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Sample records for high-affinity selective antagonist

  1. Radiolabeled high affinity peptidomimetic antagonist selectively targets {alpha}{sub v}{beta}{sub 3} receptor-positive tumor in mice

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    Jang, Beom-Su [Department of Nuclear Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Lim, Esther [Department of Radiology, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Hee Park, Seung [Department of Nuclear Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Shin, In Soo [Department of Nuclear Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Danthi, S. Narasimhan [Department of Radiology, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Hwang, In Sook [Department of Nuclear Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Le, Nhat [Department of Nuclear Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Yu, Sarah [Department of Nuclear Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Xie Jianwu [Department of Radiology, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Li, King C.P. [Department of Radiology, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Carrasquillo, Jorge A. [Department of Nuclear Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Paik, Chang H. [Department of Nuclear Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States)]. E-mail: cpaik@mail.nih.gov

    2007-05-15

    Objectives: The aim of this research was to synthesize radiolabeled peptidomimetic integrin {alpha}{sub v}{beta}{sub 3} antagonists that selectively target integrin {alpha}{sub v}{beta}{sub 3} receptor and clear rapidly from the whole body. Methods: Integrin {alpha}{sub v}{beta}{sub 3} antagonists, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino) ethyloxy]benzoyl-2-(S)-aminoethylsulfonyl-amino-{beta}-alanine (IA) and 4-[2-(3,4,5,6-tetrahydro-pyrimidin-2-ylamino)-ethyloxy]benzoyl-2-(S)-[N- (3-amino-neopenta-1-carbamyl)]-aminoethylsulfonylamino-{beta}-alanine hydrochloride (IAC), a hydrophobic carbamate derivative of IA, were conjugated with 2-p-isothiocyanatobenzyl-DOTA at the amino terminus and labeled with {sup 111}In. The {sup 111}In labeled IA and IAC were subjected to in vitro receptor binding, biodistribution and imaging studies using nude mice bearing the receptor-positive M21 human melanoma xenografts. Results: The {sup 111}In-labeled IA (40%) and -IAC (72%) specifically bound in vitro to {alpha}{sub v}{beta}{sub 3} (0.8 {mu}M) at a molar excess. This receptor binding was completely blocked by a molar excess of cold IA to {alpha}{sub v}{beta}{sub 3}. The higher receptor-binding affinity of the {sup 111}In-labeled IAC was reflected in higher tumor uptake and retention: 5.6{+-}1.4 and 4.5{+-}0.7 %ID/g vs. 3.8{+-}0.9 and 2.0{+-}0.3 %ID/g for the {sup 111}In-labeled IA at 0.33 and 2 h. The tumor uptakes were inhibited by the co-injection of 200 {mu}g of IA, indicating that the uptake was receptor mediated. These antagonists were excreted primarily via the renal system. The {sup 111}In activity retained in the whole body was quite comparable between the {sup 111}In-labeled IA (24% ID) and the {sup 111}In-labeled IAC (33% ID) at 2 h. The higher peak tumor uptake and longer retention resulted in higher tumor-to-background ratios for the {sup 111}In-labeled IAC at 2 h with 9.7, 2.3, 0.8, 1.9, 7.1, 2.2, 0.9, 3.7 and 9.9 for blood, liver, kidney, lung, heart, stomach

  2. N-Oxide analogs of WAY-100635 : new high affinity 5-HT (1A) receptor antagonists

    NARCIS (Netherlands)

    Oberwinkler - Marchais, Sandrine; Nowicki, B; Pike, VW; Halldin, C; Sandell, J; Chou, YH; Gulyas, B; Brennum, LT; Farde, L; Wikstrom, H V

    2005-01-01

    WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-pyridinyl)cyclohexanecarboxamide] 1 and its O-des-methyl derivative DWAY 2 are well-known high affinity 5-HT1A receptor antagonists. which when labeled with carbon-II (beta(+): t(1/2) 20.4min) in the carbonyl group are effective

  3. Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks

    Science.gov (United States)

    Schäffer, Lauge; Brissette, Renee E.; Spetzler, Jane C.; Pillutla, Renuka C.; Østergaard, Søren; Lennick, Michael; Brandt, Jakob; Fletcher, Paul W.; Danielsen, Gillian M.; Hsiao, Ku-Chuan; Andersen, Asser S.; Dedova, Olga; Ribel, Ulla; Hoeg-Jensen, Thomas; Hertz Hansen, Per; Blume, Arthur J.; Markussen, Jan; Goldstein, Neil I.

    2003-04-01

    Insulin is thought to elicit its effects by crosslinking the two extracellular -subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases.

  4. Effects of high affinity leptin antagonist on prolactin receptor deficient male mouse.

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    Nadège Carré

    Full Text Available Hyperprolactinemia occurs during gestation and lactation with marked hyperphagia associated with leptin resistance. Prolactin (PRL induces the expression of orexigenic neuropeptide Y (NPY in hypothalamic dorsomedial nucleus (DMH leading to hyperphagia. Along this line prolactin receptor deficient (PRLR-/- mice are resistant to obesity under high fat diet due to increased energy expenditure. As these mice have an altered food intake, our objective was to test whether leptin is responsible for these characteristics. PRLR-/- male mice and control littermates were injected subcutaneously every other day with 12 mg/kg pegylated superactive mouse leptin antagonist (PEG-SMLA for 3 weeks. We tested the effect of PEG-SMLA on body weight, food intake and metabolic parameters. The antagonist led to a rapid increase in body weight (20% but increased adipose mass in PEG-SMLA treated mice was less pronounced in PRLR-/- than in WT mice. Food intake of PEG-SMLA-injected animals increased during the first week period of the experiment but then declined to a similar level of the control animals during the second week. Interestingly, PRLR-/- mice were found to have the same bone volume than those of control mice although PEG-SMLA increased bone mass by 7% in both strains. In addition, PEG-SMLA led to insulin resistance and glucose intolerance as well as an altered lipid profile in treated mice. Altogether, these results suggest that PRLR-/- mice respond to leptin antagonist similarly to the control mice, indicating no interaction between the actions of the two hormones.

  5. Selective high-affinity polydentate ligands and methods of making such

    Energy Technology Data Exchange (ETDEWEB)

    Denardo, Sally J.; Denardo, Gerald L.; Balhorn, Rodney L.

    2018-02-06

    This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  6. Enhanced selection of high affinity DNA-reactive B cells following cyclophosphamide treatment in mice.

    Directory of Open Access Journals (Sweden)

    Daisuke Kawabata

    Full Text Available A major goal for the treatment of patients with systemic lupus erythematosus with cytotoxic therapies is the induction of long-term remission. There is, however, a paucity of information concerning the effects of these therapies on the reconstituting B cell repertoire. Since there is recent evidence suggesting that B cell lymphopenia might attenuate negative selection of autoreactive B cells, we elected to investigate the effects of cyclophosphamide on the selection of the re-emerging B cell repertoire in wild type mice and transgenic mice that express the H chain of an anti-DNA antibody. The reconstituting B cell repertoire in wild type mice contained an increased frequency of DNA-reactive B cells; in heavy chain transgenic mice, the reconstituting repertoire was characterized by an increased frequency of mature, high affinity DNA-reactive B cells and the mice expressed increased levels of serum anti-DNA antibodies. This coincided with a significant increase in serum levels of BAFF. Treatment of transgene-expressing mice with a BAFF blocking agent or with DNase to reduce exposure to autoantigen limited the expansion of high affinity DNA-reactive B cells during B cell reconstitution. These studies suggest that during B cell reconstitution, not only is negative selection of high affinity DNA-reactive B cells impaired by increased BAFF, but also that B cells escaping negative selection are positively selected by autoantigen. There are significant implications for therapy.

  7. Synthesis and biological evaluation of disubstituted N6- cyclopentyladenine analogues: The search for a neutral antagonist with high affinity for the adenosine A1 receptor

    NARCIS (Netherlands)

    Ligt, R.A.F. de; Klein, P.A.M. van der; Frijtag Drabbe Künzel, J.K. von; Lorenzen, A.; El Maate, F.A.; Fujikawa, S.; Westhoven, R. van; Hoven, T. van den; Brussee, J.; Ijzerman, A.P.

    2004-01-01

    Novel 3,8- and 8,9-disubstituted N6-cyclopentyladenine derivatives were synthesised in moderate overall yield from 6-chloropurine. The derivatives were made in an attempt to find a new neutral antagonist with high affinity for adenosine A1 receptors. N6-Cyclopentyl-9- methyladenine (N-0840) was used

  8. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

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    Wang, Deng-Liang [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Yang, Hai-Tao; Wang, Jiang-Jie [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yao, Pei-Sen [Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Pan, Ru-Jun [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yang, Chaoyong James, E-mail: cyyang@xmu.edu.cn [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Kang, De-Zhi, E-mail: kdzy99988@163.com [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  9. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    Science.gov (United States)

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  10. CC12, a high-affinity ligand for [3H]cimetidine binding, is an improgan antagonist

    NARCIS (Netherlands)

    Hough, L.H.; Nalwalk, J.B.; Phillips, J.R.; Kern, B.; Shan, Z.; Wentland, M.; de Esch, I.J.P.; Janssen, EA; Barr, T.; Stadel, R.

    2007-01-01

    Improgan, a chemical congener of cimetidine, is a highly effective non-opioid analgesic when injected into the CNS. Despite extensive characterization, neither the improgan receptor, nor a pharmacological antagonist of improgan has been previously described. Presently, the specific binding of [

  11. High-affinity aptamers selectively inhibit human nonpancreatic secretory phospholipase A2 (hnps-PLA2).

    Science.gov (United States)

    Bridonneau, P; Chang, Y F; O'Connell, D; Gill, S C; Snyder, D W; Johnson, L; Goodson, T; Herron, D K; Parma, D H

    1998-03-12

    A family of sequence-related 2'-aminopyrimidine, 2'-hydroxylpurine aptamers, developed by oligonucleotide-based combinatorial chemistry, SELEX (systematic evolution of ligand by exponential enrichment) technology, binds human nonpancreatic secretory phospholipase A2 (hnps-PLA2) with nanomolar affinities and inhibits enzymatic activity. Aptamer 15, derived from the family, binds hnps-PLA2 with a Kd equal to 1.7 +/- 0.2 nM and, in a standard chromogenic assay of enzymatic activity, inhibits hnps-PLA2 with an IC50 of 4 nM, at a mole fraction of substrate concentration of 4 x 10(-6) and a calculated Ki of 0.14 nM. Aptamer 15 is selective for hnps-PLA2, having a 25- and 2500-fold lower affinity, respectively, for the unrelated proteins human neutrophil elastase and human IgG. Contractions of guinea pig lung pleural strips induced by hnps-PLA2 are abolished by 0.3 microM aptamer 15, whereas contractions induced by arachidonic acid are not altered. The structure that is essential for binding and inhibition appears to be a 40-base hairpin/loop motif with an asymmetrical internal loop. The affinity and activity of the aptamers demonstrate the ability of the SELEX process to isolate antagonists of nonnucleic-acid-binding proteins from vast oligonucleotide combinatorial libraries.

  12. High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

    Science.gov (United States)

    Orito, N.; Umekage, S.; Sato, K.; Kawauchi, S.; Tanaka, H.; Sakai, E.; Tanaka, T.; Kikuchi, Y.

    2012-03-01

    We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be KD = 2.25×10-9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

  13. Identification of a novel snake peptide toxin displaying high affinity and antagonist behaviour for the α2-adrenoceptors.

    Science.gov (United States)

    Rouget, Céline; Quinton, Loïc; Maïga, Arhamatoulaye; Gales, Céline; Masuyer, Geoffrey; Malosse, Christian; Chamot-Rooke, Julia; Thai, Robert; Mourier, Gilles; De Pauw, Edwin; Gilles, Nicolas; Servent, Denis

    2010-11-01

    BACKGROUND AND PURPOSE Muscarinic and adrenergic G protein-coupled receptors (GPCRs) are the targets of rare peptide toxins isolated from snake or cone snail venoms. We used a screen to identify novel toxins from Dendroaspis angusticeps targeting aminergic GPCRs. These toxins may offer new candidates for the development of new tools and drugs. EXPERIMENTAL APPROACH In binding experiments with (3) H-rauwolscine, we studied the interactions of green mamba venom fractions with α(2) -adrenoceptors from rat brain synaptosomes. We isolated, sequenced and chemically synthesized a novel peptide, ρ-Da1b. This peptide was pharmacologically characterized using binding experiments and functional tests on human α(2)-adrenoceptors expressed in mammalian cells. KEY RESULTS ρ-Da1b, a 66-amino acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. Its synthetic homologue inhibited 80% of (3) H-rauwolscine binding to the three α(2)-adrenoceptor subtypes, with an affinity between 14 and 73 nM and Hill slopes close to unity. Functional experiments on α(2A) -adrenoceptor demonstrated that ρ-Da1b is an antagonist, shifting adrenaline activation curves to the right. Schild regression revealed slopes of 0.97 and 0.67 and pA(2) values of 5.93 and 5.32 for yohimbine and ρ-Da1b, respectively. CONCLUSIONS AND IMPLICATIONS ρ-Da1b is the first toxin identified to specifically interact with α(2)-adrenoceptors, extending the list of class A GPCRs sensitive to toxins. Additionally, its affinity and atypical mode of interaction open up the possibility of its use as a new pharmacological tool, in the study of the physiological roles of α(2)-adrenoceptor subtypes. British Journal of Pharmacology © 2010 The British Pharmacological Society. No claim to original French government works.

  14. Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody.

    Science.gov (United States)

    Lord, Dana M; Bird, Julie J; Honey, Denise M; Best, Annie; Park, Anna; Wei, Ronnie R; Qiu, Huawei

    2018-01-15

    Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies.

  15. Selective Distribution of a High-Affinity Plasminogen-Binding Site among Group A Streptococci Associated with Impetigo

    Science.gov (United States)

    Svensson, Mikael D.; Sjöbring, Ulf; Bessen, Debra E.

    1999-01-01

    Group A streptococci can be classified according to their tendency to cause either impetigo, pharyngitis, or both types of infection. Genotypic markers for tissue site preference lie within emm genes, which encode fibrillar surface proteins that play a key role in virulence. emm gene products (M and M-like proteins) display an extensive array of binding activities for tissue and plasma proteins of the human host. In a previous study, a high-affinity binding site for human plasmin(ogen) was mapped to the emm53 gene product. In this report, a structurally similar plasminogen-binding domain is found to be widely and selectively distributed among group A streptococci harboring the emm gene marker for the skin as the preferred tissue site for infection. The findings are highly suggestive of a central role for bacterial modulation of host plasmin(ogen) during localized infection at the epidermis. PMID:10417156

  16. Pharmacokinetics, pharmacodynamics and safety of CEP-26401, a high-affinity histamine-3 receptor antagonist, following single and multiple dosing in healthy subjects.

    Science.gov (United States)

    Spiegelstein, Ofer; Stevens, Jasper; Van Gerven, Joop; Nathan, Pradeep J; Maynard, James P; Mayleben, David W; Hellriegel, Edward; Yang, Ronghua

    2016-10-01

    CEP-26401 is a novel orally active, brain-penetrant, high-affinity histamine H3 receptor (H3R) antagonist, with potential therapeutic utility in cognition enhancement. Two randomized, double-blind, placebo-controlled dose escalation studies with single (0.02 to 5 mg) or multiple administration (0.02 to 0.5 mg once daily) of CEP-26401 were conducted in healthy subjects. Plasma and urine samples were collected to investigate CEP-26401 pharmacokinetics. Pharmacodynamic endpoints included a subset of tasks from the Cambridge Neuropsychological Test Automated Battery (CANTAB) and nocturnal polysomnography. Population pharmacokinetic-pharmacodynamic modeling was conducted on one CANTAB and one polysomnography parameter of interest. CEP-26401 was slowly absorbed (median tmax range 3-6 hours) and the mean terminal elimination half-life ranged from 24-60 hours. Steady-state plasma concentrations were achieved within six days of dosing. CEP-26401 exhibits dose- and time-independent pharmacokinetics, and renal excretion is a major elimination pathway. CEP-26401 had a dose-dependent negative effect on sleep, with some positive effects on certain CANTAB cognitive parameters seen at lower concentrations. The derived three compartment population pharmacokinetic model, with first-order absorption and elimination, accurately described the available pharmacokinetic data. CEP-26401 was generally well tolerated up to 0.5 mg/day with most common treatment related adverse events being headache and insomnia. Further clinical studies are required to establish the potential of low-dose CEP-26401 in cognition enhancement. © The Author(s) 2016.

  17. Structure-Guided Design of a Series of MCL-1 Inhibitors with High Affinity and Selectivity

    Energy Technology Data Exchange (ETDEWEB)

    Bruncko, Milan; Wang, Le; Sheppard, George S.; Phillips, Darren C.; Tahir, Stephen K.; Xue, John; Erickson, Scott; Fidanze, Steve; Fry, Elizabeth; Hasvold, Lisa; Jenkins, Gary J.; Jin, Sha; Judge, Russell A.; Kovar, Peter J.; Madar, David; Nimmer, Paul; Park, Chang; Petros, Andrew M.; Rosenberg, Saul H.; Smith, Morey L.; Song, Xiaohong; Sun, Chaohong; Tao, Zhi-Fu; Wang, Xilu; Xiao, Yu; Zhang, Haichao; Tse, Chris; Leverson, Joel D.; Elmore, Steven W.; Souers, Andrew J.

    2015-03-12

    Myeloid cell leukemia 1 (MCL-1) is a BCL-2 family protein that has been implicated in the progression and survival of multiple tumor types. Herein we report a series of MCL-1 inhibitors that emanated from a high throughput screening (HTS) hit and progressed via iterative cycles of structure-guided design. Advanced compounds from this series exhibited subnanomolar affinity for MCL-1 and excellent selectivity over other BCL-2 family proteins as well as multiple kinases and GPCRs. In a MCL-1 dependent human tumor cell line, administration of compound 30b rapidly induced caspase activation with associated loss in cell viability. The small molecules described herein thus comprise effective tools for studying MCL-1 biology.

  18. Human GIP(3-30)NH inhibits G protein-dependent as well as G protein-independent signaling and is selective for the GIP receptor with high-affinity binding to primate but not rodent GIP receptors

    DEFF Research Database (Denmark)

    Gabe, Maria Buur Nordskov; Sparre-Ulrich, Alexander Hovard; Pedersen, Mie Fabricius

    2018-01-01

    GIP(3-30)NH2is a high affinity antagonist of the GIP receptor (GIPR) in humans inhibiting insulin secretion via G protein-dependent pathways. However, its ability to inhibit G protein-independent signaling is unknown. Here we determine its action on arrestin-recruitment and receptor internalization...... using human125I-GIP(3-30)NH2. The selectivity of human GIP(3-30)NH2was examined by testing for agonistic and antagonistic properties on 62 human GPCRs. Human GIP(3-30)NH2inhibited GIP(1-42)-induced cAMP and β-arrestin 1 and 2 recruitment on the human GIPR and Schild plot analysis showed competitive...

  19. Novel high-affinity and selective biaromatic 4-substituted ¿-hydroxybutyric acid (GHB) analogues as GHB ligands

    DEFF Research Database (Denmark)

    Høg, Signe; Wellendorph, Petrine; Nielsen, Birgitte

    2008-01-01

    Gamma-hydroxybutyrate (GHB) is a metabolite of gamma-aminobutyric acid (GABA) and has been proposed to function as a neurotransmitter or neuromodulator. GHB is used in the treatment of narcolepsy and is a drug of abuse. GHB binds to both GABA(B) receptors and specific high-affinity GHB sites...

  20. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-Affinity γ-Hydroxybutyrate (GHB) Binding Sites

    DEFF Research Database (Denmark)

    Vogensen, Stine B.; Marek, Ales; Bay, Tina

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screen...

  1. "Velcro" engineering of high affinity CD47 ectodomain as signal regulatory protein α (SIRPα) antagonists that enhance antibody-dependent cellular phagocytosis.

    Science.gov (United States)

    Ho, Chia Chi M; Guo, Nan; Sockolosky, Jonathan T; Ring, Aaron M; Weiskopf, Kipp; Özkan, Engin; Mori, Yasuo; Weissman, Irving L; Garcia, K Christopher

    2015-05-15

    CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy. © 2015 by

  2. “Velcro” Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α (SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis*

    Science.gov (United States)

    Ho, Chia Chi M.; Guo, Nan; Sockolosky, Jonathan T.; Ring, Aaron M.; Weiskopf, Kipp; Özkan, Engin; Mori, Yasuo; Weissman, Irving L.; Garcia, K. Christopher

    2015-01-01

    CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the “don't-eat-me” signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined “Velcro” engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that “Velcro” engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy

  3. Site-directed selection of oligonucleotide antagonists by competitive elution.

    Science.gov (United States)

    Bridonneau, P; Chang, Y F; Buvoli, A V; O'Connell, D; Parma, D

    1999-02-01

    Oligonucleotide ligands that bind a protein or a small molecule of interest are readily isolated by in vitro selection and amplification of rare sequences from combinatorial libraries of sequence-randomized oligonucleotides (Gold et al., 1995). Classic systematic evolution of ligands by exponential enrichment (SELEX) protocols are affinity based (Tuerk and Gold, 1990), but because many problems and applications require antagonists, protocols for selecting inhibitors are both desirable and valuable. A widely applicable approach for isolating inhibitors is competitive elution with a molecule that binds the targeted molecule's active or binding site. We have used this approach to isolate antagonists of wheat germ agglutinin (WGA) from a library of 2'NH2-pyrimidine, 2'OH-purine oligonucleotides by elution with N N' N"-triacetylchitotriose, (GlcNAc)3. The highest affinity aptamers have equilibrium dissociation constants of 1 nM-20 nM for WGA, a 10(3)-10(4)-fold improvement relative to (GlcNAc)3, and unlike the carbohydrate, are highly specific. In addition to competing for binding with (GlcNAc)3, aptamers inhibit WGA-mediated agglutination of sheep erythrocytes, demonstrating that they are able to compete with natural ligands presented on the surfaces of cells. These results illustrate the feasibility of isolating high-affinity, high-specificity antagonists by competitive elution with low molecular weight, relatively low-affinity, and low-specificity small molecules.

  4. 20-125Iodo-14,15-epoxyeicosa-5(Z)-enoic acid: a high-affinity radioligand used to characterize the epoxyeicosatrienoic acid antagonist binding site.

    Science.gov (United States)

    Chen, Yuenmu; Falck, John R; Tuniki, Venugopal R; Campbell, William B

    2009-12-01

    Epoxyeicosatrienoic acids (EETs) are endothelium-derived metabolites of arachidonic acid. They relax vascular smooth muscle by membrane hyperpolarization. These actions are inhibited by the EET antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EE5ZE). We synthesized 20-(125)iodo-14,15-EE5ZE (20-(125)I-14,15-EE5ZE), a radiolabeled EET antagonist, and characterized its binding to cell membranes. 14,15-EET (10(-9)-10(-5)M) caused a concentration-related relaxation of the preconstricted bovine coronary artery and phosphorylation of p38 in U937 cells that were inhibited by 20-(125)I-14,15-EE5ZE. Specific 20-(125)I-14,15-EE5ZE binding to U937 cell membranes reached equilibrium within 5 min and remained unchanged for 30 min. The binding was saturable and reversible, and it exhibited K(D) and B(max) values of 1.11 +/- 0.13 nM and 1.13 +/- 0.04 pmol/mg protein, respectively. Guanosine 5'-O-(3-thio)triphosphate (10 muM) did not change the binding, indicating antagonist binding of the ligand. Various EETs and EET analogs (10(-10)-10(-5)M) competed for 20-(125)I-14,15-EE5ZE binding with an order of potency of 11,12-EET = 14,15-EET > 8,9-EET = 14,15-EE5ZE > 15-hydroxyeicosatetraenoic acid = 14,15-dihydroxyeicosatrienoic acid. 8,9-Dihydroxyeicosatrienoic acid and 11-hydroxyeicosatetraenoic acid did not compete for binding. The soluble and microsomal epoxide hydrolase inhibitors (1-cyclohexyl-3-dodecyl-urea, elaidamide, and 12-hydroxyl-elaidamide) and cytochrome P450 inhibitors (sulfaphenazole and proadifen) did not compete for the binding. However, two cytochrome P450 inhibitors, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) and miconazole competed for binding with K(i) of 1558 and 315 nM, respectively. Miconazole and MS-PPOH, but not proadifen, inhibited 14,15-EET-induced relaxations. These findings define an EET antagonist's binding site and support the presence of an EET receptor. The inhibition of binding by some cytochrome P450 inhibitors suggests an

  5. In vitro and in vivo evaluation of a (18F-labeled high affinity NOTA conjugated bombesin antagonist as a PET ligand for GRPR-targeted tumor imaging.

    Directory of Open Access Journals (Sweden)

    Zohreh Varasteh

    Full Text Available Expression of the gastrin-releasing peptide receptor (GRPR in prostate cancer suggests that this receptor can be used as a potential molecular target to visualize and treat these tumors. We have previously investigated an antagonist analog of bombesin (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2, RM26 conjugated to 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA via a diethylene glycol (PEG2 spacer (NOTA-P2-RM26 labeled with (68Ga and (111In. We found that this conjugate has favorable properties for in vivo imaging of GRPR-expression. The focus of this study was to develop a (18F-labelled PET agent to visualize GRPR. NOTA-P2-RM26 was labeled with (18F using aluminum-fluoride chelation. Stability, in vitro binding specificity and cellular processing tests were performed. The inhibition efficiency (IC50 of the [(natF]AlF-NOTA-P2-RM26 was compared to that of the (natGa-loaded peptide using (125I-Tyr(4-BBN as the displacement radioligand. The pharmacokinetics and in vivo binding specificity of the compound were studied. NOTA-P2-RM26 was labeled with (18F within 1 h (60-65% decay corrected radiochemical yield, 55 GBq/µmol. The radiopeptide was stable in murine serum and showed high specific binding to PC-3 cells. [(natF]AlF-NOTA-P2-RM26 showed a low nanomolar inhibition efficiency (IC50=4.4±0.8 nM. The internalization rate of the tracer was low. Less than 14% of the cell-bound radioactivity was internalized after 4 h. The biodistribution of [(18F]AlF-NOTA-P2-RM26 demonstrated rapid blood clearance, low liver uptake and low kidney retention. The tumor uptake at 3 h p.i. was 5.5±0.7 %ID/g, and the tumor-to-blood, -muscle and -bone ratios were 87±42, 159±47, 38±16, respectively. The uptake in tumors, pancreas and other GRPR-expressing organs was significantly reduced when excess amount of non-labeled peptide was co-injected. The low uptake in bone suggests a high in vivo stability of the Al-F bond. High contrast PET image was obtained 3 h p

  6. Blockade of the high-affinity noradrenaline transporter (NET) by the selective 5-HT reuptake inhibitor escitalopram: an in vivo microdialysis study in mice

    Science.gov (United States)

    Nguyen, Hai T; Guiard, Bruno P; Bacq, Alexandre; David, Denis J; David, Indira; Quesseveur, Gaël; Gautron, Sophie; Sanchez, Connie; Gardier, Alain M

    2013-01-01

    BACKGROUND AND PURPOSE Escitalopram, the S(+)-enantiomer of citalopram is the most selective 5-HT reuptake inhibitor approved. Although all 5-HT selective reuptake inhibitors (SSRIs) increase extracellular levels of 5-HT ([5-HT]ext). some also enhance, to a lesser extent, extracellular levels of noradrenaline ([NA]ext). However, the mechanisms by which SSRIs activate noradrenergic transmission in the brain remain to be determined. EXPERIMENTAL APPROACH This study examined the effects of escitalopram, on both [5-HT]ext and [NA]ext in the frontal cortex (FCx) of freely moving wild-type (WT) and mutant mice lacking the 5-HT transporter (SERT−/−) by using intracerebral microdialysis. We explored the possibilities that escitalopram enhances [NA]ext, either by a direct mechanism involving the inhibition of the low- or high-affinity noradrenaline transporters, or by an indirect mechanism promoted by [5-HT]ext elevation. The forced swim test (FST) was used to investigate whether enhancing cortical [5-HT]ext and/or [NA]ext affected the antidepressant-like activity of escitalopram. KEY RESULTS In WT mice, a single systemic administration of escitalopram produced a significant increase in cortical [5-HT]ext and [NA]ext. As expected, escitalopram failed to increase cortical [5-HT]ext in SERT−/− mice, whereas its neurochemical effects on [NA]ext persisted in these mutants. In WT mice subjected to the FST, escitalopram increased swimming parameters without affecting climbing behaviour. Finally, escitalopram, at relevant concentrations, failed to inhibit cortical noradrenaline and 5-HT uptake mediated by low-affinity monoamine transporters. CONCLUSIONS AND IMPLICATIONS These experiments suggest that escitalopram enhances, although moderately, cortical [NA]extin vivo by a direct mechanism involving the inhibition of the high-affinity noradrenaline transporter (NET). PMID:22233336

  7. Species-scanning mutagenesis of the serotonin transporter reveals residues essential in selective, high-affinity recognition of antidepressants

    DEFF Research Database (Denmark)

    Mortensen, O V; Kristensen, A S; Wiborg, O

    2001-01-01

    of the antidepressants citalopram, fluoxetine, paroxetine and imipramine were several-fold higher at hSERT compared with bSERT. No species selectivity was observed for the antidepressants fluvoxamine, and sertraline or for the psychostimulants cocaine, the cocaine analogue beta-carbomethoxy-3beta-(4-iodophenyl......, and phenylalanine-513 and to reveal molecular interactions with individual functional groups of citalopram. We suggest that methionine-180 interacts with the heterocyclic nucleus of citalopram or stabilizes the binding pocket and phenylalanine-513 to be a steric blocker of antidepressant recognition....

  8. Structure of a TCR with High Affinity for Self-antigen Reveals Basis for Escape from Negative Selection

    Energy Technology Data Exchange (ETDEWEB)

    Y Yin; Y Li; M Kerzic; R Martin; R Mariuzza

    2011-12-31

    The failure to eliminate self-reactive T cells during negative selection is a prerequisite for autoimmunity. To escape deletion, autoreactive T-cell receptors (TCRs) may form unstable complexes with self-peptide-MHC by adopting suboptimal binding topologies compared with anti-microbial TCRs. Alternatively, escape can occur by weak binding between self-peptides and MHC. We determined the structure of a human autoimmune TCR (MS2-3C8) bound to a self-peptide from myelin basic protein (MBP) and the multiple sclerosis-associated MHC molecule HLA-DR4. MBP is loosely accommodated in the HLA-DR4-binding groove, accounting for its low affinity. Conversely, MS2-3C8 binds MBP-DR4 as tightly as the most avid anti-microbial TCRs. MS2-3C8 engages self-antigen via a docking mode that resembles the optimal topology of anti-foreign TCRs, but is distinct from that of other autoreactive TCRs. Combined with a unique CDR3 conformation, this docking mode compensates for the weak binding of MBP to HLA-DR4 by maximizing interactions between MS2-3C8 and MBP. Thus, the MS2-3C8-MBP-DR4 complex reveals the basis for an alternative strategy whereby autoreactive T cells escape negative selection, yet retain the ability to initiate autoimmunity.

  9. Synthesis and in vitro evaluation of a selective antagonist and the corresponding radioligand for the prostaglandin D2 receptor CRTH2

    DEFF Research Database (Denmark)

    Ulven, Trond; Gallen, Michael J; Nielsen, Mads C

    2007-01-01

    Synthesis and preliminary in vitro biological evaluation of a selective high-affinity CRTH2 antagonist is described. The stability of an N-benzyl group facilitated synthesis of the corresponding radioligand by tritiation of a brominated precursor. The compound [(3)H]TRQ11238 represents the first ...

  10. Sexually antagonistic selection in human male homosexuality.

    Directory of Open Access Journals (Sweden)

    Andrea Camperio Ciani

    Full Text Available Several lines of evidence indicate the existence of genetic factors influencing male homosexuality and bisexuality. In spite of its relatively low frequency, the stable permanence in all human populations of this apparently detrimental trait constitutes a puzzling 'Darwinian paradox'. Furthermore, several studies have pointed out relevant asymmetries in the distribution of both male homosexuality and of female fecundity in the parental lines of homosexual vs. heterosexual males. A number of hypotheses have attempted to give an evolutionary explanation for the long-standing persistence of this trait, and for its asymmetric distribution in family lines; however a satisfactory understanding of the population genetics of male homosexuality is lacking at present. We perform a systematic mathematical analysis of the propagation and equilibrium of the putative genetic factors for male homosexuality in the population, based on the selection equation for one or two diallelic loci and Bayesian statistics for pedigree investigation. We show that only the two-locus genetic model with at least one locus on the X chromosome, and in which gene expression is sexually antagonistic (increasing female fitness but decreasing male fitness, accounts for all known empirical data. Our results help clarify the basic evolutionary dynamics of male homosexuality, establishing this as a clearly ascertained sexually antagonistic human trait.

  11. Sexually Antagonistic Selection in Human Male Homosexuality

    Science.gov (United States)

    Camperio Ciani, Andrea; Cermelli, Paolo; Zanzotto, Giovanni

    2008-01-01

    Several lines of evidence indicate the existence of genetic factors influencing male homosexuality and bisexuality. In spite of its relatively low frequency, the stable permanence in all human populations of this apparently detrimental trait constitutes a puzzling ‘Darwinian paradox’. Furthermore, several studies have pointed out relevant asymmetries in the distribution of both male homosexuality and of female fecundity in the parental lines of homosexual vs. heterosexual males. A number of hypotheses have attempted to give an evolutionary explanation for the long-standing persistence of this trait, and for its asymmetric distribution in family lines; however a satisfactory understanding of the population genetics of male homosexuality is lacking at present. We perform a systematic mathematical analysis of the propagation and equilibrium of the putative genetic factors for male homosexuality in the population, based on the selection equation for one or two diallelic loci and Bayesian statistics for pedigree investigation. We show that only the two-locus genetic model with at least one locus on the X chromosome, and in which gene expression is sexually antagonistic (increasing female fitness but decreasing male fitness), accounts for all known empirical data. Our results help clarify the basic evolutionary dynamics of male homosexuality, establishing this as a clearly ascertained sexually antagonistic human trait. PMID:18560521

  12. The ketamine analogue methoxetamine and 3- and 4-methoxy analogues of phencyclidine are high affinity and selective ligands for the glutamate NMDA receptor.

    Directory of Open Access Journals (Sweden)

    Bryan L Roth

    Full Text Available In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as 'designer drugs' and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS-2-(ethylamino-2-(3-methoxyphenylcyclohexanone and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenylcyclohexanamine and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenylcyclohexyl]piperidine and 1-[1-(4-methoxyphenylcyclohexyl]piperidine, were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects.

  13. The ketamine analogue methoxetamine and 3- and 4-methoxy analogues of phencyclidine are high affinity and selective ligands for the glutamate NMDA receptor.

    Science.gov (United States)

    Roth, Bryan L; Gibbons, Simon; Arunotayanun, Warunya; Huang, Xi-Ping; Setola, Vincent; Treble, Ric; Iversen, Les

    2013-01-01

    In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as 'designer drugs' and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS)-2-(ethylamino)-2-(3-methoxyphenyl)cyclohexanone) and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenyl)cyclohexanamine) and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenyl)cyclohexyl]piperidine and 1-[1-(4-methoxyphenyl)cyclohexyl]piperidine), were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects.

  14. Identification of a Potent and Selective Cannabinoid CB1 Receptor Antagonist from Auxarthron reticulatum.

    Science.gov (United States)

    Elsebai, Mahmoud Fahmi; Rempel, Viktor; Schnakenburg, Gregor; Kehraus, Stefan; Müller, Christa E; König, Gabriele M

    2011-11-10

    The fungus Auxarthron reticulatum derived from the marine sponge Ircinia variabilis produced the diketopiperazine alkaloid amauromine (1) and the quinolinone methyl-penicinoline (2). Compound 2 is identical to the reported methyl-marinamide, whose structure is herewith revised. In radioligand binding studies at human cannabinoid CB1 and CB2 receptors recombinantly expressed in Chinese hamster ovary (CHO) cells, amauromine (1) was found to exhibit high affinity and selectivity for the CB1 receptor (K i = 178 nM). The compound was shown to be a neutral CB1 antagonist with a K b value of 66.6 nM determined in cAMP assays. Compound 2 exhibited only weak or no effects at CB receptors. To the best of our knowledge, compound 1 is the first fungal natural product that shows affinity for cannabinoid CB1 receptors. Because of its high antagonistic potency and selectivity, it may have potential for use as a drug and/or serve as a lead structure for drug development.

  15. Brivaracetam, a selective high-affinity synaptic vesicle protein 2A (SV2A) ligand with preclinical evidence of high brain permeability and fast onset of action.

    Science.gov (United States)

    Nicolas, Jean-Marie; Hannestad, Jonas; Holden, Daniel; Kervyn, Sophie; Nabulsi, Nabeel; Tytgat, Dominique; Huang, Yiyun; Chanteux, Hugues; Staelens, Ludovicus; Matagne, Alain; Mathy, François-Xavier; Mercier, Joël; Stockis, Armel; Carson, Richard E; Klitgaard, Henrik

    2016-02-01

    Rapid distribution to the brain is a prerequisite for antiepileptic drugs used for treatment of acute seizures. The preclinical studies described here investigated the high-affinity synaptic vesicle glycoprotein 2A (SV2A) antiepileptic drug brivara-cetam (BRV) for its rate of brain penetration and its onset of action. BRV was compared with levetiracetam (LEV). In vitro permeation studies were performed using Caco-2 cells. Plasma and brain levels were measured over time after single oral dosing to audiogenic mice and were correlated with anticonvulsant activity. Tissue distribution was investigated after single dosing to rat (BRV and LEV) and dog (LEV only). Positron emission tomography (PET) displacement studies were performed in rhesus monkeys using the SV2A PET tracer [11C]UCB-J. The time course of PET tracer displacement was measured following single intravenous (IV) dosing with LEV or BRV. Rodent distribution data and physiologically based pharmacokinetic (PBPK) modeling were used to compute blood-brain barrier permeability (permeability surface area product, PS) values and then predict brain kinetics in man. In rodents, BRV consistently showed a faster entry into the brain than LEV; this correlated with a faster onset of action against seizures in audiogenic susceptible mice. The higher permeability of BRV was also demonstrated in human cells in vitro. PBPK modeling predicted that, following IV dosing to human subjects, BRV might distribute to the brain within a few minutes compared with approximately 1 h for LEV (PS of 0.315 and 0.015 ml/min/g for BRV and LEV, respectively). These data were supported by a nonhuman primate PET study showing faster SV2A occupancy by BRV compared with LEV. These preclinical data demonstrate that BRV has rapid brain entry and fast brain SV2A occupancy, consistent with the fast onset of action in the audiogenic seizure mice assay. The potential benefit of BRV for treatment of acute seizures remains to be confirmed in clinical

  16. Structural optimization of an aptamer generated from Ligand-Guided Selection (LIGS) resulted in high affinity variant toward mIgM expressed on Burkitt's lymphoma cell lines.

    Science.gov (United States)

    Zümrüt, Hasan E; Batool, Sana; Van, Nabeela; George, Shanell; Bhandari, Sanam; Mallikaratchy, Prabodhika

    2017-07-01

    Aptamers are synthetic, short nucleic acid molecules capable of specific target recognition. Aptamers are selected using a screening method termed Systematic Evolution of Ligands by Exponential enrichment (SELEX). We recently have introduced a variant of SELEX called "Ligand-Guided-Selection" (LIGS) that allows the identification of specific aptamers against known cell-surface proteins. Utilizing LIGS, we introduced three specific aptamers against membrane-bound IgM (mIgM), which is the hallmark of B cells. Out of the three aptamers selected against mIgM, an aptamer termed R1, in particular, was found to be interesting due to its ability to recognize mIgM on target cells and then block anti-IgM antibodies binding their antigen. We systematically truncated parent aptamer R1 to design shorter variants with enhanced affinity. Importantly, herein we show that the specificity of the most optimized variant of R1 aptamer is similar to that of anti-IgM antibody, indicating that the specificity of the ligand utilized in selective elution of the aptamer determines the specificity of the LIGS-generated aptamer. Furthermore, we report that truncated variants of R1 are able to recognize mIgM-positive human B lymphoma BJAB cells at physiological temperature, demonstrating that LIGS-generated aptamers could be re-optimized into higher affinity variants. Collectively, these findings show the significance of LIGS in generating highly specific aptamers with potential applications in biomedicine. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Selective agonists and antagonists of formylpeptide receptors: duplex flow cytometry and mixture-based positional scanning libraries.

    Science.gov (United States)

    Pinilla, Clemencia; Edwards, Bruce S; Appel, Jon R; Yates-Gibbins, Tina; Giulianotti, Marc A; Medina-Franco, Jose L; Young, Susan M; Santos, Radleigh G; Sklar, Larry A; Houghten, Richard A

    2013-09-01

    The formylpeptide receptor (FPR1) and formylpeptide-like 1 receptor (FPR2) are G protein-coupled receptors that are linked to acute inflammatory responses, malignant glioma stem cell metastasis, and chronic inflammation. Although several N-formyl peptides are known to bind to these receptors, more selective small-molecule, high-affinity ligands are needed for a better understanding of the physiologic roles played by these receptors. High-throughput assays using mixture-based combinatorial libraries represent a unique, highly efficient approach for rapid data acquisition and ligand identification. We report the superiority of this approach in the context of the simultaneous screening of a diverse set of mixture-based small-molecule libraries. We used a single cross-reactive peptide ligand for a duplex flow cytometric screen of FPR1 and FPR2 in color-coded cell lines. Screening 37 different mixture-based combinatorial libraries totaling more than five million small molecules (contained in 5,261 mixture samples) resulted in seven libraries that significantly inhibited activity at the receptors. Using positional scanning deconvolution, selective high-affinity (low nM K(i)) individual compounds were identified from two separate libraries, namely, pyrrolidine bis-diketopiperazine and polyphenyl urea. The most active individual compounds were characterized for their functional activities as agonists or antagonists with the most potent FPR1 agonist and FPR2 antagonist identified to date with an EC₅₀ of 131 nM (4 nM K(i)) and an IC₅₀ of 81 nM (1 nM K(i)), respectively, in intracellular Ca²⁺ response determinations. Comparative analyses of other previous screening approaches clearly illustrate the efficiency of identifying receptor selective, individual compounds from mixture-based combinatorial libraries.

  18. Design and synthesis of a novel, achiral class of highly potent and selective, orally active neurokinin-1 receptor antagonists.

    Science.gov (United States)

    Hoffmann, Torsten; Bös, Michael; Stadler, Heinz; Schnider, Patrick; Hunkeler, Walter; Godel, Thierry; Galley, Guido; Ballard, Theresa M; Higgins, Guy A; Poli, Sonia M; Sleight, Andrew J

    2006-03-01

    The discovery of a novel, achiral pyridine class of potent and orally active neurokinin-1 (NK(1)) receptor antagonists is described. The evaluation of this class is briefly outlined, leading to the identification of netupitant 21 and befetupitant 29, two new proprietary chemical entities with high affinity and excellent CNS penetration.

  19. A selective imidazobenzodiazepine antagonist of ethanol in the rat.

    Science.gov (United States)

    Suzdak, P D; Glowa, J R; Crawley, J N; Schwartz, R D; Skolnick, P; Paul, S M

    1986-12-05

    Ethanol, at pharmacologically relevant concentrations of 20 to 100 mM, stimulates gamma-aminobutyric (GABA) receptor-mediated uptake of 36Cl-labeled chlorine into isolated brain vesicles. One drug that acts at GABA-benzodiazepine receptors, the imidazobenzodiazepine Ro15-4513, has been found to be a potent antagonist of ethanol-stimulated 36Cl- uptake into brain vesicles, but it fails to antagonize either pentobarbital- or muscimol-stimulated 36Cl- uptake. Pretreatment of rats with Ro15-4513 blocks the anticonflict activity of low doses of ethanol (but not pentobarbital) as well as the behavioral intoxication observed with higher doses of ethanol. The effects of Ro15-4513 in antagonizing ethanol-stimulated 36Cl- uptake and behavior are completely blocked by benzodiazepine receptor antagonists. However, other benzodiazepine receptor inverse agonists fail to antagonize the actions of ethanol in vitro or in vivo, suggesting a novel interaction of Ro15-4513 with the GABA receptor-coupled chloride ion channel complex. The identification of a selective benzodiazepine antagonist of ethanol-stimulated 36Cl- uptake in vitro that blocks the anxiolytic and intoxicating actions of ethanol suggests that many of the neuropharmacologic actions of ethanol may be mediated via central GABA receptors.

  20. PERSPECTIVE: CHASE-AWAY SEXUAL SELECTION: ANTAGONISTIC SEDUCTION VERSUS RESISTANCE.

    Science.gov (United States)

    Holland, Brett; Rice, William R

    1998-02-01

    A model of sexual selection that leads to the evolution of exaggerated male display characters that is based on antagonistic coevolution between the sexes is described. The model is motivated by three lines of research: intersexual conflict with respect to mating, sensory exploitation, and the evolution of female resistance, as opposed to preference, for male display traits. The model generates unique predictions that permit its operation to be distinguished from other established models of sexual selection. One striking prediction is that females will frequently win the coevolutionary arms race with males, leaving them encumbered with costly ornaments that have little value except that their absence understimulates females. Examples from the literature suggest that the model may have broad application in nature. The chase-away model is a special case of the more general phenomenon of Interlocus Contest Evolution (ICE). © 1998 The Society for the Study of Evolution.

  1. Evaluation of JNJ-54717793 a Novel Brain Penetrant Selective Orexin 1 Receptor Antagonist in Two Rat Models of Panic Attack Provocation

    Directory of Open Access Journals (Sweden)

    Pascal Bonaventure

    2017-06-01

    Full Text Available Orexin neurons originating in the perifornical and lateral hypothalamic area are highly reactive to anxiogenic stimuli and have strong projections to anxiety and panic-associated circuitry. Recent studies support a role for the orexin system and in particular the orexin 1 receptor (OX1R in coordinating an integrative stress response. However, no selective OX1R antagonist has been systematically tested in two preclinical models of using panicogenic stimuli that induce panic attack in the majority of people with panic disorder, namely an acute hypercapnia-panic provocation model and a model involving chronic inhibition of GABA synthesis in the perifornical hypothalamic area followed by intravenous sodium lactate infusion. Here we report on a novel brain penetrant, selective and high affinity OX1R antagonist JNJ-54717793 (1S,2R,4R-7-([(3-fluoro-2-pyrimidin-2-ylphenylcarbonyl]-N-[5-(trifluoromethylpyrazin-2-yl]-7-azabicyclo[2.2.1]heptan-2-amine. JNJ-54717793 is a high affinity/potent OX1R antagonist and has an excellent selectivity profile including 50 fold versus the OX2R. Ex vivo receptor binding studies demonstrated that after oral administration JNJ-54717793 crossed the blood brain barrier and occupied OX1Rs in the rat brain. While JNJ-54717793 had minimal effect on spontaneous sleep in rats and in wild-type mice, its administration in OX2R knockout mice, selectively promoted rapid eye movement sleep, demonstrating target engagement and specific OX1R blockade. JNJ-54717793 attenuated CO2 and sodium lactate induced panic-like behaviors and cardiovascular responses without altering baseline locomotor or autonomic activity. These data confirm that selective OX1R antagonism may represent a novel approach of treating anxiety disorders, with no apparent sedative effects.

  2. The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies.

    Science.gov (United States)

    Rothe, Christine; Urlinger, Stefanie; Löhning, Corinna; Prassler, Josef; Stark, Yvonne; Jäger, Ute; Hubner, Bernd; Bardroff, Michael; Pradel, Ingrid; Boss, Melanie; Bittlingmaier, Renate; Bataa, Tschimegma; Frisch, Christian; Brocks, Bodo; Honegger, Annemarie; Urban, Margit

    2008-02-29

    This article describes the generation of the Human Combinatorial Antibody Library HuCAL GOLD. HuCAL GOLD is a synthetic human Fab library based on the HuCAL concept with all six complementarity-determining regions (CDRs) diversified according to the sequence and length variability of naturally rearranged human antibodies. The human antibody repertoire was analyzed in-depth, and individual CDR libraries were designed and generated for each CDR and each antibody family. Trinucleotide mixtures were used to synthesize the CDR libraries in order to ensure a high quality within HuCAL GOLD, and a beta-lactamase selection system was employed to eliminate frame-shifted clones after successive cloning of the CDR libraries. With these methods, a large, high-quality library with more than 10 billion functional Fab fragments was achieved. By using CysDisplay, the antibody fragments are displayed on the tip of the phage via a disulfide bridge between the phage coat protein pIII and the heavy chain of the antibody fragment. Efficient elution of specific phages is possible by adding reducing agents. HuCAL GOLD was challenged with a variety of different antigens and proved to be a reliable source of high-affinity human antibodies with best affinities in the picomolar range, thus functioning as an excellent source of antibodies for research, diagnostic, and therapeutic applications. Furthermore, the data presented in this article demonstrate that CysDisplay is a robust and broadly applicable display technology even for high-throughput applications.

  3. In Vitro and In Vivo Evaluation of Alexa Fluor 680-Bombesin[7–14]NH2 Peptide Conjugate, a High-Affinity Fluorescent Probe with High Selectivity for the Gastrin-Releasing Peptide Receptor

    Directory of Open Access Journals (Sweden)

    Lixin Ma

    2007-05-01

    Full Text Available Gastrin-releasing peptide (GRP receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN, a 14–amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H2N-glycylglycylglycine-BBN[7–14]NH2 peptide with the following general sequence: H2N-G-G-G-Q-W-A-V-G-H-L-M-(NH2. This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7–14]NH2 conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H2N-G-G-G-BBN[7–14]NH2 in dimethylformamide (DMF. In vitro competitive binding assays, using 125I-Tyr4-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 ± 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7–14]NH2 in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.

  4. In vitro and in vivo evaluation of Alexa Fluor 680-bombesin[7-14]NH2 peptide conjugate, a high-affinity fluorescent probe with high selectivity for the gastrin-releasing peptide receptor.

    Science.gov (United States)

    Ma, Lixin; Yu, Ping; Veerendra, Bhadrasetty; Rold, Tammy L; Retzloff, Lauren; Prasanphanich, Adam; Sieckman, Gary; Hoffman, Timothy J; Volkert, Wynn A; Smith, Charles J

    2007-01-01

    Gastrin-releasing peptide (GRP) receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN), a 14-amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H(2)N-glycylglycylglycine-BBN[7-14]NH(2) peptide with the following general sequence: H(2)N-G-G-G-Q-W-A-V-G-H-L-M-(NH(2)). This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H(2)N-G-G-G-BBN[7-14]NH(2) in dimethylformamide (DMF). In vitro competitive binding assays, using (125)I-Tyr(4)-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 +/- 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.

  5. SB223412, a neurokinin-3 receptor-selective antagonist, suppresses testosterone secretion in male guinea pigs.

    Science.gov (United States)

    Nakamura, Sho; Ito, Yoshiko; Yamamoto, Koki; Takahashi, Chudai; Dai, Mingdao; Tanahashi, Miyu; Uenoyama, Yoshihisa; Tsukamura, Hiroko; Oishi, Shinya; Maeda, Kei-Ichiro; Matsuda, Fuko

    2017-10-15

    Guinea pigs are important zoo animals and have been recommended for animal-assisted activities or therapy, however there are problems concerning testosterone inducing aggressive or sexual behaviors in male guinea pigs. Testicular testosterone secretion is regulated by pulsatile gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH) release in mammals. The mechanism generating GnRH/LH pulses is thought to be governed by kisspeptin neurons, which coexpress neurokinin B (NKB) and dynorphin A (Dyn), in the arcuate nucleus (ARC). Kisspeptin neurons in the ARC are frequently referred to as KNDy neurons. The purpose of this study was to examine whether the antagonization of NKB-neurokinin-3 receptor (NK3R) signaling can manipulate testosterone secretion in male guinea pigs. A single subcutaneous administration or 7 days of oral administration of an NK3R-selective antagonist, SB223412 (50 mg/body), significantly decreased plasma testosterone levels in male guinea pigs. In vitro binding assays confirmed that SB223412 has a high affinity to guinea pig NK3R. These results suggest that SB223412 could be used as an orally-available compound to suppress testosterone levels in male guinea pigs. Double labeling in situ hybridization of kisspeptin and either NKB or Dyn showed that kisspeptin-expressing neurons contained NKB (77.9%) or Dyn (62.3%) in the ARC, suggesting the presence of KNDy neurons in the ARC of guinea pigs. In conclusion, the present study shows that SB223412 could be a candidate compound to suppress testosterone secretion in male guinea pigs for controlling sexual and aggressive behaviors in the species. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Human Homosexuality: A Paradigmatic Arena for Sexually Antagonistic Selection?

    Science.gov (United States)

    Ciani, Andrea Camperio; Battaglia, Umberto; Zanzotto, Giovanni

    2015-01-01

    Sexual conflict likely plays a crucial role in the origin and maintenance of homosexuality in our species. Although environmental factors are known to affect human homosexual (HS) preference, sibling concordances and population patterns related to HS indicate that genetic components are also influencing this trait in humans. We argue that multilocus, partially X-linked genetic factors undergoing sexually antagonistic selection that promote maternal female fecundity at the cost of occasional male offspring homosexuality are the best candidates capable of explaining the frequency, familial clustering, and pedigree asymmetries observed in HS male proband families. This establishes male HS as a paradigmatic example of sexual conflict in human biology. HS in females, on the other hand, is currently a more elusive phenomenon from both the empirical and theoretical standpoints because of its fluidity and marked environmental influence. Genetic and epigenetic mechanisms, the latter involving sexually antagonistic components, have been hypothesized for the propagation and maintenance of female HS in the population. However, further data are needed to truly clarify the evolutionary dynamics of this trait. PMID:25635045

  7. Human homosexuality: a paradigmatic arena for sexually antagonistic selection?

    Science.gov (United States)

    Camperio Ciani, Andrea; Battaglia, Umberto; Zanzotto, Giovanni

    2015-01-29

    Sexual conflict likely plays a crucial role in the origin and maintenance of homosexuality in our species. Although environmental factors are known to affect human homosexual (HS) preference, sibling concordances and population patterns related to HS indicate that genetic components are also influencing this trait in humans. We argue that multilocus, partially X-linked genetic factors undergoing sexually antagonistic selection that promote maternal female fecundity at the cost of occasional male offspring homosexuality are the best candidates capable of explaining the frequency, familial clustering, and pedigree asymmetries observed in HS male proband families. This establishes male HS as a paradigmatic example of sexual conflict in human biology. HS in females, on the other hand, is currently a more elusive phenomenon from both the empirical and theoretical standpoints because of its fluidity and marked environmental influence. Genetic and epigenetic mechanisms, the latter involving sexually antagonistic components, have been hypothesized for the propagation and maintenance of female HS in the population. However, further data are needed to truly clarify the evolutionary dynamics of this trait. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.

  8. Intraguild predation provides a selection mechanism for bacterial antagonistic compounds

    Science.gov (United States)

    Leisner, J. J.; Haaber, J.

    2012-01-01

    Bacteriocins are bacterial proteinaceous toxins with bacteriostatic or bacteriocidal activity towards other bacteria. The current theory on their biological role concerns especially colicins, with underlying social interactions described as an example of spite. This leads to a rock–paper–scissors game between colicin producers and sensitive and resistant variants. The generality of this type of selection mechanism has previously been challenged with lactic acid bacterial (LAB) bacteriocins as an example. In the natural environment of LAB, batch cultures are the norm opposed to the natural habitats of Escherichia coli where continuous cultures are prevailing. This implies that fitness for LAB, to a large degree, is related to survival rates (bottleneck situations) rather than to growth rates. We suggest that the biological role of LAB bacteriocins is to enhance survival in the stationary growth phase by securing a supply of nutrients from lysed target cells. Thus, this social interaction is an example of selfishness rather than of spite. Specifically, it fits into an ecological model known as intraguild predation (IGP), which is a combination of competition and predation where the predator (LAB bacteriocin producer) and prey (bacteriocin susceptible bacteria) share similar and often limited resources. We hypothesize that IGP may be a common phenomenon promoting microbial production of antagonistic compounds. PMID:22951735

  9. High affinity hemoglobin and Parkinson's disease.

    Science.gov (United States)

    Graham, Jeffrey; Hobson, Douglas; Ponnampalam, Arjuna

    2014-12-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SN) region of the midbrain. Oxidative damage in this region has been shown to play an important role in the pathogenesis of this disease. Human neurons have been discovered to contain hemoglobin, with an increased concentration seen in the neurons of the SN. High affinity hemoglobin is a clinical entity resulting from mutations that create a functional increase in the binding of hemoglobin to oxygen and an inability to efficiently unload it to tissues. This can result in a number of metabolic compensatory changes, including an elevation in circulating hemoglobin and an increase in the molecule 2,3-diphosphoglycerate (2,3-DPG). Population based studies have revealed that patients with PD have elevated hemoglobin as well as 2,3-DPG levels. Based on these observations, we hypothesize that the oxidative damage seen in PD is related to an underlying high affinity hemoglobin subtype. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Comparison of the in vitro and in vivo profiles of tolterodine with those of subtype-selective muscarinic receptor antagonists.

    Science.gov (United States)

    Gillberg, P G; Sundquist, S; Nilvebrant, L

    1998-05-22

    Tolterodine [(R)-N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamine ] is a new potent and competitive muscarinic receptor antagonist developed for the treatment of urinary urge incontinence and other symptoms of overactive bladder. In vivo, tolterodine exhibits functional selectivity for the urinary bladder over salivary glands, a profile that cannot be explained in terms of selectivity for a single muscarinic receptor subtype. The aim of this study was to compare the in vitro and in vivo antimuscarinic profiles of tolterodine with those of muscarinic receptor antagonists with distinct receptor subtype-selectivity profiles: darifenacin [(S)-2-[1-[2-(2,3-dihydrobenzofuran-5-yl)ethyl]-3-pyrrolidinyl]-2,2-d iphenylacetamide; selective for muscarinic M3 receptors]; UH-AH 37 (6-chloro-5,10-dihydro-5-[(1-methyl-4-piperidinyl)acetyl]-11H-dibenzo-[b ,e][1,4]diazepine-11-one; low affinity for muscarinic M2 receptors); and AQ-RA 741 (11-([4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl)-5,11-dihydro-6H-py rido[2,3-b][1,4]benzodiazepine-6-one; high affinity for muscarinic M2 receptors). The in vitro profiles of these compounds were in agreement with previous reports; darifenacin and UH-AH 37 demonstrated selectivity for muscarinic M3/m3 over M2/m2 receptors, while the converse was observed for AQ-RA 741. In vivo, AQ-RA 741 was more potent (1.4-2.7-fold) in inhibiting urinary bladder contraction than salivation in the anaesthetised cat (i.e., a profile similar to that of tolterodine [2.5-3.3-fold]), while darifenacin and UH-AH 37 showed the reverse selectivity profile (0.6-0.8 and 0.4-0.5-fold, respectively). The results confirm that it is possible to separate the antimuscarinic effects on urinary bladder and salivary glands in vivo. The data on UH-AH 37 and darifenacin support the view that a selectivity for muscarinic M3/m3 over M2/m2 receptors may result in a more pronounced effect on salivation than on bladder contraction. The data on AQ-RA 741 may indicate

  11. Antagonistic activity of selected strains of Bacillus thuringiensis ...

    African Journals Online (AJOL)

    The aim of this work was to determine, in vitro, the antagonistic effectiveness of 60 strains of Bacillus thuringiensis against damping-off and root and stem rot caused by Rhizoctonia solani. The strains were obtained from the International Collection of Entomopathogenic Bacillus at the FCB-UANL. During the in vitro dual ...

  12. Crystal Structures of Human Orexin 2 Receptor Bound to the Subtype-Selective Antagonist EMPA.

    Science.gov (United States)

    Suno, Ryoji; Kimura, Kanako Terakado; Nakane, Takanori; Yamashita, Keitaro; Wang, Junmei; Fujiwara, Takaaki; Yamanaka, Yasuaki; Im, Dohyun; Horita, Shoichiro; Tsujimoto, Hirokazu; Tawaramoto, Maki S; Hirokawa, Takatsugu; Nango, Eriko; Tono, Kensuke; Kameshima, Takashi; Hatsui, Takaki; Joti, Yasumasa; Yabashi, Makina; Shimamoto, Keiko; Yamamoto, Masaki; Rosenbaum, Daniel M; Iwata, So; Shimamura, Tatsuro; Kobayashi, Takuya

    2018-01-02

    Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle, and are thus drug targets for the treatment of insomnia. Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source, we determined structures of human orexin 2 receptor in complex with the subtype-selective antagonist EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) at 2.30-Å and 1.96-Å resolution. In comparison with the non-subtype-selective antagonist suvorexant, EMPA contacted fewer residues through hydrogen bonds at the orthosteric site, explaining the faster dissociation rate. Comparisons among these OX 2 R structures in complex with selective antagonists and previously determined OX 1 R/OX 2 R structures bound to non-selective antagonists revealed that the residue at positions 2.61 and 3.33 were critical for the antagonist selectivity in OX 2 R. The importance of these residues for binding selectivity to OX 2 R was also revealed by molecular dynamics simulation. These results should facilitate the development of antagonists for orexin receptors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Synthesis of [[sup 3]H]tert-butyl 8-chloro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodia zepine 3-carboxylate, a selective, high affinity ligand for the diazepam insensitive (DI) subtype of the benzodiazepine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Ziqiang Gu; Costa, B.R. de; Wong, Garry; Rice, K.C.; Skolnick, Phil (National Inst. of Diabetes and Digestive and Kidney Diseases, Bethesda, MD (United States))

    1992-12-01

    The preparation of [[sup 3]H]-labelled tert-butyl 8-chloro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]benzodiazepine 3-carboxylate (TCIB, 6), a high affinity ligand for the diazepam insensitive (DI) subtype of the benzodiazepine receptor (BZR) is described. Synthesis of [[sup 3]H]TCIB was accomplished in 4 steps starting from 5-chloroisatoic anhydride. Tritium-label introduction was achieved in the final step by selective catalytic tritiolysis in 62% radiochemical yield with quantitative isotopic incorporation. (Author).

  14. Meiotic drive influences the outcome of sexually antagonistic selection at a linked locus.

    Science.gov (United States)

    Patten, M M

    2014-11-01

    Most meiotic drivers, such as the t-haplotype in Mus and the segregation distorter (SD) in Drosophila, act in a sex-specific manner, gaining a transmission advantage through one sex although suffering only the fitness costs associated with the driver in the other. Their inheritance is thus more likely through one of the two sexes, a property they share with sexually antagonistic alleles. Previous theory has shown that pairs of linked loci segregating for sexually antagonistic alleles are more likely to remain polymorphic and that linkage disequilibrium accrues between them. I probe this similarity between drive and sexual antagonism and examine the evolution of chromosomes experiencing these selection pressures simultaneously. Reminiscent of previous theory, I find that: the opportunity for polymorphism increases for a sexually antagonistic locus that is physically linked to a driving locus; the opportunity for polymorphism at a driving locus also increases when linked to a sexually antagonistic locus; and stable linkage disequilibrium accompanies any polymorphic equilibrium. Additionally, I find that drive at a linked locus favours the fixation of sexually antagonistic alleles that benefit the sex in which drive occurs. Further, I show that under certain conditions reduced recombination between these two loci is selectively favoured. These theoretical results provide clear, testable predictions about the nature of sexually antagonistic variation on driving chromosomes and have implications for the evolution of genomic architecture. © 2014 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2014 European Society For Evolutionary Biology.

  15. Identification of highly selective and potent orexin receptor 1 antagonists derived from a dual orexin receptor 1/2 antagonist based on the structural framework of pyrazoylethylbenzamide.

    Science.gov (United States)

    Futamura, Aya; Nozawa, Dai; Araki, Yuko; Tamura, Yunoshin; Tokura, Seiken; Kawamoto, Hiroshi; Tokumaru, Yuichi; Kakihara, Sora; Aoki, Takeshi; Ohtake, Norikazu

    2017-10-15

    The design, synthesis, and structure activity relationships of the novel class of pyrazolylethylbenzamide orexin receptor 1-selective antagonists are described. Further derivatization of the prototype dual orexin receptor 1/2 antagonist lead (1) by installing a (S)-methyl group into the ethyl linker moiety between the pyrazole ring and benzamide resulted in an increase of the antagonist potency against orexin receptor 1/2 receptors. Optimization of the benzamide and pyrazole parts of compounds 2 and 9b led to the identification of N-ethyl-5-fluoro-N-{(2S)-1-[5-(4-fluorophenyl)-2H-tetrazol-2-yl]propan-2-yl}-2-(pyrimidin-2-yl)benzamide (24), which exhibited excellent antagonistic activity against orexin receptor 1 with an IC50 of 2.01nM and a 265-fold selectivity for orexin receptor 1 over orexin receptor 2. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Synthesis and structure-activity relationships of piperidinylpyrrolopyridine derivatives as potent and selective H1 antagonists.

    Science.gov (United States)

    Fonquerna, Silvia; Miralpeix, Montse; Pagès, Lluís; Puig, Carles; Cardús, Arantxa; Antón, Francisca; Vilella, Dolors; Aparici, Mónica; Prieto, José; Warrellow, Graham; Beleta, Jorge; Ryder, Hamish

    2005-02-15

    The synthesis and structure-activity relationships of piperidinylpyrrolopyridines as potent and selective H(1) antagonists are discussed. It was found that the nature of the acid chain bonded to piperidine was a key feature for maintaining both the duration of action in vivo and lack of sedative properties.

  17. Novel 3-carboxy- and 3-phosphonopyrazoline amino acids as potent and selective NMDA receptor antagonists

    DEFF Research Database (Denmark)

    Conti, Paola; Pinto, Andrea; Tamborini, Lucia

    2010-01-01

    identified two highly potent and selective competitive NMDA receptor antagonists, (5S,alphaR)-1 and (5S,alphaR)-4, which exhibit good in vitro neuroprotective activity and in vivo anticonvulsant activity by i.p. administration, suggesting that these molecules may have potential use as therapeutic agents....

  18. SELECTIVITY PROFILE OF SOME RECENT MUSCARINIC ANTAGONISTS IN BOVINE AND GUINEA-PIG TRACHEA AND HEART

    NARCIS (Netherlands)

    ROFFEL, AF; HAMSTRA, JJ; ELZINGA, CRS; ZAAGSMA, J

    1994-01-01

    The functional affinities of some recently developed subtype-selective muscarinic antagonists towards bovine tracheal smooth muscle muscarinic M(3) receptors were established and compared to binding affinities for bovine cardiac M(2) and functional affinities for guinea-pig tracheal smooth muscle

  19. Discovery of LAS101057: A Potent, Selective, and Orally Efficacious A2B Adenosine Receptor Antagonist.

    Science.gov (United States)

    Eastwood, Paul; Esteve, Cristina; González, Jacob; Fonquerna, Silvia; Aiguadé, Josep; Carranco, Inés; Doménech, Teresa; Aparici, Mònica; Miralpeix, Montserrat; Albertí, Joan; Córdoba, Mónica; Fernández, Raquel; Pont, Mercè; Godessart, Núria; Prats, Neus; Loza, María Isabel; Cadavid, María Isabel; Nueda, Arsenio; Vidal, Bernat

    2011-03-10

    The structure-activity relationships for a series of pyrazine-based A2B adenosine receptor antagonists are described. From this work, LAS101057 (17), a potent, selective, and orally efficacious A2B receptor antagonist, was identified as a clinical development candidate. LAS101057 inhibits agonist-induced IL-6 production in human fibroblasts and is active in an ovalbumin (OVA)-sensitized mouse model after oral administration, reducing airway hyperresponsiveness to methacholine, Th2 cytokine production, and OVA-specific IgE levels.

  20. The F130S point mutation in the Arabidopsis high-affinity K+ transporter AtHAK5 increases K+ over Na+ and Cs+ selectivity and confers Na+ and Cs+ tolerance to yeast under heterologous expression

    Directory of Open Access Journals (Sweden)

    Fernando eAleman

    2014-09-01

    Full Text Available Potassium (K+ is an essential macronutrient required for plant growth, development and high yield production of crops. Members of group I of the KT/HAK/KUP family of transporters, such as HAK5, are key components for K+ acquisition by plant roots at low external K+ concentrations. Certain abiotic stress conditions such as salinity or Cs+-polluted soils may jeopardize plant K+ nutrition because HAK5-mediated K+ transport is inhibited by Na+ and Cs+. Here, by screening in yeast a randomly-mutated collection of AtHAK5 transporters, a new mutation in AtHAK5 sequence is identified that greatly increases Na+ tolerance. The single point mutation F130S, affecting an amino acid residue conserved in HAK5 transporters from several species, confers high salt tolerance, as well as Cs+ tolerance. This mutation increases more than 100-fold the affinity of AtHAK5 for K+ and reduces the Ki values for Na+ and Cs+, suggesting that the F130 residue may contribute to the structure of the pore region involved in K+ binding. In addition, this mutation increases the Vmax for K+. All this changes occur without increasing the amount of the AtHAK5 protein in yeast and support the idea that this residue is contributing to shape the selectivity filter of the AtHAK5 transporter.

  1. Identification of ligand-selective peptidic ActRIIB-antagonists using phage display technology.

    Science.gov (United States)

    Sakamoto, Kotaro; Kanematsu-Yamaki, Yoko; Kamada, Yusuke; Oka, Masahiro; Ohnishi, Toshiyuki; Miwa, Masanori; Asami, Taiji; Inooka, Hiroshi

    2017-09-01

    ActRIIB (activin receptor type-2B) is an activin receptor subtype constitutively expressed in the whole body, playing a role in cellular proliferation, differentiation, and metabolism. For its various physiological activities, ActRIIB interacts with activin and multiple other ligands including myostatin (MSTN), growth differentiation factor 11 (GDF11), and bone morphogenetic protein 9 (BMP9). Notably, the protein-protein interaction (PPI) between ActRIIB and MSTN negatively controls muscular development. Therefore, this PPI has been targeted for effective treatment of muscle degenerative diseases such as muscular dystrophy and sarcopenia. Here, we report the identification of ligand-selective peptidic ActRIIB-antagonists by phage display technology. Our peptides bound to the extracellular domain of ActRIIB, inhibited PPIs between ActRIIB expressed on the cell surface and its ligands, and subsequently suppressed activation of Smad that serves as the downstream signal of the ActRIIB pathway. Interestingly, these peptidic antagonists displayed different ligand selectivities; the AR2mini peptide inhibited multiple ligands (activin A, MSTN, GDF11, and BMP9), AR9 inhibited MSTN and GDF11, while AR8 selectively inhibited MSTN. This is the first report of artificial peptidic ActRIIB-antagonists possessing ligand-selectivity.

  2. Acyclic cucurbit[n]uril congeners are high affinity hosts.

    Science.gov (United States)

    Ma, Da; Zavalij, Peter Y; Isaacs, Lyle

    2010-07-16

    We present the design, synthesis via methylene bridged glycoluril tetramer building blocks, and charaterization of acyclic cucurbit[n]uril congeners that function as hosts for a wide variety of ammonium ions in water. The X-ray crystallographic characterization of the free host and its complexes with p-xylylenediamine and spermine establish the flexibility of the methylene bridged backbone of the acyclic cucurbit[n]uril congeners that allow them to adapt to the structural features of the guest. We find that the acyclic cucurbit[n]uril congeners-with their four contiguous methylene bridged glycoluril units and two aromatic o-xylylene walls bearing CO(2)H substituents-bind to ammonium ions in buffered water with values of K(a) ranging from approximately 10(5) M(-1) to greater than 10(9) M(-1). Similar to the cucurbit[n]uril family of hosts, we find that increasing the concentration of metal cations in the buffer reduces the affinity of the acyclic cucurbit[n]uril congener toward guests by competitive binding at the ureidyl C horizontal lineO portals. Although the acyclic cucurbit[n]uril congeners retain the ability to bind to ammonium ions with high affinity, they do so with lower selectivity than cucurbit[n]urils presumably do to the structural flexibility of the hosts. A methylene bridged glycoluril tetramer model compound that lacks the substituted o-xylylene walls is a much lower affinity host, which establishes the importance of these rings on the overall recognition behavior of the acyclic cucurbit[n]uril congeners. Overall, the results in this paper establish that acyclic cucurbit[n]uril receptors that contain four or more contiguous methylene bridged glycoluril units retain many of the excellent recognition properties of the cucurbit[n]uril family.

  3. Antagonistic natural and sexual selection on wing shape in a scrambling damselfly.

    Science.gov (United States)

    Outomuro, David; Söderquist, Linus; Nilsson-Örtman, Viktor; Cortázar-Chinarro, María; Lundgren, Cecilia; Johansson, Frank

    2016-07-01

    Wings are a key trait underlying the evolutionary success of birds, bats, and insects. For over a century, researchers have studied the form and function of wings to understand the determinants of flight performance. However, to understand the evolution of flight, we must comprehend not only how morphology affects performance, but also how morphology and performance affect fitness. Natural and sexual selection can either reinforce or oppose each other, but their role in flight evolution remains poorly understood. Here, we show that wing shape is under antagonistic selection with regard to sexual and natural selection in a scrambling damselfly. In a field setting, natural selection (survival) favored individuals with long and slender forewings and short and broad hindwings. In contrast, sexual selection (mating success) favored individuals with short and broad forewings and narrow-based hindwings. Both types of selection favored individuals of intermediate size. These results suggest that individuals face a trade-off between flight energetics and maneuverability and demonstrate how natural and sexual selection can operate in similar directions for some wing traits, that is, wing size, but antagonistically for others, that is, wing shape. Furthermore, they highlight the need to study flight evolution within the context of species' mating systems and mating behaviors. © 2016 The Author(s).

  4. THE INTERACTION OF SELECTIVE AND NONSELECTIVE ANTAGONISTS WITH PREJUNCTIONAL AND POSTJUNCTIONAL MUSCARINIC RECEPTOR SUBTYPES IN THE GUINEA-PIG TRACHEA

    NARCIS (Netherlands)

    TENBERGE, REJ; ROFFEL, AF; ZAAGSMA, J

    1993-01-01

    Muscarinic receptor antagonists were used to study prejunctional M2 and postjunctional M3 receptors in the isolated guinea pig trachea. The effects of four M2-selective muscarinic receptor antagonists (gallamine, methoctramine, AQ-RA 741 and AF-DX 116) were studied on twitch contractions, elicited

  5. Discovery of Potent and Highly Selective A2B Adenosine Receptor Antagonist Chemotypes.

    Science.gov (United States)

    El Maatougui, Abdelaziz; Azuaje, Jhonny; González-Gómez, Manuel; Miguez, Gabriel; Crespo, Abel; Carbajales, Carlos; Escalante, Luz; García-Mera, Xerardo; Gutiérrez-de-Terán, Hugo; Sotelo, Eddy

    2016-03-10

    Three novel families of A2B adenosine receptor antagonists were identified in the context of the structural exploration of the 3,4-dihydropyrimidin-2(1H)-one chemotype. The most appealing series contain imidazole, 1,2,4-triazole, or benzimidazole rings fused to the 2,3-positions of the parent diazinone core. The optimization process enabled identification of a highly potent (3.49 nM) A2B ligand that exhibits complete selectivity toward A1, A2A, and A3 receptors. The results of functional cAMP experiments confirmed the antagonistic behavior of representative ligands. The main SAR trends identified within the series were substantiated by a molecular modeling study based on a receptor-driven docking model constructed on the basis of the crystal structure of the human A2A receptor.

  6. High-affinity antibodies to the 1,4-dihydropyridine Ca2+-channel blockers

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, K.P.; Sharp, A.; Strom, M.; Kahl, S.D.

    1986-05-01

    Antibodies with high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers have been produced in rabbits by immunization with dihydropyridine-protein conjugates. Anti-dihydropyridine antibodies were found to specifically bind (/sup 3/H)nitrendipine, (/sup 3/H)-nimodipine, (/sup 3/H)nisoldipine, and (/sup 3/H)PN 200-110 (all 1,4-dihydropyridine Ca2+-channel blockers) with high affinity, while (/sup 3/H)verapamil, (/sup 3/H)diltiazem, and (/sup 3/H)trifluoperazine were not recognized. The average dissociation constant of the (/sup 3/H)nitrendipine-antibody complex was 0.06 (+/- 0.02) X 10(-9) M for an antiserum studied in detail and ranged from 0.01 to 0.24 X 10(-9) M for all antisera. Inhibition of (/sup 3/H)nitrendipine binding was specific for the 1,4-dihydropyridine Ca2+-channel modifiers and the concentrations required for half-maximal inhibition ranged between 0.25 and 0.90 nM. Structurally unrelated Ca2+-channel blockers, calmodulin antagonists, inactive metabolites of nitrendipine, and UV-inactivated nisoldipine did not modify (/sup 3/H)nitrendipine binding to the anti-dihydropyridine antibodies. Dihydropyridines without a bulky substituent in the 4-position of the heterocycle were able to displace (/sup 3/H)nitrendipine binding, but the concentrations required for half-maximal inhibition were greater than 800 nM. In summary, anti-dihydropyridine antibodies have been shown to have high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers and to exhibit dihydropyridine binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca2+-channel blockers.

  7. High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

    Directory of Open Access Journals (Sweden)

    Graham S Baldwin

    Full Text Available The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

  8. High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

    Science.gov (United States)

    Baldwin, Graham S; George, Graham N; Pushie, M Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

  9. Eplerenone: a selective aldosterone receptor antagonist for hypertension and heart failure.

    Science.gov (United States)

    Moore, Tera D; Nawarskas, James J; Anderson, Joe R

    2003-01-01

    Aldosterone has been implicated for many years as an important substance in the pathogenesis of heart disease. Elevated aldosterone concentrations have been documented in patients with hypertension and heart failure, leading to the use of aldosterone antagonists for the treatment of these conditions. Spironolactone has been used for nearly 2 decades for the treatment of hypertension, and more recently, has become a standard agent for the treatment of systolic heart failure. Spironolactone, however, is a nonselective antagonist of the aldosterone receptor, binding also to other steroid receptors and causing a significant percentage of patients to have sex hormone-related adverse effects such as gynecomastia. Eplerenone is the first of a new class of drugs known as selective aldosterone receptor antagonists, which selectively block the aldosterone receptor with minimal effect at other steroid receptors, thereby minimizing many of the hormonal side effects seen with spironolactone. Eplerenone has been shown to be beneficial both as monotherapy and combination therapy for lowering elevated blood pressure in patients with hypertension. The antihypertensive efficacy of eplerenone is roughly similar to that of other antihypertensive agents, although in 1 study black patients responded better with eplerenone than losartan. In addition, eplerenone has demonstrated some renoprotective effects in diabetic patients with hypertension. Recently, eplerenone was shown to significantly reduce mortality and cardiovascular morbidity in post-myocardial infarction patients with systolic heart failure currently taking standard heart failure medications. Eplerenone is generally well tolerated, although hyperkalemia with this agent is of some concern. Eplerenone is metabolized by CYP3A4 and administration with potent inhibitors of this enzyme is contraindicated because of the risk of hyperkalemia. In summary, eplerenone has proven to be beneficial in treating hypertension and post

  10. Selective depletion of uropathogenic E. coli from the gut by a FimH antagonist.

    Science.gov (United States)

    Spaulding, Caitlin N; Klein, Roger D; Ruer, Ségolène; Kau, Andrew L; Schreiber, Henry L; Cusumano, Zachary T; Dodson, Karen W; Pinkner, Jerome S; Fremont, Daved H; Janetka, James W; Remaut, Han; Gordon, Jeffrey I; Hultgren, Scott J

    2017-06-22

    Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually. Despite effective antibiotic therapy, 30-50% of patients experience recurrent UTIs. In addition, the growing prevalence of UPEC that are resistant to last-line antibiotic treatments, and more recently to carbapenems and colistin, make UTI a prime example of the antibiotic-resistance crisis and emphasize the need for new approaches to treat and prevent bacterial infections. UPEC strains establish reservoirs in the gut from which they are shed in the faeces, and can colonize the periurethral area or vagina and subsequently ascend through the urethra to the urinary tract, where they cause UTIs. UPEC isolates encode up to 16 distinct chaperone-usher pathway pili, and each pilus type may enable colonization of a habitat in the host or environment. For example, the type 1 pilus adhesin FimH binds mannose on the bladder surface, and mediates colonization of the bladder. However, little is known about the mechanisms underlying UPEC persistence in the gut. Here, using a mouse model, we show that F17-like and type 1 pili promote intestinal colonization and show distinct binding to epithelial cells distributed along colonic crypts. Phylogenomic and structural analyses reveal that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, we show that targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces intestinal colonization of genetically diverse UPEC isolates, while simultaneously treating UTI, without notably disrupting the structural configuration of the gut microbiota. By selectively depleting intestinal UPEC reservoirs, mannosides could markedly reduce the rate of UTIs and recurrent UTIs.

  11. Successful strategy for the selection of new strawberry-associated rhizobacteria antagonistic to Verticillium wilt.

    Science.gov (United States)

    Berg, G; Kurze, S; Buchner, A; Wellington, E M; Smalla, K

    2000-12-01

    In order to isolate and characterize new strawberry-associated bacteria antagonistic to the soil-borne pathogenic fungus Verticillium dahliae Kleb., rhizobacterial populations from two different strawberry species, Greenish Strawberry (Fragaria viridis) and Garden Strawberry (F. x ananassa) obtained after plating onto King's B and glycerol-arginine agar, were screened for in vitro antagonism toward V. dahliae. The proportion of isolates with antifungal activity determined in in vitro assay against V. dahliae was higher for the Garden Strawberry than for the Greenish Strawberry. From 300 isolates, 20 isolates with strong antifungal activity were selected characterized by physiological profiling and molecular fingerprinting methods. Diversity among the isolates was characterized with molecular fingerprints using amplified ribosomal DNA restriction analysis (ARDRA) and the more discriminating BOX-PCR fingerprint method. The physiological profiles were well correlated with molecular fingerprinting pattern analysis. Significant reduction of Verticillium wilt by bacterial dipping bath treatment was shown in the greenhouse and in fields naturally infested by V. dahliae. The relative increase of yield ranged from 117% (Streptomyces albidoflavus S1) to 344% (Pseudomonas fluorescens P10) in greenhouse trials, and 113% (Streptomyces albidoflavus S1) to 247% (Pseudomonas fluorescens P6) in field trials. Evaluation resulted in the selection of three effective biocontrol agents (Pseudomonas fluorescens P6, P10, and Streptomyces diastatochromogenes S9) antagonistic to the Verticillium wilt pathogen.

  12. Biocontrol of Phytophthora Blight and Anthracnose in Pepper by Sequentially Selected Antagonistic Rhizobacteria against Phytophthora capsici.

    Science.gov (United States)

    Sang, Mee Kyung; Shrestha, Anupama; Kim, Du-Yeon; Park, Kyungseok; Pak, Chun Ho; Kim, Ki Deok

    2013-06-01

    We previously developed a sequential screening procedure to select antagonistic bacterial strains against Phytophthora capsici in pepper plants. In this study, we used a modified screening procedure to select effective biocontrol strains against P. capsici; we evaluated the effect of selected strains on Phytophthora blight and anthracnose occurrence and fruit yield in pepper plants under field and plastic house conditions from 2007 to 2009. We selected four potential biocontrol strains (Pseudomonas otitidis YJR27, P. putida YJR92, Tsukamurella tyrosinosolvens YJR102, and Novosphingobium capsulatum YJR107) among 239 bacterial strains. In the 3-year field tests, all the selected strains significantly (P biocontrol activities were variable. In addition, strains YJR27, YJR92, and YJR102, in certain harvests, increased pepper fruit numbers in field tests and red fruit weights in plastic house tests. Taken together, these results indicate that the screening procedure is rapid and reliable for the selection of potential biocontrol strains against P. capsici in pepper plants. In addition, these selected strains exhibited biocontrol activities against anthracnose, and some of the strains showed plant growth-promotion activities on pepper fruit.

  13. Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases

    Energy Technology Data Exchange (ETDEWEB)

    Silveira, C.L.; Eldefrawi, A.T.; Eldefrawi, M.E. (Univ. of Maryland, Baltimore (USA))

    1990-05-01

    The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-(3H)cis-methyldioxolane ((3H)CD), which has been used to label a high affinity population of M2 receptors. A single population of sites was detected and (3H)CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with (3H)CD and considered to be of the M2 subtype. Up to 70% of the (3H)CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. (3H)CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of (3H)CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM (3H)CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting (3H)CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration.

  14. Behavioral pharmacology of AR-A000002, a novel, selective 5-hydroxytryptamine(1B) antagonist.

    Science.gov (United States)

    Hudzik, T J; Yanek, M; Porrey, T; Evenden, J; Paronis, C; Mastrangelo, M; Ryan, C; Ross, S; Stenfors, C

    2003-03-01

    The present review summarizes the behavioral pharmacology conducted to profile the anxiolytic and antidepressant potential of the selective 5-hydroxytryptamine (HT)(1B) antagonist (R)-N-[5-methyl-8-(4-methylpiperazin-1-yl)-1,2,3,4-tetrahydro-2-naphthyl]-4-morpholinobenzamide (AR-A000002). AR-A000002 functions as a 5-HT(1B) antagonist in vivo, which was shown by the antagonism of the discriminative stimulus effects in the guinea pig of the 5-HT(1B) agonist 3-(N-methylpyrrolidin-2R-ylmethyl)-5-(3-nitropyrid-2-ylamino)-lH-indole (CP135,807). Anxiolytic activity of AR-A000002 was demonstrated in the separation-induced vocalization paradigm in guinea pig pups, and in a suppressed responding procedure in pigeons and guinea pigs, but only a weak trend was noted in a suppressed responding procedure in squirrel monkeys. Antidepressant efficacy was shown in a number of paradigms. In pigeons and guinea pigs responding under a differential reinforcement of low rates schedule of reinforcement (DRL), AR-A000002 increased the number of reinforcers earned without altering the number of responses made. In guinea pigs trained under a response duration differentiation paradigm, AR-A000002 increased mean lever-press duration. Finally, AR-A000002 was shown to block escape failures in guinea pigs submitted to a learned helplessness paradigm. Taken together, these data suggest utility for 5-HT(1B) antagonists in the treatment of both anxiety and affective disorders.

  15. Selectivity of muscarinic antagonists in radioligand and in vivo experiments for the putative M1, M2 and M3 receptors.

    Science.gov (United States)

    Doods, H N; Mathy, M J; Davidesko, D; van Charldorp, K J; de Jonge, A; van Zwieten, P A

    1987-07-01

    In the present study we investigated the nature of the muscarinic receptors present in the hippocampus, sympathetic ganglia, atria and salivary glands of the rat. The heterogeneity of the muscarinic receptors was examined both in vivo and in radioligand binding experiments. To study whether the receptors present in the investigated tissues are indeed distinct subtypes we determined the potencies of antagonists in both systems. It is proposed that there are three different binding sites present in hippocampal, atrial and submandibular membranes and we suggest to classify them as M1, M2 and M3, respectively. Both in vivo and in vitro pirenzepine appears to possess high affinity for M1 receptors, whereas 4-diphenylacetoxy-N-methylpiperidine methobromide and dicyclomine show high affinity for both M1 and M3 receptors. AF-DX 116 (11-2[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one) displayed high affinity for M2 receptors.

  16. High affinity humanized antibodies without making hybridomas; immunization paired with mammalian cell display and in vitro somatic hypermutation.

    Directory of Open Access Journals (Sweden)

    Audrey D McConnell

    Full Text Available A method has been developed for the rapid generation of high-affinity humanized antibodies from immunized animals without the need to make conventional hybridomas. Rearranged IgH D(J regions were amplified from the spleen and lymph tissue of mice immunized with the human complement protein C5, fused with a limited repertoire of human germline heavy chain V-genes to form intact humanized heavy chains, and paired with a human light chain library. Completed heavy and light chains were assembled for mammalian cell surface display and transfected into HEK 293 cells co-expressing activation-induced cytidine deaminase (AID. Numerous clones were isolated by fluorescence-activated cell sorting, and affinity maturation, initiated by AID, resulted in the rapid evolution of high affinity, functional antibodies. This approach enables the efficient sampling of an immune repertoire and the direct selection and maturation of high-affinity, humanized IgGs.

  17. Structurally related nucleotides as selective agonists and antagonists at P2Y1 receptors

    Science.gov (United States)

    Jacobson, Kenneth A.; Moro, Stefano; Hoffmann, Carsten; Kim, Yong-Chul; Kim, Hak Sung; Ravi, R. Gnana; Harden, T. Kendall; Boyer, José L.

    2015-01-01

    The P2Y1 receptor responds to adenine nucleotides and is present in platelets, heart, smooth muscles prostate, ovary, and brain. A selective antagonist may be useful as an antithrombotic agent. We have analyzed the binding site of this G protein-coupled receptor using ligand design, site-directed mutagenesis, and homology modeling based on rhodopsin. We have designed and synthesized a series of deoxyadenosine 3′,5′-bisphosphate derivatives that act as antagonists, or, in some cases with small structural changes, as agonists or partial agonists. The 2-position accommodates Cl or thioethers, whereas the N6-position is limited to Me or Et. 2′-Substitution with OH or OMe increases agonist efficacy over 2′-H. Using molecular modeling of the binding site, the oxygen atoms of the ribose moiety were predicted to be non-essential, i.e. no specific H-bonds with the receptor protein appear in the model. We have, therefore, substituted this moiety with carbocylics, smaller and larger rings, conformationally constrained rings, and acyclics, with retention of affinity for the receptor. With simplified pharmacophores we are exploring the steric and electronic requirements of the receptor binding site, and the structural basis of receptor activation. PMID:11347970

  18. Selective Glucocorticoid Receptor (GR-II Antagonist Reduces Body Weight Gain in Mice

    Directory of Open Access Journals (Sweden)

    Tomoko Asagami

    2011-01-01

    Full Text Available Previous research has shown that mifepristone can prevent and reverse weight gain in animals and human subjects taking antipsychotic medications. This proof-of-concept study tested whether a more potent and selective glucocorticoid receptor antagonist could block dietary-induced weight gain and increase insulin sensitivity in mice. Ten-week-old, male, C57BL/6J mice were fed a diet containing 60% fat calories and water supplemented with 11% sucrose for 4 weeks. Groups (=8 received one of the following: CORT 108297 (80 mg/kg QD, CORT 108297 (40 mg/kg BID, mifepristone (30 mg/kg BID, rosiglitazone (10 mg/kg QD, or vehicle. Compared to mice receiving a high-fat, high-sugar diet plus vehicle, mice receiving a high-fat, high-sugar diet plus either mifepristone or CORT 108297 gained significantly less weight. At the end of the four week treatment period, mice receiving CORT 108297 40 mg/kg BID or CORT 108297 80 mg/kg QD also had significantly lower steady plasma glucose than mice receiving vehicle. However, steady state plasma glucose after treatment was not highly correlated with reduced weight gain, suggesting that the effect of the glucocorticoid receptor antagonist on insulin sensitivity may be independent of its mitigating effect on weight gain.

  19. Structure-based discovery of selective positive allosteric modulators of antagonists for the M2muscarinic acetylcholine receptor.

    Science.gov (United States)

    Korczynska, Magdalena; Clark, Mary J; Valant, Celine; Xu, Jun; Moo, Ee Von; Albold, Sabine; Weiss, Dahlia R; Torosyan, Hayarpi; Huang, Weijiao; Kruse, Andrew C; Lyda, Brent R; May, Lauren T; Baltos, Jo-Anne; Sexton, Patrick M; Kobilka, Brian K; Christopoulos, Arthur; Shoichet, Brian K; Sunahara, Roger K

    2018-02-16

    Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M 2 mAChR, seeking molecules that specifically stabilized antagonist binding. This led us to identify a positive allosteric modulator (PAM) that potentiated the antagonist N -methyl scopolamine (NMS). Structure-based optimization led to compound '628, which enhanced binding of NMS, and the drug scopolamine itself, with a cooperativity factor (α) of 5.5 and a K B of 1.1 μM, while sparing the endogenous agonist acetylcholine. NMR spectral changes determined for methionine residues reflected changes in the allosteric network. Moreover, '628 slowed the dissociation rate of NMS from the M 2 mAChR by 50-fold, an effect not observed at the other four mAChR subtypes. The specific PAM effect of '628 on NMS antagonism was conserved in functional assays, including agonist stimulation of [ 35 S]GTPγS binding and ERK 1/2 phosphorylation. Importantly, the selective allostery between '628 and NMS was retained in membranes from adult rat hypothalamus and in neonatal rat cardiomyocytes, supporting the physiological relevance of this PAM/antagonist approach. This study supports the feasibility of discovering PAMs that confer subtype selectivity to antagonists; molecules like '628 can convert an armamentarium of potent but nonselective GPCR antagonist drugs into subtype-selective reagents, thus reducing their off-target effects. Copyright © 2018 the Author(s). Published by PNAS.

  20. Preclinical pharmacology of bilastine, a new selective histamine H1 receptor antagonist: receptor selectivity and in vitro antihistaminic activity.

    Science.gov (United States)

    Corcóstegui, Reyes; Labeaga, Luis; Innerárity, Ana; Berisa, Agustin; Orjales, Aurelio

    2005-01-01

    This study aimed to establish the receptor selectivity and antihistaminic activity of bilastine, a new selective antihistamine receptor antagonist. In vitro experiments were conducted using a receptor binding screening panel and guinea-pig and rat tissues. Antihistaminic activity was determined using H1 receptor binding studies and in vitro H1 antagonism studies conducted in guinea-pig tissues and human cell lines. Receptor selectivity was established using a receptor binding screening panel and a receptor antagonism screening conducted in guinea-pig, rat and rabbit tissues. Inhibition of inflammatory mediators was determined through the Schultz-Dale reaction in sensitised guinea-pig ileum. Bilastine binds to histamine H1-receptors as indicated by its displacement of [3H]-pyrilamine from H1-receptors expressed in guinea-pig cerebellum and human embryonic kidney (HEK) cell lines. The studies conducted on guinea-pig smooth muscle demonstrated the capability of bilastine to antagonise H1-receptors. Bilastine is selective for histamine H1-receptors as shown in receptor-binding screening conducted to determine the binding capacity of bilastine to 30 different receptors. The specificity of its H1-receptor antagonistic activity was also demonstrated in a series of in vitro experiments conducted on guinea-pig and rat tissues. The results of these studies confirmed the lack of significant antagonism against serotonin, bradykinin, leukotriene D4, calcium, muscarinic M3-receptors, alpha1-adrenoceptors, beta2-adrenoceptors, and H2- and H3-receptors. The results of the in vitro Schultz-Dale reaction demonstrated that bilastine also has anti-inflammatory activity. These preclinical studies provide evidence that bilastine has H1- antihistamine activity, with high specificity for H1-receptors, and poor or no affinity for other receptors. Bilastine has also been shown to have anti-inflammatory properties.

  1. 01-ERD-111 - The Development of Synthetic High Affinity Ligands

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, J; Balhorn, R; Cosman, M; Lightstone, F; Zeller, L

    2004-02-05

    The aim of this project was to develop Synthetic High-Affinity Ligands (SHALs), which bind with high affinity and specificity to proteins of interest for national security and cancer therapy applications. The aim of producing synthetic ligands for sensory devices as an alternative to antibody-based detection assays and therapeutic agents is to overcome the drawbacks associated with antibody-based in next-generation sensors and systems. The focus area of the project was the chemical synthesis of the SHALs. The project concentrated on two different protein targets. (a) The C fragment of tetanus and botulinum toxin, potential biowarfare agents. A SHAL for tetanus or botulinum toxin would be incorporated into a sensory device for the toxins. (b) HLA-DR10, a protein found in high abundance on the surface of Non-Hodgkins Lymphoma. A SHAL specific to a tumor marker, labeled with a radionuclide, would enable the targeted delivery of radiation therapy to metastatic disease. The technical approach used to develop a SHAL for each protein target will be described in more detail below. However, in general, the development of a SHAL requires a combination of computational modeling techniques, modern nuclear magnetic resonance spectroscopy (NMR) and synthetic chemistry.

  2. Pyrazin-2(1H)-ones as a novel class of selective A3 adenosine receptor antagonists.

    Science.gov (United States)

    Azuaje, Jhonny; Carbajales, Carlos; González-Gómez, Manuel; Coelho, Alberto; Caamaño, Olga; Gutiérrez-de-Terán, Hugo; Sotelo, Eddy

    2015-01-01

    A3AR antagonists are promising drug candidates as neuroprotective agents as well as for the treatment of inflammation or glaucoma. The most widely known A3AR antagonists are derived from polyheteroaromatic scaffolds, which usually show poor pharmacokinetic properties. Accordingly, the identification of structurally simple A3AR antagonists by the exploration of novel diversity spaces is a challenging goal. A convergent and efficient Ugi-based multicomponent approach enabled the discovery of pyrazin-2(1H)-ones as a novel class of A3AR antagonists. A combined experimental/computational strategy accelerated the establishment of the most salient features of the structure-activity and structure-selectivity relationships in this series. The optimization process provided pyrazin-2(1H)-ones with improved affinity and a plausible hypothesis regarding their binding modes was proposed.

  3. Structure-Based Drug Design of Mineralocorticoid Receptor Antagonists to Explore Oxosteroid Receptor Selectivity.

    Science.gov (United States)

    Nordqvist, Anneli; O'Mahony, Gavin; Fridén-Saxin, Maria; Fredenwall, Marlene; Hogner, Anders; Granberg, Kenneth L; Aagaard, Anna; Bäckström, Stefan; Gunnarsson, Anders; Kaminski, Tim; Xue, Yafeng; Dellsén, Anita; Hansson, Eva; Hansson, Pia; Ivarsson, Ida; Karlsson, Ulla; Bamberg, Krister; Hermansson, Majlis; Georgsson, Jennie; Lindmark, Bo; Edman, Karl

    2017-01-05

    The mineralocorticoid receptor (MR) is a nuclear hormone receptor involved in the regulation of body fluid and electrolyte homeostasis. In this study we explore selectivity triggers for a series of nonsteroidal MR antagonists to improve selectivity over other members of the oxosteroid receptor family. A biaryl sulfonamide compound was identified in a high-throughput screening (HTS) campaign. The compound bound to MR with pKi =6.6, but displayed poor selectivity over the glucocorticoid receptor (GR) and the progesterone receptor (PR). Following X-ray crystallography of MR in complex with the HTS hit, a compound library was designed that explored an induced-fit hypothesis that required movement of the Met852 side chain. An improvement in MR selectivity of 11- to 79-fold over PR and 23- to 234-fold over GR was obtained. Given the U-shaped binding conformation, macrocyclizations were explored, yielding a macrocycle that bound to MR with pKi =7.3. Two protein-ligand X-ray structures were determined, confirming the hypothesized binding mode for the designed compounds. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Effect of the selective vasopressin V2 receptor antagonists in hepatic cirrhosis patients with ascites: a meta-analysis

    Directory of Open Access Journals (Sweden)

    Shao-hui TANG

    2013-07-01

    Full Text Available Objective To evaluate the efficacy and safety of selective vasopressin V2 receptor antagonists in the treatment of hepatic cirrhosis patients with ascites. Methods PubMed, EMBASE, Web of Science, The Cochrane Central Register of Controlled Trials, Database for Chinese Technical Periodical (VIP, Chinese Journal Full-Text Database (CNKI, and Wan Fang Digital Journal Full-text Database were retrieved to collect clinical randomized controlled trials of hepatic cirrhosis with ascites treated by selective vasopressin V2 receptor antagonists. Meta analysis was performed by using Review Manager 5.0. Results Nine randomized controlled trials including 1884 patients met the inclusion criteria. Meta-analysis showed that: 1 The selective vasopressin V2 receptor antagonists were associated with a significant reduction in body weight compared with placebo (WMD=–1.98kg, 95%CI:–3.24-–0.72kg, P=0.002. Treatment with selective vasopressin V2 receptor antagonists was associated with an improvement of low serum sodium concentration compared to placebo (WMD=3.74mmol/L, 95%CI: 0.91-6.58mmol/L, P=0.01. The percentage of patients with worsening ascites was higher in the group of patients treated with placebo (RR=0.51, 95%CI: 0.34-0.77, P=0.001. 2 The amplitude of increased urine volume was obviously higher in selective vasopressin V2 receptor antagonists group than in placebo group (WMD=1437.65ml, 95%CI: 649.01-2226.30ml, P=0.0004. The difference of serum creatinine in the selective vasopressin V2 receptor antagonists group was not statistically significant compared with the control group (WMD=–3.49μmol/L, 95%CI: –12.54¬5.56μmol/L, P=0.45. 3 There was no statistical significance between the two groups in the heart rate, systolic pressure, diastolic pressure and mortality (P>0.05. The rate of other adverse reactions was higher in the selective vasopressin V2 receptor antagonists group compared with that of placebo group (P=0.003. Conclusion

  5. Structure-Based Prediction of Subtype Selectivity of Histamine H3 Receptor Selective Antagonists in Clinical Trials

    DEFF Research Database (Denmark)

    Kim, Soo-Kyung; Fristrup, Peter; Abrol, Ravinder

    2011-01-01

    applications, including treatment of Alzheimer’s disease, attention deficit hyperactivity disorder (ADHD), epilepsy, and obesity.(1) However, many of these drug candidates cause undesired side effects through the cross-reactivity with other histamine receptor subtypes. In order to develop improved selectivity......Histamine receptors (HRs) are excellent drug targets for the treatment of diseases, such as schizophrenia, psychosis, depression, migraine, allergies, asthma, ulcers, and hypertension. Among them, the human H3 histamine receptor (hH3HR) antagonists have been proposed for specific therapeutic...... and activity for such treatments, it would be useful to have the three-dimensional structures for all four HRs. We report here the predicted structures of four HR subtypes (H1, H2, H3, and H4) using the GEnSeMBLE (GPCR ensemble of structures in membrane bilayer environment) Monte Carlo protocol,(2) sampling...

  6. In vitro and in vivo pharmacological characterization of the novel NK₁ receptor selective antagonist Netupitant.

    Science.gov (United States)

    Rizzi, Anna; Campi, Barbara; Camarda, Valeria; Molinari, Stefano; Cantoreggi, Sergio; Regoli, Domenico; Pietra, Claudio; Calo', Girolamo

    2012-09-01

    The novel NK(1) receptor ligand Netupitant has been characterized in vitro and in vivo. In calcium mobilization studies CHO cells expressing the human NK receptors responded to a panel of agonists with the expected order of potency. In CHO NK(1) cells Netupitant concentration-dependently antagonized the stimulatory effects of substance P (SP) showing insurmountable antagonism (pK(B) 8.87). In cells expressing NK(2) or NK(3) receptors Netupitant was inactive. In the guinea pig ileum Netupitant concentration-dependently depressed the maximal response to SP (pK(B) 7.85) and, in functional washout experiments, displayed persistent (up to 5h) antagonist effects. In mice the intrathecal injection of SP elicited the typical scratching, biting and licking response that was dose-dependently inhibited by Netupitant given intraperitoneally in the 1-10mg/kg dose range. In gerbils, foot tapping behavior evoked by the intracerebroventricular injection of a NK(1) agonist was dose-dependently counteracted by Netupitant given intraperitoneally (ID(50) 1.5mg/kg) or orally (ID(50) 0.5mg/kg). In time course experiments in gerbils Netupitant displayed long lasting effects. In all the assays Aprepitant elicited similar effects as Netupitant. These results suggest that Netupitant behaves as a brain penetrant, orally active, potent and selective NK(1) antagonist. Thus this molecule can be useful for investigating the NK(1) receptor role in the control of central and peripheral functions. Netupitant has clinical potential in conditions such as chemotherapy induced nausea and vomiting, in which the blockade of NK(1) receptors has been demonstrated valuable for patients. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Sexually antagonistic selection on genetic variation underlying both male and female same-sex sexual behavior.

    Science.gov (United States)

    Berger, David; You, Tao; Minano, Maravillas R; Grieshop, Karl; Lind, Martin I; Arnqvist, Göran; Maklakov, Alexei A

    2016-05-13

    Intralocus sexual conflict, arising from selection for different alleles at the same locus in males and females, imposes a constraint on sex-specific adaptation. Intralocus sexual conflict can be alleviated by the evolution of sex-limited genetic architectures and phenotypic expression, but pleiotropic constraints may hinder this process. Here, we explored putative intralocus sexual conflict and genetic (co)variance in a poorly understood behavior with near male-limited expression. Same-sex sexual behaviors (SSBs) generally do not conform to classic evolutionary models of adaptation but are common in male animals and have been hypothesized to result from perception errors and selection for high male mating rates. However, perspectives incorporating sex-specific selection on genes shared by males and females to explain the expression and evolution of SSBs have largely been neglected. We performed two parallel sex-limited artificial selection experiments on SSB in male and female seed beetles, followed by sex-specific assays of locomotor activity and male sex recognition (two traits hypothesized to be functionally related to SSB) and adult reproductive success (allowing us to assess fitness consequences of genetic variance in SSB and its correlated components). Our experiments reveal both shared and sex-limited genetic variance for SSB. Strikingly, genetically correlated responses in locomotor activity and male sex-recognition were associated with sexually antagonistic fitness effects, but these effects differed qualitatively between male and female selection lines, implicating intralocus sexual conflict at both male- and female-specific genetic components underlying SSB. Our study provides experimental support for the hypothesis that widespread pleiotropy generates pervasive intralocus sexual conflict governing the expression of SSBs, suggesting that SSB in one sex can occur due to the expression of genes that carry benefits in the other sex.

  8. 7-Chloroarctinone-b as a new selective PPARy antagonist potently blocks adipocyte differentiation

    National Research Council Canada - National Science Library

    Yong-tao LI Li LI Jing CHEN Tian-cen HU Jin HUANG Yue-wei GUO Hua-liang JIANG Xu SHEN

    2009-01-01

    .... In order to develop potent lead compounds for obesity treatment, we screened a natural product library for novel PPARy antagonists with inhibitory effects on adipocyte differentiation. Methods...

  9. Residues remote from the binding pocket control the antagonist selectivity towards the corticotropin-releasing factor receptor-1

    Science.gov (United States)

    Sun, Xianqiang; Cheng, Jianxin; Wang, Xu; Tang, Yun; Ågren, Hans; Tu, Yaoquan

    2015-01-01

    The corticotropin releasing factors receptor-1 and receptor-2 (CRF1R and CRF2R) are therapeutic targets for treating neurological diseases. Antagonists targeting CRF1R have been developed for the potential treatment of anxiety disorders and alcohol addiction. It has been found that antagonists targeting CRF1R always show high selectivity, although CRF1R and CRF2R share a very high rate of sequence identity. This has inspired us to study the origin of the selectivity of the antagonists. We have therefore built a homology model for CRF2R and carried out unbiased molecular dynamics and well-tempered metadynamics simulations for systems with the antagonist CP-376395 in CRF1R or CRF2R to address this issue. We found that the side chain of Tyr6.63 forms a hydrogen bond with the residue remote from the binding pocket, which allows Tyr6.63 to adopt different conformations in the two receptors and results in the presence or absence of a bottleneck controlling the antagonist binding to or dissociation from the receptors. The rotameric switch of the side chain of Tyr3566.63 allows the breaking down of the bottleneck and is a perquisite for the dissociation of CP-376395 from CRF1R.

  10. High-affinity neurotrophin receptors and ligands promote leukemogenesis

    Science.gov (United States)

    Beutel, Gernot; Rhein, Mathias; Meyer, Johann; Koenecke, Christian; Neumann, Thomas; Yang, Min; Krauter, Jürgen; von Neuhoff, Nils; Heuser, Michael; Diedrich, Helmut; Göhring, Gudrun; Wilkens, Ludwig; Schlegelberger, Brigitte; Ganser, Arnold

    2009-01-01

    Neurotrophins (NTs) and their receptors play a key role in neurogenesis and survival. The TRK (tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA, TRKB, TRKC) are high-affinity NT receptors that are expressed in a variety of human tissues. Their role in normal and malignant hematopoiesis is poorly understood. In a prospective study involving 94 adult patients we demonstrate for the first time cell-surface expression of the 3 TRKs and constitutive activation in blasts from patients with de novo or secondary acute leukemia. At least one TRK was expressed in 55% of the analyzed cases. We establish a clear correlation between the TRK expression pattern and FAB classification. Although only few point mutations were found in TRK sequences by reverse-transcriptase–polymerase chain reaction (RT-PCR), we observed coexpression of BDNF (ligand for TRKB) in more than 50% of TRKB+ cases (16/30). Activation of TRKA or TRKB by NGF and BDNF, respectively, efficiently rescued murine myeloid cells from irradiation-induced apoptosis. Coexpression of TRKB/BDNF or TRKA/NGF in murine hematopoietic cells induced leukemia. Moreover, activation of TRKs was important for survival of both human and murine leukemic cells. Our findings suggest that TRKs play an important role in leukemogenesis and may serve as a new drug target. PMID:19059881

  11. Dantrolene: A Selective Ryanodine Receptor Antagonist, Protects Against Pentylenetetrazole-Induced Seizure in Mice

    Directory of Open Access Journals (Sweden)

    Mojtaba Keshavarz

    2016-10-01

    Full Text Available Ryanodine receptor abnormalities has implicated in the generation and maintenance of seizure. Dantrolene, a selective ryanodine receptor antagonist, may be a potential drug for the prevention of seizure. Therefore, we aimed to clarify the protective effects of dantrolene against pentylenetetrazole seizure in mice. Male albino mice were received an intra-peritoneal injection of pentylenetetrazole (80 mg/kg in seven separate groups (n=8. We used dantrolene (10,20 and 40 mg/kg, caffeine (200 mg/kg, dantrolene (40 mg/kg + caffeine (200 mg/kg, diazepam (5 mg/kg as a positive control and vehicle 30 minutes before the injection of pentylenetetrazole. Then, we registered the latency time of the first seizure, the severity of seizures and the incidence of seizure and death. Kruskal-Wallis test followed by Mann-Whitney and Fisher’s exact test were used to analyze the data. Dantrolene (10,20 and 40 mg/kg significantly increased the latency time for the first seizure. Furthermore, dantrolene (20 and 40 mg/kg, but not 10 mg/kg attenuated the severity of seizures in comparison to the vehicle group. Moreover, dantrolene only at the dose of 40 mg/kg prevented from tonic-clonic seizure and death in comparison to the vehicle group. In contrast, the addition of caffeine abolished the protective effects of dantrolene on the tonic-clonic seizure/death and inhibited the beneficial effects of dantrolene on the severity of pentylenetetrazol seizures. The acute dantrolene administration produced an anticonvulsant effect in the pentylenetetrazole-induced seizure. Moreover, caffeine prevented from dantrolene anticonvulsant effects. These results may imply about ryanodine receptors and intracellular calcium roles in the generation and control of pentylenetetrazole seizure.

  12. Structure of the Human Dopamine D3 Receptor in Complex with a D2/D3 Selective Antagonist

    Energy Technology Data Exchange (ETDEWEB)

    Chien, Ellen Y.T.; Liu, Wei; Zhao, Qiang; Katritch, Vsevolod; Han, Gye Won; Hanson, Michael A.; Shi, Lei; Newman, Amy Hauck; Javitch, Jonathan A.; Cherezov, Vadim; Stevens, Raymond C. (Cornell); (Scripps); (NIDA); (Columbia); (UCSD); (Receptos)

    2010-11-30

    Dopamine modulates movement, cognition, and emotion through activation of dopamine G protein-coupled receptors in the brain. The crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride reveals important features of the ligand binding pocket and extracellular loops. On the intracellular side of the receptor, a locked conformation of the ionic lock and two distinctly different conformations of intracellular loop 2 are observed. Docking of R-22, a D3R-selective antagonist, reveals an extracellular extension of the eticlopride binding site that comprises a second binding pocket for the aryl amide of R-22, which differs between the highly homologous D2R and D3R. This difference provides direction to the design of D3R-selective agents for treating drug abuse and other neuropsychiatric indications.

  13. Mepyramine-JNJ7777120-hybrid compounds show high affinity to hH(1)R, but low affinity to hH(4)R.

    Science.gov (United States)

    Wagner, Eva; Wittmann, Hans-Joachim; Elz, Sigurd; Strasser, Andrea

    2011-11-01

    In literature, a synergism between histamine H(1) and H(4) receptor is discussed. Furthermore, it was shown, that the combined application of mepyramine, a H(1) antagonist and JNJ7777120, a H(4) receptor ligand leads to a synergistic effect in the acute murine asthma model. Thus, the aim of this study was to develop new hybrid ligands, containing one H(1) and one H(4) pharmacophor, connected by an appropriate spacer, in order to address both, H(1)R and H(4)R. Within this study, we synthesized nine hybrid compounds, which were pharmacologically characterized at hH(1)R and hH(4)R. The new compounds revealed (high) affinity to hH(1)R, but showed only low affinity to hH(4)R. Additionally, we performed molecular dynamic studies for some selected compounds at hH(1)R, in order to obtain information about the binding mode of these compounds on molecular level. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Effects of the selective 5-HT(7) receptor antagonist SB-269970 in animal models of psychosis and cognition.

    Science.gov (United States)

    Waters, Kerry A; Stean, Tania O; Hammond, Beverley; Virley, David J; Upton, Neil; Kew, James N C; Hussain, Ishrut

    2012-03-01

    The 5-hydroxytryptamine7 (5-HT7) receptor is a G-protein coupled receptor for serotonin that has been implicated in the pathophysiology of psychiatric and neurological disorders including anxiety, depression and schizophrenia. A number of studies have attempted to evaluate the potential role of the 5-HT7 receptor in schizophrenia by utilising genetic or pharmacological tools but to date these have provided conflicting results. Here we investigate the effect of a selective 5-HT7 receptor antagonist, SB-269970, in in vivo psychosis and cognition models and relate efficacy to brain exposures of the compound. SB-269970 significantly attenuated amphetamine-induced rearing and circling in rats. A similar effect was observed in an N-methyl d-aspartic acid (NMDA) receptor antagonist driven psychosis model, where SB-269970 significantly reversed phencyclidine-induced hyperlocomotion, rearing and circling; although the effect was not as robust as with the 5-HT2a receptor antagonist positive control, MDL100,907. SB-269970 also attenuated a temporal deficit in novel object recognition (NOR), indicative of an improvement in recognition memory. Pharmacokinetic analysis of plasma and brain samples taken after behavioural testing confirmed that efficacy was achieved at doses and pre-treatment times where receptor occupancy was substantial. These findings highlight the anti-psychotic and pro-cognitive potential of 5-HT7 receptor antagonists and warrant further studies to explore their therapeutic potential in schizophrenia. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. Comparison of levocabastine, a new selective H1-receptor antagonist, and disodium cromoglycate, in a nasal provocation test with allergen.

    OpenAIRE

    Kolly, M; Pécoud, A

    1986-01-01

    The effect of intranasal administration of levocabastine, a new selective H1-receptor antagonist, was investigated in a nasal provocation test (NPT) performed with allergens. The NPT allowed a quantitative estimation of the nasal allergic threshold (concentration of allergen necessary to trigger the reaction). In addition, the intensity of the three major rhinitis symptoms (obstruction, rhinorrhea and sneezing) was determined. Twelve adult patients, allergic to grass pollen, underwent a first...

  16. Novel selective thiazoleacetic acids as CRTH2 antagonists developed from in silico derived hits. Part 1

    DEFF Research Database (Denmark)

    Rist, Oystein; Grimstrup, Marie; Receveur, Jean-Marie

    2009-01-01

    Structure-activity relationships of three related series of 4-phenylthiazol-5-ylacetic acids, derived from two hits emanating from a focused library obtained by in silico screening, have been explored as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) antagonists....... Several compounds with double digit nanomolar binding affinity and full antagonistic efficacy for human CRTH2 receptor were obtained in all subclasses. The most potent compound was [2-(4-chloro-benzyl)-4-(4-phenoxy-phenyl)-thiazol-5-yl]acetic acid having an binding affinity of 3.7nM and functional...... antagonistic effect of 66nM in a BRET and 12nM in a cAMP assay with no functional activity for the other PGD2 DP receptor (27muM in cAMP)....

  17. Netupitant, a Potent and Highly Selective NK1 Receptor Antagonist, Alleviates Acetic Acid-Induced Bladder Overactivity in Anesthetized Guinea-Pigs

    OpenAIRE

    Palea, Stefano; Guilloteau, V?ronique; Rekik, Mo?z; Lovati, Emanuela; Guerard, Marc; Guardia, Maria-Alba; Lluel, Philippe; Pietra, Claudio; Yoshiyama, Mitsuharu

    2016-01-01

    Introduction. Tachykinins potently contract the isolated urinary bladder from a number of animal species and play an important role in the regulation of the micturition reflex. On the guinea-pig isolated urinary bladder we examined the effects of a new potent and selective NK1 receptor antagonist (netupitant) on the contractions induced by a selective NK1 receptor agonist, SP-methylester (SP-OMe). Moreover, the effects of netupitant and another selective NK1 antagonist (L-733,060) were studie...

  18. Treatment of restless legs syndrome with the selective AMPA receptor antagonist perampanel.

    Science.gov (United States)

    Garcia-Borreguero, Diego; Cano, Irene; Granizo, Juan Jose

    2017-06-01

    Perampanel is a selective, noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) antagonist that has been approved for the treatment of partial seizures. Here we report on the first open study evaluating its efficacy in idiopathic restless legs syndrome (RLS). The study was designed as a prospective two-month open trial. Twenty-two previously untreated patients diagnosed with idiopathic RLS began treatment with 2 mg perampanel, which was increased to 4 mg at week four if clinically necessary. Multiple Suggested Immobilization Tests (mSITs) followed by polysomnography were performed at baseline and at week eight. Severity ratings were performed every two weeks by means of the IRLS scale, and Clinical Global Index (CGI) subscale. The main endpoint was therapeutic response, defined as a 50% improvement in IRLS total score. Twenty patients completed the study. During the 8-week treatment period, the IRLS score improved from a mean (±standard deviation (SD)) 23.7 ± 4.2 to 11.5 ± 5.3. Twelve of 20 patients were full responders (improvement 50% in IRLS total score), and four responded partially. The mean effective dose of perampanel at the end of treatment was 3.8 mg/day. Treatment with perampanel also resulted in an improvement in the mean (±SD) periodic leg movement index from 27.8 ± 6.9 to 4.36 ± 2.0. Perampanel was well tolerated. The main side effects were dizziness, somnolence, headache, and irritability. These preliminary results suggest that perampanel has significant therapeutic effects on both sensory and motor symptoms. If confirmed by future controlled studies, perampanel might become a promising alternative to existing dopaminergic treatments due to its glutamatergic mechanism of action. The study provides class IV evidence supporting the therapeutic effects of perampanel in RLS/WED. Copyright © 2017. Published by Elsevier B.V.

  19. Development of prolactin receptor antagonists with reduced pH-dependence of receptor binding

    DEFF Research Database (Denmark)

    Hansen, Mathilde Johanne Kaas; Olsen, Johan Gotthardt; Bernichtein, Sophie

    2011-01-01

    Abstract The cytokine hormone prolactin has a vast number of diverse functions. Unfortunately, it also exhibits tumor growth promoting properties, which makes the development of prolactin receptor antagonists a priority. Prolactin binds to its cognate receptor with much lower affinity at low p....... From evaluation of known molecular structures of human prolactin, of the prolactin receptor and of different complexes of the two, three histidine residues in the hormone-receptor binding site 1 were selected for mutational studies. We analyzed 10 variants by circular dichroism spectroscopy, affinity...... antagonists were developed earlier and the histidine mutations were introduced within such background. The antagonistic properties were maintained and the high affinity at low pH conserved. The implications of these findings may open new areas of research in the field of prolactin cancer biology. Copyright...

  20. Selective Allosteric Antagonists for the G Protein-Coupled Receptor GPRC6A Based on the 2-Phenylindole Privileged Structure Scaffold

    DEFF Research Database (Denmark)

    Johansson, Henrik; Boesgaard, Michael Worch; Nørskov-Lauritsen, Lenea

    2015-01-01

    G protein-coupled receptors (GPCRs) represent a biological target class of fundamental importance in drug therapy. The GPRC6A receptor is a newly deorphanized class C GPCR that we recently reported for the first allosteric antagonists based on the 2-arylindole privileged structure scaffold (e.g., 1......, and 34b as antagonists at the GPRC6A receptor in the low micromolar range and show that 7 and 34b display >9-fold selectivity for the GPRC6A receptor over related GPCRs, making 7 and 34b the most potent and selective antagonists for the GPRC6A receptor reported to date....

  1. Structure-activity relationship of N-(phenylalkyl)cinnamides as novel NR2B subtype-selective NMDA receptor antagonists.

    Science.gov (United States)

    Tamiz, A P; Cai, S X; Zhou, Z L; Yuen, P W; Schelkun, R M; Whittemore, E R; Weber, E; Woodward, R M; Keana, J F

    1999-08-26

    A novel series of N-(phenylalkyl)cinnamides related to N-(4-phenylbutyl)-3,4-dihydroxy-beta-cyanocinnamide (6, an EGFR-K inhibitor with high antiproliferative activity) was synthesized and tested for antagonism at N-methyl-D-aspartate (NMDA) receptor subtypes. Potency and subunit selectivity were assayed by electrical recordings in Xenopus oocytes expressing three binary combinations of cloned rat NMDA receptor subunits: NR1A expressed in combination with either NR2A, NR2B, or NR2C. The N-(phenylalkyl)cinnamides are selective antagonists of NR1A/2B receptors. Assayed under steady-state conditions, N-(4-phenylbutyl)-4-hydroxycinnamide (16) has an IC(50) value of 77 nM and >1000-fold selectivity with respect to NR1A/2A and NR1A/2C receptors. Potency at alpha(1) adrenergic receptors is low for the four cinnamides tested. Inhibition of NR1A/2B receptors does not correlate with EGFR and ErbB2/neu tyrosine kinase inhibitor activity. The N-(phenylalkyl)cinnamide series we describe provides a novel and structurally diverse framework for designing new NR2B-selective NMDA antagonists as potential CNS therapeutics.

  2. Design, synthesis and pharmacological evaluation of indolylsulfonamide amines as potent and selective 5-HT6 receptor antagonists.

    Science.gov (United States)

    Nirogi, Ramakrishna V S; Bandyala, Thrinath Reddy; Reballi, Veena; Konda, Jagadishu Babu; Daulatabad, Anand V; Khagga, Mukkanti

    2015-02-01

    A series of N'-[3-(indole-1-sulfonyl) aryl]-N,N-dimethyl ethane-1,2-diamines and N'-[3-(indole-1-sulfonyl) aryl]-N,N-dimethyl propane-1,3-diamines was designed and synthesized as 5-HT6 receptor ligands. These compounds, when screened in a functional reporter gene-based assay, displayed potent antagonistic activity with Kb values in the range of 1.8-60 nM. The lead compound 9y has shown good ADME surrogate properties, acceptable pharmacokinetic profile and is active in animal models of cognition like novel object recognition test and Morris water maze. It was selected for detailed profiling.

  3. Effect of a new selective H1 receptor antagonist (levocabastine) in a nasal and conjunctival provocation test.

    Science.gov (United States)

    Pécoud, A; Zuber, P; Kolly, M

    1987-01-01

    Levocabastine is a new selective H1 receptor antagonist. The effect of the drug administered locally was compared to placebo in a quantified nasal and conjunctival provocation test with allergens performed in patients allergic to grass pollen. In the nasal provocation test, levocabastine was able to increase the 'reaction threshold' (dose of allergen necessary to trigger allergic symptoms) in 9 out of 12 patients; the drug inhibited rhinorrhea and sneezing, but not nasal obstruction. In the conjunctival provocation test, the 'reaction threshold' clearly increased in 10 out of 11 patients. The local administration of levocabastine might be useful in allergic rhinitis and conjunctivitis.

  4. Role of cholecystokinin and its antagonist proglumide on macronutrient selection in European sea bass Dicentrarchus labrax, L.

    Science.gov (United States)

    Rubio, V C; Sánchez-Vázquez, F J; Madrid, J A

    2008-03-18

    Teleost fish are able to adjust their energy intake when fed on pure macronutrient sources, although the exact mechanisms regulating macronutrient selection remain unknown. Since cholecystokinin (CCK) has been reported to modify macronutrient selection patterns in mammals, we explored the effect of CCK administered orally to European sea bass on the selection of separately encapsulated macronutrients. CCK doses of 0.05, 0.15 and 0.25 mg/kg BW administered in gelatine capsules for 5 consecutive days produced a significant inhibition of total food intake (21, 28 and 51%, respectively) at highest doses, evenly reducing the quantity of all the macronutrients ingested and, without affecting their relative proportions in the diet. Oral administration of proglumide, a non-specific CCK receptor antagonist, at doses of 5, 15 and 25 mg/kg BW, induced a quantitative total food intake increase of 2, 18 and 44%, respectively, and an increase of 52% in CH and 43% in P quantity ingested at highest dose. Co-administration of proglumide (25 mg/kg BW) and CCK (0.25 mg/kg BW) in a single preload capsule blocked the effects observed with CCK alone. In conclusion, orally administered CCK induced an anorexigenic effect on both total food and single macronutrient intake, an effect that is counteracted by the CCK antagonist proglumide.

  5. Label-free assessment of high-affinity antibody-antigen binding constants. Comparison of bioassay, SPR, and PEIA-ellipsometry

    NARCIS (Netherlands)

    Rispens, T.; te Velthuis, H.; Hemker, P.; Speijer, H.; Hermens, W.; Aarden, L.

    2011-01-01

    Assessment of high-affinity antibody-antigen binding parameters is important in such diverse areas as selection of therapeutic antibodies, detection of unwanted hormones in cattle and sensitive immunoassays in clinical chemistry. Label-free assessment of binding affinities is often carried out by

  6. Selective adenosine A2A receptor agonists and antagonists protect against spinal cord injury through peripheral and central effects

    Directory of Open Access Journals (Sweden)

    Esposito Emanuela

    2011-04-01

    Full Text Available Abstract Background Permanent functional deficits following spinal cord injury (SCI arise both from mechanical injury and from secondary tissue reactions involving inflammation. Enhanced release of adenosine and glutamate soon after SCI represents a component in the sequelae that may be responsible for resulting functional deficits. The role of adenosine A2A receptor in central ischemia/trauma is still to be elucidated. In our previous studies we have demonstrated that the adenosine A2A receptor-selective agonist CGS21680, systemically administered after SCI, protects from tissue damage, locomotor dysfunction and different inflammatory readouts. In this work we studied the effect of the adenosine A2A receptor antagonist SCH58261, systemically administered after SCI, on the same parameters. We investigated the hypothesis that the main action mechanism of agonists and antagonists is at peripheral or central sites. Methods Spinal trauma was induced by extradural compression of SC exposed via a four-level T5-T8 laminectomy in mouse. Three drug-dosing protocols were utilized: a short-term systemic administration by intraperitoneal injection, a chronic administration via osmotic minipump, and direct injection into the spinal cord. Results SCH58261, systemically administered (0.01 mg/kg intraperitoneal. 1, 6 and 10 hours after SCI, reduced demyelination and levels of TNF-α, Fas-L, PAR, Bax expression and activation of JNK mitogen-activated protein kinase (MAPK 24 hours after SCI. Chronic SCH58261 administration, by mini-osmotic pump delivery for 10 days, improved the neurological deficit up to 10 days after SCI. Adenosine A2A receptors are physiologically expressed in the spinal cord by astrocytes, microglia and oligodendrocytes. Soon after SCI (24 hours, these receptors showed enhanced expression in neurons. Both the A2A agonist and antagonist, administered intraperitoneally, reduced expression of the A2A receptor, ruling out the possibility that the

  7. βCCT, AN ANTAGONIST SELECTIVE FOR α1 GABAA RECEPTORS, REVERSES DIAZEPAM WITHDRAWAL-INDUCED ANXIETY IN RATS

    Science.gov (United States)

    Divljaković, Jovana; Milić, Marija; Namjoshi, Ojas A.; Tiruveedhula, Veera V.; Timić, Tamara; Cook, James M.; Savić, Miroslav M.

    2012-01-01

    The abrupt discontinuation of prolonged benzodiazepine treatment elicits a withdrawal syndrome with increased anxiety as a major symptom. The neural mechanisms underlying benzodiazepine physical dependence are still insufficiently understood. Flumazenil, the non-selective antagonist of the benzodiazepine binding site of GABAA receptors was capable of preventing and reversing the increased anxiety during benzodiazepine withdrawal in animals and humans in some, but not all studies. On the other hand, a number of data suggest that GABAA receptors containing α1 subunits are critically involved in processes developing during prolonged use of benzodiazepines, such are tolerance to sedative effects, liability to physical dependence and addiction. Hence, we investigated in the elevated plus maze the level of anxiety 24 h following 21 days of diazepam treatment and the influence of flumazenil or a preferential α1-subunit selective antagonist βCCt on diazepam withdrawal syndrome in rats. Abrupt cessation of protracted once-daily intraperitoneal administration of 2 mg/kg diazepam induced a withdrawal syndrome, measured by increased anxiety-like behavior in the elevated plus maze 24 h after treatment cessation. Acute challenge with either flumazenil (10 mg/kg) or βCCt (1.25, 5 and 20 mg/kg) alleviated the diazepam withdrawal-induced anxiety. Moreover, both antagonists induced an anxiolytic-like response close, though not identical, to that seen with acute administration of diazepam. These findings imply that the mechanism by which antagonism at GABAA receptors may reverse the withdrawal-induced anxiety involves the α1 subunit and prompt further studies aimed at linking the changes in behavior with possible adaptive changes in subunit expression and function of GABAA receptors. PMID:23149168

  8. A selective, non-peptide CRF receptor 1 antagonist prevents sodium lactate-induced acute panic-like responses.

    Science.gov (United States)

    Shekhar, Anantha; Johnson, Philip L; Fitz, Stephanie D; Nakazato, Atsuro; Chaki, Shigeyuki; Steckler, Thomas; Schmidt, Mark

    2011-04-01

    Corticotropin releasing factor (CRF) is implicated in a variety of stress-related disorders such as depression and anxiety, and blocking CRF receptors is a putative strategy for treating such disorders. Using a well-studied animal model of panic, we tested the efficacy of JNJ19567470/CRA5626, a selective, non-peptidergic CRF type 1 receptor (CRF1) antagonist (3, 10 and 40 mg/kg intraperitoneal injection), in preventing the sodium lactate (NaLac)-induced panic-like behavioural and cardiovascular responses. Adult male rats with chronic reduction of GABA levels (by inhibition of GABA synthesis with l-allyglycine, a glutamic acid decarboxylase inhibitor) in the dorsomedial/perifornical hypothalamus are highly anxious and exhibit physiological and behavioural responses to intravenous NaLac infusions similar to patients with panic disorder. These 'panic-prone' rats pre-treated with vehicle injections displayed NaLac-induced increases in autonomic responses (i.e. tachycardia and hypertensive responses), anxiety-like behaviour in the social interaction test, and flight-like increases in locomotor activity. However, systemically injecting such panic-prone rats with the highest dose of CRF1 receptor antagonist prior to NaLac infusions blocked all NaLac-induced behaviour and cardiovascular responses. These data suggest that selective CRF1 receptor antagonists could be a novel target for developing anti-panic drugs that are as effective as benzodiazepines in acute treatment of a panic attack without the deleterious side-effects (e.g. sedation and cognitive impairment) associated with benzodiazepines.

  9. The Saccharomyces cerevisiae High Affinity Phosphate Transporter Encoded by PHO84 Also Functions in Manganese Homeostasis

    National Research Council Canada - National Science Library

    Laran T. Jensen; Mispa Ajua-Alemanji; Valeria Cizewski Culotta

    2003-01-01

    ... . In a search for other genes involved in manganese homeostasis, PHO84 was identified. The PHO84 gene encodes a high affinity inorganic phosphate transporter, and we find that its disruption results in a manganese-resistant phenotype...

  10. Effects of lead on the kidney: Roles of high-affinity lead-binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    Fowler, B.A. (Univ. of Maryland, Baltimore (United States)); DuVal, G. (Univ of Maryland Medical School, Baltimore (United States))

    1991-02-01

    Lead-induced nephropathy produces both tubular and interstitial manifestations of cell injury, but the pathophysiology of these lesions is not completely understood. Delineation of the molecular factors underlying renal handling of lead is one of central importance in understanding the mechanisms of renal cell injury from this agent. Recent studies from this laboratory have identified several distinct high-affinity lead-binding proteins (PbBP) from rat kidney and brain that appear to play critical roles in the intracellular bioavailability of lead to several essential cellular processes in these target tissues at low dose levels. These studies have also shown that the real PbBP is selectively localized in only certain nephrons and only specific segments of the renal proximal tubule. The striking nephron and cell-type specificity of the localization reaction could result from physoiological differences in nephron functional activity or selective molecular uptake mechanisms/metabolism differences that act to define target cell populations in the kidney. In addition, other preliminary studies have shown that short-term, high-dose lead exposure produces increased excretion of this protein into the urine with concomitant decreases in renal concentrations.

  11. Effects of oral cetirizine, a selective H1 antagonist, on allergen- and exercise-induced bronchoconstriction in subjects with asthma.

    LENUS (Irish Health Repository)

    Gong, H

    1990-03-01

    The protective efficacy of oral cetirizine, a selective and potent H1-receptor antagonist, against the immediate bronchoconstrictive response to allergen inhalation and exercise challenge was evaluated in 16 subjects with stable, predominantly mild asthma. The subjects underwent double-blind, crossover pretreatments in randomized order in two separate protocols with (1) three daily oral doses of 20 mg of cetirizine and placebo, followed by allergen inhalation, and (2) single oral doses of cetirizine (5, 10, and 20 mg), albuterol (4 mg), and placebo, followed by exercise with cold-air inhalation. Cetirizine failed to decrease bronchial sensitivity to inhaled allergen in eight of 10 subjects. Neither cetirizine nor albuterol uniformly inhibited exercise-induced bronchoconstriction. Serum concentrations of cetirizine were consistent with systemic H1-blocking activity. Modest bronchodilation occurred after administration of cetirizine and albuterol before exercise but not after the third dose of cetirizine in the allergen protocol. One subject developed moderate drowsiness during multiple dosing with cetirizine. Thus, cetirizine, in the doses studied, is not uniformly effective in preventing allergen- or exercise-induced bronchoconstriction. Histamine is one of many mediators participating in immediate asthmatic responses, and selective H1 antagonists do not completely block these airway events. However, cetirizine may still clinically benefit some patients with asthma, such as patients with allergic rhinitis or urticaria.

  12. Isolation, identification and selection of antagonistic yeast against Alternaria alternata infection and tenuazonic acid production in wine grapes from Argentina.

    Science.gov (United States)

    Prendes, Luciana P; Merín, María G; Fontana, Ariel R; Bottini, Rubén A; Ramirez, María L; Morata de Ambrosini, Vilma I

    2017-11-08

    Epiphytic isolates with yeast characteristics from grapes of the Malbec cultivar were obtained in order to find antagonists against Alternaria alternata. From a total of 111 isolates, 82% corresponded to the yeast-like organism Aureobasidium pullulans and the rest to the non-Saccharomyces yeasts Hanseniaspora uvarum (6.3%), Metschnikowia pulcherrima or spp. (5.4%), Cryptoccocus laurentti II (2.7%), Starmerella bacilaris or Candida zemplinina (2.7%) and Rhodotorula spp. (0.9%). The 22.4% (15 out of 67) of epiphytic yeasts and yeast-like organisms evaluated were able to reduce A. alternata infection from 0.0 to 4.4% when applied 2h previous to pathogen inoculation on wounds of grape berries. From these selected strains, 14 out of 15 strains completely prevented A. alternata infection (0.0%), which implies potential for field application. All Metschnikowia (pulcherrima or spp.), S. bacillaris and almost all H. uvarum evaluated strains showed antagonist capability against A. alternata. Meanwhile, none of the lesser nutritional requirement strains belonging to A. pullulans, Cr. laurenti II and Rhodotorula spp. did. All the yeasts with capacity to prevent A. alternata infection also reduced tenuazonic acid (TA) production by 81.2 to 99.8%, finding TA levels similar to negative controls. Therefore, the epiphytic yeasts selected are promising as biological control agents against Alternaria infection and toxin production in grapes for winemaking. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Selection on an antagonistic behavioral trait can drive rapid genital coevolution in the burying beetle, Nicrophorus vespilloides.

    Science.gov (United States)

    Hopwood, Paul E; Head, Megan L; Jordan, Eleanor J; Carter, Mauricio J; Davey, Emma; Moore, Allen J; Royle, Nick J

    2016-06-01

    Male and female genital morphology varies widely across many taxa, and even among populations. Disentangling potential sources of selection on genital morphology is problematic because each sex is predicted to respond to adaptations in the other due to reproductive conflicts of interest. To test how variation in this sexual conflict trait relates to variation in genital morphology we used our previously developed artificial selection lines for high and low repeated mating rates. We selected for high and low repeated mating rates using monogamous pairings to eliminate contemporaneous female choice and male-male competition. Male and female genital shape responded rapidly to selection on repeated mating rate. High and low mating rate lines diverged from control lines after only 10 generations of selection. We also detected significant patterns of male and female genital shape coevolution among selection regimes. We argue that because our selection lines differ in sexual conflict, these results support the hypothesis that sexually antagonistic coevolution can drive the rapid divergence of genital morphology. The greatest divergence in morphology corresponded with lines in which the resolution of sexual conflict over mating rate was biased in favor of male interests. © 2016 The Author(s). Evolution published by Wiley Periodicals, Inc. on behalf of The Society for the Study of Evolution.

  14. Design and Synthesis of a Series of L-trans-4-Substituted Prolines as Selective Antagonists for the Ionotropic Glutamate Receptors Including Functional and X-ray Crystallographic Studies of New Subtype Selective Kainic Acid Receptor Subtype 1 (GluK1) Antagonist (2S,4R)-4-(2-Carboxyphenoxy)pyrrolidine-2-carboxylic Acid

    DEFF Research Database (Denmark)

    Krogsgaard-Larsen, Niels; Delgar, Claudia; Koch, Karina

    2017-01-01

    Ionotropic glutamate receptor antagonists are valuable tool compounds for studies of neurological pathways in the central nervous system. On the basis of rational ligand design, a new class of selective antagonists, represented by (2S,4R)-4-(2-carboxy-phenoxy)pyrrolidine-2-carboxylic acid (1b), f...

  15. Preparation of carbon-11 labelled prazosin, a potent and selective. cap alpha. /sub 1/-adrenoreceptor antagonist

    Energy Technology Data Exchange (ETDEWEB)

    Ehrin, E.; Crouzel, C.; Luthra, S.K.; Pike, V.W.

    1988-02-01

    The ..cap alpha../sub 1/-adrenoreceptor antagonist, Prazosin : 2-(4-(2-furoyl) piperazin-1-yl)-4-amino-6,7-dimethoxyquinazoline, has been labelled with carbon-11 for in vivo studies of ..cap alpha../sub 1/-adrenoreceptors using positron emission tomography. The preparation of (2-/sup 11/C) furoyl chloride, from cyclotron-produced (/sup 11/C) carbon dioxide, and its reaction with the secondary amine, 2-(piperazin-1-yl)-4-amino-6,7-dimethoxyquinazoline, provides a fast (35 min) route to carbon-11 labelled prazosin in high radiochemical yield (30-40%, decay-corrected) with high specific activity (26-37 GBq..mu..mol, 0.7-1.0 Ci..mu..mol).

  16. The GABAA Antagonist DPP-4-PIOL Selectively Antagonises Tonic over Phasic GABAergic Currents in Dentate Gyrus Granule Cells

    DEFF Research Database (Denmark)

    Boddum, Kim; Frølund, Bente; Kristiansen, Uffe

    2014-01-01

    GABAA receptors mediate two different types of inhibitory currents: phasic inhibitory currents when rapid and brief presynaptic GABA release activates postsynaptic GABAA receptors and tonic inhibitory currents generated by low extrasynaptic GABA levels, persistently activating extrasynaptic GABAA...... receptors. The two inhibitory current types are mediated by different subpopulations of GABAA receptors with diverse pharmacological profiles. Selective antagonism of tonic currents is of special interest as excessive tonic inhibition post-stroke has severe pathological consequences. Here we demonstrate...... that phasic and tonic GABAA receptor currents can be selectively inhibited by the antagonists SR 95531 and the 4-PIOL derivative, 4-(3,3-diphenylpropyl)-5-(4-piperidyl)-3-isoxazolol hydrobromide (DPP-4-PIOL), respectively. In dentate gyrus granule cells, SR 95531 was found approximately 4 times as potent...

  17. Selective κ opioid antagonists nor-BNI, GNTI and JDTic have low affinities for non-opioid receptors and transporters.

    Directory of Open Access Journals (Sweden)

    Thomas A Munro

    Full Text Available Nor-BNI, GNTI and JDTic induce selective κ opioid antagonism that is delayed and extremely prolonged, but some other effects are of rapid onset and brief duration. The transient effects of these compounds differ, suggesting that some of them may be mediated by other targets.In binding assays, the three antagonists showed no detectable affinity (K(i≥10 µM for most non-opioid receptors and transporters (26 of 43 tested. There was no non-opioid target for which all three compounds shared detectable affinity, or for which any two shared sub-micromolar affinity. All three compounds showed low nanomolar affinity for κ opioid receptors, with moderate selectivity over μ and δ (3 to 44-fold. Nor-BNI bound weakly to the α(2C-adrenoceptor (K(i = 630 nM. GNTI enhanced calcium mobilization by noradrenaline at the α(1A-adrenoceptor (EC₅₀ = 41 nM, but did not activate the receptor, displace radioligands, or enhance PI hydrolysis. This suggests that it is a functionally-selective allosteric enhancer. GNTI was also a weak M₁ receptor antagonist (K(B = 3.7 µM. JDTic bound to the noradrenaline transporter (K(i = 54 nM, but only weakly inhibited transport (IC₅₀ = 1.1 µM. JDTic also bound to the opioid-like receptor NOP (K(i = 12 nM, but gave little antagonism even at 30 µM. All three compounds exhibited rapid permeation and active efflux across Caco-2 cell monolayers.Across 43 non-opioid CNS targets, only GNTI exhibited a potent functional effect (allosteric enhancement of α(1A-adrenoceptors. This may contribute to GNTI's severe transient effects. Plasma concentrations of nor-BNI and GNTI may be high enough to affect some peripheral non-opioid targets. Nonetheless, κ opioid antagonism persists for weeks or months after these transient effects dissipate. With an adequate pre-administration interval, our results therefore strengthen the evidence that nor-BNI, GNTI and JDTic are highly selective κ opioid antagonists.

  18. High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination.

    Science.gov (United States)

    Kaur, Kaval; Zheng, Nai-Ying; Smith, Kenneth; Huang, Min; Li, Lie; Pauli, Noel T; Henry Dunand, Carole J; Lee, Jane-Hwei; Morrissey, Michael; Wu, Yixuan; Joachims, Michelle L; Munroe, Melissa E; Lau, Denise; Qu, Xinyan; Krammer, Florian; Wrammert, Jens; Palese, Peter; Ahmed, Rafi; James, Judith A; Wilson, Patrick C

    2015-01-01

    Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus.

  19. Enhanced membrane pore formation through high-affinity targeted antimicrobial peptides.

    Directory of Open Access Journals (Sweden)

    Christopher J Arnusch

    Full Text Available Many cationic antimicrobial peptides (AMPs target the unique lipid composition of the prokaryotic cell membrane. However, the micromolar activities common for these peptides are considered weak in comparison to nisin, which follows a targeted, pore-forming mode of action. Here we show that AMPs can be modified with a high-affinity targeting module, which enables membrane permeabilization at low concentration. Magainin 2 and a truncated peptide analog were conjugated to vancomycin using click chemistry, and could be directed towards specific membrane embedded receptors both in model membrane systems and whole cells. Compared with untargeted vesicles, a gain in permeabilization efficacy of two orders of magnitude was reached with large unilamellar vesicles that included lipid II, the target of vancomycin. The truncated vancomycin-peptide conjugate showed an increased activity against vancomycin resistant Enterococci, whereas the full-length conjugate was more active against a targeted eukaryotic cell model: lipid II containing erythrocytes. This study highlights that AMPs can be made more selective and more potent against biological membranes that contain structures that can be targeted.

  20. The chromosomal high-affinity binding sites for the Drosophila dosage compensation complex.

    Directory of Open Access Journals (Sweden)

    Tobias Straub

    2008-12-01

    Full Text Available Dosage compensation in male Drosophila relies on the X chromosome-specific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC. The principles that assure selective targeting of the DCC are unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a limited number of so-called "high-affinity sites" (HAS. Only very few such sites are known at the DNA sequence level, which has precluded the definition of DCC targeting principles. Combining RNA interference against DCC subunits, limited crosslinking, and chromatin immunoprecipitation coupled to probing high-resolution DNA microarrays, we identified a set of 131 HAS for MSL1 and MSL2 and confirmed their properties by various means. The HAS sites are distributed all over the X chromosome and are functionally important, since the extent of dosage compensation of a given gene and its proximity to a HAS are positively correlated. The sites are mainly located on non-coding parts of genes and predominantly map to regions that are devoid of nucleosomes. In contrast, the bulk of DCC binding is in coding regions and is marked by histone H3K36 methylation. Within the HAS, repetitive DNA sequences mainly based on GA and CA dinucleotides are enriched. Interestingly, DCC subcomplexes bind a small number of autosomal locations with similar features.

  1. Synthesis of site-heterologous haptens for high-affinity anti-pyraclostrobin antibody generation.

    Science.gov (United States)

    Mercader, Josep V; Agulló, Consuelo; Abad-Somovilla, Antonio; Abad-Fuentes, Antonio

    2011-03-07

    The design and synthesis of functional chemical derivatives of small organic molecules is usually a key step for the intricate production of a variety of bioconjugates. In this respect, the derivatization site at which the spacer arm is introduced in immunizing conjugates constitutes a highly critical parameter for the generation of high-affinity and selective antibodies. However, due to the usual complexity of the required synthetic procedures, the appropriate comparison of alternative tethering positions has often been neglected. In the present study, meticulous strategies were followed to prepare synthetic derivatives of pyraclostrobin with the same linkers located at diverse rationally-chosen sites. Activity appraisal of antibodies and bioconjugates was carried out by bidimensional competitive direct and indirect immunoassays, and a superior performance of two of the three synthesized haptens was found. Finally, a detailed analysis of the conformations of the target molecule and the synthesized haptens in aqueous solution was done using computer assisted molecular modeling techniques. This study suggested that the lower titers and affinities of one set of antibodies are most probably due to conformational effects of the spacer arm in the immunizing bioconjugate.

  2. High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination.

    Directory of Open Access Journals (Sweden)

    Kaval Kaur

    Full Text Available Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE. Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus.

  3. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

    Directory of Open Access Journals (Sweden)

    Weonu Choe

    2016-12-01

    Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

  4. Selection of optimal hydrate/solvate forms of a fibrinogen receptor antagonist for solid dosage development.

    Science.gov (United States)

    Bray, M L; Jahansouz, H; Kaufman, M J

    1999-01-01

    The objective of this work was to compare the physicochemical properties of four crystalline forms of the fibrinogen receptor antagonist L-738,167 [2(S)-[p-toluenesulfonyl amino]-3-[[[5,6,7,8-tetrahydro-4-oxo-5-[2-(piperidin-4-yl)ethyl] -4-H-pyrazolo[1,5-a][1,4] diazepin-2-yl] carbonyl]amino]-propionic acid] to determine the best form for use in the development of oral dosage formulations. Four crystalline forms [form A (trihydrate), form B (pentahydrate), form C, and form D] were compared using x-ray powder diffractometry, thermal analysis, and moisture sorption studies. The trihydrate, form A, was demonstrated to hydrate upon exposure to relative humidity (RH) above 50% at room temperature (25 degrees C) with conversion to the pentahydrate. The pentahydrate, form B, converted to the trihydrate at room temperature when exposed to humidity levels below 25% RH. The crystalline pentahydrate was shown to be stable to dehydration upon storage at 30 degrees C/60% RH and 40 degrees C/75% RH for 3 months. The suspension of form A or form D in water resulted in conversion to form B, the stable hydrated form in an aqueous environment. Form C has a unique crystalline structure that is stable in an aqueous environment and not subject to hydration/dehydration with changes in relative humidity and thus may offer some advantages in pharmaceutical development.

  5. Molecular Basis for Binding and Subtype Selectivity of 1,4-Benzodiazepine Antagonist Ligands of the Cholecystokinin Receptor*

    Science.gov (United States)

    Cawston, Erin E.; Lam, Polo C. H.; Harikumar, Kaleeckal G.; Dong, Maoqing; Ball, Alicja M.; Augustine, Mary Lou; Akgün, Eyup; Portoghese, Philip S.; Orry, Andrew; Abagyan, Ruben; Sexton, Patrick M.; Miller, Laurence J.

    2012-01-01

    Allosteric binding pockets in peptide-binding G protein-coupled receptors create opportunities for the development of small molecule drugs with substantial benefits over orthosteric ligands. To gain insights into molecular determinants for this pocket within type 1 and 2 cholecystokinin receptors (CCK1R and CCK2R), we prepared a series of receptor constructs in which six distinct residues in TM2, -3, -6, and -7 were reversed. Two novel iodinated CCK1R- and CCK2R-selective 1,4-benzodiazepine antagonists, differing only in stereochemistry at C3, were used. When all six residues within CCK1R were mutated to corresponding CCK2R residues, benzodiazepine selectivity was reversed, yet peptide binding selectivity was unaffected. Detailed analysis, including observations of gain of function, demonstrated that residues 6.51, 6.52, and 7.39 were most important for binding the CCK1R-selective ligand, whereas residues 2.61 and 7.39 were most important for binding CCK2R-selective ligand, although the effect of substitution of residue 2.61 was likely indirect. Ligand-guided homology modeling was applied to wild type receptors and those reversing benzodiazepine binding selectivity. The models had high predictive power in enriching known receptor-selective ligands from related decoys, indicating a high degree of precision in pocket definition. The benzodiazepines docked in similar poses in both receptors, with C3 urea substituents pointing upward, whereas different stereochemistry at C3 directed the C5 phenyl rings and N1 methyl groups into opposite orientations. The geometry of the binding pockets and specific interactions predicted for ligand docking in these models provide a molecular framework for understanding ligand selectivity at these receptor subtypes. Furthermore, the strong predictive power of these models suggests their usefulness in the discovery of lead compounds and in drug development programs. PMID:22467877

  6. Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: Characterization of an active digitonin-solubilized receptor complex

    Energy Technology Data Exchange (ETDEWEB)

    Grigoriadis, D.E.; Zaczek, R.; Pearsall, D.M.; De Souza, E.B. (National Institute on Drug Abuse, Baltimore, MD (USA))

    1989-12-01

    The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of (125I)Tyro-ovine CRF ((125I)oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for (125I) oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, (125I)oCRF labeled the same size receptor complex.

  7. Discovery of subtype selective muscarinic receptor antagonists as alternatives to atropine using in silico pharmacophore modeling and virtual screening methods.

    Science.gov (United States)

    Bhattacharjee, Apurba K; Pomponio, James W; Evans, Sarah A; Pervitsky, Dmitry; Gordon, Richard K

    2013-05-01

    Muscarinic acetylcholine receptors (mAChRs) have five known subtypes which are widely distributed in both the peripheral and central nervous system for regulation of a variety of cholinergic functions. Atropine is a well known muscarinic subtype non-specific antagonist that competitively inhibits acetylcholine (ACh) at postganglionic muscarinic sites. Atropine is used to treat organophosphate (OP) poisoning and resulting seizures in the warfighter because it competitively inhibits acetylcholine (ACh) at the muscarinic cholinergic receptors. ACh accumulates due to OP inhibition of acetylcholinesterase (AChE), the enzyme that hydrolyzes ACh. However, atropine produces several unwanted side-effects including dilated pupils, blurred vision, light sensitivity, and dry mouth. To overcome these side-effects, our goal was to find an alternative to atropine that emphasizes M1 (seizure prevention) antagonism but has minimum M2 (cardiac) and M3 (e.g., eye) antagonism so that an effective less toxic medical countermeasure may be developed to protect the warfighter against OP and other chemical warfare agents (CWAs). We adopted an in silico pharmacophore modeling strategy to develop features that are characteristics of known M1 subtype-selective compounds and used the model to identify several antagonists by screening an in-house (WRAIR-CIS) compound database. The generated model for the M1 selectivity was found to contain two hydrogen bond acceptors, one aliphatic hydrophobic, and one ring aromatic feature distributed in a 3D space. From an initial identification of about five hundred compounds, 173 compounds were selected through principal component and cluster analyses and in silico ADME/Toxicity evaluations. Next, these selected compounds were evaluated in a subtype-selective in vitro radioligand binding assay. Twenty eight of the compounds showed antimuscarinic activity. Nine compounds showed specificity for M1 receptors and low specificity for M3 receptors. The p

  8. Design and synthesis of labeled analogs of PhTX-56, a potent and selective AMPA receptor antagonist

    DEFF Research Database (Denmark)

    Andersen, Trine F; Vogensen, Stine B; Jensen, Lars S

    2005-01-01

    acetylcholine receptors. Polyamine toxins are used for the characterization of subtypes of ionotropic glutamate receptors, the Ca2+-permeable AMPA and kainate receptors. A derivative of the native polyamine toxin, philanthotoxin-56 (PhTX-56), has recently been shown to be an exceptionally potent and selective...... antagonist of Ca2+-permeable AMPA receptors. PhTX-56 and its labeled derivatives are promising tools for structure-function studies of the ion channel of the AMPA receptor. We now describe the design and synthesis of 3H-, 13C-, and 15N-labeled derivatives of PhTX-56 for molecular level studies of AMPA....... These analogs can provide detailed information on the ligand-receptor interaction. In conclusion, synthesis of labeled derivatives of PhTX-56 provides important tools for future studies of the pore-forming region of AMPA receptors....

  9. Design, synthesis, biological evaluation, and comparative docking study of 1,2,4-triazolones as CB1 receptor selective antagonists.

    Science.gov (United States)

    Han, Shuang; Zhang, Fei-Fei; Xie, Xin; Chen, Jian-Zhong

    2014-03-03

    Cannabinoids are potentially useful for the treatment of several diseases. In the present work, we report the syntheses and biological evaluations of 1,2,4-triazolone derivatives designed using a combined approach of scaffold hopping and pharmacophore-oriented method. These compounds exhibited interesting antagonistic activity to the cannabinoid CB1 receptor. The preliminary structure-activity relationships were further discussed. In addition, docking simulations were performed on the good bioactive compound 5c and the low potent compound 5d, respectively, on the basis of homology models of the CB1 and CB2 receptors, which were constructed based on human β2-adrenoreceptor and optimized in a membrane environment by MD simulations. Calculation of the binding modes gave us insights into the structural requirements for improving the cannabinoid receptor bioactivity and selectivity. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  10. Evaluation of AMG 076, a potent and selective MCHR1 antagonist, in rodent and primate obesity models.

    Science.gov (United States)

    Motani, Alykhan S; Luo, Jian; Liang, Lingming; Mihalic, Jeffrey T; Chen, Xiaoqi; Tang, Liang; Li, Leping; Jaen, Juan; Chen, Jin-Long; Dai, Kang

    2013-10-01

    Melanin-concentrating hormone (MCH) regulates food intake through activation of the receptor, MCHR1. We have identified AMG 076 as an orally bioavailable potent and selective small molecule antagonist of MCHR1. In mouse models of obesity, AMG 076 caused a reduction in body weight gain in wild-type (MCHR1+/+) but not in knockout (MCHR1-/-) mice. The body weight reduction was associated with decreases in food intake and increases in energy expenditure. Importantly, we show that these MCHR1-dependent effects of AMG 076 were also reflected in improved metabolic phenotypes, increased glucose tolerance and insulin sensitivity. Preliminary data on effects of AMG 076 in obese cynomolgus monkeys are also presented.

  11. In vivo pharmacological characterisation of bilastine, a potent and selective histamine H1 receptor antagonist.

    Science.gov (United States)

    Corcóstegui, Reyes; Labeaga, Luis; Innerárity, Ana; Berisa, Agustín; Orjales, Aurelio

    2006-01-01

    We set out to establish the in vivo histamine H(1) receptor antagonistic (antihistaminic) and antiallergic properties of bilastine. In vivo antihistaminic activity experiments consisted of measurement of: inhibition of increase in capillary permeability and reduction in microvascular extravasation and bronchospasm in rats and guinea pigs induced by histamine and other inflammatory mediators; and protection against lethality induced by histamine and other inflammatory mediators in rats. In vivo antiallergic activity experiments consisted of measurement of passive and active cutaneous anaphylactic reactions as well as type III and type IV allergic reactions in sensitised rodents. In the in vivo antihistaminic activity experiments, bilastine was shown to have a positive effect, similar to that of cetirizine and more potent than that of fexofenadine. The results of the in vivo antiallergic activity experiments showed that the properties of bilastine in this setting are similar to those observed for cetirizine and superior to fexofenadine in the model of passive cutaneous anaphylactic reaction. When active cutaneous anaphylactic reaction experiments were conducted, bilastine showed significant activity, less potent than that observed with cetirizine but superior to that of fexofenadine. Evaluation of the type III allergic reaction showed that of the antihistamines only bilastine was able to inhibit oedema in sensitised mice, although its effect in this respect was much less potent than that observed with dexamethasone. In terms of the type IV allergic reaction, neither bilastine, cetirizine nor fexofenadine significantly modified the effect caused by oxazolone. The results of our in vivo preclinical studies corroborate those obtained from previously conducted in vitro experiments of bilastine, and provide evidence that bilastine possesses antihistaminic as well as antiallergic properties, with similar potency to cetirizine and superior potency to fexofenadine.

  12. Increased orbitofrontal brain activation after administration of a selective adenosine A2A antagonist in cocaine dependent subjects

    Directory of Open Access Journals (Sweden)

    F. Gerard eMoeller

    2012-05-01

    Full Text Available Background: Positron Emission Tomography imaging studies provide evidence of reduced dopamine function in cocaine dependent subjects in the striatum, which is correlated with prefrontal cortical glucose metabolism, particularly in the orbitofrontal cortex. However, whether enhancement of dopamine in the striatum in cocaine dependent subjects would be associated with changes in prefrontal cortical brain activation is unknown. One novel class of medications that enhance dopamine function via heteromer formation with dopamine receptors in the striatum is the selective adenosine A2A receptor antagonists. This study sought to determine the effects administration of the selective adenosine A2A receptor antagonist SYN115 on brain function in cocaine dependent subjects. Methodology/Principle Findings: Twelve cocaine dependent subjects underwent two fMRI scans (one after a dose of placebo and one after a dose of 100 mg of SYN115 while performing a working memory task with 3 levels of difficulty (3, 5, and 7 digits. fMRI results showed that for 7-digit working memory activation there was significantly greater activation from SYN115 compared to placebo in portions of left (L lateral orbitofrontal cortex, L insula, and L superior and middle temporal pole. Conclusion/Significance: These findings are consistent with enhanced dopamine function in the striatum in cocaine dependent subjects via blockade of adenosine A2A receptors producing increased brain activation in the orbitofrontal cortex and other cortical regions. This suggests that at least some of the changes in brain activation in prefrontal cortical regions in cocaine dependent subjects may be related to altered striatal dopamine function, and that enhancement of dopamine function via adenosine A2A receptor blockade could be explored further for amelioration of neurobehavioral deficits associated with chronic cocaine use.

  13. GHB receptor targets in the CNS: Focus on high-affinity binding sites

    DEFF Research Database (Denmark)

    Bay, Tina; Eghorn, Laura Friis; Klein, Anders Bue

    2014-01-01

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects...... effects. In this research update, a description of the various reported receptors for GHB is provided, including GABAB receptors, certain GABAA receptor subtypes and other reported GHB receptors. The main focus will thus be on the high-affinity binding targets for GHB and their potential functional roles...

  14. High-affinity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions.

    Science.gov (United States)

    Cailliatte, Rémy; Schikora, Adam; Briat, Jean-François; Mari, Stéphane; Curie, Catherine

    2010-03-01

    In contrast with many other essential metals, the mechanisms of Mn acquisition in higher eukaryotes are seldom studied and poorly understood. We show here that Arabidopsis thaliana relies on a high-affinity uptake system to acquire Mn from the soil in conditions of low Mn availability and that this activity is catalyzed by the divalent metal transporter NRAMP1 (for Natural Resistance Associated Macrophage Protein 1). The nramp1-1 loss-of-function mutant grows poorly, contains less Mn than the wild type, and fails to take up Mn in conditions of Mn limitation, thus demonstrating that NRAMP1 is the major high-affinity Mn transporter in Arabidopsis. Based on confocal microscopy observation of an NRAMP1-green fluorescent protein fusion, we established that NRAMP1 is localized to the plasma membrane. Consistent with its function in Mn acquisition from the soil, NRAMP1 expression is restricted to the root and stimulated by Mn deficiency. Finally, we show that NRAMP1 restores the capacity of the iron-regulated transporter1 mutant to take up iron and cobalt, indicating that NRAMP1 has a broad selectivity in vivo. The role of transporters of the NRAMP family is well established in higher eukaryotes for iron but has been controversial for Mn. This study demonstrates that NRAMP1 is a physiological manganese transporter in Arabidopsis.

  15. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

    Directory of Open Access Journals (Sweden)

    Eszter Lázár-Molnár

    2017-03-01

    Full Text Available Programmed Cell Death-1 (PD-1 is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1 exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

  16. Nickel(II) Inhibits Tet-Mediated 5-Methylcytosine Oxidation by High Affinity Displacement of the Cofactor Iron(II).

    Science.gov (United States)

    Yin, Ruichuan; Mo, Jiezhen; Dai, Jiayin; Wang, Hailin

    2017-06-16

    Ten-eleven translocation (Tet) family proteins are Fe(II)- and 2-oxoglutarate-dependent dioxygenases that regulate the dynamics of DNA methylation by catalyzing the oxidation of DNA 5-methylcytosine (5mC). To exert physiologically important functions, redox-active iron chelated in the catalytic center of Tet proteins directly involves the oxidation of the multiple substrates. To understand the function and interaction network of Tet dioxygenases, it is interesting to obtain high affinity and a specific inhibitor. Surprisingly, here we found that natural Ni(II) ion can bind to the Fe(II)-chelating motif (HXD) with an affinity of 7.5-fold as high as Fe(II). Consistently, we further found that Ni(II) ion can displace the cofactor Fe(II) of Tet dioxygenases and inhibit Tet-mediated 5mC oxidation activity with an estimated IC50 of 1.2 μM. Essentially, Ni(II) can be used as a high affinity and selective inhibitor to explore the function and dynamics of Tet proteins.

  17. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Lázár-Molnár, Eszter; Scandiuzzi, Lisa; Basu, Indranil; Quinn, Thomas; Sylvestre, Eliezer; Palmieri, Edith; Ramagopal, Udupi A.; Nathenson, Stanley G.; Guha, Chandan; Almo, Steven C.

    2017-03-01

    Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

  18. Discovery of 3,4-Dihydropyrimidin-2(1H)-ones As a Novel Class of Potent and Selective A2B Adenosine Receptor Antagonists.

    Science.gov (United States)

    Crespo, Abel; El Maatougui, Abdelaziz; Biagini, Pierfrancesco; Azuaje, Jhonny; Coelho, Alberto; Brea, José; Loza, María Isabel; Cadavid, María Isabel; García-Mera, Xerardo; Gutiérrez-de-Terán, Hugo; Sotelo, Eddy

    2013-11-14

    We describe the discovery and optimization of 3,4-dihydropyrimidin-2(1H)-ones as a novel family of (nonxanthine) A2B receptor antagonists that exhibit an unusually high selectivity profile. The Biginelli-based hit optimization process enabled a thoughtful exploration of the structure-activity and structure-selectivity relationships for this chemotype, enabling the identification of ligands that combine structural simplicity with excellent hA2B AdoR affinity and remarkable selectivity profiles.

  19. Probes for narcotic receptor mediated phenomena 22. Pt.1: Synthesis and characterization of optically pure [{sup 3}H](+)-4-[({alpha}R)-{alpha}-((2S,5R)-4-propyl-2,5-dimethyl-1-pi perazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide, [{sup 3}H]SNC 121, a novel high affinity and selective ligand for delta opioid receptors

    Energy Technology Data Exchange (ETDEWEB)

    Calderon, S.N.; Bertha, C.M.; Rice, K.C. [National Inst. of Diabetes and Digestive and Kidney Diseases, Medicinal Chemistry Lab., Bethesda, MD (United States); Gutkind, J.S. [National Inst. of Dental Research, Bethesda, MD (United States); Heng Xu; Partilla, J.S.; Rothman, R.B. [National Inst. on Drug Abuse, Clinical Psychopharmacology Section, Baltimore, MD (United States)

    1996-09-01

    The synthesis of unlabelled and labelled SNC 121, a selective nonpeptide ligand for the delta opioid receptor is reported. [{sup 3}H]SNC 121 of specific activity of 26.8 Ci/mmol, was synthesized by catalytic tritiation of the optically pure precursor SNC 80. (author).

  20. Bisphenol A binds to Ras proteins and competes with guanine nucleotide exchange: implications for GTPase-selective antagonists.

    Science.gov (United States)

    Schöpel, Miriam; Jockers, Katharina F G; Düppe, Peter M; Autzen, Jasmin; Potheraveedu, Veena N; Ince, Semra; Yip, King Tuo; Heumann, Rolf; Herrmann, Christian; Scherkenbeck, Jürgen; Stoll, Raphael

    2013-12-12

    We show for the first time that bisphenol A (10) has the capacity to interact directly with K-Ras and that Rheb weakly binds to bisphenol A (10) and 4,4'-biphenol derivatives. We have characterized these interactions at atomic resolution suggesting that these compounds sterically interfere with the Sos-mediated nucleotide exchange in H- and K-Ras. We show that 4,4'-biphenol (5) selectively inhibits Rheb signaling and induces cell death suggesting that this compound might be a novel candidate for treatment of tuberous sclerosis-mediated tumor growth. Our results propose a new mode of action for bisphenol A (10) that advocates a reduced exposure to this compound in our environment. Our data may lay the foundation for the future design of GTPase-selective antagonists with higher affinity to benefit of the treatment of cancer because K-Ras inhibition is regarded to be a promising strategy with a potential therapeutic window for targeting Sos in Ras-driven tumors.

  1. Targeted Blockade of TARP-g8-Associated AMPA Receptors: Anticonvulsant Activity with the Selective Antagonist LY3130481 (CERC-611).

    Science.gov (United States)

    Witkin, Jeffrey M; Schober, Douglas A; Gleason, Scott D; Catlow, John T; Porter, Warren J; Reel, Jon; Jin, X; Hobbs, J; Gehlert, Donald; Gernert, Douglas L; Gardinier, Kevin M; Kato, Akihiko S; Ping, X; Smith, J L

    2017-11-01

    6-[(1S)-1-[1-[5-(2-hydroxyethoxy)-2-pyridyl]pyrazol-3-yl]ethyl]-3H-1,3-benzothiazol-2-one (LY3130481 or CERC-611) is a selective antagonist of AMPA receptors containing transmembrane AMPA receptor regulatory protein (TARP) g-8 that is under development for epilepsy. The present study provided a broad inquiry into its anticonvulsant properties. LY3130481 was anticonvulsant in multiple acute seizure provocation models in mice and rats. In addition, LY3130481 was effective against absence seizures in the GAERS genetic model and in the Frings mouse model. Likewise LY3130481 attenuated convulsions in mice and rats with long term induction of seizures (e.g., corneal, pentylenetetrazole, hippocampal, and amygdala kindled seizures). In slices of epileptic human cortex, LY3130481 significantly decreased neuronal firing frequencies. LY3130481 displaced from rat brain a radioligand specific for AMPA receptors associated with TARP g-8 whereas non-TARP-selective molecules did not. Binding was also observed in hippocampus freshly transected from a patient Taken as a whole, the findings reported here establish the broad anticonvulsant efficacy of LY3130481 indicating that blockade of AMPA receptors associated with TARP g-8 is sufficient for these protective effects. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. Selection of oral microbial adhesion antagonists using biotinylated Streptococcus sanguis and a human mixed oral microflora.

    Science.gov (United States)

    Guan, Y H; de Graaf, T; Lath, D L; Humphreys, S M; Marlow, I; Brook, A H

    2001-02-01

    A microtitre assay has been developed using hydroxyapatite-coated wells and Streptococcus sanguis NCTC 10904 at 10(7) cells per ml. A number of models representing toothpaste and mouthwash usage were adopted to detect the anti-adherent efficacy of a polyvinylmethylether maleic acid copolymer (PVM/MA), polyoxypropylene/polyoxyethylene block copolymer (PO/EO), two casein-derived peptides and selected silicones. The results not only confirmed the anti-adherence property of the selected components but also indicated possible molecular interactions leading to the observed performance. To account for the diversity of oral microbial cells in vivo, a further testing system was developed. This involved submerging a hydroxyapatite disc in a mixed culture of human salivary microbial cells, and exposing it to different treatments using the active component either in an aqueous dispersion or in a toothpaste. The effect of toothpastes containing PO/EO, dimethicone copoyol or PVM/MA was investigated over a 4-h incubation with microflora. These tests showed that in a toothpaste formulation the anti-adherent efficacy may be reduced when compared with an aqueous dispersion containing the same or nearly the same concentration of the active component.

  3. Evidence for a precursor of the high-affinity metastasis-associated murine laminin receptor

    DEFF Research Database (Denmark)

    Rao, C N; Castronovo, V; Schmitt, M C

    1989-01-01

    The high-affinity cellular receptor for the basement membrane component laminin is differentially expressed during tumor invasion and metastasis. A cDNA clone encoding the murine laminin receptor was isolated and identified on the basis of sequence homology to the human laminin receptor [Wewer et...

  4. High affinity calmodulin target sequence in the signalling molecule PI 3-kinase

    DEFF Research Database (Denmark)

    Fischer, R; Julsgart, J; Berchtold, M W

    1998-01-01

    M-binding peptide derived from the p110gamma isoform interacts with CaM in a calcium-dependent way. Using gel shift analysis and fluorescence spectrophotometry we discovered that the peptide forms a high affinity complex with CaM. Titration experiments using dansylated CaM gave an affinity constant of 5 n...

  5. [Peroxidative vulnerability of synaptosomal high affinity Ca++-ATPase and pharmacologic effects].

    Science.gov (United States)

    Blaschke, M; Fischer, H D; Schmidt, J

    1988-01-01

    The high affinity Ca++-ATPase participates essentially in the regulation of intrasynaptosomal calcium homeostasis. Related to posthypoxically restricted transmitter release, we examined the influence of newly-generated free radicals (ascorbic acid-ferric salt mixture) or sodium dodecyl sulfate in vitro and of a mild hypobaric hypoxia in vivo on the activity of synaptosomal high affinity Ca++-ATPase. Moreover we tested the effectiveness of piracetam, meclofenoxate hydrochloride, pyritinol and verapamil on the changed enzyme activity subsequent to a hypoxic exposure. The activity of synaptosomal high affinity Ca++-ATPase (1.04 +/- 0.03 mumol Pi/mg.h) is reduced by not more than 40% depending on the concentration of the ascorbic acid-ferric salt mixture used but is nearly totally inhibited by sodium dodecyl sulfate (0.2 mg/ml). Hypobaric hypoxia (18 h, 8.7 kPa) decreases the enzyme activity to 0.79 +/- 0.03 mumol Pi/mg.h. Piracetam, meclofenoxate hydrochloride and pyritinol are protectively effective on the decrease of enzyme activity induced by hypoxia. The results emphasize the importance of intact protein-phospholipid interactions for the enzyme activity and support relations between synaptosomal high affinity Ca++-ATPase and transmitter release.

  6. Marker assisted selection for the improvement of two antagonistic traits under mixed inheritance

    Directory of Open Access Journals (Sweden)

    Verrier Etienne

    2001-01-01

    Full Text Available Abstract A Monte Carlo simulation was used to investigate the potential of Marker Assisted Selection (MAS in a multiple-trait situation. Only additive effects were considered. The base population was assumed to be in linkage equilibrium and, next, the population was managed over 15 discrete generations, 10 males and 50 females were chosen out of the 100 candidates of each sex. Performance for two traits was simulated with an overall heritability of a given trait equal to 0.25 or 0.10 and the overall genetic correlation between traits was generally equal to -0.4 except in one case where it was equal to 0. The model involved one biallelic QTL, accounting for 10 or 20% of the genetic variance of a given trait, plus polygenes. Initial allelic frequencies at the QTL were generally equal to 0.5 but in one case were equal to 0.1 and 0.9. A marker with 120 different alleles in the 60 founder parents was simulated in the vicinity of the QTL. Two values of the recombination rate between these two loci were considered, 0.10 and 0.02. The genetic evaluation was based on a multiple-trait BLUP animal model, accounting (MAS or not (conventional BLUP for marker information. Two sets of simulations were run: (1 a "missing data"case, with males having no record for one of the traits, and (2 a "secondary trait"case, with one trait having a weight in the aggregate genotype 4 times less than the other trait and the QTL acting only on this secondary trait. In the first set, evaluation methods were found to mainly affect the accuracy of overall genetic values prediction for the trait with missing data. In comparison with BLUP, MAS led to an extra overall genetic response for the trait with missing data, which was strongly penalised under the conventional BLUP, and to a deficit in response for the other trait. This more balanced evolution of the two traits was obtained, however, at the expense of the long-term overall cumulated response for the aggregate genotype

  7. Structure-activity relationships for the antifungal activity of selective estrogen receptor antagonists related to tamoxifen.

    Directory of Open Access Journals (Sweden)

    Arielle Butts

    Full Text Available Cryptococcosis is one of the most important invasive fungal infections and is a significant contributor to the mortality associated with HIV/AIDS. As part of our program to repurpose molecules related to the selective estrogen receptor modulator (SERM tamoxifen as anti-cryptococcal agents, we have explored the structure-activity relationships of a set of structurally diverse SERMs and tamoxifen derivatives. Our data provide the first insights into the structural requirements for the antifungal activity of this scaffold. Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1 the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2 an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3 electronegative substituents on the aromatic rings modestly improved activity. Using a cell-based assay of calmodulin antagonism, we found that the anti-cryptococcal activity of the scaffold correlates with calmodulin inhibition. Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules. Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold.

  8. Antagonistic effect of disulfide-rich peptide aptamers selected by cDNA display on interleukin-6-dependent cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Nemoto, Naoto, E-mail: nemoto@fms.saitama-u.ac.jp [Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan); Innovation Center for Startups, National Institute of Advanced Industrial Science and Technology, 2-2-2 Marunouchi, Chiyoda-ku, Tokyo 100-0005 (Japan); Janusys Corporation, 508, Saitama Industrial Technology Center, Skip City, 3-12-18 Kami-Aoki, Kawaguchi, Saitama 333-0844 (Japan); Tsutsui, Chihiro [Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan); Yamaguchi, Junichi [Innovation Center for Startups, National Institute of Advanced Industrial Science and Technology, 2-2-2 Marunouchi, Chiyoda-ku, Tokyo 100-0005 (Japan); Applied Gene Technology, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Ueno, Shingo [Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan); Machida, Masayuki [Applied Gene Technology, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Kobayashi, Toshikatsu [Innovation Center for Startups, National Institute of Advanced Industrial Science and Technology, 2-2-2 Marunouchi, Chiyoda-ku, Tokyo 100-0005 (Japan); Janusys Corporation, 508, Saitama Industrial Technology Center, Skip City, 3-12-18 Kami-Aoki, Kawaguchi, Saitama 333-0844 (Japan); Sakai, Takafumi [Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan)

    2012-04-27

    Highlights: Black-Right-Pointing-Pointer Disulfide-rich peptide aptamer inhibits IL-6-dependent cell proliferation. Black-Right-Pointing-Pointer Disulfide bond of peptide aptamer is essential for its affinity to IL-6R. Black-Right-Pointing-Pointer Inhibitory effect of peptide depends on number and pattern of its disulfide bonds. -- Abstract: Several engineered protein scaffolds have been developed recently to circumvent particular disadvantages of antibodies such as their large size and complex composition, low stability, and high production costs. We previously identified peptide aptamers containing one or two disulfide-bonds as an alternative ligand to the interleukin-6 receptor (IL-6R). Peptide aptamers (32 amino acids in length) were screened from a random peptide library by in vitro peptide selection using the evolutionary molecular engineering method 'cDNA display'. In this report, the antagonistic activity of the peptide aptamers were examined by an in vitro competition enzyme-linked immunosorbent assay (ELISA) and an IL-6-dependent cell proliferation assay. The results revealed that a disulfide-rich peptide aptamer inhibited IL-6-dependent cell proliferation with similar efficacy to an anti-IL-6R monoclonal antibody.

  9. Comparison of levocabastine, a new selective H1-receptor antagonist, and disodium cromoglycate, in a nasal provocation test with allergen.

    Science.gov (United States)

    Kolly, M; Pécoud, A

    1986-10-01

    The effect of intranasal administration of levocabastine, a new selective H1-receptor antagonist, was investigated in a nasal provocation test (NPT) performed with allergens. The NPT allowed a quantitative estimation of the nasal allergic threshold (concentration of allergen necessary to trigger the reaction). In addition, the intensity of the three major rhinitis symptoms (obstruction, rhinorrhea and sneezing) was determined. Twelve adult patients, allergic to grass pollen, underwent a first NPT without pretreatment ('initial NPT'); the NPT was then repeated after the single intranasal administration of either placebo, 8 mg disodium cromoglycate (DSCG) or 0.2 mg levocabastine in a double-blind random order. The NPTs gave reproducible results since both the threshold and symptom intensities were similar in the initial NPT and in the NPT performed after placebo. The reaction threshold increased in 8/12 patients after DSCG (0.05 less than P less than 0.1) and in 9/12 patients after levocabastine (P less than 0.05). Levocabastine clearly inhibited rhinorrhea (P less than 0.001) and sneezing (P less than 0.02) but did not influence the nasal obstruction. DSCG inhibited rhinorrhea only (P less than 0.01). The intranasal administration of levocabastine might be useful in the treatment of allergic rhinitis.

  10. Autoradiographic imaging and quantification of the high-affinity GHB binding sites in rodent brain using (3)H-HOCPCA

    DEFF Research Database (Denmark)

    Klein, A B; Bay, T; Villumsen, I S

    2016-01-01

    GHB (γ-hydroxybutyric acid) is a compound endogenous to mammalian brain with high structural resemblance to GABA. GHB possesses nanomolar-micromolar affinity for a unique population of binding sites, but the exact nature of these remains elusive. In this study we utilized the highly selective GHB......, (3)H-HOCPCA displays excellent signal-to-noise ratios using rodent brain autoradiography, which makes it a valuable ligand for anatomical quantification of native GHB binding site levels. Our data confirmed that (3)H-HOCPCA labels only the high-affinity specific GHB binding site, found in high...... brain development. Due to the high sensitivity of this radioligand, we were able to detect low levels of specific binding already at E15 in mouse brain, which increased progressively until adulthood. Collectively, we show that (3)H-HOCPCA is a highly sensitive radioligand, offering advantages over...

  11. Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Martini, Lene; Schwartz, Thue W

    2005-01-01

    that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization...... for the fusion constructs was observed. We conclude that the glucagon-like peptide 1 fusion construct mimics the natural interaction of the receptor with (beta)arr2 with respect to binding peptide ligands, G protein-mediated signaling and internalization, and that this distinct molecular phenotype is reminiscent......To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting...

  12. Novel cyclic gamma-hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Greenwood, Jeremy R

    2005-01-01

    Gamma-hydroxybutyrate (GHB) is a psychotropic compound endogenous to the brain. Despite its potentially great physiological significance, its exact molecular mechanism of action is unknown. GHB is a weak agonist at GABA(B) receptors, but there is also evidence of specific GHB receptor sites......, the molecular cloning of which remains a challenge. Ligands with high affinity and specificity for the reported GHB binding site are needed for pharmacological dissection of the GHB and GABA(B) effects and for mapping the structural requirements of the GHB receptor-ligand interactions. For this purpose, we have...... analog, HOCPrCA, proved to have 10-fold higher affinity than its enantiomer. Likewise, the R-enantiomers of HOCHCA and HOCPCA selectively inhibited [3H]NCS-382 binding. The best inhibitor of these, (R)-HOCPCA, has an affinity 39 times higher than GHB and is thus among the best GHB ligands reported...

  13. Absence of high-affinity calreticulin autoantibodies in patients with systemic rheumatic diseases and coeliac disease

    DEFF Research Database (Denmark)

    Jørgensen, C S; Hansen, K B; Jacobsen, Søren

    2005-01-01

    Calreticulin has been reported to be an autoantigen in various autoimmune connective tissue diseases and in coeliac disease. Previous studies have used incubation buffers with low salt and low detergent concentrations (low stringency conditions) with serum albumin or other proteins as a blocking...... binding (high stringency conditions). Using the high stringency conditions, we screened sera from 107 patients with systemic lupus erythematosus, sera from patients with other systemic autoimmune diseases and from children with coeliac disease for the presence of high-affinity calreticulin autoantibodies...... by immunoblotting and ELISA. None of the sera contained high-affinity calreticulin antibodies. It is concluded that calreticulin is not a common autoantigen in patients with autoimmune connective tissue diseases or coeliac disease....

  14. The structural basis of a high affinity ATP binding ε subunit from a bacterial ATP synthase.

    Directory of Open Access Journals (Sweden)

    Alexander Krah

    Full Text Available The ε subunit from bacterial ATP synthases functions as an ATP sensor, preventing ATPase activity when the ATP concentration in bacterial cells crosses a certain threshold. The R103A/R115A double mutant of the ε subunit from thermophilic Bacillus PS3 has been shown to bind ATP two orders of magnitude stronger than the wild type protein. We use molecular dynamics simulations and free energy calculations to derive the structural basis of the high affinity ATP binding to the R103A/R115A double mutant. Our results suggest that the double mutant is stabilized by an enhanced hydrogen-bond network and fewer repulsive contacts in the ligand binding site. The inferred structural basis of the high affinity mutant may help to design novel nucleotide sensors based on the ε subunit from bacterial ATP synthases.

  15. Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

    OpenAIRE

    Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

    2017-01-01

    Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) te...

  16. Nuclear Choline Acetyltransferase Activates Transcription of a High-affinity Choline Transporter*

    OpenAIRE

    Matsuo, Akinori; Bellier, Jean-Pierre; Nishimura, Masaki; Yasuhara, Osamu; Saito, Naoaki; Kimura, Hiroshi

    2010-01-01

    Choline acetyltransferase (ChAT) synthesizes the neurotransmitter, acetylcholine, at cholinergic nerve terminals. ChAT contains nuclear localization signals and is also localized in the nuclei of neural and non-neuronal cells. Nuclear ChAT might have an as yet unidentified function, such as transcriptional regulation. In this study, we investigated the alteration of candidate gene transcription by ChAT. We chose high affinity choline transporter (CHT1) and vesicular acetylcholine transporter ...

  17. Functional Characteristics of the High Affinity IgG Receptor, Fc gamma RI

    NARCIS (Netherlands)

    van der Poel, Cees E.; Spaapen, Robbert M.; van de Winkel, Jan G. J.; Leusen, Jeanette H. W.

    2011-01-01

    IgG FcRs are important mediators of immunity and play a key role during Ab-based immunotherapy. Within the leukocyte IgG receptor family, only Fc gamma RI is capable of IgG binding with high affinity. Fc gamma RI exists as a complex of a ligand binding a-chain and an FcR gamma-chain. The receptors'

  18. The Saccharomyces cerevisiae high affinity phosphate transporter encoded by PHO84 also functions in manganese homeostasis.

    Science.gov (United States)

    Jensen, Laran T; Ajua-Alemanji, Mispa; Culotta, Valeria Cizewski

    2003-10-24

    In the bakers' yeast Saccharomyces cerevisiae, high affinity manganese uptake and intracellular distribution involve two members of the Nramp family of genes, SMF1 and SMF2. In a search for other genes involved in manganese homeostasis, PHO84 was identified. The PHO84 gene encodes a high affinity inorganic phosphate transporter, and we find that its disruption results in a manganese-resistant phenotype. Resistance to zinc, cobalt, and copper ions was also demonstrated for pho84Delta yeast. When challenged with high concentrations of metals, pho84Delta yeast have reduced metal ion accumulation, suggesting that resistance is due to reduced uptake of metal ions. Pho84p accounted for virtually all the manganese accumulated under metal surplus conditions, demonstrating that this transporter is the major source of excess manganese accumulation. The manganese taken in via Pho84p is indeed biologically active and can not only cause toxicity but can also be incorporated into manganese-requiring enzymes. Pho84p is essential for activating manganese enzymes in smf2Delta mutants that rely on low affinity manganese transport systems. A role for Pho84p in manganese accumulation was also identified in a standard laboratory growth medium when high affinity manganese uptake is active. Under these conditions, cells lacking both Pho84p and the high affinity Smf1p transporter accumulated low levels of manganese, although there was no major effect on activity of manganese-requiring enzymes. We conclude that Pho84p plays a role in manganese homeostasis predominantly under manganese surplus conditions and appears to be functioning as a low affinity metal transporter.

  19. Quantifying high-affinity binding of hydrophobic ligands by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Broecker, Jana; Vargas, Carolyn; Fanghänel, Jörg; Keller, Sandro

    2012-12-18

    A fast and reliable quantification of the binding thermodynamics of hydrophobic high-affinity ligands employing a new calorimetric competition experiment is described. Although isothermal titration calorimetry is the method of choice for a quantitative characterization of intermolecular interactions in solution, a reliable determination of a dissociation constant (K(D)) is typically limited to the range 100 μM > K(D) > 1 nM. Interactions displaying higher or lower K(D) values can be assessed indirectly, provided that a suitable competing ligand is available whose K(D) falls within the directly accessible affinity window. This established displacement assay, however, requires the high-affinity ligand to be soluble at high concentrations in aqueous buffer and, consequently, poses serious problems in the study of protein binding involving small-molecule ligands dissolved in organic solvents--a familiar case in many drug-discovery projects relying on compound libraries. The calorimetric competition assay introduced here overcomes this limitation, thus allowing for a detailed thermodynamic description of high-affinity receptor-ligand interactions involving poorly water-soluble compounds. Based on a single titration of receptor into a dilute mixture of the two competing ligands, this competition assay provides accurate and precise values for the dissociation constants and binding enthalpies of both high- and moderate-affinity ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation and high-affinity protein-inhibitor interactions, and explore its potential and limitations with the aid of simulations and statistical analyses.

  20. Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown.

    OpenAIRE

    Milton, N G

    1999-01-01

    Amyloid-beta (Abeta) specifically bound purified catalase with high affinity and inhibited catalase breakdown of H(2)O(2). The Abeta-induced catalase inhibition involved formation of the inactive catalase Compound II and was reversible. CatalaseAbeta interactions provide rapid functional assays for the cytotoxic domain of Abeta and suggest a mechanism for some of the observed actions of Abeta plus catalase in vitro.

  1. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    Directory of Open Access Journals (Sweden)

    Tal Noy-Porat

    2016-03-01

    Full Text Available Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1 that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  3. Molecular strategies of microbial iron assimilation: from high-affinity complexes to cofactor assembly systems.

    Science.gov (United States)

    Miethke, Marcus

    2013-01-01

    Microorganisms have to cope with restricted iron bioavailability in most environmental habitats as well as during host colonization. The continuous struggle for iron has brought forth a plethora of acquisition and assimilation strategies that share several functional and mechanistic principles. One common theme is the utilization of high-affinity chelators for extracellular iron mobilization, generally known as siderophore-dependent iron acquisition. This basic strategy is related with another central aspect of microbial iron acquisition, which is the release of the mobilized iron from extracellular sources to allow its transfer and incorporation into metabolically active proteins. A variety of mechanisms which are often coupled with high-affinity uptake have evolved to facilitate the removal of iron from siderophore ligands; however, they differ in many key aspects including substrate specificities and release efficiencies. The most sophisticated iron release pathways comprise processes of specific hydrolysis and reduction of ferric siderophores, especially in the case of high-affinity iron complexes with greatly negative redox potentials that often represent crucial factors for virulence development in bacterial and fungal pathogens. During the following steps of iron utilization, the acquired metal is transferred through intracellular trafficking pathways which may include diverse storage compartments in order to be directed to cofactor assembly systems and to final protein targeting. Several of these iron channeling routes have been described recently and provide first insights into the later steps of iron assimilation that characterize an essential part of the cellular iron homeostasis network.

  4. HLA-DRB1 may be antagonistically regulated by the coordinately evolved promoter and 3'-UTR under stabilizing selection.

    Directory of Open Access Journals (Sweden)

    Benrong Liu

    Full Text Available HLA-DRB1 is the most polymorphic MHC (major histocompatibility complex class II gene in human, and plays a crucial role in the development and function of the immune system. Extensive polymorphisms exist in the promoter and 3'-UTR of HLA-DRB1, especially a LTR (Long terminal repeat element in the promoter, which may be involved in the expression regulation. However, it remains unknown how the polymorphisms in the whole promoter region and 3'-UTR to regulate the gene expression. In this study, we investigated the extensive polymorphisms in the HLA-DRB1 promoter and 3'-UTR, and how these polymorphisms affect the gene expression in both independent and jointly manners. It was observed that most of the haplotypes in the DRB1 promoter and 3'-UTR were clustered into 4 conserved lineages (H1, H2, H3 and H4, and showed high linkage disequilibrium. Compared with H1 and H2 lineage, a LTR element in the promoter of H3 and H4 lineage significantly suppressed the promoter activity, whereas the activity of the linked 3'-UTR increased, leading to no apparent difference in the final expression product between H1/H2 and H3/H4 lineage. Nevertheless, compared with the plasmid with a promoter and 3'-UTR from the same lineage, the recombinant plasmid with a promoter from H2 and a 3'-UTR from H3 showed about double fold increased luciferase activity, Conversely, the recombinant plasmid with a promoter from H3 and a 3'-UTR from H2 resulted in about 2-fold decreased luciferase activity. These results indicate that the promoter and 3'-UTR of HLA-DRB1 may antagonistically regulate the gene expression, which may be subjected to stabilizing selection. These findings may provide a novel insight into the mechanisms of the diseases associated with HLA-DRB1 genes.

  5. Effect of netupitant, a highly selective NK₁ receptor antagonist, on the pharmacokinetics of midazolam, erythromycin, and dexamethasone.

    Science.gov (United States)

    Lanzarotti, Corinna; Rossi, Giorgia

    2013-10-01

    Netupitant is a new highly selective neurokinin-1 receptor antagonist being studied for the prevention of nausea and vomiting in patients undergoing chemotherapy. In vitro studies suggest that netupitant inhibits the cytochrome P-450 isoenzyme 3A4 (CYP3A4). Because netupitant may be used with a variety of drugs, which may be substrates of CYP3A4, two studies were designed to establish the potential risk for drug-drug interaction with three different CYP3A4 substrates: midazolam, erythromycin, and dexamethasone. Both trials were three-period crossover studies performed in healthy subjects. In the first study, 20 subjects received netupitant and either midazolam or erythromycin. In the second study, 25 subjects received netupitant and dexamethasone. Serial blood samples were collected over the course of the two studies and pharmacokinetic parameters were determined for all analytes. Netupitant, by inhibiting the CYP3A4, increased the C max and AUCinf of midazolam by 40 and 144 %, respectively, and the C max and AUCinf of erythromycin by 30 %. Netupitant was shown to increase the exposure to dexamethasone in a dose-dependent manner with the mean increase in AUC and C max by 72 and 11 %, respectively, on day 1 and by 138 and 75 %, respectively, on day 4 when co-administered with 300 mg of netupitant. The results of these studies suggest that netupitant is a moderate inhibitor of CYP3A4 and therefore, co-administration with drugs that are substrates of CYP3A4 may require dose adjustments. Treatments were well tolerated in both studies.

  6. Effects of a selective cannabinoid CB2 agonist and antagonist on intravenous nicotine self administration and reinstatement of nicotine seeking.

    Directory of Open Access Journals (Sweden)

    Islam Gamaleddin

    Full Text Available Over the last decade there have been significant advances in the discovery and understanding of the cannabinoid system along with the development of pharmacologic tools that modulate its function. Characterization of the crosstalk between nicotine addiction and the cannabinoid system may have significant implications on our understanding of the neurobiological mechanisms underlying nicotine dependence. Two types of cannabinoid receptors (CB1 and CB2 have been identified. CB1 receptors are expressed in the brain and modulate drug taking and drug seeking for various drugs of abuse, including nicotine. CB2 receptors have been recently identified in the brain and have been proposed to play a functional role in mental disorders and drug addiction. Our objective was to explore the role of CB2 receptors on intravenous nicotine self administration under two schedules of reinforcement (fixed and progressive ratio and on nicotine seeking induced by nicotine priming or by nicotine associated cues. For this, we evaluated the effects of various doses of the selective CB2 antagonist AM630 (1.25 to 5 mg/kg and CB2 agonist AM1241 (1 to 10 mg/kg on these behavioral responses in rats. Different groups of male Long Evans rats were trained to lever press for nicotine at a unit dose of 30 µg/kg/infusion. Subsequently, animals were randomized using a Latin-square design and injected with either AM1241 or AM630 using a counterbalanced within subject design. Administration of the CB2 ligands did not affect either nicotine-taking nicotine-seeking behavior. Our results do not support the involvement of CB2 receptors in nicotine-taking or nicotine-seeking behavior.

  7. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy.

    Science.gov (United States)

    Lázár-Molnár, Eszter; Scandiuzzi, Lisa; Basu, Indranil; Quinn, Thomas; Sylvestre, Eliezer; Palmieri, Edith; Ramagopal, Udupi A; Nathenson, Stanley G; Guha, Chandan; Almo, Steven C

    2017-03-01

    Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  8. High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting.

    Directory of Open Access Journals (Sweden)

    Christopher Ullman

    Full Text Available Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2. Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with 64Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model.

  9. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV

    Directory of Open Access Journals (Sweden)

    Wenjing Qi

    2016-10-01

    Full Text Available Zr(IV can form phosphate and Zr(IV (–PO32−–Zr4+– complex owing to the high affinity between Zr(IV with phosphate. Zr(IV can induce the aggregation of gold nanoparticles (AuNPs, while adenosine triphosphate(ATP can prevent Zr(IV-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRAsensor for ATP have been developed using AuNPs based on the high affinity between Zr(IVwith ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV. After the addition of ATP, ATP reacts with Zr(IV and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV, ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945 with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

  10. BU08073 a buprenorphine analogue with partial agonist activity at μ-receptors in vitro but long-lasting opioid antagonist activity in vivo in mice.

    Science.gov (United States)

    Khroyan, T V; Wu, J; Polgar, W E; Cami-Kobeci, G; Fotaki, N; Husbands, S M; Toll, L

    2015-01-01

    Buprenorphine is a potent analgesic with high affinity at μ, δ and κ and moderate affinity at nociceptin opioid (NOP) receptors. Nevertheless, NOP receptor activation modulates the in vivo activity of buprenorphine. Structure activity studies were conducted to design buprenorphine analogues with high affinity at each of these receptors and to characterize them in in vitro and in vivo assays. Compounds were tested for binding affinity and functional activity using [(35) S]GTPγS binding at each receptor and a whole-cell fluorescent assay at μ receptors. BU08073 was evaluated for antinociceptive agonist and antagonist activity and for its effects on anxiety in mice. BU08073 bound with high affinity to all opioid receptors. It had virtually no efficacy at δ, κ and NOP receptors, whereas at μ receptors, BU08073 has similar efficacy as buprenorphine in both functional assays. Alone, BU08073 has anxiogenic activity and produces very little antinociception. However, BU08073 blocks morphine and U50,488-mediated antinociception. This blockade was not evident at 1 h post-treatment, but is present at 6 h and remains for up to 3-6 days. These studies provide structural requirements for synthesis of 'universal' opioid ligands. BU08073 had high affinity for all the opioid receptors, with moderate efficacy at μ receptors and reduced efficacy at NOP receptors, a profile suggesting potential analgesic activity. However, in vivo, BU08073 had long-lasting antagonist activity, indicating that its pharmacokinetics determined both the time course of its effects and what receptor-mediated effects were observed. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The British Pharmacological Society.

  11. Identification of High Affinity Polo-like Kinase 1 (Plk1) Polo-box Domain Binding Peptides Using Oxime-based Diversification

    Science.gov (United States)

    Liu, Fa; Park, Jung-Eun; Qian, Wen-Jian; Lim, Dan; Scharow, Andrej; Berg, Thorsten; Yaffe, Michael B.; Lee, Kyung S.; Burke, Terrence R.

    2012-01-01

    In an effort to develop improved binding antagonists of the polo-like kinase 1 (Plk1) polo-box domain (PBD), we optimized interactions of the known high affinity 5-mer peptide, PLHSpT using oxime-based post-solid-phase peptide diversification of the N-terminal Pro residue. This allowed us to achieve up to two orders-of-magnitude potency enhancement. An X-ray crystal structure of the highest affinity analogue in complex with Plk1 PBD revealed new binding interactions in a hydrophobic channel that had been occluded in X-ray structures of the unliganded protein. This study represents an important example where amino acid modification by post solid-phase oxime ligation can facilitate the development of protein-protein interaction inhibitors by identifying new binding pockets that would not otherwise be accessible to coded amino acid residues. PMID:22292814

  12. Novel Peripherally Restricted Cannabinoid 1 Receptor Selective Antagonist TXX-522 with Prominent Weight-Loss Efficacy in Diet Induced Obese Mice

    Directory of Open Access Journals (Sweden)

    Wei Chen

    2017-10-01

    Full Text Available The clinical development of the first generation of globally active cannabinoid 1 receptor (CB1R antagonists was suspended because of their adverse neuropsychiatric effects. Selective blockade of peripheral CB1Rs has the potential to provide a viable strategy for the treatment of severe obesity while avoiding these central nervous system side effects. In the current study, a novel compound (TXX-522 was rationally designed based on the parent nucleus of a classical CB1R-selective antagonist/inverse agonist, rimonabant (SR141716A. Docking assays indicate that TXX-522 was bound with the CB1R in a mode similar to that of SR141716A. TXX-522 showed good binding, CB1R-selectivity (over the CB2R, and functional antagonist activities in a range of in vitro molecular and cellular assays. In vivo analysis of the steady state distribution of TXX-522 in the rat brain and blood tissues and the assay of its functional effects on CB1R activity collectively showed that TXX-522 showed minimal brain penetration. Moreover, the in vivo pharmacodynamic study further revealed that TXX-522 had good oral bioavailability and a potent anti-obesity effect, and ameliorated insulin resistance in high-fat diet-induced obese mice. No impact on food intake was observed in this model, confirming the limited brain penetration of this compound. Thus, the current study indicates that TXX-522 is a novel and potent peripherally acting selective CB1R antagonist with the potential to control obesity and related metabolic disorders.

  13. ACTH antagonists

    Directory of Open Access Journals (Sweden)

    Adrian John Clark

    2016-08-01

    Full Text Available ACTH acts via a highly selective receptor that is a member of the melanocortin receptor subfamily of type 1 G protein-coupled receptors. The ACTH receptor, also known as the melanocortin 2 receptor (MC2R is unusual in that it is absolutely dependent on a small accessory protein, melanocortin receptor accessory protein (MRAP for cell surface expression and function. ACTH is the only known naturally occurring agonist for this receptor. This lack of redundancy and high degree of ligand specificity suggests that antagonism of this receptor could provide a useful therapeutic aid and a potential investigational tool. Clinical situations in which this could be useful include (1 Cushing’s disease and ectopic ACTH syndrome – especially whilst preparing for definitive treatment of a causative tumour, or in refractory cases, or (2 congenital adrenal hyperplasia – as an adjunct to glucocorticoid replacement. A case for antagonism in other clinical situations in which there is ACTH excess can also be made. In this article we will explore the scientific and clinical case for an ACTH antagonist, and will review the evidence for existing and recently described peptides and modified peptides in this role.

  14. Selective regulation of beta 1- and beta 2-adrenoceptors in the human heart by chronic beta-adrenoceptor antagonist treatment

    NARCIS (Netherlands)

    Michel, M. C.; Pingsmann, A.; Beckeringh, J. J.; Zerkowski, H. R.; Doetsch, N.; Brodde, O. E.

    1988-01-01

    1. In 44 patients undergoing coronary artery bypass grafting, the effect of chronic administration of the beta-adrenoceptor antagonists sotalol, propranolol, pindolol, metoprolol and atenolol on beta-adrenoceptor density in right atria (containing 70% beta 1- and 30% beta 2-adrenoceptors) and in

  15. Regional distribution of high affinity binding of 3H-adenosine in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Traversa, U.; Puppini, P.; de Angelis, L.; Vertua, R.

    1984-06-01

    The high and low affinity adenosine binding sites with Kd values ranging respectively from 0.8 to 1.65 microM and from 3.1 to 13.86 microM were demonstrated in the following rat brain areas: cortex, hippocampus, striatum, cerebellum, diencephalon, and pons-medulla. Adenosine receptors involved in the high affinity binding seem to be mainly Ra-type. The analysis of the regional distribution of 3H-Adenosine showed the highest levels of specific binding in striatum and hippocampus; somewhat smaller values in cortex, cerebellum, and diencephalon, and even lower in pons-medulla.

  16. Dephosphorylation of Phytate by Using the Aspergillus niger Phytase with a High Affinity for Phytate

    OpenAIRE

    Nagashima, Tadashi; Tange, Tatsuya; Anazawa, Hideharu

    1999-01-01

    A phytase (EC 3.1.3.8) with a high affinity for phytic acid was found in Aspergillus niger SK-57 and purified to homogeneity in four steps by using ion-exchange chromatography (two types), gel filtration, and chromatofocusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme gave a single stained band at a molecular mass of approximately 60 kDa. The Michaelis constant of the enzyme for phytic acid (18.7 ± 4.6 μM) was statistically analyzed. In regard to the ort...

  17. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    Energy Technology Data Exchange (ETDEWEB)

    Yliniemelae, A.; Gynther, J. (Univ. of Kuopio (Finland)); Konschin, H.; Tylli, H. (Univ. of Helsinki (Finland)); Rouvinen, J. (Univ. of Joensuu (Finland))

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  18. Modulating Mineralocorticoid Receptor with Non-steroidal Antagonists. New Opportunities for the Development of Potent and Selective Ligands without Off-Target Side Effects.

    Science.gov (United States)

    Martín-Martínez, Mercedes; Pérez-Gordillo, Felipe L; Álvarez de la Rosa, Diego; Rodríguez, Yoel; Gerona-Navarro, Guillermo; González-Muñiz, Rosario; Zhou, Ming-Ming

    2017-04-13

    Steroidal mineralocorticoid receptor (MR) antagonists are used for treatment of a range of human diseases, but they present challenging issues of complex chemical synthesis, undesirable physical properties, and poor selectivity along with unwanted side effects. Therefore, there is a great interest in the discovery of non-steroidal ligands able to bind to the ligand-binding domain of the MR and recruit different co-regulators to produce tissue-specific therapeutic effects. Several academic groups and pharmaceutical companies have been developing a series of non-steroidal ligands that consist of different chemical scaffolds, yielding MR antagonists currently evaluated in clinical studies for the treatment of congestive heart failure, hypertension, or diabetic nephropathy. The main focus of this Perspective is to review the reported structure-activity relationships of the different series of compounds, as well as the structural studies that contribute to a better understanding of the receptor active site and are also helpful for optimization processes.

  19. Skin-targeted inhibition of PPAR β/δ by selective antagonists to treat PPAR β/δ-mediated psoriasis-like skin disease in vivo.

    Directory of Open Access Journals (Sweden)

    Katrin Hack

    Full Text Available We have previously shown that peroxisome proliferator activating receptor ß/δ (PPAR β/δ is overexpressed in psoriasis. PPAR β/δ is not present in adult epidermis of mice. Targeted expression of PPAR β/δ and activation by a selective synthetic agonist is sufficient to induce an inflammatory skin disease resembling psoriasis. Several signalling pathways dysregulated in psoriasis are replicated in this model, suggesting that PPAR β/δ activation contributes to psoriasis pathogenesis. Thus, inhibition of PPAR β/δ might harbour therapeutical potential. Since PPAR β/δ has pleiotropic functions in metabolism, skin-targeted inhibition offer the potential of reducing systemic adverse effects. Here, we report that three selective PPAR β/δ antagonists, GSK0660, compound 3 h, and GSK3787 can be formulated for topical application to the skin and that their skin concentration can be accurately quantified using ultra-high performance liquid chromatography (UPLC/mass spectrometry. These antagonists show efficacy in our transgenic mouse model in reducing psoriasis-like changes triggered by activation of PPAR β/δ. PPAR β/δ antagonists GSK0660 and compound 3 do not exhibit systemic drug accumulation after prolonged application to the skin, nor do they induce inflammatory or irritant changes. Significantly, the irreversible PPAR β/δ antagonist (GSK3787 retains efficacy when applied topically only three times per week which could be of practical clinical usefulness. Our data suggest that topical inhibition of PPAR β/δ to treat psoriasis may warrant further exploration.

  20. Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

    Science.gov (United States)

    Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

    2017-07-01

    The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

  1. Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma.

    Science.gov (United States)

    Zhang, Yi-Fan; Ho, Mitchell

    2016-09-26

    Glypican-3 (GPC3) is a cell-surface heparan sulfate proteoglycan highly expressed in hepatocellular carcinoma (HCC). We have generated a group of high-affinity mouse monoclonal antibodies targeting GPC3. Here, we report the humanization and testing of these antibodies for clinical development. We compared the affinity and cytotoxicity of recombinant immunotoxins containing mouse single-chain variable regions fused with a Pseudomonas toxin. To humanize the mouse Fvs, we grafted the combined KABAT/IMGT complementarity determining regions (CDR) into a human IgG germline framework. Interestingly, we found that the proline at position 41, a non-CDR residue in heavy chain variable regions (VH), is important for humanization of mouse antibodies. We also showed that two humanized anti-GPC3 antibodies (hYP7 and hYP9.1b) in the IgG format induced antibody-dependent cell-mediated cytotoxicity and complement-dependent-cytotoxicity in GPC3-positive cancer cells. The hYP7 antibody was tested and showed inhibition of HCC xenograft tumor growth in nude mice. This study successfully humanizes and validates high affinity anti-GPC3 antibodies and sets a foundation for future development of these antibodies in various clinical formats in the treatment of liver cancer.

  2. Choline Uptake in Agrobacterium tumefaciens by the High-Affinity ChoXWV Transporter▿

    Science.gov (United States)

    Aktas, Meriyem; Jost, Kathinka A.; Fritz, Christiane; Narberhaus, Franz

    2011-01-01

    Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called “Venus flytrap mechanism” of substrate binding. PMID:21803998

  3. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  4. Isolation of Streptomyces sp. PCB7, the first microorganism demonstrating high-affinity uptake of tropospheric H2

    National Research Council Canada - National Science Library

    Constant, Philippe; Poissant, Laurier; Villemur, Richard

    2008-01-01

    .... Studies conducted over the last three decades provide indirect evidences that H(2) soil uptake is mediated by free soil hydrogenases or by unknown microorganisms that have a high affinity for H(2...

  5. CM156, a high affinity sigma ligand, attenuates the stimulant and neurotoxic effects of methamphetamine in mice.

    Science.gov (United States)

    Kaushal, Nidhi; Seminerio, Michael J; Shaikh, Jamaluddin; Medina, Mark A; Mesangeau, Christophe; Wilson, Lisa L; McCurdy, Christopher R; Matsumoto, Rae R

    2011-01-01

    Methamphetamine (METH) is a highly addictive psychostimulant drug of abuse. Low and high dose administration of METH leads to locomotor stimulation, and dopaminergic and serotonergic neurotoxicity, respectively. The behavioral stimulant and neurotoxic effects of METH can contribute to addiction and other neuropsychiatric disorders, thus necessitating the identification of potential pharmacotherapeutics against these effects produced by METH. METH binds to σ receptors at physiologically relevant concentrations. Also, σ receptors are present on and can modulate dopaminergic and serotonergic neurons. Therefore, σ receptors provide a viable target for the development of pharmacotherapeutics against the adverse effects of METH. In the present study, CM156, a σ receptor ligand with high affinity and selectivity for σ receptors over 80 other non-σ binding sites, was evaluated against METH-induced stimulant, hyperthermic, and neurotoxic effects. Pretreatment of male, Swiss Webster mice with CM156 dose dependently attenuated the locomotor stimulation, hyperthermia, striatal dopamine and serotonin depletions, and striatal dopamine and serotonin transporter reductions produced by METH, without significant effects of CM156 on its own. These results demonstrate the ability of a highly selective σ ligand to mitigate the effects of METH. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Acute withdrawal-related hypophagia elicited by amphetamine is attenuated by pretreatment with selective dopamine D1 or D2 receptor antagonists in rats.

    Science.gov (United States)

    White, Wesley; Beyer, Jason D; White, Ilsun M

    2015-11-01

    After receiving 2.0mg/kg amphetamine, rats show two phases of reduced food intake, short-term hypophagia, during the first several hours after treatment, and longer-term hypophagia, approximately 19 to 26 h after treatment. The longer-term hypophagia may be an indicator of an acute withdrawal. This study assessed whether D1 and D2 receptor activation were important early events in the elicitation of longer-term hypophagia. Throughout a series of five-day tests, rats could lever press for food pellets for one-hour periods beginning every 3h. On test day 1, rats were given a saline pretreatment, and 15 min later they were given a saline treatment. On test day 3, they were given a pretreatment of either saline or a selective dopamine receptor antagonist, and 15 min later they were given a treatment of either saline or amphetamine (2.0mg/kg). In Experiment 1, pretreatments included 3, 12, 31, and 50 μg/kg of the selective D1 receptor antagonist SCH 23390. In Experiment 2, pretreatments included 25, 50, and 100 μg/kg of the selective D2 receptor antagonist eticlopride. Distance moved was monitored for the first 6h following pretreatment-treatment combinations to obtain an indirect behavioral measure of receptor blockade (antagonist attenuation of amphetamine hyperactivity). Food intake at each meal opportunity was monitored throughout each five day test. Patterns of food intake following day 1 saline-saline and day 3 pretreatment-treatment were compared. The combination saline-amphetamine produced short-term and longer-term hypophagia. Combinations involving antagonist-saline did not produce longer-term changes in food intake. Pretreatment with 12 to 50 μg/kg of SCH 23390 produced substantial blockade of amphetamine hyperactivity and prevented amphetamine-induced acute-withdrawal-related longer-term hypophagia. Eticlopride produced a partial blockade of longer-term hypophagia. Both D1 and D2 receptor activation are required for full expression of longer

  7. Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

    Energy Technology Data Exchange (ETDEWEB)

    Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia; Pomper, Martin G.; De Micheli, Carlo; Conti, Paola; Pinto, Andrea; Hansen, Kasper B. (JHU); (Milan); (Montana)

    2017-07-31

    NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.

  8. Methods for synthesizing tritium-labelled Idazoxan-(RX 781094) (2-(2-imidazolinyl)-1,4-benzodioxane) of high specific activity: an antagonistic radioligand selective for presynaptic. cap alpha. /sub 2/ -adrenoreceptors

    Energy Technology Data Exchange (ETDEWEB)

    Guillaumet, G.; Coudert, G.; Clement, R.; Ponchant, P.; Pichat, L.

    1986-08-01

    Four methods of syntheses of IDAZOXAN (RX 781094) (2-(2-imidazolinyl)-1,4-benzodioxane) labelled with tritium are described. This selective radioligand is a presynaptic ..cap alpha../sub 2/-adrenoreceptor antagonist.

  9. Design and Synthesis of a Series of l-trans-4-Substituted Prolines as Selective Antagonists for the Ionotropic Glutamate Receptors Including Functional and X-ray Crystallographic Studies of New Subtype Selective Kainic Acid Receptor Subtype 1 (GluK1) Antagonist (2S,4R)-4-(2-Carboxyphenoxy)pyrrolidine-2-carboxylic Acid.

    Science.gov (United States)

    Krogsgaard-Larsen, Niels; Delgar, Claudia G; Koch, Karina; Brown, Patricia M G E; Møller, Charlotte; Han, Liwei; Huynh, Tri H V; Hansen, Stinne W; Nielsen, Birgitte; Bowie, Derek; Pickering, Darryl S; Kastrup, Jette Sandholm; Frydenvang, Karla; Bunch, Lennart

    2017-01-12

    Ionotropic glutamate receptor antagonists are valuable tool compounds for studies of neurological pathways in the central nervous system. On the basis of rational ligand design, a new class of selective antagonists, represented by (2S,4R)-4-(2-carboxyphenoxy)pyrrolidine-2-carboxylic acid (1b), for cloned homomeric kainic acid receptors subtype 1 (GluK1) was attained (Ki = 4 μM). In a functional assay, 1b displayed full antagonist activity with IC50 = 6 ± 2 μM. A crystal structure was obtained of 1b when bound in the ligand binding domain of GluK1. A domain opening of 13-14° was seen compared to the structure with glutamate, consistent with 1b being an antagonist. A structure-activity relationship study showed that the chemical nature of the tethering atom (C, O, or S) linking the pyrrolidine ring and the phenyl ring plays a key role in the receptor selectivity profile and that substituents on the phenyl ring are well accommodated by the GluK1 receptor.

  10. Selective and rapid monitoring of dual platelet inhibition by aspirin and P2Y12 antagonists by using multiple electrode aggregometry

    Directory of Open Access Journals (Sweden)

    Lorenz Reinhard

    2010-05-01

    Full Text Available Abstract Background Poor platelet inhibition by aspirin or clopidogrel has been associated with adverse outcomes in patients with cardiovascular diseases. A reliable and facile assay to measure platelet inhibition after treatment with aspirin and a P2Y12 antagonist is lacking. Multiple electrode aggregometry (MEA, which is being increasingly used in clinical studies, is sensitive to platelet inhibition by aspirin and clopidogrel, but a critical evaluation of MEA monitoring of dual anti-platelet therapy with aspirin and P2Y12 antagonists is missing. Design and Methods By performing in vitro and ex vivo experiments, we evaluated in healthy subjects the feasibility of using MEA to monitor platelet inhibition of P2Y12 antagonists (clopidogrel in vivo, cangrelor in vitro and aspirin (100 mg per day in vivo, and 1 mM or 5.4 mM in vitro alone, and in combination. Statistical analyses were performed by the Mann-Whitney rank sum test, student' t-test, analysis of variance followed by the Holm-Sidak test, where appropriate. Results ADP-induced platelet aggregation in hirudin-anticoagulated blood was inhibited by 99.3 ± 1.4% by in vitro addition of cangrelor (100 nM; p 95% and 100 ± 3.2%, respectively (p in vitro or ex vivo. Oral intake of clopidogrel did not significantly reduce AA-induced aggregation, but P2Y12 blockade by cangrelor (100 nM in vitro diminished AA-stimulated aggregation by 53 ± 26% (p Conclusions Selective platelet inhibition by aspirin and P2Y12 antagonists alone and in combination can be rapidly measured by MEA. We suggest that dual anti-platelet therapy with these two types of anti-platelet drugs can be optimized individually by measuring platelet responsiveness to ADP and AA with MEA before and after drug intake.

  11. Peptidomics applied: A new strategy for development of selective antagonists/agonists of insect pyrokinin (FXPRLamide family using a novel conformational-mimetic motif

    Directory of Open Access Journals (Sweden)

    Ronald J. Nachman

    2014-06-01

    Full Text Available Applied peptidomics: A PK active core analog incorporating a novel transPro conformational-mimetic motif, the dihydroimidazole moiety, was found to demonstrate pure, selective agonism in pyrokinin (PK family bioassays. A second PK core analog incorporating the dihydroimidazole moiety proved to be an antagonist of the diapause-termination activity of another PK assay. The dihydroimidazole analogs feature a modification adjacent to the primary tissue-bound peptidase hydrolysis site that is expected to enhance biostability over natural PK peptides identified by peptidomics. The research identifies a novel scaffold to design mimetic PK analogs as potential environmentally favorable pest management agents capable of disrupting PK-regulated systems.

  12. An integrated QSAR modeling approach to explore the structure-property and selectivity relationships of N-benzoyl-L-biphenylalanines as integrin antagonists.

    Science.gov (United States)

    Amin, Sk Abdul; Adhikari, Nilanjan; Bhargava, Sonam; Gayen, Shovanlal; Jha, Tarun

    2017-11-17

    Integrins [Formula: see text] and [Formula: see text] are important targets to treat different inflammatory diseases, such as multiple sclerosis, inflammatory bowel diseases, rheumatoid arthritis, atherosclerosis, and asthma. Despite being valuable targets, only a few work has been reported to date regarding molecular modeling studies on these integrins. Not only that, none of these reports addressed the selectivity issue between integrins [Formula: see text] and [Formula: see text]. Therefore, a major challenge regarding the design and discovery of selective integrin antagonists remains. In this study, a series of 142 N-benzoyl-L-biphenylalanines having both integrin [Formula: see text] and [Formula: see text] inhibitory activities were considered for a variety of QSAR approaches including regression and classification-based 2D-QSARs, Hologram QSARs, 3D-QSAR CoMFA and CoMSIA studies to identify the structural requirements of these integrin antagonists. All these QSAR models were statistically validated and subsequently correlated with each other to get a detailed understanding of the activity and selectivity profiles of these molecules.

  13. A high affinity recombinant antibody to the human EphA3 receptor with enhanced ADCC activity.

    Science.gov (United States)

    Tomasevic, Nenad; Luehrsen, Kenneth; Baer, Mark; Palath, Varghese; Martinez, David; Williams, Jason; Yi, Christina; Sujatha-Bhaskar, Swathi; Lanke, Rohini; Leung, John; Ching, Wendy; Lee, Andreia; Bai, Lu; Yarranton, Geoffrey; Bebbington, Christopher

    2014-12-01

    EphA3 is expressed in solid tumors and leukemias and is an attractive target for the therapy. We have generated a panel of Humaneered® antibodies to the ligand-binding domain using a Fab epitope-focused library that has the same specificity as monoclonal antibody mIIIA4. A high-affinity antibody was selected that competes with the mIIIA4 antibody for binding to EphA3 and has an improved affinity of ∼1 nM. In order to generate an antibody with potent cell-killing activity the variable regions were assembled with human IgG1k constant regions and expressed in a Chinese hamster ovary (CHO) cell line deficient in fucosyl transferase. Non-fucosylated antibodies have been reported to have enhanced binding affinity for the IgG receptor CD16a (FcγRIIIa). The affinity of the antibody for recombinant CD16a was enhanced approximately 10-fold. This resulted in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity against EphA3-expressing leukemic cells, providing a potent antibody for the evaluation as a therapeutic agent.

  14. VNARs: An Ancient and Unique Repertoire of Molecules That Deliver Small, Soluble, Stable and High Affinity Binders of Proteins

    Directory of Open Access Journals (Sweden)

    Caroline Barelle

    2015-09-01

    Full Text Available At 420 million years, the variable domain of New Antigen Receptors or VNARs are undoubtedly the oldest (and smallest antigen binding single domains identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established and has served to provide a greater understanding of the evolution of humoral immunity; their cellular components and processes as well as the underlying genetic organization and molecular control mechanisms. Intriguingly, unlike the variable domain of the camelid heavy chain antibodies or VHH, VNARs do not conform to all of the characteristic properties of classical antibodies with an ancestral origin that clearly distinguishes them from true immunoglobulin antibodies. However, this uniqueness of their origin only adds to their potential as next generation therapeutic biologics with their structural and functional attributes and commercial freedom all enhancing their profile and current success. In fact their small size, remarkable stability, molecular flexibility and solubility, together with their high affinity and selectivity for target, all reinforce the potential of these domains as drug candidates. The purpose of this review is to provide an overview of the existing basic biology of these unique domains, to highlight the drug-like properties of VNARs and describe current progress in their journey towards the clinic.

  15. Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

    Science.gov (United States)

    Rannversson, Hafsteinn; Andersen, Jacob; Sørensen, Lena; Bang-Andersen, Benny; Park, Minyoung; Huber, Thomas; Sakmar, Thomas P.; Strømgaard, Kristian

    2016-01-01

    Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslinking unnatural amino acids (UAAs) into 75 different positions in hSERT. UAAs are incorporated with high specificity, and functionally active transporters have similar transport properties and pharmacological profiles compared with wild-type transporters. We employ ultraviolet-induced crosslinking with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within the central substrate-binding site of hSERT, while no crosslinking is observed at the vestibular-binding site. Taken together, our data provide direct evidence for defining the high-affinity antidepressant binding site in hSERT. PMID:27089947

  16. A linker peptide with high affinity towards silica-containing materials.

    Science.gov (United States)

    Sunna, Anwar; Chi, Fei; Bergquist, Peter L

    2013-06-25

    A peptide sequence with affinity to silica-containing materials was fused to a truncated form of Streptococcus strain G148 Protein G. The resulting recombinant Linker-Protein G (LPG) was produced in Escherichia coli and purified to apparent homogeneity. It displayed high affinity towards two natural clinoptilolite zeolites. The LPG also displayed high binding affinity towards commercial-grade synthetic zeolite, silica and silica-containing materials. A commercial sample of the truncated Protein G and a basic protein, both without the linker, did not bind to natural or synthetic zeolites or silica. We conclude that the zeolite-binding affinity is mediated by the linker peptide sequence. As a consequence, these data may imply that the binding affinity is directed to the SiO2 component rather than to the atomic orientation on the zeolite crystal surface as previously assumed. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Expression of the Arabidopsis high-affinity hexose transporter STP13 correlates with programmed cell death

    DEFF Research Database (Denmark)

    Nørholm, Morten Helge Hauberg; Nour-Eldin, Hussam H; Brodersen, Peter

    2006-01-01

    We report the biochemical characterization in Xenopus oocytes of the Arabidopsis thaliana membrane protein, STP13, as a high affinity, hexose-specific H(+)-symporter. Studies with kinase activators suggest that it is negatively regulated by phosphorylation. STP13 promoter GFP reporter lines show ......13 in PCD is supported by microarray data from e.g. plants undergoing senescence and a strong correlation between STP13 transcripts and the PCD phenotype in different accelerated cell death (acd11) mutants....... GFP expression only in the vascular tissue in emerging petals under non-stressed conditions. Quantitative PCR and the pSTP13-GFP plants show induction of STP13 in programmed cell death (PCD) obtained by treatments with the fungal toxin fumonisin B1 and the pathogen Pseudomonas syringae. A role for STP...

  18. Triazoloquinazolinediones as novel high affinity ligands for the benzodiazepine site of GABA(A) receptors

    DEFF Research Database (Denmark)

    Nilsson, Jakob; Gidlöf, Ritha; Nielsen, Elsebet Østergaard

    2011-01-01

    Based on a pharmacophore model of the benzodiazepine-binding site of GABA(A) receptors, a series of 2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c]quinazoline-3,5-diones (structure type I) were designed, synthesized, and identified as high-affinity ligands of the binding site. For several compounds, K......(i) values of around 0.20nM were determined. They show a structural resemblance with the previously described 2-phenyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones (II) and 2-phenyl-[1,2,4]triazolo[1,5-a]quinoxalin-4(5H)-one (III). The 9-bromo substituted compounds 8a-d were prepared in an 8-step synthesis...

  19. Dephosphorylation of phytate by using the Aspergillus niger phytase with a high affinity for phytate.

    Science.gov (United States)

    Nagashima, T; Tange, T; Anazawa, H

    1999-10-01

    A phytase (EC 3.1.3.8) with a high affinity for phytic acid was found in Aspergillus niger SK-57 and purified to homogeneity in four steps by using ion-exchange chromatography (two types), gel filtration, and chromatofocusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme gave a single stained band at a molecular mass of approximately 60 kDa. The Michaelis constant of the enzyme for phytic acid (18.7 +/- 4.6 microM) was statistically analyzed. In regard to the orthophosphate released from phytic acid, a significant difference between a low K(m) phytase from A. niger SK-57 and a high K(m) phytase from Aspergillus ficuum was recognized.

  20. Cationic polymer brush-modified cellulose nanocrystals for high-affinity virus binding

    Science.gov (United States)

    Rosilo, Henna; McKee, Jason R.; Kontturi, Eero; Koho, Tiia; Hytönen, Vesa P.; Ikkala, Olli; Kostiainen, Mauri A.

    2014-09-01

    Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications.Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface

  1. Kinetics and autoradiography of high affinity uptake of serotonin by primary astrocyte cultures

    Energy Technology Data Exchange (ETDEWEB)

    Katz, D.M.; Kimelberg, H.K.

    1985-07-01

    Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; (/sup 3/H)-5-HT). At concentrations in the range of 0.01 to 0.7 microM (/sup 3/H)-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM (/sup 3/H)-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of (/sup 3/H)-5-HT/mg of protein/4 min for the Na+-dependent component. In the absence of Na+ the uptake was nonsaturable. Omission of the monoamine oxidase inhibitor pargyline markedly reduced the Na+-dependent component of (/sup 3/H)-5-HT uptake but had a negligible effect on the Na+-independent component. This suggest significant oxidative deamination of serotonin after it has been taken up by the high affinity system, followed by release of its metabolite. The authors estimated that this system enabled the cells to concentrate (/sup 3/H)-5-HT up to 44-fold at an external (/sup 3/H)-5-HT concentration of 10(-7) M. Inhibition of (/sup 3/H)-5-HT uptake by a number of clinically effective antidepressants was also consistent with a specific high affinity uptake mechanism for 5-HT, the order of effectiveness of inhibition being chlorimipramine greater than fluoxetine greater than imipramine = amitriptyline greater than desmethylimipramine greater than iprindole greater than mianserin. Uptake of (/sup 3/H)-5-HT was dependent on the presence of Cl- as well as Na+ in the medium, and the effect of omission of both ions was nonadditive. Varying the concentration of K+ in the media from 1 to 50 mM had a limited effect on (/sup 3/H)-5-HT uptake.

  2. Selection and differentiation of Bacillus spp. Antagonistic to Fusarium oxysporum f.sp. lycopersici and Alternaria solani infecting Tomato.

    Science.gov (United States)

    Shanmugam, Veerubommu; Atri, Kamini; Gupta, Samriti; Kanoujia, Nandina; Naruka, Digvijay Singh

    2011-03-01

    Antagonistic Bacillus spp. displaying in vitro production of siderophore, chitinase, and β-1,3-glucanase were identified from dual culture assays. In independent greenhouse studies, seed bacterization and soil application of Bacillus atrophaeus S2BC-2 challenge inoculated with Fusarium oxysporum f.sp. lycopersici (FOL) and Alternaria solani (AS) recorded low percent disease index of 25.3 and 28.7, respectively, over nonbacterised pathogen control (44.3 and 56.4). The low disease incidence corroborated with tomato growth promotion with high vigor index (8,041.2) and fresh plant weight (82.5 g) on challenge inoculation with FOL. Analysis of root and leaf samples in rhizobacterial treatment challenged with FOL and AS revealed maximum induction of chitinase (1.9 and 1.7 U/mg of protein, respectively) and β-1,3-glucanase (23.5 and 19.2 U/mg of protein, respectively). In native gel activity assays, the rhizobacterial treatment on challenge inoculation strongly expressed three high intensity PO isoforms along with one low intensity isoform. In studies on genetic diversity of the Bacillus strains by repetitive extragenomic palindromic-polymerase chain reaction (REP-PCR) and amplified rDNA restriction analysis (ARDRA) patterns, ARDRA was more highly discriminant than REP-PCR and allowed grouping of the strains and differentiation of the antagonistic strains from other isolates.

  3. Selective and nonselective serotonin antagonists block the aversive stimulus properties of MK212 and m-chlorophenylpiperazine (mCPP) in mice.

    Science.gov (United States)

    Walker, Ellen A; Kohut, Stephen J; Hass, Richard W; Brown, Edward K; Prabandham, Anupama; Lefever, Timothy

    2005-12-01

    Serotonin(2C) (5-HT(2C)) receptors have been implicated to treat mood disorders such as depression and anxiety. In the present study, the capacities of two 5-HT(2C) agonists, MK212 and mCPP, to produce conditioned taste aversions in mice were evaluated. On two training days, Swiss-Webster male mice (19-34g) were trained to associate the flavor of a novel solution with the injection of various doses of MK212 or mCPP. On two alternate training days, mice were trained to associate a different flavored solution with an injection of saline. For testing, both flavored solutions were presented simultaneously and an avoidance of the MK212 or mCPP-paired solution indicated conditioned taste aversion. Robust conditioned taste aversions were observed to solutions paired with 1.0 or 10mg/kg MK212 or mCPP. Acquisition of conditioned taste aversions was blocked by nonselective serotonin antagonists cyproheptadine, bromo-LSD, metergoline, methysergide and mianserin. Selective 5-HT(2B/2C) antagonist SB206,553 blocked both MK212- and mCPP-induced conditioned taste aversion although selective 5-HT(2B/2C) antagonist SB200,646 only blocked mCPP-induced conditioned taste aversion. In a single-bottle procedure, MK212, bromo-LSD, and mianserin failed to alter acquisition rate of a LiCl-induced conditioned taste aversion. Taken together, these data indicate that the serotonin agonists MK212 and mCPP produce conditioned taste aversion and that these effects are mediated predominantly through 5-HT(2C) receptors.

  4. Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs

    Science.gov (United States)

    Munday, Jane C.; Eze, Anthonius A.; Baker, Nicola; Glover, Lucy; Clucas, Caroline; Aguinaga Andrés, David; Natto, Manal J.; Teka, Ibrahim A.; McDonald, Jennifer; Lee, Rebecca S.; Graf, Fabrice E.; Ludin, Philipp; Burchmore, Richard J. S.; Turner, C. Michael R.; Tait, Andy; MacLeod, Annette; Mäser, Pascal; Barrett, Michael P.; Horn, David; De Koning, Harry P.

    2014-01-01

    Objectives Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. Methods The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. Results All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. Conclusions TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter. PMID:24235095

  5. Post-training dopamine receptor agonists and antagonists affect memory storage in mice irrespective of their selectivity for D1 or D2 receptors.

    Science.gov (United States)

    Castellano, C; Cestari, V; Cabib, S; Puglisi-Allegra, S

    1991-11-01

    Post-training administration of the selective D1 and D2 agonists SKF 38393 and LY 171555 dose-dependently facilitated retention of an inhibitory avoidance response in mice, while the selective D1 or D2 antagonists SCH 23390 and (-)sulpiride produced an impairment of retention. These effects are not to be ascribed to a nonspecific action of the drugs on retention performance, as the latencies during the retention test of those mice that had not received a footshock during the training were not increased by the post-training drug administration. The effects on retention performance induced by DA agonists and antagonists seem to be due to an effect on memory consolidation, since they have been observed when drugs were given at short, but not at long, periods of time after training. These results showing a similar role of D1 and D2 receptor types on memory storage appear not to be consistent with a body of neuropharmacological, neurophysiological, and behavioral evidence pointing to a different functional role of these types of DA receptors. This discrepancy is discussed in terms of possible involvement of different brain systems, peripheral systems, or possible second messenger processes activated by the two receptor types and leading to similar effects on memory processes.

  6. Determining pharmacological selectivity of the kappa opioid receptor antagonist LY2456302 using pupillometry as a translational biomarker in rat and human.

    Science.gov (United States)

    Rorick-Kehn, Linda M; Witcher, Jennifer W; Lowe, Stephen L; Gonzales, Celedon R; Weller, Mary Ann; Bell, Robert L; Hart, John C; Need, Anne B; McKinzie, Jamie H; Statnick, Michael A; Suico, Jeffrey G; McKinzie, David L; Tauscher-Wisniewski, Sitra; Mitch, Charles H; Stoltz, Randall R; Wong, Conrad J

    2014-10-31

    Selective kappa opioid receptor antagonism is a promising experimental strategy for the treatment of depression. The kappa opioid receptor antagonist, LY2456302, exhibits ~30-fold higher affinity for kappa opioid receptors over mu opioid receptors, which is the next closest identified pharmacology. Here, we determined kappa opioid receptor pharmacological selectivity of LY2456302 by assessing mu opioid receptor antagonism using translational pupillometry in rats and humans. In rats, morphine-induced mydriasis was completely blocked by the nonselective opioid receptor antagonist naloxone (3mg/kg, which produced 90% mu opioid receptor occupancy), while 100 and 300 mg/kg LY2456302 (which produced 56% and 87% mu opioid receptor occupancy, respectively) only partially blocked morphine-induced mydriasis. In humans, fentanyl-induced miosis was completely blocked by 50mg naltrexone, and LY2456302 dose-dependently blocked miosis at 25 and 60 mg (minimal-to-no blockade at 4-10mg). We demonstrate, for the first time, the use of translational pupillometry in the context of receptor occupancy to identify a clinical dose of LY2456302 achieving maximal kappa opioid receptor occupancy without evidence of significant mu receptor antagonism. © The Author 2015. Published by Oxford University Press on behalf of CINP.

  7. Acute D3 Antagonist GSK598809 Selectively Enhances Neural Response During Monetary Reward Anticipation in Drug and Alcohol Dependence.

    Science.gov (United States)

    Murphy, Anna; Nestor, Liam J; McGonigle, John; Paterson, Louise; Boyapati, Venkataramana; Ersche, Karen D; Flechais, Remy; Kuchibatla, Shankar; Metastasio, Antonio; Orban, Csaba; Passetti, Filippo; Reed, Laurence; Smith, Dana; Suckling, John; Taylor, Eleanor; Robbins, Trevor W; Lingford-Hughes, Anne; Nutt, David J; Deakin, John Fw; Elliott, Rebecca

    2017-04-01

    Evidence suggests that disturbances in neurobiological mechanisms of reward and inhibitory control maintain addiction and provoke relapse during abstinence. Abnormalities within the dopamine system may contribute to these disturbances and pharmacologically targeting the D3 dopamine receptor (DRD3) is therefore of significant clinical interest. We used functional magnetic resonance imaging to investigate the acute effects of the DRD3 antagonist GSK598809 on anticipatory reward processing, using the monetary incentive delay task (MIDT), and response inhibition using the Go/No-Go task (GNGT). A double-blind, placebo-controlled, crossover design approach was used in abstinent alcohol dependent, abstinent poly-drug dependent and healthy control volunteers. For the MIDT, there was evidence of blunted ventral striatal response to reward in the poly-drug-dependent group under placebo. GSK598809 normalized ventral striatal reward response and enhanced response in the DRD3-rich regions of the ventral pallidum and substantia nigra. Exploratory investigations suggested that the effects of GSK598809 were mainly driven by those with primary dependence on alcohol but not on opiates. Taken together, these findings suggest that GSK598809 may remediate reward deficits in substance dependence. For the GNGT, enhanced response in the inferior frontal cortex of the poly-drug group was found. However, there were no effects of GSK598809 on the neural network underlying response inhibition nor were there any behavioral drug effects on response inhibition. GSK598809 modulated the neural network underlying reward anticipation but not response inhibition, suggesting that DRD3 antagonists may restore reward deficits in addiction.

  8. Anorectic, thermogenic and anti-obesity activity of a selective orexin-1 receptor antagonist in ob/ob mice.

    Science.gov (United States)

    Haynes, Andrea C; Chapman, Helen; Taylor, Colleen; Moore, Gary B T; Cawthorne, Michael A; Tadayyon, Mohammad; Clapham, John C; Arch, Jonathan R S

    2002-03-15

    A single dose of the orexin-1 (OX1) receptor antagonist 1-(2-methylbenzoxazol-6-yl)-3-[1,5] naphthyridin-4-yl urea hydrochloride (SB-334867-A) reduces orexin-A-induced feeding and natural feeding in Sprague Dawley rats. In this study, the anti-obesity effects of SB-334867-A were determined in genetically obese (ob/ob) mice dosed with SB-334867-A (30 mg/kg, i.p.) once daily for 7 days, and then twice daily for a further 7 days. SB-334867-A reduced cumulative food intake and body weight gain over 14 days. Total fat mass gain, determined by Dual Emission X-ray Absorptiometry, was reduced, while gain in fat-free mass was unchanged. Fasting (5 h) blood glucose was also reduced at the end of the study, with a trend to reduced plasma insulin. Interscapular brown adipose tissue (BAT) weight was reduced, the tissue was noticeably darker in colour and quantitative PCR (TaqMan) analysis of this tissue showed a trend to an increase in uncoupling protein-1 mRNA expression, suggesting that SB-334867-A might stimulate thermogenesis. This was confirmed in a separate study in which a single dose of SB-334867-A (30 mg/kg, i.p.) increased metabolic rate over 4 h in ob/ob mice. OX1 receptor mRNA was detected in BAT, and its expression was increased by 58% by treatment with SB-334867-A. This is the first demonstration that OX1 receptor antagonists have potential as both anti-obesity and anti-diabetic agents.

  9. Targeting CXCR4 by a selective peptide antagonist modulates tumor microenvironment and microglia reactivity in a human glioblastoma model.

    Science.gov (United States)

    Mercurio, Laura; Ajmone-Cat, Maria Antonietta; Cecchetti, Serena; Ricci, Alessandro; Bozzuto, Giuseppina; Molinari, Agnese; Manni, Isabella; Pollo, Bianca; Scala, Stefania; Carpinelli, Giulia; Minghetti, Luisa

    2016-03-25

    The CXCL12/CXCR4 pathway regulates tumor cell proliferation, metastasis, angiogenesis and the tumor-microenvironment cross-talk in several solid tumors, including glioblastoma (GBM), the most common and fatal brain cancer. In the present study, we evaluated the effects of peptide R, a new specific CXCR4 antagonist that we recently developed by a ligand-based approach, in an in vitro and in vivo model of GBM. The well-characterized CXCR4 antagonist Plerixafor was also included in the study. The effects of peptide R on CXCR4 expression, cell survival and migration were assessed on the human glioblastoma cell line U87MG exposed to CXCL12, by immunofluorescence and western blotting, MTT assay, flow cytometry and transwell chamber migration assay. Peptide R was then tested in vivo, by using U87MG intracranial xenografts in CD1 nude mice. Peptide R was administered for 23 days since cell implantation and tumor volume was assessed by magnetic resonance imaging (MRI) at 4.7 T. Glioma associated microglia/macrophage (GAMs) polarization (anti-tumor M1 versus pro-tumor M2 phenotypes) and expressions of vascular endothelial growth factor (VEGF) and CD31 were assessed by immunohistochemistry and immunofluorescence. We found that peptide R impairs the metabolic activity and cell proliferation of human U87MG cells and stably reduces CXCR4 expression and cell migration in response to CXCL12 in vitro. In the orthotopic U87MG model, peptide R reduced tumor cellularity, promoted M1 features of GAMs and astrogliosis, and hindered intra-tumor vasculature. Our findings suggest that targeting CXCR4 by peptide R might represent a novel therapeutic approach against GBM, and contribute to the rationale to further explore in more complex pre-clinical settings the therapeutic potential of peptide R, alone or in combination with standard therapies of GBM.

  10. Selection of P2Y12 antagonist, treatment initiation, and predictors of high on-treatment platelet reactivity in a "Real World" registry.

    Science.gov (United States)

    Motovska, Zuzana; Ondrakova, Martina; Bednar, Frantisek; Knot, Jiri; Ulman, Jaroslav; Maly, Marek

    2015-06-01

    The present study aimed to compare characteristics related to selection of a P2Y₁₂ antagonist, investigate initiation of therapy with new-generation drugs, and identify predictors of high on-treatment platelet reactivity (HTPR) in patients with acute coronary syndrome treated with stent percutaneous coronary intervention (PCI). Data from 589 patients in the LAPCOR (Laboratory AntiPlatelet efficacy and Clinical Outcome Registry; ClinicalTrials.gov Identifier: NCT02264912) registry was analyzed. P2Y₁₂ receptor antagonist efficacy was measured by VASP phosphorylation 24 ± 4 hours after a loading dose of clopidogrel (600 mg, N=407), prasugrel (60 mg, N=106), or ticagrelor (180 mg, N=76) and expressed by platelet reactivity index (PRI). HTPR was defined as PRI ≥50%. Patients treated with prasugrel were significantly younger and had significantly higher hemoglobin levels than those who received clopidogrel or ticagrelor, while chronic kidney disease was significantly more prevalent in the ticagrelor group. Almost all invasively managed patients given new-generation drugs received a loading dose after coronary angiography. Mean residual PRI and HTPR were significantly higher after clopidogrel (44.2 ± 23.1% and 42.2%, respectively) vs. prasugrel (17.7 ± 18.0% and 9.4%, respectively) or ticagrelor (18.8 ± 17.0% and 7.9%, respectively; all pmean platelet volume (p=0.03). Safety is the most important aspect under consideration in choosing new agents for an individual patient. Other than platelet count and mean platelet volume, factors known as predictors of higher platelet reactivity, did not influence the efficacy of new-generation P2Y₁₂ receptor antagonists. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Melatonin Administration Alters Nicotine Preference Consumption via Signaling Through High-Affinity Melatonin Receptors

    Science.gov (United States)

    Horton, William J.; Gissel, Hannah J.; Saboy, Jennifer E.; Wright, Kenneth P.; Stitzel, Jerry A.

    2015-01-01

    Rationale While it is known that tobacco use varies across the 24-hour day, the time-of-day effects are poorly understood. Findings from several previous studies indicate a potential role for melatonin in these time-of-day effects; however the specific underlying mechanisms have not been well characterized. Understanding of these mechanisms may lead to potential novel smoking cessation treatments. Objective Examine the role of melatonin and melatonin receptors in nicotine free choice consumption Methods A two-bottle oral nicotine choice paradigm was utilized with melatonin supplementation in melatonin deficient mice (C57BL/6J) or without melatonin supplementation in mice proficient at melatonin synthesis (C3H/Ibg) compared to melatonin proficient mice lacking both or one of the high affinity melatonin receptors (MT1 and MT2; double null mutant DM, or MT1 or MT2). Preference for bitter and sweet tastants also was assessed in wild type and MT1 and MT2 DM mice. Finally, home cage locomotor monitoring was performed to determine the effect of melatonin administration on activity patterns. Results Supplemental melatonin in drinking water significantly reduced free-choice nicotine consumption in C57BL/6J mice, which do not produce endogenous melatonin, while not altering activity patterns. Independently, genetic deletion of both MT1 and MT2 receptors in a melatonin proficient mouse strain (C3H) resulted in significantly more nicotine consumption than controls. However single genetic deletion of either the MT1 or MT2 receptor alone did not result in increased nicotine consumption. Deletion of MT1 and MT2 did not impact taste preference. Conclusions This study demonstrates that nicotine consumption can be affected by exogenous or endogenous melatonin and requires at least one of the high-affinity melatonin receptors. The fact that expression of either the MT1 or MT2 melatonin receptor is sufficient to maintain lower nicotine consumption suggests functional overlap and

  12. Melatonin administration alters nicotine preference consumption via signaling through high-affinity melatonin receptors.

    Science.gov (United States)

    Horton, William J; Gissel, Hannah J; Saboy, Jennifer E; Wright, Kenneth P; Stitzel, Jerry A

    2015-07-01

    While it is known that tobacco use varies across the 24-h day, the time-of-day effects are poorly understood. Findings from several previous studies indicate a potential role for melatonin in these time-of-day effects; however, the specific underlying mechanisms have not been well characterized. Understanding of these mechanisms may lead to potential novel smoking cessation treatments. The objective of this study is examine the role of melatonin and melatonin receptors in nicotine free-choice consumption A two-bottle oral nicotine choice paradigm was utilized with melatonin supplementation in melatonin-deficient mice (C57BL/6J) or without melatonin supplementation in mice proficient at melatonin synthesis (C3H/Ibg) compared to melatonin-proficient mice lacking both or one of the high-affinity melatonin receptors (MT1 and MT2; double-null mutant DM, or MT1 or MT2). Preference for bitter and sweet tastants also was assessed in wild-type and MT1 and MT2 DM mice. Finally, home cage locomotor monitoring was performed to determine the effect of melatonin administration on activity patterns. Supplemental melatonin in drinking water significantly reduced free-choice nicotine consumption in C57BL/6J mice, which do not produce endogenous melatonin, while not altering activity patterns. Independently, genetic deletion of both MT1 and MT2 receptors in a melatonin-proficient mouse strain (C3H) resulted in significantly more nicotine consumption than controls. However, single genetic deletion of either the MT1 or MT2 receptor alone did not result in increased nicotine consumption. Deletion of MT1 and MT2 did not impact taste preference. This study demonstrates that nicotine consumption can be affected by exogenous or endogenous melatonin and requires at least one of the high-affinity melatonin receptors. The fact that expression of either the MT1 or MT2 melatonin receptor is sufficient to maintain lower nicotine consumption suggests functional overlap and potential mechanistic

  13. High-Affinity Manganese Uptake by the Metal Transporter NRAMP1 Is Essential for Arabidopsis Growth in Low Manganese Conditions[C][W

    Science.gov (United States)

    Cailliatte, Rémy; Schikora, Adam; Briat, Jean-François; Mari, Stéphane; Curie, Catherine

    2010-01-01

    In contrast with many other essential metals, the mechanisms of Mn acquisition in higher eukaryotes are seldom studied and poorly understood. We show here that Arabidopsis thaliana relies on a high-affinity uptake system to acquire Mn from the soil in conditions of low Mn availability and that this activity is catalyzed by the divalent metal transporter NRAMP1 (for Natural Resistance Associated Macrophage Protein 1). The nramp1-1 loss-of-function mutant grows poorly, contains less Mn than the wild type, and fails to take up Mn in conditions of Mn limitation, thus demonstrating that NRAMP1 is the major high-affinity Mn transporter in Arabidopsis. Based on confocal microscopy observation of an NRAMP1-green fluorescent protein fusion, we established that NRAMP1 is localized to the plasma membrane. Consistent with its function in Mn acquisition from the soil, NRAMP1 expression is restricted to the root and stimulated by Mn deficiency. Finally, we show that NRAMP1 restores the capacity of the iron-regulated transporter1 mutant to take up iron and cobalt, indicating that NRAMP1 has a broad selectivity in vivo. The role of transporters of the NRAMP family is well established in higher eukaryotes for iron but has been controversial for Mn. This study demonstrates that NRAMP1 is a physiological manganese transporter in Arabidopsis. PMID:20228245

  14. In silico investigation of PARP-1 catalytic domains in holo and apo states for the design of high-affinity PARP-1 inhibitors.

    Science.gov (United States)

    Salmas, Ramin Ekhteiari; Unlu, Ayhan; Yurtsever, Mine; Noskov, Sergei Y; Durdagi, Serdar

    2016-01-01

    The rational design of high-affinity inhibitors of poly-ADP-ribose polymerase-1 (PARP-1) is at the heart of modern anti-cancer drug design. While relevance of enzyme to DNA repair processes in cellular environment is firmly established, the structural and functional understanding of the main determinants for high-affinity ligands controlling PARP-1 activity is still lacking. The conserved active site of PARP-1 represents an ideal target for inhibitors and may offer a novel target at the treatment of breast cancer. To fill the gap in the structural knowledge, we report on the combination of molecular dynamics (MD) simulations, principal component analysis (PCA), and conformational analysis that analyzes in great details novel binding mode for a number of inhibitors at the PARP-1. While optimization of the binding affinity for original target is an important goal in the drug design, many of the promising molecules for treatment of the breast cancer are plagued by significant cardiotoxicity. One of the most common side-effects reported for a number of polymerase inhibitors is its off-target interactions with cardiac ion channels and hERG1 channel, in particular. Thus, selected candidate PARP-1 inhibitors were also screened in silico at the central cavities of hERG1 potassium ion channel.

  15. Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody.

    Science.gov (United States)

    Bedzyk, W D; Weidner, K M; Denzin, L K; Johnson, L S; Hardman, K D; Pantoliano, M W; Asel, E D; Voss, E W

    1990-10-25

    Single-chain antibody of the (NH2) VL-linker-VH (COOH) design, was constructed based on prototype high affinity anti-fluorescein monoclonal antibody (mAb) 4-4-20. Purified single-chain antibody (SCA) 4-4-20/212 was studied relative to Ig mAb 4-4-20 in terms of ligand binding, kinetics, idiotypy, metatypy, and stability in denaturing agents. Ligand-binding data correlated with metatypic relatedness of the liganded site. Anti-metatypic reagents reacted preferentially with the liganded conformer of the 4-4-20 antibody active site and were unreactive with free ligand and the non-liganded (idiotypic) state. All results were consistent with the conclusion that SCA 4-4-20/212, with a 14-amino acid linker folded into a native conformational state that closely simulated the prototypical mAb. Furthermore, GndHCl unfolding and refolding studies demonstrated H and L chain variable domain intrinsic stability between SCA 4-4-20/212 and a 50 kDa antigen-binding fragment were nearly identical. This suggested CH1 and CL domain interactions may be more prevalent in V region molecular dynamics than structure.

  16. Early signs of pathological cognitive aging in mice lacking high-affinity nicotinic receptors.

    Directory of Open Access Journals (Sweden)

    Eleni eKonsolaki

    2016-04-01

    Full Text Available In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. Α deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-, which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioural signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviours, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm and extend the hypothesis that β2-/- animals exhibit age-related cognitive impairments, manifested in both spatial learning and recognition memory tasks. In addition, we reveal deficits in spontaneous behaviour and habituation processes earlier in life. To our knowledge, this is the first study to perform an extensive behavioural examination of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioural changes to global dementia due to the combined effect of the neuropathology and aging.

  17. Identification of LAG3 high affinity aptamers by HT-SELEX and Conserved Motif Accumulation (CMA.

    Directory of Open Access Journals (Sweden)

    Mario Martínez Soldevilla

    Full Text Available LAG3 receptor belongs to a family of immune-checkpoints expressed in T lymphocytes and other cells of the immune system. It plays an important role as a rheostat of the immune response. Focus on this receptor as a potential therapeutic target in cancer immunotherapy has been underscored after the success of other immune-checkpoint blockade strategies in clinical trials. LAG3 showcases the interest in the field of autoimmunity as several studies show that LAG3-targeting antibodies can also be used for the treatment of autoimmune diseases. In this work we describe the identification of a high-affinity LAG3 aptamer by High Throughput Sequencing SELEX in combination with a study of potential conserved binding modes according to sequence conservation by using 2D-structure prediction and 3D-RNA modeling using Rosetta. The aptamer with the highest accumulation of these conserved sequence motifs displays the highest affinity to LAG3 recombinant soluble proteins and binds to LAG3-expressing lymphocytes. The aptamer described herein has the potential to be used as a therapeutic agent, as it enhances the threshold of T-cell activation. Nonetheless, in future applications, it could also be engineered for treatment of autoimmune diseases by target depletion of LAG3-effector T lymphocytes.

  18. A high-affinity putrescine-cadaverine transporter from Trypanosoma cruzi

    Science.gov (United States)

    Hasne, Marie-Pierre; Coppens, Isabelle; Soysa, Radika; Ullman, Buddy

    2011-01-01

    Summary Whereas mammalian cells and most other organisms can synthesize polyamines from basic amino acids, the protozoan parasite Trypanosoma cruzi is incapable of polyamine biosynthesis de novo and therefore obligatorily relies upon putrescine acquisition from the host to meet its nutritional requirements. The cell surface proteins that mediate polyamine transport into T. cruzi, as well as most eukaryotes, however, have by-in-large eluded discovery at the molecular level. Here we report the identification and functional characterization of two polyamine transporters, TcPOT1.1 and TcPOT1.2, encoded by alleles from two T. cruzi haplotypes. Overexpression of the TcPOT1.1 and TcPOT1.2 genes in T. cruzi epimastigotes revealed that TcPOT1.1 and TcPOT1.2 were high-affinity transporters that recognized both putrescine and cadaverine but not spermidine or spermine. Furthermore, the activities and subcellular locations of both TcPOT1.1 and TcPOT1.2 in intact parasites were profoundly influenced by extracellular putrescine availability. These results establish TcPOT1.1 and TcPOT1.2 as key components of the T. cruzi polyamine transport pathway, an indispensable nutritional function for the parasite that may be amenable to therapeutic manipulation. PMID:20149109

  19. In silico design of high-affinity ligands for the immobilization of inulinase.

    Science.gov (United States)

    Holyavka, M G; Kondratyev, M S; Samchenko, A A; Kabanov, A V; Komarov, V M; Artyukhov, V G

    2016-04-01

    Using computer modeling, virtual screening of high-affinity ligands for immobilization of inulinase - an enzyme that cleaves inulin and fructose-containing polymers to fructose - has been performed. The inulinase molecule from Aspergillus ficuum (pdb: 3SC7) taken from the database of protein structures was used as a protein model and the target for flexible docking. The set of ligands studied included simple sugars (activators, inhibitors, products of enzymatic catalysis), as well as high-molecular weight compounds (polycation and polyanion exchange resins, glycoproteins, phenylalanine-proline peptide, polylactate, and caffeine). Based on the comparative analysis of the values of the total energy and the localization of ligand binding sites, we made several assumptions concerning the mechanisms of interaction of the suggested matrices for the immobilization of enzyme molecules and the structural features of such complexes. It was also assumed that the candidates for immobilization agents meeting the industrial requirements may be glycoproteins, for which we propose an additional incorporation of cysteine residues into their structure, aimed to create disulfide «anchors» to the surface. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Glutaraldehyde pretreatment blocks temperature-induced high-affinity (/sup 3/H) tryptamine binding

    Energy Technology Data Exchange (ETDEWEB)

    Serikyaku, S.; Ishitani, R.

    1988-01-01

    The effect of glutaraldehyde (and Azure A) on temperature-sensitive high-affinity (/sup 3/H) tryptamine binding was investigated in rat brain synaptic plasma membranes. In the 0.01-0.1 % concentration range, the glutaraldehyde pretreatment preferentially inhibited only the above-mentioned portion of the binding, whereas the posttreatment of this reagent had no effect. On the other hand, in cases of pretreatment or posttreatment, a concentration of glutaraldehyde as high as 0.1 % was inactive on the basal (/sup 3/H) ligand binding capacity of the membranes. Furthermore, it was revealed that the Scatchard plot of (/sup 3/H) tryptamine binding in membranes pretreated with glutaraldehyde conformed to a straight line, as did a similar plot of temperature-independent binding. And, it was interesting to find that the binding parameters (K/sub D/ and B/sub max/ values) of both samples corresponded closely to each other. On the contrary, in all concentrations, Azure A affected nonspecifically both the temperature-dependent and the independent (/sup 3/H) tryptamine binding to the same degree, regardless of whether or not there was pretreatment or posttreatment. 17 references, 2 figures, 1 table.

  1. Productive common light chain libraries yield diverse panels of high affinity bispecific antibodies

    Science.gov (United States)

    Van Blarcom, Thomas; Melton, Zea; Cheung, Wai Ling; Wagstrom, Chris; McDonough, Dan; Valle Oseguera, Cendy; Ding, Sheng; Rossi, Andrea; Potluri, Shobha; Sundar, Purnima; Sirota, Marina; Yan, Yu; Jones, Jeffrey; Roe-Zurz, Zygy; Srivatsa Srinivasan, Surabhi; Zhai, Wenwu; Pons, Jaume; Rajpal, Arvind; Chaparro-Riggers, Javier

    2018-01-01

    ABSTRACT The commercial success of bispecific antibodies generally has been hindered by the complexities associated with generating appropriate molecules for both research scale and large scale manufacturing purposes. Bispecific IgG (BsIgG) based on two antibodies that use an identical common light chain can be combined with a minimal set of Fc mutations to drive heavy chain heterodimerization in order to address these challenges. However, the facile generation of common light chain antibodies with properties similar to traditional monoclonal antibodies has not been demonstrated and they have only been used sparingly. Here, we describe the design of a synthetic human antibody library based on common light chains to generate antibodies with biochemical and biophysical properties that are indistinguishable to traditional therapeutic monoclonal antibodies. We used this library to generate diverse panels of well-behaved, high affinity antibodies toward a variety of epitopes across multiple antigens, including mouse 4-1BB, a therapeutically important T cell costimulatory receptor. Over 200 BsIgG toward 4-1BB were generated using an automated purification method we developed that enables milligram-scale production of BsIgG. This approach allowed us to identify antibodies with a wide range of agonistic activity that are being used to further investigate the therapeutic potential of antibodies targeting one or more epitopes of 4-1BB. PMID:29227213

  2. Function and Regulation of the Plant COPT Family of High-Affinity Copper Transport Proteins

    Directory of Open Access Journals (Sweden)

    Sergi Puig

    2014-01-01

    Full Text Available Copper (Cu is an essential micronutrient for all eukaryotes because it participates as a redox active cofactor in multiple biological processes, including mitochondrial respiration, photosynthesis, oxidative stress protection, and iron (Fe transport. In eukaryotic cells, Cu transport toward the cytoplasm is mediated by the conserved CTR/COPT family of high-affinity Cu transport proteins. This outlook paper reviews the contribution of our research group to the characterization of the function played by the Arabidopsis thaliana COPT1–6 family of proteins in plant Cu homeostasis. Our studies indicate that the different tissue specificity, Cu-regulated expression, and subcellular localization dictate COPT-specialized contribution to plant Cu transport and distribution. By characterizing lack-of-function Arabidopsis mutant lines, we conclude that COPT1 mediates root Cu acquisition, COPT6 facilitates shoot Cu distribution, and COPT5 mobilizes Cu from storage organelles. Furthermore, our work with copt2 mutant and COPT-overexpressing plants has also uncovered Cu connections with Fe homeostasis and the circadian clock, respectively. Future studies on the interaction between COPT transporters and other components of the Cu homeostasis network will improve our knowledge of plant Cu acquisition, distribution, regulation, and utilization by Cu-proteins.

  3. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    Energy Technology Data Exchange (ETDEWEB)

    Nye, J.S.

    1988-01-01

    The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.

  4. A bioinformatics search for selective histamine h4 receptor antagonists through structure-based virtual screening strategies.

    Science.gov (United States)

    Christopher, Fenila; Thangam, Elden Berla; Suresh, Muthaiyan Xavier

    2012-05-01

    The prevalence of allergic disease is increasing dramatically in the developed world. Studies of allergic diseases have clearly demonstrated that histamine plays an important role in the pathogenesis of the early-phase allergic response. Histamine effects are mediated by H1, H2, H3, and H4 receptors. The presence of the histamine H4 receptors on leukocytes and mast cells suggests that the new histamine receptor H4 plays an important role in the modulation of the immune system. Thus, histamine H4 receptor is an attractive target for anti-allergic therapy. In our present study, we have generated a histamine H4 receptor model using I-TASSER based on human B2-adrenergic G-protein-coupled receptor. Structurally similar compounds of the three known antagonists JNJ777120, thioperamide, and Vuf6002 were retrieved from PubChem, and database was prepared. Virtual screening of those databases was performed, and six compounds with high docking score were identified. Also the binding mode revealed that all the six compounds had interaction with Asp94 of the receptor. Our results serve as a starting point in the development of novel lead compounds in anti-allergic therapy. © 2012 John Wiley & Sons A/S.

  5. The selective cysteinyl leukotriene receptor 1 (CysLT1R) antagonist montelukast regulates extracellular matrix remodeling.

    Science.gov (United States)

    Peng, Jingli; Zhou, Hezheng; Kuang, Guoping; Xie, Lilian; Tian, Tao; Liu, Ru

    2017-03-11

    Scar formation after filtration surgery of glaucoma is mainly caused by excessive synthesis of new extracellular matrix (ECM) and contraction of subconjunctival tissue mediated by human Tenon fibroblasts (HTFs) and the transforming growth factor (TGF-β1). Montelukast, a potent and specific cysteinyl leukotriene receptor 1 (cysLT1R) antagonist, is a licensed drug clinically used for the treatment of bronchial asthma. In this study, we investigated the effects of montelukast on the contractility of HTFs cultured in a three-dimensional collagen gel. We found that cysLT1R was expressed in HTFs. Interestingly, the expression of cysLT1R was increased in response to TGF-β1 in a dose dependent manner, suggesting its potential role in TGF-β1 induced fibrosis. Importantly, we found that montelukast inhibited TGF-β1-induced collagen gel contraction mediated by HTFs in a concentration- and time-dependent manner. In addition, TGF-β1-induced expression of MMP-1 and MMP-3, generation of fibronectin and type I collagen production, focal adhesion kinase (FAK) and paxillin phosphorylation in HTFs were also ameliorated by montelukast in a dose dependent manner. These results suggested that montelukast might provide therapeutic possibilities for inhibition of scar formation after such surgery. Copyright © 2017. Published by Elsevier Inc.

  6. Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components.

    Science.gov (United States)

    Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, U A

    2014-10-01

    Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd 's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and impact of the study: Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. © 2014 Soma Logic, Inc. published by John Wiley & Sons Ltd On behalf of the society for Applied Microbiology.

  7. Subtype-selective modulation of human beta 1- and beta 2-adrenoceptor function by beta-adrenoceptor agonists and antagonists

    NARCIS (Netherlands)

    Brodde, O. E.; Daul, A.; Michel, M. C.

    1990-01-01

    In healthy volunteers a 14-day treatment with the selective beta 1-adrenoceptor agonist xamoterol (2 x 200 mg/day) desensitized beta 1-adrenoceptor-mediated physiological effects, but did not affect beta 2-adrenoceptor-mediated effects; in contrast, a 9-day treatment with the selective beta

  8. Efficient synthesis of novel NK1 receptor antagonists: selective 1,4-addition of grignard reagents to 6-chloronicotinic acid derivatives.

    Science.gov (United States)

    Hoffmann-Emery, Fabienne; Hilpert, Hans; Scalone, Michelangelo; Waldmeier, Pius

    2006-03-03

    A new efficient synthesis of two novel classes of NK1 receptor antagonists, among them befetupitant and netupitant, starting from 6-chloronicotinic acid is described. The introduction of the o-tolyl substituent at C4 of the pyridine ring was achieved by a one-pot selective 1,4-Grignard addition/oxidation sequence to 6-chloronicotinic acid or a derivative of it. The scope of this addition/oxidation sequence was examined. It was also shown that the carboxylic function can be converted to a methyl amino group by a Hofmann rearrangement followed by reduction. Furthermore, a new high-yielding synthesis of 2-(3,5-bistrifluoromethylphenyl)-2-methyl propionic acid based on the carbonylation of the tertiary alcohol obtained by Grignard addition of 3,5-bis(trifluoromethyl)bromobenzene to acetone was established.

  9. Inhibitory effect of the selective serotonin 5-HT₃ receptor antagonist ramosetron on duodenal acidification-induced gastric hypersensitivity in rats.

    Science.gov (United States)

    Nakata-Fukuda, Mari; Hirata, Takuya; Keto, Yoshihiro; Yamano, Mayumi; Yokoyama, Toshihide; Uchiyama, Yasuo

    2014-05-15

    Irritable bowel syndrome (IBS) and functional dyspepsia (FD) are both functional gastrointestinal disorders and frequently co-occur in patients. While one cause of FD appears to be gastric hypersensitivity, whether the hypersensitivity is affected by IBS treatments remains unclear, given the lack of appropriate animal models for testing. Here, we established an experimental model of duodenal acidification-induced gastric hypersensitivity in conscious rats. The model involved duodenal acidification induced by the infusion of hydrochloric acid into the proximal duodenum, with the nociceptive response being determined as the change in mean arterial pressure (MAP) during gastric distension via an indwelling latex balloon. Using our model we evaluated the effects of duodenal acidification, increased distension pressure, and orally administered therapeutic agents for IBS with diarrhea (IBS-D). Duodenal acidification enhanced the pressor response during gastric distension, and pretreatment with the opioid κ-receptor agonist fedotozine (10mg/kg, intra-arterial) inhibited the pressor response. Pressure levels of 15-60 mm Hg increased MAP in response to gastric distension. The serotonin 5-HT3 receptor antagonist ramosetron (30 μg/kg) inhibited MAP increase induced by duodenal acidification, with no other IBS-D therapeutic agents showing any effect. In contrast, the serotonin 5-HT3 receptor agonist m-chlorophenylbiguanide (1mg/kg) significantly enhanced the pressor response during gastric distension. These findings indicate that the serotonin 5-HT3 receptor plays a key role in duodenal acidification-induced gastric hypersensitivity in rats, suggesting that ramosetron may reduce FD symptoms by ameliorating sensitized gastric perception. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Effects of the Selective α5-GABAAR Antagonist S44819 on Excitability in the Human Brain: A TMS-EMG and TMS-EEG Phase I Study.

    Science.gov (United States)

    Darmani, Ghazaleh; Zipser, Carl M; Böhmer, Gabriele M; Deschet, Karine; Müller-Dahlhaus, Florian; Belardinelli, Paolo; Schwab, Matthias; Ziemann, Ulf

    2016-12-07

    Alpha-5 gamma-aminobutyric acid type A receptors (α5-GABAARs) are located extrasynaptically, regulate neuronal excitability through tonic inhibition, and are fundamentally important for processes such as plasticity and learning. For example, pharmacological blockade of α5-GABAAR in mice with ischemic stroke improved recovery of function by normalizing exaggerated perilesional α5-GABAAR-dependent tonic inhibition. S44819 is a novel competitive selective antagonist of the α5-GABAAR at the GABA-binding site. Pharmacological modulation of α5-GABAAR-mediated tonic inhibition has never been investigated in the human brain. Here, we used transcranial magnetic stimulation (TMS) to test the effects of a single oral dose of 50 and 100 mg of S44819 on electromyographic (EMG) and electroencephalographic (EEG) measures of cortical excitability in 18 healthy young adults in a randomized, double-blinded, placebo-controlled, crossover phase I study. A dose of 100 mg, but not 50 mg, of S44819 decreased active motor threshold, the intensity needed to produce a motor evoked potential of 0.5 mV, and the amplitude of the N45, a GABAAergic component of the TMS-evoked EEG response. The peak serum concentration of 100 mg S44819 correlated directly with the decrease in N45 amplitude. Short-interval intracortical inhibition, a TMS-EMG measure of synaptic GABAAergic inhibition, and other components of the TMS-evoked EEG response remained unaffected. These findings provide first time evidence that the specific α5-GABAAR antagonist S44819 reached human cortex to impose an increase in cortical excitability. These data warrant further development of S44819 in a human clinical trial to test its efficacy in enhancing recovery of function after ischemic stroke. The extrasynaptic α-5 gamma-aminobutyric acid type A receptor (α5-GABAAR) regulates neuronal excitability through tonic inhibition in the mammalian brain. Tonic inhibition is important for many fundamental processes such as

  11. The physiological significance of HKT1, a Na{sup +} - coupled high affinity K{sup +} transporter in `Triticum aestivum`

    Energy Technology Data Exchange (ETDEWEB)

    Box, S.; Schachtman, D.P. [University of Adelaide, SA (Australia). Department of Botany

    1997-12-31

    Full text: Several mechanisms for high affinity K{sup +} uptake by higher plants have been proposed:-an ATP-energised K:+ pump, a K{sup +}/H{sup +} antiport and a H{sup +}coupled carrier. Recently, a Na{sup +}--coupled high affinity K{sup +} transporter, HKT1, was isolated from wheat roots. Whilst Na{sup +}K{sup +} symports have been described in charophyte algae, the cloning of HKT1 from wheat is the first, evidence that this type d transport mechanism may function in higher plants. Is the activity of HKT1 an important mechanism involved in K{sup +} acquisition by wheat? The aim of this study was to assess the physiological significance of Na{sup +}- coupled high affinity K{sup +} uptake in T. aestivum. To determine whether HKT1 plays a significant role in wheat growth, we measured the dry weights and ion content of plants grown in a range of [K{sup +}], with and without Na{sup +}. To directly assess the activity of Na{sup +}- coupled K{sup +} transport, {sup 86}Rb{sup +} and {sup 22}Na{sup +} flux analyses were performed on the elongation zones and whole roots of intact seedlings, expressing a high affinity K{sup +} uptake system. The results of these growth and tracer flux studies will be discussed in relation to the expression of the gene encoding HKT1 in T. aestivum

  12. Are basophil histamine release and high affinity IgE receptor expression involved in asymptomatic skin sensitization?

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Assing, K; Jensen, Lone Hummelshøj

    2006-01-01

    Immunoglobulin (Ig)E-sensitized persons with positive skin prick test, but no allergy symptoms, are classified as being asymptomatic skin sensitized (AS). The allergic type 1 disease is dependant on IgE binding to the high affinity IgE-receptor (FcepsilonRI) expressed on basophils and mast cells...

  13. A rhodamine-labeled citalopram analogue as a high-affinity fluorescent probe for the serotonin transporter

    DEFF Research Database (Denmark)

    Zhang, Peng; Jørgensen, Trine Nygaard; Løland, Claus Juul

    2013-01-01

    A novel fluorescent ligand was synthesized as a high-affinity, high specificity probe for visualizing the serotonin transporter (SERT). The rhodamine fluorophore was extended from an aniline substitution on the 5-position of the dihydroisobenzofuran ring of citalopram (2, 1-(3-(dimethylamino)prop...

  14. A dualistic conformational response to substrate binding in the human serotonin transporter reveals a high affinity state for serotonin

    DEFF Research Database (Denmark)

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida

    2015-01-01

    that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation...

  15. Acute oral administration of the novel, competitive and selective glucocorticoid receptor antagonist ORG 34517 reduces the severity of ethanol withdrawal and related hypothalamic-pituitary-adrenal axis activation.

    Science.gov (United States)

    Reynolds, Anna R; Saunders, Meredith A; Brewton, Honoree' W; Winchester, Sydney R; Elgumati, Ibrahim S; Prendergast, Mark A

    2015-09-01

    The development of ethanol dependence is associated with alterations in hypothalamic-pituitary-adrenal (HPA) axis and activation of type II glucocorticoid receptors (GR). These effects may contribute to withdrawal-associated anxiety, craving and relapse to drinking. The present studies examined acute and oral administration of the novel, selective and competitive GR antagonist ORG 34517 on the severity of ethanol withdrawal. Adult, male Sprague-Dawley rats were administered ethanol (4g/kg/i.g.) twice daily for 5 days followed by 2 days of withdrawal for 1, 2 or 3 consecutive cycles. Blood ethanol levels (BELs) were determined at 0930 on Day 4 of each week, while blood corticosterone levels (BCLs) were obtained at 11:00hours on the first day of each ethanol withdrawal. During early withdrawal, subjects received oral administration of ORG 345617 (60mg/kg/i.g.) or a placebo and withdrawal was monitored. Peak BELs of 225.52mg/dl were observed during the third week. Withdrawal from three cycles of the regimen produced marked behavioral abnormalities (e.g., aggression, rigidity, and hypoactivity) and significant increases in BCLs of ethanol-dependent subjects. Acute, oral administration of ORG 34517 during early withdrawal significantly reduced both the severity of ethanol withdrawal, as reflected in reduced rigidity, aggression, and hypoactivity, and elevations in BCL without producing any sedative-like effects. The present findings demonstrate that repeated ethanol exposure and withdrawal is associated with significant behavioral abnormalities and dysregulation of HPA axis activation. Further these data suggest that selective GR antagonists should be further considered as putative pharmacotherapies for treatment of ethanol dependence. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  16. Hydroxyproline-induced Helical Disruption in Conantokin Rl-B Affects Subunit-selective Antagonistic Activities toward Ion Channels of N-Methyl-d-aspartate Receptors*

    Science.gov (United States)

    Kunda, Shailaja; Yuan, Yue; Balsara, Rashna D.; Zajicek, Jaroslav; Castellino, Francis J.

    2015-01-01

    Conantokins are ∼20-amino acid peptides present in predatory marine snail venoms that function as allosteric antagonists of ion channels of the N-methyl-d-aspartate receptor (NMDAR). These peptides possess a high percentage of post-/co-translationally modified amino acids, particularly γ-carboxyglutamate (Gla). Appropriately spaced Gla residues allow binding of functional divalent cations, which induces end-to-end α-helices in many conantokins. A smaller number of these peptides additionally contain 4-hydroxyproline (Hyp). Hyp should prevent adoption of the metal ion-induced full α-helix, with unknown functional consequences. To address this disparity, as well as the role of Hyp in conantokins, we have solved the high resolution three-dimensional solution structure of a Gla/Hyp-containing 18-residue conantokin, conRl-B, by high field NMR spectroscopy. We show that Hyp10 disrupts only a small region of the α-helix of the Mn2+·peptide complex, which displays cation-induced α-helices on each terminus of the peptide. The function of conRl-B was examined by measuring its inhibition of NMDA/Gly-mediated current through NMDAR ion channels in mouse cortical neurons. The conRl-B displays high inhibitory selectivity for subclasses of NMDARs that contain the functionally important GluN2B subunit. Replacement of Hyp10 with N8Q results in a Mg2+-complexed end-to-end α-helix, accompanied by attenuation of NMDAR inhibitory activity. However, replacement of Hyp10 with Pro10 allowed the resulting peptide to retain its inhibitory property but diminished its GluN2B specificity. Thus, these modified amino acids, in specific peptide backbones, play critical roles in their subunit-selective inhibition of NMDAR ion channels, a finding that can be employed to design NMDAR antagonists that function at ion channels of distinct NMDAR subclasses. PMID:26048991

  17. A selective neurokinin-1 receptor antagonist in chronic PTSD: a randomized, double-blind, placebo-controlled, proof-of-concept trial.

    Science.gov (United States)

    Mathew, Sanjay J; Vythilingam, Meena; Murrough, James W; Zarate, Carlos A; Feder, Adriana; Luckenbaugh, David A; Kinkead, Becky; Parides, Michael K; Trist, David G; Bani, Massimo S; Bettica, Paolo U; Ratti, Emiliangelo M; Charney, Dennis S

    2011-03-01

    The substance P-neurokinin-1 receptor (SP-NK(1)R) system has been extensively studied in experimental models of stress, fear, and reward. Elevated cerebrospinal fluid (CSF) SP levels were reported previously in combat-related PTSD. No medication specifically targeting this system has been tested in PTSD. This proof-of-concept randomized, double-blind, placebo-controlled trial evaluated the selective NK(1)R antagonist GR205171 in predominately civilian PTSD. Following a 2-week placebo lead-in, 39 outpatients with chronic PTSD and a Clinician-Administered PTSD Scale (CAPS) score ≥50 were randomized to a fixed dose of GR205171 (N=20) or placebo (N=19) for 8weeks. The primary endpoint was mean change from baseline to endpoint in the total CAPS score. Response rate (≥50% reduction in baseline CAPS) and safety/tolerability were secondary endpoints. CSF SP concentrations were measured in a subgroup of patients prior to randomization. There was significant improvement in the mean CAPS total score across all patients over time, but no significant difference was found between GR205171 and placebo. Likewise, there was no significant effect of drug on the proportion of responders [40% GR205171 versus 21% placebo (p=0.30)]. An exploratory analysis showed that GR205171 treatment was associated with significant improvement compared to placebo on the CAPS hyperarousal symptom cluster. GR205171 was well-tolerated, with no discontinuations due to adverse events. CSF SP concentrations were positively correlated with baseline CAPS severity. The selective NK(1)R antagonist GR205171 had fewer adverse effects but was not significantly superior to placebo in the short-term treatment of chronic PTSD. (ClinicalTrials.gov Identifier: NCT 00211861, NCT 00383786). Published by Elsevier B.V.

  18. Selectivity is species-dependent: Characterization of standard agonists and antagonists at human, rat, and mouse adenosine receptors

    National Research Council Canada - National Science Library

    Alnouri, Mohamad Wessam; Jepards, Stephan; Casari, Alessandro; Schiedel, Anke C; Hinz, Sonja; Müller, Christa E

    2015-01-01

    Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species...

  19. Novel screening system for high-affinity ligand of heredity vitamin D-resistant rickets-associated vitamin D receptor mutant R274L using bioluminescent sensor.

    Science.gov (United States)

    Mano, Hiroki; Nishikawa, Miyu; Yasuda, Kaori; Ikushiro, Shinichi; Saito, Nozomi; Sawada, Daisuke; Honzawa, Shinobu; Takano, Masashi; Kittaka, Atsushi; Sakaki, Toshiyuki

    2017-03-01

    Hereditary vitamin D-resistant rickets (HVDRR) is caused by mutations in the vitamin D receptor (VDR) gene. Arg274 located in the ligand binding domain (LBD) of VDR is responsible for anchoring 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) by forming a hydrogen bond with the 1α-hydroxyl group of 1α,25(OH)2D3. The Arg274Leu (R274L) mutation identified in patients with HVDRR causes a 1000-fold decrease in the affinity for 1α,25(OH)2D3, and dramatically reduces vitamin D- related gene expression. Recently, we successfully constructed fusion proteins consisting of split-luciferase and LBD of the VDR. The chimeric protein LucC-LBD-LucN, which displays the C-terminal domain of luciferase (LucC) at its N-terminus, can detect and discriminate between VDR agonists and antagonists. The LucC-LBD (R274L)-LucN was constructed to screen high-affinity ligands for the mutant VDR (R274L). Of the 33 vitamin D analogs, 5 showed much higher affinities for the mutant VDR (R274L) than 1α,25(OH)2D3, and 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-(OH)2D3 showed the highest affinity. These compounds might be potential therapeutics for HVDRR caused by the mutant VDR (R274L). Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. High Affinity vs. Native Fibronectin in the Modulation of αvβ3 Integrin Conformational Dynamics: Insights from Computational Analyses and Implications for Molecular Design.

    Directory of Open Access Journals (Sweden)

    Antonella Paladino

    2017-01-01

    Full Text Available Understanding how binding events modulate functional motions of multidomain proteins is a major issue in chemical biology. We address several aspects of this problem by analyzing the differential dynamics of αvβ3 integrin bound to wild type (wtFN10, agonist or high affinity (hFN10, antagonist mutants of fibronectin. We compare the dynamics of complexes from large-scale domain motions to inter-residue coordinated fluctuations to characterize the distinctive traits of conformational evolution and shed light on the determinants of differential αvβ3 activation induced by different FN sequences. We propose an allosteric model for ligand-based integrin modulation: the conserved integrin binding pocket anchors the ligand, while different residues on the two FN10's act as the drivers that reorganize relevant interaction networks, guiding the shift towards inactive (hFN10-bound or active states (wtFN10-bound. We discuss the implications of results for the design of integrin inhibitors.

  1. Design and synthesis of novel, potent and selective hypoxanthine analogs as adenosine A1 receptor antagonists and their biological evaluation.

    Science.gov (United States)

    Koul, Summon; Ramdas, Vidya; Barawkar, Dinesh A; Waman, Yogesh B; Prasad, Neela; Madadi, Santosh Kumar; Shejul, Yogesh D; Bonagiri, Rajesh; Basu, Sujay; Menon, Suraj; Reddy, Srinivasa B; Chaturvedi, Sandhya; Chennamaneni, Srinivas Rao; Bedse, Gaurav; Thakare, Rhishikesh; Gundu, Jayasagar; Chaudhary, Sumit; De, Siddhartha; Meru, Ashwinkumar V; Palle, Venkata; Chugh, Anita; Mookhtiar, Kasim A

    2017-03-15

    Multipronged approach was used to synthesize a library of diverse C-8 cyclopentyl hypoxanthine analogs from a common intermediate III. Several potent and selective compounds were identified and evaluated for pharmacokinetic (PK) properties in Wistar rats. One of the compounds 14 with acceptable PK parameters was selected for testing in in vivo primary acute diuresis model. The compound demonstrated significant diuretic activity in this model. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Analysis of high-affinity binding of protein kinase R to double-stranded RNA.

    Science.gov (United States)

    Husain, Bushra; Mukerji, Ishita; Cole, James L

    2012-11-06

    Protein kinase R (PKR) is an interferon-induced kinase that plays a pivotal role in the innate immunity response to viral infection. PKR is activated upon binding to double-stranded RNA (dsRNA). Our previous analysis of binding of PKR to dsRNAs ranging from 20 to 40 bp supports a dimerization model for activation in which 30 bp represents the minimal length required to bind two PKR monomers and activate PKR via autophosphorylation. These studies were complicated by the formation of protein-RNA aggregates, particularly at low salt concentrations using longer dsRNAs. Here, we have taken advantage of the enhanced sensitivity afforded using fluorescence-detected analytical ultracentrifugation to reduce the RNA concentrations from micromolar to nanomolar. Under these conditions, we are able to characterize high-affinity binding of PKR to longer dsRNAs in 75 mM NaCl. The PKR binding stoichiometries are increased at lower salt concentrations but remain lower than those previously obtained for the dsRNA binding domain. The dependence of the limiting PKR binding stoichiometries on dsRNA length does not conform to standard models for nonspecific binding and suggests that binding to longer sequences occurs via a different binding mode with a larger site size. Although dimerization plays a key role in the PKR activation mechanism, the ability of shorter dsRNAs to bind two PKR monomers is not sufficient to induce autophosphorylation. We propose that activation of PKR by longer RNAs is correlated with an alternative binding mode in which both of the dsRNA binding motifs contact the RNA, inducing PKR to dimerize via a direct interaction of the kinase domains.

  3. Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT.

    Directory of Open Access Journals (Sweden)

    Katherine J Fishwick

    Full Text Available Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer's Disease (AD, and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylcholine (ACh is closely linked to the activity of the high-affinity choline transporter protein (CHT, but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase in cells significantly lowers phospho-PKB/Akt levels and decreases choline uptake activity. We show total internal reflection microscopy (TIRF imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization.

  4. Integrin alphaVbeta6 is a high-affinity receptor for coxsackievirus A9.

    Science.gov (United States)

    Heikkilä, Outi; Susi, Petri; Stanway, Glyn; Hyypiä, Timo

    2009-01-01

    Coxsackievirus A9 (CAV9), a member of the genus Enterovirus in the family Picornaviridae, possesses an integrin-binding arginine-glycine-aspartic acid (RGD) motif in the C terminus of VP1 capsid protein. CAV9 has been shown to utilize integrins alphaVbeta3 and alphaVbeta6 as primary receptors for cell attachment. While CAV9 RGD-mutants (RGE and RGDdel) are capable of infecting rhabdomyosarcoma (RD) cell line, they grow very poorly in an epithelial lung carcinoma cell line (A549). In this study, the relationships between CAV9 infectivity in A549 and RD cells, receptor expression and integrin binding were analysed. A549 cells were shown to express both integrins alphaVbeta3 and alphaVbeta6, whereas alphaVbeta6 expression was not detected on the RD cells. Native CAV9 but not RGE and RGDdel mutants bound efficiently to immobilized alphaVbeta3 and alphaVbeta6. Adhesion of CAV9 but not RGE/RGDdel to A549 cells was also significantly higher than to RD cells. In contrast, no affinity or adhesion of bacterially produced VP1 proteins to the integrins or to the cells was detected. Function-blocking antibodies against alphaV-integrins blocked CAV9 but not CAV9-RGDdel infectivity, indicating that the viruses use different internalization routes; this may explain the differential infection kinetics of CAV9 and RGDdel. In an affinity assay, soluble alphaVbeta6, but not alphaVbeta3, bound to immobilized CAV9. Similarly, only soluble alphaVbeta6 blocked virus infectivity. These data suggest that CAV9 binding to alphaVbeta6 is a high-affinity interaction, which may indicate its importance in clinical infections; this remains to be determined.

  5. Effects of a newly developed potent orexin-2 receptor-selective antagonist Compound1m on sleep/wake states in mice

    Directory of Open Access Journals (Sweden)

    Keishi eEtori

    2014-01-01

    Full Text Available Orexins (also known as hypocretins, which are hypothalamic neuropeptides, play critical roles in the regulation of sleep/wakefulness states by activating two G-protein coupled receptors (GPCRs, orexin 1 (OX1R and orexin 2 receptors (OX2R. In order to know the difference between effects of OX2R-selective antagonists (2-SORA and dual orexin receptor antagonists (DORA, and to understand the mechanisms underlying orexin-mediated regulation of sleep/wakefulness states, we examined the effects of a newly developed 2-SORA, Compound 1m (C1m, and a DORA, suvorexant, on sleep/wakefulness states in C57BL/6J mice. After oral administration in the dark period, both C1m and suvorexant exhibited potent sleep-promoting properties with similar efficacy in a dose-dependent manner. While C1m did not increase NREM and REM sleep episode durations, suvorexant induced longer episode durations of NREM and REM sleep as compared with both the vehicle- and C1m-administered groups. When compounds were injected during light period, C1m did not show a significant change in sleep/wakefulness states in the light period, whereas suvorexant slightly but significantly increased the sleep time. We also found that C1m did not affect the time of REM sleep, while suvorexant markedly increased it. This suggests that although OX1R-mediated pathway plays a pivotal role in promoting wakefulness, OX1R-mediated pathway also plays an additional role. OX1R-mediated pathway also plays a role in suppression of REM sleep. Fos-immunostaining showed that both compounds affected the activity of arousal-related neurons with different patterns. These results suggest partly overlapping and partly distinct roles of orexin receptors in the regulation of sleep/wakefulness states.

  6. A potent and selective calcitonin gene-related peptide (CGRP) receptor antagonist, MK-8825, inhibits responses to nociceptive trigeminal activation: Role of CGRP in orofacial pain.

    Science.gov (United States)

    Romero-Reyes, Marcela; Pardi, Vanessa; Akerman, Simon

    2015-09-01

    Temporomandibular disorders (TMDs) are orofacial pains within the trigeminal distribution, which involve the masticatory musculature, the temporomandibular joint or both. Their pathophysiology remains unclear, as inflammatory mediators are thought to be involved, and clinically TMD presents pain and sometimes limitation of function, but often appears without gross indications of local inflammation, such as visible edema, redness and increase in temperature. Calcitonin gene-related peptide (CGRP) has been implicated in other pain disorders with trigeminal distribution, such as migraine, of which TMD shares a significant co-morbidity. CGRP causes activation and sensitization of trigeminal primary afferent neurons, independent of any inflammatory mechanisms, and thus may also be involved in TMD. Here we used a small molecule, selective CGRP receptor antagonist, MK-8825, to dissect the role of CGRP in inducing spontaneous nociceptive facial grooming behaviors, neuronal activation in the trigeminal nucleus, and systemic release of pro-inflammatory cytokines, in a mouse model of acute orofacial masseteric muscle pain that we have developed, as a surrogate of acute TMD. We show that CFA masseteric injection causes significant spontaneous orofacial pain behaviors, neuronal activation in the trigeminal nucleus, and release of interleukin-6 (IL-6). In mice pre-treated with MK-8825 there is a significant reduction in these spontaneous orofacial pain behaviors. Also, at 2 and 24h after CFA injection the level of Fos immunoreactivity in the trigeminal nucleus, used as a marker of neuronal activation, was much lower on both ipsilateral and contralateral sides after pre-treatment with MK-8825. There was no effect of MK-8825 on the release of IL-6. These data suggest that CGRP may be involved in TMD pathophysiology, but not via inflammatory mechanisms, at least in the acute stage. Furthermore, CGRP receptor antagonists may have therapeutic efficacy in the treatment of TMD, as they

  7. Novel 5-HT5A receptor antagonists ameliorate scopolamine-induced working memory deficit in mice and reference memory impairment in aged rats

    Directory of Open Access Journals (Sweden)

    Mayako Yamazaki

    2015-03-01

    Full Text Available Despite the human 5-HT5A receptor being cloned in 1994, the biological function of this receptor has not been extensively characterized due to a lack of specific ligands. We recently reported that the selective 5-HT5A receptor antagonist ASP5736 ameliorated cognitive impairment in several animal models of schizophrenia. Given that areas of the brain with high levels of 5-HT5A receptor expression, such as the hippocampus and cerebral cortex, have important functions in cognition and memory, we evaluated the chemically diverse, potent and brain-penetrating 5-HT5A receptor antagonists ASP5736, AS2030680, and AS2674723 in rodent models of cognitive dysfunction associated with dementia. Each of these compounds exhibited a high affinity for recombinant 5-HT5A receptors that was comparable to that of the non-selective ligand of this receptor, lysergic acid diethylamide (LSD. Although each compound had a low affinity for other receptors, 5-HT5A was the only receptor for which all three compounds had a high affinity. Each of the three compounds ameliorated scopolamine-induced working memory deficit in mice and improved reference memory impairment in aged rats at similar doses. Further, ASP5736 decreased the binding of LSD to 5-HT5A receptors in the olfactory bulb of rats in a dose-dependent manner and occupied 15%–50% of brain 5-HT5A receptors at behaviorally effective doses. These results indicate that the 5-HT5A receptor is involved in learning and memory and that treatment with 5-HT5A receptor antagonists might be broadly effective for cognitive impairment associated with not only schizophrenia but also dementia.

  8. Characterization of high affinity (/sup 3/H)triazolam binding in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Earle, M.; Concas, A.; Yamamura, H.I.

    1986-03-01

    The hypnotic Triazolam (TZ), a triazolo (1,4)-benzodiazepine, displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Specific binding properties of this recently tritiated TZ were characterized. The authors major objectives were the direct measurement of the temperature dependence and the GABA effect on (/sup 3/H)TZ binding. Saturation studies showed a shift to lower affinity at 37/sup 0/C (K/sub d/ = 0.25 +/- 0.01 nM at O/sup 0/C; K/sub d/ = 1.46 +/- 0.03 nM at 37/sup 0/C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. at 0/sup 0/C and 1001 +/- 43 fmoles/mg prot. at 37/sup 0/C). Inhibition studies showed that (/sup 3/H)TZ binding displayed no GABA shift at 0/sup 0/C(K/sub i/ 0.37 +/- 0.03 nM/- GABA and K/sub i/ = 0.55 +/- 0.13 nM/+GABA) but a nearly two-fold shift was apparent at 37/sup 0/C (K/sub i/ = 2.92 +/- 0.2 nM/-GABA; K/sub i/ = 1.37 +/- 0.11 mM/+GABA). These results were also confirmed by saturation studies in the presence or absence of GABA showing a shift to higher affinity in the presence of GABA only at 37/sup 0/C. In Ro 15-1788/(/sup 3/H)TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on (/sup 3/H)TZ binding at both temperatures. In conclusion (/sup 3/H)TZ binding showed an extremely high affinity for benzodiazepine receptors. In contrast to reported literature, the findings suggest that TZ interacts with benzodiazepine receptors similar to other benzodiazepine agonists.

  9. Twins in spirit part II: DOTATATE and high-affinity DOTATATE - the clinical experience

    Energy Technology Data Exchange (ETDEWEB)

    Brogsitter, Claudia; Zoephel, Klaus; Hartmann, Holger; Kotzerke, Joerg [Technische Universitaet Dresden, Department of Nuclear Medicine, Dresden (Germany); Schottelius, Margret; Wester, Hans-Juergen [Technische Universitaet Muenchen, Pharmaceutical Radiochemistry and Department of Nuclear Medicine, Muenchen (Germany)

    2014-06-15

    Over recent decades interest in diagnosis and treatment of neuroendocrine tumours (NET) has steadily grown. The basis for diagnosis and therapy of NET with radiolabelled somatostatin (hsst) analogues is the variable overexpression of hsst receptors (hsst1-5 receptors). We hypothesized that radiometal derivatives of DOTA-iodo-Tyr{sup 3}-octreotide analogues might be excellent candidates for somatostatin receptor imaging. We therefore explored the diagnostic potential of {sup 68}Ga-DOTA-iodo-Tyr{sup 3}-octreotate [{sup 68}Ga-DOTA,3-iodo-Tyr{sup 3},Thr{sup 8}]octreotide ({sup 68}Ga-HA-DOTATATE; HA, high-affinity) compared to the established {sup 68}Ga-DOTA-Tyr{sup 3}-octreotate ({sup 68}Ga-DOTATATE) in vivo. The study included 23 patients with known somatostatin receptor-positive metastases from NETs, thyroid cancer or glomus tumours who were investigated with both {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE. A patient-based and a lesion-based comparative analysis was carried out of normal tissue distribution and lesion detectability in a qualitative and a semiquantitative manner. {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE showed comparable uptake in the liver (SUV{sub mean} 8.9 ± 2.2 vs. 9.3 ± 2.5, n.s.), renal cortex (SUV{sub mean} 13.3 ± 3.9 vs. 14.5 ± 3.7, n.s.) and spleen (SUV{sub mean} 24.0 ± 6.7 vs. 22.9 ± 7.3, n.s.). A somewhat higher pituitary uptake was found with {sup 68}Ga-HA-DOTATATE (SUV{sub mean} 6.3 ± 1.8 vs. 5.4 ± 2.1, p < 0.05). On a lesion-by-lesion basis a total of 344 lesions were detected. {sup 68}Ga-HA-DOTATATE demonstrated 328 lesions (95.3 % of total lesions seen), and {sup 68}Ga-DOTATATE demonstrated 332 lesions (96.4 %). The mean SUV{sub max} of all lesions was not significantly different between {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE (17.8 ± 11.4 vs. 16.7 ± 10.7, n.s.). Our analysis demonstrated very good concordance between {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE PET data. As the availability and use of {sup

  10. NETUPITANT, A POTENT AND HIGHLY SELECTIVE NK1 RECEPTOR ANTAGONIST, ALLEVIATES ACETIC ACID-INDUCED BLADDER OVERACTIVITY IN ANESTHETIZED GUINEA-PIGS

    Directory of Open Access Journals (Sweden)

    Stefano Palea

    2016-08-01

    Full Text Available Introduction. Tachykinins potently contract the isolated urinary bladder from a number of animal species and play an important role in the regulation of the micturition reflex. On the guinea-pig isolated urinary bladder we examined the effects of a new potent and selective NK1 receptor antagonist (netupitant on the contractions induced by a selective NK1 receptor agonist, SP-methylester (SP-OMe. Moreover, the effects of netupitant and another selective NK1 antagonist (L-733,060 were studied in anesthetized guinea-pigs using two experimental models, the isovolumetric bladder contractions and a model of bladder overactivity induced by intravesical administration of acetic acid (AA. Methods and Results. Detrusor muscle strips were mounted in 5 mL organ baths and isometric contractions to cumulative concentrations of SP-OME were recorded before and after incubation with increasing concentrations of netupitant. In anesthetized female guinea-pigs, reflex bladder activity was examined under isovolumetric conditions with the bladder distended with saline or during cystometry using intravesical infusion of acetic acid (AA. After a 30 min stabilization period, netupitant (0.1-3 mg/kg, i.v. or L-733,060 (3-10 mg/kg, i.v. were administered. In the detrusor muscle, netupitant produced a concentration-dependent inhibition (mean pKB = 9.24 of the responses to SP-OMe. Under isovolumetric conditions, netupitant or L-733,060 reduced bladder contraction frequency in a dose-dependent manner, but neither drug changed bladder contraction amplitude. In the AA model, netupitant dose-dependently increased intercontraction interval (ICI but had no effect on the amplitude of micturition (AM. L-733,060 dose-dependently increased ICI also but this effect was paralleled by a significant reduction of AM. Conclusion. Netupitant decreases the frequency of reflex bladder contractions without altering their amplitude, suggesting that this drug targets the afferent limb of the

  11. Netupitant, a Potent and Highly Selective NK1 Receptor Antagonist, Alleviates Acetic Acid-Induced Bladder Overactivity in Anesthetized Guinea-Pigs.

    Science.gov (United States)

    Palea, Stefano; Guilloteau, Véronique; Rekik, Moéz; Lovati, Emanuela; Guerard, Marc; Guardia, Maria-Alba; Lluel, Philippe; Pietra, Claudio; Yoshiyama, Mitsuharu

    2016-01-01

    Introduction. Tachykinins potently contract the isolated urinary bladder from a number of animal species and play an important role in the regulation of the micturition reflex. On the guinea-pig isolated urinary bladder we examined the effects of a new potent and selective NK1 receptor antagonist (netupitant) on the contractions induced by a selective NK1 receptor agonist, SP-methylester (SP-OMe). Moreover, the effects of netupitant and another selective NK1 antagonist (L-733,060) were studied in anesthetized guinea-pigs using two experimental models, the isovolumetric bladder contractions and a model of bladder overactivity induced by intravesical administration of acetic acid (AA). Methods and Results. Detrusor muscle strips were mounted in 5 mL organ baths and isometric contractions to cumulative concentrations of SP-OME were recorded before and after incubation with increasing concentrations of netupitant. In anesthetized female guinea-pigs, reflex bladder activity was examined under isovolumetric conditions with the bladder distended with saline or during cystometry using intravesical infusion of AA. After a 30 min stabilization period, netupitant (0.1-3 mg/kg, i.v.) or L-733,060 (3-10 mg/kg, i.v.) were administered. In the detrusor muscle, netupitant produced a concentration-dependent inhibition (mean pKB = 9.24) of the responses to SP-OMe. Under isovolumetric conditions, netupitant or L-733,060 reduced bladder contraction frequency in a dose-dependent manner, but neither drug changed bladder contraction amplitude. In the AA model, netupitant dose-dependently increased intercontraction interval (ICI) but had no effect on the amplitude of micturition (AM). L-733,060 dose-dependently increased ICI also but this effect was paralleled by a significant reduction of AM. Conclusion. Netupitant decreases the frequency of reflex bladder contractions without altering their amplitude, suggesting that this drug targets the afferent limb of the micturition reflex circuit

  12. An improved method to prepare an injectable microemulsion of the galanin-receptor 3 selective antagonist, SNAP 37889, using Kolliphor(®) HS 15.

    Science.gov (United States)

    Scheller, Karlene J; Williams, Spencer J; Lawrence, Andrew J; Jarrott, Bevyn; Djouma, Elvan

    2014-01-01

    Research into the galanin-3 (GAL3) receptor has many challenges, including the lack of commercially available selective ligands. While the identification of non-peptidergic GAL3 receptor-selective antagonists, 1-phenyl-3-[3-(trifluoromethyl)phenyl]iminoindol-2-one (SNAP 37889) and 1-[3-(2-pyrrolidin-1-ylethoxy)phenyl]-3-[3-(trifluoromethyl)phenyl]iminoindol-2-one (SNAP 398299) have implicated a role for GAL3 receptors in anxiety, depression and drug-seeking behaviour, a major limitation of their use is poor aqueous solubility. Previously we have used 5% dimethylsulfoxide (DMSO) with 1% hydroxypropylmethyl cellulose in saline to dissolve SNAP 37889 for intraperitoneal (i.p.) injections of rats; however this produced a micro-suspension that was not ideal. The injectable formulation of SNAP 37889 was improved as follows:•30% (w/v) Kolliphor(®) HS 15 (Solutol HS(®) 15) and sodium phosphate buffer (0.01 M, pH 7.4) were used as vehicles.•A smooth glass mortar and pestle was used to triturate the Kolliphor(®) HS 15 and SNAP 37889 into a paste before addition to the sodium phosphate buffer at room temperature (RT).•The resulting mixture was vortexed until the paste was fully dissolved and the microemulsion was allowed to sit for 20 min to allow air bubbles to coalesce.

  13. High Affinity Binding of Chp1 Chromodomain to K9 Methylated Histone H3 is Required to Establish Centromeric Hterochromatin

    Energy Technology Data Exchange (ETDEWEB)

    Schalch, T.; Job, G; Noffsinger, V; Shanker, S; Kuscu, C; Joshua-Tor, L; Partridge, J

    2009-01-01

    In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced.

  14. Ginkgolide X is a potent antagonist of anionic Cys-loop receptors with a unique selectivity profile at glycine receptors

    DEFF Research Database (Denmark)

    Jensen, Anders Asbjørn; Bergmann, Marianne Lerbæk; Sander, Tommy

    2010-01-01

    The novel ginkgolide analog ginkgolide X was characterized functionally at human glycine and gamma-aminobutyric acid type A receptors (GlyRs and GABAARs, respectively) in the fluorescence-based FLIPR Membrane Potential assay. The compound inhibited the signalling of all GABAAR subtypes included...... and alpha2beta subtypes at concentrations up to 300 muM. Thus, the functional properties of the compound were significantly different from those of the naturally occuring ginkgolides A, B, C, J and M but similar to those of picrotoxin. In a mutagenesis study the 6' M2 residues in the GlyR ion channel were...... identified as the primary molecular determinant of the selectivity profile of ginkgolide X, and a 6' M2 ring consisting of five Thr residues was found to be of key importance for its activity at the GABAAR. Conformational analysis and docking of low-energy conformations of the native ginkgolide...

  15. Functional characterization of the high affinity IgG Receptor : making heads and tails of FcγRI

    NARCIS (Netherlands)

    van der Poel, C.E.

    2011-01-01

    This thesis focuses on human FcγRI, a high affinity receptor for antibodies of the IgG isotype. IgG is the most abundant antibody type in blood and all currently FDA approved therapeutic antibodies are of the IgG isotype. FcγRI, a member of the activating Fcγ receptors, exists as a complex of a

  16. Benzodiazepines have high-affinity binding sites and induce melanogenesis in B16/C3 melanoma cells.

    OpenAIRE

    Matthew, E; Laskin, J D; Zimmerman, E A; Weinstein, I B; Hsu, K C; Engelhardt, D L

    1981-01-01

    We found that two markers of differentiation, tyrosinase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) activity and melanin synthesis, are induced by diazepam in B16/C3 mouse melanoma cells. We also demonstrated high-affinity binding sites for [3H]diazepam in these cells by radioreceptor assay, and we visualized binding to the cell surface by fluorescence microscopy with a benzodiazepine analog conjugated to a fluorescein-labeled protein. Our studies also showed tha...

  17. α4βδ GABA receptors are high-affinity targets for γ-hydroxybutyric acid (GHB)

    DEFF Research Database (Denmark)

    Absalom, N.; Karim, N.; Eghorn, L.F.

    2012-01-01

    γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinit...... and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism....

  18. Hydroxyproline-induced Helical Disruption in Conantokin Rl-B Affects Subunit-selective Antagonistic Activities toward Ion Channels of N-Methyl-d-aspartate Receptors.

    Science.gov (United States)

    Kunda, Shailaja; Yuan, Yue; Balsara, Rashna D; Zajicek, Jaroslav; Castellino, Francis J

    2015-07-17

    Conantokins are ~20-amino acid peptides present in predatory marine snail venoms that function as allosteric antagonists of ion channels of the N-methyl-d-aspartate receptor (NMDAR). These peptides possess a high percentage of post-/co-translationally modified amino acids, particularly γ-carboxyglutamate (Gla). Appropriately spaced Gla residues allow binding of functional divalent cations, which induces end-to-end α-helices in many conantokins. A smaller number of these peptides additionally contain 4-hydroxyproline (Hyp). Hyp should prevent adoption of the metal ion-induced full α-helix, with unknown functional consequences. To address this disparity, as well as the role of Hyp in conantokins, we have solved the high resolution three-dimensional solution structure of a Gla/Hyp-containing 18-residue conantokin, conRl-B, by high field NMR spectroscopy. We show that Hyp(10) disrupts only a small region of the α-helix of the Mn(2+)·peptide complex, which displays cation-induced α-helices on each terminus of the peptide. The function of conRl-B was examined by measuring its inhibition of NMDA/Gly-mediated current through NMDAR ion channels in mouse cortical neurons. The conRl-B displays high inhibitory selectivity for subclasses of NMDARs that contain the functionally important GluN2B subunit. Replacement of Hyp(10) with N(8)Q results in a Mg(2+)-complexed end-to-end α-helix, accompanied by attenuation of NMDAR inhibitory activity. However, replacement of Hyp(10) with Pro(10) allowed the resulting peptide to retain its inhibitory property but diminished its GluN2B specificity. Thus, these modified amino acids, in specific peptide backbones, play critical roles in their subunit-selective inhibition of NMDAR ion channels, a finding that can be employed to design NMDAR antagonists that function at ion channels of distinct NMDAR subclasses. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Glucose uptake and growth of glucose-limited chemostat cultures of Aspergillus niger and a disruptant lacking MstA, a high-affinity glucose transporter

    NARCIS (Netherlands)

    Jorgensen, T.R.; vanKuyk, P.A.; Poulsen, B.R.; Ruijter, G.J.G.; Visser, J.; Iversen, J.J.L.

    2007-01-01

    This is a study of high-affinity glucose uptake in Aspergillus niger and the effect of disruption of a high-affinity monosaccharide-transporter gene, mstA. The substrate saturation constant (K-s) of a reference strain was about 15 mu M in glucose-limited chemostat culture. Disruption of mstA

  20. Radiosynthesis and Evaluation of [(11)C]3-Hydroxycyclopent-1-enecarboxylic Acid as Potential PET Ligand for the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Jensen, Claus H; Hansen, Hanne D; Bay, Tina

    2017-01-01

    γ-Hydroxybutyric acid (GHB) is an endogenous neuroactive substance and proposed neurotransmitter with affinity for both low- and high-affinity binding sites. A radioligand with high and specific affinity toward the high-affinity GHB binding site would be a unique tool toward a more complete...

  1. High-affinity glucose transport in Aspergillus nidulans is mediated by the products of two related but differentially expressed genes.

    Directory of Open Access Journals (Sweden)

    Josep V Forment

    Full Text Available Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation.

  2. Synthesis and pharmacology of 3-isoxazolol amino acids as selective antagonists at group I metabotropic glutamic acid receptors

    DEFF Research Database (Denmark)

    Madsen, U; Bräuner-Osborne, H; Frydenvang, Karla Andrea

    2001-01-01

    GluRs), the few analogues of (RS)-2-amino-3-(3-hydroxy-5-isoxazolyl)propionic acid [HIBO, (RS)-4] so far known typically interact with iGluRs as well as metabotropic Glu receptors (mGluRs). We here report the synthesis and pharmacology of a series of 4-substituted analogues of HIBO. The hexyl analogue 9 was shown......Using ibotenic acid (2) as a lead, two series of 3-isoxazolol amino acid ligands for (S)-glutamic acid (Glu, 1) receptors have been developed. Whereas analogues of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid [AMPA, (RS)-3] interact selectively with ionotropic Glu receptors (i...... to originate in (S)-11 (EC(50) = 395 microM, K(b) = 86 and 90 microM, respectively). Compound 9, administered icv, but not sc, was shown to protect mice against convulsions induced by N-methyl-D-aspartic acid (NMDA). Compounds 9 and 11 were resolved using chiral HPLC, and the configurational assignments...

  3. Chronic administration of the selective P2X3, P2X2/3 receptor antagonist, A-317491, transiently attenuates cancer-induced bone pain in mice

    DEFF Research Database (Denmark)

    Hansen, Rikke Rie; Nasser, Arafat; Falk, Sarah

    2012-01-01

    The purinergic P2X3 and P2X2/3 receptors are in the peripheral nervous system almost exclusively confined to afferent sensory neurons, where they are found both at peripheral and central synapses. The P2X3 receptor is implicated in both neuropathic and inflammatory pain. However, the role of the P2......X3 receptor in chronic cancer-induced bone pain is less known. Here we investigated the effect of systemic acute and chronic administration of the selective P2X3, P2X2/3 receptor antagonist (5-[[[(3-Phenoxyphenyl)methyl][(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl]-1......,2,4-benzenetricarboxylic acid sodium salt hydrate) (A-317491) in a murine model of cancer-induced bone pain. Chronic administration of A-317491 (30µmol/kgs.c., b.i.d.) resulted in a transient attenuation of pain related behaviours in the early stage of the bone cancer model, but had no effect in the late and more...

  4. RC-3095, a Selective Gastrin-Releasing Peptide Receptor Antagonist, Does Not Protect the Lungs in an Experimental Model of Lung Ischemia-Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Vera L. Oliveira-Freitas

    2015-01-01

    Full Text Available RC-3095, a selective GRPR antagonist, has been shown to have anti-inflammatory properties in different models of inflammation. However, its protective effect on lungs submitted to lung ischemia-reperfusion injury has not been addressed before. Then, we administrated RC-3095 intravenously before and after lung reperfusion using an animal model of lung ischemia-reperfusion injury (LIRI by clamping the pulmonary hilum. Twenty Wistar rats were subjected to an experimental model in four groups: SHAM, ischemia-reperfusion (IR, RC-Pre, and RC-Post. The final mean arterial pressure significantly decreased in IR and RC-Pre compared to their values before reperfusion (P<0.001. The RC-Post group showed significant decrease of partial pressure of arterial oxygen at the end of the observation when compared to baseline (P=0.005. Caspase-9 activity was significantly higher in the RC-Post as compared to the other groups (P<0.013. No significant differences were observed in eNOS activity among the groups. The groups RC-Pre and RC-Post did not show any significant decrease in IL-1β (P=0.159 and TNF-α (P=0.260, as compared to IR. The histological score showed no significant differences among the groups. In conclusion, RC-3095 does not demonstrate a protective effect in our LIRI model. Additionally, its use after reperfusion seems to potentiate cell damage, stimulating apoptosis.

  5. Role of neurokinin 1 receptors in dextran sulfate-induced colitis: studies with gene-deleted mice and the selective receptor antagonist netupitant.

    Science.gov (United States)

    Szitter, István; Pintér, Erika; Perkecz, Anikó; Kemény, Agnes; Kun, József; Kereskai, László; Pietra, Claudio; Quinn, John P; Zimmer, Andreas; Berger, Alexandra; Paige, Christopher J; Helyes, Zsuzsanna

    2014-05-01

    The function of the neurokinin 1 (NK1) receptor was investigated in the DSS-induced mouse colitis model using NK1 receptor-deficient mice and the selective antagonist netupitant. Colitis was induced by oral administration of 20 mg/ml DSS solution for 7 days in C57BL/6 and Tacr1 KO animals (n = 5-7). During the induction, one-half of the C57BL/6 and Tacr1 KO group received one daily dose of 6 mg/kg netupitant, administered intraperitoneally, the other half of the group received saline, respectively. Disease activity index (DAI), on the basis of stool consistency, blood and weight loss, was determined over 7 days. Histological evaluation, myeloperoxidase (MPO) measurement, cytokine concentrations and receptor expression analysis were performed on the colon samples. NK1 receptors are up-regulated in the colon in response to DSS treatment. DSS increased DAI, histopathological scores, BLC, sICAM-1, IFN-γ, IL-16 and JE in wildtype mice, which were significantly reduced in NK1 receptor-deficient ones. NK1 receptor antagonism with netupitant significantly diminished DAI, inflammatory histopathological alterations, BLC, IFN-γ, IL-13 and IL-16 in wildtype mice, but not in the NK1-deficient ones. MPO was similarly elevated and netupitant significantly decreased its activity in both groups. NK1 receptor antagonism could be beneficial for colitis via inhibiting different inflammatory mechanisms.

  6. Comparison of two different starting multiple dose gonadotropin-releasing hormone antagonist protocols in a selected group of in vitro fertilization-embryo transfer patients.

    Science.gov (United States)

    Escudero, Ernesto; Bosch, Ernesto; Crespo, Juana; Simón, Carlos; Remohí, José; Pellicer, Antonio

    2004-03-01

    To compare the efficacy of two starting protocols of multiple dose GnRH antagonists (GnRH-a). Prospective randomized controlled study. In vitro fertilization-embryo transfer program at the Instituto Valenciano de Infertilidad, Valencia, Spain. One hundred nine patients undergoing controlled ovarian hyperstimulation (COH) with recombinant gonadotropins and GnRH-a (0.25 mg/d). Patients started GnRH-a administration on stimulation day 6 (group 1) or when the leading follicle reached a mean diameter of 14 mm (group 2). Implantation and pregnancy rates; serum E(2) and LH levels during ovarian stimulation; days of stimulation and GnRH-a administration. Days needed for ovarian stimulation were similar in both groups but there was a significant difference when comparing days of GnRH-a administration. Serum E(2) and LH followed similar curves in both groups. Implantation and pregnancy rates were 23.7% and 44.4 % in group 1 and 28.6% and 50.9 % in group 2 (P=not significant [NS]). The efficacy of the two starting protocols of the multiple dose GnRH-a evaluated in this study is similar; however, this remark can only be drawn for a selected group of patients.

  7. A Selective Novel Peroxisome Proliferator–Activated Receptor (PPAR)-α Antagonist Induces Apoptosis and Inhibits Proliferation of CLL Cells In Vitro and In Vivo

    Science.gov (United States)

    Messmer, Davorka; Lorrain, Kymmy; Stebbins, Karin; Bravo, Yalda; Stock, Nicholas; Cabrera, Geraldine; Correa, Lucia; Chen, Austin; Jacintho, Jason; Chiorazzi, Nicholas; Yan, Xiao Jie; Spaner, David; Prasit, Peppi; Lorrain, Daniel

    2015-01-01

    Tumor-specific metabolic changes can reveal new therapeutic targets. Our findings implicate a supporting role for fatty acid metabolism in chronic lymphocytic leukemia (CLL) cell survival. Peroxisome proliferator–activated receptor (PPAR)-α, a major transcriptional regulator of fatty acid oxidation, was recently shown to be upregulated in CLL. To evaluate PPARα as a potential therapeutic target, we developed a highly selective, potent small molecule antagonist of PPARα, NXT629. NXT629 inhibited agonist-induced transcription of PPARα-regulated genes, demonstrating target engagement in CLL cells. Furthermore, NXT629 induced apoptosis of CLL cells even in the presence of a protective microenvironment. To mimic the proliferative lymphoid compartment of CLL, we examined the activity of NXT629 on CLL cells that were stimulated to proliferate in vitro. NXT629 reduced the number of leukemia cells undergoing cell division. In addition, in two xenograft mouse models of CLL (one a model for nondividing and one for dividing CLL), NXT629 reduced the number of viable CLL cells in vivo. Overall, these results suggest that fatty acid metabolism promotes survival and proliferation of primary CLL cells and that inhibiting PPARα gene regulation could be a new therapeutic approach to treating CLL. PMID:26070013

  8. A Selective Novel Peroxisome Proliferator-Activated Receptor (PPAR)-α Antagonist Induces Apoptosis and Inhibits Proliferation of CLL Cells In Vitro and In Vivo.

    Science.gov (United States)

    Messmer, Davorka; Lorrain, Kymmy; Stebbins, Karin; Bravo, Yalda; Stock, Nicholas; Cabrera, Geraldine; Correa, Lucia; Chen, Austin; Jacintho, Jason; Chiorazzi, Nicholas; Yan, Xiao Jie; Spaner, David; Prasit, Peppi; Lorrain, Daniel

    2015-06-09

    Tumor-specific metabolic changes can reveal new therapeutic targets. Our findings implicate a supporting role for fatty acid metabolism in chronic lymphocytic leukemia (CLL) cell survival. Peroxisome proliferator-activated receptor (PPAR)-α, a major transcriptional regulator of fatty acid oxidation, was recently shown to be upregulated in CLL. To evaluate PPARα as a potential therapeutic target, we developed a highly selective, potent small molecule antagonist of PPARα, NXT629. NXT629 inhibited agonist-induced transcription of PPARα-regulated genes, demonstrating target engagement in CLL cells. Furthermore, NXT629 induced apoptosis of CLL cells even in the presence of a protective microenvironment. To mimic the proliferative lymphoid compartment of CLL, we examined the activity of NXT629 on CLL cells that were stimulated to proliferate in vitro. NXT629 reduced the number of leukemia cells undergoing cell division. In addition, in two xenograft mouse models of CLL (one a model for nondividing and one for dividing CLL), NXT629 reduced the number of viable CLL cells in vivo. Overall, these results suggest that fatty acid metabolism promotes survival and proliferation of primary CLL cells and that inhibiting PPARα gene regulation could be a new therapeutic approach to treating CLL.

  9. Protective effect of caffeine and a selective A2A receptor antagonist on impairment of memory and oxidative stress of aged rats.

    Science.gov (United States)

    Leite, Marlon Régis; Wilhelm, Ethel A; Jesse, Cristiano R; Brandão, Ricardo; Nogueira, Cristina Wayne

    2011-04-01

    In this study, the effects of caffeine (CAF) and SCH58261, a selective A(2A) receptor antagonist, on memory impairment and oxidative stress generated by aging in rats were investigated. Young and aged rats were treated daily per 10 days with CAF (30 mg/kg p.o.) or SCH58261 (0.5mg/kg, p.o.) or vehicle (1 ml/kg p.o.). Rats were trained and tested in a novel object recognition task. After the behavioral test, ascorbic acid and oxygen and nitrogen reactive species levels as well as Na(+)K(+) ATPase activity were determined in rat brain. The results demonstrated that the age-related memory deficit was reversed by treatment with CAF or SCH58261. Treatment with CAF or SCH58261 significantly normalized oxygen and nitrogen reactive species levels increased in brains of aged rats. Na(+)K(+) ATPase activity inhibited in brains of aged rats was also normalized by CAF or SCH58261 treatment. A decrease in basal ascorbic acid levels in brains of aged rats was not changed by CAF or SCH58261. These results demonstrated that CAF and SCH58261, modulators of adenosinergic receptors, were able to reverse age-associated memory impairment and to partially reduce oxidative stress. Copyright © 2010 Elsevier Inc. All rights reserved.

  10. High-Affinity DNA Aptamer Generation Targeting von Willebrand Factor A1-Domain by Genetic Alphabet Expansion for Systematic Evolution of Ligands by Exponential Enrichment Using Two Types of Libraries Composed of Five Different Bases.

    Science.gov (United States)

    Matsunaga, Ken-Ichiro; Kimoto, Michiko; Hirao, Ichiro

    2017-01-11

    The novel evolutionary engineering method ExSELEX (genetic alphabet expansion for systematic evolution of ligands by exponential enrichment) provides high-affinity DNA aptamers that specifically bind to target molecules, by introducing an artificial hydrophobic base analogue as a fifth component into DNA aptamers. Here, we present a newer version of ExSELEX, using a library with completely randomized sequences consisting of five components: four natural bases and one unnatural hydrophobic base, 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). In contrast to the limited number of Ds-containing sequence combinations in our previous library, the increased complexity of the new randomized library could improve the success rates of high-affinity aptamer generation. To this end, we developed a sequencing method for each clone in the enriched library after several rounds of selection. Using the improved library, we generated a Ds-containing DNA aptamer targeting von Willebrand factor A1-domain (vWF) with significantly higher affinity (KD = 75 pM), relative to those generated by the initial version of ExSELEX, as well as that of the known DNA aptamer consisting of only the natural bases. In addition, the Ds-containing DNA aptamer was stabilized by introducing a mini-hairpin DNA resistant to nucleases, without any loss of affinity (KD = 61 pM). This new version is expected to consistently produce high-affinity DNA aptamers.

  11. Administration of a selective β2 adrenergic receptor antagonist exacerbates neuropathology and cognitive deficits in a mouse model of Alzheimer's disease.

    Science.gov (United States)

    Branca, Caterina; Wisely, Elena V; Hartman, Lauren K; Caccamo, Antonella; Oddo, Salvatore

    2014-12-01

    Currently, there are no available approaches to cure or slow down the progression of Alzheimer's disease (AD), which is characterized by the accumulation of extracellular amyloid-β (Aβ) deposits and intraneuronal tangles that comprised hyperphosphorylated tau. The β2 adrenergic receptors (β2ARs) are expressed throughout the cortex and hippocampus and play a key role in cognitive functions. Alterations in the function of these receptors have been linked to AD; however, these data remain controversial as apparent contradicting reports have been published. Given the current demographics of growing elderly population and the high likelihood of concurrent β-blocker use for other chronic conditions, more studies into the role of this receptor in AD animal models are needed. Here, we show that administration of ICI 118,551 (ICI), a selective β2AR antagonist, exacerbates cognitive deficits in a mouse model of AD, the 3xTg-AD mice. Neuropathologically, ICI increased Aβ levels and Aβ plaque burden. Concomitantly, ICI-treated 3xTg-AD mice showed an increase in tau phosphorylation and accumulation. Mechanistically, these changes were linked to an increase in amyloidogenic amyloid precursor protein processing. These results suggest that under the conditions used here, selective pharmacologic inhibition of β2ARs has detrimental effects on AD-like pathology in mice. Overall, these studies strengthen the notion that the link between β2ARs and AD is likely highly complex and suggest caution in generalizing the beneficial effects of β blockers on AD. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Insights from the fungus Fusarium oxysporum point to high affinity glucose transporters as targets for enhancing ethanol production from lignocellulose.

    Directory of Open Access Journals (Sweden)

    Shahin S Ali

    Full Text Available Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km((glucose was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing.

  13. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen; Nakano, Hiroyasu

    2017-09-21

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

  14. High-affinity CCK receptors are coupled to phospholipase A2 pathways to mediate pancreatic amylase secretion.

    Science.gov (United States)

    Tsunoda, Y; Owyang, C

    1995-09-01

    It is well recognized that JMV-180, a cholecystokinin (CCK) analogue, acts as an agonist on the high-affinity CCK receptor in pancreatic acinar cells. It caused Ca2+ oscillations and amylase secretion in a manner independent of the phospholipase C-inositol 1,4,5-trisphosphate (IP3) pathway. We investigated the mechanism by which the high-affinity CCK receptor utilizes IP3-independent Ca2+ signal transduction to mediate amylase secretion. JMV-180 (1-1,000 nM)-stimulated Ca2+ oscillations and amylase secretion were significantly inhibited by the phospholipase A2 (PLA2) inhibitor, ONO-RS-082 (10 microM). Using streptolysin O-permeabilized cells, we showed that a porcine pancreatic anti-PLA2 antibody from rabbit serum (250 ng/ml) inhibited JMV-180-stimulated amylase secretion. In contrast to CCK octapeptide, JMV-180 (1 nM-10 microM) had no effect on intracellular IP3 levels. These concentrations of JMV-180 did, however, increase intracellular levels of arachidonic acid (AA) metabolite by 2.5-fold in a biphasic manner. Application of exogenous AA (10 microM) released 60% of ATP-incorporated 45Ca2+ from permeabilized pancreatic acini within 3 min in a transient manner. We also showed that active phorbol ester (100 nM) inhibited Ca2+ oscillations and amylase secretion stimulated by JMV-180 (10 nM) or CCK-OPE (100 nM). Application of Mn2+ (2 mM) to superfused acini resulted in a rapid quench of fura 2 fluorescence during 10 nM JMV-180 stimulation, suggesting an involvement of extracellular Ca2+ influx. However, the major source of Ca2+ utilized for oscillations during high-affinity CCK receptor activation was intracellular. In conclusion, we have demonstrated that the high-affinity CCK receptors are coupled to PLA2 pathways to produce AA, which mediates cytosolic Ca2+ oscillation and monophasic amylase secretion, in rat pancreatic acinar cells.

  15. The selective orexin receptor 1 antagonist ACT-335827 in a rat model of diet-induced obesity associated with metabolic syndrome

    Directory of Open Access Journals (Sweden)

    Michel Alexander Steiner

    2013-12-01

    Full Text Available The orexin system regulates feeding, nutrient metabolism and energy homeostasis. Acute pharmacological blockade of orexin receptor 1 (OXR-1 in rodents induces satiety and reduces normal and palatable food intake. Genetic OXR-1 deletion in mice improves hyperglycemia under high-fat (HF diet conditions. Here we investigated the effects of chronic treatment with the novel selective OXR-1 antagonist ACT-335827 in a rat model of diet-induced obesity (DIO associated with metabolic syndrome (MetS. Rats were fed either standard chow (SC or a cafeteria (CAF diet comprised of intermittent human snacks and a constant free choice between a HF/sweet (HF/S diet and SC for 13 weeks. Thereafter the SC group was treated with vehicle (for 4 weeks and the CAF group was divided into a vehicle and an ACT-335827 treatment group. Energy and water intake, food preference, and indicators of MetS (abdominal obesity, glucose homeostasis, plasma lipids, and blood pressure were monitored. Hippocampus-dependent memory, which can be impaired by DIO, was assessed. CAF diet fed rats treated with ACT-335827 consumed less of the HF/S diet and more of the SC, but did not change their snack or total kcal intake compared to vehicle-treated rats. ACT-335827 increased water intake and the high-density lipoprotein associated cholesterol proportion of total circulating cholesterol. ACT-335827 slightly increased body weight gain (4% versus controls and feed efficiency in the absence of hyperphagia. These effects were not associated with significant changes in the elevated fasting glucose and triglyceride (TG plasma levels, glucose intolerance, elevated blood pressure, and adiposity due to CAF diet consumption. Neither CAF diet consumption alone nor ACT-335827 affected memory. In conclusion, the main metabolic characteristics associated with DIO and MetS in rats remained unaffected by chronic ACT-335827 treatment, suggesting that pharmacological OXR-1 blockade has minimal impact in this

  16. Antitumour activity of the glycoengineered type II anti-CD20 antibody obinutuzumab (GA101) in combination with the MDM2-selective antagonist idasanutlin (RG7388).

    Science.gov (United States)

    Herting, Frank; Herter, Sylvia; Friess, Thomas; Muth, Gunther; Bacac, Marina; Sulcova, Jitka; Umana, Pablo; Dangl, Markus; Klein, Christian

    2016-11-01

    To investigate whether the glycoengineered type II anti-CD20 monoclonal antibody obinutuzumab (GA101) combined with the selective MDM2 antagonist idasanutlin (RG7388) offers superior efficacy to monotherapy in treating B-lymphoid malignancies in preclinical models. The combined effect of obinutuzumab or rituximab plus idasanutlin on direct cell death/apoptosis induction and antibody-dependent cellular cytotoxicity (ADCC) was evaluated using p53 wild-type Z-138 and DoHH-2 lymphoma cells. Furthermore, whole blood B-cell depletion was analysed, and tumour growth inhibition was evaluated in subcutaneous xenograft models. Idasanutlin induced concentration-dependent death of Z-138 and DoHH-2 cells. At concentrations >10-100 nm, idasanutlin enhanced obinutuzumab-induced death of DoHH-2 and Z-138 cells without negatively impacting obinutuzumab-mediated ADCC, natural killer cell activation or whole blood B-cell depletion. In the Z-138 xenograft model, a suboptimal dose of obinutuzumab with idasanutlin yielded substantial tumour growth inhibition and prolonged survival in a time-to-event analysis. In the DoHH-2 model, idasanutlin plus obinutuzumab showed superior tumour growth inhibition to idasanutlin plus rituximab. Obinutuzumab plus idasanutlin enhanced cell death of p53 wild-type tumour cells vs. rituximab plus idasanutlin without affecting obinutuzumab-mediated ADCC or B-cell depletion and showed robust antitumour efficacy in xenograft models, strongly supporting the investigation of this combination in clinical trials. © 2016 The Authors. European Journal of Haematology Published by John Wiley & Sons Ltd.

  17. Efficacy and tolerability of tropisetron in primary fibromyalgia--a highly selective and competitive 5-HT3 receptor antagonist. German Fibromyalgia Study Group.

    Science.gov (United States)

    Färber, L; Stratz, T; Brückle, W; Späth, M; Pongratz, D; Lautenschläger, J; Kötter, I; Zöller, B; Peter, H H; Neeck, G; Alten, R; Müller, W

    2000-01-01

    Based on a potential role for serotonin receptors in fibromyalgia, we investigated the efficacy and tolerability of treatment with tropisetron, a highly selective, competitive inhibitor of the 5-HT3 receptor. In this prospective, multicenter, double-blind, parallel-group, dose-finding study, 418 patients suffering from primary fibromyalgia (ACR criteria) were randomly assigned to receive either placebo, 5 mg, 10 mg or 15 mg tropisetron once daily, respectively. The duration of treatment was 10 days. The clinical response was measured by changes in pain-score, visual analog scale (VAS), and the number of painful tender-points. Treatment with 5 mg tropisetron resulted in a significantly higher response rate (39.2%) when compared with placebo (26.2%) (p=0.033). The absolute reduction in pain-score was -13.5% for 5 mg tropisetron, -13.0% for 10 mg tropisetron, and -6.3% for placebo (pVAS, while a clear trend in terms of clinical benefit was seen with 10 mg tropisetron. The number of painful tender-points was also reduced significantly (p=0.002) in the 5 mg tropisetron group. Of interest, during the 12-month follow-up period, pain intensity of responders on 5 mg and 10 mg tropisetron was still markedly below baseline. The treatment was well tolerated, with gastro-intestinal complaints being the most frequently reported side effects, in keeping with the known safety profile for 5-HT3 receptor antagonists. This study demonstrates the efficacy of short-term treatment with 5 mg tropisetron once daily in primary fibromyalgia. Treatment was well tolerated and prolonged clinical benefits were seen.

  18. Complex stimulus properties of LSD: a drug discrimination study with alpha 2-adrenoceptor agonists and antagonists.

    Science.gov (United States)

    Marona-Lewicka, D; Nichols, D E

    1995-08-01

    The influence of several alpha 2-adrenergic agents on the discriminative stimulus (DS) properties of lysergic acid diethylamide (LSD) was studied in rats trained to discriminate 0.08 mg/kg (186 nmol/kg) of LSD from saline in a two-lever operant paradigm. Only yohimbine fully mimicked LSD with an ED50 of 2.05 mg/kg (5.24 mumol/kg). Yohimbine's 5-HT1A agonist properties may be responsible for this substitution. Other alpha 2-adrenoceptor antagonists, idazoxan with an agonist/antagonist profile at 5-HT1A receptors and RS 26026-197, a highly selective alpha 2-adrenoceptor antagonist, failed to produce substitution. Clonidine, an alpha 2-adrenoceptor agonist, did not substitute for LSD but the response rate was dose-dependently reduced. None of the alpha 2-adrenergic agents used for pretreatment before LSD inhibited the response to the LSD training dose. Coadministration of clonidine with LSD produced a leftward shift of the dose-response relationship of LSD without a significant change in the slope of the dose-response line. Simultaneous administration of alpha 2-adrenergic agents with LSD shifted the dose-response curve to the left only when the adrenergic agent also possessed at least moderate affinity for the 5-HT1A receptor. In addition, radioligand competition experiments were performed that showed LSD to have relatively high affinity (Ki = 37 nM) for [3H]clonidine-labeled sites in rat cortex with lower affinity for [3H]yohimbine labeled sites. While previous studies have suggested that the nature of the LSD cue may be essentially expressed by 5-HT2 receptor activation, the present data show that this cue can be modulated by effects of LSD at 5-HT1A and at other monoamine neurotransmitter receptors.

  19. Premature aging phenotype in mice lacking high affinity nicotinic receptors: region specific changes in layer V pyramidal cell morphology

    Directory of Open Access Journals (Sweden)

    Eleni Konsolaki

    2014-02-01

    accelerated cognitive aging, based on structural alterations and spatial learning deficits only evident in old animals (Zoli et al., 1999; Picciotto and Zoli, 2002. However a systematic comparison of neuronal microanatomy in adult and aged animals has not been done to date. In the present study adult (4-6months and old (22-24months WT and β2-/- animals were used to examine the respective contributions of age and genotype on neuronal structure. We focus on layer V pyramidal cells because: (i they constitute the main cortical output (DeFelipe and Farinas, 1992; Romand et al., 2011 (ii they are often reported to exhibit increased sensitivity to aging (Nakamura et al., 1985; Baskys et al., 1990; De Brabander et al., 1998; Turner et al., 2005; (iii they possess a high density of cholinergic terminals (Houser et al., 1985 and, in contrast to layer III cells, they exhibit strong presynaptic modulation by β2 containing nAChRs and are activated by nAChR stimulation (Poorthuis et al., 2013; hence they would be a sensitive readout for the lack of high affinity nicotinic receptors. Furthermore, to examine the degree of age-related vulnerability across distinct cortical areas we used YFP-H mice that express yellow fluorescent protein (YFP in specific populations of thick-tufted layer V pyramidal neurons across the cortical mantle (Feng et al., 2000; Sugino et al., 2006. We used mutants crossed with YFP+ mice in order to have the same labeled populations in both genotypes, and we examined cells in primary visual cortex (V1 and anterior cingulate cortex (ACC, two cortical regions that receive similar cholinergic inputs (McKinney et al., 1983; Jacobowitz and Creed, 1983; Everitt and Robbins, 1997; Laplante et al., 2005 but have distinct cytoarchitecture and functional role (Elston et al., 2005. We ask whether neurons in old β2-/- mice exhibit greater structural deficits than aged-matched controls and whether deficits appear in old age or are already present earlier. Brains from 21 adult

  20. A putative high affinity phosphate transporter, CmPT1, enhances tolerance to Pi deficiency of chrysanthemum.

    Science.gov (United States)

    Liu, Peng; Chen, Sumei; Song, Aiping; Zhao, Shuang; Fang, Weimin; Guan, Zhiyong; Liao, Yuan; Jiang, Jiafu; Chen, Fadi

    2014-01-10

    Inorganic phosphate (Pi) is essential for plant growth, and phosphorus deficiency is a main limiting factor in plant development. Its acquisition is largely mediated by Pht1 transporters, a family of plasma membrane-located proteins. Chrysanthemum is one of the most important ornamental plants, its productivity is usually compromised when grown in phosphate deficient soils, but the study of phosphate transporters in chrysanthemum is limited. We described the isolation from chrysanthemum of a homolog of the Phosphate Transporter 1 (PT1) family. Its predicted product is a protein with 12 transmembrane domains, highly homologous with other high affinity plant Pi transporters. Real-time quantitative PCR analysis revealed that the gene was transcribed strongly in the root, weakly in the stem and below the level of detection in the leaf of chrysanthemum plants growing in either sufficient or deficient Pi conditions. Transcript abundance was greatly enhanced in Pi-starved roots. A complementation assay in yeast showed that CmPT1 partially compensated for the absence of phosphate transporter activity in yeast strain MB192. The estimated Km of CmPT1 was 35.2 μM. Under both Pi sufficient and deficient conditions, transgenic plants constitutively expressing CmPT1 grew taller than the non-transformed wild type, produced a greater volume of roots, accumulated more biomass and took up more phosphate. CmPT1 encodes a typical, root-expressed, high affinity phosphate transporter, plays an important role in coping Pi deficiency of chrysanthemum plants.

  1. Identification of a high-affinity Ca sup 2+ pump associated with endocytotic vesicles in Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    Milne, J.L.; Coukell, M.B. (York Univ., North York, Ontario (Canada))

    1989-11-01

    In the cellular slime mold Dictyostelium discoideum, changes in free cytosolic Ca{sup 2+} are thought to regulate certain processes during cell aggregation and differentiation. To understand the mechanisms controlling free Ca{sup 2+} levels in this organism, the authors previously isolated and characterized an ATP/Mg{sup 2+}-dependent, high-affinity Ca{sup 2+} pump which appeared to be a component of inside-out plasma membrane vesicles. In this report, they demonstrate that a high-affinity Ca{sup 2+} pump, with properties virtually identical to the isolated pump, can be detected in filipin- or digitonin-permeabilized cells of Dictyostelium. Moreover, Ca{sup 2+}-pumping vesicles, which migrate on Percoll/KCl gradients like the vesicles identified earlier, can be isolated from the permeabilized cells. Results of additional experiments suggest that this intracellular Ca{sup 2+} transporter is associated with a high-capacity non-IP{sub 3}-releasable Ca{sup 2+} store which is generated by endocytosis. A possible role for this store in maintaining Ca{sup 2+} homeostasis in Dictyostelium is discussed.

  2. Specific recognition of the C-terminal end of A beta 42 by a high affinity monoclonal antibody

    DEFF Research Database (Denmark)

    Axelsen, Trine Veje; Holm, Arne; Birkelund, Svend

    2009-01-01

    The neurotoxic peptide A beta(42) is derived from the amyloid precursor protein by proteolytic cleavage and is deposited in the brain of patients suffering from Alzheimer's disease (AD). In this study we generate a high affinity monoclonal antibody that targets the C-terminal end of A beta(42......) with high specificity. By this is meant that the paratope of the antibody must enclose the C-terminal end of A beta(42) including the carboxy-group of amino acid 42, and not just recognize a linear epitope in the C-terminal part of A beta. This has been accomplished by using a unique antigen construct made...... by the Ligand Presenting Assembly technology (LPA technology). This strategy results in dimeric presentation of the free C-terminal end of A beta(42). The generated Mab A beta1.1 is indeed specific for the C-terminal end of A beta(42) to which it binds with high affinity. Mab A beta1.1 recognizes the epitope...

  3. High Affinity, Developability and Functional Size: The Holy Grail of Combinatorial Antibody Library Generation

    Directory of Open Access Journals (Sweden)

    Kathrin Tissot

    2011-05-01

    Full Text Available Since the initial description of phage display technology for the generation of human antibodies, a variety of selection methods has been developed. The most critical parameter for all in vitro-based approaches is the quality of the antibody library. Concurrent evolution of the libraries has allowed display and selection technologies to reveal their full potential. They come in different flavors, from naïve to fully synthetic and differ in terms of size, quality, method of preparation, framework and CDR composition. Early on, the focus has mainly been on affinities and thus on library size and diversity. Subsequently, the increased awareness of developability and cost of goods as important success factors has spurred efforts to generate libraries with improved biophysical properties and favorable production characteristics. More recently a major focus on reduction of unwanted side effects through reduced immunogenicity and improved overall biophysical behavior has led to a re-evaluation of library design.

  4. Balancing selected medication costs with total number of daily injections: a preference analysis of GnRH-agonist and antagonist protocols by IVF patients.

    Science.gov (United States)

    Sills, E Scott; Collins, Gary S; Salem, Shala A; Jones, Christopher A; Peck, Alison C; Salem, Rifaat D

    2012-08-30

    During in vitro fertilization (IVF), fertility patients are expected to self-administer many injections as part of this treatment. While newer medications have been developed to substantially reduce the number of these injections, such agents are typically much more expensive. Considering these differences in both cost and number of injections, this study compared patient preferences between GnRH-agonist and GnRH-antagonist based protocols in IVF. Data were collected by voluntary, anonymous questionnaire at first consultation appointment. Patient opinion concerning total number of s.c. injections as a function of non-reimbursed patient cost associated with GnRH-agonist [A] and GnRH-antagonist [B] protocols in IVF was studied. Completed questionnaires (n = 71) revealed a mean +/- SD patient age of 34 +/- 4.1 yrs. Most (83.1%) had no prior IVF experience; 2.8% reported another medical condition requiring self-administration of subcutaneous medication(s). When out-of-pocket cost for [A] and [B] were identical, preference for [B] was registered by 50.7% patients. The tendency to favor protocol [B] was weaker among patients with a health occupation. Estimated patient costs for [A] and [B] were $259.82 +/- 11.75 and $654.55 +/- 106.34, respectively (p medication costs for GnRH-antagonist IVF vs. GnRH-agonist IVF protocols. A conditional preference to minimize downregulation (using GnRH-antagonist) was noted among some, but not all, IVF patient sub-groups. Compared to IVF patients with a health occupation, the preference for GnRH-antagonist was weaker than for other patients. While reducing total number of injections by using GnRH-antagonist is a desirable goal, it appears this advantage is not perceived equally by all IVF patients and its utility is likely discounted heavily by patients when nonreimbursed medication costs reach a critical level.

  5. A new serotonin 5-HT6 receptor antagonist with procognitive activity - Importance of a halogen bond interaction to stabilize the binding

    Science.gov (United States)

    González-Vera, Juan A.; Medina, Rocío A.; Martín-Fontecha, Mar; Gonzalez, Angel; de La Fuente, Tania; Vázquez-Villa, Henar; García-Cárceles, Javier; Botta, Joaquín; McCormick, Peter J.; Benhamú, Bellinda; Pardo, Leonardo; López-Rodríguez, María L.

    2017-01-01

    Serotonin 5-HT6 receptor has been proposed as a promising therapeutic target for cognition enhancement though the development of new antagonists is still needed to validate these molecules as a drug class for the treatment of Alzheimer’s disease and other pathologies associated with memory deficiency. As part of our efforts to target the 5-HT6 receptor, new benzimidazole-based compounds have been designed and synthesized. Site-directed mutagenesis and homology models show the importance of a halogen bond interaction between a chlorine atom of the new class of 5-HT6 receptor antagonists identified herein and a backbone carbonyl group in transmembrane domain 4. In vitro pharmacological characterization of 5-HT6 receptor antagonist 7 indicates high affinity and selectivity over a panel of receptors including 5-HT2B subtype and hERG channel, which suggests no major cardiac issues. Compound 7 exhibited in vivo procognitive activity (1 mg/kg, ip) in the novel object recognition task as a model of memory deficit.

  6. Combinatorial optimization of cystine-knot peptides towards high-affinity inhibitors of human matriptase-1.

    Directory of Open Access Journals (Sweden)

    Bernhard Glotzbach

    Full Text Available Cystine-knot miniproteins define a class of bioactive molecules with several thousand natural members. Their eponymous motif comprises a rigid structured core formed by six disulfide-connected cysteine residues, which accounts for its exceptional stability towards thermic or proteolytic degradation. Since they display a remarkable sequence tolerance within their disulfide-connected loops, these molecules are considered promising frameworks for peptide-based pharmaceuticals. Natural open-chain cystine-knot trypsin inhibitors of the MCoTI (Momordica cochinchinensis trypsin inhibitor and SOTI (Spinacia oleracea trypsin inhibitor families served as starting points for the generation of inhibitors of matriptase-1, a type II transmembrane serine protease with possible clinical relevance in cancer and arthritic therapy. Yeast surface-displayed libraries of miniproteins were used to select unique and potent matriptase-1 inhibitors. To this end, a knowledge-based library design was applied that makes use of detailed information on binding and folding behavior of cystine-knot peptides. Five inhibitor variants, four of the MCoTI family and one of the SOTI family, were identified, chemically synthesized and oxidatively folded towards the bioactive conformation. Enzyme assays revealed inhibition constants in the low nanomolar range for all candidates. One subnanomolar binder (Ki = 0.83 nM with an inverted selectivity towards trypsin and matriptase-1 was identified.

  7. Correlation of receptor occupancy of metabotropic glutamate receptor subtype 1 (mGluR1) in mouse brain with in vivo activity of allosteric mGluR1 antagonists.

    Science.gov (United States)

    Suzuki, Gentaroh; Kawagoe-Takaki, Hiroko; Inoue, Takao; Kimura, Toshifumi; Hikichi, Hirohiko; Murai, Takashi; Satow, Akio; Hata, Mikiko; Maehara, Shunsuke; Ito, Satoru; Kawamoto, Hiroshi; Ozaki, Satoshi; Ohta, Hisashi

    2009-07-01

    The aim of this study was to clarify the relationship between receptor occupancy and in vivo pharmacological activity of mGluR1 antagonists. The tritiated mGluR1-selective allosteric antagonist [(3)H]FTIDC (4-[1-(2-fluoropyridin-3-yl)-5-methyl-1H-1,2,3-triazol-4-yl]-N-isopropyl-N-methyl-3,6-dihydropyridine-1(2H)-carboxamide) was identified as a radioligand having high affinity for mGluR1-expressing CHO cells (K(D) = 2.1 nM) and mouse cerebellum (K(D) = 3.7 nM). [(3)H]FTIDC bound to mGluR1 was displaced by structurally unrelated allosteric antagonists, suggesting there is a mutual binding pocket shared with different allosteric antagonists. The binding specificity of [(3)H]FTIDC for mGluR1 in brain sections was demonstrated by the lack of significant binding to brain sections prepared from mGluR1-knockout mice. Ex vivo receptor occupancy with [(3)H]FTIDC revealed that the receptor occupancy level by FTIDC correlated well with FTIDC dosage and plasma concentration. Intracerebroventricular administration of (S)-3,5-dihydroxyphenylglycine is known to elicit face washing behavior that is mainly mediated by mGluR1. Inhibition of this behavioral change by FTIDC correlated with the receptor occupancy level of mGluR1 in the brain. A linear relationship between the receptor occupancy and in vivo activity was also demonstrated using structurally diverse mGluR1 antagonists. The receptor occupancy assays could help provide guidelines for selecting appropriate doses of allosteric mGluR1 antagonist for examining the function of mGluR1 in vivo.

  8. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

    DEFF Research Database (Denmark)

    Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Kudryavtsev, Denis

    2016-01-01

    to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover...

  9. Z944, a Novel Selective T-Type Calcium Channel Antagonist Delays the Progression of Seizures in the Amygdala Kindling Model.

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    Pablo Miguel Casillas-Espinosa

    Full Text Available Temporal lobe epilepsy (TLE is the most common form of drug resistant epilepsy. Current treatment is symptomatic, suppressing seizures, but has no disease modifying effect on epileptogenesis. We examined the effects of Z944, a potent T-type calcium channel antagonist, as an anti-seizure agent and against the progression of kindling in the amygdala kindling model of TLE. The anti-seizure efficacy of Z944 (5mg/kg, 10mg/kg, 30mg/kg and 100mg/kg was assessed in fully kindled rats (5 class V seizures as compared to vehicle, ethosuximide (ETX, 100mg/kg and carbamazepine (30mg/kg. Each animal received the seven treatments in a randomised manner. Seizure class and duration elicited by six post-drug stimulations was determined. To investigate for effects in delaying the progression of kindling, naive animals received Z944 (30mg/kg, ETX (100mg/kg or vehicle 30-minutes prior to each kindling stimulation up to a maximum of 30 stimulations, with seizure class and duration recorded after each stimulation. At the completion of drug treatment, CaV3.1, CaV3.2 and CaV3.3 mRNA expression levels were assessed in the hippocampus and amygdala using qPCR. Z944 was not effective at suppressing seizures in fully kindled rats compared to vehicle. Animals receiving Z944 required significantly more stimulations to evoke a class III (p<0.05, IV (p<0.01 or V (p<0.0001 seizure, and to reach a fully kindled state (p<0.01, than animals receiving vehicle. There was no significant difference in the mRNA expression of the T-type Ca2+ channels in the hippocampus or amygdala. Our results show that selectively targeting T-type Ca2+ channels with Z944 inhibits the progression of amygdala kindling. This could be a potential for a new therapeutic intervention to mitigate the development and progression of epilepsy.

  10. Enantiomers of HA-966 (3-amino-1-hydroxypyrrolid-2-one) exhibit distinct central nervous system effects: (+)-HA-966 is a selective glycine/N-methyl-D-aspartate receptor antagonist, but (-)-HA-966 is a potent gamma-butyrolactone-like sedative

    Energy Technology Data Exchange (ETDEWEB)

    Singh, L.; Donald, A.E.; Foster, A.C.; Hutson, P.H.; Iversen, L.L.; Iversen, S.D.; Kemp, J.A.; Leeson, P.D.; Marshall, G.R.; Oles, R.J.; Priestley, T.; Thorn, L.; Tricklebank, M.D.; Vass, C.A.; Williams, B.J. (Merck Sharp and Dohme Research Labs., Essex (England))

    1990-01-01

    The antagonist effect of {+-}-3-amino-1-hydroxypyrrolid-2-one (HA-966) at the N-methyl-D-aspartate (NMDA) receptor occurs through a selective interaction with the glycine modulatory site within the receptor complex. When the enantiomers of {+-}-HA-966 were resolved, the (R)-(+)-enantiomer was found to be a selective glycine/NMDA receptor antagonist, a property that accounts for its anticonvulsant activity in vivo. In contrast, the (S)-(-)-enantiomer was only weakly active as an NMDA-receptor antagonist, but nevertheless it possessed a marked sedative and muscle relaxant action in vivo. In radioligand binding experiments, (+)-HA-966 inhibited strychnine-insensitive ({sup 3}H)glycine binding to rat cerebral cortex synaptic membranes with an IC{sub 50} of 12.5 {mu}M, whereas (-)-HA-966 had an IC{sub 50} value of 339 {mu}M. In mice, (+)-HA-966 antagonized sound and N-methyl-DL-aspartic acid (NMDLA)-induced seizures. The coadministration of D-serine dose-dependently antagonized the anticonvulsant effect of a submaximal dose of (+)-HA-966 against NMDLA-induced seizures. The sedative/ataxic effect of racemic HA-966 was mainly attributable to the (-)-enantiomer. It is suggested that, as in the case of the sedative {gamma}-butyrolactone, disruption of striatal dopaminergic mechanisms may be responsible for this action.

  11. Kinetic Characterization of a Panel of High-Affinity Monoclonal Antibodies Targeting Ricin and Recombinant Re-Formatting for Biosensor Applications

    Directory of Open Access Journals (Sweden)

    Michelle Cummins

    2014-05-01

    Full Text Available Ricin is a potent glycoprotein toxin that is structurally composed of two subunits joined via a disulfide bond: a ~30 kDa subunit A (RTA and a ~32 kDa subunit B (RTB. There are fears of ricin being used as a weapon for warfare and terrorism and, as such, there is an increasing need for the development of immunodiagnostic reagents targeted towards this toxin. This article describes the production and characterization of a panel of six ricin-specific monoclonal IgG antibodies (mAbs, previously selected based upon their ability to inhibit ricin-mediated killing of cultured cells. Subsequent epitope binding analysis using the surface plasmon resonance (SPR array biosensor (ProteOn XPR36 indicated three distinct, non-competitive binding epitopes (“bins”. The association (ka and dissociation (kd rate constants and binding affinities (KD of each of the mAbs to ricin were also determined by SPR using Biacore T100 instrument. Affinities (KD ranged from 0.1 nM to 9 nM. We present the coding sequences of the variable domains of the six mAbs, the expression, kinetic and cytotoxicity assays for two recombinant Fab (rFab fragments and demonstrate a rFab affinity improvement by chain-shuffling. Together, these antibodies and constituent rFabs represent a panel of reagents for high-affinity recognition of ricin with potential national security biosensor applications.

  12. An in vitro-identified high-affinity nucleosome-positioning signal is capable of transiently positioning a nucleosome in vivo

    Directory of Open Access Journals (Sweden)

    Gracey Lia E

    2010-07-01

    Full Text Available Abstract Background The physiological function of eukaryotic DNA occurs in the context of nucleosomal arrays that can expose or obscure defined segments of the genome. Certain DNA sequences are capable of strongly positioning a nucleosome in vitro, suggesting the possibility that favorable intrinsic signals might reproducibly structure chromatin segments. As high-throughput sequencing analyses of nucleosome coverage in vitro and in vivo have become possible, a vigorous debate has arisen over the degree to which intrinsic DNA:nucleosome affinities orchestrate the in vivo positions of nucleosomes, thereby controlling physical accessibility of specific sequences in DNA. Results We describe here the in vivo consequences of placing a synthetic high-affinity nucleosome-positioning signal, the 601 sequence, into a DNA plasmid vector in mice. Strikingly, the 601 sequence was sufficient to position nucleosomes during an early phase after introduction of the DNA into the mice (when the plasmid vector transgene was active. This positioning capability was transient, with a loss of strong positioning at a later time point when the transgenes had become silent. Conclusions These results demonstrate an ability of DNA sequences selected solely for nucleosome affinity to organize chromatin in vivo, and the ability of other mechanisms to overcome these interactions in a dynamic nuclear environment.

  13. High affinity dopamine D2 receptor radioligands. 1. Regional rat brain distribution of iodinated benzamides

    Energy Technology Data Exchange (ETDEWEB)

    Kessler, R.M.; Ansari, M.S.; de Paulis, T.; Schmidt, D.E.; Clanton, J.A.; Smith, H.E.; Manning, R.G.; Gillespie, D.; Ebert, M.H. (Vanderbilt University School of Medicine, Nashville, TN (USA))

    1991-08-01

    Five 125I-labeled substituted benzamides, which are close structural analogues of (S)-sulpiride, eticlopride, and isoremoxipride, were evaluated for their selective in vivo uptake into dopamine D2 receptor rich tissue of the rat brain. Iodopride (KD 0.88 nM), an iodine substituted benzamide structurally related to sulpiride, displayed a maximal striatum: cerebellar uptake ratio of 7.6. Demonstration of saturation of the receptor with (125I)iodopride in striatum required uptake in frontal cortex to be used, rather than cerebellar uptake, to define nonspecific binding. Two other ligands structurally related to eticlopride, iclopride (KD 0.23 nM) and itopride (KD 0.16 nM), displayed maximal striatal: cerebellar uptake ratios of 9.8 and 3.3, respectively. The most potent ligands, epidepride (KD 0.057 nM) and ioxipride (KD 0.070 nM) showed striatal:cerebellar uptake ratios of 234 and 65, respectively. The observed uptake ratios correlated poorly with the affinity constants for the dopamine D2 receptor alone, but were highly correlated (r = 0.92) with the product of the receptor dissociation constant (KD) and the apparent lipophilicity (kw), as determined by reverse-phase HPLC at pH 7.5. Total striatal uptake also appeared dependent on lipophilicity, with maximal uptake occurring for ligands having log kw 2.4-2.8.

  14. Three homologous subunits form a high affinity peptide-gated ion channel in Hydra

    DEFF Research Database (Denmark)

    Dürrnagel, Stefan; Kuhn, Anne; Tsiairis, Charisios D

    2010-01-01

    Recently, three ion channel subunits of the degenerin (DEG)/epithelial Na(+) channel (ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na(+) channels (HyNaCs) 2-4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated...... by the neuropeptides Hydra-RFamides I and II. The HyNaC2/3 channel is so far the only cloned ionotropic receptor from cnidarians and, together with the related ionotropic receptor FMRFamide-activated Na(+) channel (FaNaC) from snails, the only known peptide-gated ionotropic receptor. The HyNaC2/3 channel has pore...... properties, like a low Na(+) selectivity and a low amiloride affinity, that are different from other channels of the DEG/ENaC gene family, suggesting that a component of the native Hydra channel might still be lacking. Here, we report the cloning of a new ion channel subunit from Hydra, HyNaC5. The new...

  15. High-level amikacin resistance in Pseudomonas aeruginosa associated with a 3'-phosphotransferase with high affinity for amikacin.

    Science.gov (United States)

    Torres, C; Perlin, M H; Baquero, F; Lerner, D L; Lerner, S A

    2000-08-01

    This work describes the characterization of the phosphotransferase enzymatic activity responsible for amikacin resistance in two clinical Pseudomona aeruginosa strains, isolated from a hospital that used amikacin as first-line aminoglycoside. Amikacin-resistant P. aeruginosa PA40 and PA43 (MIC: 128 mg/l) were shown to have APH activity with a substrate profile similar to that of APH(3')-VI. The enzyme from P. aeruginosa PA40 was purified to > 70% homogeneity. The Km of amikacin for this enzyme was 1.4 microM, the Vmax/Km ratio for amikacin was higher than for the other aminoglycosides tested and PCR and DNA sequencing ruled out the presence of aph(3')-IIps. Amikacin resistance in this strain was, therefore, associated with APH(3')-VI and the high affinity of this enzyme for amikacin could explain the high-level resistance that we observed.

  16. Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics

    Science.gov (United States)

    Aweda, Tolulope A.; Meares, Claude F.

    2011-01-01

    For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (KA >108 M−1; KD titration calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate. PMID:21964396

  17. Substance P downregulates expression of the high affinity IgE receptor (FcepsilonRI) by human mast cells.

    Science.gov (United States)

    McCary, Christine; Tancowny, Brian P; Catalli, Adriana; Grammer, Leslie C; Harris, Kathleen E; Schleimer, Robert P; Kulka, Marianna

    2010-03-30

    The effect of the neuropeptide substance P (SP) on human mast cell (MC) phenotype is poorly understood. In this study, SP effects on human MC expression of the high affinity IgE receptor (FcepsilonRI) were characterized. SP downregulated expression of FcepsilonRI mRNA and protein by approximately 50% and in a concentration dependent manner, the effect was partially mediated by engagement of the neurokinin-1 receptor (NK1R) and resulted in reduced mast cell activation. Sensitization of MC with IgE prior to SP exposure protected MC from SP-mediated FcepsilonRI downregulation. SP release may inhibit MC responses to allergens and these results may have implications in neuroinflammatiion and stress. Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.

  18. Substance P downregulates expression of the high affinity IgE receptor (FcεRI) by human mast cells

    Science.gov (United States)

    McCary, Christine; Tancowny, Brian P.; Catalli, Adriana; Grammer, Leslie C.; Harris, Kathleen E.; Schleimer, Robert P.; Kulka, Marianna

    2013-01-01

    The effect of the neuropeptide substance P (SP) on human mast cell (MC) phenotype is poorly understood. In this study, SP effects on human MC expression of the high affinity IgE receptor (FcεRI) were characterized. SP downregulated expression of FcεRI mRNA and protein by approximately 50% and in a concentration dependent manner, the effect was partially mediated by engagement of the neurokinin-1 receptor (NK1R) and resulted in reduced mast cell activation. Sensitization of MC with IgE prior to SP exposure protected MC from SP-mediated FcεRI downregulation. SP release may inhibit MC responses to allergens and these results may have implications in neuroinflammatiion and stress. PMID:20117843

  19. Tsetse salivary gland proteins 1 and 2 are high affinity nucleic acid binding proteins with residual nuclease activity.

    Directory of Open Access Journals (Sweden)

    Guy Caljon

    Full Text Available Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2 display DNA/RNA non-specific, high affinity nucleic acid binding with K(D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.

  20. Three homologous subunits form a high affinity peptide-gated ion channel in Hydra.

    Science.gov (United States)

    Dürrnagel, Stefan; Kuhn, Anne; Tsiairis, Charisios D; Williamson, Michael; Kalbacher, Hubert; Grimmelikhuijzen, Cornelis J P; Holstein, Thomas W; Gründer, Stefan

    2010-04-16

    Recently, three ion channel subunits of the degenerin (DEG)/epithelial Na(+) channel (ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na(+) channels (HyNaCs) 2-4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated by the neuropeptides Hydra-RFamides I and II. The HyNaC2/3 channel is so far the only cloned ionotropic receptor from cnidarians and, together with the related ionotropic receptor FMRFamide-activated Na(+) channel (FaNaC) from snails, the only known peptide-gated ionotropic receptor. The HyNaC2/3 channel has pore properties, like a low Na(+) selectivity and a low amiloride affinity, that are different from other channels of the DEG/ENaC gene family, suggesting that a component of the native Hydra channel might still be lacking. Here, we report the cloning of a new ion channel subunit from Hydra, HyNaC5. The new subunit is closely related to HyNaC2 and -3 and co-localizes with HyNaC2 and -3 to the base of the tentacles. Coexpression in Xenopus oocytes of HyNaC5 with HyNaC2 and -3 largely increases current amplitude after peptide stimulation and affinity of the channel to Hydra-RFamides I and II. Moreover, the HyNaC2/3/5 channel has altered pore properties and amiloride affinity, more similarly to other DEG/ENaC channels. Collectively, our results suggest that the three homologous subunits HyNaC2, -3, and -5 form a peptide-gated ion channel in Hydra that could contribute to fast synaptic transmission.

  1. Histones have high affinity for the glomerular basement membrane. Relevance for immune complex formation in lupus nephritis

    Energy Technology Data Exchange (ETDEWEB)

    Schmiedeke, T.M.; Stoeckl, F.W.W.; Weber, R.; Sugisaki, Y.; Batsford, S.R.; Vogt, A.

    1989-06-01

    An effort has been made to integrate insights on charge-based interactions in immune complex glomerulonephritis with nuclear antigen involvement in lupus nephritis. Attention was focussed on the histones, a group of highly cationic nuclear constituents, which could be expected to bind to fixed anionic sites present in the glomerular basement membrane (GBM). We demonstrated that all histone subfractions, prepared according to Johns, have a high affinity for GBM and the basement membrane of peritubular capillaries. Tissue uptake of /sup 125/I-labeled histones was measured by injecting 200 micrograms of each fraction into the left kidney via the aorta and measuring organ uptake after 15 min. In glomeruli isolated from the left kidneys, the following quantities of histones were found: f1, 13 micrograms; f2a (f2al + f2a2), 17 micrograms; f2b, 17 micrograms; and f3, 32 micrograms. Kinetic studies of glomerular binding showed that f1 disappeared much more rapidly than f2a. The high affinity of histones (pI between 10.5 and 11.0; mol wt 10,000-22,000) for the GBM correlates well with their ability to form aggregates (mol wt greater than 100,000) for comparison lysozyme (pI 11, mol wt 14,000), which does not aggregate spontaneously bound poorly (0.4 micrograms in isolated glomeruli). The quantity of histones and lysozyme found in the isolated glomeruli paralleled their in vitro affinity for a Heparin-Sepharose column (gradient elution studies). This gel matrix contains the sulfated, highly anionic polysaccharide heparin, which is similar to the negatively charged heparan sulfate present in the GBM. Lysozyme eluted with 0.15 M NaCl, f1 with 1 M NaCl, and f2a, f2b, and f3 could not be fully desorbed even with 2 M NaCl; 6 M guanidine-HCl was necessary.

  2. Molecular identification of high-affinity glutamate transporters in sheep and cattle forestomach, intestine, liver, kidney, and pancreas.

    Science.gov (United States)

    Howell, J A; Matthews, A D; Swanson, K C; Harmon, D L; Matthews, J C

    2001-05-01

    Glutamate metabolism is essential to support many facets of metabolism. The objective of this study was to determine the tissue distribution of glutamate transporters known to support the tissue metabolism of glutamate. The expression of proteins capable of high-affinity glutamate transport (system X-(AG)) by epithelia isolated from the rumen, omasum, duodenum, jejunum, ileum, cecum, and colon and homogenates of liver, kidney, and pancreatic tissues from wethers (n = 4; BW = 28.4 +/- 8.4 kg) and steers (n = 3; BW = 426 +/- 32.3 kg) fed forage-based diets was evaluated by immunoblot analysis. Proteins EAAC1 (62, 93 kDa) and GLT-1 (142, 188, >202 kDa) were expressed by every tissue examined. In contrast, GLAST1 (140 kDa) was expressed only by the pancreas, and EAAT4 (67 kDa) was detected only in sheep brain. To corroborate protein expression data, the presence and size of transporter mRNA in ileal, liver, and pancreatic homogenates were evaluated by Northern analysis. GLAST1 mRNA (2.4, 4.3 kb) was detected only in the pancreas, whereas EAAC1 (2.2, 2.8 kb) and GLT-1 (12.1 kb) mRNA transcripts were detected in all three tissues. The expression of EAAT4 and GLT-1 mRNA was confirmed by reverse transcriptase-polymerase chain reaction analyses. Sequencing of the resulting partial-length ovine GLT-1 cDNA revealed 100% identity with the rat homolog. Overall, these data demonstrate that sheep and cattle share the same pattern of system X-(AG) transporter expression, which differed among tissues and transporter isoforms. Accordingly, these data provide the fundamental knowledge to initiate research that determines whether the expression of high-affinity glutamate transporters by ruminants is sensitive to ontogenic and(or) dietary regulation.

  3. High affinity antigen recognition of the dual specific variants of herceptin is entropy-driven in spite of structural plasticity.

    Directory of Open Access Journals (Sweden)

    Jenny Bostrom

    Full Text Available The antigen-binding site of Herceptin, an anti-human Epidermal Growth Factor Receptor 2 (HER2 antibody, was engineered to add a second specificity toward Vascular Endothelial Growth Factor (VEGF to create a high affinity two-in-one antibody bH1. Crystal structures of bH1 in complex with either antigen showed that, in comparison to Herceptin, this antibody exhibited greater conformational variability, also called "structural plasticity". Here, we analyzed the biophysical and thermodynamic properties of the dual specific variants of Herceptin to understand how a single antibody binds two unrelated protein antigens. We showed that while bH1 and the affinity-improved bH1-44, in particular, maintained many properties of Herceptin including binding affinity, kinetics and the use of residues for antigen recognition, they differed in the binding thermodynamics. The interactions of bH1 and its variants with both antigens were characterized by large favorable entropy changes whereas the Herceptin/HER2 interaction involved a large favorable enthalpy change. By dissecting the total entropy change and the energy barrier for dual interaction, we determined that the significant structural plasticity of the bH1 antibodies demanded by the dual specificity did not translate into the expected increase of entropic penalty relative to Herceptin. Clearly, dual antigen recognition of the Herceptin variants involves divergent antibody conformations of nearly equivalent energetic states. Hence, increasing the structural plasticity of an antigen-binding site without increasing the entropic cost may play a role for antibodies to evolve multi-specificity. Our report represents the first comprehensive biophysical analysis of a high affinity dual specific antibody binding two unrelated protein antigens, furthering our understanding of the thermodynamics that drive the vast antigen recognition capacity of the antibody repertoire.

  4. The role of valence on the high-affinity binding of Griffonia simplicifolia isolectins to type A human erythrocytes.

    Science.gov (United States)

    Knibbs, R N; Takagaki, M; Blake, D A; Goldstein, I J

    1998-12-01

    The Griffonia simplicifolia-I (GS-I) isolectins have been used to probe the effect of lectin valence on their high-affinity binding to human erythrocytes. These tetrameric lectins are composed of A and B subunits and constitute a series of five isolectins (A4, A3B, A2B2, AB3, B4). The A subunit is specific for alpha-D-GalNAc end groups and binds to the blood type A determinant GalNAcalpha1, as well as to terminal alpha-D-Gal groups found on type B cells. The B subunit is specific for alpha-D-Gal end groups, and binds very specifically to type B erythrocytes. This series of isolectins is tetravalent (A4), trivalent (A3B), divalent (A2B2), and monovalent (AB3) for type A erythrocytes; thus, this system provides the opportunity to examine the effect of lectin valency on the association constants of these GS-I isolectins binding to cells. Cell binding experiments carried out using 125I-labeled GS-I isolectins and type A human erythrocytes allowed us to demonstrate that (1) the association constant of the isolectin monovalent for alpha-D-GalNAc (AB3) is virtually identical to its association constant for the haptenic sugar methyl-N-acetyl-alpha-D-galactosaminide, reported previously, and (2) the association constant of the GS-I isolectins for human type A erythrocytes increases with increasing valency of the isolectin. These results indicate that the increased affinity displayed by the GS-I isolectins for human type A erythrocytes is dependent on their multivalency, and not on an extended binding site nor on nonspecific, or noncarbohydrate, interactions of the lectin with the cell surface. These findings should be of general relevance to understanding the high-affinity interactions observed between other multivalent proteins and multivalent ligands (e.g., cell surfaces).

  5. Nerve growth factor, brain-derived neurotrophic factor and their high-affinity receptors are overexpressed in extramammary Paget's disease.

    Science.gov (United States)

    Qian, Yue; Takeuchi, Satoshi; Chen, Shan-Juan; Dugu, Long; Tsuji, Gaku; Xie, Lining; Nakahara, Takeshi; Moroi, Yoichi; Tu, Ya-Ting; Furue, Masutaka

    2010-11-01

    Neurotrophin (NT) systems appear to play important roles in the pathogenesis of several tumors, but their expression in extramammary Paget's disease (EPD) has not been investigated. Thirty-four paraffin-embedded EPD specimens (32 primary EPD and 2 metastatic to lymph nodes) were subject to immunohistochemical staining for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT3, NT4, their high-affinity receptors (TrkA, TrkB and TrkC) and the common low-affinity receptor, p75 NT receptor (p75). All 34 EPD specimens, including 2 metastatic to lymph nodes, showed cytoplasmic overexpression of NGF, BDNF, TrkA and TrkB. The expression (% positive cells) of NGF, BDNF, NT3, NT4, TrkA and TrkB (81.6 ± 14.9, 86.0 ± 10.4, 89.6 ± 14.9, 87.8 ± 17.9, 83 ± 14.4 and 86.2 ± 11.7%) in EPD was significantly higher than in normal skin (21.6 ± 6.5, 27.6 ± 4.5, 19.7 ± 10.1, 8.2 ± 10.0, 25.0 ± 5.3 and 25.4 ± 6.4%), and the expression of these factors in invasive EPD was significantly higher than in noninvasive EPD. Interestingly, Paget cells were negative for p75 and TrkC in all the 34 EPD specimens. These results suggest that overexpression of NGF, BDNF and their high-affinity receptors (TrkA and TrkB) might play a role in the pathogenesis of EPD. Copyright © 2010 John Wiley & Sons A/S.

  6. Balancing selected medication costs with total number of daily injections: a preference analysis of GnRH-agonist and antagonist protocols by IVF patients

    Directory of Open Access Journals (Sweden)

    Sills E

    2012-08-01

    Full Text Available Abstract Background During in vitro fertilization (IVF, fertility patients are expected to self-administer many injections as part of this treatment. While newer medications have been developed to substantially reduce the number of these injections, such agents are typically much more expensive. Considering these differences in both cost and number of injections, this study compared patient preferences between GnRH-agonist and GnRH-antagonist based protocols in IVF. Methods Data were collected by voluntary, anonymous questionnaire at first consultation appointment. Patient opinion concerning total number of s.c. injections as a function of non-reimbursed patient cost associated with GnRH-agonist [A] and GnRH-antagonist [B] protocols in IVF was studied. Results Completed questionnaires (n = 71 revealed a mean +/− SD patient age of 34 +/− 4.1 yrs. Most (83.1% had no prior IVF experience; 2.8% reported another medical condition requiring self-administration of subcutaneous medication(s. When out-of-pocket cost for [A] and [B] were identical, preference for [B] was registered by 50.7% patients. The tendency to favor protocol [B] was weaker among patients with a health occupation. Estimated patient costs for [A] and [B] were $259.82 +/− 11.75 and $654.55 +/− 106.34, respectively (p  Conclusions This investigation found consistently higher non-reimbursed direct medication costs for GnRH-antagonist IVF vs. GnRH-agonist IVF protocols. A conditional preference to minimize downregulation (using GnRH-antagonist was noted among some, but not all, IVF patient sub-groups. Compared to IVF patients with a health occupation, the preference for GnRH-antagonist was weaker than for other patients. While reducing total number of injections by using GnRH-antagonist is a desirable goal, it appears this advantage is not perceived equally by all IVF patients and its utility is likely discounted heavily by patients when nonreimbursed medication costs

  7. Identification of a nonbasic melanin hormone receptor 1 antagonist as an antiobesity clinical candidate.

    Science.gov (United States)

    Washburn, William N; Manfredi, Mark; Devasthale, Pratik; Zhao, Guohua; Ahmad, Saleem; Hernandez, Andres; Robl, Jeffrey A; Wang, Wei; Mignone, James; Wang, Zhenghua; Ngu, Khehyong; Pelleymounter, Mary Ann; Longhi, Daniel; Zhao, Rulin; Wang, Bei; Huang, Ning; Flynn, Neil; Azzara, Anthony V; Barrish, Joel C; Rohrbach, Kenneth; Devenny, James J; Rooney, Suzanne; Thomas, Michael; Glick, Susan; Godonis, Helen E; Harvey, Susan J; Cullen, Mary Jane; Zhang, Hongwei; Caporuscio, Christian; Stetsko, Paul; Grubb, Mary; Maxwell, Brad D; Yang, Hong; Apedo, Atsu; Gemzik, Brian; Janovitz, Evan B; Huang, Christine; Zhang, Lisa; Freeden, Chris; Murphy, Brian J

    2014-09-25

    Identification of MCHR1 antagonists with a preclinical safety profile to support clinical evaluation as antiobesity agents has been a challenge. Our finding that a basic moiety is not required for MCHR1 antagonists to achieve high affinity allowed us to explore structures less prone to off-target activities such as hERG inhibition. We report the SAR evolution of hydroxylated thienopyrimidinone ethers culminating in the identification of 27 (BMS-819881), which entered obesity clinical trials as the phosphate ester prodrug 35 (BMS-830216).

  8. Dopamine transporter-dependent and -independent striatal binding of the benztropine analog JHW 007, a cocaine antagonist with low abuse liability

    Science.gov (United States)

    The benztropine analog JHW 007 displays high affinity for the dopamine transporter (DAT), but unlike typical DAT ligands, has relatively low abuse liability and blocks effects of cocaine,including its self-administration. To determine sites responsible for the cocaine-antagonist effects of JHW 007, ...

  9. Stereoselective synthesis and biodistribution of potent [11C]-labeled antagonists for positron emission tomography imaging of muscarinic receptors in the airways.

    NARCIS (Netherlands)

    Visser, T.J; van Waarde, Aaren; Jansen, T.J H; Visser, G.M; van der Mark, T.W; Kraan, J; Ensing, K; Vaalburg, W.

    1997-01-01

    Quantitation of muscarinic receptors in the lungs in vivo with positron emission tomography (PET) is of clinical interest. For that purpose we decided to develop [C-11]-labeled ligands with a high affinity (K-D <0.1 nM). Three quaternary muscarinic antagonists, racemic N-methylpiperidin-4-yl

  10. A novel and selective melanin-concentrating hormone receptor 1 antagonist ameliorates obesity and hepatic steatosis in diet-induced obese rodent models.

    Science.gov (United States)

    Kawata, Yayoi; Okuda, Shoki; Hotta, Natsu; Igawa, Hideyuki; Takahashi, Masashi; Ikoma, Minoru; Kasai, Shizuo; Ando, Ayumi; Satomi, Yoshinori; Nishida, Mayumi; Nakayama, Masaharu; Yamamoto, Syunsuke; Nagisa, Yasutaka; Takekawa, Shiro

    2017-02-05

    Melanin-concentrating hormone (MCH), a cyclic neuropeptide expressed predominantly in the lateral hypothalamus, plays an important role in the control of feeding behavior and energy homeostasis. Mice lacking MCH or MCH1 receptor are resistant to diet-induced obesity (DIO) and MCH1 receptor antagonists show potent anti-obesity effects in preclinical studies, indicating that MCH1 receptor is a promising target for anti-obesity drugs. Moreover, recent studies have suggested the potential of MCH1 receptor antagonists for treatment of non-alcoholic fatty liver disease (NAFLD). In the present study, we show the anti-obesity and anti-hepatosteatosis effect of our novel MCH1 receptor antagonist, Compound A. Repeated oral administration of Compound A resulted in dose-dependent body weight reduction and had an anorectic effect in DIO mice. The body weight lowering effect of Compound A was more potent than that of pair-feeding. Compound A also reduced lipid content and the expression level of lipogenesis-, inflammation-, and fibrosis-related genes in the liver of DIO mice. Conversely, intracerebroventricular infusion of MCH caused induction of hepatic steatosis as well as increase in body weight in high-fat diet-fed wild type mice, but not MCH1 receptor knockout mice. The pair-feeding study revealed the MCH-MCH1 receptor system affects hepatic steatosis through a mechanism that is independent of body weight change. Metabolome analysis demonstrated that Compound A upregulated lipid metabolism-related molecules, such as acylcarnitines and cardiolipins, in the liver. These findings suggest that our novel MCH1 receptor antagonist, Compound A, exerts its beneficial therapeutic effect on NAFLD and obesity through a central MCH-MCH1 receptor pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Modulation of Neurological Deficits and Expression of Glutamate Receptors during Experimental Autoimmune Encephalomyelitis after Treatment with Selected Antagonists of Glutamate Receptors

    Directory of Open Access Journals (Sweden)

    Grzegorz Sulkowski

    2013-01-01

    Full Text Available The aim of our investigation was to characterize the role of group I mGluRs and NMDA receptors in pathomechanisms of experimental autoimmune encephalomyelitis (EAE, the rodent model of MS. We tested the effects of LY 367385 (S-2-methyl-4-carboxyphenylglycine, a competitive antagonist of mGluR1, MPEP (2-methyl-6-(phenylethynyl-pyridine, an antagonist of mGluR5, and the uncompetitive NMDA receptor antagonists amantadine and memantine on modulation of neurological deficits observed in rats with EAE. The neurological symptoms of EAE started at 10-11 days post-injection (d.p.i. and peaked after 12-13 d.p.i. The protein levels of mGluRs and NMDA did not increase in early phases of EAE (4 d.p.i., but starting from 8 d.p.i. to 25 d.p.i., we observed a significant elevation of mGluR1 and mGluR5 protein expression by about 20% and NMDA protein expression by about 10% over the control at 25 d.p.i. The changes in protein levels were accompanied by changes in mRNA expression of group I mGluRs and NMDARs. During the late disease phase (20–25 d.p.i., the mRNA expression levels reached 300% of control values. In contrast, treatment with individual receptor antagonists resulted in a reduction of mRNA levels relative to untreated animals.

  12. S179D prolactin: antagonistic agony!

    Science.gov (United States)

    Walker, Ameae M

    2007-09-30

    The aims of this review are three-fold: first, to collate what is known about the production and activities of phosphorylated prolactin (PRL), the latter largely, but not exclusively, as illustrated through the use of the molecular mimic, S179D PRL; second, to apply this and related knowledge to produce an updated model of prolactin-receptor interactions that may apply to other members of this cytokine super-family; and third, to promote a shift in the current paradigm for the development of clinically important growth antagonists. This third aim explains the title since, based on results with S179D PRL, it is proposed that agents which signal to antagonistic ends may be better therapeutics than pure antagonists-hence antagonistic agony. Since S179D PRL is not a pure antagonist, we have proposed the term selective prolactin receptor modulator (SPeRM) for this and like molecules.

  13. Selective Adsorption of Tetrahydropalmatine by a Molecularly ...

    African Journals Online (AJOL)

    NICO

    1School of Chemistry and Chemical Engineering, Guangxi University for Nationalities, Nanning 530006, P.R. China. 2Guangxi ... Selective adsorption experiments demonstrated the high affinity and THP selectiv- ... Molecularly imprinted polymer, tetrahydropalmatine, selective adsorption, microcalorimetry, modified rosin. 1.

  14. Aquaretic effect of lixivaptan, an oral, non-peptide, selective V2 receptor vasopressin antagonist, in New York Heart Association functional class II and III chronic heart failure patients.

    Science.gov (United States)

    Abraham, William T; Shamshirsaz, Alireza A; McFann, Kim; Oren, Ron M; Schrier, Robert W

    2006-04-18

    The purpose of this study was to examine the renal effects of a V2 receptor arginine vasopressin (AVP) antagonist in heart failure. Arginine vasopressin has been implicated in the renal water retention and dilutional hyponatremia associated with chronic heart failure. We examined the effects of the oral, non-peptide, selective V2 receptor antagonist lixivaptan in 42 diuretic-requiring patients with mild-to-moderate heart failure in a randomized, double-blind, placebo-controlled, ascending single-dose study. After overnight fluid deprivation, patients received single-blind placebo on day -1 (baseline) and double-blind study medication (placebo [n = 12] or lixivaptan 10, 30, 75, 150, 250, or 400 mg [n = 5 per dose group]) on day 1, followed by 4 h of continued fluid restriction and additional 20 h with ad libitum fluid intake. At all but the 10-mg dose, lixivaptan produced a significant and dose-related increase in urine volume over 4 h, compared with placebo. During 24 h, increases in urine volume ranged from 1.8 l with placebo to 3.9 l after the 400-mg lixivaptan dose (p heart failure and suggest that the V2 receptor antagonist lixivaptan may be a promising therapeutic agent for the treatment of heart failure.

  15. The dietary polyphenols trans-resveratrol and curcumin selectively bind human CB1 cannabinoid receptors with nanomolar affinities and function as antagonists/inverse agonists.

    Science.gov (United States)

    Seely, Kathryn A; Levi, Mark S; Prather, Paul L

    2009-07-01

    The dietary polyphenols trans-resveratrol [5-[(1E)-2-(4-hydroxyphenyl)ethenyl]-1,3-benzenediol; found in red wine] and curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1E,6E-heptadiene-3,5-dione] (found in curry powders) exert anti-inflammatory and antioxidant effects via poorly defined mechanisms. It is interesting that cannabinoids, derived from the marijuana plant (Cannabis sativa), produce similar protective effects via CB1 and CB2 receptors. We examined whether trans-resveratrol, curcumin, and ASC-J9 [1,7-bis(3,4-dimethoxyphenyl)-5-hydroxy-1E,4E,6E-heptatriene-3-one] (a curcumin analog) act as ligands at cannabinoid receptors. All three bind to human (h) CB1 and mouse CB1 receptors with nanomolar affinities, displaying only micromolar affinities for hCB2 receptors. Characteristic of inverse agonists, the polyphenols inhibit basal G-protein activity in membranes prepared from Chinese hamster ovary (CHO)-hCB1 cells or mouse brain that is reversed by a neutral CB1 antagonist. Furthermore, they competitively antagonize G-protein activation produced by a CB1 agonist. In intact CHO-hCB1 cells, the polyphenols act as neutral antagonists, producing no effect when tested alone, whereas competitively antagonizing CB1 agonist mediated inhibition of adenylyl cyclase activity. Confirming their neutral antagonist profile in cells, the polyphenols similarly attenuate stimulation of adenylyl cyclase activity produced by a CB1 inverse agonist. In mice, the polyphenols dose-dependently reverse acute hypothermia produced by a CB1 agonist. Upon repeated administration, the polyphenols also reduce body weight in mice similar to that produced by a CB1 antagonist/inverse agonist. Finally, trans-resveratrol and curcumin share common structural motifs with other known cannabinoid receptor ligands. Collectively, we suggest that trans-resveratrol and curcumin act as antagonists/inverse agonists at CB1 receptors at dietary relevant concentrations. Therefore, these polyphenols and their

  16. Synthesis of tricyclic indole-2-carboxylic [correction of caboxylic] acids as potent NMDA-glycine antagonists.

    Science.gov (United States)

    Katayama, S; Ae, N; Nagata, R

    2001-05-18

    The practical synthesis of a series of tricyclic indole-2-carboxylic acids, 7-chloro-3-arylaminocarbonylmethyl-1,3,4,5-tetrahydrobenz[cd]indole-2-carboxylic acids, as a new class of potent NMDA-glycine antagonists is described. The synthetic route to the key intermediate 12a comprises a regioselective iodination of 4-chloro-2-nitrotoluene, modified Reissert indole synthesis, Jeffery's Heck-type reaction with allyl alcohol, Wittig-Horner-Emmons reaction, and iodination at the indole C-3 position. The key step in the route is an intramolecular cyclization of 12a to give the tricyclic indole structure. Two methods of cyclization, (1) an intramolecular radical cyclization of 12a and (2) a sequence of intramolecular Heck reaction of 12a followed by a 1,4-reduction, were performed. The resulting tricyclic indole diester 13a was selectively hydrolyzed to afford the desired tricyclic indole monocarboxylic acid 16 on a multihundred gram scale without any chromatographic purifications. Optical resolution of 16 to (-)-isomer 17 and (+)-isomer 18 was carried out, and the resulting isomers were derivatized, respectively. Evaluation of the optically active derivatives for affinity to the NMDA-glycine binding site using the radio ligand binding assay with [(3)H]-5,7-dichlorokynurenic acid revealed that the derivatives of (-)-isomer 17 were more potent than the others and that especially substituted anilide (-)-isomer 24 (K(i) = 0.8 nM) showed high affinity.

  17. NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype

    DEFF Research Database (Denmark)

    Martini, Lene; Hastrup, Hanne; Holst, Birgitte

    2002-01-01

    against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed...

  18. Preparation of a novel antiserum to aromatase with high affinity and specificity: Its clinicopathological significance on breast cancer tissue.

    Science.gov (United States)

    Kanomata, Naoki; Matsuura, Shiro; Nomura, Tsunehisa; Kurebayashi, Junichi; Mori, Taisuke; Kitawaki, Jo; Moriya, Takuya

    2017-01-01

    Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 statuses. This antiserum will be applicable to clinicopathological examination of aromatase in addition to ER and PgR for an appropriate use of aromatase inhibitor on the treatment of breast cancer. Further studies on the relationship between Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and

  19. Mitogenic effects of urokinase on melanoma cells are independent of high affinity binding to the urokinase receptor.

    Science.gov (United States)

    Koopman, J L; Slomp, J; de Bart, A C; Quax, P H; Verheijen, J H

    1998-12-11

    The structural and functional properties of the urokinase-type plasminogen activator (u-PA) that are involved in the mitogenic effect of this proteolytic enzyme on human melanoma cells M14 and IF6 and the role of the u-PA receptor (u-PAR) in transducing this signal were analyzed. Native u-PA purified from urine induced a mitogenic response in quiescent IF6 and M14 cells that ranged from 25 to 40% of the mitogenic response obtained by fetal calf serum. The half-maximum response in M14 and IF6 cells was reached at u-PA concentrations of approximately 35 and 60 nM, respectively. Blocking the proteolytic activity of u-PA resulted in a 30% decrease of the mitogenic effect, whereas inhibition of plasmin activity did not alter the mitogenic effect. No mitogenic response was elicited by low molecular weight u-PA, lacking the growth factor domain and the kringle domain. The ATF domain of u-PA induced a mitogenic response that was similar to complete u-PA. Defucosylated ATF and recombinant u-PA purified from Escherichia coli lacking all post-translational modifications did not induce a mitogenic response. Blocking the interaction of u-PA with u-PAR, using a specific monoclonal antibody, did not alter the mitogenic effect induced by u-PA. The binding of radiolabeled u-PA to M14 and IF6 cells was characterized by high affinity binding mediated by u-PAR and low affinity binding to an unknown binding site. These results demonstrate that proteolytically inactive u-PA is able to induce a mitogenic response in quiescent melanoma cells in vitro by a mechanism that involves the ATF domain but is independent of high affinity binding to u-PAR. Furthermore, it suggests that u-PA is able to bind with low affinity to a hitherto unidentified membrane associated protein that could be involved in u-PA-induced signal transduction.

  20. Design, synthesis and in vitro pharmacology of GluK1 and GluK3 antagonists. Studies towards the design of subtype-selective antagonists through 2-carboxyethyl-phenylalanines with substituents interacting with non-conserved residues in the GluK binding sites.

    Science.gov (United States)

    Sköld, Niklas; Nielsen, Birgitte; Olsen, Jacob; Han, Liwei; Olsen, Lars; Madsen, Ulf; Kristensen, Jesper L; Pickering, Darryl S; Johansen, Tommy N

    2014-10-01

    In order to identify compounds selective for the GluK1 and GluK3 subtypes of kainate receptors we have designed and synthesized a series of (S)-2-amino-3-((2-carboxyethyl)phenyl)propanoic acid analogs with hydrogen bond donating and accepting substituents on the aromatic ring. Based on crystal structures of GluK1 in complex with related ligands, the compounds were designed to explore possible interactions with non-conserved residues outside the glutamate ligand binding site and challenge the water binding network. Apart from obtaining GluK1 selective antagonists one analog with a phenyl-substituted urea (compound 31) showed some preference for GluK3 over GluK1-receptors. Docking studies indicate that this preference may be attributed to contacts between the NH of the urea substituent and non-conserved Ser741 and Ser761 residues. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Non-basic azolotriazinone MCHR1 antagonists for the treatment of obesity: An empirical brain-exposures-driven candidate selection for in vivo efficacy studies.

    Science.gov (United States)

    Devasthale, Pratik; Wang, Wei; Mignone, James; Renduchintala, Kishore; Radhakrishnan, Sridhar; Dhanapal, Jayanthi; Selvaraj, Jagannath; Kuppusamy, Rajesh; Pelleymounter, Mary Ann; Longhi, Daniel; Huang, Ning; Flynn, Neil; Azzara, Anthony V; Rohrbach, Kenneth; Devenny, James; Rooney, Suzanne; Thomas, Michael; Glick, Susan; Godonis, Helen; Harvey, Susan; Cullen, Mary Jane; Zhang, Hongwei; Caporuscio, Christian; Stetsko, Paul; Grubb, Mary; Huang, Christine; Zhang, Lisa; Freeden, Chris; Murphy, Brian J; Robl, Jeffrey A; Washburn, William N

    2015-10-15

    Non-basic azolotriazinones were explored using an empirical free brain exposures-driven approach to identify potent MCHR1 antagonists for evaluation in in vivo efficacy studies. An optimized lead from this series, 1j (rMCHR1 Ki=1.8 nM), demonstrated a 6.9% reduction in weight gain relative to vehicle in a rat model at 30 mg/kg after 4 days of once-daily oral treatment as a glycine prodrug. Despite a promising efficacy profile, an assessment of the biliary toxicity risk of this compound rendered this compound non-progressible. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI

    OpenAIRE

    Sabban, Sari; Ye, Hongtu; Helm, Birgit

    2014-01-01

    The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development ...

  3. Development of a potent DOTA-conjugated bombesin antagonist for targeting GRPr-positive tumours

    Energy Technology Data Exchange (ETDEWEB)

    Mansi, Rosalba; Maecke, Helmut R. [University Hospital Basel, Division of Radiological Chemistry, Basel (Switzerland); University of Freiburg, Department of Nuclear Medicine, Freiburg (Germany); Wang, Xuejuan [University Hospital Basel, Division of Radiological Chemistry, Basel (Switzerland); Forrer, Flavio [University Hospital Basel, Institute of Nuclear Medicine, Basel (Switzerland); Erasmus Medical Centre, Nuclear Medicine, Rotterdam (Netherlands); Waser, Beatrice; Cescato, Renzo; Reubi, Jean Claude [University of Berne, Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, Berne (Switzerland); Graham, Keith; Borkowski, Sandra [Bayer Schering Pharma AG, Global Drug Discovery, Berlin (Germany)

    2011-01-15

    Radiolabelled somatostatin-based antagonists show a higher uptake in tumour-bearing mouse models than agonists of similar or even distinctly higher receptor affinity. Very similar results were obtained with another family of G protein-coupled receptor ligands, the bombesin family. We describe a new conjugate, RM2, with the chelator DOTA coupled to D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH{sub 2} via the cationic spacer 4-amino-1-carboxymethyl-piperidine for labelling with radiometals such as {sup 111}In and {sup 68}Ga. RM2 was synthesized on a solid support and evaluated in vitro in PC-3 cells. IC{sub 50} and K{sub d} values were determined. The antagonist potency was evaluated by immunofluorescence-based internalization and Ca{sup 2+} mobilization assays. Biodistribution studies were performed in PC-3 and LNCaP tumour-bearing mice with {sup 111}In-RM2 and {sup 68}Ga-RM2, respectively. PET/CT studies were performed on PC-3 and LNCaP tumour-bearing nude mice with {sup 68}Ga-RM2. RM2 and {sup 111}In-RM2 are high-affinity and selective ligands for the GRP receptor (7.7{+-}3.3 nmol/l for RM2; 9.3{+-}3.3 nmol/l for {sup nat}In-RM2). The potent antagonistic properties were confirmed by an immunofluorescence-based internalization and Ca{sup 2+} mobilization assays. {sup 68}Ga- and {sup 111}In-RM2 showed high and specific uptake in both the tumour and the pancreas. Uptake in the tumour remained high (15.2{+-}4.8%IA/g at 1 h; 11.7{+-}2.4%IA/g at 4 h), whereas a relatively fast washout from the pancreas and the other abdominal organs was observed. Uptake in the pancreas decreased rapidly from 22.6{+-}4.7%IA/g at 1 h to 1.5{+-}0.5%IA/g at 4 h. RM2 was shown to be a potent GRPr antagonist. Pharmacokinetics and imaging studies indicate that {sup 111}In-RM2 and {sup 68}Ga-RM2 are ideal candidates for clinical SPECT and PET studies. (orig.)

  4. ZipA binds to FtsZ with high affinity and enhances the stability of FtsZ protofilaments.

    Directory of Open Access Journals (Sweden)

    Anuradha Kuchibhatla

    Full Text Available A bacterial membrane protein ZipA that tethers FtsZ to the membrane is known to promote FtsZ assembly. In this study, the binding of ZipA to FtsZ was monitored using fluorescence spectroscopy. ZipA was found to bind to FtsZ with high affinities at three different (6.0, 6.8 and 8.0 pHs, albeit the binding affinity decreased with increasing pH. Further, thick bundles of FtsZ protofilaments were observed in the presence of ZipA under the pH conditions used in this study indicating that ZipA can promote FtsZ assembly and stabilize FtsZ polymers under unfavorable conditions. Bis-ANS, a hydrophobic probe, decreased the interaction of FtsZ and ZipA indicating that the interaction between FtsZ and ZipA is hydrophobic in nature. ZipA prevented the dilution induced disassembly of FtsZ polymers suggesting that it stabilizes FtsZ protofilaments. Fluorescein isothiocyanate-labeled ZipA was found to be uniformly distributed along the length of the FtsZ protofilaments indicating that ZipA stabilizes FtsZ protofilaments by cross-linking them.

  5. Fragile X mental retardation protein recognition of G quadruplex structure per se is sufficient for high affinity binding to RNA.

    Science.gov (United States)

    Bole, Medhavi; Menon, Lakshmi; Mihailescu, Mihaela-Rita

    2008-12-01

    Fragile X syndrome, the most common form of inherited mental retardation is caused by the expansion of a CGG trinucleotide repeat in the fragile X mental retardation 1 (fmr1) gene. The abnormal expansion of the CGG repeat causes hypermethylation and subsequent silencing of the fmr1 gene, resulting in the loss of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine rich region (RGG box) to bind to messenger RNAs that form G quadruplex structures. Several studies reported that the G quadruplex RNA recognition alone is not sufficient for FMRP RGG box binding and that an additional stem and/or a G quadruplex-stem junction region may also be important in recognition. In this study we have used biophysical methods such as fluorescence, UV, CD and NMR spectroscopy to demonstrate that the recognition of the RNA G quadruplex structure per se, in the absence of a stem region, is sufficient for the FMRP high affinity and specific binding. These findings indicate that the presence of a stem structure in some of the FMRP G quadruplex forming mRNAs is not a requirement for protein recognition as previously believed, but rather for the proper formation of the correct RNA G quadruplex structure recognized by FMRP.

  6. G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity.

    Science.gov (United States)

    Tatsumi, Kasumi; Sakashita, Gyosuke; Nariai, Yuko; Okazaki, Kosuke; Kato, Hiroaki; Obayashi, Eiji; Yoshida, Hisashi; Sugiyama, Kanako; Park, Sam-Yong; Sekine, Joji; Urano, Takeshi

    2017-03-07

    The recognition specificity of monoclonal antibodies (mAbs) has made mAbs among the most frequently used tools in both basic science research and in clinical diagnosis and therapies. Precise determination of the epitope allows the development of epitope tag systems to be used with recombinant proteins for various purposes. Here we describe a new family of tag derived from the epitope recognized by a highly specific mAb G196. The minimal epitope was identified as the five amino acid sequence Asp-Leu-Val-Pro-Arg. Permutation analysis was used to characterize the binding requirements of mAb G196, and the variable regions of the mAb G196 were identified and structurally analyzed by X-ray crystallography. Isothermal titration calorimetry revealed the high affinity (Kd = 1.25 nM) of the mAb G196/G196-epitope peptide interaction, and G196-tag was used to detect several recombinant cytosolic and nuclear proteins in human and yeast cells. mAb G196 is valuable for developing a new peptide tagging system for cell biology and biochemistry research.

  7. New high affinity monoclonal antibodies recognize non-overlapping epitopes on mesothelin for monitoring and treating mesothelioma.

    Science.gov (United States)

    Zhang, Yi-Fan; Phung, Yen; Gao, Wei; Kawa, Seiji; Hassan, Raffit; Pastan, Ira; Ho, Mitchell

    2015-05-21

    Mesothelin is an emerging cell surface target in mesothelioma and other solid tumors. Most antibody drug candidates recognize highly immunogenic Region I (296-390) on mesothelin. Here, we report a group of high-affinity non-Region I rabbit monoclonal antibodies. These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin. One pair of antibodies (YP218 and YP223) is suitable to detect soluble mesothelin in a sandwich ELISA with high sensitivity. The new assay can also be used to measure serum mesothelin concentration in mesothelioma patients, indicating its potential use for monitoring patients treated with current antibody therapies targeting Region I. The antibodies are highly specific and sensitive in immunostaining of mesothelioma. To explore their use in tumor therapy, we have generated the immunotoxins based on the Fv of these antibodies. One immunotoxin (YP218 Fv-PE38) exhibits potent anti-tumor cytotoxicity towards primary mesothelioma cell lines in vitro and an NCI-H226 xenograft tumor in mice. Furthermore, we have engineered a humanized YP218 Fv that retains full binding affinity for mesothelin-expressing cancer cells. In conclusion, with their unique binding properties, these antibodies may be promising candidates for monitoring and treating mesothelioma and other mesothelin-expressing cancers.

  8. A Dualistic Conformational Response to Substrate Binding in the Human Serotonin Transporter Reveals a High Affinity State for Serotonin*

    Science.gov (United States)

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida; Wiborg, Ove; Sinning, Steffen

    2015-01-01

    Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT. PMID:25614630

  9. ZrFsy1, a high-affinity fructose/H+ symporter from fructophilic yeast Zygosaccharomyces rouxii.

    Directory of Open Access Journals (Sweden)

    Maria José Leandro

    Full Text Available Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H(+ symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H(+ symporter, with Km 0.45 ± 0.07 mM and Vmax 0.57 ± 0.02 mmol h(-1 (gdw(-1. We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2% and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H(+ symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system.

  10. ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis.

    Science.gov (United States)

    Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Rajulu, Charukesi; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier; Rubio, Vicente

    2015-09-01

    Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. © 2015 American Society of Plant Biologists. All rights reserved.

  11. ZK91587: a novel synthetic antimineralocorticoid displays high affinity for corticosterone (type I) receptors in the rat hippocampus

    Energy Technology Data Exchange (ETDEWEB)

    Sutanto, W.; de Kloet, E.R.

    1988-01-01

    In vitro cytosol binding assays have shown the properties of binding of a novel steroid, ZK91587 (15..beta.., 16..beta..b-methylene-mexrenone) in the brain of rats. Scatchard and Woolf analyses of the binding data reveal the binding of (/sup 3/H) ZK91587 to the total hippocampal coritcosteroid receptor sites with high affinity, and low capacity. When 100-fold excess RU28362 was included simultaneously with (/sup 3/H) ZK91587, the labelled steroid binds with the same affinity and capacity. Relative binding affinities (RBA) of various steroids for the Type I or Type II corticosteroid receptor in these animals are: Type I: ZK91587 = corticosterone (B) > cortisol (F); Type II: B > F >>> ZK91587. In the binding kinetic study, ZK91587 has a high association rate of binding in the rat. The steroid dissociates following a one slope pattern, indicating, the present data demonstrate that in the rat hippocampus, ZK91587 binds specifically to the Type I (corticosterone-preferring/mineralocorticoid-like receptor.

  12. Ectomycorrhiza-mediated repression of the high-affinity ammonium importer gene AmAMT2 in Amanita muscaria.

    Science.gov (United States)

    Willmann, Anita; Weiss, Michael; Nehls, Uwe

    2007-02-01

    A main function of ectomycorrhizas, a symbiosis between certain soil fungi and fine roots of woody plants, is the exchange of plant-derived carbohydrates for fungus-derived nutrients. As it is required in large amounts, nitrogen is of special interest. A gene (AmAMT2) coding for a putative fungal ammonium importer was identified in an EST project of functional Amanita muscaria/poplar ectomycorrhizas. Heterologous expression of the entire AmAMT2 coding region in yeast revealed the corresponding protein to be a high-affinity ammonium importer. In axenically grown Amanita hyphae AmAMT2 expression was strongly repressed by nitrogen, independent of whether the offered nitrogen source was transported by AmAMT2 or not. In functional ectomycorrhizas the AmAMT2 transcript level was further decreased in both hyphal networks (sheath and Hartig net), while extraradical hyphae revealed strong gene expression. Together our data suggest that (1) AmAMT2 expression is regulated by the endogenous nitrogen content of hyphae and (2) fungal hyphae in ectomycorrhizas are well supported with nitrogen even when the extraradical mycelium is nitrogen limited. As a consequence of AmAMT2 repression in mycorrhizas, ammonium can be suggested as a potential nitrogen source delivered by fungal hyphae in symbiosis.

  13. Structural determinants for high-affinity binding in a Nedd4 WW3* domain-Comm PY motif complex.

    Science.gov (United States)

    Kanelis, Voula; Bruce, M Christine; Skrynnikov, Nikolai R; Rotin, Daniela; Forman-Kay, Julie D

    2006-03-01

    Interactions between the WW domains of Drosophila Nedd4 (dNedd4) and Commissureless (Comm) PY motifs promote axon crossing at the CNS midline and muscle synaptogenesis. Here we report the solution structure of the dNedd4 WW3* domain complexed to the second PY motif (227'TGLPSYDEALH237') of Comm. Unexpectedly, there are interactions between WW3* and ligand residues both N- and C-terminal to the PY motif. Residues Y232'-L236' form a helical turn, following the PPII helical PY motif. Mutagenesis and binding studies confirm the importance of these extensive contacts, not simultaneously observed in other WW domain complexes, and identify a variable loop in WW3* responsible for its high-affinity interaction. These studies expand our general understanding of the molecular determinants involved in WW domain-ligand recognition. In addition, they provide insights into the specific regulation of dNedd4-mediated ubiquitination of Comm and subsequent internalization of Comm or the Comm/Roundabout complex, critical for CNS and muscle development.

  14. Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of [alpha]- and PPII-helices

    Energy Technology Data Exchange (ETDEWEB)

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Patel, Manisha H.; Robinette, Rebekah A.; Crowley, Paula J.; Michalek, Suzanne; Brady, L. Jeannine; Deivanayagam, Champion (Cornell); (UAB); (Florida)

    2010-08-18

    Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 {angstrom}) crystal structure of the A{sub 3}VP{sub 1} fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 {angstrom}) through the interaction of two noncontiguous regions in the primary sequence. The A{sub 3} repeat of the alanine-rich domain adopts an extended {alpha}-helix that intertwines with the P{sub 1} repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A{sub 3} and P{sub 1} helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.

  15. Expression of NGF, BDNF and their high-affinity receptors in ovine mammary glands during development and lactation.

    Science.gov (United States)

    Colitti, Monica

    2015-12-01

    The distribution of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and their high-affinity tyrosine kinase receptors TrkA and TrkB was investigated by immunohistochemical method in the mammary gland of ewes from prepubertal stage to involution. NGF and BDNF protein expressions were strong during development of glands at prepubertal stage and during pregnancy and decreased during lactation and involution. The expressions localized in both stromal and parenchymal cells of developing gland were mainly arranged in the apical side of secretory cells during lactation. These observations were also confirmed at transcriptional level by RT-PCR analyses. The highest expression of all genes significantly occurred at prepubertal stage. NGF was then down-regulated from pregnancy to involution, and no statistical differences were observed among these stages. The receptor TrkA was also under-expressed from pregnancy to involution, and its expression significantly differed between pregnancy and 30 days of lactation and also between 30 and 60 days of lactation. BDNF was significantly down-regulated at 60 days of lactation in comparison with prepubertal stage and again between pregnancy and 30 days of lactation. The relative abundance of its receptor, TrkB, showed also a significant down-regulation at 60 days of lactation in comparison with pregnancy and involution. Among the myriad of other molecular signals involved in the mammary gland cycle, the local production of neuropeptides and their receptors could be of interest in understanding their potential role in mammary biology.

  16. Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

    DEFF Research Database (Denmark)

    Kandra, L.; Abou Hachem, Maher; Gyemant, G.

    2006-01-01

    as binding barriers. Barley a-amylase I mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile...

  17. High-affinity human leucocyte antigen class I binding variola-derived peptides induce CD4(+) T cell responses more than 30 years post-vaccinia virus vaccination

    DEFF Research Database (Denmark)

    Wang, M.; Tang, Sheila Tuyet; Lund, Ole

    2009-01-01

    Interferon-gamma secreting T lymphocytes against pox virus-derived synthetic 9-mer peptides were tested by enzyme-linked immunospot in peripheral blood of individuals vaccinated with vaccinia virus more than 30 years ago. The peptides were characterized biochemically as high-affinity human...

  18. A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

    DEFF Research Database (Denmark)

    Bach, Anders*; Clausen, Bettina H; Møller, Magda

    2012-01-01

    Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-d-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors...

  19. Amino propynyl benzoic acid building block in rigid spacers of divalent ligands binding to the Syk SH2 domains with equally high affinity as the natural ligand

    NARCIS (Netherlands)

    Dekker, Frank J; de Mol, Nico J; Fischer, Marcel J E; Liskamp, Rob M J; Dekker, Frank

    2003-01-01

    The construction of rigid spacers composed of amino propynyl benzoic acid building blocks is described. These spacers were used to link two phosphopeptide ligand sites towards obtaining divalent ligands with a high affinity for Syk tandem SH2 domains, which are important in signal transduction. The

  20. Cytokine-induced immune complex binding to the high-affinity IgG receptor, FcγRI, in the presence of monomeric IgG

    NARCIS (Netherlands)

    van der Poel, C.E.; Karssemeijer, R.A.; Boross, P.; van der Linden, J.A.; Blokland - Fromme, M.; van de Winkel, J.G.J.; Leusen, J.H.W.

    2010-01-01

    FcγRI is the sole high-affinity immunoglobulin G (IgG) receptor on leukocytes. Its role in immunity and the clearance of opsonized particles has been challenged, as the receptor function may well be hindered by serum IgG. Here, we document immune complex binding by FcγRI to be readily enhanced by

  1. RNA Aptamer Binds Heparin-Binding Epidermal Growth Factor-Like Growth Factor with High Affinity and Specificity and Neutralizes Its Activity

    Directory of Open Access Journals (Sweden)

    Masaki Yamato

    2017-09-01

    Conclusion: We identified a novel RNA aptamer that bound with high affinity and specificity to rhHB-EGF and potently inhibited the rhHB-EGF-mediated phosphorylation of EGFR. The anti-HB-EGF aptamer may be a promising therapeutic agent for specifically neutralizing HB-EGF signaling.

  2. High-Affinity Sites Form an Interaction Network to Facilitate Spreading of the MSL Complex across the X Chromosome in Drosophila

    NARCIS (Netherlands)

    Ramírez, Fidel; Lingg, Thomas; Toscano, Sarah; Lam, Kin Chung; Georgiev, Plamen; Chung, Ho-Ryun; Lajoie, Bryan R; de Wit, Elzo; Zhan, Ye; de Laat, Wouter; Dekker, Job; Manke, Thomas; Akhtar, Asifa

    2015-01-01

    Dosage compensation mechanisms provide a paradigm to study the contribution of chromosomal conformation toward targeting and spreading of epigenetic regulators over a specific chromosome. By using Hi-C and 4C analyses, we show that high-affinity sites (HAS), landing platforms of the male-specific

  3. Identification of the magnesium-binding domain of the high affinity ATP binding-site of the Bacillus subtilis and Escherichia coli seca protein

    NARCIS (Netherlands)

    van der Wolk, J.P.W.; Klose, M; de Wit, Janny; Blaauwen, T.den; Freudl, R; Driessen, A.J.M.

    1995-01-01

    The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane. The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA. This domain

  4. Soil carbon content and relative abundance of high affinity H2-oxidizing bacteria predict atmospheric H2 soil uptake activity better than soil microbial community composition

    NARCIS (Netherlands)

    Khdhiri, Mondher; Hesse, Laura; Popa, Maria Elena; Quiza, Liliana; Lalonde, Isabelle; Meredith, Laura K.; Röckmann, Thomas; Constant, Philippe

    2015-01-01

    Soil-atmosphere exchange of H2 is controlled by gas diffusion and the microbial production and oxidation activities in soil. Among these parameters, the H2 oxidation activity catalyzed by soil microorganisms harboring high affinity hydrogenase is the most difficult variable to parameterize because

  5. Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity

    Science.gov (United States)

    Algimantas P. Valaitis; Jeremy L. Jenkins; Mi Kyong Lee; Donald H. Dean; Karen J. Garner

    2001-01-01

    BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were...

  6. Novel radioiodinated {gamma}-hydroxybutyric acid analogues for radiolabeling and Photolinking of high-affinity {gamma}-hydroxybutyric acid binding sites

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Sabbatini, Paola

    2010-01-01

    ¿-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low- and high-affinity sites in the mammalian brain. To target the specific GHB binding sites, we have developed a (125)I-labeled GHB analog and characterized its binding in rat brain...

  7. Novel Radioiodinated γ-Hydroxybutyric Acid Analogues for Radiolabeling and Photolinking of High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Sabbatini, Paola

    2010-01-01

    γ-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low- and high-affinity sites in the mammalian brain. To target the specific GHB binding sites, we have developed a 125I-labeled GHB analog and characterized its binding in rat brain...

  8. High-affinity NO(3-)-H+ cotransport in the fungus Neurospora: induction and control by pH and membrane voltage.

    Science.gov (United States)

    Blatt, M R; Maurousset, L; Meharg, A A

    1997-11-01

    High-affinity nitrate transport was examined in intact hyphae of Neurospora crassa using electrophysiological recordings to characterize the response of the plasma membrane to NO3- challenge and to quantify transport activity. The NO3(-)-associated membrane current was determined using a three electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in hyphae transferred to NO3(-)-free, N-limited medium for 15 hr, and in hyphae grown in the absence of a nitrogen source after a single 2-min exposure to 100 microM NO3-. In the latter, induction showed a latency of 40-80 min and rose in scalar fashion with full transport activity measurable approx. 100 min after first exposure to NO3-; it was marked by the appearance of a pronounced sensitivity of membrane voltage to extracellular NO3- additions which, after induction, resulted in reversible membrane depolarizations of (+)54-85 mV in the presence of 50 microM NO3-; and it was suppressed when NH4+ was present during the first, inductive exposure to NO3-. Voltage clamp measurements carried out immediately before and following NO3- additions showed that the NO3(-)-evoked depolarizations were the consequence of an inward-directed current that appeared in parallel with the depolarizations across the entire range of accessible voltages (-400 to +100 mV). Measurements of NO3- uptake using NO3(-)-selective macroelectrodes indicated a charge stoichiometry for NO3- transport of 1(+):1(NO3-) with common K(m) and Jmax values around 25 microM and 75 pmol NO3- cm-2sec-1, respectively, and combined measurements of pHo and [NO3-]o showed a net uptake of approx. 1 H+ with each NO3- anion. Analysis of the NO3- current demonstrated a pronounced voltage sensitivity within the normal physiological range between -300 and -100 mV as well as interactions between the kinetic parameters of membrane voltage, pHo and [NO3

  9. OsHAK1, a High-Affinity Potassium Transporter, Positively Regulates Responses to Drought Stress in Rice

    Directory of Open Access Journals (Sweden)

    Guang Chen

    2017-11-01

    Full Text Available Drought is one of the environmental factors that severely restrict plant distribution and crop production. Recently, we reported that the high-affinity potassium transporter OsHAK1 plays important roles in K acquisition and translocation in rice over low and high K concentration ranges, however, knowledge on the regulatory roles of OsHAK1 in osmotic/drought stress is limited. Here, transcript levels of OsHAK1 were found transiently elevated by water deficit in roots and shoots, consistent with the enhanced GUS activity in transgenic plants under stress. Under drought conditions, OsHAK1 knockout mutants (KO presented lower tolerance to the stress and displayed stunted growth at both the vegetative and reproductive stages. Phenotypic analysis of OsHAK1 overexpression seedlings (Ox demonstrated that they present better tolerance to drought stress than wild-type (WT. Compared to WT seedlings, OsHAK1 overexpressors had lower level of lipid peroxidation, higher activities of antioxidant enzymes (POX and CAT and higher proline accumulation. Furthermore, qPCR analysis revealed that OsHAK1 act as a positive regulator of the expression of stress-responsive genes as well as of two well-known rice channel genes (OsTPKb and OsAKT1 involved in K homeostasis and stress responses in transgenic plants under dehydration. Most important, OsHAK1-Ox plants displayed enhanced drought tolerance at the reproductive stage, resulting in 35% more grain yield than WT under drought conditions, and without exhibiting significant differences under normal growth conditions. Consequently, OsHAK1 can be considered to be used in molecular breeding for improvement of drought tolerance in rice.

  10. Proanthocyanidin oxidation of Arabidopsis seeds is altered in mutant of the high-affinity nitrate transporter NRT2.7

    Science.gov (United States)

    David, Laure C.; Dechorgnat, Julie; Ferrario-Méry, Sylvie

    2014-01-01

    NRT2.7 is a seed-specific high-affinity nitrate transporter controlling nitrate content in Arabidopsis mature seeds. The objective of this work was to analyse further the consequences of the nrt2.7 mutation for the seed metabolism. This work describes a new phenotype for the nrt2.7-2 mutant allele in the Wassilewskija accession, which exhibited a distinctive pale-brown seed coat that is usually associated with a defect in flavonoid oxidation. Indeed, this phenotype resembled those of tt10 mutant seeds defective in the laccase-like enzyme TT10/LAC15, which is involved in the oxidative polymerization of flavonoids such as the proantocyanidins (PAs) (i.e. epicatechin monomers and PA oligomers) and flavonol glycosides. nrt2.7-2 and tt10-2 mutant seeds displayed the same higher accumulation of PAs, but were partially distinct, since flavonol glycoside accumulation was not affected in the nrt2.7-2 seeds. Moreover, measurement of in situ laccase activity excluded a possibility of the nrt2.7-2 mutation affecting the TT10 enzymic activity at the early stage of seed development. Functional complementation of the nrt2.7-2 mutant by overexpression of a full-length NRT2.7 cDNA clearly demonstrated the link between the nrt2.7 mutation and the PA phenotype. However, the PA-related phenotype of nrt2.7-2 seeds was not strictly correlated to the nitrate content of seeds. No correlation was observed when nitrate was lowered in seeds due to limited nitrate nutrition of plants or to lower nitrate storage capacity in leaves of clca mutants deficient in the vacuolar anionic channel CLCa. All together, the results highlight a hitherto-unknown function of NRT2.7 in PA accumulation/oxidation. PMID:24532452

  11. Dephosphorylation and quantification of organic phosphorus in poultry litter by purified phytic-acid high affinity Aspergillus phosphohydrolases.

    Science.gov (United States)

    Dao, Thanh H; Hoang, Khanh Q

    2008-08-01

    Extracellular phosphohydrolases mediate the dephosphorylation of phosphoesters and influence bioavailability and loss of agricultural P to the environment to pose risks of impairment of sensitive aquatic ecosystems. Induction and culture of five strains of Aspergillus were conducted to develop a source of high-affinity and robust phosphohydrolases for detecting environmental P and quantifying bioactive P pools in heterogeneous environmental specimens. Enzyme stability and activity against organic P in poultry litter were evaluated in 71 samples collected across poultry producing regions of Arkansas, Maryland, and Oklahoma of the US Differences existed in strains' adaptability to fermentation medium as they showed a wide range of phytate-degrading activity. Phosphohydrolases from Aspergillus ficuum had highest activity when the strain was cultured on a primarily chemical medium, compared to Aspergillus oryzae which preferred a wheat bran-based organic medium. Kinetics parameters of A. ficuum enzymes (K(m)=210 microM; V(max) of 407 nmol s(-1)) indicated phytic acid-degrading potential equivalent to that of commercial preparations. Purified A. ficuum phosphohydrolases effectively quantified litter bioactive P pools, showing that organic P occurred at an average of 54 (+/-14)% of total P, compared to inorganic phosphates, which averaged 41 (+/-12)%. Litter management and land application options must consider the high water-extractable and organic P concentrations and the biological availability of the organic enzyme-labile P pool. Robustness of A. ficuum enzymes and simplicity of the in situ ligand-based enzyme assay may thus increase routine assessment of litter bioactive P composition to sense for on-farm accumulation of such environmentally-sensitive P forms.

  12. High affinity anti-TIM-3 and anti-KIR monoclonal antibodies cloned from healthy human individuals.

    Directory of Open Access Journals (Sweden)

    Stefan Ryser

    Full Text Available We report here the cloning of native high affinity anti-TIM-3 and anti-KIR IgG monoclonal antibodies (mAbs from peripheral blood mononuclear cells (PBMC of healthy human donors. The cells that express these mAbs are rare, present at a frequency of less than one per 105 memory B-cells. Using our proprietary multiplexed screening and cloning technology CellSpot™ we assessed the presence of memory B-cells reactive to foreign and endogenous disease-associated antigens within the same individual. When comparing the frequencies of antigen-specific memory B-cells analyzed in over 20 screening campaigns, we found a strong correlation of the presence of anti-TIM-3 memory B-cells with memory B-cells expressing mAbs against three disease-associated antigens: (i bacterial DNABII proteins that are a marker for Gram negative and Gram positive bacterial infections, (ii hemagglutinin (HA of influenza virus and (iii the extracellular domain of anaplastic lymphoma kinase (ALK. One of the native anti-KIR mAbs has similar characteristics as lirilumab, an anti-KIR mAb derived from immunization of humanized transgenic mice that is in ongoing clinical trials. It is interesting to speculate that these native anti-TIM-3 and anti-KIR antibodies may function as natural regulatory antibodies, analogous to the pharmacological use in cancer treatment of engineered antibodies against the same targets. Further characterization studies are needed to define the mechanisms through which these native antibodies may function in healthy and disease conditions.

  13. Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

    Directory of Open Access Journals (Sweden)

    Thomas Stockner

    Full Text Available The high-resolution crystal structure of the leucine transporter (LeuT is frequently used as a template for homology models of the dopamine transporter (DAT. Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii LeuT and DAT share a rather low overall sequence identity (22% and (iii the extracellular loop 2 (EL2 of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

  14. Evaluation of the full evaporation technique for quantitative analysis of high boiling compounds with high affinity for apolar matrices.

    Science.gov (United States)

    van Boxtel, Niels; Wolfs, Kris; van Schepdael, Ann; Adams, Erwin

    2014-06-27

    In order to reduce inaccuracies due to possible matrix effects in conventional static headspace-gas chromatography (sHS-GC), it is standard practice to match the composition of calibration standards towards the composition of the sample to be analysed by adding blank matrix. However, the latter is not always available and in that case the full evaporation technique (FET) could be a solution. With FET a small sample volume is introduced in a HS vial and compounds of interest are completely evaporated. Hence no equilibrium between the condensed phase and vapour phase exists. Without the existence of an equilibrium, matrix effects are less likely to occur. Another issue often encountered with sHS-sampling is that low vapour pressure compounds with a high affinity for the dilution medium show a limited sensitivity. FET has proven to be an appropriate solution to address this problem too. In this work, the applicability of FET for the quantitative analysis of high boiling compounds in different complex apolar matrices is examined. Data show that FET is an excellent tool to overcome matrix effects often encountered with conventional sHS analysis. The tested method shows excellent accuracy with recovery values around 100% as well as repeatability with RSD values around 1% for the quantification of high boiling compounds (bp>200°C) such as camphor, menthol, methyl salicylate and ethyl salicylate in various matrices. LOQ values were found to be around 0.3μg per vial. Following validation of the technique, several topical pharmaceutical formulations like ThermoCream(®), Reflexspray(®), Vicks Vaporub(®) and Radosalil(®) were examined. For the latter, a comparison has been made with a sHS-method described in literature. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Identification and Tumour-Binding Properties of a Peptide with High Affinity to the Disialoganglioside GD2.

    Directory of Open Access Journals (Sweden)

    Jan Müller

    Full Text Available Neuroectodermal tumours are characterized by aberrant processing of disialogangliosides concomitant with high expression of GD2 or GD3 on cell surfaces. Antibodies targeting GD2 are already in clinical use for therapy of neuroblastoma, a solid tumour of early childhood. Here, we set out to identify peptides with high affinity to human disialoganglioside GD2. To this end, we performed a combined in vivo and in vitro screen using a recombinant phage displayed peptide library. We isolated a phage displaying the peptide sequence WHWRLPS that specifically binds to the human disialoganglioside GD2. Binding specificity was confirmed by mutational scanning and by comparative analyses using structurally related disialogangliosides. In vivo, significant enrichment of phage binding to xenografts of human neuroblastoma cells in mice was observed. Tumour-specific phage accumulation could be blocked by intravenous coinjection of the corresponding peptide. Comparative pharmacokinetic analyses revealed higher specific accumulation of 68Ga-labelled GD2-binding peptide compared to 111In-labelled peptide in xenografts of human neuroblastoma. In contrast to 124I-MIBG, which is currently evaluated as a neuroblastoma marker in PET/CT, 68Ga-labelled GD2-specific peptide spared the thyroid but was enriched in the kidneys, which could be partially blocked by infusion of amino acids.In summary, we here report on a novel tumour-homing peptide that specifically binds to the disialoganglioside GD2, accumulates in xenografts of neuroblastoma cells in mice and bears the potential for tumour detection using PET/CT. Thus, this peptide may serve as a new scaffold for diagnosing GD2-positive tumours of neuroectodermal origin.

  16. Synthetic 1,2,3-triazole-linked glycoconjugates bind with high affinity to human galectin-3.

    Science.gov (United States)

    Marchiori, Marcelo Fiori; Souto, Dênio Emanuel Pires; Bortot, Leandro Oliveira; Pereira, João Francisco; Kubota, Lauro Tatsuo; Cummings, Richard D; Dias-Baruffi, Marcelo; Carvalho, Ivone; Campo, Vanessa Leiria

    2015-07-01

    This work describes the synthesis of the 1,2,3-triazole amino acid-derived-3-O-galactosides 1-6 and the 1,2,3-triazole di-lactose-derived glycoconjugate 7 as potential galectin-3 inhibitors. The target compounds were synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition reaction ('click chemistry') between the azido-derived amino acids N3-ThrOBn, N3-PheOBn, N3-N-Boc-TrpOBn, N3-N-Boc-LysOBn, N3-O-tBu-AspOBn and N3-l-TyrOH, and the corresponding alkyne-based sugar 3-O-propynyl-GalOMe, as well as by click chemistry reaction between the azido-lactose and 2-propynyl lactose. Surface plasmon resonance (SPR) assays showed that all synthetic glycoconjugates 1-7 bound to galectin-3 with high affinity, but the highest binders were the amino acids-derived glycoconjugates 2 (KD 7.96μM) and 4 (KD 4.56μM), and the divalent lactoside 7 (KD1 0.15μM/KD2 19μM). Molecular modeling results were in agreement with SPR assays, since more stable interactions with galectin-3 were identified for glycoconjugates 2, 4 and 7. Regarding compounds 2 and 4, they established specific cation-π (Arg144) and ionic (Asp148) interactions, whereas glycoconjugate 7 was capable to bridge two independent galectin-3 CRDs, creating a non-covalent cross-link between two monomers and, thus, reaching a submicromolar affinity towards galectin-3. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Comparative protection against rat intestinal reperfusion injury by a new inhibitor of sPLA2, COX-1 and COX-2 selective inhibitors, and an LTC4 receptor antagonist

    Science.gov (United States)

    Arumugam, Thiruma V; Arnold, Naomi; Proctor, Lavinia M; Newman, Michelle; Reid, Robert C; Hansford, Karl A; Fairlie, David P; Shiels, Ian A; Taylor, Stephen M

    2003-01-01

    A new group IIa sPLA2 inhibitor was compared with selective inhibitors of COX-1, COX-2 and an LTC4 antagonist for effects on local and remote tissue injuries following ischaemia and reperfusion (I/R) of the small intestine in rats. In an acute model of ischaemia (30 min) and reperfusion (150 min) injury in the absence of inhibitors, there was significant intestinal haemorrhage, oedema and mucosal damage, neutropenia, elevated serum levels of aspartate aminotransferase (AST) and hypotension. Preischaemic treatment with the inhibitor of sPLA2 (Group IIa), at 5 mg kg−1 i.v. or 10 mg kg−1 p.o. significantly inhibited I/R-induced neutropenia, the elevation of serum levels of AST, intestinal oedema and hypotension. Pretreatment with the COX-2 inhibitor celebrex (10 mg kg−1 i.v.) and the LTC4 antagonist zafirlukast (1 mg kg−1 i.v.) also showed marked improvement with I/R-induced AST, oedema and neutropenia. Hypotension was only reduced by the LTC4 antagonist. The COX-1 inhibitor flunixin (1 mg kg−1 i.v.) did not effect improvement in the markers of tissue injury. Histological examination of rat I/R injury showed that all of the drugs offered some protection to the mucosal layer damage compared to no drug treatment. Given i.v., the sPLA2 inhibitor was more effective than either the COX-1 or COX-2 inhibitors in preventing rat I/R injury. These results indicate that a potent new inhibitor of sPLA2 (group IIa) protects the rat small intestine from I/R injury after oral or intravenous administration. COX-2 and LTC4 inhibitors also showed some beneficial effects against intestinal I/R injury. Our study suggests that sPLA2 (Group IIa) may have a pathogenic role in intestinal I/R in rats. PMID:12967936

  18. Inhibition of A2A Adenosine Receptor Signaling in Cancer Cells Proliferation by the Novel Antagonist TP455

    Directory of Open Access Journals (Sweden)

    Stefania Gessi

    2017-12-01

    Full Text Available Several evidences indicate that the ubiquitous nucleoside adenosine, acting through A1, A2A, A2B, and A3 receptor (AR subtypes, plays crucial roles in tumor development. Adenosine has contrasting effects on cell proliferation depending on the engagement of different receptor subtypes in various tumors. The involvement of A2AARs in human A375 melanoma, as well as in human A549 lung and rat MRMT1 breast carcinoma proliferation has been evaluated in view of the availability of a novel A2AAR antagonist, with high affinity and selectivity, named as 2-(2-furanyl-N5-(2-methoxybenzyl[1,3]thiazolo[5,4-d]pyrimidine-5,7-diammine (TP455. Specifically, the signaling pathways triggered in the cancer cells of different origin and the antagonist effect of TP455 were investigated. The A2AAR protein expression was evaluated through receptor binding assays. Furthermore, the effect of A2AAR activation on cell proliferation at 24, 48 and 72 hours was studied. The selective A2AAR agonist 2-p-(2-carboxyethylphenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride (CGS21680, concentration-dependently induced cell proliferation in A375, A549, and MRMT1 cancer cells and the effect was potently antagonized by the A2AAR antagonist TP455, as well as by the reference A2AAR blocker 4-(2-[7-amino-2-(2-furyl[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethylphenol (ZM241385. As for the signaling pathway recruited in this response we demonstrated that, by using the specific inhibitors of signal transduction pathways, the effect of A2AAR stimulation was induced through phospholipase C (PLC and protein kinase C-delta (PKC-δ. In addition, we evaluated, through the AlphaScreen SureFire phospho(p protein assay, the kinases enrolled by A2AAR to stimulate cell proliferation and we found the involvement of protein kinase B (AKT, extracellular regulated kinases (ERK1/2, and c-Jun N-terminal kinases (JNKs. Indeed, we demonstrated that the CGS21680 stimulatory effect on kinases was

  19. The Rice High-Affinity K+ Transporter OsHKT2;4 Mediates Mg2+ Homeostasis under High-Mg2+ Conditions in Transgenic Arabidopsis

    Directory of Open Access Journals (Sweden)

    Chi Zhang

    2017-10-01

    Full Text Available Rice (Oryza sativa; background Nipponbare contains nine HKT (high-affinity K+ transport-like genes encoding membrane proteins belonging to the superfamily of Ktr/TRK/HKT. OsHKTs have been proposed to include four selectivity filter-pore-forming domains homologous to the bacterial K+ channel KcsA, and are separated into OsHKT1s with Na+-selective activity and OsHKT2s with Na+-K+ symport activity. As a member of the OsHKT2 subfamily, OsHKT2;4 renders Mg2+ and Ca2+ permeability for yeast cells and Xenopus laevis oocytes, besides K+ and Na+. However, physiological functions related to Mg2+in planta have not yet been identified. Here we report that OsHKT2;4 from rice (O. sativa; background Nipponbare functions as a low-affinity Mg2+ transporter to mediate Mg2+ homeostasis in plants under high-Mg2+ environments. Using the functional complementation assay in Mg2+-uptake deficient Salmonella typhimurium strains MM281 and electrophysiological analysis in X. laevis oocytes, we found that OsHKT2;4 could rescue the growth of MM281 in Mg2+-deficient conditions and induced the Mg2+ currents in oocytes at millimolar range of Mg2+. Additionally, overexpression of OsHKT2;4 to Arabidopsis mutant lines with a knockout of AtMGT6, a gene encoding the transporter protein necessary for Mg2+ adaptation in Arabidopsis, caused the Mg2+ toxicity to the leaves under the high-Mg2+ stress, but not under low-Mg2+ environments. Moreover, this Mg2+ toxicity symptom resulted from the excessive Mg2+ translocation from roots to shoots, and was relieved by the increase in supplemental Ca2+. Together, our results demonstrated that OsHKT2;4 is a low-affinity Mg2+ transporter responsible for Mg2+ transport to aerials in plants under high-Mg2+ conditions.

  20. Design, synthesis and in vitro pharmacology of GluK1 and GluK3 antagonists. Studies towards the design of subtype-selective antagonists through 2-carboxyethyl-phenylalanines with substituents interacting with non-conserved residues in the GluK binding sites

    DEFF Research Database (Denmark)

    Sköld, Niklas; Nielsen, Birgitte; Olsen, Jakob

    2014-01-01

    In order to identify compounds selective for the GluK1 and GluK3 subtypes of kainate receptors we have designed and synthesized a series of (S)-2-amino-3-((2-carboxyethyl)phenyl)propanoic acid analogs with hydrogen bond donating and accepting substituents on the aromatic ring. Based on crystal st......-substituted urea (compound 31) showed some preference for GluK3 over GluK1-receptors. Docking studies indicate that this preference may be attributed to contacts between the NH of the urea substituent and non-conserved Ser741 and Ser761 residues.......In order to identify compounds selective for the GluK1 and GluK3 subtypes of kainate receptors we have designed and synthesized a series of (S)-2-amino-3-((2-carboxyethyl)phenyl)propanoic acid analogs with hydrogen bond donating and accepting substituents on the aromatic ring. Based on crystal...... structures of GluK1 in complex with related ligands, the compounds were designed to explore possible interactions with non-conserved residues outside the glutamate ligand binding site and challenge the water binding network. Apart from obtaining GluK1 selective antagonists one analog with a phenyl...

  1. Rupatadine: a new selective histamine H1 receptor and platelet-activating factor (PAF) antagonist. A review of pharmacological profile and clinical management of allergic rhinitis.

    Science.gov (United States)

    Izquierdo, Iñaki; Merlos, Manuel; García-Rafanell, Julián

    2003-06-01

    Rupatadine is a new agent for the management of diseases with allergic inflammatory conditions, such as seasonal and perennial rhinitis. The pharmacological profile of rupatadine offers particular benefits in terms of a strong antagonist activity towards both histamine H1 receptors and platelet-activating factor (PAF) receptors. Rupatadine has a rapid onset of action, and its long-lasting effect (>24 h) permits once-daily dosing. Rupatadine should not be used in combination with the cytochrome P450 inhibitors, such as erythromycin or ketoconazole, due to an increase in AUC and Cmax for rupatadine, although no clinically relevant adverse events have been reported. In addition, rupatadine, at the recommended dose of 10 mg, has been shown to be free of sedative effects and not to cause significant changes in the corrected QT interval in special populations, including the elderly, nor when coadministered with erythromycin or ketoconazole. Preclinical data have also shown that rupatadine and its main active metabolites did not interfere with cloned human HERG channel and did not affect in vitro isolated dog Purkinje fibers at concentrations at least 2000 times greater than those obtained with therapeutic doses in humans. Rupatadine is clinically effective in relieving symptoms in patients with seasonal and perennial allergic rhinitis. Newly published data on its efficacy and safety suggest that this compound may improve the nasal and non-nasal symptoms in comparison to other currently available second generation H1 receptor antihistamines. 2003 Prous Science. All rights reserved.

  2. EMA401: an old antagonist of the AT2R for a new indication in neuropathic pain

    Directory of Open Access Journals (Sweden)

    Keppel Hesselink JM

    2017-02-01

    Full Text Available Jan M Keppel Hesselink,1 Michael E Schatman2 1Institute of Neuropathic Pain, Bosch en Duin, the Netherlands; 2Department of Public Health and Community Medicine, Tufts University School of Medicine, Boston, MA, USA Abstract: EMA401 is an old molecule, synthesized by Parke-Davis in the last century and characterized at that time as an AT2R antagonist. Professor Maree Smith and her group from the University of Queensland (Australia patented the drug and many related derivatives and other compounds with high affinity for the AT2R for the indication neuropathic pain in 2004, an example of drug repositioning. After some years of university work, the Australian biotech company Spinifex Pharmaceuticals took over further research and development and characterized the S-enantiomer, code name EMA401, and related compounds in a variety of animal models for neuropathic and cancer pain. EMA401 was selected as the lead compound, based on high selectivity for the AT2R and good oral bioavailability (33%. EMA401, however, was only administered once in a chronic neuropathic pain model, and no data have been published in other pain models, or during steady state, although such data were available for the racemate EMA400 and some related compounds (EMA200, EMA400. A pilot phase IIa study demonstrated the efficacy and safety of the drug taken twice daily as two capsules of 50 mg (400 mg/day. In 2015, Novartis took over the clinical development. Two phase IIb studies designed by Spinifex Pharmaceuticals were put on hold, probably because Novartis wanted to improve the clinical design or collect additional preclinical data. Further data are eagerly awaited, especially since EMA401 is first-in-class in neuropathic pain. Keywords: angiotensin II type 2 receptor, antagonist, neuropathic pain, EMA401, novel, development, drug repositioning 

  3. {sup 99m}Tc(CO){sub 3}-DTMA bombesin conjugates having high affinity for the GRP receptor

    Energy Technology Data Exchange (ETDEWEB)

    Lane, Stephanie R. [Research Division, Harry S. Truman Memorial Veterans' Hospital, Columbia, MO 65201 (United States); Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States); Veerendra, Bhadrasetty [Department of Radiology, University of Missouri-Columbia School of Medicine, Columbia, MO 65211 (United States); Rold, Tammy L. [Department of Internal Medicine, University of Missouri-Columbia School of Medicine, Columbia, MO 65211 (United States); Sieckman, Gary L. [Research Division, Harry S. Truman Memorial Veterans' Hospital, Columbia, MO 65201 (United States); Hoffman, Timothy J. [Research Division, Harry S. Truman Memorial Veterans' Hospital, Columbia, MO 65201 (United States); Radiopharmaceutical Sciences Institute, University of Missouri-Columbia School of Medicine, Columbia, MO 65211 (United States); Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States); Jurisson, Silvia S. [Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States); Smith, Charles J. [Department of Radiology, University of Missouri-Columbia School of Medicine, Columbia, MO 65211 (United States); Research Division, Harry S. Truman Memorial Veterans' Hospital, Columbia, MO 65201 (United States); University of Missouri Research Reactor Center, University of Missouri-Columbia, Columbia, MO 65211 (United States); Radiopharmaceutical Sciences Institute, University of Missouri-Columbia School of Medicine, Columbia, MO 65211 (United States)], E-mail: smithcj@health.missouri.edu

    2008-04-15

    Introduction: Targeted diagnosis of specific human cancer types continues to be of significant interest in nuclear medicine. {sup 99m}Tc is ideally suited as a diagnostic radiometal for in vivo tumor targeting due to its ideal physical characteristics and diverse labeling chemistries in numerous oxidation states. Methods: In this study, we report a synthetic approach toward design of a new tridentate amine ligand for the organometallic aqua-ion [{sup 99m}Tc(H{sub 2}O){sub 3}(CO){sub 3}]{sup +}. The new chelating ligand framework, 2-(N,N'-Bis(tert-butoxycarbonyl)diethylenetriamine) acetic acid (DTMA), was synthesized from a diethylenetriamine precursor and fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy ({sup 1}H and {sup 13}C). DTMA was conjugated to H{sub 2}N-(X)-BBN(7-14)NH{sub 2}, where X=an amino acid or aliphatic pharmacokinetic modifier and BBN=bombesin peptide, by means of solid phase peptide synthesis. DTMA-(X)-BBN(7-14)NH{sub 2} conjugates were purified by reversed-phase high-performance chromatography and characterized by electrospray-ionization mass spectrometry. Results: The new conjugates were radiolabeled with [{sup 99m}Tc(H{sub 2}O){sub 3}(CO){sub 3}]{sup +} produced via Isolink radiolabeling kits to produce [{sup 99m}Tc(CO){sub 3}-DTMA-(X)-BBN(7-14)NH{sub 2}]. Radiolabeled conjugates were purified by reversed-phase high-performance chromatography. Effective receptor binding behavior was evaluated in vitro and in vivo. Conclusions: [{sup 99m}Tc(CO){sub 3}-DTMA-(X)-BBN(7-14)NH{sub 2}] conjugates displayed very high affinity for the gastrin releasing peptide receptor in vitro and in vivo. Therefore, these conjugates hold some propensity to be investigated as molecular imaging agents that specifically target human cancers uniquely expressing the gastrin releasing peptide receptor subtypes.

  4. A high affinity conformational state on VLA integrin heterodimers induced by an anti-beta 1 chain monoclonal antibody.

    Science.gov (United States)

    Arroyo, A G; García-Pardo, A; Sánchez-Madrid, F

    1993-05-05

    The VLA integrin subfamily includes receptors for extracellular matrix proteins as well as receptors involved in cell-cell adhesive interactions. We have previously described the up-regulation of VLA integrin-mediated cell attachment to different ligands by the anti-beta 1 TS2/16 monoclonal antibody (mAb) (Arroyo, A. G., Sánchez-Mateos, P., Campanero, M. R., Martín-Padura, I., Dejana, E., and Sánchez-Madrid, F. (1992) J. Cell Biol. 117, 659-670). In this report, we have investigated the mechanism involved in this regulatory effect. The anti-beta 1-mediated regulatory effect on cell adhesion did not require "de novo" protein synthesis, since it was not affected by pretreatment with either cycloheximide or actinomycin D. To quantitate the effect of the regulatory anti-beta 1 TS2/16 mAb on the affinity of VLA-5 for its ligand, an RGD-containing fragment of fibronectin (FN80), we performed binding studies of radiolabeled soluble FN80 to U-937 cells. The affinity of VLA-5 for FN80 was enhanced about 4-fold in the presence of TS2/16 mAb (Kd = 0.98 +/- 0.07 microM) compared to the functionally irrelevant anti-beta 1 Alex 1/4 mAb (Kd = 4.23 +/- 0.92 microM), whereas no alteration in the number of binding sites was observed. Indeed, the anti-beta 1 TS2/16 mAb is inducing this high affinity state on VLA heterodimers by a direct change on the conformation of these receptors as demonstrated by affinity chromatography analysis using extracellular matrix proteins covalently bound to Sepharose. The yield of VLA-5 fibronectin receptor bound to FN80-Sepharose columns was strongly increased upon treatment of U-937 cell lysates with mAb TS2/16. Moreover, higher concentrations of EDTA were required for eluting the VLA-5 integrin from this matrix. This up-regulatory effect was also observed with F(ab')2 and Fab fragments of the anti-beta 1 TS2/16 mAb, and was also exerted on the purified VLA-5 receptor. Similarly, the yield of VLA-2 retained on a collagen I-Sepharose column was

  5. Dopamine D2 receptor radiotracers [{sup 11}C](+)-PHNO and [{sup 3}H]raclopride are indistinguishably inhibited by D2 agonists and antagonists ex vivo

    Energy Technology Data Exchange (ETDEWEB)

    McCormick, Patrick N. [Institute of Medical Science, University of Toronto, Toronto, Ontario, M5S 1A8 (Canada)], E-mail: patrick.mccormick@camhpet.ca; Kapur, Shitij [Department of Psychiatry, University of Toronto, Toronto, Ontario, M5S 1A8 (Canada); PET Center, Center for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Seeman, Philip [Department of Psychiatry, University of Toronto, Toronto, Ontario, M5S 1A8 (Canada); Department of Pharmacology, University of Toronto, Toronto, Ontario, M5S 1A8 (Canada); Wilson, Alan A. [Department of Psychiatry, University of Toronto, Toronto, Ontario, M5S 1A8 (Canada); PET Center, Center for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada)

    2008-01-15

    Introduction: In vitro, the dopamine D2 receptor exists in two states, with high and low affinity for agonists. The high-affinity state is the physiologically active state thought to be involved in dopaminergic illnesses such as schizophrenia. The positron emission tomography radiotracer [{sup 11}C](+)-PHNO ([{sup 11}C](+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4] oxazin-9-o l), being a D2 agonist, should selectively label the high-affinity state at tracer dose and therefore be more susceptible to competition by agonist as compared to the antagonist [{sup 3}H]raclopride, which binds to both affinity states. Methods: We tested this prediction using ex vivo dual-radiotracer experiments in conscious rats. D2 antagonists (haloperidol or clozapine), a partial agonist (aripiprazole), a full agonist [(-)-NPA] or the dopamine-releasing drug amphetamine (AMPH) were administered to rats prior to an intravenous coinjection of [{sup 11}C](+)-PHNO and [{sup 3}H]raclopride. Rats were sacrificed 60 min after radiotracer injection. Striatum, cerebellum and plasma samples were counted for {sup 11}C and {sup 3}H. The specific binding ratio {l_brace}SBR, i.e., [%ID/g (striatum)-%ID/g (cerebellum)]/(%ID/g (cerebellum){r_brace} was used as the outcome measure. Results: In response to D2 antagonists, partial agonist or full agonist, [{sup 11}C](+)-PHNO and [{sup 3}H]raclopride SBRs responded indistinguishably in terms of both ED{sub 50} and Hill slope (e.g., (-)-NPA ED{sub 50} values are 0.027 and 0.023 mg/kg for [{sup 11}C](+)-PHNO and [{sup 3}H]raclopride, respectively). In response to AMPH challenge, [{sup 11}C](+)-PHNO and [{sup 3}H]raclopride SBRs were inhibited to the same degree. Conclusions: We have shown that the SBRs of [{sup 11}C](+)-PHNO- and [{sup 3}H]raclopride do not differ in their response to agonist challenge. These results do not support predictions of the in vivo binding behavior of a D2 agonist radiotracer and cast some doubt on the in vivo

  6. Trimeric gp120-specific bovine monoclonal antibodies require cysteine and aromatic residues in CDRH3 for high affinity binding to HIV Env.

    Science.gov (United States)

    Heydarchi, Behnaz; Center, Rob J; Bebbington, Jonathan; Cuthbertson, Jack; Gonelli, Christopher; Khoury, Georges; Mackenzie, Charlene; Lichtfuss, Marit; Rawlin, Grant; Muller, Brian; Purcell, Damian

    2017-04-01

    We isolated HIV-1 Envelope (Env)-specific memory B cells from a cow that had developed high titer polyclonal immunoglobulin G (IgG) with broad neutralizing activity after a long duration vaccination with HIV-1AD8 Env gp140 trimers. We cloned the bovine IgG matched heavy (H) and light (L) chain variable (V) genes from these memory B cells and constructed IgG monoclonal antibodies (mAbs) with either a human constant (C)-region/bovine V-region chimeric or fully bovine C and V regions. Among 42 selected Ig+ memory B cells, two mAbs (6A and 8C) showed high affinity binding to gp140 Env. Characterization of both the fully bovine and human chimeric isoforms of these two mAbs revealed them as highly type-specific and capable of binding only to soluble AD8 uncleaved gp140 trimers and covalently stabilized AD8 SOSIP gp140 cleaved trimers, but not monomeric gp120. Genomic sequence analysis of the V genes showed the third heavy complementarity-determining region (CDRH3) of 6A mAb was 21 amino acids in length while 8C CDRH3 was 14 amino acids long. The entire V heavy (VH) region was 27% and 25% diverged for 6A and 8C, respectively, from the best matched germline V genes available, and the CDRH3 regions of 6A and 8C were 47.62% and 78.57% somatically mutated, respectively, suggesting a high level of somatic hypermutation compared with CDRH3 of other species. Alanine mutagenesis of the VH genes of 6A and 8C, showed that CDRH3 cysteine and tryptophan amino acids were crucial for antigen binding. Therefore, these bovine vaccine-induced anti-HIV antibodies shared some of the notable structural features of elite human broadly neutralizing antibodies, such as CDRH3 size and somatic mutation during affinity-maturation. However, while the 6A and 8C mAbs inhibited soluble CD4 binding to gp140 Env, they did not recapitulate the neutralizing activity of the polyclonal antibodies against HIV infection.

  7. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

    OpenAIRE

    Naiwen Cui; Huidan Zhang; Nils Schneider; Ye Tao; Haruichi Asahara; Zhiyi Sun; Yamei Cai; Koehler, Stephan A.; de Greef, Tom F. A.; Alireza Abbaspourrad; Weitz, David A; Shaorong Chong

    2016-01-01

    Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-...

  8. Zinc deficiency up-regulates expression of high-affinity phosphate transporter genes in both phosphate-sufficient and -deficient barley roots.

    Science.gov (United States)

    Huang, C; Barker, S J; Langridge, P; Smith, F W; Graham, R D

    2000-09-01

    Phosphate (P) is taken up by plants through high-affinity P transporter proteins embedded in the plasma membrane of certain cell types in plant roots. Expression of the genes that encode these transporters responds to the P status of the plants, and their transcription is normally tightly controlled. However, this tight control of P uptake is lost under Zn deficiency, leading to very high accumulation of P in plants. We examined the effect of plant Zn status on the expression of the genes encoding the HVPT1 and HVPT2 high-affinity P transporters in barley (Hordeum vulgare L. cv Weeah) roots. The results show that the expression of these genes is intimately linked to the Zn status of the plants. Zn deficiency induced the expression of genes encoding these P transporters in plants grown in either P-sufficient or -deficient conditions. Moreover, the role of Zn in the regulation of these genes is specific in that it cannot be replaced by manganese (a divalent cation similar to Zn). It appears that Zn plays a specific role in the signal transduction pathway responsible for the regulation of genes encoding high-affinity P transporters in plant roots. The significance of Zn involvement in the regulation of genes involved in P uptake is discussed.

  9. A fluorescent protein scaffold for presenting structurally constrained peptides provides an effective screening system to identify high affinity target-binding peptides.

    Directory of Open Access Journals (Sweden)

    Tetsuya Kadonosono

    Full Text Available Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131-L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides.

  10. A fluorescent protein scaffold for presenting structurally constrained peptides provides an effective screening system to identify high affinity target-binding peptides.

    Science.gov (United States)

    Kadonosono, Tetsuya; Yabe, Etsuri; Furuta, Tadaomi; Yamano, Akihiro; Tsubaki, Takuya; Sekine, Takuya; Kuchimaru, Takahiro; Sakurai, Minoru; Kizaka-Kondoh, Shinae

    2014-01-01

    Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131-L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2)-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides.

  11. Opioid Antagonist Impedes Exposure.

    Science.gov (United States)

    Merluzzi, Thomas V.; And Others

    1991-01-01

    Thirty spider-phobic adults underwent exposure to 17 phobic-related, graded performance tests. Fifteen subjects were assigned to naltrexone, an opioid antagonist, and 15 were assigned to placebo. Naltrexone had a significant effect on exposure, with naltrexone subjects taking significantly longer to complete first 10 steps of exposure and with…

  12. Non-Selective Cannabinoid Receptor Antagonists, Hinokiresinols Reduce Infiltration of Microglia/Macrophages into Ischemic Brain Lesions in Rat via Modulating 2-Arachidonolyglycerol-Induced Migration and Mitochondrial Activity.

    Directory of Open Access Journals (Sweden)

    Angela M A Anthony Jalin

    Full Text Available Growing evidence suggests that therapeutic strategies to modulate the post-ischemic inflammatory responses are promising approaches to improve stroke outcome. Although the endocannabinoid system has been emerged as an endogenous therapeutic target to regulate inflammation after stroke insult, the downstream mechanisms and their potentials for therapeutic intervention remain controversial. Here we identified trans- and cis-hinokiresinols as novel non-selective antagonists for two G-protein-coupled cannabinoid receptors, cannabinoid receptor type 1 and type 2. The Electric Cell-substrate Impedance Sensing and Boyden chamber migration assays using primary microglial cultures revealed that both hinokiresinols significantly inhibited an endocannabinoid, 2-arachidonoylglycerol-induced migration. Hinokiresinols modulated 2-arachidonoylglycerol-induced mitochondrial bioenergetics in microglia as evidenced by inhibition of ATP turnover and reduction in respiratory capacity, thereby resulting in impaired migration activity. In rats subjected to transient middle cerebral artery occlusion (1.5-h followed by 24-h reperfusion, post-ischemic treatment with hinokiresinols (2 and 7-h after the onset of ischemia, 10 mg/kg significantly reduced cerebral infarct and infiltration of ED1-positive microglial/macrophage cells into cerebral ischemic lesions in vivo. Co-administration of exogenous 2-AG (1 mg/kg, i.v., single dose at 2 h after starting MCAO abolished the protective effect of trans-hinokiresionol. These results suggest that hinokiresinols may serve as stroke treatment by targeting the endocannabinoid system. Alteration of mitochondrial bioenergetics and consequent inhibition of inflammatory cells migration may be a novel mechanism underlying anti-ischemic effects conferred by cannabinoid receptor antagonists.

  13. Seleção de antagonistas fúngicos a Fusarium solani e Fusarium oxysporum em substrato comercial para mudas Selection of fungi antagonistic to Fusarium solani and Fusarium oxysporum in commercial substrate for seedlings

    Directory of Open Access Journals (Sweden)

    Luciana Zago Ethur

    2007-12-01

    Full Text Available Testes in vitro são geralmente utilizados para a seleção inicial de agentes de biocontrole contra fungos de solo, faltando metodologias que utilizem solo e/ou substrato. O objetivo deste trabalho foi realizar a seleção massal de isolados fúngicos antagônicos a F. solani e F. oxysporum em substrato comercial para mudas. Foram realizados dois experimentos com os patógenos F. solani e F. oxysporum e com 98 possíveis antagonistas fúngicos, dos gêneros Penicillium claviforme, Penicillium, Aspergillus e Cladosporium. A suspensão dos patógenos foi inserida no substrato, em copos plásticos, sendo acrescentada, cinco dias depois, a suspensão dos demais fungos. Avaliou-se o número de unidades formadoras de colônia de F. solani e F. oxysporum por grama de substrato após nove dias. Dos 98 isolados utilizados contra F. solani, 43 % não diferiram da testemunha e 57% reduziram o seu desenvolvimento em substrato, sendo que os três melhores isolados fúngicos foram do gênero Penicillium claviforme. Os três isolados de Penicillium claviforme selecionados para F. solani também foram eficientes para F. oxysporum.Tests in vitro are usually used for the initial selection of biocontrol agents against soil fungi, lacking methodologies using soil and/or substrate. The objective of this research was to accomplish the mass selection of fungi isolates antagonistic to F. solani and F. oxysporum in commercial substrate for seedlings. Two experiments were conducted, with the pathogens F. solani and F. oxysporum, and 98 possible antagonistic fungi of the genera Penicillium claviforme, Penicillium, Aspergillus and Cladosporium. The suspension of the pathogens was inoculated in the substrate, in plastic cups, and the suspension of the other fungi was added five days later. The number of colony-forming unit of F. solani and F. oxysporum/g of substrate was counted after nine days. Of the 98 isolates used against F. solani, 43% did not differ from the control

  14. Evaluation of (Z)-2-((1-benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one analogues as novel, high affinity ligands for CB1 and CB2 cannabinoid receptors.

    Science.gov (United States)

    Madadi, Nikhil Reddy; Penthala, Narsimha Reddy; Brents, Lisa K; Ford, Benjamin M; Prather, Paul L; Crooks, Peter A

    2013-04-01

    A small library of N-benzyl indolequinuclidinone (IQD) analogs has been identified as a novel class of cannabinoid ligands. The affinity and selectivity of these IQDs for the two established cannabinoid receptor subtypes, CB1 and CB2, was evaluated. Compounds 8 (R=R(2)=H, R(1)=F) and 13 (R=COOCH3, R(1)=R(2)=H) exhibited high affinity for CB2 receptors with Ki values of 1.33 and 2.50 nM, respectively, and had lower affinities for the CB1 receptor (Ki values of 9.23 and 85.7 nM, respectively). Compound 13 had the highest selectivity of all the compounds examined, and represents a potent cannabinoid ligand with 34-times greater selectivity for CB2R over CB1R. These findings are significant for future drug development, given recent reports demonstrating beneficial use of cannabinoid ligands in a wide variety of human disease states including drug abuse, depression, schizophrenia, inflammation, chronic pain, obesity, osteoporosis and cancer. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

    Directory of Open Access Journals (Sweden)

    Rui Liu

    2016-06-01

    Full Text Available Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples.

  16. The High-Affinity Phosphate Transporter GmPT5 Regulates Phosphate Transport to Nodules and Nodulation in Soybean1[W][OA

    Science.gov (United States)

    Qin, Lu; Zhao, Jing; Tian, Jiang; Chen, Liyu; Sun, Zhaoan; Guo, Yongxiang; Lu, Xing; Gu, Mian; Xu, Guohua; Liao, Hong

    2012-01-01

    Legume biological nitrogen (N) fixation is the most important N source in agroecosystems, but it is also a process requiring a considerable amount of phosphorus (P). Therefore, developing legume varieties with effective N2 fixation under P-limited conditions could have profound significance for improving agricultural sustainability. We show here that inoculation with effective rhizobial strains enhanced soybean (Glycine max) N2 fixation and P nutrition in the field as well as in hydroponics. Furthermore, we identified and characterized a nodule high-affinity phosphate (Pi) transporter gene, GmPT5, whose expression was elevated in response to low P. Yeast heterologous expression verified that GmPT5 was indeed a high-affinity Pi transporter. Localization of GmPT5 expression based on β-glucuronidase staining in soybean composite plants with transgenic roots and nodules showed that GmPT5 expression occurred principally in the junction area between roots and young nodules and in the nodule vascular bundles for juvenile and mature nodules, implying that GmPT5 might function in transporting Pi from the root vascular system into nodules. Overexpression or knockdown of GmPT5 in transgenic composite soybean plants altered nodulation and plant growth performance, which was partially dependent on P supply. Through both in situ and in vitro 33P uptake assays using transgenic soybean roots and nodules, we demonstrated that GmPT5 mainly functions in transporting Pi from roots to nodules, especially under P-limited conditions. We conclude that the high-affinity Pi transporter, GmPT5, controls Pi entry from roots to nodules, is critical for maintaining Pi homeostasis in nodules, and subsequently regulates soybean nodulation and growth performance. PMID:22740613

  17. Sequence specific and high affinity recognition of 5′-ACGCGT-3′ by rationally designed pyrrole-imidazole H-pin polyamides: Thermodynamic and structural studies

    Science.gov (United States)

    Mackay, Hilary; Brown, Toni; Uthe, Peter B.; Westrate, Laura; Sielaff, Alan; Jones, Justin; Lajiness, James P.; Kluza, Jerome; O’Hare, Caroline; Nguyen, Binh; Davis, Zach; Bruce, Chrystal; Wilson, W. David; Hartley, John A.; Lee, Moses

    2013-01-01

    in general agreement with ΔCp values determined from changes in the solvent accessible surface areas using complexes of the H-pins bound to (5′-CCACGCGTGG)2. According to the models, the H-pins fit snugly in the minor groove and the linker comfortably holds both polyamide portions in place, with the oxygen atoms pointing into the solvent. In summary, the H-pin polyamide provides an important molecular design motif for the discovery of future generations of programmable small molecules capable of binding to target DNA sequences with high affinity and selectivity. PMID:18819814

  18. Sequence specific and high affinity recognition of 5'-ACGCGT-3' by rationally designed pyrrole-imidazole H-pin polyamides: thermodynamic and structural studies.

    Science.gov (United States)

    Mackay, Hilary; Brown, Toni; Uthe, Peter B; Westrate, Laura; Sielaff, Alan; Jones, Justin; Lajiness, James P; Kluza, Jerome; O'Hare, Caroline; Nguyen, Binh; Davis, Zach; Bruce, Chrystal; Wilson, W David; Hartley, John A; Lee, Moses

    2008-10-15

    agreement with DeltaC(p) values determined from changes in the solvent accessible surface areas using complexes of the H-pins bound to (5'-CCACGCGTGG)(2). According to the models, the H-pins fit snugly in the minor groove and the linker comfortably holds both polyamide portions in place, with the oxygen atoms pointing into the solvent. In summary, the H-pin polyamide provides an important molecular design motif for the discovery of future generations of programmable small molecules capable of binding to target DNA sequences with high affinity and selectivity.

  19. A Mixed Glucocorticoid/Mineralocorticoid Selective Modulator With Dominant Antagonism in the Male Rat Brain.

    Science.gov (United States)

    Atucha, Erika; Zalachoras, Ioannis; van den Heuvel, José K; van Weert, Lisa T C M; Melchers, Diana; Mol, Isabel M; Belanoff, Joseph K; Houtman, René; Hunt, Hazel; Roozendaal, Benno; Meijer, Onno C

    2015-11-01

    Adrenal glucocorticoid hormones are potent modulators of brain function in the context of acute and chronic stress. Both mineralocorticoid (MRs) and glucocorticoid receptors (GRs) can mediate these effects. We studied the brain effects of a novel ligand, C118335, with high affinity for GRs and modest affinity for MRs. In vitro profiling of receptor-coregulator interactions suggested that the compound is a "selective modulator" type compound for GRs that can have both agonistic and antagonistic effects. Its molecular profile for MRs was highly similar to those of the full antagonists spironolactone and eplerenone. C118335 showed predominantly antagonistic effects on hippocampal mRNA regulation of known glucocorticoid target genes. Likewise, systemic administration of C118335 blocked the GR-mediated posttraining corticosterone-induced enhancement of memory consolidation in an inhibitory avoidance task. Posttraining administration of C118335, however, gave a strong and dose-dependent impairment of memory consolidation that, surprisingly, reflected involvement of MRs and not GRs. Finally, C118335 treatment acutely suppressed the hypothalamus-pituitary-adrenal axis as measured by plasma corticosterone levels. Mixed GR/MR ligands, such as C118335, can be used to unravel the mechanisms of glucocorticoid signaling. The compound is also a prototype of mixed GR/MR ligands that might alleviate the harmful effects of chronic overexposure to endogenous glucocorticoids.

  20. Discerning the Role of the Hydroxyproline Residue in the Structure of Conantokin Rl-B and Its Role in GluN2B Subunit-Selective Antagonistic Activity toward N-Methyl-d-Aspartate Receptors.

    Science.gov (United States)

    Yuan, Yue; Balsara, Rashna D; Zajicek, Jaroslav; Kunda, Shailaja; Castellino, Francis J

    2016-12-27

    Conantokins (con) are short γ-carboxyglutamate (Gla)-containing polypeptides expressed by marine snails that function as antagonists of N-methyl-d-aspartate receptor (NMDAR) ion channels. The Gla residues govern structural conformations and antagonistic activities of the conantokins. In addition to Gla, some conantokins, e.g., conRl-B, also contain a hydroxyproline (HyP or O) residue, which in this case is centrally located in the peptide at position 10. Because conRl-B specifically inhibits ion channels of GluN2B subunit-containing heterotetrameric NMDARs, we evaluated the unusual role of HyP(10) in this effect. To accomplish this goal, we examined synthetic variants of conRl-B in which HyP(10) was either deleted (conRl-B[ΔO(10)]) or replaced with alanine (conRl-B[O(10)A]) or proline (conRl-B[O(10)P]). The solution structures of these variants were determined by nuclear magnetic resonance spectroscopy. Deletion of HyP(10), or replacement of HyP(10) with Ala(10), attenuated the distortion in the central region of the apo-conRl-B helix and allowed Mg(2+)-complexed end-to-end α-helix formation. The inhibitory properties of these variants were assessed by measuring NMDA/Gly-stimulated intracellular Ca(2+) influx in mice neurons. ConRl-B[O(10)P] retained its NMDAR ion channel inhibitory activity in wild-type (WT) neurons but lost its GluN2B specificity, whereas conRl-B[ΔO(10)] showed overall diminished inhibitory function. ConRl-B[O(10)A] showed attenuated inhibitory function but retained its GluN2B specificity. Thus, HyP(10) plays a critical role in maintaining the structural integrity of conRl-B, which can be correlated with its GluN2B subunit-selective inhibition. Weakened inhibition by conRl-B was also observed in neurons lacking either the GluN2C or GluN2D subunit, compared to WT neurons. This suggests that GluN2C and GluN2D are also required for inhibition by conRl-B.

  1. High-affinity cooperative Ca2+binding by MICU1-MICU2 serves as an on-off switch for the uniporter.

    Science.gov (United States)

    Kamer, Kimberli J; Grabarek, Zenon; Mootha, Vamsi K

    2017-08-01

    The mitochondrial calcium uniporter is a Ca 2+ -activated Ca 2+ channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca 2+ signals. It is regulated by two paralogous EF-hand proteins-MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca 2+ We reconstitute the MICU1-MICU2 heterodimer and demonstrate that it binds Ca 2+ cooperatively with high affinity. We discover that both MICU1 and MICU2 exhibit affinity for the mitochondria-specific lipid cardiolipin. We determine the minimum Ca 2+ concentration required for disinhibition of the uniporter in permeabilized cells and report a close match with the Ca 2+ -binding affinity of MICU1-MICU2. We conclude that cooperative, high-affinity interaction of the MICU1-MICU2 complex with Ca 2+ serves as an on-off switch, leading to a tightly controlled channel, capable of responding directly to cytosolic Ca 2+ signals. © 2017 The Authors.

  2. In vivo effector functions of high-affinity mouse IgG receptor FcγRI in disease and therapy models.

    Science.gov (United States)

    Gillis, Caitlin M; Zenatti, Priscila P; Mancardi, David A; Beutier, Héloïse; Fiette, Laurence; Macdonald, Lynn E; Murphy, Andrew J; Celli, Susanna; Bousso, Philippe; Jönsson, Friederike; Bruhns, Pierre

    2017-06-01

    Two activating mouse IgG receptors (FcγRs) have the ability to bind monomeric IgG, the high-affinity mouse FcγRI and FcγRIV. Despite high circulating levels of IgG, reports using FcγRI(-/-) or FcγRIV(-/-) mice or FcγRIV-blocking antibodies implicate these receptors in IgG-induced disease severity or therapeutic Ab efficacy. From these studies, however, one cannot conclude on the effector capabilities of a given receptor, because different activating FcγRs possess redundant properties in vivo, and cooperation between FcγRs may occur, or priming phenomena. To help resolve these uncertainties, we used mice expressing only FcγRI to determine its intrinsic properties in vivo. FcγRI(only) mice were sensitive to IgG-induced autoimmune thrombocytopenia and anti-CD20 and anti-tumour immunotherapy, but resistant to IgG-induced autoimmune arthritis, anaphylaxis and airway inflammation. Our results show that the in vivo roles of FcγRI are more restricted than initially reported using FcγRI(-/-) mice, but confirm effector capabilities for this high-affinity IgG receptor in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri.

    Science.gov (United States)

    Nie, Laiyin; Grell, Ernst; Malviya, Viveka Nand; Xie, Hao; Wang, Jingkang; Michel, Hartmut

    2016-07-22

    Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H(+) or Na(+) electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4',6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications

    Science.gov (United States)

    Imaduwage, Kasun P.; Go, Eden P.; Zhu, Zhikai; Desaire, Heather

    2016-11-01

    A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have Ki values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods.

  5. Assessing high affinity binding to HLA-DQ2.5 by a novel peptide library based approach

    DEFF Research Database (Denmark)

    Jüse, Ulrike; Arntzen, Magnus; Højrup, Peter

    2011-01-01

    to HLA are then isolated by size exclusion chromatography and sequenced by tandem mass spectrometry online coupled to liquid chromatography. The MS/MS data are subsequently searched against a library defined database using a search engine such as Mascot, followed by manual inspection of the results. We...... used two dodecamer and two decamer peptide libraries and HLA-DQ2.5 to test possibilities and limits of this method. The selected sequences which we identified in the fraction eluted from HLA-DQ2.5 showed a higher average of their predicted binding affinity values compared to the original peptide...

  6. Antibacterial and antagonistic activity of selected traditional ...

    African Journals Online (AJOL)

    ... east Wollega administrative zone of and were analyzed for their anti-methanol extracts of some medicinal Streptococcus pyogenes, Streptococcus microbial activities. The diethyl ether and plants have inhibited bacterial strains; pneumonia, Streptococcus agalacticeae, Pathogens Antibacterial activity Salmonella tphyrium, ...

  7. N-phenylalkyl-substituted tropane analogs of boat conformation with high selectivity for the dopamine versus serotonin transporter.

    Science.gov (United States)

    Prakash, K R; Tamiz, A P; Araldi, G L; Zhang, M; Johnson, K M; Kozikowski, A P

    1999-12-06

    A series of N-phenylalkyl-substituted tropane analogs of boat conformation was synthesized, and these tropanes were evaluated for their ability to inhibit high affinity uptake of dopamine (DA) and serotonin (5-HT) into striatal nerve endings (synaptosomes). Some of these compounds exhibit high affinity for the DA transporter with a 5-HT/DA transporter selectivity ratio of >50.

  8. NFM Cross-Reactivity to MOG Does Not Expand a Critical Threshold Level of High-Affinity T Cells Necessary for Onset of Demyelinating Disease.

    Science.gov (United States)

    Blanchfield, Lori; Sabatino, Joseph J; Lawrence, Laurel; Evavold, Brian D

    2017-10-15

    Of interest to the etiology of demyelinating autoimmune disease is the potential to aberrantly activate CD4(+) T cells due to cross-recognition of multiple self-epitopes such as has been suggested for myelin oligodendrocyte glycoprotein epitope 35-55 (MOG35-55) and neurofilament medium protein epitope 15-35 (NFM15-35). NFM15-35 is immunogenic in C57BL/6 mice but fails to induce demyelinating disease by polyclonal T cells despite having the same TCR contact residues as MOG35-55, a known encephalitogenic Ag. Despite reported cross-reactivity with MOG-specific T cells, the polyclonal response to NFM15-35 did not expand threshold numbers of MOG38-49 tetramer-positive T cells. Furthermore, NFM lacked functional synergy with MOG to promote experimental autoimmune encephalomyelitis because NFM-deficient synonymous with knockout mice developed an identical disease course to wild-type mice after challenge with MOG35-55 Single-cell analysis of encephalitogenic T cells using the peptide:MHC monomer-based two-dimensional micropipette adhesion frequency assay confirmed that NFM was not a critical Ag driving demyelinating disease because NFM18-30-specific T cells in the CNS were predominantly reactive to MOG38-49 The absence of NFM contribution to disease allowed mapping of the amino acids required for encephalitogenicity and expansion of high-affinity, MOG-specific T cells that defined the polyclonal response. Alterations of N-terminal residues outside of the NFM15-35 core nonamer promoted expansion of high-affinity, MOG38-49 tetramer-positive T cells and promoted consistent experimental autoimmune encephalomyelitis induction, unlike mice challenged with NFM15-35 Although NFM15-35 is immunogenic and cross-reactive with MOG at the polyclonal level, it fails to expand a threshold level of encephalitogenic, high-affinity MOG-specific T cells. Copyright © 2017 by The American Association of Immunologists, Inc.

  9. Comparison of high affinity binding of {sup 3}H-proadifen and {sup 3}H-(-)-cocaine t rat liver membranes

    Energy Technology Data Exchange (ETDEWEB)

    Ross, S.B. [Astra Arcus AB, Dept. of Neuropharmacology, Soedertaelje (Sweden)

    1995-06-01

    The characteristics of the binding of {sup 3}H-proadifen to rat liver membranes were studied and compared to those of {sup 3}H-cocaine. It was found that {sup 3}H-proadifen was bound reversibly with high affinity (K{sub D}=1.8{+-}0.5 nM) and large capacity (B{sub max}=2010{+-}340 pmol/g wet tissue) to liver membranes. The corresponding values for the {sup 3}H-cocaine binding were 3.5 nM and 1000 pmol/g wet tissue. The binding of {sup 3}H-proadifen was mainly localised to the microsomal fraction. The number of binding sites was not increased by treatment of rats with phenobarbitone. With 1 {mu}M CdCl{sub 2} in the incubation buffer it was possible to differentiate between two {sup 3}H-cocaine binding sites with K{sub d} values of 1.6 and 7.7 nM and B{sub max} values of 280 and 940 pmol/g wet liver tissue. S-(-)-Alaproclate inhibited the binding of {sup 3}H-proadifen and {sup 3}H-cocaine inhibited the binding of {sup 3}H-proadifen (IC{sub 50}=10 nM) and proadifen that of {sup 3}H-cocaine (IC{sub 50}=1 nM). There was a high correlation coefficient (r{sub r}=0.972; P<0.01; n=12) in the Spearman rank test between the inhibitory potencies of compounds examined in both systems. Beside some potent alaproclate analogues a couple of compounds had moderately high affinity (IC{sub 50}=100-500 nM): chloroquine, phenoxybenzamine, amitriptyline, ajmaline, remoxipride, imipramine and (-)-alaprenolol. CdCl{sub 2}, ZnCl{sub 2} and CuCl{sub 2} inhibited the binding of both ligands with low Hill coefficients, indicating heterogeneous binding sites. The inhibition curve of Cd{sup 2+} on the cocaine binding was biphasic with a high affinity part around 50 nM and a low affinity part at 15{mu}M. The similarity of the characteristics of the binding of these ligands with that of {sup 3}H-alaproclate is discussed. It is suggested that all three compounds bind to the same sites, although additional binding sites seem to exist for proadifen. (au) (9 refs.).

  10. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Moran, Jeffery H. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Arkansas Department of Public Health, Public Health Laboratory, Little Rock, AR 72205 (United States); Prather, Paul L., E-mail: pratherpaull@uams.edu [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States)

    2013-06-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB{sub 1}Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB{sub 2}Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB{sub 2}Rs (hCB{sub 2}Rs). The affinity of cannabinoids for hCB{sub 2}Rs was determined by competition binding studies employing CHO-hCB{sub 2} membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB{sub 2} cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB{sub 2}Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB{sub 2}Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB{sub 2}R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB{sub 2}Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB{sub 2}Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB{sub 1} and CB{sub 2}Rs. - Highlights: • JWH-018

  11. Discovery, SAR, and Radiolabeling of Halogenated Benzimidazole Carboxamide Antagonists as Useful Tools for (alpha)4(beta)1 Integrin Expressed on T- and B-cell Lymphomas

    Energy Technology Data Exchange (ETDEWEB)

    Carpenter, R D; Natarajan, A; Lau, E Y; Andrei, M; Solano, D M; Lightstone, F C; DeNardo, S J; Lam, K S; Kurth, M J

    2010-02-08

    The cell surface receptor {alpha}{sub 4}{beta}{sub 1} integrin is an attractive yet poorly understood target for selective diagnosis and treatment of T- and B-cell lymphomas. This report focuses on the rapid microwave preparation of medicinally pertinent benzimidazole heterocycles, structure-activity relationships (SAR) of novel halobenzimidazole carboxamide antagonists 3-6, and preliminary biological evaluation of radioiodinated agents 7, 8, and 18. The I-125 derivative 18 had good tumor uptake (12 {+-} 1% ID/g at 24 h; 4.5 {+-} 1% ID/g at 48 h) and tumor:kidney ratio ({approx}4:1 at 24 h; 2.5:1 at 48 h) in xenograft murine models of B-cell lymphoma. Molecular homology models of {alpha}{sub 4}{beta}{sub 1} integrin have predicted that docked halobenzimidazole carboxamides have the halogen atom in a suitable orientation for halogen-hydrogen bonding. These high affinity ({approx} pM binding) halogenated ligands are attractive tools for medicinal and biological use; the fluoro and iodo derivatives are potential radiodiagnostic ({sup 18}F) or radiotherapeutic ({sup 131}I) agents, whereas the chloro and bromo analogues could provide structural insight into integrin-ligand interactions through photoaffinity cross-linking/mass spectroscopy experiments, as well as co-crystallization X-ray studies.

  12. A tyrosine kinase inhibitor-based high-affinity PET radiopharmaceutical targets vascular endothelial growth factor receptor.

    Science.gov (United States)

    Li, Feng; Jiang, Sheng; Zu, Youli; Lee, Daniel Y; Li, Zheng

    2014-09-01

    Tyrosine kinase receptors including vascular endothelial growth factor receptor (VEGFR) have gained significant attention as pharmacologic targets. However, clinical evaluation of small-molecule drugs or biologics that target these pathways has so far yielded mixed results in a variety of solid tumors. The reasons for response variability remain unknown, including the temporal and spatial patterns of receptor tyrosine kinase expression. Methods to detect and quantify the presence of such cellular receptors would greatly facilitate drug development and therapy response assessment. We aimed to generate specific imaging agents as potential companion diagnostics that could also be used for targeted radionuclide therapy. Here, we report on the synthesis and initial preclinical performance of (64)Cu-labeled probes that were based on the kinase inhibitor already in clinical use, vandetanib (ZD6474), as a VEGFR-selective theranostic radiopharmaceutical. A monomeric (ZD-G1) and a dimeric (ZD-G2) derivative of ZD6474 were synthesized and conjugated with DOTA for chelation with (64)Cu to produce the probes (64)Cu-DOTA-ZD-G1 and (64)Cu-DOTA-ZD-G2. The binding affinity and specificity to VEGFR were measured using U-87 MG cells known to overexpress VEGFR. Small-animal PET and biodistribution studies were performed with (64)Cu-labeled probes (3-4 MBq) intravenously administered in U-87 MG tumor-bearing mice with or without coinjection of unlabeled ZD-G2 for up to 24 h after injection. Receptor-binding assays yielded a mean equilibrium dissociation constant of 44.7 and 0.45 nM for monomeric and dimeric forms, respectively, indicating a synergistic effect in VEGFR affinity by multivalency. Small-animal PET/CT imaging showed rapid tumor accumulation of (64)Cu-DOTA-ZD-G2, with excellent tumor-to-normal tissue contrast by 24 h. Coinjection of the (64)Cu-DOTA-ZD-G2 with 50 nmol (60 μg) of nonradioactive ZD-G2 effectively blocked tumor uptake. A (64)Cu-labeled probe derived from an

  13. High-Affinity "Click" RGD Peptidomimetics as Radiolabeled Probes for Imaging αvβ3Integrin.

    Science.gov (United States)

    Piras, Monica; Testa, Andrea; Fleming, Ian N; Dall'Angelo, Sergio; Andriu, Alexandra; Menta, Sergio; Mori, Mattia; Brown, Gavin D; Forster, Duncan; Williams, Kaye J; Zanda, Matteo

    2017-07-20

    Nonpeptidic Arg-Gly-Asp (RGD)-mimic ligands were designed and synthesized by click chemistry between an arginine-azide mimic and an aspartic acid-alkyne mimic. Some of these molecules combine excellent in vitro properties (high α v β 3 affinity, selectivity, drug-like logD, high metabolic stability) with a variety of radiolabeling options (e.g., tritium and fluorine-18, plus compatibility with radio-iodination), not requiring the use of chelators or prosthetic groups. The binding mode of the resulting triazole RGD mimics to α v β 3 or α IIb β 3 receptors was investigated by molecular modeling simulations. Lead compound 12 was successfully radiofluorinated and used for in vivo positron emission tomography/computed tomography (PET/CT) studies in U87 tumor models, which showed only modest tumor uptake and retention, owing to rapid excretion. These results demonstrate that the novel click RGD mimics are excellent radiolabeled probes for in vitro and cell-based studies on α v β 3 integrin, whereas further optimization of their pharmacokinetic and dynamic profiles is necessary for successful use in in vivo imaging. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. High-resolution crystal structure of human Mapkap kinase 3 in complex with a high affinity ligand.

    Science.gov (United States)

    Cheng, Robert; Felicetti, Brunella; Palan, Shilpa; Toogood-Johnson, Ian; Scheich, Christoph; Barker, John; Whittaker, Mark; Hesterkamp, Thomas

    2010-01-01

    The Mapkap kinases 2 and 3 (MK2 and MK3) have been implicated in intracellular signaling pathways leading to the production of the pro-inflammatory cytokine tumor necrosis factor alpha. MK2 has been pursued by the biopharmaceutical industry for many years for the development of a small molecule anti-inflammatory treatment and drug-like inhibitors have been described. The development of some of these compounds, however, has been slowed by the absence of a high-resolution crystal structure of MK2. Herein we present a high-resolution (1.9 A) crystal structure of the highly homologous MK3 in complex with a pharmaceutical lead compound. While all of the canonical features of Ser/Thr kinases in general and MK2 in particular are recapitulated in MK3, the detailed analysis of the binding interaction of the drug-like ligand within the adenine binding pocket allows relevant conclusions to be drawn for the further design of potent and selective drug candidates.

  15. Effect of netupitant, a highly selective NK₁ receptor antagonist, on the pharmacokinetics of palonosetron and impact of the fixed dose combination of netupitant and palonosetron when coadministered with ketoconazole, rifampicin, and oral contraceptives.

    Science.gov (United States)

    Calcagnile, Selma; Lanzarotti, Corinna; Rossi, Giorgia; Henriksson, Anders; Kammerer, Klaus Peter; Timmer, Wolfgang

    2013-10-01

    Neurokinin-1 receptor antagonists (NK1 RAs) are commonly coadministered with a 5-HT3 RA such as palonosetron to prevent nausea and vomiting induced by chemotherapy. Netupitant, a new highly selective NK1 RA, is both a substrate for and a moderate inhibitor of CYP3A4. Three studies were designed to evaluate the potential drug-drug interaction of netupitant with palonosetron and of the fixed dose combination of netupitant and palonosetron, NEPA, with an inhibitor (ketoconazole), an inducer (rifampicin) and a substrate (oral contraceptives) of CYP3A4. Study 1 was a three-way crossover in 18 healthy subjects receiving netupitant alone, palonosetron alone, and the combination of both antiemetics. Studies 2 and 3 were two-way crossover trials where healthy subjects received NEPA (the fixed dose combination of netupitant and palonosetron). In study 2, 36 subjects received NEPA alone (day 1) and in combination with ketoconazole or rifampicin. In study 3, 24 healthy women received ethinylestradiol/levonorgestrel alone or in combination with NEPA (day 1). There were no significant pharmacokinetic interactions between netupitant and palonosetron. Ketoconazole increased netupitant area under curve (AUC) by 140 % and C max by 25 %. Rifampicin decreased netupitant AUC by 83 % and C max by 62 %. NEPA did not significantly affect exposure to ethinylestradiol, while systemic exposure to levonorgestrel increased by 40 %, but this was not considered clinically relevant. There were no clinically relevant interactions between netupitant and palonosetron, or between NEPA and oral contraceptives. The coadministration of NEPA with inhibitors or inducers of CYP3A4 may require dose adjustments. Treatments were well tolerated.

  16. The ketamine analogue methoxetamine and 3- and 4-methoxy analogues of phencyclidine are high affinity and selective ligands for the glutamate NMDA receptor

    National Research Council Canada - National Science Library

    Roth, Bryan L; Gibbons, Simon; Arunotayanun, Warunya; Huang, Xi-Ping; Setola, Vincent; Treble, Ric; Iversen, Les

    2013-01-01

    In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as 'designer drugs' and compared them to the parent substances via the resources...

  17. Characterisation of [11C]PR04.MZ in Papio anubis baboon: A selective high-affinity radioligand for quantitative imaging of the dopamine transporter

    Energy Technology Data Exchange (ETDEWEB)

    Riss P. J.; Fowler J.; Riss, P.J.; Hooker, J.M.; Shea, C.; Xu, Y.; Carter, P.; Warner, D.; Ferrari V.; Kim, S.W.; Aigbirhio, F.I.; Fowler, J.S.; Roesch, F.

    2011-10-25

    N-(4-fluorobut-2-yn-1-yl)-2{beta}-carbomethoxy-3{beta}-(4{prime}-tolyl)nortropane (PR04.MZ, 1) is a PET radioligand for the non-invasive exploration of the function of the cerebral dopamine transporter (DAT). A reliable automated process for routine production of the carbon-11 labelled analogue [{sup 11}C]PR04.MZ ([{sup 11}C]-1) has been developed using GMP compliant equipment. An adult female Papioanubis baboon was studied using a test-retest protocol with [{sup 11}C]-1 in order to assess test-retest reliability, metabolism and CNS distribution profile of the tracer in non-human primates. Blood sampling was performed throughout the studies for determination of the free fraction in plasma (fP), plasma input functions and metabolic degradation of the radiotracer [{sup 11}C]-1. Time-activity curves were derived for the putamen, the caudate nucleus, the ventral striatum, the midbrain and the cerebellum. Distribution volumes (VT) and non-displaceable binding potentials (BPND) for various brain regions and the blood were obtained from kinetic modelling. [{sup 11}C]-1 shows promising results as aselective marker of the presynaptic dopamine transporter. With the reliable visualisation of the extra-striatal dopaminergic neurons and no indication on labelled metabolites, the tracer provides excellent potential for translation into man.

  18. The HLA-DP2 protein binds the immunodominant epitope from myelin basic protein, MBP85-99, with high affinity

    DEFF Research Database (Denmark)

    Hansen, B E; Nielsen, Claus Henrik; Madsen, H O

    2011-01-01

    Myelin basic protein (MBP) is a candidate autoantigen in multiple sclerosis (MS). The immunodominant epitope for T-cell responses is assigned to the amino acid sequence MBP84-102, which binds to human leukocyte antigen (HLA)-DR2a (DRB5*0101) and HLA-DR2b (DRB1*1501) of the HLA-DR2 haplotype...... as the HLA-DRB1*1501, where the MBP89V is preferred as the p1 anchor. Notably, full-length MBP was able to compete for peptide binding with an affinity similar to that seen for the high-affinity binding peptides, DRa170-83 and IIP53-65. In summary, the HLA-DP2 molecule binds the immunodominant epitope in MS...

  19. Interrogating the Molecular Basis for Substrate Recognition in Serotonin and Dopamine Transporters with High-Affinity Substrate-Based Bivalent Ligands

    DEFF Research Database (Denmark)

    Andersen, Jacob; Ladefoged, Lucy Kate; Kristensen, Trine N. Bjerre

    2016-01-01

    The transporters for the neurotransmitters serotonin and dopamine (SERT and DAT, respectively) are targets for drugs used in the treatment of mental disorders and widely used drugs of abuse. Studies of prokaryotic homologues have advanced our structural understanding of SERT and DAT, but it still...... remains enigmatic whether the human transporters contain one or two high-affinity substrate binding sites. We have designed and employed 24 bivalent ligands possessing a highly systematic combination of substrate moieties (serotonin and/or dopamine) and aliphatic or poly(ethylene glycol) spacers to reveal...... insight into substrate recognition in SERT and DAT. An optimized bivalent ligand comprising two serotonin moieties binds SERT with 3,800-fold increased affinity compared to that of serotonin, suggesting that the human transporters have two distinct substrate binding sites. We show that the bivalent...

  20. The P-glycoprotein (ABCB1) linker domain encodes high-affinity binding sequences to alpha- and beta-tubulins.

    Science.gov (United States)

    Georges, Elias

    2007-06-26

    P-Glycoprotein (or ABCB1) has been shown to cause multidrug resistance in tumor cell lines selected with lipophilic anticancer drugs. ABCB1 encodes a duplicated molecule with two hydrophobic and hydrophilic domains linked by a highly charged region of approximately 90 amino acids, the "linker domain" with as yet unknown function(s). In this report, we demonstrate a role for this domain in binding to other cellular proteins. Using overlapping hexapeptides that encode the entire amino acid sequence of the linker domain of human ABCB1, we show a direct and specific binding between sequences in the linker domain and several intracellular proteins. Three different polypeptide sequences [617EKGIYFKLVTM627 (LDS617-627), 657SRSSLIRKRSTRRSVRGSQA676 (LDS657-676), and 693PVSFWRIMKLNLT705 (LDS693-705)] in the linker domain interacted tightly with several proteins with apparent molecular masses of approximately 80, 57, and 30 kDa. Interestingly, only the 57 kDa protein (or P57) interacted with all three different sequences of the linker domain. Purification and partial N-terminal amino acid sequencing of P57 showed that it encodes the N-terminal amino acids of alpha- and beta-tubulins. The identity of the P57 interacting protein as tubulins was further confirmed by Western blotting using monoclonal antibodies to alpha- and beta-tubulin. Taken together, the results of this study provide the first evidence for ABCB1 protein interaction mediated by sequences in the linker domain. These findings are likely to provide further insight into the functions of ABCB1 in normal and drug resistant tumor cells.

  1. A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

    Directory of Open Access Journals (Sweden)

    Jonathan S Wall

    Full Text Available Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

  2. Land-Use Influences the Distribution and Activity of High Affinity CO-Oxidizing Bacteria Associated to type I-coxL Genotype in Soil

    Directory of Open Access Journals (Sweden)

    Liliana eQuiza

    2014-06-01

    Full Text Available Soil carboxydovore bacteria are the biological sink of atmospheric carbon monoxide (CO. The initial oxidation of CO is catalyzed by a CO-dehydrogenase (CODH, and the gene coxL encodes the large subunit of the enzyme. Only a few carboxydovore isolates were shown to oxidize atmospheric CO and little is known about the potential impact of global change on the ecophysiology of this functional group. The main objective of this study was to assess the impact of land-use and soil properties on coxL gene diversity and identify molecular indicators for the soil uptake of atmospheric CO. Soil samples were collected in three neighboring sites encompassing different land-use types, namely deciduous forest, larch plantation and maize field. CO uptake activity was related to total carbon and nitrogen content in soil, with the highest activity observed in deciduous forest. An extensive coxL database was assembled to optimize a PCR detection assay targeting sequences belonging to functional type I-CODH and hypothetical type II-CODH. Fully replicated coxL gene libraries unveiled a unique molecular signature in deciduous forest soil, with enrichment of type I sequences. Genetic profiles of larch and maize monocultures were not statistically different and showed higher level of coxL gene richness than deciduous forest. Soil water content and CO uptake activity explained 38% of the variation of coxL gene profiles in a canonical ordination analysis, leading to the identification of sequences belonging to the δ-Proteobacteria cluster as indicator for high affinity CO uptake activity. Enrichment of type I and δ-Proteobacteria coxL sequences in deciduous forest were confirmed by qPCR in an independent soil survey. CO uptake activity in model carboxydovore bacteria suggested that a significant fraction of detected putative high affinity CO oxidizers were active in soil. Land-use was a driving force separating coxL diversity in deciduous forest from monocultures.

  3. Metal ion blockage of tritium incorporation into gamma-carboxyglutamic acid of prothrombin. Stoichiometry of gamma-carboxyglutamic acid to Gd3+ for the high affinity sites

    Energy Technology Data Exchange (ETDEWEB)

    Bajaj, S.P.; Saini, R.; Katz, A.; Cai, G.Z.; Maki, S.L.; Brodsky, G.L.

    1988-07-15

    Prothrombin possesses two high affinity and four low affinity gamma-carboxyglutamic acid (Gla)-dependent gadolinium binding sites. Earlier work has shown that tritium can be specifically incorporated at the gamma-carbon of Gla in proteins at pH 5. In the present work we show that inclusion of saturating concentrations of Ca2+ in nondenaturing buffer systems ranging from pH 5.5 to 8.5 prevents the exchange of tritium into all 10 Gla residues of prothrombin. Similarly, saturating concentrations of Gd3+ prevent tritium incorporation into Gla at pH 5.5. Positive cooperativity was observed for the binding of Gd3+ to human prothrombin (at pH 5.5) for the two high affinity sites (Kd congruent to 35 nM). The four low affinity sites bind Gd3+ with a Kd congruent to 5 microM. Incubation of prothrombin ranging in concentrations from 10 to 40 microM with 2 eq of Gd3+ at pH 5.5 prevents 5.7 (average of seven determinations) Gla residues from tritium incorporation. Sedimentation velocity experiments conducted at pH 5.5 indicate that prothrombin in the presence of saturating concentrations of Gd3+ polymerizes, most likely, to a trimer. Further, in the presence of 2 eq of Gd3+, calculated percent weight average concentration of monomer prothrombin is congruent to 100% at 10 microM, approximately equal to 95% at 20 microM, and congruento to 80% at 40 microM protein concentration. Thus, it appears that under conditions in which prothrombin primarily exists as a monomer, occupancy of the initial two metal binding sites by Gd3+ involves six Gla residues.

  4. Single Nucleotide Polymorphisms of the High Affinity IgG Receptor FcγRI Reduce Immune Complex Binding and Downstream Effector Functions.

    Science.gov (United States)

    Brandsma, Arianne M; Ten Broeke, Toine; van Dueren den Hollander, Evelien; Caniels, Thomas G; Kardol-Hoefnagel, Tineke; Kuball, Jürgen; Leusen, Jeanette H W

    2017-10-01

    Binding of IgG Abs to FcγRs on immune cells induces FcγR cross-linking that leads to cellular effector functions, such as phagocytosis, Ab-dependent cellular cytotoxicity, and cytokine release. However, polymorphisms in low affinity FcγRs have been associated with altered avidity toward IgG, thereby substantially impacting clinical outcomes of multimodular therapy when targeting cancer or autoimmune diseases with mAbs as well as the frequency and severity of autoimmune diseases. In this context, we investigated the consequences of three nonsynonymous single nucleotide polymorphisms (SNPs) for the high affinity receptor for IgG, FcγRI. Only SNP V39I, located in the extracellular domain of FcγRI, reduces immune-complex binding of FcγRI whereas monomeric IgG binding is unaffected. This leads to reduced FcγRI effector functions, including Fc receptor γ-chain signaling and intracellular calcium mobilization. SNPs I301M and I338T, located in the transmembrane or intracellular domain, respectively, have no influence on monomeric IgG or immune complex binding, but FcRγ signaling is decreased for both SNPs, especially for I338T. We also found that the frequency of these SNPs in a cohort of healthy Dutch individuals is very low within the population. To our knowledge, this study addresses for the first time the biological consequences of SNPs in the high affinity FcγR, and reveals reduction in several FcγRI functions, which have the potential to alter efficacy of therapeutic Abs. Copyright © 2017 by The American Association of Immunologists, Inc.

  5. Naturally occurring tyrosine kinase inserts block high affinity binding of phospholipase C gamma and Shc to TrkC and neurotrophin-3 signaling.

    Science.gov (United States)

    Guiton, M; Gunn-Moore, F J; Glass, D J; Geis, D R; Yancopoulos, G D; Tavaré, J M

    1995-09-01

    Neurotrophin-3 binds to the receptor tyrosine kinase, TrkC. Several naturally occurring splice variants of TrkC exist including those with 14- and 39-amino acid inserts within the tyrosine kinase homology region. When expressed in fibroblasts, full-length TrkC, but not the kinase insert variants, mediated neurotrophin-3-stimulated cell proliferation. We investigated the molecular basis of this signaling defect. The kinase inserts blocked the ability of TrkC to mediate neurotrophin-3 stimulated c-myc and c-fos transcription and activation of the AP-1 transcriptional complex. In cells expressing full-length TrkC, neurotrophin-3 promoted a sustained activation of mitogen-activated protein kinase; TrkC containing kinase inserts only mediated transient activation of mitogen-activated protein kinase. The kinase inserts specifically blocked neurotrophin-3-stimulated autophosphorylation of the phospholipase C gamma binding site on TrkC (tyrosine 789) resulting in a severe reduction in phospholipase C gamma association with TrkC and its tyrosine phosphorylation. Neurotrophin-3-stimulated phosphorylation of the Shc binding site (tyrosine 485) on TrkC, and tyrosine phosphorylation of Shc itself, was unaffected by the kinase inserts; however, the kinase inserts blocked high affinity Shc association with TrkC. It is proposed that the lack of high affinity binding of Shc and/or phospholipase C gamma to the TrkC kinase insert variants may be responsible for the inability of these variants to bring about a full biological response in fibroblasts.

  6. In vivo neutralization of α-cobratoxin with high-affinity llama single-domain antibodies (VHHs and a VHH-Fc antibody.

    Directory of Open Access Journals (Sweden)

    Gabrielle Richard

    Full Text Available Small recombinant antibody fragments (e.g. scFvs and VHHs, which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab'2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α-Cbtx, the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2 was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α-Cbtx. Mouse α-Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20 and the VHH2-Fc antibody effectively neutralized lethality induced by α-Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy.

  7. Isolation of a high affinity Bet v 1-specific IgG-derived ScFv from a subject vaccinated with hypoallergenic Bet v 1 fragments.

    Science.gov (United States)

    Gadermaier, Elisabeth; Marth, Katharina; Lupinek, Christian; Campana, Raffaela; Hofer, Gerhard; Blatt, Katharina; Smiljkovic, Dubravka; Roder, Uwe; Focke-Tejkl, Margarete; Vrtala, Susanne; Keller, Walter; Valent, Peter; Valenta, Rudolf; Flicker, Sabine

    2018-01-09

    Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild type allergens. However, so far no treatment-induced IgG antibodies have been characterized. To clone, express and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a non-allergic subject. A phage-displayed combinatorial single chain fragment (ScFv) library was constructed from blood of the immunized subject and screened for Bet v 1-reactive antibody fragments. ScFvs were tested for specificity and cross-reactivity to native Bet v 1 and related pollen and food allergens and epitope mapping was performed. Germline ancestor genes of the antibody were analyzed with the ImMunoGeneTics (IMGT) database. The affinity to Bet v 1 and cross-reactive allergens was determined by surface plasmon resonance measurements. The ability to inhibit patients' IgE binding to ELISA plate-bound allergens and allergen-induced basophil activation was assessed. A combinatorial ScFv library was obtained from the vaccinated donor after three injections with the Bet v 1 fragments. Despite being almost in germline configuration, ScFv (clone H3-1) reacted with high affinity to native Bet v 1 and homologous allergens, inhibited allergic patients' polyclonal IgE binding to Bet v 1 and partially suppressed allergen-induced basophil activation. Immunization with unfolded hypoallergenic allergen derivatives induces high affinity antibodies even in non-allergic subjects which recognize the folded wild-type allergens and inhibit polyclonal IgE binding of allergic patients. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  8. Land-use influences the distribution and activity of high affinity CO-oxidizing bacteria associated to type I-coxL genotype in soil.

    Science.gov (United States)

    Quiza, Liliana; Lalonde, Isabelle; Guertin, Claude; Constant, Philippe

    2014-01-01

    Soil carboxydovore bacteria are the biological sink of atmospheric carbon monoxide (CO). The initial oxidation of CO is catalyzed by a CO-dehydrogenase (CODH), and the gene coxL encodes the large subunit of the enzyme. Only a few carboxydovore isolates were shown to oxidize atmospheric CO and little is known about the potential impact of global change on the ecophysiology of this functional group. The main objective of this study was to assess the impact of land-use and soil properties on coxL gene diversity and identify molecular indicators for the soil uptake of atmospheric CO. Soil samples were collected in three neighboring sites encompassing different land-use types, namely deciduous forest, larch plantation and maize field. CO uptake activity was related to total carbon and nitrogen content in soil, with the highest activity observed in deciduous forest. An extensive coxL database was assembled to optimize a PCR detection assay targeting sequences belonging to functional type I-CODH and hypothetical type II-CODH. Fully replicated coxL gene libraries unveiled a unique molecular signature in deciduous forest soil, with enrichment of type I sequences. Genetic profiles of larch and maize monocultures were not statistically different and showed higher level of coxL gene richness than deciduous forest. Soil water content and CO uptake activity explained 38% of the variation of coxL gene profiles in a canonical ordination analysis, leading to the identification of sequences belonging to the δ-Proteobacteria cluster as indicator for high affinity CO uptake activity. Enrichment of type I and δ-Proteobacteria coxL sequences in deciduous forest were confirmed by qPCR in an independent soil survey. CO uptake activity in model carboxydovore bacteria suggested that a significant fraction of detected putative high affinity CO oxidizers were active in soil. Land-use was a driving force separating coxL diversity in deciduous forest from monocultures.

  9. GABA-agonists induce the formation of low-affinity GABA-receptors on cultured cerebellar granule cells via preexisting high affinity GABA receptors

    DEFF Research Database (Denmark)

    Belhage, B; Meier, E; Schousboe, A

    1986-01-01

    -tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP, 150 microM) or THIP plus the antagonist bicuculline methobromide (150 microM of each) or in the absence of the agonist or antagonist. Membranes isolated from granule cells cultured in a medium without the GABA agonist revealed a single binding site for GABA......The kinetics of specific GABA-binding to membranes isolated from cerebellar granule cells, cultured for 12 days from dissociated cerebella of 7-day-old rats was studied using [3H]GABA as the ligand. The granule cells were cultured in the presence of the specific GABA receptor agonist 4, 5, 6, 7...

  10. Full domain closure of the ligand-binding core of the ionotropic glutamate receptor iGluR5 induced by the high affinity agonist dysiherbaine and the functional antagonist 8,9-dideoxyneodysiherbaine

    DEFF Research Database (Denmark)

    Frydenvang, Karla Andrea; Lash, L Leanne; Naur, Peter

    2009-01-01

    The prevailing structural model for ligand activation of ionotropic glutamate receptors posits that agonist efficacy arises from the stability and magnitude of induced domain closure in the ligand-binding core structure. Here we describe an exception to the correlation between ligand efficacy...... and domain closure. A weakly efficacious partial agonist of very low potency for homomeric iGluR5 kainate receptors, 8,9-dideoxy-neodysiherbaine (MSVIII-19), induced a fully closed iGluR5 ligand-binding core. The degree of relative domain closure, ~30 degrees , was similar to that we resolved...... to inter-domain hydrogen bonds residues Glu441 and Ser721 in the iGluR5-S1S2 structure. The weaker interactions of MSVIII-19 with iGluR5 compared to DH, together with altered stability of the inter-domain interaction, may be responsible for the apparent uncoupling of domain closure and channel opening...

  11. Antagonistic parent-offspring co-adaptation.

    Directory of Open Access Journals (Sweden)

    Mathias Kölliker

    Full Text Available BACKGROUND: In species across taxa, offspring have means to influence parental investment (PI. PI thus evolves as an interacting phenotype and indirect genetic effects may strongly affect the co-evolutionary dynamics of offspring and parental behaviors. Evolutionary theory focused on explaining how exaggerated offspring solicitation can be understood as resolution of parent-offspring conflict, but the evolutionary origin and diversification of different forms of family interactions remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: In contrast to previous theory that largely uses a static approach to predict how "offspring individuals" and "parental individuals" should interact given conflict over PI, we present a dynamic theoretical framework of antagonistic selection on the PI individuals obtain/take as offspring and the PI they provide as parents to maximize individual lifetime reproductive success; we analyze a deterministic and a stochastic version of this dynamic framework. We show that a zone for equivalent co-adaptation outcomes exists in which stable levels of PI can evolve and be maintained despite fast strategy transitions and ongoing co-evolutionary dynamics. Under antagonistic co-adaptation, cost-free solicitation can evolve as an adaptation to emerging preferences in parents. CONCLUSIONS/SIGNIFICANCE: We show that antagonistic selection across the offspring and parental life-stage of individuals favors co-adapted offspring and parental behavior within a zone of equivalent outcomes. This antagonistic parent-offspring co-adaptation does not require solicitation to be costly, allows for rapid divergence and evolutionary novelty and potentially explains the origin and diversification of the observed provisioning forms in family life.

  12. Characterization of the muscarinic receptor subtype(s) mediating contraction of the guinea-pig lung strip and inhibition of acetylcholine release in the guinea-pig trachea with the selective muscarinic receptor antagonist tripitramine

    NARCIS (Netherlands)

    Roffel, A.F; Davids, J.H; Elzinga, C.R S; Wolf, D; Zaagsma, Hans; Kilbinger, H

    1 The muscarinic receptor subtypes mediating contraction of the guinea-pig lung strip and inhibition of the release of acetylcholine from cholinergic vagus nerve endings in the guinea-pig trachea in vitro have previously been characterized as M-2-like, i.e. having antagonist affinity profiles that

  13. Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues

    Energy Technology Data Exchange (ETDEWEB)

    Waser, Beatrice; Reubi, Jean Claude [University of Berne, Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, PO Box 62, Berne (Switzerland)

    2014-06-15

    Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the {sup 125}iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer {sup 125}I-GLP-1(7-36)amide. Receptor autoradiography studies with {sup 125}I-GLP-1(7-36)amide agonist or {sup 125}I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. The antagonist {sup 125}I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer {sup 125}I-GLP-1(7-36)amide. For comparison, {sup 125}I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. The GLP-1 receptor antagonist exendin(9-39) labelled with {sup 125}I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients. (orig.)

  14. Distinctions between non-peptide angiotensin II AT1-receptor antagonists

    Directory of Open Access Journals (Sweden)

    Georges Vauquelin

    2001-03-01

    Full Text Available A far-reaching understanding of the molecular action mechanism of AT1-receptor antagonists (AIIAs was obtained by using CHO cells expressing transfected human AT 1-receptors. In this model, direct [3H]-antagonist binding and inhibition of agonist-induced responses (inositol phosphate accumulation can be measured under identical experimental conditions. Whereas preincubation with a surmountable AIIA (losartan causes parallel shifts of the angiotensin II (Ang II concentration-response curve, insurmountable antagonists also cause partial (i.e., 30% for irbesartan, 50% for valsartan, 70% for EXP3174, to almost complete (95% for candesartan reductions of the maximal response. The main conclusions are that all investigated antagonists are competitive with respect to Ang II. They bind to a common or overlapping site on the receptor in a mutually exclusive way. Insurmountable inhibition is related to the slow dissociation rate of the antagonist-receptor complex (t 1/2 of 7 minutes for irbesartan, 17 minutes for valsartan, 30 minutes for EXP3174 and 120 minutes for candesartan. Antagonist-bound AT1-receptors can adopt a fast and a slow reversible state. This is responsible for the partial nature of the insurmountable inhibition. The long-lasting effect of candesartan, the active metabolite of candesartan cilexetil, in vascular smooth muscle contraction studies, as well as in in vivo experiments on rat and in clinical studies, is compatible with its slow dissociation from, and continuous recycling between AT1-receptors. This recycling, or `rebinding' takes place because of the very high affinity of candesartan for the AT1-receptor.

  15. Pharmacokinetics, pharmacodynamics and safety of QGE031 (ligelizumab), a novel high-affinity anti-IgE antibody, in atopic subjects

    Science.gov (United States)

    Arm, J P; Bottoli, I; Skerjanec, A; Floch, D; Groenewegen, A; Maahs, S; Owen, C E; Jones, I; Lowe, P J

    2014-01-01

    Background Using a monoclonal antibody with greater affinity for IgE than omalizumab, we examined whether more complete suppression of IgE provided greater pharmacodynamic effects, including suppression of skin prick responses to allergen. Objective To explore the pharmacokinetics, pharmacodynamics and safety of QGE031 (ligelizumab), a novel high-affinity humanized monoclonal IgG1κ anti-IgE. Methods Preclinical assessments and two randomized, placebo-controlled, double-blind clinical trials were conducted in atopic subjects. The first trial administered single doses of QGE031 (0.1–10 mg/kg) or placebo intravenously, while the second trial administered two to four doses of QGE031 (0.2– 4 mg/kg) or placebo subcutaneously at 2-week intervals. Both trials included an open-label omalizumab arm. Results Sixty of 73 (82%) and 96 of 110 (87%) subjects completed the intravenous and subcutaneous studies, respectively. Exposure to QGE031 and its half-life depended on the QGE031 dose and serum IgE level. QGE031 had a biexponential pharmacokinetic profile after intravenous administration and a terminal half-life of approximately 20 days. QGE031 demonstrated dose- and time-dependent suppression of free IgE, basophil FcεRI and basophil surface IgE superior in extent (free IgE and surface IgE) and duration to omalizumab. At Day 85, 6 weeks after the last dose, skin prick wheal responses to allergen were suppressed by > 95% and 41% in subjects treated subcutaneously with QGE031 (2 mg/kg) or omalizumab, respectively (P < 0.001). Urticaria was observed in QGE031- and placebo-treated subjects and was accompanied by systemic symptoms in one subject treated with 10 mg/kg intravenous QGE031. There were no serious adverse events. Conclusion and Clinical Relevance These first clinical data for QGE031, a high-affinity IgG1κ anti-IgE, demonstrate that increased suppression of free IgE compared with omalizumab translated to superior pharmacodynamic effects in atopic subjects

  16. Design and Investigation of a [18F]-Labeled Benzamide Derivative as a High Affinity Dual Sigma Receptor Subtype Radioligand for Prostate Tumor Imaging.

    Science.gov (United States)

    Yang, Dongzhi; Comeau, Anthony; Bowen, Wayne D; Mach, Robert H; Ross, Brian D; Hong, Hao; Van Dort, Marcian E

    2017-03-06

    High overexpression of sigma (σ) receptors (σ1 and σ2 subtypes) in a variety of human solid tumors has prompted the development of σ receptor-targeting radioligands, as imaging agents for tumor detection. A majority of these radioligands to date target the σ2 receptor, a potential marker of tumor proliferative status. The identification of approximately equal proportions of both σ receptor subtypes in prostate tumors suggests that a high affinity, dual σ receptor-targeting radioligand could potentially provide enhanced tumor targeting efficacy in prostate cancer. To accomplish this goal, we designed a series of ligands which bind to both σ receptor subtypes with high affinity. Ligand 3a in this series, displaying optimal dual σ receptor subtype affinity (σ1, 6.3 nM; σ2, 10.2 nM) was radiolabeled with fluorine-18 (18F) to give [18F]3a and evaluated as a σ receptor-targeting radioligand in the mouse PC-3 prostate tumor model. Cellular assays with PC-3 cells demonstrated that a major proportion of [18F]3a was localized to cell surface σ receptors, while ∼10% of [18F]3a was internalized within cells after incubation for 3.5 h. Serial PET imaging in mice bearing PC-3 tumors revealed that uptake of [18F]3a was 1.6 ± 0.8, 4.4 ± 0.3, and 3.6 ± 0.6% ID/g (% injection dose per gram) in σ receptor-positive prostate tumors at 15 min, 1.5 h, and 3.5 h postinjection, respectively (n = 3) resulting in clear tumor visualization. Blocking studies conducted with haloperidol (a nonselective inhibitor for both σ receptor subtypes) confirmed that the uptake of [18F]3a was σ receptor-mediated. Histology analysis confirmed similar expression of σ1 and σ2 in PC-3 tumors which was significantly greater than its expression in normal organs/tissues such as liver, kidney, and muscle. Metabolite studies revealed that >50% of radioactivity in PC-3 tumors at 30 min postinjection represented intact [18F]3a. Prominent σ receptor-specific uptake of [18F]3a in prostate tumors

  17. Genome data mining and soil survey for the novel group 5 [NiFe]-hydrogenase to explore the diversity and ecological importance of presumptive high-affinity H(2)-oxidizing bacteria.

    Science.gov (United States)

    Constant, Philippe; Chowdhury, Soumitra Paul; Hesse, Laura; Pratscher, Jennifer; Conrad, Ralf

    2011-09-01

    Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H(2) possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H(2)-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H(2) oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 10(6) to 10(8) hhyL gene copies g (dry weight)(-1). Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H(2)-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H(2) oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H(2) by soil, because high-affinity H(2) oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur.

  18. Facilitation and inhibition of male rat ejaculatory behaviour by the respective 5-HT1A and 5-HT1B receptor agonists 8-OH-DPAT and anpirtoline, as evidenced by use of the corresponding new and selective receptor antagonists NAD-299 and NAS-181

    Science.gov (United States)

    Hillegaart, Viveka; Ahlenius, Sven

    1998-01-01

    Ejaculatory problems and anorgasmia are well-known side-effects of the SSRI antidepressants, and a pharmacologically induced increase in serotonergic neurotransmission inhibits ejaculatory behaviour in the rat. In the present study the role of 5-HT1A and 5-HT1B receptors in the mediation of male rat ejaculatory behaviour was examined by use of selective agonists and antagonists acting at these 5-HT receptor subtypes.The 5-HT1A receptor agonist 8-OH-DPAT (0.25–4.00 μmol kg−1 s.c.) produced an expected facilitation of the male rat ejaculatory behaviour, and this effect was fully antagonized by pretreatment with the new selective 5-HT1A receptor antagonist (R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide hydrogen (2R,3R) tartrate monohydrate (NAD-299) (1.0 μmol kg−1 s.c.). NAD-299 by itself (0.75–3.00 μmol kg−1 s.c.) did not affect the male rat ejaculatory behaviour.The 5-HT1B receptor agonist anpirtoline (0.25–4.00 μmol kg−1 s.c.) produced a dose-dependent inhibition of the male rat ejaculatory behaviour, and this effect was fully antagonized by pretreatment with the 5-HT1B receptor antagonist isamoltane (16 μmol kg−1 s.c.) as well as by the new and selective antagonist (R)-(+)-2-(3-morpholinomethyl-2H-chromene-8-yl)oxymethylmorpholino methansulphonate (NAS-181) (16 μmol kg−1 s.c.). Isamoltane (1.0–16.0 μmol kg−1 s.c.) and NAD-181 (1.0–16.0 μmol kg−1 s.c.) had no, or weakly facilitatory effects on the male rat ejaculatory behaviour. The non-selective 5-HT1 receptor antagonist (−)-pindolol (8 μmol kg−1 s.c.), did not antagonize the inhibition produced by anpirtoline.The present results demonstrate opposite effects, facilitation and inhibition, of male rat ejaculatory behaviour by stimulation of 5-HT1A and 5-HT1B receptors, respectively, suggesting that the SSRI-induced inhibition of male ejaculatory dysfunction is due to 5-HT1B receptor stimulation. PMID:9886765

  19. GABAA/Benzodiazepine receptor binding in patients with schizophrenia using [11C]Ro15-4513, a radioligand with relatively high affinity for alpha5 subunit.

    Science.gov (United States)

    Asai, Yoshiyuki; Takano, Akihiro; Ito, Hiroshi; Okubo, Yoshiro; Matsuura, Masato; Otsuka, Akihiko; Takahashi, Hidehiko; Ando, Tomomichi; Ito, Shigeo; Arakawa, Ryosuke; Asai, Kunihiko; Suhara, Tetsuya

    2008-02-01

    Dysfunction of the GABA system is considered to play a role in the pathology of schizophrenia. Individual subunits of GABA(A)/Benzodiazepine (BZ) receptor complex have been revealed to have different functional properties. alpha5 subunit was reported to be related to learning and memory. Changes of alpha5 subunit in schizophrenia were reported in postmortem studies, but the results were inconsistent. In this study, we examined GABA(A)/BZ receptor using [(11)C]Ro15-4513, which has relatively high affinity for alpha5 subunit, and its relation to clinical symptoms in patients with schizophrenia. [(11)C]Ro15-4513 bindings of 11 patients with schizophrenia (6 drug-naïve and 5 drug-free) were compared with those of 12 age-matched healthy control subjects using positron emission tomography. Symptoms were assessed using the Positive and Negative Syndrome Scale. [(11)C]Ro15-4513 binding was quantified by binding potential (BP) obtained by the reference tissue model. [(11)C]Ro15-4513 binding in the prefrontal cortex and hippocampus was negatively correlated with negative symptom scores in patients with schizophrenia, although there was no significant difference in BP between patients and controls. GABA(A)/BZ receptor including alpha5 subunit in the prefrontal cortex and hippocampus might be involved in the pathophysiology of negative symptoms of schizophrenia.

  20. Binding site number variation and high-affinity binding consensus of Myb-SANT-like transcription factor Adf-1 in Drosophilidae.

    Science.gov (United States)

    Lang, Michael; Juan, Elvira

    2010-10-01

    There is a growing interest in the evolution of transcription factor binding sites and corresponding functional change of transcriptional regulation. In this context, we have examined the structural changes of the ADF-1 binding sites at the Adh promoters of Drosophila funebris and D. virilis. We detected an expanded footprinted region in D. funebris that contains various adjacent binding sites with different binding affinities. ADF-1 was described to direct sequence-specific DNA binding to sites consisting of the multiple trinucleotide repeat . The ADF-1 recognition sites with high binding affinity differ from this trinucleotide repeat consensus sequence and a new consensus sequence is proposed for the high-affinity ADF-1 binding sites. In vitro transcription experiments with the D. funebris and D. virilis ADF-1 binding regions revealed that stronger ADF-1 binding to the expanded D. funebris ADF-1 binding region only moderately lead to increased transcriptional activity of the Adh gene. The potential of this regional expansion is discussed in the context of different ADF-1 cellular concentrations and maintenance of the ADF-1 stimulus. Altogether, evolutionary change of ADF-1 binding regions involves both, rearrangements of complex binding site cluster and also nucleotide substitutions within sites that lead to different binding affinities.

  1. High-affinity choline uptake (HACU) and choline acetyltransferase (ChAT) activity in neuronal cultures for mechanistic and drug discovery studies.

    Science.gov (United States)

    Ray, Balmiki; Bailey, Jason A; Simon, Jay R; Lahiri, Debomoy K

    2012-07-01

    Acetylcholine (ACh) is the neurotransmitter used by cholinergic neurons at the neuromuscular junction, in parasympathetic peripheral nerve terminals, and in important memory-related circuits in the brain, and takes part in other critical functions. ACh is synthesized from choline and acetyl coenzyme A by the enzyme choline acetyltransferase (ChAT). The formation of ACh in cholinergic nerve terminals requires the transport of choline into cells from the extracellular space and the activity of ChAT. High-affinity choline uptake (HACU) represents the majority of choline uptake into the nerve terminal and is the acutely regulated, rate-limiting step in ACh synthesis. HACU can be differentiated from nonspecific choline uptake by inhibition of the choline transporter with hemicholinium. Several methods have been described previously to measure HACU and ChAT activity simultaneously in synaptosomes, but a well-documented protocol for cultured cells is lacking. We describe a procedure for simultaneous measurement of HACU and ChAT in cultured cells by simple radionuclide-based techniques. Using this procedure, we have quantitatively determined HACU and ChAT activity in cholinergically differentiated human neuroblastoma (SK-N-SH) cells. These simple methods can be used for neurochemical and drug discovery studies relevant to several disorders, including Alzheimer's disease, myasthenia gravis, and cardiovascular disease.

  2. Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Warner, Marvin G.; Grate, Jay W.; Tyler, Abby J.; Ozanich, Richard M.; Miller, Keith D.; Lou, Jianlong; Marks, James D.; Bruckner-Lea, Cindy J.

    2009-09-01

    A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment was demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.

  3. The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1, Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo

    Directory of Open Access Journals (Sweden)

    Tatiana P. Sankova

    2017-11-01

    Full Text Available There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in Escherichia coli cells was constructed from glutathione-S-transferase (GST and the N-terminal domain (ectodomain of human high affinity copper transporter CTR1 (hNdCTR1, which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and metallic silver. In this bacterial population, filamentous bacteria with a length of about 10 µm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell’s copper metabolism and its chelating properties are discussed.

  4. The Mitochondrial Metallochaperone SCO1 Is Required to Sustain Expression of the High-Affinity Copper Transporter CTR1 and Preserve Copper Homeostasis

    Directory of Open Access Journals (Sweden)

    Christopher J. Hlynialuk

    2015-02-01

    Full Text Available Human SCO1 fulfills essential roles in cytochrome c oxidase (COX assembly and the regulation of copper (Cu homeostasis, yet it remains unclear why pathogenic mutations in this gene cause such clinically heterogeneous forms of disease. Here, we establish a Sco1 mouse model of human disease and show that ablation of Sco1 expression in the liver is lethal owing to severe COX and Cu deficiencies. We further demonstrate that the Cu deficiency is explained by a functional connection between SCO1 and CTR1, the high-affinity transporter that imports Cu into the cell. CTR1 is rapidly degraded in the absence of SCO1 protein, and we show that its levels are restored in Sco1−/− mouse embryonic fibroblasts upon inhibition of the proteasome. These data suggest that mitochondrial signaling through SCO1 provides a post-translational mechanism to regulate CTR1-dependent Cu import into the cell, and they further underpin the importance of mitochondria in cellular Cu homeostasis.

  5. Induction of high-affinity IgE receptor on lung dendritic cells during viral infection leads to mucous cell metaplasia.

    Science.gov (United States)

    Grayson, Mitchell H; Cheung, Dorothy; Rohlfing, Michelle M; Kitchens, Robert; Spiegel, Daniel E; Tucker, Jennifer; Battaile, John T; Alevy, Yael; Yan, Le; Agapov, Eugene; Kim, Edy Y; Holtzman, Michael J

    2007-10-29

    Respiratory viral infections are associated with an increased risk of asthma, but how acute Th1 antiviral immune responses lead to chronic inflammatory Th2 disease remains undefined. We define a novel pathway that links transient viral infection to chronic lung disease with dendritic cell (DC) expression of the high-affinity IgE receptor (FcepsilonRIalpha). In a mouse model of virus-induced chronic lung disease, in which Sendai virus triggered a switch to persistent mucous cell metaplasia and airway hyperreactivity after clearance of replicating virus, we found that FceRIa(-/-) mice no longer developed mucous cell metaplasia. Viral infection induced IgE-independent, type I IFN receptor-dependent expression of FcepsilonRIalpha on mouse lung DCs. Cross-linking DC FcepsilonRIalpha resulted in the production of the T cell chemoattractant CCL28. FceRIa(-/-) mice had decreased CCL28 and recruitment of IL-13-producing CD4(+) T cells to the lung after viral infection. Transfer of wild-type DCs to FceRIa(-/-) mice restored these events, whereas blockade of CCL28 inhibited mucous cell metaplasia. Therefore, lung DC expression of FcepsilonRIalpha is part of the antiviral response that recruits CD4(+) T cells and drives mucous cell metaplasia, thus linking antiviral responses to allergic/asthmatic Th2 responses.

  6. Characterization of dendritic cell phenotype in allergic conjunctiva: increased expression of Fc(epsilon)RI, the high-affinity receptor for immunoglobulin E.

    Science.gov (United States)

    Manzouri, B; Ohbayashi, M; Leonardi, A; Larkin, D F P; Ono, S J

    2009-11-01

    Dendritic cells (DCs) express the high-affinity receptor for IgE (Fc(epsilon)RI) on their surface, which may enhance their ability to capture and internalize antigens for presentation to T-lymphocytes. The aim of this study was to determine if expression of Fc(epsilon)RI(+) DCs is increased in the conjunctivae of vernal keratoconjunctivitis (VKC) patients compared with those of normal controls. Conjunctival biopsies were obtained from non-atopic and VKC patients. Double immunohistochemical staining was carried out using antibodies against Fc(epsilon)RI and the CD1a antigen, a DC marker. The double-positive cells were counted in five representative fields of view for each conjunctival sample. Fc(epsilon)RI(+) CD1a(+) cells were present in significantly higher numbers in VKC conjunctivae compared with normal controls (mean cell count of 21.3 in VKC vs5.0 in controls, PRI-expressing DCs tended to be confined to the epithelial layer or the superficial substantia propria, but in the VKC samples these Fc(epsilon)RI(+) cells were mainly concentrated in the deeper substantia propria. Fc(epsilon)RI(+) DC numbers are elevated in the conjunctivae of VKC patients, a finding consistent with the results of other studies focusing on atopic conditions. Elevated expression of Fc(epsilon)RI on DCs would facilitate antigen presentation and enhance T-cell priming, thereby contributing to ocular symptoms.

  7. Botrytis cinerea can import and utilize nucleosides in salvage and catabolism and BcENT functions as high affinity nucleoside transporter.

    Science.gov (United States)

    Daumann, Manuel; Golfier, Philippe; Knüppel, Nathalie; Hahn, Matthias; Möhlmann, Torsten

    2016-08-01

    Nucleotide de novo synthesis is an essential pathway in nearly all organisms. Transport processes as well as salvage and catabolism of nucleotides and pathway intermediates are required to balance nucleotide pools. We have analysed the genome of the fungal plant pathogen Botrytis cinerea for genes involved in nucleotide metabolism and found a complete set of genes necessary for purine and pyrimidine uptake and salvage based on homology of the gene products to corresponding proteins from Aspergillus nidulans. Candidate genes required for a complete purine catabolic sequence were identified in addition. These analyses were complemented by growth tests showing functional transport and salvage activity for pyrimidines. Growth of B. cinerea mycelium in nitrogen free medium could be restored by addition of purines, indicating the presence of a functional purine catabolism, whereas pyrimidines did not support growth. Bcin07g05490 (BcENT) was identified as sole member of the equilibrative nucleoside transporter (ENT) family. The protein synthesized in Saccharomyces cerevisiae revealed high affinity transport of adenosine (KM = 6.81 μM) and uridine (KM=9.04 μM). Furthermore, a BcENT knockout mutant was generated and tested in a range of growth and infection assays. These results provide detailed insight in the use of externally supplied nucleobases and nucleosides by B. cinerea. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  8. Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status

    Science.gov (United States)

    Bao, Aili; Liang, Zhijun; Zhao, Zhuqing; Cai, Hongmei

    2015-01-01

    AMT1-3 encodes the high affinity NH4+ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants. PMID:25915023

  9. Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status

    Directory of Open Access Journals (Sweden)

    Aili Bao

    2015-04-01

    Full Text Available AMT1-3 encodes the high affinity NH4+ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants.

  10. LysGH15B, the SH3b domain of staphylococcal phage endolysin LysGH15, retains high affinity to staphylococci.

    Science.gov (United States)

    Gu, Jingmin; Lu, Rong; Liu, Xiaohe; Han, Wenyu; Lei, Liancheng; Gao, Yu; Zhao, Honglei; Li, Yue; Diao, Yuwen

    2011-12-01

    LysGH15, a phage endolysin, exhibits a particularly broad lytic spectrum against Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA). Sequence analysis reveals that this endolysin contains a C-terminal cell wall binding domain (SH3b), which causes the endolysin to bind to host strains. In this study, the substrate binding affinity of the SH3b domain (LysGH15B) was evaluated. A fusion protein of LysGH15B and green fluorescent protein (LysGH15B-GFP) were cloned and expressed in Escherichia coli. Laser scanning confocal microscopy was used to detect the fluorescence of the treated cells irradiated at different excitation wavelengths and to determine the binding activity of LysGH15B-GFP and GFP. We found that LysGH15B-GFP not only generated green fluorescence, but, more importantly, also displayed specific affinity to staphylococcal isolates, especially MRSA. In contrast, the single GFP did not display any binding activity. The high affinity was attributed to the portion of LysGH15B and the binding activity of the fusion protein was specific to staphylococci. This study provides an insight into the SH3b domain of LysGH15. The specific binding activity may cause LysGH15B to serve as an anchoring device, and offer an alternative approach for cell surface attachment onto staphylococci.

  11. The glucose sensor-like protein Hxs1 is a high-affinity glucose transporter and required for virulence in Cryptococcus neoformans.

    Directory of Open Access Journals (Sweden)

    Tong-Bao Liu

    Full Text Available Cryptococcus is a major fungal pathogen that frequently causes systemic infection in patients with compromised immunity. Glucose, an important signal molecule and the preferred carbon source for Cryptococcus, plays a critical role in fungal development and virulence. Cryptococcus contains more than 50 genes sharing high sequence homology with hexose transporters in Saccharomyces cerevisiae. However, there is no report on their function in glucose sensing or transport. In this study, we investigated two hexose transporter-like proteins (Hxs1 and Hxs2 in Cryptococcus that share the highest sequence identity with the glucose sensors Snf3 and Rgt2 in S. cerevisiae. The expression of HXS1 is repressed by high glucose, while the HXS2 expression is not regulated by glucose. Functional studies showed that Hxs1 is required for fungal resistance to oxidative stress and fungal virulence. The hxs1Δ mutant exhibited a significant reduction in glucose uptake activity, indicating that Hxs1 is required for glucose uptake. Heterologous expression of Cryptococcus HXS1 rendered the S. cerevisiae mutant lacking all 20 hexose transporters a high glucose uptake activity, demonstrating that Hxs1 functions as a glucose transporter. Heterologous expression of HXS1 in the snf3Δ rgt2Δ double mutant did not complement its growth in YPD medium containing the respiration inhibitor antimycin A, suggesting that Hxs1 may not function as a glucose sensor. Taken together, our results demonstrate that Hxs1 is a high-affinity glucose transporter and required for fungal virulence.

  12. The Glucose Sensor-Like Protein Hxs1 Is a High-Affinity Glucose Transporter and Required for Virulence in Cryptococcus neoformans

    Science.gov (United States)

    Baker, Gregory M.; Fahmy, Hany; Jiang, Linghuo; Xue, Chaoyang

    2013-01-01

    Cryptococcus is a major fungal pathogen that frequently causes systemic infection in patients with compromised immunity. Glucose, an important signal molecule and the preferred carbon source for Cryptococcus, plays a critical role in fungal development and virulence. Cryptococcus contains more than 50 genes sharing high sequence homology with hexose transporters in Saccharomyces cerevisiae. However, there is no report on their function in glucose sensing or transport. In this study, we investigated two hexose transporter-like proteins (Hxs1 and Hxs2) in Cryptococcus that share the highest sequence identity with the glucose sensors Snf3 and Rgt2 in S. cerevisiae. The expression of HXS1 is repressed by high glucose, while the HXS2 expression is not regulated by glucose. Functional studies showed that Hxs1 is required for fungal resistance to oxidative stress and fungal virulence. The hxs1Δ mutant exhibited a significant reduction in glucose uptake activity, indicating that Hxs1 is required for glucose uptake. Heterologous expression of Cryptococcus HXS1 rendered the S. cerevisiae mutant lacking all 20 hexose transporters a high glucose uptake activity, demonstrating that Hxs1 functions as a glucose transporter. Heterologous expression of HXS1 in the snf3Δ rgt2Δ double mutant did not complement its growth in YPD medium containing the respiration inhibitor antimycin A, suggesting that Hxs1 may not function as a glucose sensor. Taken together, our results demonstrate that Hxs1 is a high-affinity glucose transporter and required for fungal virulence. PMID:23691177

  13. The glucose sensor-like protein Hxs1 is a high-affinity glucose transporter and required for virulence in Cryptococcus neoformans.

    Science.gov (United States)

    Liu, Tong-Bao; Wang, Yina; Baker, Gregory M; Fahmy, Hany; Jiang, Linghuo; Xue, Chaoyang

    2013-01-01

    Cryptococcus is a major fungal pathogen that frequently causes systemic infection in patients with compromised immunity. Glucose, an important signal molecule and the preferred carbon source for Cryptococcus, plays a critical role in fungal development and virulence. Cryptococcus contains more than 50 genes sharing high sequence homology with hexose transporters in Saccharomyces cerevisiae. However, there is no report on their function in glucose sensing or transport. In this study, we investigated two hexose transporter-like proteins (Hxs1 and Hxs2) in Cryptococcus that share the highest sequence identity with the glucose sensors Snf3 and Rgt2 in S. cerevisiae. The expression of HXS1 is repressed by high glucose, while the HXS2 expression is not regulated by glucose. Functional studies showed that Hxs1 is required for fungal resistance to oxidative stress and fungal virulence. The hxs1Δ mutant exhibited a significant reduction in glucose uptake activity, indicating that Hxs1 is required for glucose uptake. Heterologous expression of Cryptococcus HXS1 rendered the S. cerevisiae mutant lacking all 20 hexose transporters a high glucose uptake activity, demonstrating that Hxs1 functions as a glucose transporter. Heterologous expression of HXS1 in the snf3Δ rgt2Δ double mutant did not complement its growth in YPD medium containing the respiration inhibitor antimycin A, suggesting that Hxs1 may not function as a glucose sensor. Taken together, our results demonstrate that Hxs1 is a high-affinity glucose transporter and required for fungal virulence.

  14. H2-saturation of high affinity H2-oxidizing bacteria alters the ecological niche of soil microorganisms unevenly among taxonomic groups

    Directory of Open Access Journals (Sweden)

    Sarah Piché-Choquette

    2016-03-01

    Full Text Available Soil microbial communities are continuously exposed to H2 diffusing into the soil from the atmosphere. N2-fixing nodules represent a peculiar microniche in soil where H2 can reach concentrations up to 20,000 fold higher than in the global atmosphere (0.530 ppmv. In this study, we investigated the impact of H2 exposure on soil bacterial community structure using dynamic microcosm chambers simulating soil H2 exposure from the atmosphere and N2-fixing nodules. Biphasic kinetic parameters governing H2 oxidation activity in soil changed drastically upon elevated H2 exposure, corresponding to a slight but significant decay of high affinity H2-oxidizing bacteria population, accompanied by an enrichment or activation of microorganisms displaying low-affinity for H2. In contrast to previous studies that unveiled limited response by a few species, the relative abundance of 958 bacterial ribotypes distributed among various taxonomic groups, rather than a few distinct taxa, was influenced by H2 exposure. Furthermore, correlation networks showed important alterations of ribotype covariation in response to H2 exposure, suggesting that H2 affects microbe-microbe interactions in soil. Taken together, our results demonstrate that H2-rich environments exert a direct influence on soil H2-oxidizing bacteria in addition to indirect effects on other members of the bacterial communities.

  15. Isolation of a high affinity neutralizing monoclonal antibody against 2009 pandemic H1N1 virus that binds at the 'Sa' antigenic site.

    Directory of Open Access Journals (Sweden)

    Nachiket Shembekar

    Full Text Available Influenza virus evades host immunity through antigenic drift and shift, and continues to circulate in the human population causing periodic outbreaks including the recent 2009 pandemic. A large segment of the population was potentially susceptible to this novel strain of virus. Historically, monoclonal antibodies (MAbs have been fundamental tools for diagnosis and epitope mapping of influenza viruses and their importance as an alternate treatment option is also being realized. The current study describes isolation of a high affinity (K(D = 2.1±0.4 pM murine MAb, MA2077 that binds specifically to the hemagglutinin (HA surface glycoprotein of the pandemic virus. The antibody neutralized the 2009 pandemic H1N1 virus in an in vitro microneutralization assay (IC(50 = 0.08 µg/ml. MA2077 also showed hemagglutination inhibition activity (HI titre of 0.50 µg/ml against the pandemic virus. In a competition ELISA, MA2077 competed with the binding site of the human MAb, 2D1 (isolated from a survivor of the 1918 Spanish flu pandemic on pandemic H1N1 HA. Epitope mapping studies using yeast cell-surface display of a stable HA1 fragment, wherein 'Sa' and 'Sb' sites were independently mutated, localized the binding site of MA2077 within the 'Sa' antigenic site. These studies will facilitate our understanding of antigen antibody interaction in the context of neutralization of the pandemic influenza virus.

  16. Premature Aging Phenotype in Mice Lacking High-Affinity Nicotinic Receptors: Region-Specific Changes in Layer V Pyramidal Cell Morphology.

    Science.gov (United States)

    Konsolaki, Eleni; Skaliora, Irini

    2015-08-01

    The mechanisms by which aging leads to alterations in brain structure and cognitive deficits are unclear. Α deficient cholinergic system has been implicated as one of the main factors that could confer a heightened vulnerability to the aging process, and mice lacking high-affinity nicotinic receptors (β2(-/-)) have been proposed as an animal model of accelerated cognitive aging. To date, however, age-related changes in neuronal microanatomy have not been studied in these mice. In the present study, we examine the neuronal structure of yellow fluorescent protein (YFP(+)) layer V neurons in 2 cytoarchitectonically distinct cortical regions in wild-type (WT) and β2(-/-) animals. We find that (1) substantial morphological differences exist between YFP(+) cells of the anterior cingulate cortex (ACC) and primary visual cortex (V1), in both genotypes; (2) in WT animals, ACC cells are more susceptible to aging compared with cells in V1; and (3) β2 deletion is associated with a regionally and temporally specific increase in vulnerability to aging. ACC cells exhibit a prematurely aged phenotype already at 4-6 months, whereas V1 cells are spared in adulthood but strongly affected in old animals. Collectively, our data reveal region-specific synergistic effects of aging and genotype and suggest distinct vulnerabilities in V1 and ACC neurons. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Evaluation of the Agonist PET Radioligand [11C]GR103545 to Image Kappa Opioid Receptor in Humans: Kinetic Model Selection, Test-Retest Reproducibility and Receptor Occupancy by the Antagonist PF-04455242

    Science.gov (United States)

    Naganawa, Mika; Jacobsen, Leslie K.; Zheng, Ming-Qiang; Lin, Shu-Fei; Banerjee, Anindita; Byon, Wonkyung; Weinzimmer, David; Tomasi, Giampaolo; Nabulsi, Nabeel; Grimwood, Sarah; Badura, Lori L.; Carson, Richard E.; McCarthy, Timothy J.; Huang, Yiyun

    2014-01-01

    Introduction Kappa opioid receptors (KOR) are implicated in several brain disorders. In this report, a first-in-human Positron Emission Tomography (PET) study was conducted with the potent and selective KOR agonist tracer, [11C]GR103545, to determine an appropriate kinetic model for analysis of PET imaging data and assess the test-retest reproducibility of model-derived binding parameters. The non-displaceable distribution volume (VND) was estimated from a blocking study with naltrexone. In addition, KOR occupancy of PF-04455242, a selective KOR antagonist that is active in preclinical models of depression, was also investigated. Methods For determination of a kinetic model and evaluation of test-retest reproducibility, 11 subjects were scanned twice with [11C]GR103545. Seven subjects were scanned before and 75 min after oral administration of naltrexone (150 mg). For the KOR occupancy study, six subjects were scanned at baseline and 1.5 h and 8 h after an oral dose of PF-04455242 (15 mg, n = 1 and 30 mg, n = 5). Metabolite-corrected arterial input functions were measured and all scans were 150 min in duration. Regional time-activity curves (TACs) were analyzed with 1- and 2-tissue compartment models (1TC and 2TC) and the multilinear analysis (MA1) method to derive regional volume of distribution (VT). Relative test-retest variability (TRV), absolute test-retest variability (aTRV) and intra-class coefficient (ICC) were calculated to assess test-retest reproducibility of regional VT. Occupancy plots were computed for blocking studies to estimate occupancy and VND. The half maximal inhibitory concentration (IC50) of PF-04455242 was determined from occupancies and drug concentrations in plasma. [11C]GR103545 in vivo KD was also estimated. Results Regional TACs were well described by the 2TC model and MA1. However, 2TC VT was sometimes estimated with high standard error. Thus MA1 was the model of choice. Test-retest variability was ~15%, depending on the outcome

  18. Effects of muscarinic receptor antagonists on cocaine discrimination in wild-type mice and in muscarinic receptor M1, M2, and M4 receptor knockout mice

    DEFF Research Database (Denmark)

    Joseph, Lauren; Thomsen, Morgane

    2017-01-01

    Muscarinic M1/M4 receptor stimulation can reduce abuse-related effects of cocaine and may represent avenues for treating cocaine addiction. Muscarinic antagonists can mimic and enhance effects of cocaine, including discriminative stimulus (SD) effects, but the receptor subtypes mediating those...... effects are not known. A better understanding of the complex cocaine/muscarinic interactions is needed to evaluate and develop potential muscarinic-based medications. Here, knockout mice lacking M1, M2, or M4 receptors (M1-/-, M2-/-, M4-/-), as well as control wild-type mice and outbred Swiss-Webster mice...... with cocaine. In intact animals, antagonists with high affinity at M1/M4 receptors partially substituted for cocaine and increased the SD effect of cocaine, while M2-preferring antagonists did not substitute, and reduced the SD effect of cocaine. The cocaine-like effects of scopolamine were absent in M1...

  19. Abiotic conditions affect floral antagonists and mutualists of Impatiens capensis (Balsaminaceae).

    Science.gov (United States)

    Soper Gorden, Nicole L; Adler, Lynn S

    2013-04-01

    While the effect of abiotic factors on leaf herbivory is well known, the relative importance of abiotic conditions influencing both mutualists and antagonists is less well understood. Species interactions could enhance or reduce the direct effects of abiotic factors, depending on how mutualists and antagonists respond to abiotic conditions. We manipulated soil nutrients and shade in a factorial design and measured soil moisture in the annual Impatiens capensis. We then measured interactions with mutualists (two pollinating species) and antagonists (herbivores, florivores, nectar thieves, and flower bud gallers), as well as plant growth, floral rewards, and plant reproduction. Fertilizer increased plant growth, floral attractiveness, mutualist and antagonist interactions, and plant reproduction. Shade had no effects, and soil moisture was negatively associated with plant growth and reproduction. All effects were additive. Mutualist and antagonist floral interactions both increased on fertilized plants, but antagonists increased at a greater rate, leading to a larger ratio of antagonist to mutualist interactions on fertilized plants. Despite having more antagonists, fertilized plants still had significantly higher reproduction, suggesting higher tolerance to antagonists. Abiotic effects can have consistent effects on antagonists and mutualists, and on both floral and leaf antagonists. However, tolerance to antagonisms increased in favorable conditions. Thus, the direct positive effects of favorable abiotic conditions on plants outweighed negative indirect effects via increased antagonisms, which may lead to selection to grow in high-nutrient microsites in spite of increased herbivory.

  20. Synthesis of N-substituted 4-(4-hydroxyphenyl)piperidines, 4-(4-hydroxybenzyl)piperidines, and (+/-)-3-(4-hydroxyphenyl)pyrrolidines: selective antagonists at the 1A/2B NMDA receptor subtype.

    Science.gov (United States)

    Guzikowski, A P; Tamiz, A P; Acosta-Burruel, M; Hong-Bae, S; Cai, S X; Hawkinson, J E; Keana, J F; Kesten, S R; Shipp, C T; Tran, M; Whittemore, E R; Woodward, R M; Wright, J L; Zhou, Z L

    2000-03-09

    Antagonists at the 1A/2B subtype of the NMDA receptor (NR1A/2B) are typically small molecules that consist of a 4-benzyl- or a 4-phenylpiperidine with an omega-phenylalkyl substituent on the heterocyclic nitrogen. Many of these antagonists, for example ifenprodil (1), incorporate a 4-hydroxy substituent on the omega-phenyl group. In this study, the position of this 4-hydroxy substituent was transferred from the omega-phenyl group to the benzyl or phenyl group located on the 4-position of the piperidine ring. Analogues incorporating pyrrolidine in lieu of piperidine were also prepared. Electrical recordings using cloned receptors expressed in Xenopus oocytes show that high-potency antagonists at the NR1A/2B subtype are obtained employing N-(omega-phenylalkyl)-substituted 4-(4-hydroxyphenyl)piperidine, 4-(4-hydroxybenzyl)piperidine, and (+/-)-3-(4-hydroxyphenyl)pyrrolidine as exemplified by 21 (IC(50) = 0.022 microM), 33 (IC(50) = 0.059 microM), and 40 (IC(50) = 0.017 microM), respectively. These high-potency antagonists are >1000 times more potent at the NR1A/2B subtype than at either the NR1A/2A or NR1A/2C subtypes. The binding affinities of 21 at alpha(1)-adrenergic receptors ([(3)H]prazosin, IC(50) = 0.54 microM) and dopamine D2 receptors ([(3)H]raclopride, IC(50) = 1.2 microM) are reduced by incorporating a hydroxy group onto the 4-position of the piperidine ring and the beta-carbon of the N-alkyl spacer to give (+/-)-27: IC(50) NR1A/2B, 0.026; alpha(1), 14; D2, 105 microM. The high-potency phenolic antagonist 21 and its low-potency O-methylated analogue 18 are both potent anticonvulsants in a mouse maximal electroshock-induced seizure (MES) study (ED(50) (iv) = 0.23 and 0.56 mg/kg, respectively). These data indicate that such compounds penetrate the blood-brain barrier but their MES activity may not be related to NMDA receptor antagonism.

  1. alpha2-Adrenoceptor antagonists reverse the 5-HT2 receptor antagonist suppression of head-twitch behavior in mice.

    Science.gov (United States)

    Matsumoto, K; Mizowaki, M; Thongpraditchote, S; Murakami, Y; Watanabe, H

    1997-03-01

    The alpha2-adrenoceptor agonist clonidine, as well as 5-HT2 receptor antagonists, reportedly suppress 5-HT2 receptor-mediated head-twitch behavior. We investigated the effect of alpha2-adrenoceptor antagonists on the suppressive action of 5-HT2 receptor antagonists in mice pretreated with the noradrenaline toxin 6-hydroxydopamine (6-OHDA) or the 5-HT synthesis inhibitor p-chlorophenylalanine (p-CPA). In normal mice, idazoxan (0.08-0.2 mg/kg, IP) or yohimbine (0.2-2.0 mg/kg, IP), both alpha2-adrenoceptor antagonists, had no effect on the head-twitch response caused by 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT; 16 mg/kg, IP), but idazoxan significantly enhanced the response at 0.5 mg/kg. On the other hand, these alpha2-adrenoceptor antagonists, at doses that had no effect on the basal number of head-twitches (idazoxan 0.2 mg/kg and yohimbine 0.5 mg/kg), significantly attenuated not only the suppressive effect of clonidine (0.01 mg/kg, IP) on head-twitch response but also that of the 5-HT2 receptor antagonist ritanserin (0.03 mg/kg, IP). Moreover, idazoxan (0.2 mg/kg) also significantly reversed the inhibition by 0.01 mg/kg (IP) ketanserin, a selective 5-HT2 receptor antagonist. Pretreatment with 6-OHDA plus nomifensine but not with p-CPA significantly attenuated the effect of idazoxan (0.2-0.5 mg/kg) on the ritanserin inhibition of the head-twitch response. Prazosin, an alpha1-adrenoceptor antagonist, dose-dependently suppressed the response, and the effect of prazosin (1.25 mg/kg) was significantly attenuated by 0.5 mg/kg idazoxan. These results indicate that endogenous noradrenaline is involved in the apparent antagonistic interaction between selective alpha2-adrenoceptor antagonists and 5-HT2 receptor antagonists in the head-twitch response, and suggest that noradrenaline stimulation of alpha1-adrenoceptors may be involved in this apparent antagonism.

  2. Design, Synthesis, and in Vitro Pharmacology of New Radiolabeled γ-Hydroxybutyric Acid Analogues Including Photolabile Analogues with Irreversible Binding to the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Sabbatini, Paola; Wellendorph, Petrine; Høg, Signe

    2010-01-01

    γ-Hydroxybutyric acid (GHB) is a psychotropic compound endogenous to the brain. Despite its potential physiological significance, the complete molecular mechanisms of action remain unexplained. To facilitate the isolation and identification of the high-affinity GHB binding site, we herein report...

  3. AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists

    Directory of Open Access Journals (Sweden)

    Terry W. Moody

    2017-07-01

    Full Text Available While peptide antagonists for the gastrin-releasing peptide receptor (BB2R, neuromedin B receptor (BB1R, and bombesin (BB receptor subtype-3 (BRS-3 exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA1 binds with high affinity to the BB1R, BB2R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB1R, BB2R, and BRS-3 with similar affinity (Ki = 1.4–10.8 µM. AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca2+ in human lung cancer cells transfected with BB1R, BB2R, and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells, which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells that have BBR. The results indicate that AM-37 and ST-36 function as small molecule BB receptor antagonists.

  4. The UL8 subunit of the helicase-primase complex of herpes simplex virus promotes DNA annealing and has a high affinity for replication forks.

    Science.gov (United States)

    Bermek, Oya; Weller, Sandra K; Griffith, Jack D

    2017-09-22

    During lytic infection, herpes simplex virus (HSV) DNA is replicated by a mechanism involving DNA recombination. For instance, replication of the HSV-1 genome produces X- and Y-branched structures, reminiscent of recombination intermediates. HSV-1's replication machinery includes a trimeric helicase-primase composed of helicase (UL5) and primase (UL52) subunits and a third subunit, UL8. UL8 has been reported to stimulate the helicase and primase activities of the complex in the presence of ICP8, an HSV-1 protein that functions as an annealase, a protein that binds complementary single-stranded DNA (ssDNA) and facilitates its annealing to duplex DNA. UL8 also influences the intracellular localization of the UL5/UL52 subunits, but UL8's catalytic activities are not known. In this study we used a combination of biochemical techniques and transmission electron microscopy. First, we report that UL8 alone forms protein filaments in solution. Moreover, we also found that UL8 binds to ssDNAs >50-nucletides long and promotes the annealing of complementary ssDNA to generate highly branched duplex DNA structures. Finally, UL8 has a very high affinity for replication fork structures containing a gap in the lagging strand as short as 15 nucleotides, suggesting that UL8 may aid in directing or loading the trimeric complex onto a replication fork. The properties of UL8 uncovered here suggest that UL8 may be involved in the generation of the X- and Y-branched structures that are the hallmarks of HSV replication. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome

    Science.gov (United States)

    Draberova, Lubica; Paulenda, Tomas; Halova, Ivana; Potuckova, Lucie; Bugajev, Viktor; Bambouskova, Monika; Tumova, Magda; Draber, Petr

    2015-01-01

    Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of Fc

  6. FSY1, a horizontally transferred gene in the Saccharomyces cerevisiae EC1118 wine yeast strain, encodes a high-affinity fructose/H+ symporter.

    Science.gov (United States)

    Galeote, Virginie; Novo, Maïté; Salema-Oom, Madalena; Brion, Christian; Valério, Elisabete; Gonçalves, Paula; Dequin, Sylvie

    2010-12-01

    Transport of glucose and fructose in the yeast Saccharomyces cerevisiae plays a crucial role in controlling the rate of wine fermentation. In S. cerevisiae, hexoses are transported by facilitated diffusion via hexose carriers (Hxt), which prefer glucose to fructose. However, utilization of fructose by wine yeast is critically important at the end of fermentation. Here, we report the characterization of a fructose transporter recently identified by sequencing the genome of the commercial wine yeast strain EC1118 and found in many other wine yeasts. This transporter is designated Fsy1p because of its homology with the Saccharomyces pastorianus fructose/H(+) symporter Fsy1p. A strain obtained by transformation of the V5 hxt1-7Δ mutant with FSY1 grew well on fructose, but to a much lesser extent on glucose as the sole carbon source. Sugar uptake and symport experiments showed that FSY1 encodes a proton-coupled symporter with high affinity for fructose (K(m) 0.24±0.04mM). Using real-time RT-PCR, we also investigated the expression pattern of FSY1 in EC1118 growing on various carbon sources. FSY1 was repressed by high concentrations of glucose or fructose and was highly expressed on ethanol as the sole carbon source. The characteristics of this transporter indicate that its acquisition could confer a significant advantage to S. cerevisiae during the wine fermentation process. This transporter is a good example of acquisition of a new function in yeast by horizontal gene transfer.

  7. Single-chain site-specific mutations of fluorescein-amino acid contact residues in high affinity monoclonal antibody 4-4-20.

    Science.gov (United States)

    Denzin, L K; Whitlow, M; Voss, E W

    1991-07-25

    Previous crystallographic studies of high affinity anti-fluorescein monoclonal antibody 4-4-20 (Ka = 1.7 x 10(10) M-1) complexed with fluorescyl ligand resolved active site contact residues involved in binding. For better definition of the relative roles of three light chain antigen contact residues (L27dhis, L32tyr and L34arg), four site-specific mutations (L27dhis to L27lys, L32tyr to L32phe, and L34arg to L34lys and L34his) were generated and expressed in single-chain antigen binding derivatives of monoclonal antibody 4-4-20 containing two different polypeptide linkers (SCA 4-4-20/205c, 25 amino acids and SCA 4-4-20/212, 14 amino acids). Results showed that L27dhis and L32tyr were necessary for wild type binding affinities, however, were not required for near-wild type Qmax values (where Qmax is the maximum fluoroscein fluorescence quenching expressed as percent). Tyrosine L32 which hydrogen bonds with ligand was also characterized at the haptenic level through the use of 9-hydroxyphenylfluoron which lacks the carboxyl group to which L32 tyrosine forms a hydrogen bond. Results demonstrated that wild type SCA and mutant L32phe possessed similar HPF binding characteristics. Active site contact residue L34arg was important for fluorescein quenching maxima and binding affinity (L34his mutant), however, substitution of lysine for arginine at L34 did not have a significant effect on observed Qmax value. In addition, substitutions had no effect on structural and topological characteristics, since all mutants retained similar idiotypic and metatypic properties. Finally, two linkers were comparatively examined to determine relative contributions to mutant binding properties and stability. No linker effects were observed. Collectively, these results verified the importance of these light chain fluorescein contact residues in the binding pocket of monoclonal antibody 4-4-20.

  8. Development of a high-affinity peptide that prevents phospholemman (PLM) inhibition of the sodium/calcium exchanger 1 (NCX1).

    Science.gov (United States)

    Wanichawan, Pimthanya; Hodne, Kjetil; Hafver, Tandekile Lubelwana; Lunde, Marianne; Martinsen, Marita; Louch, William Edward; Sejersted, Ole Mathias; Carlson, Cathrine Rein

    2016-08-01

    NCX1 (Na(+)/Ca(2+) exchanger 1) is an important regulator of intracellular Ca(2+) and a potential therapeutic target for brain ischaemia and for diastolic heart failure with preserved ejection fraction. PLM (phospholemman), a substrate for protein kinases A and C, has been suggested to regulate NCX1 activity. However, although several studies have demonstrated that binding of phosphorylated PLM (pSer(68)-PLM) leads to NCX1 inhibition, other studies have failed to demonstrate a functional interaction of these proteins. In the present study, we aimed to analyse the biological function of the pSer(68)-PLM-NCX1 interaction by developing high-affinity blocking peptides. PLM was observed to co-fractionate and co-immunoprecipitate with NCX1 in rat left ventricle, and in co-transfected HEK (human embryonic kidney)-293 cells. For the first time, the NCX1-PLM interaction was also demonstrated in the brain. PLM binding sites on NCX1 were mapped to two regions by peptide array assays, containing the previously reported PASKT and QKHPD motifs. Conversely, the two NCX1 regions bound identical sequences in the cytoplasmic domain of PLM, suggesting that NCX1-PASKT and NCX1-QKHPD might bind to each PLM monomer. Using two-dimensional peptide arrays of the native NCX1 sequence KHPDKEIEQLIELANYQVLS revealed that double substitution of tyrosine for positions 1 and 4 (K1Y and D4Y) enhanced pSer(68)-PLM binding 8-fold. The optimized peptide blocked binding of NCX1-PASKT and NCX1-QKHPD to PLM and reversed PLM(S68D) inhibition of NCX1 activity (both forward and reverse mode) in HEK-293 cells. Altogether our data indicate that PLM interacts directly with NCX1 and inhibits NCX1 activity when phosphorylated at Ser(68). © 2016 The Author(s).

  9. High-affinity nitrate/nitrite transporter genes (Nrt2) in Tisochrysis lutea: identification and expression analyses reveal some interesting specificities of Haptophyta microalgae.

    Science.gov (United States)

    Charrier, Aurélie; Bérard, Jean-Baptiste; Bougaran, Gaël; Carrier, Grégory; Lukomska, Ewa; Schreiber, Nathalie; Fournier, Flora; Charrier, Aurélie F; Rouxel, Catherine; Garnier, Matthieu; Cadoret, Jean-Paul; Saint-Jean, Bruno

    2015-08-01

    Microalgae have a diversity of industrial applications such as feed, food ingredients, depuration processes and energy. However, microalgal production costs could be substantially improved by controlling nutrient intake. Accordingly, a better understanding of microalgal nitrogen metabolism is essential. Using in silico analysis from transcriptomic data concerning the microalgae Tisochrysis lutea, four genes encoding putative high-affinity nitrate/nitrite transporters (TlNrt2) were identified. Unlike most of the land plants and microalgae, cloning of genomic sequences and their alignment with complementary DNA (cDNA) sequences did not reveal the presence of introns in all TlNrt2 genes. The deduced TlNRT2 protein sequences showed similarities to NRT2 proteins of other phyla such as land plants and green algae. However, some interesting specificities only known among Haptophyta were also revealed, especially an additional sequence of 100 amino acids forming an atypical extracellular loop located between transmembrane domains 9 and 10 and the function of which remains to be elucidated. Analyses of individual TlNrt2 gene expression with different nitrogen sources and concentrations were performed. TlNrt2.1 and TlNrt2.3 were strongly induced by low NO3 (-) concentration and repressed by NH4 (+) substrate and were classified as inducible genes. TlNrt2.2 was characterized by a constitutive pattern whatever the substrate. Finally, TlNrt2.4 displayed an atypical response that was not reported earlier in literature. Interestingly, expression of TlNrt2.4 was rather related to internal nitrogen quota level than external nitrogen concentration. This first study on nitrogen metabolism of T. lutea opens avenues for future investigations on the function of these genes and their implication for industrial applications. © 2015 Scandinavian Plant Physiology Society.

  10. Certain photooxidized derivatives of tryptophan bind with very high affinity to the Ah receptor and are likely to be endogenous signal substances

    Energy Technology Data Exchange (ETDEWEB)

    Rannug, A.; Rannug, U.; Rosenkranz, H.S.; Winqvist, L.; Westerholm, R.; Agurell, E.; Grafstroem, A.K.

    1987-11-15

    The purpose of the present study was to determine whether ultraviolet light (UV) irradiation of amino acids produces compounds with affinity for the Ah receptor. Aqueous solutions of L-tryptophan were exposed to radiation from an unfiltered high-pressure mercury lamp. The photoproducts formed were solvent-extracted or concentrated on Sep-Pak C18 cartridges. The concentrated extracts or eluants were treated for their ability to compete with /sup 3/H-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Binding was assayed in liver cytosolic preparations from Sprague-Dawley rats using a technique based on hydroxylapatite separation. Photoproducts with receptor affinity were formed in a time-dependent manner. Histidine and tryptamine also gave products upon UV irradiation that competed with TCDD. Commercial tryptophan, at least aged, contained trace amounts of impurities with receptor affinity. Analysis by TLC and high-pressure liquid chromatography of the photo-products of tryptophan showed a minimum of three different binding compounds. Two of the products were studied in greater detail. One of them, showing UV absorbance and yellow fluorescence, gave a molecular ion (M+) of 284 and the other gave M+ 312 but showed little UV absorption and fluorescence. The concentration, based on mass spectrometry quantifications, of the two compounds that displaced more than 50% of TCDD was found to be extremely low, giving Kd values of 0.44 nM (M+ 312) and 0.07 nM (M+ 284). The existence of high affinity receptors for oxidized amino acids is postulated and their possible role in the proliferative cellular responses to TCDD and tryptophan is discussed briefly.

  11. Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome.

    Science.gov (United States)

    Draberova, Lubica; Paulenda, Tomas; Halova, Ivana; Potuckova, Lucie; Bugajev, Viktor; Bambouskova, Monika; Tumova, Magda; Draber, Petr

    2015-01-01

    Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of Fc

  12. Synthesis of hapten and preparation of specific polyclonal antibody with high affinity for lenalidomide, the potent drug for treatment of multiple myeloma

    Directory of Open Access Journals (Sweden)

    Darwish Ibrahim A

    2012-10-01

    Full Text Available Abstract Background For therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND, the potent drug for treatment of multiple myeloma (MM, a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma. Results In this study, a hapten of LND (N-glutaryl-LND was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND was confirmed by mass, 1H-NMR, and 13C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA and keyhole limpet hemocyanin (KLH proteins by ethyl-3-(3-dimethylaminopropyl carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy. Conclusions The high affinity of the antibody (IC50 = 10 ng/mL will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.

  13. The Employment of Leukotriene Antagonists in Cutaneous Diseases Belonging to Allergological Field

    Directory of Open Access Journals (Sweden)

    Eustachio Nettis

    2010-01-01

    Full Text Available Leukotrienes (LTs are potent biological proinflammatory mediators. LTC4, LTD4, and LTE4 are more frequently involved in chronic inflammatory responses and exert their actions binding to a cysteinyl-LT 1 (CysLT1 receptor and a cysteinyl-LT 2 (CysLT2 receptor. LTs receptor antagonists available for clinical use demonstrate high-affinity binding to the CysLT1 receptor. In this paper the employment of anti-LTs in allergic cutaneous diseases is analyzed showing that several studies have recently reported a beneficial effects of these agents (montelukast and zafirlukast as well as zileuton for the treatment of some allergic cutaneous related diseases-like chronic urticaria and atopic eczema although their proper application remains to be established.

  14. Cannabinoid CB1 receptor antagonists in therapeutic and structural perspectives.

    Science.gov (United States)

    Lange, Jos H M; Kruse, Chris G

    2008-01-01

    The observed antiobesity effect of rimonabant (1) in a pharmacological rodent model 10 years ago has led to a surge in the search for novel cannabinoid CB1 antagonists as a new therapeutic target for the treatment of obesity. Rimonabant showed clinical efficacy in the treatment of obesity and also improved cardiovascular and metabolic risk factors. Cannabinoid CB1 receptor antagonists have also good prospects in other therapeutic areas, including smoking and alcohol addiction as well as cognitive impairment. Solvay's research achievements in this fast-moving field are reported in relation with the current state of the art. Several medicinal chemistry strategies have been pursued. The application of the concept of conformational constraint led to the discovery of more rigid analogs of the prototypic CB1 receptor antagonist rimonabant. Replacement of the central heterocyclic pyrazole ring in rimonabant yielded imidazoles, triazoles, and thiazoles as selective CB1 receptor antagonists. Dedicated medium-throughput screening efforts delivered one 3,4-diarylpyrazoline hit. Its poor pharmacokinetic properties were successfully optimized which led to the discovery of orally active and highly CB1/CB2 receptor selective analogs in this series. Regioisomeric 1,5-diarylpyrazolines, 1,2-diarylimidazolines, and water-soluble imidazoles have been designed as novel CB1 receptor antagonist structure classes. Copyright 2008 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.

  15. Functionalized Congeners of P2Y1 Receptor Antagonists:

    Energy Technology Data Exchange (ETDEWEB)

    de Castro, Sonia [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health; Maruoka, Hiroshi [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health; Hong, Kunlun [ORNL; Kilbey, II, S Michael [ORNL; Costanzi, Stefano [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health; Hechler, Béatrice [University of Strasbourg; Gachet, Christian [EFS-Alsace, Strasbourg, France; Harden, T. Kendall [University of North Carolina School of Medicine; Jacobson, Kenneth A. [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health

    2010-01-01

    The P2Y{sub 1} receptor is a prothrombotic G protein-coupled receptor (GPCR) activated by ADP. Preference for the North (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of the P2Y{sub 1} receptor was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute. A series of covalently linkable N{sup 6}-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphates containing extended 2-alkynyl chains was designed, and binding affinity at the human (h) P2Y{sub 1} receptor determined. The chain of these functionalized congeners contained hydrophilic moieties, a reactive substituent, or biotin, linked via an amide. Variation of the chain length and position of an intermediate amide group revealed high affinity of carboxylic congener 8 (K{sub i} 23 nM) and extended amine congener 15 (K{sub i} 132 nM), both having a 2-(1-pentynoyl) group. A biotin conjugate 18 containing an extended {epsilon}-aminocaproyl spacer chain exhibited higher affinity than a shorter biotinylated analogue. Alternatively, click coupling of terminal alkynes of homologous 2-dialkynyl nucleotide derivatives to alkyl azido groups produced triazole derivatives that bound to the P2Y{sub 1} receptor following deprotection of the bisphosphate groups. The preservation of receptor affinity of the functionalized congeners was consistent with new P2Y{sub 1} receptor modeling and ligand docking. Attempted P2Y{sub 1} antagonist conjugation to PAMAM dendrimer carriers by amide formation or palladium-catalyzed reaction between an alkyne on the dendrimer and a 2-iodopurine-derivatized nucleotide was unsuccessful. A dialkynyl intermediate containing the chain length favored in receptor binding was conjugated to an azide-derivatized dendrimer, and the conjugate inhibited ADP-promoted human platelet aggregation. This is the first example of attaching a strategically functionalized P2Y receptor

  16. The novel dopamine D3 receptor antagonists/partial agonists CAB2-015 and BAK4-54 inhibit oxycodone-taking and oxycodone-seeking behavior in rats.

    Science.gov (United States)

    You, Zhi-Bing; Gao, Jun-Tao; Bi, Guo-Hua; He, Yi; Boateng, Comfort; Cao, Jianjing; Gardner, Eliot L; Newman, Amy Hauck; Xi, Zheng-Xiong

    2017-11-01

    The use of prescription opioid analgesics, particularly oxycodone, has dramatically increased, and parallels escalated opioid abuse and drug-related deaths worldwide. Understanding the molecular mechanisms underlying the development of opioid dependence and expanding treatment options to counter prescription opioid abuse has become a critical public health matter. In the present study, we first evaluated the reinforcing effects of oxycodone in a rat model of self-administration and then explored the potential utility of two novel high affinity dopamine D3 receptor (D3R) antagonists/partial agonists, CAB2-015 and BAK4-54, for treatment of prescription opioid abuse and dependence. We found that rats acquired oxycodone self-administration rapidly within a range of unit doses that was similar to that for heroin, confirming that oxycodone has significant abuse potential. Strikingly, pretreatment with either CAB2-015 or BAK4-54 (0.4-10 mg/kg, i.p.) dose-dependently decreased oxycodone self-administration, and shifted the oxycodone dose-response curve downward. Repeated pretreatment with CAB2-015 or BAK4-54 (0.4-4 mg/kg) facilitated extinction and inhibited oxycodone-induced reinstatement of drug-seeking behavior. In addition, pretreatment with CAB2-015 or BAK4-54 (4-10 mg/kg) also dose-dependently decreased oxycodone-enhanced locomotor activity, but only CAB2-015 decreased oral sucrose self-administration. These data suggest that D3R antagonists may be suitable alternatives or adjunctive to opioid-based medications currently used clinically in treating opioid addiction and that the D3R-selective ligands (CAB2-015 or BAK4-54) provide new lead molecules for development. Published by Elsevier Ltd.

  17. Excitatory amino acid receptor antagonists

    DEFF Research Database (Denmark)

    Johansen, T N; Frydenvang, Karla Andrea; Ebert, B

    1997-01-01

    We have previously shown that (RS)-2-amino-2-(5-tert-butyl-3-hydroxyisoxazol-4-yl)acetic acid (ATAA) is an antagonist at N-methyl-D-aspartic acid (NMDA) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors. We have now resolved ATAA via diastereomeric salt formation...

  18. Clinical Development of Histamine H4 Receptor Antagonists.

    Science.gov (United States)

    Thurmond, Robin L; Venable, Jennifer; Savall, Brad; La, David; Snook, Sandra; Dunford, Paul J; Edwards, James P

    2017-01-01

    The discovery of the histamine H4 receptor (H4R) provided a new avenue for the exploration of the physiological role of histamine, as well as providing a new drug target for the development of novel antihistamines. The first step in this process was the identification of selective antagonists to help unravel the pharmacology of the H4R relative to other histamine receptors. The discovery of the selective H4R antagonist JNJ 7777120 was vital for showing a role for the H4R in inflammation and pruritus. While this compound has been very successful as a tool for understanding the function of the receptor, it has drawbacks, including a short in vivo half-life and hypoadrenocorticism toxicity in rats and dogs, that prevented advancing it into clinical studies. Further research let to the discovery of JNJ 39758979, which, similar to JNJ 7777120, was a potent and selective H4R antagonist and showed anti-inflammatory and anti-pruritic activity preclinically. JNJ 39758979 advanced into human clinical studies and showed efficacy in reducing experimental pruritus and in patients with atopic dermatitis. However, development of this compound was terminated due to the occurrence of drug-induced agranulocytosis. This was overcome by developing another H4R antagonist with a different chemical structure, toreforant, that does not appear to have this side effect. Toreforant has been tested in clinical studies in patients with rheumatoid arthritis, asthma, or psoriasis. In conclusions there have been many H4R antagonists reported in the literature, but only a few have been studied in humans underscoring the difficulty in finding ligands with all of the properties necessary for testing in the clinic. Nevertheless, the clinical data to date suggests that H4R antagonists can be beneficial in treating atopic dermatitis and pruritus.

  19. PdeH, a high-affinity cAMP phosphodiesterase, is a key regulator of asexual and pathogenic differentiation in Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Ravikrishna Ramanujam

    2010-05-01

    Full Text Available Cyclic AMP-dependent pathways mediate the communication between external stimuli and the intracellular signaling machinery, thereby influencing important aspects of cellular growth, morphogenesis and differentiation. Crucial to proper function and robustness of these signaling cascades is the strict regulation and maintenance of intracellular levels of cAMP through a fine balance between biosynthesis (by adenylate cyclases and hydrolysis (by cAMP phosphodiesterases. We functionally characterized gene-deletion mutants of a high-affinity (PdeH and a low-affinity (PdeL cAMP phosphodiesterase in order to gain insights into the spatial and temporal regulation of cAMP signaling in the rice-blast fungus Magnaporthe oryzae. In contrast to the expendable PdeL function, the PdeH activity was found to be a key regulator of asexual and pathogenic development in M. oryzae. Loss of PdeH led to increased accumulation of intracellular cAMP during vegetative and infectious growth. Furthermore, the pdeHDelta showed enhanced conidiation (2-3 fold, precocious appressorial development, loss of surface dependency during pathogenesis, and highly reduced in planta growth and host colonization. A pdeHDelta pdeLDelta mutant showed reduced conidiation, exhibited dramatically increased (approximately 10 fold cAMP levels relative to the wild type, and was completely defective in virulence. Exogenous addition of 8-Br-cAMP to the wild type simulated the pdeHDelta defects in conidiation as well as in planta growth and development. While a fully functional GFP-PdeH was cytosolic but associated dynamically with the plasma membrane and vesicular compartments, the GFP-PdeL localized predominantly to the nucleus. Based on data from cAMP measurements and Real-Time RTPCR, we uncover a PdeH-dependent biphasic regulation of cAMP levels during early and late stages of appressorial development in M. oryzae. We propose that PdeH-mediated sustenance and dynamic regulation of cAMP signaling

  20. Nuclear DNA sensor IFI16 as circulating protein in autoimmune diseases is a signal of damage that impairs endothelial cells through high-affinity membrane binding.

    Directory of Open Access Journals (Sweden)

    Francesca Gugliesi

    Full Text Available IFI16, a nuclear pathogenic DNA sensor induced by several pro-inflammatory cytokines, is a multifaceted protein with various functions. It is also a target for autoantibodies as specific antibodies have been demonstrated in the sera of patients affected by systemic autoimmune diseases. Following transfection of virus-derived DNA, or treatment with UVB, IFI16 delocalizes from the nucleus to the cytoplasm and is then eventually released into the extracellular milieu. In this study, using an in-house capture enzyme-linked immunsorbent assay we demonstrate that significant levels of IFI16 protein can also exist as circulating form in the sera of autoimmune patients. We also show that the rIFI16 protein, when added in-vitro to endothelial cells, does not affect cell viability, but severely limits their tubulogenesis and transwell migration activities. These inhibitory effects are fully reversed in the presence of anti-IFI16 N-terminal antibodies, indicating that its extracellular activity resides within the N-terminus. It was further demonstrated that endogenous IFI16 released by apoptotic cells bind neighboring cells in a co-culture. Immunofluorescence assays revealed existence of high-affinity binding sites on the plasma membrane of endothelial cells. Free recombinant IFI16 binds these sites on HUVEC with dissociation constant of 2.7 nM, radioiodinated and unlabeled IFI16 compete for binding sites, with inhibition constant (Ki of 14.43 nM and half maximal inhibitory concentration (IC50 of 67.88 nM; these data allow us to estimate the presence of 250,000 to 450,000 specific binding sites per cell. Corroborating the results from functional assays, this binding could be completely inhibited using anti-IFI16 N-terminal antibody, but not with an antibody raised against the IFI16 C-terminal. Altogether, these data demonstrate that IFI16 may exist as circulating protein in the sera of autoimmune patients which binds endothelial cells causing damage

  1. Adsorption and photophysics of fullerene C60 at liquid-zeolite particle interfaces: unusually high affinity for hydrophobic, ultrastabilized zeolite Y.

    Science.gov (United States)

    Ellison, Eric H

    2006-06-15

    O2 at the Y901-toluene interface were 18, 9, and 3 times lower, respectively, relative to rate constants in solution. These differences point out that the approach of molecular quenchers to C60 at the interface is more hindered for larger molecules, an expected result for C60 located in half-supercage bowls. The high affinity of fullerenes for hydrophobic zeolite Y provides a strategy for organizing fullerenes at interfaces and for studies of fullerene photochemistry.

  2. Sexually antagonistic polymorphism in simultaneous hermaphrodites

    Science.gov (United States)

    Jordan, Crispin Y.; Connallon, Tim

    2015-01-01

    In hermaphrodites, pleiotropic genetic tradeoffs between female and male reproductive functions can lead to sexually antagonistic (SA) selection, where individual alleles have conflicting fitness effects on each sex function. While an extensive theory of SA selection exists for dioecious species, these results have not been generalized to hermaphrodites. We develop population genetic models of SA selection in simultaneous hermaphrodites, and evaluate effects of dominance, selection on each sex function, self-fertilization, and population size, on the maintenance of polymorphism. Under obligate outcrossing, hermaphrodite model predictions converge exactly with those of dioecious populations. Self-fertilization in hermaphrodites generates three points of divergence with dioecious theory. First, opportunities for stable polymorphism decline sharply and become less sensitive to dominance with increased selfing. Second, selfing introduces an asymmetry in the relative importance of selection through male versus female reproductive functions, expands the parameter space favorable for the evolutionary invasion of female-beneficial alleles, and restricts invasion criteria for male-beneficial alleles. Finally, contrary to models of unconditionally beneficial alleles, selfing decreases genetic hitchhiking effects of invading SA alleles, and should therefore decrease these population genetic signals of SA polymorphisms. We discuss implications of SA selection in hermaphrodites, including its potential role in the evolution of “selfing syndromes”. PMID:25311368

  3. Histamine H4 receptor antagonists: the new antihistamines?

    Science.gov (United States)

    Fung-Leung, Wai-Ping; Thurmond, Robin L; Ling, Ping; Karlsson, Lars

    2004-11-01

    Antihistamines (histamine H1 receptor antagonists) are a mainstay treatment for atopic allergy, yet they are only partially effective in relieving the symptoms of the disease. They also have very limited value for the treatment of asthma, despite the well-characterized bronchoconstrictory effects of histamine. The recent discovery of a fourth histamine receptor (H4), and the realization that it is exclusively expressed on hematopoietic cell types that are most implicated in the development and symptomatology of allergy and asthma, suggests that pharmacological targeting of the H4 receptor, either alone or in combination with H1 receptor antagonists, may prove useful for treating both allergy and asthma. Here we review the known biology associated with the H4 receptor, as well the effects of a highly selective H1 receptor antagonist.

  4. In vivo binding, pharmacokinetics and metabolism of the selective M{sub 2} muscarinic antagonists [{sup 3}H]AF-DX 116 and [{sup 3}H]AF-DX 384 in the anesthetized rat

    Energy Technology Data Exchange (ETDEWEB)

    Mickala, Patrick; Boutin, Herve; Bellanger, Cecile; Chevalier, Cyril; MacKenzie, Eric T.; Dauphin, Francois

    1996-02-01

    The pharmacokinetics, in vivo binding and metabolism of two M{sub 2} muscarinic receptor antagonists, [{sup 3}H]AF-DX 116 and [{sup 3}H]AF-DX 384, were studied in anesthetized rats, which received either the tracer alone or following a saturating injection of atropine. Both radioligands were cleared from the circulation with distribution half-lives of 17 and 14 sec and elimination half-lives of 17 and 40 min for [{sup 3}H]AF-DX 116 and [{sup 3}H]AF-DX 384, respectively. A radioactive distribution, predominant in peripheral organs when compared to brain, was found at each time studied after tracer injection. Atropine-displaceable tracer uptake was evidenced at 20-40 min in brain (31%), submandibular glands (26%), spleen (37%) and notably heart (55%) for [{sup 3}H]AF-DX 116 but only in heart (50%) for [{sup 3}H]AF-DX 384 at 10-20 min. Regional brain sampling revealed a relatively uniform distribution of [{sup 3}H]AF-DX 384 and a -45% atropine saturation effect (i.e., specific binding) in the thalamus 20 min after injection. Sequential thin-layer chromatographic studies performed on tissue extracts demonstrated the rapid appearance of labeled metabolites of both radiotracers in brain (but less so in liver) and especially in cardiac tissues, where almost 70% of total radioactivity still corresponded to authentic tracer 40 min after injection. Thus, based on their low blood-brain barrier permeability and the high presence of labeled metabolites in the central nervous system, AF-DX 116 and AF-DX 384 might be more helpful in the study of M{sub 2} muscarinic receptors present in heart rather than brain. Labeled with positron emittors, these M{sub 2} antagonists might be applicable to the pathophysiological study of disease states, such as cardiomyopathies.

  5. Differential regulation of leptin transport by the choroid plexus and blood-brain barrier and high affinity transport systems for entry into hypothalamus and across the blood-cerebrospinal fluid barrier.

    Science.gov (United States)

    Zlokovic, B V; Jovanovic, S; Miao, W; Samara, S; Verma, S; Farrell, C L

    2000-04-01

    Leptin is a circulating hormone that controls food intake and energy homeostasis. Little is known about leptin entry into the central nervous system (CNS). The blood-cerebrospinal fluid (CSF) barrier at the choroid plexus and the blood-brain barrier (BBB) at the cerebral endothelium are two major controlling sites for entry of circulating proteins into the brain. In the present study, we characterized leptin transport across the blood-CSF barrier and the BBB by using a brain perfusion model in lean rats. Rapid, high-affinity transport systems mediated leptin uptake by the hypothalamus (KM = 0.2 ng/ml) a