of HTS hit 2a which led to the discovery of 4-anilino-N-methylthieno[3,2-d]pyrimidines and 4-anilino-N-methylthieno[2,3-d]pyrimidines as potent apoptosis inducers. Compounds 5d and 5e were the most potent with EC(50) values of 0.008 and 0.004microM in T47D human breast cancer cells, respectively. Compound 5d was found to be highly active in the MX-1 breast cancer model. Functionally, compounds 5d and 5e both induced apoptosis through inhibition of tubulin polymerization.

Agonist properties of a stable hexapeptide analog of neurotensin, N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1).

PubMed

Akunne, H C; Demattos, S B; Whetzel, S Z; Wustrow, D J; Davis, D M; Wise, L D; Cody, W L; Pugsley, T A; Heffner, T G

1995-04-18

The major signal transduction pathway for neurotensin (NT) receptors is the G-protein-dependent stimulation of phospholipase C, leading to the mobilization of intracellular free Ca2+ ([Ca2+]i) and the stimulation of cyclic GMP. We investigated the functional actions of an analog of NT(8-13), N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1), and other NT related analogs by quantitative measurement of the cytosolic free Ca2+ concentration in HT-29 (human colonic adenocarcinoma) cells using the Ca(2+)-sensitive dye fura-2/AM and by effects on cyclic GMP levels in rat cerebellar slices. The NT receptor binding affinities for these analogs to HT-29 cell membranes and newborn (10-day-old) mouse brain membranes were also investigated. Data obtained from HT-29 cell and mouse brain membrane preparations showed saturable single high-affinity sites and binding densities (Bmax) of 130.2 and 87.5 fmol/mg protein, respectively. The respective KD values were 0.47 and 0.39 nM, and the Hill coefficients were 0.99 and 0.92. The low-affinity levocabastine-sensitive site was not present (K1 > 10,000) in either membrane preparation. Although the correlation of binding between HT-29 cell membranes and mouse brain membranes was quite significant (r = 0.92), some of the reference agents had lower binding affinities in the HT-29 cell membranes. The metabolically stable compound NT1 plus other NT analogs and related peptides [NT, NT(8-13), xenopsin, neuromedin N, NT(9-13), kinetensin and (D-Trp11)-NT] increased intracellular Ca2+ levels in HT-29 cells, indicating NT receptor agonist properties. The effect of NT1 in mobilizing [Ca2+]i blocked by SR 48692, a non-peptide NT antagonist. Receptor binding affinities of NT analogs to HT-29 cell membranes were positively correlated with potencies for mobilizing intracellular calcium in the same cells. In addition, NT1 increased cyclic GMP levels in rat cerebellar slices, confirming the latter findings of its NT agonist action. These results substantiate

Structure-guided development of dual β2 adrenergic/dopamine D2 receptor agonists.

PubMed

Weichert, Dietmar; Stanek, Markus; Hübner, Harald; Gmeiner, Peter

2016-06-15

Aiming to discover dual-acting β2 adrenergic/dopamine D2 receptor ligands, a structure-guided approach for the evolution of GPCR agonists that address multiple targets was elaborated. Starting from GPCR crystal structures, we describe the design, synthesis and biological investigation of a defined set of compounds leading to the identification of the benzoxazinone (R)-3, which shows agonist properties at the adrenergic β2 receptor and substantial G protein-promoted activation at the D2 receptor. This directed approach yielded molecular probes with tuned dual activity. The congener desOH-3 devoid of the benzylic hydroxyl function was shown to be a β2 adrenergic antagonist/D2 receptor agonist with Ki values in the low nanomolar range. The compounds may serve as a promising starting point for the investigation and treatment of neurological disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

Determination of the molecular structure of tetrakis(trimethylsilyl)hydrazine, N 2(SiMe 3) 4, in the gas phase by electron diffraction

NASA Astrophysics Data System (ADS)

Brain, Paul T.; Irving, Iain A.; Rankin, David W. H.; Robertson, Heather E.; Leung, Wing-Por; Bühl, Michael

1997-09-01

The gas-phase molecular structure of tetrakis(trimethylsily)hydrazine, N 2(SiMe 3) 4, in the gas phase has been determined by electron diffraction. The skeleton consists of two planar NSi 2 fragments which are not coplanar, but twisted 86.9(38)° relative to one another. This conformation, combined with the arrangements of the trimethylsilyl groups, gives the molecule overall D2 symmetry, and ensures that there are no major steric interactions between the groups. Ab initio calculations at the {SCF}/{6-31 G∗ } level confirm that D2 symmetry is favoured. The bond lengths ( ra) of 143.3(16) pm for NN. 175.9(5) pm for SiN and 187.5(2) pm for SiC, and the SiNSi and CSiC angles of 124.1(15)° and 108.8(7) respectively, also indicate that this is not a severely strained molecule, although the SiN distance is quite long, being comparable to that observed in N(SiMe 3) 3.

Finite size effect on hydrogen bond cooperativity in (Ala)n polypeptides: A DFT study using numeric atom-centered orbitals

NASA Astrophysics Data System (ADS)

Blum, Volker; Ireta, Joel; Scheffler, Matthias

2007-03-01

An accurate representation of the energetic contribution Ehb of hydrogen bonds to structure formation is paramount to understand the secondary structure stability of proteins, both qualitatively and quantitatively. However, Ehb depends strongly on its environment, and even on the surrounding peptide conformation itself. For instance, a short α-helical polypeptide (Ala)4 can not be stabilized by its single hydrogen bond, whereas an infinite α-helical chain (Ala)∞ is clearly energetically stable over a fully extended conformation. We here use all-electron density functional calculations in the PBE generalized gradient approximation by a recently developed, computationally efficient numeric atom-centered orbital based code^1 to investigate this H-bond cooperativity that is intrinsic to Alanine-based polypeptides (Ala)n (n=1-20,∞). We compare finite and infinite prototypical helical conformations (α, π, 310) on equal footing, with both neutral and ionic termination for finite (Ala)n peptides. Moderately sized NAO basis sets allow to capture Ehb with meV accuracy, revealing a clear jump in Ehb (cooperativity) when two H-bonds first appear in line, followed by slower and more continuous increase of Ehb towards n->∞. ^1 V. Blum, R. Gehrke, P. Havu, V. Havu, M. Scheffler, The FHI Ab Initio Molecular Simulations (aims) Project, Fritz-Haber-Institut, Berlin (2006).

Expanding the Chemistry of Actinide Metallocene Bromides. Synthesis, Properties and Molecular Structures of the Tetravalent and Trivalent Uranium Bromide Complexes: (C 5Me 4R) 2UBr 2, (C 5Me 4R) 2U(O-2,6- iPr 2C 6H 3)(Br), and [K(THF)][(C 5Me 4R) 2UBr 2] (R = Me, Et)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lichtscheidl, Alejandro Gaston; Pagano, Justin K.; Scott, Brian Lindley

The organometallic uranium species (C 5Me 4R) 2UBr 2 (R = Me, Et) were obtained by treating their chloride analogues (C 5Me 4R) 2UCl 2 (R = Me, Et) with Me 3SiBr. Treatment of (C 5Me 4R) 2UCl 2 and (C 5Me 4R) 2UBr 2 (R = Me, Et) with K(O-2,6- iPr 2C 6H 3) afforded the halide aryloxide mixed-ligand complexes (C 5Me 4R) 2U(O-2,6- iPr 2C 6H 3)(X) (R = Me, Et; X = Cl, Br). Complexes (C 5Me 4R) 2U(O-2,6- iPr 2C 6H 3)(Br) (R = Me, Et) can also be synthesized by treating (C 5Me 4R) 2U(O-2,6-more » iPr 2C 6H 3)(Cl) (R = Me, Et) with Me 3SiBr, respectively. Reduction of (C 5Me 4R) 2UCl 2 and (C 5Me 4R) 2UBr 2 (R = Me, Et) with KC 8 led to isolation of uranium(III) “ate” species [K(THF)][(C 5Me 5) 2UX 2] (X = Cl, Br) and [K(THF) 0.5][(C 5Me 4Et) 2UX 2] (X = Cl, Br), which can be converted to the neutral complexes (C 5Me 4R) 2U[N(SiMe 3) 2] (R = Me, Et). Analyses by nuclear magnetic resonance spectroscopy, X-ray crystallography, and elemental analysis are also presented.« less

Expanding the Chemistry of Actinide Metallocene Bromides. Synthesis, Properties and Molecular Structures of the Tetravalent and Trivalent Uranium Bromide Complexes: (C 5Me 4R) 2UBr 2, (C 5Me 4R) 2U(O-2,6- iPr 2C 6H 3)(Br), and [K(THF)][(C 5Me 4R) 2UBr 2] (R = Me, Et)

DOE PAGES

Lichtscheidl, Alejandro Gaston; Pagano, Justin K.; Scott, Brian Lindley; ...

2016-01-06

The organometallic uranium species (C 5Me 4R) 2UBr 2 (R = Me, Et) were obtained by treating their chloride analogues (C 5Me 4R) 2UCl 2 (R = Me, Et) with Me 3SiBr. Treatment of (C 5Me 4R) 2UCl 2 and (C 5Me 4R) 2UBr 2 (R = Me, Et) with K(O-2,6- iPr 2C 6H 3) afforded the halide aryloxide mixed-ligand complexes (C 5Me 4R) 2U(O-2,6- iPr 2C 6H 3)(X) (R = Me, Et; X = Cl, Br). Complexes (C 5Me 4R) 2U(O-2,6- iPr 2C 6H 3)(Br) (R = Me, Et) can also be synthesized by treating (C 5Me 4R) 2U(O-2,6-more » iPr 2C 6H 3)(Cl) (R = Me, Et) with Me 3SiBr, respectively. Reduction of (C 5Me 4R) 2UCl 2 and (C 5Me 4R) 2UBr 2 (R = Me, Et) with KC 8 led to isolation of uranium(III) “ate” species [K(THF)][(C 5Me 5) 2UX 2] (X = Cl, Br) and [K(THF) 0.5][(C 5Me 4Et) 2UX 2] (X = Cl, Br), which can be converted to the neutral complexes (C 5Me 4R) 2U[N(SiMe 3) 2] (R = Me, Et). Analyses by nuclear magnetic resonance spectroscopy, X-ray crystallography, and elemental analysis are also presented.« less

4,2’:6’,4”- and 3,2’:6’,3”-Terpyridines: The Conflict between Well-Defined Vectorial Properties and Serendipity in the Assembly of 1D-, 2D- and 3D-Architectures

PubMed Central

Klein, Y. Maximilian; Prescimone, Alessandro; Constable, Edwin C.

2017-01-01

A comparative investigation of the coordination assemblies formed between Co(NCS)2 and two monotopic 4,2’:6’,4’’-terpyridine (4,2’:6’,4”-tpy) ligands or two related ditopic ligands is reported. Crystals were grown by layering MeOH solutions of Co(NCS)2 over a CHCl3 or 1,2-C6H4Cl2 solution of the respective ligand at room temperature. With 4’-(2-methylpyrimidin-5-yl)-4,2’:6’,4”-terpyridine (6), the 1D-coordination polymer {[Co2(NCS)4(MeOH)4(6)2]∙2MeOH∙8H2O}n assembles with 6 coordinating only through the outer N-donors of the 4,2’:6’,4”-tpy unit; coordination by the MeOH solvent blocks two cobalt coordination sites preventing propagation in a higher-dimensional network. A combination of Co(NCS)2 and 1-(4,2‘:6’,4”-terpyridin-4’-yl)ferrocene (7) leads to {[Co(NCS)2(7)2]∙4CHCl3}n which contains a (4,4) net; the 2D-sheets associate through π-stacking interactions between ferrocenyl and pyridyl units. A 3D-framework is achieved through use of the ditopic ligand 1,4-bis(npropoxy)-2,5-bis(4,2’:6’,4”-terpyridin-4’-yl)benzene (8) which acts as a 4-connecting node in {[Co(NCS)2(8)2]∙2C6H4Cl2}n; the combination of metal and ligand planar 4-connecting nodes results in a {65.8} cds net. For a comparison with the coordinating abilities of the previously reported 1,4-bis(noctoxy)-2,5-bis(4,2’:6’,4”-terpyridin-4’-yl)benzene (3), a more flexible analogue 9 was prepared. {[Co(NCS)2(9)]∙2CHCl3}n contains a (4,4) net defined by both metal and ligand planar 4-connecting nodes. The noctoxy tails of 9 protrude from each side of the (4,4) net and thread through adjacent sheets; the arene-attached noctoxy chains associate through a combination of van der Waals and C–H...π interactions. PMID:28773088

[Dmt(1)]DALDA analogues with enhanced μ opioid agonist potency and with a mixed μ/κ opioid activity profile.

PubMed

Bai, Longxiang; Li, Ziyuan; Chen, Jiajia; Chung, Nga N; Wilkes, Brian C; Li, Tingyou; Schiller, Peter W

2014-04-01

Analogues of [Dmt(1)]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2; Dmt=2',6'-dimethyltyrosine), a potent μ opioid agonist peptide with mitochondria-targeted antioxidant activity, were prepared by replacing Phe(3) with various 2',6'-dialkylated Phe analogues, including 2',6'-dimethylphenylalanine (Dmp), 2',4',6'-trimethylphenylalanine (Tmp), 2'-isopropyl-6'-methylphenylalanine (Imp) and 2'-ethyl-6'-methylphenylalanine (Emp), or with the bulky amino acids 3'-(1-naphthyl)alanine (1-Nal), 3'-(2-naphthyl)alanine (2-Nal) or Trp. Several compounds showed significantly increased μ agonist potency, retained μ receptor selectivity and are of interest as drug candidates for neuropathic pain treatment. Surprisingly, the Dmp(3)-, Imp(3)-, Emp(3)- and 1-Nal(3)-containing analogues showed much increased κ receptor binding affinity and had mixed μ/κ properties. In these cases, molecular dynamics studies indicated conformational preorganization of the unbound peptide ligands due to rotational restriction around the C(β)C(γ) bond of the Xxx(3) residue, in correlation with the observed κ receptor binding enhancement. Compounds with a mixed μ/κ opioid activity profile are known to have therapeutic potential for treatment of cocaine abuse. Copyright © 2014 Elsevier Ltd. All rights reserved.

Pharmacophore, QSAR, and binding mode studies of substrates of human cytochrome P450 2D6 (CYP2D6) using molecular docking and virtual mutations and an application to chinese herbal medicine screening.

PubMed

Mo, Sui-Lin; Liu, Wei-Feng; Li, Chun-Guang; Zhou, Zhi-Wei; Luo, Hai-Bin; Chew, Helen; Liang, Jun; Zhou, Shu-Feng

2012-07-01

The highly polymorphic human cytochrome P450 2D6 (CYP2D6) metabolizes about 25% of currently used drugs. In this study, we have explored the interaction of a large number of substrates (n = 120) with wild-type and mutated CYP2D6 by molecular docking using the CDOCKER module. Before we conducted the molecular docking and virtual mutations, the pharmacophore and QSAR models of CYP2D6 substrates were developed and validated. Finally, we explored the interaction of a traditional Chinese herbal formula, Fangjifuling decoction, with CYP2D6 by virtual screening. The optimized pharmacophore model derived from 20 substrates of CYP2D6 contained two hydrophobic features and one hydrogen bond acceptor feature, giving a relevance ratio of 76% when a validation set of substrates were tested. However, our QSAR models gave poor prediction of the binding affinity of substrates. Our docking study demonstrated that 117 out of 120 substrates could be docked into the active site of CYP2D6. Forty one out of 117 substrates (35.04%) formed hydrogen bonds with various active site residues of CYP2D6 and 53 (45.30%) substrates formed a strong π-π interaction with Phe120 (53/54), with only carvedilol showing π-π interaction with Phe483. The active site residues involving hydrogen bond formation with substrates included Leu213, Lys214, Glu216, Ser217, Gln244, Asp301, Ser304, Ala305, Phe483, and Phe484. Furthermore, the CDOCKER algorithm was further applied to study the impact of mutations of 28 active site residues (mostly non-conserved) of CYP2D6 on substrate binding modes using five probe substrates including bufuralol, debrisoquine, dextromethorphan, sparteine, and tramadol. All mutations of the residues examined altered the hydrogen bond formation and/or aromatic interactions, depending on the probe used in molecular docking. Apparent changes of the binding modes have been observed with the Glu216Asp and Asp301Glu mutants. Overall, 60 compounds out of 130 from Fangjifuling decoction

In vivo dopamine agonist properties of rotigotine: Role of D1 and D2 receptors.

PubMed

Fenu, Sandro; Espa, Elena; Pisanu, Augusta; Di Chiara, Gaetano

2016-10-05

Rotigotine acts in vitro as a full agonist of dopamine D1 receptors at concentrations almost superimposable to those at which it acts on D2 receptors. However in vivo evidence of the differences between the agonist activity of rotigotine at D1 receptors from that on the D2 receptors has not been provided yet. In order to test the ability of rotigotine to stimulate dopamine D1 and D2 receptors in vivo, we studied the effect of SCH39166 and eticlopride, selective dopamine D1 and D2/D3 receptor antagonists respectively, on rotigotine-induced contralateral turning behavior in 6-hydroxydopamine lesioned rats. Furthermore, the expression of the immediate-early gene c-fos in the caudate-putamen, was evaluated. As a comparison, we tested the D2/D3 agonist pramipexole. In primed rats, rotigotine (0.035, 0.1 and 0.35mg/kg) induced dose-dependent contralateral turning. Turning induced by 0.1mg/kg of rotigotine was reduced by pretreatment with the D1 antagonist SCH39166 and the D2 antagonist eticlopride. In drug-naive rats, rotigotine was less effective in eliciting turning but SCH39166 still reduced turning induced by rotigotine (0.35mg/kg). Pramipexole induced contralateral turning only in primed rats. SCH39166 potentiated and eticlopride abolished pramipexole-induced turning. Rotigotine induced Fos expression in the caudate-putamen and SCH39166 completely blocked it. Pramipexole failed to induce Fos. These results indicate that rotigotine acts in vivo as an agonist of D1 and D2 receptors while pramipexole is devoid of D1 activity in vivo. Given their differing DA receptor profiles, rotigotine and pramipexole might differ in their spectrum of application to the therapy of Parkinson's disease. Copyright © 2016 Elsevier B.V. All rights reserved.

Enhancement of Attentional Performance by Selective Stimulation of α4β2* nAChRs: Underlying Cholinergic Mechanisms

PubMed Central

Howe, William M; Ji, Jinzhao; Parikh, Vinay; Williams, Sarah; Mocaër, Elisabeth; Trocmé-Thibierge, Caryn; Sarter, Martin

2010-01-01

Impairments in attention are a major component of the cognitive symptoms of neuropsychiatric and neurodegenerative disorders. Using an operant sustained attention task (SAT), including a distractor condition (dSAT), we assessed the putative pro-attentional effects of the selective α4β2* nicotinic acetylcholine receptor (nAChR) agonist S 38232 in comparison with the non-selective agonist nicotine. Neither drug benefited SAT performance. However, in interaction with the increased task demands implemented by distractor presentation, the selective agonist, but not nicotine, enhanced the detection of signals during the post-distractor recovery period. This effect is consistent with the hypothesis that second-long increases in cholinergic activity (‘transients') mediate the detection of cues and that nAChR agonists augment such transients. Electrochemical recordings of prefrontal cholinergic transients evoked by S 38232 and nicotine indicated that the α4β2* nAChR agonist evoked cholinergic transients that were characterized by a faster rise time and more rapid decay than those evoked by nicotine. Blockade of the α7 nAChR ‘sharpens' nicotine-evoked transients; therefore, we determined the effects of co-administration of nicotine and the α7 nAChR antagonist methyllycaconitine on dSAT performance. Compared with vehicle and nicotine alone, this combined treatment significantly enhanced the detection of signals. These results indicate that compared with nicotine, α4β2* nAChR agonists significantly enhance attentional performance and that the dSAT represents a useful behavioral screening tool. The combined behavioral and electrochemical evidence supports the hypothesis that nAChR agonist-evoked cholinergic transients, which are characterized by rapid rise time and fast decay, predict robust drug-induced enhancement of attentional performance. PMID:20147893

Agonist properties of N,N-dimethyltryptamine at serotonin 5-HT2A and 5-HT2C receptors.

PubMed

Smith, R L; Canton, H; Barrett, R J; Sanders-Bush, E

1998-11-01

Extensive behavioral and biochemical evidence suggests an agonist role at the 5-HT2A receptor, and perhaps the 5-HT2C receptor, in the mechanism of action of hallucinogenic drugs. However the published in vitro pharmacological properties of N,N-dimethyltryptamine (DMT), an hallucinogenic tryptamine analog, are not consistent with this hypothesis. We, therefore, undertook an extensive investigation into the properties of DMT at 5-HT2A and 5-HT2C receptors. In fibroblasts transfected with the 5-HT2A receptor or the 5-HT2C receptor, DMT activated the major intracellular signaling pathway (phosphoinositide hydrolysis) to an extent comparable to that produced by serotonin. Because drug efficacy changes with receptor density and cellular microenvironment, we also examined the properties of DMT in native preparations using a behavioral and biochemical approach. Rats were trained to discriminate an antagonist ketanserin from an agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in a two-lever choice paradigm. Pharmacological studies showed that responding on the DOI and ketanserin lever reflected agonist and antagonist activity at 5-HT2A receptors, and hence, was a suitable model for evaluating the in vivo functional properties of DMT. Like other 5-HT2A receptor agonists, DMT substituted fully for DOI. Intact choroid plexus was used to evaluate the agonist properties at endogenous 5-HT2C receptors; DMT was a partial agonist at 5-HT2C receptors in this native preparation. Thus, we conclude that DMT behaves as an agonist at both 5-HT2A and 5-HT2A receptors. One difference was evident in that the 5-HT2C, but not the 5-HT2A, receptor showed a profound desensitization to DMT over time. This difference is interesting in light of the recent report that the hallucinogenic activity of DMT does not tolerate in humans and suggests the 5-HT2C receptor plays a less prominent role in the action of DMT.

Phosphodiesterase 4 Inhibitors Attenuate the Asthma Phenotype Produced by β2-Adrenoceptor Agonists in Phenylethanolamine N-Methyltransferase-Knockout Mice.

PubMed

Forkuo, Gloria S; Kim, Hosu; Thanawala, Vaidehi J; Al-Sawalha, Nour; Valdez, Daniel; Joshi, Radhika; Parra, Sergio; Pera, Tonio; Gonnella, Patricia A; Knoll, Brian J; Walker, Julia K L; Penn, Raymond B; Bond, Richard A

2016-08-01

Mice lacking the endogenous β2-adrenoceptor (β2AR) agonist epinephrine (phenylethanolamine N-methyltransferase [PNMT]-knockout mice) are resistant to developing an "asthma-like" phenotype in an ovalbumin sensitization and challenge (Ova S/C) model, and chronic administration of β2AR agonists to PNMT-KO mice restores the phenotype. Based on these and other studies showing differential effects of various β2AR ligands on the asthma phenotype, we have speculated that the permissive effect of endogenous epinephrine and exogenous β2AR agonists on allergic lung inflammation can be explained by qualitative β2AR signaling. The β2AR can signal through at least two pathways: the canonical Gαs-cAMP pathway and a β-arrestin-dependent pathway. Previous studies suggest that β-arrestin-2 is required for allergic lung inflammation. On the other hand, cell-based assays suggest antiinflammatory effects of Gαs-cAMP signaling. This study was designed to test whether the in vitro antiinflammatory effects of phosphodiesterase 4 inhibitors, known to increase intracellular cAMP in multiple airway cell types, attenuate the asthma-like phenotype produced by the β2AR agonists formoterol and salmeterol in vivo in PNMT-KO mice, based on the hypothesis that skewing β2AR signaling toward Gαs-cAMP pathway is beneficial. Airway inflammatory cells, epithelial mucus production, and airway hyperresponsiveness were quantified. In Ova S/C PNMT-KO mice, formoterol and salmeterol restored the asthma-like phenotype comparable to Ova S/C wild-type mice. However, coadministration of either roflumilast or rolipram attenuated this formoterol- or salmeterol-driven phenotype in Ova S/C PNMT-KO. These findings suggest that amplification of β2AR-mediated cAMP by phosphodiesterase 4 inhibitors attenuates the asthma-like phenotype promoted by β-agonists.

F15063, a compound with D2/D3 antagonist, 5-HT1A agonist and D4 partial agonist properties: (II) Activity in models of positive symptoms of schizophrenia

PubMed Central

Depoortère, R; Bardin, L; Auclair, A L; Kleven, M S; Prinssen, E; Colpaert, F; Vacher, B; Newman-Tancredi, A

2007-01-01

Background and purpose: F15063 is a high affinity D2/D3 antagonist, D4 partial agonist, and high efficacy 5-HT1A agonist, with little affinity (40-fold lower than for D2 receptors) at other central targets. Here, the profile of F15063 was evaluated in models of positive symptoms of schizophrenia and motor side-effects. Experimental approach: Rodent behavioural tests were based on reversal of hyperactivity induced by psychostimulants and on measures of induction of catalepsy and ‘serotonin syndrome'. Key results: F15063 potently (ED50s: 0.23 to 1.10 mg kg−1 i.p.) reversed methylphenidate-induced stereotyped behaviors, blocked d-amphetamine and ketamine hyperlocomotion, attenuated apomorphine-induced prepulse inhibition (PPI) deficits, and was active in the conditioned avoidance test. In mice, it reversed apomorphine-induced climbing (ED50 = 0.30 mg kg−1 i.p.). F15063, owing to its 5-HT1A agonism, did not produce (ED50 > 40 mg kg−1 i.p.) catalepsy in rats and mice, a behavior predictive of occurrence of extra-pyramidal syndrome (EPS) in man. This absence of cataleptogenic activity was maintained upon sub-chronic treatment of rats for 5 days at 40 mg kg−1 p.o. Furthermore, F15063 did not induce the ‘serotonin syndrome' in rats (flat body posture and forepaw treading: ED50 >32 mg kg−1 i.p.). Conclusions and implications: F15063 conformed to the profile of an atypical antipsychotic, with potent actions in models of hyperdopaminergic activity but without inducing catalepsy. These data suggest that F15063 may display potent antipsychotic actions with low EPS liability. This profile is complemented by a favourable profile in rodent models of negative symptoms and cognitive deficits of schizophrenia (companion paper). PMID:17375086

1,2,3-Triazole Rings as a Disulfide Bond Mimetic in Chimeric AGRP-Melanocortin Peptides: Design, Synthesis, and Functional Characterization.

PubMed

Tala, Srinivasa R; Singh, Anamika; Lensing, Cody J; Schnell, Sathya M; Freeman, Katie T; Rocca, James R; Haskell-Luevano, Carrie

2018-05-16

The melanocortin system is involved in the regulation of complex physiological functions, including energy and weight homeostasis, feeding behavior, inflammation, sexual function, pigmentation, and exocrine gland function. The five melanocortin receptors that belong to the superfamily of G protein-coupled receptors (GPCRs) are regulated by endogenously expressed agonists and antagonists. The aim of this study was to explore the potential of replacing the disulfide bridge in chimeric AGRP-melanocortin peptide Tyr-c[Cys-His-d-Phe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH 2 (1) with 1,2,3-triazole moieties. A series of 1,2,3-triazole-bridged peptidomimetics were designed, synthesized, and pharmacologically evaluated at the mouse melanocortin receptors. The ligands possessed nanomolar to micromolar agonist cAMP signaling potency. A key finding was that the disulfide bond in peptide 1 can be replaced with the monotriazole ring with minimal effect on the functional activity at the melanocortin receptors. The 1,5-disubstituted triazole-bridged peptide 6 showed equipotent functional activity at the mMC3R and modest 5-fold decreased agonist potency at the mMC4R compared to those of 1. Interestingly, the 1,4- and 1,5-disubstituted isomers of the triazole ring resulted in different selectivities at the receptor subtypes, indicating subtle structural features that may be exploited in the generation of selective melanocortin ligands. Introducing cyclic and acyclic bis-triazole moieties into chimeric AGRP template 1 generally decreased agonist activity. These results will be useful for the further design of neuronal chemical probes for the melanocortin receptors as well as in other receptor systems.

Hallucinogenic 5-HT2AR agonists LSD and DOI enhance dopamine D2R protomer recognition and signaling of D2-5-HT2A heteroreceptor complexes.

PubMed

Borroto-Escuela, Dasiel O; Romero-Fernandez, Wilber; Narvaez, Manuel; Oflijan, Julia; Agnati, Luigi F; Fuxe, Kjell

2014-01-03

Dopamine D2LR-serotonin 5-HT2AR heteromers were demonstrated in HEK293 cells after cotransfection of the two receptors and shown to have bidirectional receptor-receptor interactions. In the current study the existence of D2L-5-HT2A heteroreceptor complexes was demonstrated also in discrete regions of the ventral and dorsal striatum with in situ proximity ligation assays (PLA). The hallucinogenic 5-HT2AR agonists LSD and DOI but not the standard 5-HT2AR agonist TCB2 and 5-HT significantly increased the density of D2like antagonist (3)H-raclopride binding sites and significantly reduced the pKiH values of the high affinity D2R agonist binding sites in (3)H-raclopride/DA competition experiments. Similar results were obtained in HEK293 cells and in ventral striatum. The effects of the hallucinogenic 5-HT2AR agonists on D2R density and affinity were blocked by the 5-HT2A antagonist ketanserin. In a forskolin-induced CRE-luciferase reporter gene assay using cotransfected but not D2R singly transfected HEK293 cells DOI and LSD but not TCB2 significantly enhanced the D2LR agonist quinpirole induced inhibition of CRE-luciferase activity. Haloperidol blocked the effects of both quinpirole alone and the enhancing actions of DOI and LSD while ketanserin only blocked the enhancing actions of DOI and LSD. The mechanism for the allosteric enhancement of the D2R protomer recognition and signalling observed is likely mediated by a biased agonist action of the hallucinogenic 5-HT2AR agonists at the orthosteric site of the 5-HT2AR protomer. This mechanism may contribute to the psychotic actions of LSD and DOI and the D2-5-HT2A heteroreceptor complex may thus be a target for the psychotic actions of hallunicogenic 5-HT2A agonists. Copyright © 2013 Elsevier Inc. All rights reserved.

Autoradiographic evidence for two classes of mu opioid binding sites in rat brain using (/sup 125/I)FK33824

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rothman, R.B.; Jacobson, A.E.; Rice, K.C.

1987-11-01

Previous studies demonstrated that pretreatment of brain membranes with the irreversible mu antagonist, beta-funaltrexamine (beta-FNA), partially eliminated mu binding sites (25,35), consistent with the existence of two mu binding sites distinguished by beta-FNA. This paper tests the hypothesis that the FNA-sensitive and FNA-insensitive mu binding sites have different anatomical distributions in rat brain. Prior to autoradiographic visualization of mu binding sites, (/sup 3/H)oxymorphone, (/sup 3/H)D-ala2-MePhe4, Gly-ol5-enkephalin (DAGO), and (/sup 125/I)D-ala2-Me-Phe4-met(o)-ol)enkephalin (FK33824) were shown to selectively label mu binding sites using slide mounted sections of molded minced rat brain. As found using membranes, beta-FNA eliminated only a portion of mu bindingmore » sites. Autoradiographic visualization of mu binding sites using the mu-selective ligand (/sup 125/I)FK33824 in control and FNA-treated sections of rat brain demonstrated that the proportion of mu binding sites sensitive to beta-FNA varied across regions of the brain, particularly the dorsal thalamus, ventrobasal complex and the hypothalamus, providing anatomical data supporting the existence of two classes of mu binding sites in rat brain.« less

Interaction of ( sup 3 H)MK-801 with multiple states of the N-methyl-D-aspartate receptor complex of rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Javitt, D.C.; Zukin, S.R.

1989-01-01

N-Methyl-D-aspartate (N-Me-D-Asp) and phencyclidine receptors interactively mediate central nervous system processes including psychotomimetic effects of drugs as well as neurodegenerative, cognitive, and developmental events. To elucidate the mechanism of this interaction, effects of N-Me-D-Asp agonists and antagonists and of glycine-like agents upon binding of the radiolabeled phencyclidine receptor ligand ({sup 3}H)MK-801 were determined in rat brain. Scatchard analysis revealed two discrete components of ({sup 3}H)MK-801 binding after 4 hr of incubation. Incubation in the presence of L-glutamate led to an increase in apparent densities but not in affinities of both components of ({sup 3}H)MK-801 binding as well as conversion ofmore » sites from apparent low to high affinity. Incubation in the presence of combined D-serine and L-glutamate led to an increase in the apparent density of high-affinity ({sup 3}H)MK-801 binding compared with incubation in the presence of either L-glutamate or D-serine alone. These data support a model in which phencyclidine receptor ligands bind differentially to closed as well as open conformations of the N-Me-D-Asp receptor complex and in which glycine-like agents permit or facilitate agonist-induced conversion of N-Me-D-Asp receptors from closed to open conformations.« less

Effect of thiorphan on response of the guinea-pig gallbladder to tachykinins.

PubMed

Maggi, C A; Patacchini, R; Renzi, D; Santicioli, P; Regoli, D; Rovero, P; Drapeau, G; Surrenti, C; Meli, A

1989-06-08

Tachykinins produced a concentration-related contraction of the isolated guinea-pig gallbladder, with a rank order of potency neurokinin A (NKA) greater than Arg-neurokinin B = neurokinin B (NKB) greater than substance P (SP). Only the effect of SP was potentiated by thiorphan (0.1-10 microM). A significant enhancement of the response to SP was also produced by captopril (1 microM). [Nle10]NKA-(4-10) and [beta-Ala8]NKA-(4-10), selective NK-2 receptor agonists, were active, whereas [Pro9]SP sulfone (selective NK-1 agonist) was almost ineffective. [MePhe7]NKB (selective NK-3 agonist) had some activity but only at high concentrations. Septide was almost ineffective and DiMeC7 had an action comparable to that of [MePhe7]NKB. None of the effects induced by these synthetic tachykinin analogs were significantly potentiated by thiorphan. Capsaicin (10 microM) produced a contraction which was unaffected by thiorphan. Both capsaicin and NKA-induced contractions were antagonized by Spantide at concentrations (5-10 microM) which had no effect against the atropine-sensitive contractions produced by electrical field stimulation. Capsaicin (1 microM) produced a consistent release of SP-like immunoreactivity (SP-LI) and a second application of the drug had no further effect, indicating complete desensitization. SP-LI release by capsaicin was almost doubled in the presence of thiorphan. These findings indicate that NK-2 and possibly some NK-3 receptors mediate the contractile response of the guinea-pig gallbladder to tachykinins. Both exogenous and endogenous (released by capsaicin) SP were degraded to a significant extent in this organ via a thiorphan-sensitive mechanism, the identity of which remains to be established.

Potent μ-Opioid Receptor Agonists from Cyclic Peptides Tyr-c[D-Lys-Xxx-Tyr-Gly]: Synthesis, Biological, and Structural Evaluation.

PubMed

Li, Yangmei; Cazares, Margret; Wu, Jinhua; Houghten, Richard A; Toll, Laurence; Dooley, Colette

2016-02-11

To optimize the structure of a μ-opioid receptor ligand, analogs H-Tyr-c[D-Lys-Xxx-Tyr-Gly] were synthesized and their biological activity was tested. The analog containing a Phe(3) was identified as not only exhibiting binding affinity 14-fold higher than the original hit but also producing agonist activity 3-fold more potent than morphine. NMR study suggested that a trans conformation at D-Lys(2)-Xxx(3) is crucial for these cyclic peptides to maintain high affinity, selectivity, and functional activity toward the μ-opioid receptor.

Pharmacological Characterization of 30 Human Melanocortin-4 Receptor Polymorphisms with the Endogenous Proopiomelanocortin Derived Agonists, Synthetic Agonists, and the Endogenous Agouti-Related Protein (AGRP) Antagonist

PubMed Central

Xiang, Zhimin; Proneth, Bettina; Dirain, Marvin L.; Litherland, Sally A.; Haskell-Luevano, Carrie

2010-01-01

The melanocortin-4 receptor (MC4R) is a G-protein coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic bio marker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and non-obese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [α-, β, γ2-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87-132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4R's and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface expression by flow

Selective synthesis of cis- and trans-[(NHC(Me))2PtCl2] and [NHC(Me)Pt(cod)Cl][NHC(Me)PtCl3] using NHC(Me)SiCl4.

PubMed

Lewis-Alleyne, Lesley C; Bassil, Bassem S; Böttcher, Tobias; Röschenthaler, Gerd-Volker

2014-11-14

NHC(Me)SiCl4 (NHC(Me) = 1,3-dimethylimidazolidin-2-ylidene) was used to synthesise novel NHC(Me)-Pt(ii) complexes. An atypical trans-cis isomerisation process was also achieved for [(NHC(Me))2PtCl2], while the synthesis of the unique double-complex salt [(NHC(Me))Pt(cod)Cl] [(NHC(Me))PtCl3] (cod = 1,5-cyclooctadiene) revealed the first-ever N-heterocyclic carbene analogue of the Cossa's salt anion.

Structural Modifications of Neuroprotective Anti-Parkinsonian (−)-N6-(2-(4-(Biphenyl-4-yl)piperazin-1-yl)-ethyl)-N6-propyl-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine (D-264): An Effort toward the Improvement of in Vivo Efficacy of the Parent Molecule

PubMed Central

2015-01-01

In our overall goal to develop multifunctional dopamine D2/D3 agonist drugs for the treatment of Parkinson’s disease (PD), we previously synthesized potent D3 preferring agonist D-264 (1a), which exhibited neuroprotective properties in two animal models of PD. To enhance the in vivo efficacy of 1a, a structure–activity relationship study was carried out. Competitive binding and [35S]GTPγS functional assays identified compound (−)-9b as one of the lead molecules with preferential D3 agonist activity (EC50(GTPγS); D3 = 0.10 nM; D2/D3 (EC50): 159). Compounds (−)-9b and (−)-8b exhibited high in vivo activity in two PD animal models, reserpinized and 6-hydroxydopamine (OHDA)-induced unilateral lesioned rats. On the other hand, 1a failed to show any in vivo activity in these models unless the compound was dissolved in 5–10% beta-hydroxy propyl cyclodextrin solution. Lead compounds exhibited appreciable radical scavenging activity. In vitro experiments with dopaminergic MN9D cells indicated neuroprotection by both 1a and (−)-9b from toxicity of MPP+. PMID:24471976

Precision imaging of 4.4 MeV gamma rays using a 3-D position sensitive Compton camera.

PubMed

Koide, Ayako; Kataoka, Jun; Masuda, Takamitsu; Mochizuki, Saku; Taya, Takanori; Sueoka, Koki; Tagawa, Leo; Fujieda, Kazuya; Maruhashi, Takuya; Kurihara, Takuya; Inaniwa, Taku

2018-05-25

Imaging of nuclear gamma-ray lines in the 1-10 MeV range is far from being established in both medical and physical applications. In proton therapy, 4.4 MeV gamma rays are emitted from the excited nucleus of either 12 C* or 11 B* and are considered good indicators of dose delivery and/or range verification. Further, in gamma-ray astronomy, 4.4 MeV gamma rays are produced by cosmic ray interactions in the interstellar medium, and can thus be used to probe nucleothynthesis in the universe. In this paper, we present a high-precision image of 4.4 MeV gamma rays taken by newly developed 3-D position sensitive Compton camera (3D-PSCC). To mimic the situation in proton therapy, we first irradiated water, PMMA and Ca(OH)2 with a 70 MeV proton beam, then we identified various nuclear lines with the HPGe detector. The 4.4 MeV gamma rays constitute a broad peak, including single and double escape peaks. Thus, by setting an energy window of 3D-PSCC from 3 to 5 MeV, we show that a gamma ray image sharply concentrates near the Bragg peak, as expected from the minimum energy threshold and sharp peak profile in the cross section of 12 C(p,p) 12 C*.

The bifunctional μ opioid agonist/antioxidant [Dmt(1)]DALDA is a superior analgesic in an animal model of complex regional pain syndrome-type i.

PubMed

Schiller, Peter W; Nguyen, Thi M-D; Saray, Amy; Poon, Annie Wing Hoi; Laferrière, André; Coderre, Terence J

2015-11-18

Reactive oxygen species (ROS) play an important role in the development of complex regional pain syndrome-Type I (CRPS-I), as also demonstrated with the chronic post ischemia pain (CPIP) animal model of CRPS-I. We show that morphine and the antioxidant N-acetylcysteine (NAC) act synergistically to reduce mechanical allodynia in CPIP rats. The tetrapeptide amide [Dmt(1)]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2) is a potent and selective μ opioid receptor (MOR) agonist with favorable pharmacokinetic properties and with antioxidant activity due to its N-terminal Dmt (2',6'-dimethyltyrosine) residue. In the CPIP model, [Dmt(1)]DALDA was 15-fold more potent than morphine in reversing mechanical allodynia and 4.5-fold more potent as analgesic in the heat algesia test. The results indicate that bifunctional compounds with MOR agonist/antioxidant activity have therapeutic potential for the treatment of CRPS-I.

PET Imaging of D2/3 agonist binding in healthy human subjects with the radiotracer [11C]-N-propyl-nor-apomorphine (NPA): preliminary evaluation and reproducibility studies

PubMed Central

Narendran, Rajesh; Frankle, W. Gordon; Mason, N. Scott; Laymon, Charles M.; Lopresti, Brian J; Price, Julie C.; Kendro, Steve; Vora, Shivangi; Litschge, Maralee; Mountz, James M.; Mathis, Chester A.

2009-01-01

Objective (-)-N-[11C]-Propyl-norapomorphine (NPA) is a full dopamine D2/3 receptor agonist radiotracer suitable for imaging D2/3 receptors configured in a state of high affinity for agonists using Positron Emission Tomography (PET). The aim of the present study was to define the optimal analytic method to derive accurate and reliable D2/3 receptor parameters with [11C]NPA. Methods Six healthy subjects (4 females/2 males) underwent two [11C]NPA scans in the same day. D2/3 receptor binding parameters were estimated using kinetic analysis (using 1- and 2- tissue compartment models) as well as simplified reference tissue method in the three functional subdivisions of the striatum (associative striatum, AST; limbic striatum LST and sensorimotor striatum SMST). The test-retest variability and intraclass correlation coefficient were assessed for distribution volume (VT), binding potential relative to plasma concentration (BPP), and binding potential relative to nondisplaceable uptake (BPND) Results A two-tissue compartment kinetic model adequately described the functional subdivisions of the striatum as well as cerebellum time-activity data. The reproducibility of VT was excellent (≤ 10%) in all regions, for this approach. The reproducibility of both BPP (≤ 12%) and BPND (≤ 10%) was also excellent. The intraclass correlation coefficient of BPP and BPND were acceptable as well (> 0.75) in the three functional subdivisions of the striatum. Although SRTM led to an underestimation of BPND values relative to that estimated by kinetic analysis by 8 to 13%, the values derived using both the methods were reasonably well correlated (r2 = 0.89, n = 84). Both methods were similarly effective at detecting the differences in [11C]NPA BPND between subjects. Conclusion The results of this study indicate that [11C]NPA can be used to measure D2/3 receptors configured in a state of high affinity for the agonists with high reliability and reproducibility in the functional subdivisions

Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer

PubMed Central

Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I.; Lluís, Carme; Cortés, Antoni; Volkow, Nora D.; Schiffmann, Serge N.; Ferré, Sergi; Casadó, Vicent

2015-01-01

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain. PMID:26100888

Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer.

PubMed

Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Cortés, Antoni; Volkow, Nora D; Schiffmann, Serge N; Ferré, Sergi; Casadó, Vicent

2015-07-07

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain.

The novel antidyskinetic drug sarizotan elicits different functional responses at human D2-like dopamine receptors.

PubMed

Kuzhikandathil, Eldo V; Bartoszyk, Gerd D

2006-09-01

Sarizotan (EMD 128130) is a chromane derivative that exhibits affinity at serotonin and dopamine receptors. Sarizotan effectively suppresses levodopa-induced dyskinesia in primate and rodent models of Parkinson's disease, and tardive dyskinesia in a rodent model. Results from clinical trials suggest that sarizotan significantly alleviates levodopa-induced dyskinesia. The functional effects of sarizotan on individual dopamine receptor subtypes are not known. Here we report the functional effects of sarizotan on human D2-like dopamine receptors (D2S, D2L, D3, D4.2 and D4.4) individually expressed in the AtT-20 neuroendocrine cell line. Using the coupling of D2-like dopamine receptors to G-protein coupled inward rectifier potassium channels we determined that sarizotan is a full agonist at D3 and D4.4 receptors (EC50=5.6 and 5.4 nM, respectively) but a partial agonist at D2S, D2L and D4.2 receptors (EC50=29, 23 and 4.5 nM, respectively). Consistent with its partial agonist property, sarizotan is an antagonist at D2S and D2L receptors (IC50=52 and 121 nM, respectively). Using the coupling of D2-like dopamine receptors to adenylyl cyclase we determined that sarizotan is a full agonist at D2L, D3, D4.2 and D4.4 receptors (EC50=0.51, 0.47, 0.48 and 0.23 nM, respectively) but a partial agonist at D2S receptors (EC50=0.6 nM).

The structures of marialite (Me[subscript 6]) and meionite (Me[subscript 93]) in space groups P4[subscript 2]/n and I4/m, and the absence of phase transitions in the scapolite series

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antao, Sytle M.; Hassan, Ishmael; Calgary)

2014-05-28

The crystal structures of marialite (Me{sub 6}) from Badakhshan, Afghanistan and meionite (Me{sub 93}) from Mt. Vesuvius, Italy were obtained using synchrotron high-resolution powder X-ray diffraction (HRPXRD) data and Rietveld structure refinements. Their structures were refined in space groups I4/m and P42/n, and similar results were obtained. The Me{sub 6} sample has a formula Ca{sub 0.24}Na{sub 3.37}K{sub 0.24}[Al{sub 3.16}Si{sub 8.84}O{sub 24}]Cl{sub 0.84}(CO{sub 3}){sub 0.15}, and its unit-cell parameters are a = 12.047555(7), c = 7.563210(6) {angstrom}, and V = 1097.751(1) {angstrom}{sup 3}. The average (T1-O) distances are 1.599(1) {angstrom} in I4/m and 1.600(2) {angstrom} in P4{sub 2}/n, indicating that themore » T1 site contains only Si atoms. In P4{sub 2}/n, the average distances of (T2-O) = 1.655(2) and (T3-O) = 1.664(2) {angstrom} are distinct and are not equal to each other. However, the mean (T2,3-O) = 1.659(2) {angstrom} in P4{sub 2}/n and is identical to the (T2-O) = 1.659(1) {angstrom} in I4/m. The (M-O) [7] = 2.754(1) {angstrom} (M site is coordinated to seven framework O atoms) and M-A = 2.914(1) {angstrom}; these distances are identical in both space groups. The Me{sub 93} sample has a formula of Na{sub 0.29}Ca{sub 3.76}[Al{sub 5.54}Si{sub 6.46}O{sub 24}]Cl{sub 0.05}(SO{sub 4}){sub 0.02}(CO{sub 3}){sub 0.93}, and its unit-cell parameters are a = 12.19882(1), c = 7.576954(8) {angstrom}, and V = 1127.535(2) {angstrom}{sup 3}. A similar examination of the Me{sub 93} sample also shows that both space groups give similar results; however, the C-O distance is more reasonable in P4{sub 2}/n than in I4/m. Refining the scapolite structure near Me{sub 0} or Me{sub 100} in I4/m forces the T2 and T3 sites (both with multiplicity 8 in P4{sub 2}/n) to be equivalent and form the T2' site (with multiplicity 16 in I4/m), but (T2-O) is not equal to (T3-O) in P4{sub 2}/n. Using different space groups for different regions across the series implies phase

Detection of diastereomer peptides as the intermediates generating D-amino acids during acid hydrolysis of peptides.

PubMed

Miyamoto, Tetsuya; Sekine, Masae; Ogawa, Tetsuhiro; Hidaka, Makoto; Watanabe, Hidenori; Homma, Hiroshi; Masaki, Haruhiko

2016-11-01

In this study, we investigated whether the amino acid residues within peptides were isomerized (and the peptides converted to diastereomers) during the early stages of acid hydrolysis. We demonstrate that the model dipeptides L-Ala-L-Phe and L-Phe-L-Ala are epimerized to produce the corresponding diastereomers at a very early stage, prior to their acid hydrolytic cleavage to amino acids. Furthermore, the sequence-inverted dipeptides were generated via formation of a diketopiperazine during hydrolytic incubation, and these dipeptides were also epimerized. The proportion of diastereomers increased rapidly during incubation for 0.5-2 h. During acid hydrolysis, C-terminal residues of the model dipeptides were isomerized faster than N-terminal residues, consistent with the observation that the D-amino acid values of the C-terminal residues determined by the 0 h-extrapolating method were larger than those of the N-terminal residues. Thus, the artificial D-amino acid contents determined by the 0 h-extrapolating method appear to be products of the isomerization of amino acid residues during acid hydrolysis.

Agonists and antagonists for P2 receptors

PubMed Central

Jacobson, Kenneth A.; Costanzi, Stefano; Joshi, Bhalchandra V.; Besada, Pedro; Shin, Dae Hong; Ko, Hyojin; Ivanov, Andrei A.; Mamedova, Liaman

2015-01-01

Recent work has identified nucleotide agonists selective for P2Y1, P2Y2 and P2Y6 receptors and nucleotide antagonists selective for P2Y1, P2Y12 and P2X1 receptors. Selective non-nucleotide antagonists have been reported for P2Y1, P2Y2, P2Y6, P2Y12, P2Y13, P2X2/3/P2X3 and P2X7 receptors. For example, the dinucleotide INS 37217 (Up4dC) potently activates the P2Y2 receptor, and the non-nucleotide antagonist A-317491 is selective for P2X2/3/P2X3 receptors. Nucleotide analogues in which the ribose moiety is substituted by a variety of novel ring systems, including conformation-ally locked moieties, have been synthesized as ligands for P2Y receptors. The focus on conformational factors of the ribose-like moiety allows the inclusion of general modifications that lead to enhanced potency and selectivity. At P2Y1,2,4,11 receptors, there is a preference for the North conformation as indicated with (N)-methanocarba analogues. The P2Y1 antagonist MRS2500 inhibited ADP-induced human platelet aggregation with an IC50 of 0.95 nM. MRS2365, an (N)-methanocarba analogue of 2-MeSADP, displayed potency (EC50) of 0.4 nM at the P2Y1 receptor, with >10 000-fold selectivity in comparison to P2Y12 and P2Y13 receptors. At P2Y6 receptors there is a dramatic preference for the South conformation. Three-dimensional structures of P2Y receptors have been deduced from structure activity relationships (SAR), mutagenesis and modelling studies. Detailed three-dimensional structures of P2X receptors have not yet been proposed. PMID:16805423

Effects of melanocortin-4 receptor agonists and antagonists on expression of genes related to reproduction in spotted scat, Scatophagus argus.

PubMed

Jiang, Dong-Neng; Li, Jian-Tao; Tao, Ya-Xiong; Chen, Hua-Pu; Deng, Si-Ping; Zhu, Chun-Hua; Li, Guang-Li

2017-05-01

Melanocortin-4 receptor (Mc4r) function related to reproduction in fish has not been extensively investigated. Here, we report on gene expression changes by real-time PCR following treatment with Mc4r agonists and antagonists in the spotted scat (Scatophagus argus). Using in vitro incubated hypothalamus, the Mc4r nonselective agonist NDP-MSH ([Nle 4 , D-Phe 7 ]-α-melanocyte stimulating hormone; 10 -6 M) and selective agonist THIQ (N-[(3R)-1, 2, 3, 4-Tetrahydroisoquinolinium-3-ylcarbonyl]- (1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl) piperidin-1-yl]-2-oxoethylamine; 10 -7 M) significantly increased the expression of gnrh (Gonadotropin releasing hormone), while the Mc4r nonselective antagonist SHU9119 (Ac-Nle-[Asp-His-DPhe/DNal(2')-Arg-Trp-Lys]-NH2; 10 -6 M) and selective antagonist Ipsen 5i (compound 5i synthesized in Ipsen Research Laboratories; 10 -6 M) significantly inhibited gnrh expression after 3 h of incubation. In incubated pituitary tissue, NDP-MSH and THIQ significantly increased the expression of fshb (Follicle-stimulating hormone beta subunit) and lhb (Luteinizing hormone beta subunit), while SHU9119 and Ipsen 5i significantly decreased fshb and lhb expression after 3 h of incubation. During the in vivo experiment, THIQ (1 mg/kg bw) significantly increased gnrh expression in hypothalamic tissue, as well as the fshb and lhb expression in pituitary tissue 12 h after abdominal injection. Furthermore, Ipsen 5i (1 mg/kg bw) significantly inhibited gnrh expression in hypothalamic tissue, as well as fshb and lhb gene expression in pituitary tissue 12 h after abdominal injection. In summary, Mc4r singling appears to stimulate gnrh expression in the hypothalamus, thereby modulating the synthesis of Fsh and Lh in the pituitary. In addition, Mc4r also appears to directly regulate fshb and lhb levels in the pituitary in spotted scat. Our study suggests that Mc4r, through the hypothalamus and pituitary, participates in reproductive

Nucleus accumbens opioid, GABaergic, and dopaminergic modulation of palatable food motivation: contrasting effects revealed by a progressive ratio study in the rat.

PubMed

Zhang, Min; Balmadrid, Christian; Kelley, Ann E

2003-04-01

The current studies were designed to evaluate whether incentive motivation for palatable food is altered after manipulations of opioid, GABAergic, and dopaminergic transmission within the nucleus accumbens. A progressive ratio schedule was used to measure lever-pressing for sugar pellets after microinfusion of drugs into the nucleus accumbens in non-food-deprived rats. The mu opioid agonist D-Ala2, NMe-Phe4, Glyo15-enkephalin and the indirect dopamine agonist amphetamine induced a marked increase in break point and correct lever-presses; the GABA(A) agonist muscimol did not affect breakpoint or lever-presses. The data suggest that opioid, dopaminergic, and GABAergic systems within the accumbens differentially modulate food-seeking behavior through mechanisms related to hedonic evaluation of food, incentive salience, and control of motor feeding circuits, respectively.

Synthesis of the (N2)3- radical from Y2+ and its protonolysis reactivity to form (N2H2)2- via the Y[N(SiMe3)2]3/KC8 reduction system.

PubMed

Fang, Ming; Lee, David S; Ziller, Joseph W; Doedens, Robert J; Bates, Jefferson E; Furche, Filipp; Evans, William J

2011-03-23

Examination of the Y[N(SiMe(3))(2)](3)/KC(8) reduction system that allowed isolation of the (N(2))(3-) radical has led to the first evidence of Y(2+) in solution. The deep-blue solutions obtained from Y[N(SiMe(3))(2)](3) and KC(8) in THF at -35 °C under argon have EPR spectra containing a doublet at g(iso) = 1.976 with a 110 G hyperfine coupling constant. The solutions react with N(2) to generate (N(2))(2-) and (N(2))(3-) complexes {[(Me(3)Si)(2)N](2)(THF)Y}(2)(μ-η(2):η(2)-N(2)) (1) and {[(Me(3)Si)(2)N](2)(THF)Y}(2)(μ-η(2):η(2)-N(2))[K(THF)(6)] (2), respectively, and demonstrate that the Y[N(SiMe(3))(2)](3)/KC(8) reaction can proceed through an Y(2+) intermediate. The reactivity of (N(2))(3-) radical with proton sources was probed for the first time for comparison with the (N(2))(2-) and (N(2))(4-) chemistry. Complex 2 reacts with [Et(3)NH][BPh(4)] to form {[(Me(3)Si)(2)N](2)(THF)Y}(2)(μ-N(2)H(2)), the first lanthanide (N(2)H(2))(2-) complex derived from dinitrogen, as well as 1 as a byproduct, consistent with radical disproportionation reactivity.

Sodium ion modulates D2 receptor characteristics of dopamine agonist and antagonist binding sites in striatum and retina

PubMed Central

Makman, Maynard H.; Dvorkin, B.; Klein, Patrice N.

1982-01-01

Sodium ion (Na+) influences binding of both dopamine agonists and antagonists to D2 receptors in striatum and retina. Also, Na+ markedly potentiates the loss of high-affinity agonist binding due to the GTP analogue p[NH]ppG. 2-Amino-6, 7-dihydroxy-1,2,3,4-tetrahydro[5,8-3H]naphthalene ([3H]ADTN) binds exclusively to an agonist conformation of D2 receptor in both striatum and retina, distinct from the antagonist conformation labeled by [3H]spiroperidol or [3H]domperidone in striatum or by [3H]spiroperidol in retina. Na+ is not required for interaction of [3H]ADTN or antagonist radioligand sites with the selective D2 agonist LY-141865, the D2 antagonist domperidone, or nonselective dopamine agonists or antagonists; however, Na+ is necessary for high affinity interaction of those radioligand sites with the D2 antagonists molindone and metoclopramide. With Na+ present, striatal sites for [3H]ADTN, [3H]spiroperidol, and [3H]domperidone have similar affinities for antagonists but only [3H]ADTN sites have high affinity for agonists. Na+ further decreases the low affinity of dopamine agonists for [3H]spiroperidol binding sites. Also, Na+ enhances [3H]spiroperidol and decreases [3H]ADTN binding. Na+ alone causes bound [3H]ADTN to dissociate from at least 30% of striatal and 50% of retinal sites, and with Na+ present [3H]ADTN rapidly dissociates from the remaining sites upon addition of p[NH]ppG. It is proposed that D2 receptors in striatum and retina exist in distinct but interconvertible conformational states, with different properties depending on the presence or absence of Na+ and of guanine nucleotide. PMID:6213964

Selective Adsorption Resonances in the Scattering of n-H2 p-H2 n-D2 and o-D2 from Ag(111)

NASA Astrophysics Data System (ADS)

Yu, Chien-Fan; Whaley, K. Birgitta; Hogg, Charles S.; Sibener, Steven J.

1983-12-01

Diffractive and rotationally mediated selective adsorption scattering resonances are reported for n-H2 p-H2 n-D2 and o-D2 on Ag(111). Small resonance shifts and line-width differences are observed between n-H2 and p-H2 indicating a weak orientation dependence of the laterally averaged H2/Ag(111) potential. The p-H2 and o-D2 levels were used to determine the isotropic component of this potential, yielding a well depth of ~ 32 meV.

Molecular Characterization of Enterococcus faecalis N06-0364 with Low-Level Vancomycin Resistance Harboring a Novel d-Ala-d-Ser Gene Cluster, vanL▿

PubMed Central

Boyd, David A.; Willey, Barbara M.; Fawcett, Darlene; Gillani, Nazira; Mulvey, Michael R.

2008-01-01

Enterococcus faecalis N06-0364, exhibiting a vancomycin MIC of 8 μg/ml, was found to harbor a novel d-Ala-d-Ser gene cluster, designated vanL. The vanL gene cluster was similar in organization to the vanC operon, but the VanT serine racemase was encoded by two separate genes, vanTmL (membrane binding) and vanTrL (racemase). PMID:18458129

Methadone Reverses Analgesic Tolerance Induced by Morphine Pretreatment

PubMed Central

Posa, Luca; Accarie, Alison; Marie, Nicolas

2016-01-01

Background: Opiates such as morphine are the most powerful analgesics, but their protracted use is restrained by the development of tolerance to analgesic effects. Recent works suggest that tolerance to morphine might be due to its inability to promote mu opioid receptor endocytosis, and the co-injection of morphine with a mu opioid receptor internalizing agonist like [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin reduces tolerance to morphine. So far, no studies have been conducted to evaluate the ability of methadone to reduce morphine tolerance in morphine-pretreated animals, a treatment sequence that could be encountered in opiate rotation protocol. We investigated the ability of methadone (a mu opioid receptor internalizing agonist used in therapy) to reverse morphine tolerance and the associated cellular mechanisms in the periaqueductal gray matter, a region involved in pain control. Methods: We measured analgesic response following a challenge dose of morphine in the hot plate test and investigated regulation of mu opioid receptor (coupling and endocytosis) and some cellular mechanisms involved in tolerance such as adenylate cyclase superactivation and changes in N-methyl-d-aspartate receptor subunits expression and phosphorylation state. Results: A chronic treatment with morphine promoted tolerance to its analgesic effects and was associated with a lack of mu opioid receptor endocytosis, adenylate cyclase overshoot, NR2A and NR2B downregulation, and phosphorylation of NR1. We reported that a methadone treatment in morphine-treated mice reversed morphine tolerance to analgesia by promoting mu opioid receptor endocytosis and blocking cellular mechanisms of tolerance. Conclusions: Our data might lead to rational strategies to tackle opiate tolerance in the frame of opiate rotation. PMID:26390873

Ligand-induced μ opioid receptor internalization in enteric neurons following chronic treatment with the opiate fentanyl.

PubMed

Anselmi, Laura; Jaramillo, Ingrid; Palacios, Michelle; Huynh, Jennifer; Sternini, Catia

2013-06-01

Morphine differs from most opiates its poor ability to internalize μ opioid receptors (μORs). However, chronic treatment with morphine produces adaptational changes at the dynamin level, which enhance the efficiency of acute morphine stimulation to promote μOR internalization in enteric neurons. This study tested the effect of chronic treatment with fentanyl, a μOR-internalizing agonist, on ligand-induced endocytosis and the expression of the intracellular trafficking proteins, dynamin and β-arrestin, in enteric neurons using organotypic cultures of the guinea pig ileum. In enteric neurons from guinea pigs chronically treated with fentanyl, μOR immunoreactivity was predominantly at the cell surface after acute exposure to morphine with a low level of μOR translocation, slightly higher than in neurons from naïve animals. This internalization was not due to morphine's direct effect, because it was also observed in neurons exposed to medium alone. By contrast, D-Ala2-N-Me-Phe4-Gly-ol5-enkephalin (DAMGO), a potent μOR-internalizing agonist, induced pronounced and rapid μOR endocytosis in enteric neurons from animals chronically treated with fentanyl or from naïve animals. Chronic fentanyl treatment did not alter dynamin or β-arrestin expression. These findings indicate that prolonged activation of μORs with an internalizing agonist such as fentanyl does not enhance the ability of acute morphine to trigger μOR endocytosis or induce changes in intracellular trafficking proteins, as observed with prolonged activation of μORs with a poorly internalizing agonist such as morphine. Cellular adaptations induced by chronic opiate treatment might be ligand dependent and vary with the agonist efficiency to induce receptor internalization. Copyright © 2013 Wiley Periodicals, Inc.

μ-Opioid Receptor-Mediated Inhibition of Intercalated Neurons and Effect on Synaptic Transmission to the Central Amygdala.

PubMed

Blaesse, Peter; Goedecke, Lena; Bazelot, Michaël; Capogna, Marco; Pape, Hans-Christian; Jüngling, Kay

2015-05-13

The amygdala is a key region for the processing of information underlying fear, anxiety, and fear extinction. Within the local neuronal networks of the amygdala, a population of inhibitory, intercalated neurons (ITCs) modulates the flow of information among various nuclei of amygdala, including the basal nucleus (BA) and the centromedial nucleus (CeM) of the amygdala. These ITCs have been shown to be important during fear extinction and are target of a variety of neurotransmitters and neuropeptides. Here we provide evidence that the activation of μ-opioid receptors (MORs) by the specific agonist DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin) hyperpolarizes medially located ITCs (mITCs) in acute brain slices of mice. Moreover, we use whole-cell patch-clamp recordings in combination with local electrical stimulation or glutamate uncaging to analyze the effect of MOR activation on local microcircuits. We show that the GABAergic transmission between mITCs and CeM neurons is attenuated by DAMGO, whereas the glutamatergic transmission on CeM neurons and mITCs is unaffected. Furthermore, MOR activation induced by theta burst stimulation in BA suppresses plastic changes of feedforward inhibitory transmission onto CeM neurons as revealed by the MOR antagonist CTAP d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2. In summary, the mITCs constitute a target for the opioid system, and therefore, the activation of MOR in ITCs might play a central role in the modulation of the information processing between the basolateral complex of the amygdala and central nuclei of the amygdala. Copyright © 2015 Blaesse, Goedecke et al.

Opposing Effects of Dopamine D1- and D2-Like Agonists on Intracranial Self-Stimulation in Male Rats

PubMed Central

Lazenka, Matthew F.; Legakis, Luke P.; Negus, S. Stevens

2016-01-01

Dopamine acts through dopamine type 1 receptors (comprised of D1 and D5 subtypes) and dopamine type 2 receptors (comprised of D2, D3 and D4 subtypes). Intracranial self-stimulation (ICSS) is one experimental procedure that can be used to evaluate abuse-related effects of drugs targeting dopamine receptors. This study evaluated effects of dopamine receptor ligands on ICSS in rats using experimental procedures that have been used previously to examine abused indirect dopamine agonists such as cocaine and amphetamine. Male Sprague-Dawley rats responded under a fixed-ratio 1 schedule for electrical stimulation of the medial forebrain bundle, and frequency of stimulation varied from 56–158 Hz in 0.05 log increments during each experimental session. Drug potency and time course were determined for the D1 ligands A77636, SKF82958, SKF38393, fenoldopam and SCH39166 and the D2/3 ligands sumanirole, apomorphine, quinpirole, PD128907, pramipexole, aripiprazole, eticolopride and PG01037. The high-efficacy D1 agonists A77636 and SKF82958 produced dose-dependent, time-dependent, and abuse-related facilitation of ICSS. Lower efficacy D1 ligands and all D2/3 ligands failed to facilitate ICSS at any dose or pretreatment time. A mixture of SKF82958 and quinpirole produced a mixture of effects produced by each drug alone. Quinpirole also failed to facilitate ICSS after regimens of repeated treatment with either quinpirole or cocaine. These studies provide more evidence for divergent effects of dopamine D1- and D2-family agonists on ICSS procedure in rats and suggest that ICSS may be a useful complement to other approaches for preclinical abuse potential assessment, in part because of the reproducibility of results. PMID:26987070

D1 receptor agonist improves sleep-wake parameters in experimental parkinsonism.

PubMed

Hyacinthe, Carole; Barraud, Quentin; Tison, François; Bezard, Erwan; Ghorayeb, Imad

2014-03-01

Both excessive daytime sleepiness (EDS) and rapid eye movement (REM) sleep deregulation are part of Parkinson's disease (PD) non-motor symptoms and may complicate dopamine replacement therapy. We report here that dopamine agonists act differentially on sleep architecture in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine macaque monkey. Continuous sleep and wake electroencephalographic monitoring revealed no effect of the selective dopamine D2 receptor agonist quinpirole on EDS, whereas the selective dopamine D1 receptor agonist SKF38393 efficiently alleviated EDS and restored REM sleep to baseline values. The present results question the relevance of abandoning D1 receptor agonist treatment in PD as it might actually improve sleep-related disorders. Copyright © 2013 Elsevier Inc. All rights reserved.

Evaluation of 11C-Me-NB1 as a Potential PET Radioligand for Measuring GluN2B-Containing NMDA Receptors, Drug Occupancy, and Receptor Cross Talk.

PubMed

Krämer, Stefanie D; Betzel, Thomas; Mu, Linjing; Haider, Ahmed; Herde, Adrienne Müller; Boninsegni, Anna K; Keller, Claudia; Szermerski, Marina; Schibli, Roger; Wünsch, Bernhard; Ametamey, Simon M

2018-04-01

Clinical and preclinical research with modulators at the N -methyl-d-aspartate (NMDA) receptor GluN2B N-terminal domain (NTD) aims for the treatment of various neurologic diseases. The interpretation of the results is hampered by the lack of a suitable NMDA PET tracer for assessing the receptor occupancy of potential drugs. We have developed 11 C-Me-NB1 as a PET tracer for imaging GluN1/GluN2B-containing NMDA receptors and used it to investigate in rats the dose-dependent receptor occupancy of eliprodil, a GluN2B NTD modulator. Methods: 11 C-Me-NB1 was synthesized and characterized by in vitro displacement binding experiments with rat brain membranes, in vitro autoradiography, and blocking and displacement experiments by PET and PET kinetic modeling. Receptor occupancy by eliprodil was studied by PET with 11 C-Me-NB1. Results: 11 C-Me-NB1 was synthesized at 290 ± 90 GBq/μmol molar activity, 7.4 ± 1.9 GBq total activity at the end of synthesis ( n = 17), and more than 99% radiochemical purity. 11 C-Me-NB1 binding in rat brain was blocked in vitro and in vivo by the NTD modulators Ro-25-6981 and eliprodil. Half-maximal receptor occupancy by eliprodil occurred at 1.5 μg/kg. At 1 mg/kg of eliprodil, a dose with reported neuroprotective effects, more than 99.5% of binding sites were occupied. In vitro, 11 C-Me-NB1 binding was independent of the σ-1 receptor (Sigma1R), and the Sigma1R agonist (+)-pentazocine did not compete for high-affinity binding. In vivo, a 2.5 mg/kg dose of (+)-pentazocine abolished 11 C-Me-NB1-specific binding, indicating an indirect effect of Sigma1R on 11 C-Me-NB1 binding. Conclusion: 11 C-Me-NB1 is suitable for the in vivo imaging of NMDA GluN1/GluN2B receptors and the assessment of receptor occupancy by NTD modulators. GluN1/GluN2B NMDA receptors are fully occupied at neuroprotective doses of eliprodil. Furthermore, 11 C-Me-NB1 enables imaging of GluN1/GluN2B NMDA receptor cross talk. © 2018 by the Society of Nuclear Medicine and

Tuning the reactivity in classic low-spin d6 rhenium(I) tricarbonyl radiopharmaceutical synthon by selective bidentate ligand variation (L,L'-Bid; L,L'= N,N', N,O, and O,O' donor atom sets) in fac-[Re(CO)3(L,L'-Bid)(MeOH)]n complexes.

PubMed

Schutte, Marietjie; Kemp, Gerdus; Visser, Hendrik G; Roodt, Andreas

2011-12-19

A range of fac-[Re(CO)(3)(L,L'-Bid)(H(2)O)](n) (L,L'-Bid = neutral or monoanionic bidentate ligands with varied L,L' donor atoms, N,N', N,O, or O,O': 1,10-phenanthroline, 2,2'-bipydine, 2-picolinate, 2-quinolinate, 2,4-dipicolinate, 2,4-diquinolinate, tribromotropolonate, and hydroxyflavonate; n = 0, +1) has been synthesized and the aqua/methanol substitution has been investigated. The complexes were characterized by UV-vis, IR and NMR spectroscopy and X-ray crystallographic studies of the compounds fac-[Re(CO)(3)(Phen)(H(2)O)]NO(3)·0.5Phen, fac-[Re(CO)(3)(2,4-dQuinH)(H(2)O)]·H(2)O, fac-[Re(CO)(3)(2,4-dQuinH)Py]Py, and fac-[Re(CO)(3)(Flav)(CH(3)OH)]·CH(3)OH are reported. A four order-of-magnitude of activation for the methanol substitution is induced as manifested by the second order rate constants with (N,N'-Bid) < (N,O-Bid) < (O,O'-Bid). Forward and reverse rate and stability constants from slow and stopped-flow UV/vis measurements (k(1), M(-1) s(-1); k(-1), s(-1); K(1), M(-1)) for bromide anions as entering nucleophile are as follows: fac-[Re(CO)(3)(Phen)(MeOH)](+) (50 ± 3) × 10(-3), (5.9 ± 0.3) × 10(-4), 84 ± 7; fac-[Re(CO)(3)(2,4-dPicoH)(MeOH)] (15.7 ± 0.2) × 10(-3), (6.3 ± 0.8) × 10(-4), 25 ± 3; fac-[Re(CO)(3)(TropBr(3))(MeOH)] (7.06 ± 0.04) × 10(-2), (4 ± 1) × 10(-3), 18 ± 4; fac-[Re(CO)(3)(Flav)(MeOH)] 7.2 ± 0.3, 3.17 ± 0.09, 2.5 ± 2. Activation parameters (ΔH(k1)(++), kJmol(-1); ΔS(k1)(), J K(-1) mol(-1)) from Eyring plots for entering nucleophiles as indicated are as follows: fac-[Re(CO)(3)(Phen)(MeOH)](+) iodide 70 ± 1, -35 ± 3; fac-[Re(CO)(3)(2,4-dPico)(MeOH)] bromide 80.8 ± 6, -8 ± 2; fac-[Re(CO)(3)(Flav)(MeOH)] bromide 52 ± 5, -52 ± 15. A dissociative interchange mechanism is proposed. © 2011 American Chemical Society

Evaluation of biological activities of new LH-RH antagonists (T-series) in male and female rats.

PubMed

Pinski, J; Yano, T; Janaky, T; Nagy, A; Juhasz, A; Bokser, L; Groot, K; Schally, A V

1993-01-01

A series of new highly potent LH-RH antagonists (T-series) has been synthesized in our laboratory. Among these analogs, antagonists [Ac-D-Nal(2), D-Phe(4Cl)2, D-Pal(3)3, D-Lys(A2pr(Car)2)6, D-Ala10]LH-RH (T-140); [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Lys(A2pr(Ac)2)6, D-Ala10]LH-RH (T-148); [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Lys(A2pr(For)2)6, D-Ala10]LH-RH (T-151) and [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Lys(A2bu(For)2)6, D-Ala10]LH-RH (T-159) were the most powerful. Antagonists T-140, T-148 and T-151 produced a complete blockade of ovulation in normal cycling rats at a dose of 1.5 micrograms/rat and antagonist T-159 at a dose of only 0.75 micrograms/rat. The inhibitory effects of compounds T-148, T-151 and T-159 on gonadotropin and sex steroid secretion were investigated in male and female rats. To determine their effect on LH levels in castrated male and ovariectomized female rats, T-148, T-151 and T-159 were injected subcutaneously in doses of 0.625 and 2.5 micrograms/rat. Blood samples were taken at different intervals for 48 h. All three compounds at either dose caused a significant (P < 0.01) decrease in LH levels for more than 6 h. Significant (P < 0.01) inhibition of LH lasted for more than 24 h following a dose of 2.5 micrograms sc of all 3 antagonists in both male and female rats. Serum FSH levels were also suppressed significantly for more than 48 h in castrated male rats by all three antagonists at a dose of 5 micrograms/rat sc.(ABSTRACT TRUNCATED AT 250 WORDS)

The energy spectrum of neutrons from 7Li(d,n)8Be reaction at deuteron energy 2.9 MeV

NASA Astrophysics Data System (ADS)

Mitrofanov, Konstantin V.; Piksaikin, Vladimir M.; Zolotarev, Konstantin I.; Egorov, Andrey S.; Gremyachkin, Dmitrii E.

2017-09-01

The neutron beams generated at the electrostatic accelerators using nuclear reactions T(p,n)3He, D(d,n)3He, 7Li(p,n)7Be, T(d,n)4He, 7Li(d,n)8Be, 9Be(d,n)10B are widely used in neutron physics and in many practical applications. Among these reactions the least studied reactions are 7Li(d,n)8Be and 9Be(d,n)10B. The present work is devoted to the measurement of the neutron spectrum from 7Li(d,n)8Be reaction at 0∘ angle to the deuteron beam axis on the electrostatic accelerator Tandetron (JSC "SSC RF - IPPE") using activation method and a stilbene crystal scintillation detector. The first time ever 7Li(d,n)8Be reaction was measured by activation method. The target was a thick lithium layer on metallic backing. The energy of the incident deuteron was 2.9 MeV. As activation detectors a wide range of nuclear reactions were used: 27Al(n,p)27Mg, 27Al(n,α)24Na, 113In(n,n')113mIn, 115In(n,n')115mIn, 115In(n,γ)116mIn, 58Ni(n,p)58mCo, 58Ni(n,2n)57Ni, 197Au(n,γ)198Au, 197Au(n,2n)196Au, 59Co(n,p)59Fe, 59Co(n,2n)58m+gCo, 59Co (n,g)60Co. Measurement of the induced gamma-activity was carried out using HPGe detector Canberra GX5019 [1]. The up-to-date evaluations of the cross sections for these reactions were used in processing of the data. The program STAYSL was used to unfold the energy spectra. The neutron spectra obtained by activation detectors is consistent with the corresponding data measured by a stilbene crystal scintillation detector within their uncertainties.

PDK1/Akt/PDE4D axis identified as a target for asthma remedy synergistic with β2 AR agonists by a natural agent arctigenin.

PubMed

Fang, R; Cui, Q; Sun, J; Duan, X; Ma, X; Wang, W; Cheng, B; Liu, Y; Hou, Y; Bai, G

2015-12-01

Asthma is a heterogenetic disorder characterized by chronic inflammation with variable airflow obstruction and airway hyper-responsiveness. As the most potent and popular bronchodilators, β2 adrenergic receptor (β2 AR) agonists bind to the β2 ARs that are coupled via a stimulatory G protein to adenylyl cyclase, thereby improving cAMP accumulation and resulting in airway smooth muscle relaxation. We previously demonstrated arctigenin had a synergistic function with the β2 AR agonist, but the target for this remained elusive. Chemical proteomics capturing was used to enrich and uncover the target of arctigenin in human bronchial smooth muscle cells, and reverse docking and molecular dynamic stimulation were performed to evaluate the binding of arctigenin and its target. In vitro enzyme activities and protein levels were demonstrated with special kits and Western blotting. Finally, guinea pig tracheal muscle segregation and ex vivo function were analysed. Arctigenin bound to PDK1 with an ideal binding free energy -25.45 kcal/mol and inhibited PDK1 kinase activity without changing its protein level. Additionally, arctigenin reduced PKB/Akt-induced phosphorylation of PDE4D, which was first identified in this study. Attenuation of PDE4D resulted in cAMP accumulation in human bronchial smooth muscle. The inhibition of PDK1 showed a synergistic function with β2 AR agonists and relaxed the constriction of segregated guinea pig tracheal muscle. The PDK1/Akt/PDE4D axis serves as a novel asthma target, which may benefit airflow obstruction. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation

PubMed Central

Zoabi, Muhammad; Nadar-Ponniah, Prathamesh T.; Khoury-Haddad, Hanan; Usaj, Marko; Budowski-Tal, Inbal; Haran, Tali; Henn, Arnon; Mandel-Gutfreund, Yael; Ayoub, Nabieh

2014-01-01

The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin. PMID:25378304

Suppression of meiosis of male germ cells by an antagonist of luteinizing hormone-releasing hormone.

PubMed Central

Szende, B; Redding, T W; Schally, A V

1990-01-01

Male nude mice were implanted with osmotic minipumps releasing 50 micrograms of a potent antagonist of luteinizing hormone-releasing hormone (LH-RH) per day [N-Ac-[D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-RH] (SB-75) [Nal(2), 3-(2-naphthyl)alanine; Phe(pCl), 4-chlorophenylalanine; Pal(3), 3-(3-pyridyl)alanine; Cit, citrulline], or they were treated with s.c. injections of SB-75 (25 micrograms twice a day). Another group of nude mice received an injection of microcapsules of the agonist [D-Trp6]LH-RH liberating 25 micrograms/day. One month after the initiation of treatment, the testicular weights were significantly reduced and the blood testosterone values were at castration levels in all treated groups. Histologically, only the testicles of the mice treated with SB-75 released from minipumps showed a significant decrease of meiosis. The most advanced forms of germ cells were spermatogonia in 26%, spermatocytes in 17%, and round spermatids in 35% of the seminiferous tubules. Only 22% of the tubules contained elongated spermatids. The suppression of meiotic activity by this modern LH-RH antagonist can possibly be used for the development of methods for male contraception and for the protection of germ cells against the damage caused by cytotoxic drugs and x-radiation. Images PMID:2405399

Tachykinin receptors in the guinea-pig renal pelvis: activation by exogenous and endogenous tachykinins.

PubMed

Maggi, C A; Patacchini, R; Eglezos, A; Quartara, L; Giuliani, S; Giachetti, A

1992-09-01

1. The contractile response to substance P, neurokinin A, selective agonists for the NK1, NK2 and NK3 tachykinin receptors and the activity of receptor-selective antagonists has been investigated in circular muscle strips of the guinea-pig isolated renal pelvis in the presence of indomethacin (3 microM). 2. Neurokinin A was the most potent agonist tested, being about 32 times more potent than substance P. The action of both substance P and neurokinin A was enhanced by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The selective NK2 receptor agonist [beta Ala8] neurokinin A (4-10), was slightly less potent and effective than neurokinin A itself. The selective NK1 receptor agonist [Sar9] substance P sulphone was effective at low (nM) concentrations but its maximal effect did not exceed 30% of maximal response to substance P or neurokinin A. The NK3-selective agonist [MePhe7] neurokinin B was effective only at high (microM) concentrations. 3. The pseudopeptide derivative of neurokinin A(4-10), MDL 28,564, displayed a clear-cut agonist character, although it was less potent than neurokinin A. 4. The responses to roughly equieffective (25-35% of maximal response) concentrations of [beta Ala8] neurokinin A (4-10), MDL 28,564 and [MePhe7] neurokinin B were antagonized to a similar extent by MEN 10,376 (3 microM), a selective NK2 tachykinin receptor antagonist, while the response to [Sar9] substance P sulphone was unchanged. 5. The response to [Sar9] substance P sulphone was inhibited by the NK1 receptor-selective antagonist, GR 82,334 (3 microM) while the response to [beta Ala8] neurokinin A (4-10) was unchanged. 6. The selective NK2 receptor antagonists MEN 10,376, L 659,877 and R 396 antagonized competitively the response to [PAla8] neurokinin A (4-10) with the following rank order of potency (pA2 values in parentheses): MEN 10,376 (7.41)>L 659,877 (7.15)>R 396 (6.43). MEN 10,376 and L 659,877 also competitively antagonized the response to

X-ray diffraction studies of enkephalins. Crystal structure of [(4'-bromo) Phe4,Leu5]enkephalin.

PubMed Central

Ishida, T; Kenmotsu, M; Mino, Y; Inoue, M; Fujiwara, T; Tomita, K; Kimura, T; Sakakibara, S

1984-01-01

In order to investigate the structure-activity relationship of [Leu5]- and [Met5]enkephalins, [(4'-bromo)Phe4, Leu5]-, [(4'-bromo)Phe4, Met5]- and [Met5] enkephalins were synthesized and crystallized. The crystal structure of [(4'-bromo) Phe4, Leu5]- enkephalin was determined by X-ray diffraction method using the heavy atom method and refined to R = 0.092 by the least-squares method. The molecule in this crystal took essentially the same type I' beta-turn conformation found in [Leu5]enkephalin [Smith & Griffin (1978) Science 199, 1214-1216). On the other hand, the preliminary three-dimensional Patterson analyses showed that the most probable conformations of [(4'-bromo)Phe4,Met5]- and [Met5]enkephalins are both the dimeric extended forms. Based on these insights, the biologically active conformation of enkephalin was discussed in relation to the mu- and delta-receptors. PMID:6721829

A Novel Aminotetralin-Type Serotonin (5-HT) 2C Receptor-Specific Agonist and 5-HT2A Competitive Antagonist/5-HT2B Inverse Agonist with Preclinical Efficacy for Psychoses

PubMed Central

Morgan, Drake; Felsing, Daniel; Kondabolu, Krishnakanth; Rowland, Neil E.; Robertson, Kimberly L.; Sakhuja, Rajeev; Booth, Raymond G.

2014-01-01

Development of 5-HT2C agonists for treatment of neuropsychiatric disorders, including psychoses, substance abuse, and obesity, has been fraught with difficulties, because the vast majority of reported 5-HT2C selective agonists also activate 5-HT2A and/or 5-HT2B receptors, potentially causing hallucinations and/or cardiac valvulopathy. Herein is described a novel, potent, and efficacious human 5-HT2C receptor agonist, (−)-trans-(2S,4R)-4-(3′[meta]-bromophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (−)-MBP), that is a competitive antagonist and inverse agonist at human 5-HT2A and 5-HT2B receptors, respectively. (−)-MBP has efficacy comparable to the prototypical second-generation antipsychotic drug clozapine in three C57Bl/6 mouse models of drug-induced psychoses: the head-twitch response elicited by [2,5]-dimethoxy-4-iodoamphetamine; hyperlocomotion induced by MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (dizocilpine maleate)]; and hyperlocomotion induced by amphetamine. (−)-MBP, however, does not alter locomotion when administered alone, distinguishing it from clozapine, which suppresses locomotion. Finally, consumption of highly palatable food by mice was not increased by (−)-MBP at a dose that produced at least 50% maximal efficacy in the psychoses models. Compared with (−)-MBP, the enantiomer (+)-MBP was much less active across in vitro affinity and functional assays using mouse and human receptors and also translated in vivo with comparably lower potency and efficacy. Results indicate a 5-HT2C receptor-specific agonist, such as (−)-MBP, may be pharmacotherapeutic for psychoses, without liability for obesity, hallucinations, heart disease, sedation, or motoric disorders. PMID:24563531

Effects of monoamine oxidase inhibitor and cytochrome P450 2D6 status on 5-methoxy-N,N-dimethyltryptamine metabolism and pharmacokinetics.

PubMed

Shen, Hong-Wu; Wu, Chao; Jiang, Xi-Ling; Yu, Ai-Ming

2010-07-01

5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a natural psychoactive indolealkylamine drug that has been used for recreational purpose. Our previous study revealed that polymorphic cytochrome P450 2D6 (CYP2D6) catalyzed 5-MeO-DMT O-demethylation to produce active metabolite bufotenine, while 5-MeO-DMT is mainly inactivated through deamination pathway mediated by monoamine oxidase (MAO). This study, therefore, aimed to investigate the impact of CYP2D6 genotype/phenotype status and MAO inhibitor (MAOI) on 5-MeO-DMT metabolism and pharmacokinetics. Enzyme kinetic studies using recombinant CYP2D6 allelic isozymes showed that CYP2D6.2 and CYP2D6.10 exhibited 2.6- and 40-fold lower catalytic efficiency (V(max)/K(m)), respectively, in producing bufotenine from 5-MeO-DMT, compared with wild-type CYP2D6.1. When co-incubated with MAOI pargyline, 5-MeO-DMT O-demethylation in 10 human liver microsomes showed significantly strong correlation with bufuralol 1'-hydroxylase activities (R(2)=0.98; P<0.0001) and CYP2D6 contents (R(2)=0.77; P=0.0007), whereas no appreciable correlations with enzymatic activities of other P450 enzymes. Furthermore, concurrent MAOI harmaline sharply reduced 5-MeO-DMT depletion and increased bufotenine formation in human CYP2D6 extensive metabolizer hepatocytes. In vivo studies in wild-type and CYP2D6-humanized (Tg-CYP2D6) mouse models showed that Tg-CYP2D6 mice receiving the same dose of 5-MeO-DMT (20mg/kg, i.p.) had 60% higher systemic exposure to metabolite bufotenine. In addition, pretreatment of harmaline (5mg/kg, i.p.) led to 3.6- and 4.4-fold higher systemic exposure to 5-MeO-DMT (2mg/kg, i.p.), and 9.9- and 6.1-fold higher systemic exposure to bufotenine in Tg-CYP2D6 and wild-type mice, respectively. These findings indicate that MAOI largely affects 5-MeO-DMT metabolism and pharmacokinetics, as well as bufotenine formation that is mediated by CYP2D6. (c) 2010 Elsevier Inc. All rights reserved.

Synthesis, spectroscopic characterization, and determination of the solution association energy of the dimer [Co{N(SiMe3)2}2]2: magnetic studies of low-coordinate Co(II) silylamides [Co{N(SiMe3)2}2L] (L = PMe3, pyridine, and THF) and related species that reveal evidence of very large zero-field splittings.

PubMed

Bryan, Aimee M; Long, Gary J; Grandjean, Fernande; Power, Philip P

2013-10-21

The synthesis, magnetic, and spectroscopic characteristics of the synthetically useful dimeric cobalt(II) silylamide complex [Co{N(SiMe3)2}2]2 (1) and several of its Lewis base complexes have been investigated. Variable-temperature nuclear magnetic resonance (NMR) spectroscopy of 1 showed that it exists in a monomer-dimer equilibrium in benzene solution and has an association energy (ΔGreacn) of -0.30(20) kcal mol(-1) at 300 K. Magnetic data for the polycrystalline, red-brown [Co{N(SiMe3)2}2]2 (1) showed that it displays strong antiferromagnetic exchange coupling, expressed as -2JexS1S2, between the two S = (3)/2 cobalt(II) centers with a Jex value of -215(5) cm(-1), which is consistent with its bridged dimeric structure in the solid state. The electronic spectrum of 1 in solution is reported for the first time, and it is shown that earlier reports of the melting point, synthesis, electronic spectrum, and magnetic studies of the monomer "Co{N(SiMe3)2}2" are consistent with those of the bright green-colored tetrahydrofuran (THF) complex [Co{N(SiMe3)2}2(THF)] (4). Treatment of 1 with various Lewis bases yielded monomeric three-coordinated species-[Co{N(SiMe3)2}2(PMe3)] (2), and [Co{N(SiMe3)2}2(THF)] (4), as well as the previously reported [Co{N(SiMe3)2}2(py)] (3)-and the four-coordinated species [Co{N(SiMe3)2}2(py)2] (5) in good yields. The paramagnetic complexes 2-4 were characterized by electronic and (1)H NMR spectroscopy, and by X-ray crystallography in the case of 2 and 4. Magnetic studies of 2-5 and of the known three-coordinated cobalt(II) species [Na(12-crown-4)2][Co{N(SiMe3)2}3] (6) showed that they have considerably larger χMT products and, hence, magnetic moments, than the spin-only values of 1.875 emu K mol(-1) and 3.87 μB, which is indicative of a significant zero-field splitting and g-tensor anisotropy resulting from the pseudo-trigonal crystal field. A fit of χMT for 2-6 yields a large g-tensor anisotropy, large negative D-values (between -62 cm(-1

Purification and characterization of enantioselective N-acetyl-β-Phe acylases from Burkholderia sp. AJ110349.

PubMed

Imabayashi, Yuki; Suzuki, Shun'ichi; Kawasaki, Hisashi; Nakamatsu, Tsuyoshi

2016-01-01

For the production of enantiopure β-amino acids, enantioselective resolution of N-acyl β-amino acids using acylases, especially those recognizing N-acetyl-β-amino acids, is one of the most attractive methods. Burkholderia sp. AJ110349 had been reported to exhibit either (R)- or (S)-enantiomer selective N-acetyl-β-Phe amidohydrolyzing activity, and in this study, both (R)- and (S)-enantioselective N-acetyl-β-Phe acylases were purified to be electrophoretically pure and determined the sequences, respectively. They were quite different in terms of enantioselectivities and in their amino acids sequences and molecular weights. Although both the purified acylases were confirmed to catalyze N-acetyl hydrolyzing activities, neither of them show sequence similarities to the N-acetyl-α-amino acid acylases reported thus far. Both (R)- and (S)-enantioselective N-acetyl-β-Phe acylase were expressed in Escherichia coli. Using these recombinant strains, enantiomerically pure (R)-β-Phe (>99% ee) and (S)-β-Phe (>99% ee) were obtained from the racemic substrate.

Effects of monoamine oxidase inhibitor and cytochrome P450 2D6 status on 5-Methoxy-N,N-dimethyltryptamine Metabolism and Pharmacokinetics

PubMed Central

Shen, Hong-Wu; Wu, Chao; Jiang, Xi-Ling; Yu, Ai-Ming

2010-01-01

5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a natural psychoactive indolealkylamine drug that has been used for recreational purpose. Our previous study revealed that polymorphic cytochrome P450 2D6 (CYP2D6) catalyzed 5-MeO-DMT O-demethylation to produce active metabolite bufotenine, while 5-MeO-DMT is mainly inactivated through deamination pathway mediated by monoamine oxidase (MAO). This study, therefore, aimed to investigate the impact of CYP2D6 genotype/phenotype status and MAO inhibitor (MAOI) on 5-MeO-DMT metabolism and pharmacokinetics. Enzyme kinetic studies using recombinant CYP2D6 allelic isozymes showed that CYP2D6.2 and CYP2D6.10 exhibited 2.6- and 40-fold lower catalytic efficiency (Vmax/Km), respectively, in producing bufotenine from 5-MeO-DMT, compared with wild-type CYP2D6.1. When co-incubated with MAOI pargyline, 5-MeO-DMT O-demethylation in 10 human liver microsomes showed significantly strong correlation with bufuralol 1’-hydroxylase activities (R² = 0.98; p < 0.0001) and CYP2D6 contents (R² = 0.77; p = 0.0007), whereas no appreciable correlations with enzymatic activities of other P450 enzymes. Furthermore, concurrent MAOI harmaline sharply reduced 5-MeO-DMT depletion and increased bufotenine formation in human CYP2D6 extensive metabolizer hepatocytes. In vivo studies in wild-type and CYP2D6-humanized (Tg-CYP2D6) mouse models showed that Tg-CYP2D6 mice receiving the same dose of 5-MeO-DMT (20 mg/kg, i.p.) had 60% higher systemic exposure to metabolite bufotenine. In addition, pre-treatment of harmaline (5 mg/kg, i.p.) led to 3.6- and 4.4-fold higher systemic exposure to 5-MeO-DMT (2 mg/kg, i.p.), and 9.9- and 6.1-fold higher systemic exposure to bufotenine in Tg-CYP2D6 and wild-type mice, respectively. These findings indicate that MAOI largely affects 5-MeO-DMT metabolism and pharmacokinetics, as well as bufotenine formation that is mediated by CYP2D6. PMID:20206139

Structural and computational analysis of peptide recognition mechanism of class-C type penicillin binding protein, alkaline D-peptidase from Bacillus cereus DF4-B

PubMed Central

Nakano, Shogo; Okazaki, Seiji; Ishitsubo, Erika; Kawahara, Nobuhiro; Komeda, Hidenobu; Tokiwa, Hiroaki; Asano, Yasuhisa

2015-01-01

Alkaline D-peptidase from Bacillus cereus DF4-B, called ADP, is a D-stereospecific endopeptidase reacting with oligopeptides containing D-phenylalanine (D-Phe) at N-terminal penultimate residue. ADP has attracted increasing attention because it is useful as a catalyst for synthesis of D-Phe oligopeptides or, with the help of substrate mimetics, L-amino acid peptides and proteins. Structure and functional analysis of ADP is expected to elucidate molecular mechanism of ADP. In this study, the crystal structure of ADP (apo) form was determined at 2.1 Å resolution. The fold of ADP is similar to that of the class C penicillin-binding proteins of type-AmpH. Docking simulations and fragment molecular orbital analyses of two peptides, (D-Phe)4 and (D-Phe)2-(L-Phe)2, with the putative substrate binding sites of ADP indicated that the P1 residue of the peptide interacts with hydrophobic residues at the S1 site of ADP. Furthermore, molecular dynamics simulation of ADP for 50 nsec suggested that the ADP forms large cavity at the active site. Formation of the cavity suggested that the ADP has open state in the solution. For the ADP, having the open state is convenient to bind the peptides having bulky side chain, such as (D-Phe)4. Taken together, we predicted peptide recognition mechanism of ADP. PMID:26370172

Effect of bombesin receptor subtype-3 and its synthetic agonist on signaling, glucose transport and metabolism in myocytes from patients with obesity and type 2 diabetes

PubMed Central

GONZÁLEZ, NIEVES; MARTÍN-DUCE, ANTONIO; MARTÍNEZ-ARRIETA, FÉLIX; MORENO-VILLEGAS, ZAIDA; PORTAL-NÚÑEZ, SERGIO; SANZ, RAÚL; EGIDO, JESÚS

2015-01-01

Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein-coupled receptor (GPCR) member of the bombesin receptor family. Several studies have suggested an association between obesity, alterations in glucose metabolism, diabetes and the BRS-3 receptor. In this study, we focused on patients simultaneously diagnosed with obesity and type 2 diabetes (OB/T2D). The analysis of BRS-3 expression in the skeletal muscle of these patients revealed a marked decrease in the expression of BRS-3 at the mRNA (23.6±1.3-fold downregulation, p<0.0001) and protein level (49±7% decrease, p<0.05) compared to the normal patients (no obesity and diabetes). Moreover, in cultured primary myocytes from patients with OB/T2D, the synthetic BRS-3 agonist, [D-Try6,β-Ala11,Phe13,Nle14]bombesin6–14, significantly increased the phosphorylation levels of mitogen-activated protein kinase (MAPK), p90RSK1, protein kinase B (PKB) and p70s6K. Specifically, the ligand at 10−11 M induced the maximal phosphorylation of MAPKs (p42, 159±15% of the control; p44, 166±11% of the control; p<0.0001) and p90RSK1 (148±2% of the control, p<0.0001). The basal phosphorylation levels of all kinases were reduced (p<0.05) in the patients with OB/T2D compared to the normal patients. Furthermore, the BRS-3 agonist stimulated glucose transport, which was already detected at 10−12 M (133±9% of the control), reached maximal levels at 10−11 M (160±9%, p<0.0001) and was maintained at up to 10−8 M (overall mean, 153±7%; p<0.007). This effect was less promiment than that attained with 10−8 M insulin (202±9%, p=0.009). The effect of the agonist on glycogen synthase a activity achieved the maximum effect at 10−11 M (165±16% of the control; p<0.0001), which did not differ from that observed with higher concentrations of the agonist. These results suggest that muscle cells isolated from patients with OB/T2D have extremely high sensitivity to the synthetic ligand, and the effects are particularly observed on

Psychedelic 5-methoxy-N,N-dimethyltryptamine: metabolism, pharmacokinetics, drug interactions, and pharmacological actions.

PubMed

Shen, Hong-Wu; Jiang, Xi-Ling; Winter, Jerrold C; Yu, Ai-Ming

2010-10-01

5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) belongs to a group of naturally-occurring psychoactive indolealkylamine drugs. It acts as a nonselective serotonin (5-HT) agonist and causes many physiological and behavioral changes. 5-MeO-DMT is O-demethylated by polymorphic cytochrome P450 2D6 (CYP2D6) to an active metabolite, bufotenine, while it is mainly inactivated through the deamination pathway mediated by monoamine oxidase A (MAO-A). 5-MeO-DMT is often used with MAO-A inhibitors such as harmaline. Concurrent use of harmaline reduces 5-MeO-DMT deamination metabolism and leads to a prolonged and increased exposure to the parent drug 5-MeO-DMT, as well as the active metabolite bufotenine. Harmaline, 5-MeO-DMT and bufotenine act agonistically on serotonergic systems and may result in hyperserotonergic effects or serotonin toxicity. Interestingly, CYP2D6 also has important contribution to harmaline metabolism, and CYP2D6 genetic polymorphism may cause considerable variability in the metabolism, pharmacokinetics and dynamics of harmaline and its interaction with 5-MeO-DMT. Therefore, this review summarizes recent findings on biotransformation, pharmacokinetics, and pharmacological actions of 5-MeO-DMT. In addition, the pharmacokinetic and pharmacodynamic drug-drug interactions between harmaline and 5-MeO-DMT, potential involvement of CYP2D6 pharmacogenetics, and risks of 5-MeO-DMT intoxication are discussed.

Psychedelic 5-Methoxy-N,N-dimethyltryptamine: Metabolism, Pharmacokinetics, Drug Interactions, and Pharmacological Actions

PubMed Central

Shen, Hong-Wu; Jiang, Xi-Ling; Winter, Jerrold C.; Yu, Ai-Ming

2011-01-01

5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) belongs to a group of naturally-occurring psychoactive indolealkylamine drugs. It acts as a nonselective serotonin (5-HT) agonist and causes many physiological and behavioral changes. 5-MeO-DMT is O-demethylated by polymorphic cytochrome P450 2D6 (CYP2D6) to an active metabolite, bufotenine, while it is mainly inactivated through the deamination pathway mediated by monoamine oxidase A (MAO-A). 5-MeO-DMT is often used with MAO-A inhibitors such as harmaline. Concurrent use of harmaline reduces 5-MeO-DMT deamination metabolism and leads to a prolonged and increased exposure to the parent drug 5-MeO-DMT, as well as the active metabolite bufotenine. Harmaline, 5-MeO-DMT and bufotenine act agonistically on serotonergic systems and may result in hyperserotonergic effects or serotonin toxicity. Interestingly, CYP2D6 also has important contribution to harmaline metabolism, and CYP2D6 genetic polymorphism may cause considerable variability in the metabolism, pharmacokinetics and dynamics of harmaline and its interaction with 5-MeO-DMT. Therefore, this review summarizes recent findings on biotransformation, pharmacokinetics, and pharmacological actions of 5-MeO-DMT. In addition, the pharmacokinetic and pharmacodynamic drug-drug interactions between harmaline and 5-MeO-DMT, potential involvement of CYP2D6 pharmacogenetics, and risks of 5-MeO-DMT intoxication are discussed. PMID:20942780

Sodium sulfite pH-buffering effect for improved xylose-phenylalanine conversion to N-(1-deoxy-d-xylulos-1-yl)-phenylalanine during an aqueous Maillard reaction.

PubMed

Cui, Heping; Duhoranimana, Emmanuel; Karangwa, Eric; Jia, Chengsheng; Zhang, Xiaoming

2018-04-25

The yield of the Maillard reaction intermediate (MRI), prepared in aqueous medium, is usually unsatisfactory. However, the addition of sodium sulfite could improve the conversion of xylose-phenylalanine (Xyl-Phe) to the MRI (N-(1-deoxy-d-xylulos-1-yl)-phenylalanine) in aqueous medium. Sodium sulfite (Na 2 SO 3 ) showed a significant pH-buffering effect during the Maillard reaction, which accounted for its facilitation of the N-(1-deoxy-d-xylulos-1-yl)-phenylalanine yield. The results revealed that the pH could be maintained at a relatively high level (above 7.0) for an optimized pH-buffering effect when Na 2 SO 3 (4.0%) was added before the reaction of Xyl-Phe. Thus, the conversion of Xyl-Phe to N-(1-deoxy-d-xylulos-1-yl)-phenylalanine increased from 47.23% to 74.86%. Furthermore, the addition moment of Na 2 SO 3 and corresponding solution pH were crucial factors in regulating the pH-buffering effect of Na 2 SO 3 on N-(1-deoxy-d-xylulos-1-yl)-phenylalanine yield. Based on the pH-buffering effect of Na 2 SO 3 and maintaining the optimal pH 7.4 relatively stable, the conversion of Xyl-Phe to N-(1-deoxy-d-xylulos-1-yl)-phenylalanine was successfully improved. Copyright © 2017 Elsevier Ltd. All rights reserved.

Design and Synthesis of N6-Substituted-4′-thioadenosine-5′-uronamides As Potent and Selective Human A3 Adenosine Receptor Agonists

PubMed Central

Choi, Won Jun; Lee, Hyuk Woo; Kim, Hea Ok; Chinn, Moshe; Gao, Zhan-Guo; Patel, Amit; Jacobson, Kenneth A.; Moon, Hyung Ryong; Jung, Young Hoon; Jeong, Lak Shin

2009-01-01

On the basis of a bioisosteric rationale, 4′-thionucleoside analogues of IB-MECA, which is a potent and selective A3 adenosine receptor agonist (AR), were synthesized from d-gulonic acid γ-lactone. The 4′-thio analogue (5h) of IB-MECA showed extremely high binding affinity (Ki = 0.25 nM) at the human A3AR and was more potent than IB-MECA (Ki = 1.4 nM). Bulky substituents at the 5′-uronamide position, such as cyclohexyl and 2- methylbenzyl, in this series of 2-H nucleoside derivatives were tolerated in A3AR binding, although small alkyl analogues were more potent. PMID:19879151

Mechanisms of inverse agonist action at D2 dopamine receptors

PubMed Central

Roberts, David J; Strange, Philip G

2005-01-01

Mechanisms of inverse agonist action at the D2(short) dopamine receptor have been examined. Discrimination of G-protein-coupled and -uncoupled forms of the receptor by inverse agonists was examined in competition ligand-binding studies versus the agonist [3H]NPA at a concentration labelling both G-protein-coupled and -uncoupled receptors. Competition of inverse agonists versus [3H]NPA gave data that were fitted best by a two-binding site model in the absence of GTP but by a one-binding site model in the presence of GTP. Ki values were derived from the competition data for binding of the inverse agonists to G-protein-uncoupled and -coupled receptors. Kcoupled and Kuncoupled were statistically different for the set of compounds tested (ANOVA) but the individual values were different in a post hoc test only for (+)-butaclamol. These observations were supported by simulations of these competition experiments according to the extended ternary complex model. Inverse agonist efficacy of the ligands was assessed from their ability to reduce agonist-independent [35S]GTPγS binding to varying degrees in concentration–response curves. Inverse agonism by (+)-butaclamol and spiperone occurred at higher potency when GDP was added to assays, whereas the potency of (−)-sulpiride was unaffected. These data show that some inverse agonists ((+)-butaclamol, spiperone) achieve inverse agonism by stabilising the uncoupled form of the receptor at the expense of the coupled form. For other compounds tested, we were unable to define the mechanism. PMID:15735658

The mGlu(2/3) agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate, is anti- and proconvulsant in DBA/2 mice.

PubMed

Moldrich, R X; Talebi, A; Beart, P M; Chapman, A G; Meldrum, B S

2001-02-16

The anticonvulsant activity of the selective group II metabotropic glutamate receptor (mGlu) agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC) has been evaluated in chemoconvulsant and sound-induced models of epileptic seizures in DBA/2 mice. 2R,4R-APDC (> or =10 nmol, intracerebroventricularly (i.c.v.), -15 min) transiently reduced sound-induced seizure activity including clonic seizures to 40% of vehicle at 20 nmol (i.c.v.) and 30% of vehicle at 100 mg/kg (intraperitoneally (i.p.), -15 min). 2R,4R-APDC inhibited clonic seizures induced by the group III mGlu antagonist (R,S)-alpha-methylserine-O-phosphate (2.5 micromol, i.c.v.) when co-injected at 20-40 nmol and inhibited limbic seizure activity induced by the mGlu(1/5) agonist (R,S)-3,5-dihydroxyphenylglycine (1.5 micromol, i.c.v.) when co-injected at 10-40 nmol. A reversal of the anticonvulsant activity of 2R,4R-APDC was observed at (>20 nmol) in each of the chemoconvulsant and sound-induced models of epileptic seizures. 2R,4R-APDC (0.1-1 micromol, i.c.v.) induced stimulus-independent, rapid and dose-dependent clonic seizures. Selective mGlu(2/3) agonists represent a novel class of potential anti-epileptic drugs, however due to the proconvulsant activity observed here, 2R,4R-APDC is obviously limited in this regard.

Hypothalamic neuropeptide Y (NPY) and the attenuation of hyperphagia in streptozotocin diabetic rats treated with dopamine D1/D2 agonists

PubMed Central

Kuo, Dong-Yih

2006-01-01

Dopamine is an appetite suppressant, while neuropeptide Y (NPY), an appetite stimulant in the brain, is reported to be involved in anorectic action induced by a combined administration of D1/D2 agonists in normal rats. In diabetic rats, however, these factors have not been studied. Rats (including normal, diabetic and insulin-treated diabetic rats) were given daily injections of saline or D1/D2 agonists for 6 days. Changes in food intake and hypothalamic NPY content of these rats were assessed and compared. The D1/D2 agonist-induced anorectic responses were altered in diabetic rats compared to normal rats treated similarly. Both the anorectic response on the first day of dosing and the tolerant response on the subsequent days were attenuated. This alteration was independent of the neuroendocrine disturbance on feeding behavior since the basic pattern of food intake during the time course of a 24-h day/night cycle was similar in normal and diabetic rats; the decrease of food intake following drug treatment was only shown at the initial interval of 0–6 h in both groups of rats. However, this alteration coincided with changes in NPY content following D1/D2 coadministration. The replacement of insulin in diabetic rats could normalize both NPY content and D1/D2 agonist-induced anorexia. It is demonstrated that the response of D1/D2 agonist-induced appetite suppression is attenuated in diabetic rats compared to normal rats and that elevated hypothalamic NPY content may contribute to this alteration. PMID:16702993

Borel Summability of Perturbative Series in 4D N=2 and 5D N=1 Supersymmetric Theories.

PubMed

Honda, Masazumi

2016-05-27

We study weak coupling perturbative series in 4D N=2 and 5D N=1 supersymmetric gauge theories with Lagrangians. We prove that the perturbative series of these theories in the zero-instanton sector are Borel summable for various observables. Our result for the 4D N=2 case supports an expectation from a recent proposal on a semiclassical realization of infrared renormalons in QCD-like theories, where the semiclassical solution does not exist in N=2 theories and the perturbative series are unambiguous, namely, Borel summable. We also prove that the perturbative series in an arbitrary number of instanton sectors are Borel summable for a wide class of theories. It turns out that exact results can be obtained by summing over the Borel resummations with every instanton number.

Competitive antagonists discriminate between NK2 tachykinin receptor subtypes.

PubMed Central

Maggi, C. A.; Patacchini, R.; Giuliani, S.; Rovero, P.; Dion, S.; Regoli, D.; Giachetti, A.; Meli, A.

1990-01-01

1. We have compared the ability of various tachykinins and selective tachykinin receptor agonists to induce contraction of the endothelium-denuded rabbit pulmonary artery (RPA) and hamster trachea (HT) and have estimated the affinity of some newly developed NK2 selective antagonists in the same tissues. 2. In confirmation of previous findings, experiments with the agonists indicated that NK2 receptors are the main if not the sole mediators of the response to tachykinins in both RPA and HT. No evidence for significant degradation of neurokinin A (NKA) was found in either tissue when experiments were repeated in the presence of a mixture of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 3. The peptide antagonists tested were: Peptide I = [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10); Peptide II = [Tyr5, D-Trp6,8,9, Arg10]-NKA(3-10); Peptide III = Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2. The three peptides produced a concentration-dependent rightward shift of the concentration-response curve to NKA in both RPA and HT with no significant depression of the maximal response attainable. The slopes of the Schild plots were not significantly different from unity, indicating a competitive antagonism. Peptides I and II were about 100 times more potent in the RPA than in the HT, while Peptide III was about 100 times more potent in the HT than RPA. 4. The pA2 values obtained in these two tissues with the three antagonists were not significantly different when tested in the absence or presence of peptidase inhibitors, or when a selective NK2 receptor agonist, [beta Ala8]-NKA(4-10) was used instead of NKA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2167737

Substance P-induced respiratory excitation is blunted by delta-receptor specific opioids in the rat medulla oblongata.

PubMed

Chen, Z; Hedner, J; Hedner, T

1996-06-01

The effects of substance P (SP) and the naturally occurring met-enkephalin and the synthetic mu-specific opioid agonist, DAGO (Tyr-D-Ala-Gly-N-Methy-Phe-Gly-ol) and the delta-specific opioid agonist DADL (Tyr-D-Ala-Gly-Phe-D-Leu) on basal ventilation were investigated in halothane-anaesthetized rats. Local injections of SP (0.75-1.5 nmol) in the ventrolateral medulla oblongata (VLM), e.g. nucleus paragigantocellularis, and nucleus reticularis lateralis increased ventilation because of an elevation of tidal volume. Met-enkephalin induced a short-lasting ventilatory depression mainly because of a depression of tidal volume. Activation of delta- and mu-opioid receptors in the VLM by local application of DADL and DAGO, respectively, induced ventilatory depression, which was later in onset and more long-lasting. Local administration of met-enkephalin into the VLM also produced a long-lasting inhibition of the SP-induced ventilatory excitation. A similar blockade of the SP-induced excitatory ventilatory response could be elicited by DADL but not by DAGO. This antagonistic effect was attenuated by local application of the delta-opioid receptor antagonist ICI 154. 129. We conclude that the naturally occurring met-enkephalin as well as synthetic mu- and delta-specific enkephalin analogues (DAGO and DADL, respectively) in VLM depress basal ventilation by an effect on inspiratory drive. There is a functional antagonism between activation of delta-opioid receptors and SP receptors into the VLM in respect to respiratory regulation.

Characterization of the tachykinin NK2 receptor in the human bronchus: influence of amastatin-sensitive metabolic pathways.

PubMed Central

Astolfi, M.; Treggiari, S.; Giachetti, A.; Meini, S.; Maggi, C. A.; Manzini, S.

1994-01-01

1. The aim of this study was to characterize the tachykinin NK2 receptor subtype mediating the spasmogenic response in the human isolated bronchus. The motor response to neurokinin A (NKA) and the selective NK2 agonist [beta Ala8]NKA(4-10), as well as the antagonistic effects of cyclic (L659,877) and linear (MEN 10376) peptide NK2 antagonists were assessed in the presence or absence of amastatin (an inhibitor of aminopeptidases A and M). 2. NKA was more potent than [beta Ala8]NKA(4-10) in eliciting bronchoconstriction (pD2 being 7,43 and 6,87 respectively). In the presence of amastatin (1 microM), the estimated affinity of [beta Ala8]NKA(4-10), but not that of NKA, was significantly increased to yield a pD2 of 7,44. 3. L659,877 and MEN 10376 inhibited [beta Ala8]NKA(4-10)-induced contraction with similar affinities; pA2 values were 5.7 +/- 0.22 and 6.3 +/- 0.32, respectively. Amastatin (1 microM) increased the potency of MEN 10376 to 7.28 +/- 0.46, whereas that of L659,877 was unaffected. 4. In the presence of amastatin the pseudopeptide MDL 28,564 behaved as a partial agonist. 5. We conclude that the NK2 receptor subtype present in the human bronchus has properties similar to those described for the circular muscle of the human colon and thus may be classified as a 'NK2A' subtype. We show that the apparent potency of peptides, bearing N-terminal acidic residues, is influenced by an amastatin-sensitive peptidase, possibly aminopeptidase A. PMID:8004400

Characterization of the tachykinin NK2 receptor in the human bronchus: influence of amastatin-sensitive metabolic pathways.

PubMed

Astolfi, M; Treggiari, S; Giachetti, A; Meini, S; Maggi, C A; Manzini, S

1994-02-01

1. The aim of this study was to characterize the tachykinin NK2 receptor subtype mediating the spasmogenic response in the human isolated bronchus. The motor response to neurokinin A (NKA) and the selective NK2 agonist [beta Ala8]NKA(4-10), as well as the antagonistic effects of cyclic (L659,877) and linear (MEN 10376) peptide NK2 antagonists were assessed in the presence or absence of amastatin (an inhibitor of aminopeptidases A and M). 2. NKA was more potent than [beta Ala8]NKA(4-10) in eliciting bronchoconstriction (pD2 being 7,43 and 6,87 respectively). In the presence of amastatin (1 microM), the estimated affinity of [beta Ala8]NKA(4-10), but not that of NKA, was significantly increased to yield a pD2 of 7,44. 3. L659,877 and MEN 10376 inhibited [beta Ala8]NKA(4-10)-induced contraction with similar affinities; pA2 values were 5.7 +/- 0.22 and 6.3 +/- 0.32, respectively. Amastatin (1 microM) increased the potency of MEN 10376 to 7.28 +/- 0.46, whereas that of L659,877 was unaffected. 4. In the presence of amastatin the pseudopeptide MDL 28,564 behaved as a partial agonist. 5. We conclude that the NK2 receptor subtype present in the human bronchus has properties similar to those described for the circular muscle of the human colon and thus may be classified as a 'NK2A' subtype. We show that the apparent potency of peptides, bearing N-terminal acidic residues, is influenced by an amastatin-sensitive peptidase, possibly aminopeptidase A.

Wheel running reduces high-fat diet intake, preference and mu-opioid agonist stimulated intake

PubMed Central

Liang, Nu-Chu; Bello, Nicholas T.; Moran, Timothy H.