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Sample records for nucleoside triphosphatase activity

  1. Characterization of nucleoside triphosphatase activity in isolated pea nuclei and its photoreversible regulation by light

    NASA Technical Reports Server (NTRS)

    Chen, Y. R.; Roux, S. J.

    1986-01-01

    A nucleoside triphosphatase (NTPase) present in highly purified preparations of pea nuclei was partially characterized. The activity of this enzyme was stimulated by divalent cations (Mg2+ = Mn2+ > Ca2+), but was not affected by the monovalent cations, Na+ and K+. The Mg(2+)-dependent activity was further stimulated by concentrations of Ca2+ in the low micromolar range. It could catalyze the hydrolysis of ATP, GTP, UTP, and CTP, all with a pH optimum of 7.5. The nuclear NTPase activity was not inhibited by vanadate, oligomycin, or nitrate, but was inhibited by relatively low concentrations of quercetin and the calmodulin inhibitor, compound 48/80. The NTPase was stimulated more than 50% by red light, and this effect was reversed by subsequent irradiation with far-red light. The photoreversibility of the stimulation indicated that the photoreceptor for this response was phytochrome, an important regulator of photomorphogenesis and gene expression in plants.

  2. Nucleoside triphosphatase and RNA helicase activities associated with GB virus B nonstructural protein 3.

    PubMed

    Zhong, W; Ingravallo, P; Wright-Minogue, J; Skelton, A; Uss, A S; Chase, R; Yao, N; Lau, J Y; Hong, Z

    1999-09-01

    GB virus B (GBV-B) is a positive-stranded RNA virus that belongs to the Flaviviridae family. This virus is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species). Nonstructural protein 3 (NS3) of GBV-B contains sequence motifs predictive of three enzymatic activities: serine protease, nucleoside triphosphatase (NTPase), and RNA helicase. The N-terminal serine protease has been characterized and shown to share similar substrate specificity with the HCV NS3 protease. In this report, a full-length GBV-B NS3 protein was expressed in Escherichia coli and purified to homogeneity. This recombinant protein was shown to possess polynucleotide-stimulated NTPase and double-stranded RNA (dsRNA) unwinding activities. Both activities were abolished by a single amino acid substitution, from the Lys (K) residue in the conserved walker motif A (or Ia) "AXXXXGK(210)S" to an Ala (A), confirming that they are intrinsic to GBV-B NS3. Kinetic parameters (K(m) and k(cat)) for hydrolysis of various NTPs or dNTPs were obtained. The dsRNA unwinding activity depends on the presence of divalent metal ions and ATP and requires an RNA duplex substrate with 3' unpaired regions (RNAs with 5' unpaired regions only or with blunt ends are not suitable substrates for this enzyme). This indicates that GBV-B NS3 RNA helicase unwinds dsRNA in the 3' to 5' direction. Direct interaction of the GBV-B NS3 protein with a single-stranded RNA was established using a gel-based RNA bandshift assay. Finally, a homology model of GBV-B NS3 RNA helicase domain based on the 3-dimensional structure of the HCV NS3 helicase that shows a great similarity in overall structure and surface charge distribution between the two proteins was proposed. PMID:10497107

  3. Characterization of a ribonuclease-sensitive nucleoside triphosphatase activity from HeLa nuclei.

    PubMed Central

    Richardson, L V; Richardson, J P

    1985-01-01

    Approximately one-third of the total ATP-hydrolysis activity in isolated HeLa nuclei is sensitive to RNAase (ribonuclease). This activity is selectively extracted with pulse-labelled RNA. In the extracts it co-sediments with various particles with sedimentation coefficients from 10S to 50S, but especially with 24S and 40S particles. ATP hydrolysis by the isolated particles was inhibited extensively (greater than 80%) by RNAase A, heparin and 0.2 M-NaCl. The activity of RNAase-treated particles was recovered when poly(A) was added, but not when DNA was added. The isolated particles exhibited RNAase-sensitive hydrolysis activities for dATP, GTP, CTP and UTP as well as for ATP, and the UTPase activity in the extracts showed nearly the same sedimentation distribution as the ATPase activity. When samples of isolated particles were irradiated with u.v. light in the presence of [alpha-32P]ATP, a 39 kDa polypeptide with a broad distribution from 10S to 50S like that of the ATPase and a 55 kDa polypeptide with a sharp distribution at 24S were photolabelled. Taken together, the data suggest that ATP-hydrolysis activity found in nuclear ribonucleoprotein subfractions appears to be the result of one or two RNA-dependent NTPases that are normally associated with endogenous RNA in a wide variety of particles. Images Fig. 3. PMID:2408602

  4. The major nucleoside triphosphatase in pea (Pisum sativum L.) nuclei and in rat liver nuclei share common epitopes also present in nuclear lamins

    NASA Technical Reports Server (NTRS)

    Tong, C. G.; Dauwalder, M.; Clawson, G. A.; Hatem, C. L.; Roux, S. J.

    1993-01-01

    The major nucleoside triphosphatase (NTPase) activities in mammalian and pea (Pisum sativum L.) nuclei are associated with enzymes that are very similar both biochemically and immunochemically. The major NTPase from rat liver nuclei appears to be a 46-kD enzyme that represents the N-terminal portion of lamins A and C, two lamina proteins that apparently arise from the same gene by alternate splicing. Monoclonal antibody (MAb) G2, raised to human lamin C, both immunoprecipitates the major (47 kD) NTPase in pea nuclei and recognizes it in western blot analyses. A polyclonal antibody preparation raised to the 47-kD pea NTPase (pc480) reacts with the same lamin bands that are recognized by MAb G2 in mammalian nuclei. The pc480 antibodies also bind to the same lamin-like bands in pea nuclear envelope-matrix preparations that are recognized by G2 and three other MAbs known to bind to mammalian lamins. In immunofluorescence assays, pc480 and anti-lamin antibodies stain both cytoplasmic and nuclear antigens in plant cells, with slightly enhanced staining along the periphery of the nuclei. These results indicate that the pea and rat liver NTPases are structurally similar and that, in pea nuclei as in rat liver nuclei, the major NTPase is probably derived from a lamin precursor by proteolysis.

  5. Ethacrynic acid inhibitable Ca2+ and Mg2+-activated membrane adenosine triphosphatase in rat mast cells.

    PubMed Central

    Magro, A M

    1977-01-01

    A crude plasma membrane fraction from the homogenate of purified rat mast cells demonstrates a high degree of Ca2+-dependent and Mg2+-dependent adenosine triphosphatase (ATPase) activity. The microsomal and mitochondrial fractions show negligible amounts of the Ca2+ and Mg2+-activated ATPases. The broad ATPase inhibitor, ethacrynic acid, effectively blocks the mast cell ATPase activity while ouabain demonstrates little inhibitory effect. Correspondingly, ethacrynic acid inhibits histamine release from antigen-challenged mast cells while ouabain does not. Both ATPase inhibition and histamine release inhibition by ethacrynic acid require the presence of the olefinic bond in the ethacrynic acid molecule. PMID:75076

  6. Mapping the triphosphatase active site of baculovirus mRNA capping enzyme LEF4 and evidence for a two-metal mechanism

    PubMed Central

    Martins, Alexandra; Shuman, Stewart

    2003-01-01

    The 464-amino acid baculovirus LEF4 protein is a bifunctional mRNA capping enzyme with triphosphatase and guanylyltransferase activities. The N-terminal half of LEF4 constitutes an autonomous triphosphatase catalytic domain. The LEF4 triphosphatase belongs to a family of metal-dependent phosphohydrolases, which includes the RNA triphosphatases of fungi, protozoa, Chlorella virus and poxviruses. The family is defined by two glutamate-containing motifs (A and C), which form a metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight stranded β barrel (the so-called ‘triphosphate tunnel’). Here we probed whether baculovirus LEF4 is a member of the tunnel subfamily, via mutational mapping of amino acids required for triphosphatase activity. We identified four new essential side chains in LEF4 via alanine scanning and illuminated structure–activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight five acidic and four basic amino acids that are likely to comprise the LEF4 triphosphatase active site (Glu9, Glu11, Arg51, Arg53, Glu97, Lys126, Arg179, Glu181 and Glu183). These nine essential residues are conserved in LEF4 orthologs from all strains of baculoviruses. We discerned no pattern of clustering of the catalytic residues of the baculovirus triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). However, there is similarity to the active site of vaccinia RNA triphosphatase. We infer that the baculovirus and poxvirus triphosphatases are a distinct lineage within the metal-dependent RNA triphosphatase family. Synergistic activation of the LEF4 triphosphatase by manganese and magnesium suggests a two-metal mechanism of γ phosphate hydrolysis. PMID:12595553

  7. The distribution of sodium-potassium-activated adenosine triphosphatase in medulla and cortex of the kidney

    PubMed Central

    Hendler, Ernesto D.; Torretti, Jorge; Epstein, Franklin H.

    1971-01-01

    The activity of sodium-potassium-activated adenosine triphosphatase (Na-K-ATPase) is considerably higher in homogenates of outer medulla than in the cortex or papilla of the kidney. The enzyme has similar kinetic characteristics in both cortex and medulla, and binds ouabain in the same proportion. The discrepancy in enzymatic activity is not paralleled by similar change in the activity of adenyl cyclase, 5′nucleotidase, glucose-6-phosphatase, or succinic dehydrogenase. Na-K-ATPase is also higher in distal convoluted tubules (ventral slices) than in the proximal tubules (dorsal slices) of the kidney of Amphiuma. The high concentration of Na-K-ATPase in the red medulla of the kidney is probably related to the presence here of the thick ascending limb of the loop of Henle, and this has important implications with regard to the mechanism of sodium reabsorption by different portions of the nephron. PMID:4325313

  8. New hypoxanthine nucleosides with RNA antiviral activity.

    PubMed

    Nair, V; Ussery, M A

    1992-08-01

    A series of novel C-2 functionalized hypoxanthine and purine ribonucleosides have been synthesized and evaluated against exotic RNA viruses of the family or genus alpha, arena, flavi, and rhabdo. Both specific and broad-spectrum antiviral activities were discovered but only with hypoxanthine nucleosides. PMID:1444325

  9. The RNA triphosphatase domain of L protein of Rinderpest virus exhibits pyrophosphatase and tripolyphosphatase activities.

    PubMed

    Singh, Piyush Kumar; Subbarao, Shaila Melkote

    2016-10-01

    L protein of the Rinderpest virus, an archetypal paramyxovirus possesses RNA-dependent RNA polymerase activity which transcribes the genome into mRNAs as well as replicates the RNA genome. The protein also possesses RNA triphosphatase (RTPase), guanylyltransferase (GTase) and methyltransferase enzyme activities responsible for capping the mRNAs in a conventional pathway similar to that of the host pathway. Subsequent to the earlier characterization of the GTase activity of L protein and identification of the RTPase domain of the L protein, we report here, additional enzymatic activities associated with the RTPase domain. We have characterized the pyrophosphatase and tripolyphosphatase activities of the L-RTPase domain which are metal-dependent and proceed much faster than the RTPase activity. Interestingly, the mutant proteins E1645A and E1647A abrogated the pyrophosphatase and tripolyphosphatase significantly, indicating a strong overlap of the active sites of these activities with that of RTPase. We discuss the likely role of GTase-associated L protein pyrophosphatase in the polymerase function. We also discuss a possible biological role for the tripolyphosphatase activity hitherto considered insignificant for the viruses possessing such activity. PMID:27170418

  10. Na+-K+-activated adenosine triphosphatase and intestinal electrolyte transport. Effect of adrenal steroids.

    PubMed Central

    Charney, A N; Kinsey, M D; Myers, L; Gainnella, R A; Gots, R E

    1975-01-01

    Sodium-potassium-activated adenosine triphosphatase (Na-K-ATPase) is associated with electrolyte transport in many tissues. To help delineate its role in intestinal transport, changes in rat intestinal electrolyte and water transport induced by injecting methylprednisolone acetate 3 mg/100 g or deoxycorticosterone acetate (DOCA) 0.5 mg/100 g per day for 3 days were correlated with changes in Na-K-ATPase activity. Methylprednisolone increased sodium and water absorption, potassium secretion, transmural potential difference, and Na-K-ATPase activity in the jejunum, ileum, and colon. Examination of isolated epithelial cells demonstrated that the jejunal and ileal increase in Na-K-ATPase occurred in both the villus tip and crypermeability, Mg-ATPase, and adenylate cyclase activities were unchanged by methylprednisolone. DOCA increased sodium and water absorption, potassium secretion, transmural potential difference, and Na-K-ATPase activity in the colon alone. Colonic Mg-ATPase and adenylate cyclase activities were unaffected. Jejunal and ileal enzyme activity, electrolyte transport, and permeability were unchanged by DOCA. Methylprednisolone and DOCA were not additive in their effect on colonic Na-K-ATPase activity. Methylprednisolone and DOCA increased electrolyte and water transport and Na-K-ATPase activity concomitantly in specific segments of small intestine and colon. These data are consistent with an important role for Na-K-ATPase in intestinal electrolyte and water transport. PMID:125764

  11. Nucleosides Accelerate Inflammatory Osteolysis, Acting as Distinct Innate Immune Activators

    PubMed Central

    Pan, George; Zheng, Rui; Yang, Pingar; Li, Yao; Clancy, John P.; Liu, Jianzhong; Feng, Xu; Garber, David A; Spearman, Paul; McDonald, Jay M

    2015-01-01

    The innate immune system and its components play an important role in the pathogenesis of inflammatory bone destruction. Blockade of inflammatory cytokines does not completely arrest bone erosion, suggesting that other mediators also may be involved in osteolysis. Previously we showed that nucleosides promote osteoclastogenesis and bone-resorption activity in the presence of receptor activator for nuclear factor κB ligand (RANKL) in vitro. The studies described here further demonstrate that selected nucleosides and nucleoside analogues accelerate bone destruction in mice immunized with collagen II alone (CII) but also further enhance bone erosion in mice immunized by collagen II plus complete Freund's adjuvant (CII + CFA). Abundant osteoclasts are accumulated in destructive joints. These data indicate that nucleosides act as innate immune activators distinct from CFA, synergistically accelerating osteoclast formation and inflammatory osteolysis. The potential roles of the surface triggering receptor expressed on myeloid cells (TREM) and the intracellular inflammasome in nucleoside-enhanced osteoclastogenesis have been studied. These observations provide new insight into the pathogenesis and underlying mechanism of bone destruction in inflammatory autoimmune osteoarthritis. PMID:21472777

  12. Macrophages require different nucleoside transport systems for proliferation and activation.

    PubMed

    Soler, C; García-Manteiga, J; Valdés, R; Xaus, J; Comalada, M; Casado, F J; Pastor-Anglada, M; Celada, A; Felipe, A

    2001-09-01

    To evaluate the mechanisms involved in macrophage proliferation and activation, we studied the regulation of the nucleoside transport systems. In murine bone marrow-derived macrophages, the nucleosides required for DNA and RNA synthesis are recruited from the extracellular medium. M-CSF induced macrophage proliferation and DNA and RNA synthesis, whereas interferon gamma (IFN-gamma) led to activation, blocked proliferation, and induced only RNA synthesis. Macrophages express at least the concentrative systems N1 and N2 (CNT2 and CNT1 genes, respectively) and the equilibrative systems es and ei (ENT1 and ENT2 genes, respectively). Incubation with M-CSF only up-regulated the equilibrative system es. Inhibition of this transport system blocked M-CSF-dependent proliferation. Treatment with IFN-gamma only induced the concentrative N1 and N2 systems. IFN-gamma also down-regulated the increased expression of the es equilibrative system induced by M-CSF. Thus, macrophage proliferation and activation require selective regulation of nucleoside transporters and may respond to specific requirements for DNA and RNA synthesis. This report also shows that the nucleoside transporters are critical for macrophage proliferation and activation. PMID:11532978

  13. The VirB4 Family of Proposed Traffic Nucleoside Triphosphatases: Common Motifs in Plasmid RP4 TrbE Are Essential for Conjugation and Phage Adsorption

    PubMed Central

    Rabel, Christian; Grahn, A. Marika; Lurz, Rudi; Lanka, Erich

    2003-01-01

    Proteins of the VirB4 family are encoded by conjugative plasmids and by type IV secretion systems, which specify macromolecule export machineries related to conjugation systems. The central feature of VirB4 proteins is a nucleotide binding site. In this study, we asked whether members of the VirB4 protein family have similarities in their primary structures and whether these proteins hydrolyze nucleotides. A multiple-sequence alignment of 19 members of the VirB4 protein family revealed striking overall similarities. We defined four common motifs and one conserved domain. One member of this protein family, TrbE of plasmid RP4, was genetically characterized by site-directed mutagenesis. Most mutations in trbE resulted in complete loss of its activities, which eliminated pilus production, propagation of plasmid-specific phages, and DNA transfer ability in Escherichia coli. Biochemical studies of a soluble derivative of RP4 TrbE and of the full-length homologous protein R388 TrwK revealed that the purified forms of these members of the VirB4 protein family do not hydrolyze ATP or GTP and behave as monomers in solution. PMID:12533481

  14. Effects of lead and natriuretic hormone on kinetics of sodium-potassium-activated adenosine triphosphatase: possible relevance to hypertension.

    PubMed Central

    Weiler, E; Khalil-Manesh, F; Gonick, H

    1988-01-01

    Inhibition of vascular smooth muscle sodium-potassium-activated adenosine triphosphatase (Na-K-ATPase) has been postulated as a central mechanism in enhancing vascular contractility. In the present study, kinetics of inhibition of Na-K-ATPase by lead, ouabain, and natriuretic hormone (NH) was studied in a purified hog cerebral cortex enzyme preparation. Determination of I50 values for lead, ouabain, and NH revealed that NH is the most potent inhibitor of the enzyme system (0.8 x 10(-6) M ouabain equivalents). Kinetic analyses indicated that lead and NH exhibited different inhibitory mechanisms. The inhibition by lead was noncompetitive with respect to potassium and competitive with respect to sodium and MgATP. Natriuretic hormone was noncompetitive with respect to potassium, uncompetitive with respect to MgATP, and exhibited no inhibitory effect with respect to sodium. Synergism between lead and NH in the inhibition of Na-K-ATPase raises the possibility that lead may be a contributory factor in hypertension via this mechanism. PMID:2849538

  15. Semaphorin 3A activates the guanosine triphosphatase Rab5 to promote growth cone collapse and organize callosal axon projections.

    PubMed

    Wu, Kong-Yan; He, Miao; Hou, Qiong-Qiong; Sheng, Ai-Li; Yuan, Lei; Liu, Fei; Liu, Wen-Wen; Li, Guangpu; Jiang, Xing-Yu; Luo, Zhen-Ge

    2014-01-01

    Axon guidance (pathfinding) wires the brain during development and is regulated by various attractive and repulsive cues. Semaphorin 3A (Sema3A) is a repulsive cue, inducing the collapse of axon growth cones. In the mammalian forebrain, the corpus callosum is the major commissure that transmits information flow between the two hemispheres, and contralateral axons assemble into well-defined tracts. We found that the patterning of callosal axon projections in rodent layer II and III (L2/3) cortical neurons in response to Sema3A was mediated by the activation of Rab5, a small guanosine triphosphatase (GTPase) that mediates endocytosis, through the membrane fusion protein Rabaptin-5 and the Rab5 guanine nucleotide exchange factor (GEF) Rabex-5. Rabaptin-5 bound directly to Plexin-A1 in the Sema3A receptor complex [an obligate heterodimer formed by Plexin-A1 and neuropilin 1 (NP1)]; Sema3A enhanced this interaction in cultured neurons. Rabaptin-5 bridged the interaction between Rab5 and Plexin-A1. Sema3A stimulated endocytosis from the cell surface of callosal axon growth cones. In utero electroporation to reduce Rab5 or Rabaptin-5 impaired axon fasciculation or caused mistargeting of L2/3 callosal projections in rats. Overexpression of Rabaptin-5 or Rab5 rescued the defective callosal axon fasciculation or mistargeting of callosal axons caused by the loss of Sema3A-Plexin-A1 signaling in rats expressing dominant-negative Plexin-A1 or in NP1-deficient mice. Thus, our findings suggest that Rab5, its effector Rabaptin-5, and its regulator Rabex-5 mediate Sema3A-induced axon guidance during brain development. PMID:25161316

  16. Semaphorin 3A activates the guanosine triphosphatase Rab5 to promote growth cone collapse and organize callosal axon projections

    PubMed Central

    Wu, Kong-Yan; He, Miao; Hou, Qiong-Qiong; Sheng, Ai-Li; Yuan, Lei; Liu, Fei; Liu, Wen-Wen; Li, Guangpu; Jiang, Xing-Yu; Luo, Zhen-Ge

    2015-01-01

    Axon guidance (pathfinding) wires the brain during development and is regulated by various attractive and repulsive cues. Semaphorin 3A (Sema3A) is a repulsive cue, inducing the collapse of axon growth cones. In the mammalian forebrain, the corpus callosum is the major commissure that transmits information flow between the two hemispheres, and contralateral axons assemble into well-defined tracts. We found that the patterning of callosal axon projections in rodent layer II and III (L2/3) cortical neurons in response to Sema3A was mediated by the activation of Rab5, a small guanosine triphosphatase (GTPase) that mediates endocytosis, through the membrane fusion protein Rabaptin-5 and the Rab5 guanine nucleotide exchange factor (GEF) Rabex-5. Rabaptin-5 bound directly to Plexin-A1 in the Sema3A receptor complex [an obligate heterodimer formed by Plexin-A1 and neuropilin 1 (NP1)]; Sema3A enhanced this interaction in cultured neurons. Rabaptin-5 bridged the interaction between Rab5 and Plexin-A1. Sema3A stimulated endocytosis from the cell surface of callosal axon growth cones. In utero electroporation to reduce Rab5 or Rabaptin-5 impaired axon fasciculation or caused mistargeting of L2/3 callosal projections in rats. Over-expression of Rabaptin-5 or Rab5 rescued the defective callosal axon fasciculation or mistargeting of callosal axons caused by the loss of Sema3A–Plexin-A1 signaling in rats expressing dominant-negative Plexin-A1 or in NP1-deficient mice. Thus, our findings suggest that Rab5, its effector Rabaptin-5, and its regulator Rabex-5 mediate Sema3A-induced axon guidance during brain development. PMID:25161316

  17. Localization of calcium stimulated adenosine triphosphatase activity in blood vessels of the skeleton

    NASA Technical Reports Server (NTRS)

    Doty, S. B.

    1985-01-01

    Alkaline phosphatase is an enzyme found in bone forming cells which decreases in certain bones as a result of hypogravity or non-weight bearing. This enzyme can also hydrolyze adenosine triphosphate. Therefore, an effort was made to localize calcium-stimulated ATPase by cytochemistry to determine whether altered bone cell activity might be related to changing calcium levels which occur during hypogravity. The results indicate that Ca(++)-ATPase is largely found along the endothelium and basal lamina of blood vessels, and not found in bone forming cells. This suggests that calcium regulation in the vicinity of bone formation may be modulated by the vasculature of the area.

  18. Halobacterial adenosine triphosphatases and the adenosine triphosphatase from Halobacterium saccharovorum

    NASA Technical Reports Server (NTRS)

    Kristjansson, Hordur; Sadler, Martha H.; Hochstein, Lawrence I.

    1986-01-01

    Membranes prepared from various members of the genus Halobacterium contained a Triton X-l00 activated adenosine triphosphatase. The enzyme from Halobacterium saccharovorum was unstable in solutions of low ionic strength and maximally active in the presence of 3.5 M NaCl. A variety of nucleotide triphosphates was hydrolyzed. MgADP, the product of ATP hydrolysis, was not hydrolyzed and was a competitive inhibitor with respect to MgATP. The enzyme from H. saccharovorum was composed of at least 2 and possibly 4 subunits. The 83-kDa and 60-kDa subunits represented about 90 percent of total protein. The 60-kDa subunit reacted with dicyclohexyl-carbodiimide when inhibition was carried out in an acidic medium. The enzyme from H. saccharovorum, possesses properties of an F(1)F(0) as well as an E(1)E(2) ATPase.

  19. Synthesis, antiviral activity, cytotoxicity and cellular pharmacology of l-3'-azido-2',3'-dideoxypurine nucleosides.

    PubMed

    Zhang, Hong-Wang; Detorio, Mervi; Herman, Brian D; Solomon, Sarah; Bassit, Leda; Nettles, James H; Obikhod, Aleksandr; Tao, Si-Jia; Mellors, John W; Sluis-Cremer, Nicolas; Coats, Steven J; Schinazi, Raymond F

    2011-09-01

    Microwave-assisted optimized transglycosylation reactions were used to prepare eleven modified l-3'-azido-2',3'-dideoxypurine nucleosides. These l-nucleoside analogs were evaluated against HIV and hepatitis B virus. The l-3'-azido-2',3'-dideoxypurines nucleosides were metabolized to nucleoside 5'-triphosphates in primary human lymphocytes, but exhibited weak or no antiviral activity against HIV-1. The nucleosides were also inactive against HBV in HepG2 cells. Pre-steady state kinetic experiments demonstrated that the l-3'-azido-2',3'-dideoxypurine triphosphates could be incorporated by purified HIV-1 reverse transcriptase, although their catalytic efficiency (k(pol)/K(d)) of incorporation was low. Interestingly, a phosphoramidate prodrug of l-3'-azido-2',3'-dideoxyadenosine exhibited anti-HIV-1 activity without significant toxicity. PMID:21700368

  20. Structural and functional characterization of a noncanonical nucleoside triphosphate pyrophosphatase from Thermotoga maritima

    SciTech Connect

    Awwad, Khaldeyah; Desai, Anna; Smith, Clyde; Sommerhalter, Monika

    2013-02-01

    A 2.15 Å resolution crystal structure of TM0159 with bound IMP and enzyme-kinetic data are presented. This noncanonical nucleoside triphosphatase from T. maritima helps to maintain a correct pool of DNA and RNA precursor molecules. The hyperthermophilic bacterium Thermotoga maritima has a noncanonical nucleoside triphosphatase that catalyzes the conversion of inosine triphosphate (ITP), deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP) into inosine monophosphate (IMP), deoxyinosine monophosphate (IMP) and xanthosine monophosphate (XMP), respectively. The k{sub cat}/K{sub m} values determined at 323 and 353 K fall between 1.31 × 10{sup 4} and 7.80 × 10{sup 4} M{sup −1} s{sup −1}. ITP and dITP are slightly preferred over XTP. Activity towards canonical nucleoside triphosphates (ATP and GTP) was not detected. The enzyme has an absolute requirement for Mg{sup 2+} as a cofactor and has a preference for alkaline conditions. A protein X-ray structure of the enzyme with bound IMP was obtained at 2.15 Å resolution. The active site houses a well conserved network of residues that are critical for substrate recognition and catalysis. The crystal structure shows a tetramer with two possible dimer interfaces. One of these interfaces strongly resembles the dimer interface that is found in the structures of other noncanonical nucleoside pyrophosphatases from human (human ITPase) and archaea (Mj0226 and PhNTPase)

  1. The Role of Flexible Loops in Folding, Trafficking and Activity of Equilibrative Nucleoside Transporters

    PubMed Central

    Aseervatham, Jaya; Tran, Lucky; Machaca, Khaled; Boudker, Olga

    2015-01-01

    Equilibrative nucleoside transporters (ENTs) are integral membrane proteins, which reside in plasma membranes of all eukaryotic cells and mediate thermodynamically downhill transport of nucleosides. This process is essential for nucleoside recycling, and also plays a key role in terminating adenosine-mediated cellular signaling. Furthermore, ENTs mediate the uptake of many drugs, including anticancer and antiviral nucleoside analogues. The structure and mechanism, by which ENTs catalyze trans-membrane transport of their substrates, remain unknown. To identify the core of the transporter needed for stability, activity, and for its correct trafficking to the plasma membrane, we have expressed human ENT deletion mutants in Xenopus laevis oocytes and determined their localization, transport properties and susceptibility to inhibition. We found that the carboxyl terminal trans-membrane segments are essential for correct protein folding and trafficking. In contrast, the soluble extracellular and intracellular loops appear to be dispensable, and must be involved in the fine-tuning of transport regulation. PMID:26406980

  2. The Role of Flexible Loops in Folding, Trafficking and Activity of Equilibrative Nucleoside Transporters.

    PubMed

    Aseervatham, Jaya; Tran, Lucky; Machaca, Khaled; Boudker, Olga

    2015-01-01

    Equilibrative nucleoside transporters (ENTs) are integral membrane proteins, which reside in plasma membranes of all eukaryotic cells and mediate thermodynamically downhill transport of nucleosides. This process is essential for nucleoside recycling, and also plays a key role in terminating adenosine-mediated cellular signaling. Furthermore, ENTs mediate the uptake of many drugs, including anticancer and antiviral nucleoside analogues. The structure and mechanism, by which ENTs catalyze trans-membrane transport of their substrates, remain unknown. To identify the core of the transporter needed for stability, activity, and for its correct trafficking to the plasma membrane, we have expressed human ENT deletion mutants in Xenopus laevis oocytes and determined their localization, transport properties and susceptibility to inhibition. We found that the carboxyl terminal trans-membrane segments are essential for correct protein folding and trafficking. In contrast, the soluble extracellular and intracellular loops appear to be dispensable, and must be involved in the fine-tuning of transport regulation. PMID:26406980

  3. Solution conformations of nucleoside analogues exhibiting antiviral activity against human immunodeficiency virus

    NASA Astrophysics Data System (ADS)

    Dijkstra, Sandra; Benevides, James M.; Thomas, George J.

    1991-01-01

    The molecular-conformational basis for HIV-1 antiviral activity of dideoxynucleoside analogues is unknown. A recent proposal by van Roey [1] that furanose sugar puckering in the C2' -endo family (namely C3' -exo) may account for the enhanced anti-HIV-1 activity of azidothymidine (AZT), dideoxythymidine (ddT) and dideoxycytidine (ddC) has been tested by conformational analysis of these and related agents, using laser Raman spectroscopy of their solutions and crystal structures. The results show that nucleoside analogues exhibiting anti-HIV-1 activity, including AZT, ddT and ddC, exist in solution with C3' -endo as the predominating sugar pucker. The C3' -endo solution conformations differ fundamentally from the C3' -exo conformations observed in the corresponding crystal structures. Accordingly, the crystal conformation cannot be responsible for enhanced recognition of these agents, either by nucleoside kinase or reverse transcriptase, as a mechanism to explain antiviral activity. The present findings suggest that C3' -endo sugear pucker, rather than C3' -exo pucker, or other puckers of the C2' -endo family, is more probably the required conformation for antivaral activity. The present work also shows that nucleoside phosphorylation does not, in general, change the preferred solution conformation of a nucleoside. Therefore, C3' -endo sugar pucker is likely to be the preferred conformation for both nucleoside kinase and reverse transcriptase recognition. In this study, the list of thymidine nucleoside conformation markers available from Raman spectra is extended and additional group frequency assignments for C3' -azido, C3' -deoxy and related nucleoside derivatives are provided.

  4. Transforming growth factor-β inhibits IQ motif containing guanosine triphosphatase activating protein 1 expression in lung fibroblasts via the nuclear factor-κB signaling pathway.

    PubMed

    Zong, Chuanyue; Zhang, Xianlong; Xie, Ying; Cheng, Jiawen

    2015-07-01

    IQ motif containing guanosine triphosphatase activating protein 1 (IQGAP1) is associated with idiopathic pulmonary fibrogenesis (IPF); however, characterization of the expression of IQGAP1 in lung fibroblasts has remained elusive. The present study therefore evaluated IQGAP1 expression in mouse and human lung fibroblasts under fibrotic conditions via western blot analysis. It was revealed that IQGAP1 expression levels were significantly decreased in lung fibroblasts isolated from bleomycin-challenged mice than in those of control mice. Transforming growth factor-β (TGF-β) induced differentiation, as well as decreased expression of IQGAP1 in WI-38 cells human lung fibroblasts. Furthermore, inhibition of nuclear factor (NF)-κB activation restored the TGF-β-induced inhibition of IQGAP1 expression in WI-38 cells. In lysophosphatidic acid (LPA)-challenged WI-38 cells, the expression of IQGAP1 was also decreased, while neutralized anti-TGF-β antibody treatment restored the LPA-induced inhibition of IQGAP1 expression. These data indicated that TGF-β inhibited IQGAP1 expression in lung fibroblasts via the NF-κB signaling pathway, presenting a potential novel therapeutic target for the treatment of IPF. PMID:25684348

  5. Scopadulcic acid B, a new tetracyclic diterpenoid from Scoparia dulcis L. Its structure, H+, K(+)-adenosine triphosphatase inhibitory activity and pharmacokinetic behaviour in rats.

    PubMed

    Hayashi, T; Okamura, K; Kakemi, M; Asano, S; Mizutani, M; Takeguchi, N; Kawasaki, M; Tezuka, Y; Kikuchi, T; Morita, N

    1990-10-01

    The structure of scopadulcic acid B (2, SDB), a major ingredient of the Paraguayan herb "Typychá kuratũ" (Scoparia dulcis L.), was elucidated mainly by comparison of its spectral data with that of scopadulcic acid A (1). SDB inhibited both the K(+)-dependent adenosine triphosphatase (ATPase) activity of a hog gastric proton pump (H+, K(+)-ATPase) with a value of 20-30 microM for IC50 and proton transport into gastric vesicles. Pharmacokinetic studies of SDB in rats indicated that plasma SDB concentrations after i.v. injection of the sodium salt of SDB (SDB-Na) were described reasonably well by a two-compartment open model with Michaelis-Menten elimination kinetics. Plasma concentrations after oral administration of SDB-Na or SDB showed a much slower decline than what was expected following the i. v. study. It was suggested that the sustained plasma level of SDB after oral administration of SDB-Na or SDB was accounted for by relatively slow but efficient gastro-intestinal absorption in rats. PMID:1963813

  6. Spectrum of activity and mechanisms of resistance of various nucleoside derivatives against gammaherpesviruses.

    PubMed

    Coen, Natacha; Duraffour, Sophie; Topalis, Dimitri; Snoeck, Robert; Andrei, Graciela

    2014-12-01

    The susceptibilities of gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and animal rhadinoviruses, to various nucleoside analogs was investigated in this work. Besides examining the antiviral activities and modes of action of antivirals currently marketed for the treatment of alpha- and/or betaherpesvirus infections (including acyclovir, ganciclovir, penciclovir, foscarnet, and brivudin), we also investigated the structure-activity relationship of various 5-substituted uridine and cytidine molecules. The antiviral efficacy of nucleoside derivatives bearing substitutions at the 5 position was decreased if the bromovinyl was replaced by chlorovinyl. 1-β-D-Arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU), a nucleoside with an arabinose configuration of the sugar ring, exhibited no inhibitory effect against rhadinoviruses but was active against EBV. On the other hand, the fluoroarabinose cytidine analog 2'-fluoro-5-iodo-aracytosine (FIAC) showed high selectivity indices against gammaherpesviruses that were comparable to those of brivudin. Additionally, we selected brivudin- and acyclovir-resistant rhadinoviruses in vitro and characterized them by phenotypic and genotypic (i.e., sequencing of the viral thymidine kinase, protein kinase, and DNA polymerase) analysis. Here, we reveal key amino acids in these enzymes that play an important role in substrate recognition. Our data on drug susceptibility profiles of the different animal gammaherpesvirus mutants highlighted cross-resistance patterns and indicated that pyrimidine nucleoside derivatives are phosphorylated by the viral thymidine kinase and purine nucleosides are preferentially activated by the gammaherpesvirus protein kinase. PMID:25267682

  7. Synthesis of optically pure dioxolane nucleosides and their anti-HIV activity

    SciTech Connect

    Siddigui, M.A.; Evans, C.; Jin, H.L.; Tse, A.; Brown, W.; Nguyen-Ba, N.; Mansour, T.S.; Cameron, J.M.

    1993-12-31

    The clinical candidate 3TC, 1, possessing non-natural absolute stereochemistry is a potent and non-toxic inhibitor of a key enzyme, reverse transcriptase, involved in the replicative cycle of the HIV. Selective inhibition of both HIV and HBV is seen. In view of the authors` interest in finding correlation between stereochemistry and antiviral activity, several enantiomerically pure dioxolane nucleosides, 2, were synthesized and assayed. The discussion will focus on (a) the synthesis of optically pure dioxolane sugars from L-ascorbic acid, (b) enzymatic resolution of purine dioxolane nucleosides, (c) anti HIV-1 activity in MT-4 cells.

  8. Synthesis and in vitro growth inhibitory activity of novel silyl- and trityl-modified nucleosides.

    PubMed

    Panayides, Jenny-Lee; Mathieu, Véronique; Banuls, Laetitia Moreno Y; Apostolellis, Helen; Dahan-Farkas, Nurit; Davids, Hajierah; Harmse, Leonie; Rey, M E Christine; Green, Ivan R; Pelly, Stephen C; Kiss, Robert; Kornienko, Alexander; van Otterlo, Willem A L

    2016-06-15

    Seventeen silyl- and trityl-modified (5'-O- and 3',5'-di-O-) nucleosides were synthesized with the aim of investigating the in vitro antiproliferative activities of these nucleoside derivatives. A subset of the compounds was evaluated at a fixed concentration of 100μM against a small panel of tumor cell lines (HL-60, K-562, Jurkat, Caco-2 and HT-29). The entire set was also tested at varying concentrations against two human glioma lines (U373 and Hs683) to obtain GI50 values, with the best results being values of ∼25μM. PMID:27157005

  9. Chemical synthesis of nucleoside-gamma-[32P]triphosphates of high specific activity.

    PubMed

    Janecka, A; Panusz, H; Pankowski, J; Koziołkiewicz, W

    1980-01-01

    A simple chemical procedure for the preparation of four common ribonucleoside 5-gamma-[32P]triphosphates of high specific activity (up to 10 Ci/mmole) based on the condensation of orthophosphoric acid with the corresponding nucleoside 5-diphosphate in the presence of ethyl chloroformate as well as the methods of purification and identification of the products are described. PMID:7375446

  10. Oxidative Stress Parameters and Erythrocyte Membrane Adenosine Triphosphatase Activities in Streptozotocin-induced Diabetic Rats Administered Aqueous Preparation of Kalanchoe Pinnata Leaves

    PubMed Central

    Menon, Nikhil; Sparks, Jean; Omoruyi, Felix O.

    2016-01-01

    Background: Diabetes mellitus is a chronic metabolic disease that according to the World Health Organization affects more than 382 million people. The rise in diabetes mellitus coupled with the lack of an effective treatment has led many to investigate medicinal plants to identify a viable alternative. Objective: To evaluate red blood cell (RBC) membrane adenosine triphosphatase (ATPase) activities and antioxidant levels in streptozotocin-induced diabetic rats administered aqueous preparation of Kalanchoe pinnata leaves. Materials and Methods: Diabetes mellitus was induced in rats by a single administration of streptozotocin (60 mg/kg). Diabetic rats were then treated with aqueous K. pinnata preparation (three mature leaves ~ 9.96 g/70 kg body weight or about 0.14 g/kg body weight/day) for 30 days. Serum glucose, RBC membrane ATPase activities, and antioxidant levels were determined. Results: We noted weight loss and reduced food consumption in the treated diabetic group. Serum glucose levels were reduced in the treated diabetic group compared to the other groups. Superoxide dismutase activity and glutathione levels were not significantly elevated in the treated group compared to the diabetic group. However, serum catalase activity was significantly (P < 0.05) increased in the treated diabetic group compared to the other groups. Serum thiobarbituric acid reactive substances were not significantly altered among the groups. There was a significant (P < 0.05) increase in Mg2+ ATPase activity and a nonsignificant increase in Na+/K+ ATPase activity in the RBC membrane of the treated diabetic group compared to the diabetic group. Conclusion: The consumption of aqueous preparation of K. pinnata may accrue benefits in the management of diabetes by lowering oxidative stress often associated with the disease and improving the availability of cellular magnesium through an increase in the magnesium ATPase pump in the RBC membrane for increased cellular metabolism of glucose

  11. Antiproliferative activity of bicyclic benzimidazole nucleosides: synthesis, DNA-binding and cell cycle analysis.

    PubMed

    Sontakke, Vyankat A; Lawande, Pravin P; Kate, Anup N; Khan, Ayesha; Joshi, Rakesh; Kumbhar, Anupa A; Shinde, Vaishali S

    2016-04-26

    An efficient route was developed for synthesis of bicyclic benzimidazole nucleosides from readily available d-glucose. The key reactions were Vörbruggen glycosylation and ring closing metathesis (RCM). Primarily, to understand the mode of DNA binding, we performed a molecular docking study and the binding was found to be in the minor groove region. Based on the proposed binding model, UV-visible and fluorescence spectroscopic techniques using calf thymus DNA (CT-DNA) demonstrated a non-intercalative mode of binding. Antiproliferative activity of nucleosides was tested against MCF-7 and MDA-MB-231 breast cancer cell lines and found to be active at low micromolar concentrations. Compounds and displayed significant antiproliferative activity as compared to and with the reference anticancer drug, doxorubicin. Cell cycle analysis showed that nucleoside induced cell cycle arrest at the S-phase. Confocal microscopy has been performed to validate the induction of cellular apoptosis. Based on these findings, such modified bicyclic benzimidazole nucleosides will make a significant contribution to the development of anticancer drugs. PMID:27074628

  12. [Synthesis, conformation, and spectroscopy of nucleoside analogues concerning their antiviral activity].

    PubMed

    Kuśmierek, Jarosław T; Stolarski, Ryszard

    2015-01-01

    Chemically modified analogues of nucleosides and nucleotides, have been thoroughly investigated since the discovery of DNA double helix by Watson and Crick in 1953 (Nature 171: 737). Chemical structures, first of all tautomerism, of the nucleic acid bases, as well as the conformations of the nucleic acids constituents, determine the secondary and tertiary structures of DNA and RNA polymers. Similarly, structural and dynamic parameters of nucleoside derivatives determine their biological activity in mutagenesis, neoplastic transformation, as well as antiviral or anticancer properties. In this review, a multidisciplinary approach of Prof. David Shugar's group is presented in the studies on nucleosides and nucleotides. It consists in chemical syntheses of suitable analogues, measurements of physicochemical and spectral parameters, conformational analysis by means of nuclear magnetic resonance (NMR) and X-ray diffraction, as well as characteristics of the nucleoside analogues as inhibitors of some selected, target enzymes, crucial in respect to antiviral activity of the analogues. These long-lasting studies follows upon the line of the main paradigm of molecular biophysics, i. e. structure-activity relationship. PMID:26677575

  13. Effects of vaccinia virus uracil DNA glycosylase catalytic site and deoxyuridine triphosphatase deletion mutations individually and together on replication in active and quiescent cells and pathogenesis in mice

    PubMed Central

    De Silva, Frank S; Moss, Bernard

    2008-01-01

    Background Low levels of uracil in DNA result from misincorporation of dUMP or cytosine deamination. Vaccinia virus (VACV), the prototype poxvirus, encodes two enzymes that can potentially reduce the amount of uracil in DNA. Deoxyuridine triphosphatase (dUTPase) hydrolyzes dUTP, generating dUMP for biosynthesis of thymidine nucleotides while decreasing the availability of dUTP for misincorporation; uracil DNA glycosylase (UNG) cleaves uracil N-glycosylic bonds in DNA initiating base excision repair. Studies with actively dividing cells showed that the VACV UNG protein is required for DNA replication but the UNG catalytic site is not, whereas the dUTPase gene can be deleted without impairing virus replication. Recombinant VACV with an UNG catalytic site mutation was attenuated in vivo, while a dUTPase deletion mutant was not. However, the importance of the two enzymes for replication in quiescent cells, their possible synergy and roles in virulence have not been fully assessed. Results VACV mutants lacking the gene encoding dUTPase or with catalytic site mutations in UNG and double UNG/dUTPase mutants were constructed. Replication of UNG and UNG/dUTPase mutants were slightly reduced compared to wild type or the dUTPase mutant in actively dividing cells. Viral DNA replication was reduced about one-third under these conditions. After high multiplicity infection of quiescent fibroblasts, yields of wild type and mutant viruses were decreased by 2-logs with relative differences similar to those observed in active fibroblasts. However, under low multiplicity multi-step growth conditions in quiescent fibroblasts, replication of the dUTPase/UNG mutant was delayed and 5-fold lower than that of either single mutant or parental virus. This difference was exacerbated by 1-day serial passages on quiescent fibroblasts, resulting in 2- to 3-logs lower titer of the double mutant compared to the parental and single mutant viruses. Each mutant was more attenuated than a revertant

  14. Crystal Structure of Baculovirus RNA Triphosphatase Complexed with Phosphate

    SciTech Connect

    Changela, Anita; Martin, Alexandra; Shuman, Stewart; Mondragon, Alfonso

    2010-03-05

    Baculovirus RNA 5'-triphosphatase (BVP) exemplifies a family of RNA-specific cysteine phosphatases that includes the RNA triphosphatase domains of metazoan and plant mRNA capping enzymes. Here we report the crystal structure of BVP in a phosphate-bound state at 1.5 {angstrom} resolution. BVP adopts the characteristic cysteine-phosphatase {alpha}/{beta} fold and binds two phosphate ions in the active site region, one of which is proposed to mimic the phosphate of the product complex after hydrolysis of the covalent phosphoenzyme intermediate. The crystal structure highlights the role of backbone amides and side chains of the P-loop motif {sup 118}HCTHGXNRT{sup 126} in binding the cleavable phosphate and stabilizing the transition state. Comparison of the BVP structure to the apoenzyme of mammalian RNA triphosphatase reveals a concerted movement of the Arg-125 side chain (to engage the phosphate directly) and closure of an associated surface loop over the phosphate in the active site. The structure highlights a direct catalytic role of Asn-124, which is the signature P-loop residue of the RNA triphosphatase family and a likely determinant of the specificity of BVP for hydrolysis of phosphoanhydride linkages.

  15. Magnetic resonance and kinetic studies of the mechanism of membrane-bound sodium and potassium ion- activated adenosine triphosphatase.

    PubMed

    Grisham, C M; Mildvan, A S

    1975-01-01

    EPR and water proton relaxation rate (1/T1) studies of partially (40%) and "fully" (90%) purified preparations of membrane-bound (Na+ + K+) activated ATPase from sheep kidney indicate one tight binding site for Mn2+ per enzyme dimer, with a dissociation constant (KD = 0.88 muM) in agreement with the kinetically determined activator constant, identifying this Mn2+-binding site as the active site of the ATPase. Competition studies indicate that Mg2+ binds at this site with a dissociation constant of 1 mM in agreement with its activator constant. Inorganic phosphate and methylphosphonate bind to the enzyme-Mn2+ complex with similar high affinities and decrease 1/T1 of water protons due to a decrease from four to three in the number of rapidly exchanging water protons in the coordination sphere of enzyme-bound Mn2+. The relative effectiveness of Na+ and K+ in facilitating ternary complex formation with HPO2-4 and CH3PO2-3 as a function of pH indicates that Na+ induces the phosphate monoanion to interact with enzyme-bound Mn2+. Thus protonation of an enzyme-bound phosphoryl group would convert a K+-binding site to a Na+-binding site. Dissociation constants for K+ and Na+, estimated from NMR titrations, agreed with kinetically determined activator constants of these ions consistent with binding to the active site. Parallel 32Pi-binding studies show negligible formation (less than 7%) of a covalent E-P complex under these conditions, indicating that the NMR method has detected an additional noncovalent intermediate in ion transport. Ouabain, which increases the extent of phosphorylation of the enzyme to 24% at pH 7.8 and to 106% at pH 6.1, produced further decreases in 1/T1 of water protons. Preliminary 31P- relaxation studies of CH3PO2-3 in the presence of ATPase and Mn2+ yield an Mn to P distance (6.9 +/- 0.5 A) suggesting a second sphere enzyme-Mn-ligand-CH3PO2-3 complex. Previous kinetic studies have shown that T1+ substitutes for K+ in the activation of the enzyme

  16. Pre-steady-state studies of the adenosine triphosphatase activity of coupled submitochondrial particles. Regulation by ADP.

    PubMed

    Martins, O B; Tuena de Gómez-Puyou, M; Gómez-Puyou, A

    1988-09-20

    ATPase activities were measured in 10 mM MgCl2, 5 mM ATP, 1 mM ADP, and 1 microM FCCP with submitochondrial particles from bovine heart that had been stimulated by delta mu H+-forming substrates and with particles whose natural inhibitor protein was partially removed by heating. The activities were not linear with time. With both particles, the rate of ATP hydrolysis in the 7-fold greater than that in the steady state. Pre-steady-state and steady-state kinetic studies showed that the decrease of ATPase activity was due to the binding of ADP in a high-affinity site of the enzyme (K0.5 of 10 microM). Inhibition of ATP hydrolysis was accompanied by the binding of approximately 1 mol of ADP/mol of particulate F1; 10 microM ADP gave half-maximal binding. ADP could be replaced by IDP, but with an affinity 50-fold lower (K0.5 of 0.5 mM). Maximal inhibition by ADP and IDP was achieved in less than 5 s. Inhibition was enhanced by uncouplers. Even in the presence of pyruvate kinase and phosphoenolpyruvate, the rates of hydrolysis were about 2.5-fold higher in the first seconds of reaction than in the steady state. This decrease of ATPase activity also correlated with the binding of nearly 1 mol of ADP/mol of F1. This inhibitory ADP remained bound to the enzyme after several thousand turnovers. Apparently, it is possible to observe maximal rates of hydrolysis only in the first few catalytic cycles of the enzyme. PMID:2974725

  17. Cytokinin Nucleosides - Natural Compounds with a Unique Spectrum of Biological Activities.

    PubMed

    Drenichev, Mikhail S; Oslovsky, Vladimir E; Mikhailov, Sergey N

    2016-01-01

    Cytokinin nucleosides exhibit antitumor, antiviral, antiprotozoal, blood pressure reducing, anti-inflammatory, and antipsychotic activity. These compounds also influence platelet aggregation and exhibit some other biological activities. Cytokinins are N6-substituted adenines and represent an important group of phytohormones with diverse biochemical functions in plants, stimulating cell division and plant growth. The main structural feature of cytokinin nucleosides is the presence of a hydrophobic hydrocarbon moiety at the N6-position of adenosine. This moiety is responsible for a difference in physicochemical and biological properties as compared to adenosine. 1-N-Tuberculosinyladenosine and N6-tuberculosinyladenosine are specifically produced by Mycobacterium tuberculosis as components of the plasmatic membrane, thus making them attractive targets for clinical test development. Structurally related compounds were found in marine organisms. It has been shown also that tRNA contains N6-isoprenyladenosine and some other related compounds. This review summarizes the structural features, biological activity, and the synthesis of cytokinin nucleosides and some of their closely related derivatives such as cytokinins and terpene derivatives of adenine. PMID:27086793

  18. The crystal structure and activity of a putative trypanosomal nucleoside phosphorylase reveal it to be a homodimeric uridine phosphorylase

    PubMed Central

    Larson, Eric T.; Mudeppa, Devaraja G.; Gillespie, J. Robert; Mueller, Natascha; Napuli, Alberto J.; Arif, Jennifer A.; Ross, Jenni; Arakaki, Tracy L.; Lauricella, Angela; DeTitta, George; Luft, Joseph; Zucker, Frank; Verlinde, Christophe L. M. J.; Fan, Erkang; Van Voorhis, Wesley C.; Buckner, Frederick S.; Rathod, Pradipsinh K.; Hol, Wim G. J.; Merritt, Ethan A.

    2010-01-01

    Purine nucleoside phosphorylases and uridine phosphorylases are closely related enzymes involved in purine and pyrimidine salvage, respectively, which catalyze the removal of the ribosyl moiety from nucleosides so that the nucleotide base may be recycled. Parasitic protozoa generally are incapable of de novo purine biosynthesis so the purine salvage pathway is of potential therapeutic interest. Information about pyrimidine biosynthesis in these organisms is much more limited. Though all seem to carry at least a subset of enzymes from each pathway, the dependency on de novo pyrimidine synthesis versus salvage varies from organism to organism and even from one growth stage to another. We have structurally and biochemically characterized a putative nucleoside phosphorylase from the pathogenic protozoan Trypanosoma brucei and find that it is a homodimeric uridine phosphorylase. This is the first characterization of a uridine phosphorylase from a trypanosomal source despite this activity being observed decades ago. Although this gene was broadly annotated as a putative nucleoside phosphorylase, it was widely inferred to be a purine nucleoside phosphorylase. Our characterization of this trypanosomal enzyme shows that it is possible to distinguish between purine and uridine phosphorylase activity at the sequence level based on the absence or presence of a characteristic uridine phosphorylase-specificity insert. We suggest that this recognizable feature may aid in proper annotation of the substrate specificity of enzymes in the nucleoside phosphorylase family. PMID:20070944

  19. Anti‐flavivirus Activity of Different Tritylated Pyrimidine and Purine Nucleoside Analogues

    PubMed Central

    Serpi, Michaela; Slusarczyk, Magdalena; Ferrari, Valentina; Pertusati, Fabrizio; Meneghesso, Silvia; Derudas, Marco; Farleigh, Laura; Zanetta, Paola; Bugert, Joachim

    2016-01-01

    Abstract A series of tritylated and dimethoxytritylated analogues of selected pyrimidine and purine nucleosides were synthesized and evaluated for their in vitro inhibitory activity against two important members of the genus Flavivirus in the Flaviviridae family, the yellow fever (YFV) and dengue viruses (DENV). Among all compounds tested, the 5′‐O‐tritylated and the 5′‐O‐dimethoxytritylated 5‐fluorouridine derivatives exerted potency against YFV. Interestingly in the series of purine analogues, the 5′O, N‐bis‐tritylated fludarabine derivative revealed strong inhibitory activity against DENV at μm concentrations, however significantly weaker potency against YFV. PMID:27551659

  20. Anti-flavivirus Activity of Different Tritylated Pyrimidine and Purine Nucleoside Analogues.

    PubMed

    McGuigan, Christopher; Serpi, Michaela; Slusarczyk, Magdalena; Ferrari, Valentina; Pertusati, Fabrizio; Meneghesso, Silvia; Derudas, Marco; Farleigh, Laura; Zanetta, Paola; Bugert, Joachim

    2016-06-01

    A series of tritylated and dimethoxytritylated analogues of selected pyrimidine and purine nucleosides were synthesized and evaluated for their in vitro inhibitory activity against two important members of the genus Flavivirus in the Flaviviridae family, the yellow fever (YFV) and dengue viruses (DENV). Among all compounds tested, the 5'-O-tritylated and the 5'-O-dimethoxytritylated 5-fluorouridine derivatives exerted potency against YFV. Interestingly in the series of purine analogues, the 5'O, N-bis-tritylated fludarabine derivative revealed strong inhibitory activity against DENV at μm concentrations, however significantly weaker potency against YFV. PMID:27551659

  1. Functional proteomics of adenosine triphosphatase system in the rat striatum during aging☆

    PubMed Central

    Villa, Roberto Federico; Ferrari, Federica; Gorini, Antonella

    2012-01-01

    The maximum rates of adenosine triphosphatase (ATPase) systems related to energy consumption were systematically evaluated in synaptic plasma membranes isolated from the striata of male Wistar rats aged 2, 6, 12, 18, and 24 months, because of their key role in presynaptic nerve ending homeostasis. The following enzyme activities were evaluated: sodium-potassium-magnesium adenosine triphosphatase (Na+, K+, Mg2+-ATPase); ouabain-insensitive magnesium adenosine triphosphatase (Mg2+-ATPase); sodium-potassium adenosine triphosphatase (Na+, K+-ATPase); direct magnesium adenosine triphosphatase (Mg2+-ATPase); calcium-magnesium adenosine triphosphatase (Ca2+, Mg2+-ATPase); and acetylcholinesterase. The results showed that Na+, K+-ATPase decreased at 18 and 24 months, Ca2+, Mg2+-ATPase and acetylcholinesterase decreased from 6 months, while Mg2+-ATPase was unmodified. Therefore, ATPases vary independently during aging, suggesting that the ATPase enzyme systems are of neuropathological and pharmacological importance. This could be considered as an experimental model to study regeneration processes, because of the age-dependent modifications of specific synaptic plasma membranes. ATPases cause selective changes in some cerebral functions, especially bioenergetic systems. This could be of physiopathological significance, particularly in many central nervous system diseases, where, during regenerative processes, energy availability is essential. PMID:25806051

  2. Synthesis and anticancer activities of novel 8-azapurine carbocyclic nucleoside hydrazones.

    PubMed

    Wang, Yeming; Yan, Hong; Ma, Chao; Lu, Dan

    2015-10-15

    A series of novel 8-azapurine carbocyclic nucleoside hydrazones were synthesized through a useful procedure starting from amino alcohol and pyrimido dichloride. All the products were characterized by (1)H NMR, (13)C NMR and HRMS spectral analysis and the stereochemical structure of key intermediate was also confirmed by a single crystal X-ray diffraction crystallographic analysis. Moreover, the anticancer activities were evaluated in vitro against human liver cancer Huh-7 cell line and human breast cancer A549 cell line. PMID:26364944

  3. Broad-spectrum antiviral and cytocidal activity of cyclopentenylcytosine, a carbocyclic nucleoside targeted at CTP synthetase.

    PubMed

    De Clercq, E; Murase, J; Marquez, V E

    1991-06-15

    Cyclopentenylcytosine (Ce-Cyd) is a broad-spectrum antiviral agent active against DNA viruses [herpes (cytomegalo), pox (vaccinia)], (+)RNA viruses [picorna (polio, Coxsackie, rhino), toga (Sindbis, Semliki forest), corona], (-)RNA viruses [orthomyxo (influenza), paramyxo (parainfluenza, measles), arena (Junin, Tacaribe), rhabdo (vesicular stomatitis)] and (+/-)RNA viruses (reo). Ce-Cyd is a more potent antiviral agent than its saturated counterpart, cyclopentylcytosine (carbodine, C-Cyd). Ce-Cyd also has potent cytocidal activity against a number of tumor cell lines. The putative target enzyme for both the antiviral and antitumor action of Ce-Cyd is assumed to be the CTP synthetase that converts UTP to CTP. In keeping with this hypothesis was the finding that the antiviral and cytocidal effects of Ce-Cyd are readily reversed by Cyd and, to a lesser extent, Urd, but not by other nucleosides such as dThd or dCyd. In contrast, pyrazofurin and 6-azauridine, two nucleoside analogues that are assumed to interfere with OMP decarboxylase, another enzyme involved in the biosynthesis of pyrimidine ribonucleotides, potentiate the cytocidal activity of Ce-Cyd. Ce-Cyd should be further pursued, as such and in combination with OMP decarboxylase inhibitors, for its therapeutic potential in the treatment of both viral and neoplastic diseases. PMID:1710119

  4. Escherichia coli mutants deficient in deoxyuridine triphosphatase.

    PubMed Central

    Hochhauser, S J; Weiss, B

    1978-01-01

    Mutants deficient in deoxyuridine triphosphatase (dUTPase) were identified by enzyme assays of randomly chosen heavily mutagenized clones. Five mutants of independent origin were obtained. One mutant produced a thermolabile enzyme, and it was presumed to have a mutation in the structural gene for dUTPase, designated dut. The most deficient mutant had the following associated phenotypes: less than 1% of parental dUTPase activity, prolonged generation time, increased sensitivity to 5'-fluorodeoxyuridine, increased rate of spontaneous mutation, increased rate of recombination (hyper-Rec), an inhibition of growth in the presence of 2 mM uracil, and a decreased ability to support the growth of phage P1 (but not T4 or lambda). This mutation also appeared to be incompatible with pyrE mutations. A revertant selected by its faster growth had regained dUTPase activity and lost its hyper-Rec phenotype. Many of the properties of the dut mutants are compatible with their presumed increased incorporation of uracil into DNA and the subsequent transient breakage of the DNA by excision repair. PMID:148458

  5. Investigation of the active site and the conformational stability of nucleoside diphosphate kinase by site-directed mutagenesis.

    PubMed

    Tepper, A D; Dammann, H; Bominaar, A A; Véron, M

    1994-12-23

    Nucleoside-diphosphate kinase (EC 2.7.4.6) catalyzes phosphate exchange between nucleoside triphosphates and nucleoside diphosphates. Its 17 kDa subunits are highly conserved throughout evolution in both sequence and tertiary structure. Using site-directed mutagenesis we investigated the function of 8 amino acids (Lys16, Tyr56, Arg92, Thr98, Arg109, Asn119, Ser124, and Glu133) that are totally conserved among all nucleoside diphosphate kinases known to date. The mutant proteins all show decreased specific activity and support roles for these residues in catalysis, substrate binding, or both, as was previously proposed on the basis of the x-ray structure (Moréra, S., Lascu, I., Dumas, C., LeBras, G., Briozzo, P., Véron, M., and Janin, J. (1994) Biochemistry 33, 459-467). Furthermore, residues Lys16, Arg109, and Asn 119 were identified to play important roles in conformational stability or subunit interactions. We show that Lys16 and Asn119 form a rigid structure that is important for enzymatic function and that Arg109, known to interact with the phosphate moiety of the substrate, also plays an important role in subunit association. The dual roles of Lys16, Arg109, and Asn119 in both substrate binding and subunit assembly provide further evidence for a functional coupling between catalytic activity and quaternary structure in nucleoside diphosphate kinase. PMID:7798215

  6. Synthesis and Anti-HCV Activity of 4-Methoxy-7H-Pyrrolo[2,3-d] Pyrimidine Carbocyclic Nucleosides.

    PubMed

    Thiyagarajan, Anandarajan; Toyama, Masaaki; Baba, Masanori; Sharon, Ashoke; Bal, Chandralata

    2016-06-01

    The present study includes the exploration of new possible nucleoside mimetics based on 4-methoxy-7H-pyrrolo[2,3-d]pyrimidine carbocyclic nucleosides (4a-g), which were synthesized by 10-15 synthetic steps and characterized adequately. We report the anti-HCV activities and cytotoxicities of 4a-g. Compound 4a was analyzed by single crystal X-ray diffraction which showed some puckering in the cyclopentene ring with a 2'-endo conformation and anti-base disposition (χ = -125.7°). PMID:27152477

  7. Nanomolar Inhibitors of Trypanosoma brucei RNA Triphosphatase

    PubMed Central

    Smith, Paul; Ho, C. Kiong; Takagi, Yuko; Djaballah, Hakim

    2016-01-01

    ABSTRACT Eukaryal taxa differ with respect to the structure and mechanism of the RNA triphosphatase (RTPase) component of the mRNA capping apparatus. Protozoa, fungi, and certain DNA viruses have a metal-dependent RTPase that belongs to the triphosphate tunnel metalloenzyme (TTM) superfamily. Because the structures, active sites, and chemical mechanisms of the TTM-type RTPases differ from those of mammalian RTPases, the TTM RTPases are potential targets for antiprotozoal, antifungal, and antiviral drug discovery. Here, we employed RNA interference (RNAi) knockdown methods to show that Trypanosoma brucei RTPase Cet1 (TbCet1) is necessary for proliferation of procyclic cells in culture. We then conducted a high-throughput biochemical screen for small-molecule inhibitors of the phosphohydrolase activity of TbCet1. We identified several classes of chemicals—including chlorogenic acids, phenolic glycopyranosides, flavonoids, and other phenolics—that inhibit TbCet1 with nanomolar to low-micromolar 50% inhibitory concentrations (IC50s). We confirmed the activity of these compounds, and tested various analogs thereof, by direct manual assays of TbCet1 phosphohydrolase activity. The most potent nanomolar inhibitors included tetracaffeoylquinic acid, 5-galloylgalloylquinic acid, pentagalloylglucose, rosmarinic acid, and miquelianin. TbCet1 inhibitors were less active (or inactive) against the orthologous TTM-type RTPases of mimivirus, baculovirus, and budding yeast (Saccharomyces cerevisiae). Our results affirm that a TTM RTPase is subject to potent inhibition by small molecules, with the caveat that parallel screens against TTM RTPases from multiple different pathogens may be required to fully probe the chemical space of TTM inhibition. PMID:26908574

  8. A metal-containing nucleoside that possesses both therapeutic and diagnostic activity against cancer.

    PubMed

    Choi, Jung-Suk; Maity, Ayan; Gray, Thomas; Berdis, Anthony J

    2015-04-10

    Nucleoside transport is an essential process that helps maintain the hyperproliferative state of most cancer cells. As such, it represents an important target for developing diagnostic and therapeutic agents that can effectively detect and treat cancer, respectively. This report describes the development of a metal-containing nucleoside designated Ir(III)-PPY nucleoside that displays both therapeutic and diagnostic properties against the human epidermal carcinoma cell line KB3-1. The cytotoxic effects of Ir(III)-PPY nucleoside are both time- and dose-dependent. Flow cytometry analyses validate that the nucleoside analog causes apoptosis by blocking cell cycle progression at G2/M. Fluorescent microscopy studies show rapid accumulation in the cytoplasm within 4 h. However, more significant accumulation is observed in the nucleus and mitochondria after 24 h. This localization is consistent with the ability of the metal-containing nucleoside to influence cell cycle progression at G2/M. Mitochondrial depletion is also observed after longer incubations (Δt ∼48 h), and this effect may produce additional cytotoxic effects. siRNA knockdown experiments demonstrate that the nucleoside transporter, hENT1, plays a key role in the cellular entry of Ir(III)-PPY nucleoside. Collectively, these data provide evidence for the development of a metal-containing nucleoside that functions as a combined therapeutic and diagnostic agent against cancer. PMID:25713072

  9. Versatile synthesis of oxime-containing acyclic nucleoside phosphonates--synthetic solutions and antiviral activity.

    PubMed

    Solyev, Pavel N; Jasko, Maxim V; Kleymenova, Alla A; Kukhanova, Marina K; Kochetkov, Sergey N

    2015-11-28

    New oxime-containing acyclic nucleoside phosphonates 9-{2-[(phosphonomethyl)oximino]ethyl}adenine (1), -guanine (2) and 9-{2-[(phosphonomethyl)oximino]propyl}adenine (3) with wide spectrum activity against different types of viruses were synthesized. The key intermediate, diethyl aminooxymethylphosphonate, was obtained by the Mitsunobu reaction. Modified conditions for the by-product separation (without chromatography and distillation) allowed us to obtain 85% yield of the aminooxy intermediate. The impact of DBU and Cs2CO3 on the N(9)/N(7) product ratio for adenine and guanine alkylation was studied. A convenient procedure for aminooxy group detection was found. The synthesized phosphonates were tested and they appeared to display moderate activity against different types of viruses (HIV, herpes viruses in cell cultures, and hepatitis C virus in the replicon system) without toxicity up to 1000 μM. PMID:26383895

  10. Arginine kinase shows nucleoside diphosphate kinase-like activity toward deoxythymidine diphosphate.

    PubMed

    Lopez-Zavala, Alonso A; Sotelo-Mundo, Rogerio R; Hernandez-Flores, Jose M; Lugo-Sanchez, Maria E; Sugich-Miranda, Rocio; Garcia-Orozco, Karina D

    2016-06-01

    Arginine kinase (AK) (ATP: L-arginine phosphotransferase, E.C. 2.7.3.3) catalyzes the reversible transfer of ATP γ-phosphate group to L-arginine to synthetize phospho-arginine as a high-energy storage. Previous studies suggest additional roles for AK in cellular processes. Since AK is found only in invertebrates and it is homologous to creatine kinase from vertebrates, the objective of this work was to demonstrate nucleoside diphosphate kinase-like activity for shrimp AK. For this, AK from marine shrimp Litopenaeus vannamei (LvAK) was purified and its activity was assayed for phosphorylation of TDP using ATP as phosphate donor. Moreover, by using high-pressure liquid chromatography (HPLC) the phosphate transfer reaction was followed. Also, LvAK tryptophan fluorescence emission changes were detected by dTDP titration, suggesting that the hydrophobic environment of Trp 221, which is located in the top of the active site, is perturbed upon dTDP binding. The kinetic constants for both substrates Arg and dTDP were calculated by isothermal titration calorimetry (ITC). Besides, docking calculations suggested that dTDP could bind LvAK in the same cavity where ATP bind, and LvAK basic residues (Arg124, 126 and 309) stabilize the dTDP phosphate groups and the pyrimidine base interact with His284 and Ser122. These results suggest that LvAK bind and phosphorylate dTDP being ATP the phosphate donor, thus describing a novel alternate nucleoside diphosphate kinase-like activity for this enzyme. PMID:27072556

  11. Mitochondrial adenosine triphosphatase of the fission yeast, Schizosaccharomyces pombe 972h-. Changes in activity and oligomycin-sensitivity during the cell cycle of catabolite-repressed and -de-repressed cells.

    PubMed Central

    Edwards, S W; Lloyd, D

    1977-01-01

    1. Changes in activity of ATPase (adenosine triphosphatase) during the cell cycle of Schizosaccharomyces pombe were analysed in cell-free extracts of cells harvested from different stages of growth of synchronous cultures and also after cell-cycle fractionation. 2. Oligomycin-sensitive ATPase oscillates in both glucose-repressed synchronous cultures and shows four maxima of activity approximately equally spaced through the cell cycle. The amplitude of the oscillations accounts for between 13 and 80% of the total activity at different times in the cell cycle. 3. Oligomycin sensitivity varies over a fourfold range at different stages of the cell cycle. 4. The periodicity of maximum oligomycin sensitivity is one-quarter of a cell cycle. 5. These results were confirmed for the first three-quarters of the cell cycle by cell-cycle fractionation. 6. In cells growing synchronously with glycerol, ATPase activity increases in a stepwise pattern, with two steps per cell cycle; the first of these occurs at 0.54 of the cell cycle and the second at 0.95. 7. These results are discussed in relation to previously obtained data on the development of mitochondrial activities during the cell cycle. PMID:139890

  12. Structural and functional characterization of a noncanonical nucleoside triphosphate pyrophosphatase from Thermotoga maritima

    PubMed Central

    Awwad, Khaldeyah; Desai, Anna; Smith, Clyde; Sommerhalter, Monika

    2013-01-01

    The hyperthermophilic bacterium Thermotoga maritima has a noncanonical nucleoside triphosphatase that catalyzes the conversion of inosine triphosphate (ITP), deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP) into inosine monophosphate (IMP), deoxyinosine monophosphate (IMP) and xanthosine monophosphate (XMP), respectively. The k cat/K m values determined at 323 and 353 K fall between 1.31 × 104 and 7.80 × 104  M −1 s−1. ITP and dITP are slightly preferred over XTP. Activity towards canonical nucleoside triphosphates (ATP and GTP) was not detected. The enzyme has an absolute requirement for Mg2+ as a cofactor and has a preference for alkaline conditions. A protein X-ray structure of the enzyme with bound IMP was obtained at 2.15 Å resolution. The active site houses a well conserved network of residues that are critical for substrate recognition and catalysis. The crystal structure shows a tetramer with two possible dimer interfaces. One of these interfaces strongly resembles the dimer interface that is found in the structures of other noncanonical nucleoside pyrophosphatases from human (human ITPase) and archaea (Mj0226 and PhNTPase). PMID:23385455

  13. Adenosine triphosphatases of thermophilic archaeal double-stranded DNA viruses

    PubMed Central

    2014-01-01

    Adenosine triphosphatases (ATPases) of double-stranded (ds) DNA archaeal viruses are structurally related to the AAA+ hexameric helicases and translocases. These ATPases have been implicated in viral life cycle functions such as DNA entry into the host, and viral genome packaging into preformed procapsids. We summarize bioinformatical analyses of a wide range of archaeal ATPases, and review the biochemical and structural properties of those archaeal ATPases that have measurable ATPase activity. We discuss their potential roles in genome delivery into the host, virus assembly and genome packaging in comparison to hexameric helicases and packaging motors from bacteriophages. PMID:25105011

  14. E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) of Leishmania amazonensis inhibits macrophage activation.

    PubMed

    Gomes, Rodrigo Saar; de Carvalho, Luana Cristina Faria; de Souza Vasconcellos, Raphael; Fietto, Juliana Lopes Rangel; Afonso, Luís Carlos Crocco

    2015-04-01

    Leishmania amazonensis, the causal agent of diffuse cutaneous leishmaniasis, is known for its ability to modulate the host immune response. Because a relationship between ectonucleotidase activity and the ability of Leishmania to generate injury in C57BL/6 mice has been demonstrated, in this study we evaluated the involvement of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) activity of L. amazonensis in the process of infection of J774-macrophages. Our results show that high-activity parasites show increased survival rate in LPS/IFN-γ-activated cells, by inhibiting the host-cell NO production. Conversely, inhibition of E-NTPDase activity reduces the parasite survival rates, an effect associated with increased macrophage NO production. E-NTPDase activity generates substrate for the production of extracellular adenosine, which binds to A2B receptors and reduces IL-12 and TNF-α produced by activated macrophages, thus inhibiting NO production. These results indicate that E-NTPDase activity is important for survival of L. amazonensis within macrophages, showing the role of the enzyme in modulating macrophage response and lower NO production, which ultimately favors infection. Our results point to a new mechanism of L. amazonensis infection that may pave the way for the development of new treatments for this neglected disease. PMID:25554487

  15. Structural Insight into Activation Mechanism of Toxoplasma gondii Nucleoside Triphosphate Diphosphohydrolases by Disulfide Reduction*

    PubMed Central

    Krug, Ulrike; Zebisch, Matthias; Krauss, Michel; Sträter, Norbert

    2012-01-01

    The intracellular parasite Toxoplasma gondii produces two nucleoside triphosphate diphosphohydrolases (NTPDase1 and -3). These tetrameric, cysteine-rich enzymes require activation by reductive cleavage of a hitherto unknown disulfide bond. Despite a 97% sequence identity, both isozymes differ largely in their ability to hydrolyze ATP and ADP. Here, we present crystal structures of inactive NTPDase3 as an apo form and in complex with the product AMP to resolutions of 2.0 and 2.2 Å, respectively. We find that the enzyme is present in an open conformation that precludes productive substrate binding and catalysis. The cysteine bridge 258–268 is identified to be responsible for locking of activity. Crystal structures of constitutively active variants of NTPDase1 and -3 generated by mutation of Cys258–Cys268 show that opening of the regulatory cysteine bridge induces a pronounced contraction of the whole tetramer. This is accompanied by a 12° domain closure motion resulting in the correct arrangement of all active site residues. A complex structure of activated NTPDase3 with a non-hydrolyzable ATP analog and the cofactor Mg2+ to a resolution of 2.85 Å indicates that catalytic differences between the NTPDases are primarily dictated by differences in positioning of the adenine base caused by substitution of Arg492 and Glu493 in NTPDase1 by glycines in NTPDase3. PMID:22130673

  16. Leishmania amazonensis: Biological and biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities.

    PubMed

    Pinheiro, Carla M; Martins-Duarte, Erica S; Ferraro, Rodrigo B; Fonseca de Souza, André Luíz; Gomes, Marta T; Lopes, Angela H C S; Vannier-Santos, Marcos A; Santos, André L S; Meyer-Fernandes, José R

    2006-09-01

    The presence of Leishmania amazonensis ecto-nucleoside triphosphate triphosphohydrolase activities was demonstrated using antibodies against different NTPDase members by Western blotting, flow cytometry, and immunoelectron microscopy analysis. Living promastigote cells sequentially hydrolyzed the ATP molecule generating ADP, AMP, and adenosine, indicating that this surface enzyme may play a role in the salvage of purines from the extracellular medium. The L. amazonensis ecto-NTPDase activities were insensitive to Triton X-100, but they were enhanced by divalent cations, such as Mg(2+). In addition, the ecto-NTPDase activities decreased with time for 96 h when promastigotes were grown in vitro. On the other hand, these activities increased considerably when measured in living amastigote forms. Furthermore, the treatment with adenosine, a mediator of several relevant biological phenomena, induced a decrease in the reactivity with anti-CD39 antibody, raised against mammalian E-NTPDase, probably because of down regulation in the L. amazonensis ecto-NTPDase expression. Also, adenosine and anti-NTPDase antibodies induced a significant diminishing in the interaction between promastigotes of L. amazonensis and mouse peritoneal macrophages. PMID:16603157

  17. Ester prodrugs of acyclic nucleoside thiophosphonates compared to phosphonates: synthesis, antiviral activity and decomposition study.

    PubMed

    Roux, Loïc; Priet, Stéphane; Payrot, Nadine; Weck, Clément; Fournier, Maëlenn; Zoulim, Fabien; Balzarini, Jan; Canard, Bruno; Alvarez, Karine

    2013-05-01

    9-[2-(Thiophosphonomethoxy)ethyl]adenine [S-PMEA, 8] and (R)-9-[2-(Thiophosphonomethoxy)propyl]adenine [S-PMPA, 9] are acyclic nucleoside thiophosphonates we described recently that display the same antiviral spectrum (DNA viruses) as approved and potent phosphonates PMEA and (R)-PMPA. Here, we describe the synthesis, antiviral activities in infected cell cultures and decomposition study of bis(pivaloyloxymethoxy)-S-PMEA [Bis-POM-S-PMEA, 13] and bis(isopropyloxymethylcarbonyl)-S-PMPA [Bis-POC-S-PMPA, 14] as orally bioavailable prodrugs of the S-PMEA 8 and S-PMPA 9, in comparison to the equivalent "non-thio" derivatives [Bis-POM-PMEA, 11] and [Bis-POC-PMPA, 12]. Compounds 11, 12, 13 and 14 were evaluated for their in vitro antiviral activity against HIV-1-, HIV-2-, HBV- and a broad panel of DNA viruses, and found to exhibit moderate to potent antiviral activity. In order to determine the decomposition pathway of the prodrugs 11, 12, 13 and 14 into parent compounds PMEA, PMPA, 8 and 9, kinetic data and decomposition pathways in several media are presented. As expected, bis-POM-S-PMEA 13 and bis-POC-S-PMPA 14 behaved as prodrugs of S-PMEA 8 and S-PMPA 9. However, thiophosphonates 8 and 9 were released very smoothly in cell extracts, in contrast to the release of PMEA and PMPA from "non-thio" prodrugs 11 and 12. PMID:23603046

  18. Amphiphilic cationic nanogels as brain-targeted carriers for activated nucleoside reverse transcriptase inhibitors

    PubMed Central

    Warren, G; Makarov, E; Lu, Y; Senanayake, T; Rivera, K; Gorantla, S; Poluektova, LY; Vinogradov, SV

    2015-01-01

    Progress in AIDS treatment shifted emphasis towards limiting adverse effects of antiviral drugs while improving the treatment of hard-to-reach viral reservoirs. Many therapeutic nucleoside reverse transcriptase inhibitors (NRTI) have a limited access to the central nervous system (CNS). Increased NRTI levels induced various complications during the therapy, including neurotoxicity, due to the NRTI toxicity to mitochondria. Here, we describe an innovative design of biodegradable cationic cholesterol-ε-polylysine nanogel carriers for delivery of triphosphorylated NRTIs that demonstrated high anti-HIV activity along with low neurotoxicity, warranting minimal side effects following systemic administration. Efficient CNS targeting was achieved by nanogel modification with brain-specific peptide vectors. Novel dual and triple-drug nanoformulations, analogous to therapeutic NRTI cocktails, displayed equal or higher antiviral activity in HIV-infected macrophages compared to free drugs. Our results suggest potential alternative approach to HIV-1 treatment focused on the effective nanodrug delivery to viral reservoirs in the CNS and reduced neurotoxicity. PMID:25559020

  19. Influence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence

    PubMed Central

    Santos, Ramon F.; Pôssa, Marcela A. S.; Bastos, Matheus S.; Guedes, Paulo M. M.; Almeida, Márcia R.; DeMarco, Ricardo; Verjovski-Almeida, Sergio; Bahia, Maria T.; Fietto, Juliana L. R.

    2009-01-01

    Background The protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family) from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models. Methodology/Principal Findings We followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes) obtained during sequential sub-cultivation in mammalian cells. ATPase/ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin), or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites. Conclusions/Significance Our results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target candidates in chemotherapy

  20. Nucleoside Inhibitors of Zika Virus.

    PubMed

    Eyer, Luděk; Nencka, Radim; Huvarová, Ivana; Palus, Martin; Joao Alves, Maria; Gould, Ernest A; De Clercq, Erik; Růžek, Daniel

    2016-09-01

    There is growing evidence that Zika virus (ZIKV) can cause devastating infant brain defects and other neurological disorders in humans. However, no specific antiviral therapy is available at present. We tested a series of 2'-C- or 2'-O-methyl-substituted nucleosides, 2'-C-fluoro-2'-C-methyl-substituted nucleosides, 3'-O-methyl-substituted nucleosides, 3'-deoxynucleosides, derivatives with 4'-C-azido substitution, heterobase-modified nucleosides, and neplanocins for their ability to inhibit ZIKV replication in cell culture. Antiviral activity was identified when 2'-C-methylated nucleosides were tested, suggesting that these compounds might represent promising lead candidates for further development of specific antivirals against ZIKV. PMID:27234417

  1. Purine nucleoside phosphorylase and xanthine oxidase activities in erythrocytes and plasma from marine, semiaquatic and terrestrial mammals.

    PubMed

    López-Cruz, Roberto I; Pérez-Milicua, Myrna Barjau; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal-Vertiz, Jaime A; de la Rosa, Alejandro; Vázquez-Medina, José P; Zenteno-Savín, Tania

    2014-05-01

    Purine nucleoside phosphorylase (PNP) and xanthine oxidase (XO) are key enzymes involved in the purine salvage pathway. PNP metabolizes purine bases to synthetize purine nucleotides whereas XO catalyzes the oxidation of purines to uric acid. In humans, PNP activity is reported to be high in erythrocytes and XO activity to be low in plasma; however, XO activity increases after ischemic events. XO activity in plasma of northern elephant seals has been reported during prolonged fasting and rest and voluntary associated apneas. The objective of this study was to analyze circulating PNP and XO activities in marine mammals adapted to tolerate repeated cycles of ischemia/reperfusion associated with diving (bottlenose dolphin, northern elephant seal) in comparison with semiaquatic (river otter) and terrestrial mammals (human, pig). PNP activities in plasma and erythrocytes, as well as XO activity in plasma, from all species were quantified by spectrophotometry. No clear relationship in circulating PNP or XO activity could be established between marine, semiaquatic and terrestrial mammals. Erythrocytes from bottlenose dolphins and humans are highly permeable to nucleosides and glucose, intraerythrocyte PNP activity may be related to a release of purine nucleotides from the liver. High-energy costs will probably mean a higher ATP degradation rate in river otters, as compared to northern elephant seals or dolphins. Lower erythrocyte PNP activity and elevated plasma XO activity in northern elephant seal could be associated with fasting and/or sleep- and dive-associated apneas. PMID:24530799

  2. The functional unit of calcium-plus-magnesium-ion-dependent adenosine triphosphatase from sarcoplasmic reticulum. The aggregational state of the deoxycholate-solubilized protein in an enzymically active form

    PubMed Central

    Jørgensen, Karl E.; Lind, Kirsten E.; Røigaard-Petersen, Hans; Møller, Jesper V.

    1978-01-01

    Vesicles consisting of (Ca2++Mg2+)-dependent ATPase (adenosine triphosphatase), and lipid were prepared from sarcoplasmic reticulum of rabbit skeletal muscle. As with non-ionic detergents [le Maire, Møller & Tanford (1976) Biochemistry 15, 2336–2342] the (Ca2++Mg2+)-dependent ATPase after solubilization by deoxycholate showed a pronounced tendency to form oligomers in gel-chromatographic experiments, when eluted in the presence of deoxycholate and phosphatidylcholine. To evaluate the functional significance of oligomer formation the properties of enzymically active preparations of ATPase, solubilized by deoxycholate, were studied. Such preparations were obtained at a protein concentration of 2.5mg/ml in the presence of a high salt concentration (0.4m-KCl) and sucrose (0.3m) in the solubilization medium. Analytical ultracentrifugation of solubilized ATPase showed one protein boundary moving at the same rate as gel-chromatographically prepared monomeric ATPase (s20,w=6.0S). From simultaneous measurements of the diffusion coefficient an apparent molecular weight of 133000 was calculated, consistent with solubilization of ATPase in predominantly monomeric form. The enzymic activity of deoxycholate-solubilized ATPase when measured directly in the solubilization medium at optimal Ca2+ and MgATP concentrations was about 35–50% of that of vesicular ATPase. The dependence of enzymic activity on MgATP concentration indicated that the solubilized ATPase retained high-affinity binding of MgATP, but the presence of high concentrations of the nucleotide did not stimulate activity further, in contrast with that of vesicular ATPase. The dependence of enzymic activity on the free Ca2+ concentration was essentially the same for both solubilized and vesicular forms, indicating that interaction of ATPase with more than one molecule of Ca2+ is required for enzyme activity. Solubilized enzyme at 20°C was phosphorylated to about the same degree as vesicular ATPase. It is concluded

  3. Interferon-gamma regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways.

    PubMed

    Soler, Concepció; Felipe, Antonio; García-Manteiga, José; Serra, Maria; Guillén-Gómez, Elena; Casado, F Javier; MacLeod, Carol; Modolell, Manuel; Pastor-Anglada, Marçal; Celada, Antonio

    2003-11-01

    The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-gamma (interferon-gamma). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-gamma was studied using macrophages from STAT1 knockout mice. IFN-gamma triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-gamma is required for DNA synthesis. Interestingly, IFN-gamma led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-gamma up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-gamma is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-gamma in macrophages. PMID:12868960

  4. Silicon(IV) phthalocyanines substituted axially with different nucleoside moieties. Effects of nucleoside type on the photosensitizing efficiencies and in vitro photodynamic activities.

    PubMed

    Zheng, Bi-Yuan; Shen, Xiao-Min; Zhao, Dong-Mei; Cai, Yi-Bin; Ke, Mei-Rong; Huang, Jian-Dong

    2016-06-01

    A series of new silicon(IV) phthalocyanines (SiPcs) di-substituted axially with different nucleoside moieties have been synthesized and evaluated for their singlet oxygen quantum yields (ΦΔ) and in vitro photodynamic activities. The adenosine-substituted SiPc shows a lower photosensitizing efficiency (ΦΔ=0.35) than the uridine- and cytidine-substituted analogs (ΦΔ=0.42-0.44), while the guanosine-substituted SiPc exhibits a weakest singlet oxygen generation efficiency with a ΦΔ value down to 0.03. On the other hand, replacing axial adenosines with chloro-modified adenosines and purines can result in the increase of photogenerating singlet oxygen efficiencies of SiPcs. The formed SiPcs 1 and 2, which contain monochloro-modified adenosines and dichloro-modified purines respectively, appear as efficient photosensitizers with ΦΔ of 0.42-0.44. Both compounds 1 and 2 present high photocytotoxicities against HepG2 and BGC823 cancer cells with IC50 values ranging from 9nM to 33nM. The photocytotoxicities of these two compounds are remarkably higher than the well-known anticancer photosensitizer, chlorin e6 (IC50=752nM against HepG2 cells) in the same condition. As revealed by confocal microscopy, for both cell lines, compound 1 can essentially bind to mitochondria, while compound 2 is just partially localized in mitochondria. In addition, the two compounds induce cell death of HepG2 cells likely through apoptosis. PMID:27085051

  5. Effects of chronic growth hormone hypersecretion on intrinsic contractility, energetics, isomyosin pattern, and myosin adenosine triphosphatase activity of rat left ventricle.

    PubMed Central

    Timsit, J; Riou, B; Bertherat, J; Wisnewsky, C; Kato, N S; Weisberg, A S; Lubetzki, J; Lecarpentier, Y; Winegrad, S; Mercadier, J J

    1990-01-01

    We studied papillary muscle mechanics and energetics, myosin phenotype, and ATPase activities in left ventricles from rats bearing a growth hormone (GH)--secreting tumor. 18 wk after tumor induction, animals exhibited a dramatic increase in body weight (+101% vs. controls) but no change in the ventricular weight/body weight ratio. The maximum isometric force of papillary muscles normalized per cross-sectional area rose markedly (+42%, P less than 0.05 vs. controls), whereas the maximum unloaded shortening velocity did not change. This was observed despite a marked isomyosin shift towards V3 (32 +/- 5% vs. 8 +/- 2% in controls, P less than 0.001). Increased curvature of the force-velocity relationship (+64%, P less than 0.05 vs. controls) indicated that the muscles contracted more economically, suggesting the involvement of V3 myosin. Total calcium- and actin-activated myosin ATPase activities assayed on quickly frozen left ventricular sections were similar in tumor-bearing rats and in controls. After alkaline preincubation, these activities only decreased in tumor-bearing rats, demonstrating that V3 enzymatic sites were involved in total ATPase activity. These data demonstrate that chronic GH hypersecretion in the rat leads to a unique pattern of myocardial adaptation which allows the muscle to improve its contractile performance and economy simultaneously, thanks to myosin phenoconversion and an increase in the number of active enzymatic sites. Images PMID:2143510

  6. Improved purification of brine-shrimp (Artemia saline) (Na+ + K+)-activated adenosine triphosphatase and amino-acid and carbohydrate analyses of the isolated subunits.

    PubMed

    Peterson, G L; Hokin, L E

    1980-10-15

    Purification of the (Na+ + K+)-activated ATPase has been improved 2-fold the respect to both purity and yield over the previous method [Peterson, Ewing, Hootman & Conte (1978) J. Biol. Chem. 253, 4762-4770] by using Lubrol WX and non-denaturing concentrations of sodium dodecyl sulphate (SDS). The enzyme was purified 200-fold over the homogenate. The preparation had a specific activity of about 600 mumol of Pi/h per mg of protein, and was about 60% pure according to quantification of Coomassie Blue-stained SDS/polyacrylamide gels. The yield of purified enzyme was about 10 mg of protein per 100g of dry brine-shrimp (Artemia salina) cysts. The method is highly suitable for purification either on a small scale (10-25g of dry cysts) or on a large scale (900g of dry cysts) and methods are described for both. The large (Na+ + K+)-activated ATPase subunit (alpha-subunit) was isolated in pure form by SDS-gel filtration on Bio-Gel A 1.5m. The small subunit (beta-subunit) was eluted with other contaminating proteins on the Bio-Gel column, but was isolated in pure form by extraction from SDS/polyacrylamide gels. The amino acid and carbohydrate compositions of both subunits are reported. The alpha-subunit contained 5.2% carbohydrate by weight, and the beta-subunit 9.2%. Sialic acid was absent from both subunits. PMID:6272692

  7. Characterization of nucleoside triphosphate diphosphohydrolase activity in Trichomonas gallinae and the influence of penicillin and streptomycin in extracellular nucleotide hydrolysis.

    PubMed

    Borges, Fernanda Pires; de Brum Vieira, Patrícia; Wiltuschnig, Renata C M; Tasca, Tiana; De Carli, Geraldo Attilio; Bonan, Carla Denise

    2008-06-01

    Here we described an nucleoside triphosphate diphosphohydrolase (NTPDase) activity in living trophozoites of Trichomonas gallinae. The enzyme hydrolyzes a variety of purine and pyrimidine nucleoside di- and triphosphates in an optimum pH range of 6.0-8.0. This enzyme activity was activated by high concentrations of divalent cations, such as calcium and magnesium. Contaminant activities were ruled out because the enzyme was not inhibited by classical inhibitors of ATPases (ouabain, 5.0 mM sodium azide, oligomycin) and alkaline phosphatases (levamisole). A significant inhibition of ATP hydrolysis (38%) was observed in the presence of 20 mM sodium azide. Sodium orthovanadate inhibited ATP and ADP hydrolysis (24% and 78%), respectively. The apparent K(M) (Michaelis constant) values were 667.62+/-13 microM for ATP and 125+/-5.3 microM for ADP. V(max) (maximum velocity) values were 0.44+/-0.007 nmol Pi min(-1) per 10(6) trichomonads and 0.91+/-0.12 nmol Pi min(-1) per 10(6) trichomonads for ATP and ADP, respectively. Moreover, we showed a marked decrease in ATP, ADP and AMP hydrolysis when the parasites were grown in the presence of penicillin and streptomycin. The existence of an NTPDase activity in T. gallinae may be involved in pathogenicity, protecting the parasite from the cytolytic effects of the extracellular nucleotides. PMID:18422631

  8. Enzymatic synthesis of acyclic nucleoside thiophosphonate diphosphates: effect of the α-phosphorus configuration on HIV-1 RT activity.

    PubMed

    Priet, Stéphane; Roux, Loic; Saez-Ayala, Magali; Ferron, François; Canard, Bruno; Alvarez, Karine

    2015-05-01

    The acyclic nucleosides thiophosphonates (9-[2-(thiophosphonomethoxy)ethyl]adenine (S-PMEA) and (R)-9-[2-(thiophosphonomethoxy)propyl]adenine (S-PMPA), exhibit antiviral activity against HIV-1, -2 and HBV. Their diphosphate forms S-PMEApp and S-PMPApp, synthesized as stereoisomeric mixture, are potent inhibitors of wild-type (WT) HIV-1 RT. Understanding HIV-1 RT stereoselectivity, however, awaits resolution of the diphosphate forms into defined stereoisomers. To this aim, thiophosphonate monophosphates S-PMEAp and S-PMPAp were synthesized and used in a stereocontrolled enzyme-catalyzed phosphoryl transfer reaction involving either nucleoside diphosphate kinase (NDPK) or creatine kinase (CK) to obtain thiophosphonate diphosphates as separated isomers. We then quantified substrate preference of recombinant WT HIV-1 RT toward pure stereoisomers using in vitro steady-state kinetic analyses. The crystal structure of a complex between Dictyostelium NDPK and S-PMPApp at 2.32Å allowed to determine the absolute configuration at the α-phosphorus atom in relation to the stereo-preference of studied enzymes. The RP isomer of S-PMPApp and S-PMEApp are the preferred substrate over SP for both NDPK and HIV-1 RT. PMID:25766862

  9. Base-Modified Nucleosides: Etheno Derivatives

    PubMed Central

    Jahnz-Wechmann, Zofia; Framski, Grzegorz R.; Januszczyk, Piotr A.; Boryski, Jerzy

    2016-01-01

    This review presents synthesis and chemistry of nucleoside analogs, possessing an additional fused, heterocyclic ring of the “etheno” type, such as 1,N6-ethenoadenosine, 1,N4-ethenocytidine, 1,N2-ethenoguanosine, and other related derivatives. Formation of ethenonucleosides, in the presence of α-halocarbonyl reagents and their mechanism, stability, and degradation, reactions of substitution and transglycosylation, as well as their application in the nucleoside synthesis, have been described. Some of the discussed compounds may be applied as chemotherapeutic agents in antiviral and anticancer treatment, acting as pro-nucleosides of already known, biologically active nucleoside analogs. PMID:27200341

  10. Development of a new HPLC method using fluorescence detection without derivatization for determining purine nucleoside phosphorylase activity in human plasma.

    PubMed

    Giuliani, Patricia; Zuccarini, Mariachiara; Buccella, Silvana; Rossini, Margherita; D'Alimonte, Iolanda; Ciccarelli, Renata; Marzo, Matteo; Marzo, Antonio; Di Iorio, Patrizia; Caciagli, Francesco

    2016-01-15

    Purine nucleoside phosphorylase (PNP) activity is involved in cell survival and function, since PNP is a key enzyme in the purine metabolic pathway where it catalyzes the phosphorolysis of the nucleosides to the corresponding nucleobases. Its dysfunction has been found in relevant pathological conditions (such as inflammation and cancer), so the detection of PNP activity in plasma could represent an attractive marker for early diagnosis or assessment of disease progression. Thus the aim of this study was to develop a simple, fast and sensitive HPLC method for the determination of PNP activity in plasma. The separation was achieved on a Phenomenex Kinetex PFP column using 0.1% formic acid in water and methanol as mobile phases in gradient elution mode at a flow rate of 1ml/min and purine compounds were detected using UV absorption and fluorescence. The analysis was fast since the run was achieved within 13min. This method improved the separation of the different purines, allowing the UV-based quantification of the natural PNP substrates (inosine and guanosine) or products (hypoxanthine and guanine) and its subsequent metabolic products (xanthine and uric acid) with a good precision and accuracy. The most interesting innovation is the simultaneous use of a fluorescence detector (excitation/emission wavelength of 260/375nm) that allowed the quantification of guanosine and guanine without derivatization. Compared with UV, the fluorescence detection improved the sensitivity for guanine detection by about 10-fold and abolished almost completely the baseline noise due to the presence of plasma in the enzymatic reaction mixture. Thus, the validated method allowed an excellent evaluation of PNP activity in plasma which could be useful as an indicator of several pathological conditions. PMID:26720700

  11. Hybridization specificity, enzymatic activity and biological (Ha-ras) activity of oligonucleotides containing 2,4-dideoxy-beta-D-erythro-hexopyranosyl nucleosides.

    PubMed Central

    Augustyns, K; Godard, G; Hendrix, C; Van Aerschot, A; Rozenski, J; Saison-Behmoaras, T; Herdewijn, P

    1993-01-01

    Antisense oligonucleotides with a 2,4-dideoxyhexopyranosyl nucleoside incorporated at the 3'-end and at a mutation site of the Ha-ras oncogene mRNA were synthesized. Melting temperature studies revealed that an A*-G mismatch is more stable than an A*-T mismatch with these hexopyranosyl nucleosides incorporated at the mutation site. The oligonucleotides are stable against enzymatic degradation. RNase H mediated cleavage studies revealed selective cleavage of mutated Ha-ras mRNA. The oligonucleotide containing two pyranose nucleosides at the penultimate position activates RNase H more strongly than natural oligonucleotides. No correlation, however, was found between DNA - DNA or RNA - DNA melting temperatures and RNase H mediated cleavage capacity. Although the A*-G mismatch gives more stable hybridization than the A*-T base pairing, only the oligonucleotides containing an A*-T base pair are recognized by RNase H. This modification is situated 3 base pairs upstream to the cleavage site. Finally, the double pyranose modified oligonucleotide was able to reduce the growth of T24 cells (bladder carcinoma) while the unmodified antisense oligonucleotide was not. Images PMID:7694231

  12. The Nucleoside Analog BMS-986001 Shows Greater In Vitro Activity against HIV-2 than against HIV-1

    PubMed Central

    Raugi, Dana N.; Wu, Vincent H.; Leong, Sally S.; Parker, Kate M.; Oakes, Mariah K.; Sow, Papa Salif; Ba, Selly; Seydi, Moussa; Gottlieb, Geoffrey S.

    2015-01-01

    Treatment options for individuals infected with human immunodeficiency virus type 2 (HIV-2) are restricted by the intrinsic resistance of the virus to nonnucleoside reverse transcriptase inhibitors (NNRTIs) and the reduced susceptibility of HIV-2 to several protease inhibitors (PIs) used in antiretroviral therapy (ART). In an effort to identify new antiretrovirals for HIV-2 treatment, we evaluated the in vitro activity of the investigational nucleoside analog BMS-986001 (2′,3′-didehydro-3′-deoxy-4′-ethynylthymidine; also known as censavudine, festinavir, OBP-601, 4′-ethynyl stavudine, or 4′-ethynyl-d4T). In single-cycle assays, BMS-986001 inhibited HIV-2 isolates from treatment-naive individuals, with 50% effective concentrations (EC50s) ranging from 30 to 81 nM. In contrast, EC50s for group M and O isolates of HIV-1 ranged from 450 to 890 nM. Across all isolates tested, the average EC50 for HIV-2 was 9.5-fold lower than that for HIV-1 (64 ± 18 nM versus 610 ± 200 nM, respectively; mean ± standard deviation). BMS-986001 also exhibited full activity against HIV-2 variants whose genomes encoded the single amino acid changes K65R and Q151M in reverse transcriptase, whereas the M184V mutant was 15-fold more resistant to the drug than the parental HIV-2ROD9 strain. Taken together, our findings show that BMS-986001 is an effective inhibitor of HIV-2 replication. To our knowledge, BMS-986001 is the first nucleoside analog that, when tested against a diverse collection of HIV-1 and HIV-2 isolates, exhibits more potent activity against HIV-2 than against HIV-1 in culture. PMID:26392486

  13. The Nucleoside Analog BMS-986001 Shows Greater In Vitro Activity against HIV-2 than against HIV-1.

    PubMed

    Smith, Robert A; Raugi, Dana N; Wu, Vincent H; Leong, Sally S; Parker, Kate M; Oakes, Mariah K; Sow, Papa Salif; Ba, Selly; Seydi, Moussa; Gottlieb, Geoffrey S

    2015-12-01

    Treatment options for individuals infected with human immunodeficiency virus type 2 (HIV-2) are restricted by the intrinsic resistance of the virus to nonnucleoside reverse transcriptase inhibitors (NNRTIs) and the reduced susceptibility of HIV-2 to several protease inhibitors (PIs) used in antiretroviral therapy (ART). In an effort to identify new antiretrovirals for HIV-2 treatment, we evaluated the in vitro activity of the investigational nucleoside analog BMS-986001 (2',3'-didehydro-3'-deoxy-4'-ethynylthymidine; also known as censavudine, festinavir, OBP-601, 4'-ethynyl stavudine, or 4'-ethynyl-d4T). In single-cycle assays, BMS-986001 inhibited HIV-2 isolates from treatment-naive individuals, with 50% effective concentrations (EC50s) ranging from 30 to 81 nM. In contrast, EC50s for group M and O isolates of HIV-1 ranged from 450 to 890 nM. Across all isolates tested, the average EC50 for HIV-2 was 9.5-fold lower than that for HIV-1 (64 ± 18 nM versus 610 ± 200 nM, respectively; mean ± standard deviation). BMS-986001 also exhibited full activity against HIV-2 variants whose genomes encoded the single amino acid changes K65R and Q151M in reverse transcriptase, whereas the M184V mutant was 15-fold more resistant to the drug than the parental HIV-2ROD9 strain. Taken together, our findings show that BMS-986001 is an effective inhibitor of HIV-2 replication. To our knowledge, BMS-986001 is the first nucleoside analog that, when tested against a diverse collection of HIV-1 and HIV-2 isolates, exhibits more potent activity against HIV-2 than against HIV-1 in culture. PMID:26392486

  14. Antimalarial activity of prodrugs of N-branched acyclic nucleoside phosphonate inhibitors of 6-oxopurine phosphoribosyltransferases.

    PubMed

    Hocková, Dana; Janeba, Zlatko; Naesens, Lieve; Edstein, Michael D; Chavchich, Marina; Keough, Dianne T; Guddat, Luke W

    2015-09-01

    Acyclic nucleoside phosphonates (ANPs) that contain a 6-oxopurine base are good inhibitors of the human and Plasmodium falciparum 6-oxopurine phosphoribosyltransferases (PRTs), key enzymes of the purine salvage pathway. Chemical modifications, based on the crystal structures of several inhibitors in complex with the human PRTase, led to the design of a new class of inhibitors--the aza-ANPs. Because of the negative charges of the phosphonic acid moiety, their ability to cross cell membranes is, however, limited. Thus, phosphoramidate prodrugs of the aza-ANPs were prepared to improve permeability. These prodrugs arrest parasitemia with IC50 values in the micromolar range against Plasmodium falciparum-infected erythrocyte cultures (both chloroquine-sensitive and chloroquine-resistant Pf strains). The prodrugs exhibit low cytotoxicity in several human cell lines. Thus, they fulfill two essential criteria to qualify them as promising antimalarial drug leads. PMID:26275679

  15. Direct synthesis of imino-C-nucleoside analogues and other biologically active iminosugars

    PubMed Central

    Bergeron-Brlek, Milan; Meanwell, Michael; Britton, Robert

    2015-01-01

    Iminosugars have attracted increasing attention as chemical probes, chaperones and leads for drug discovery. Despite several clinical successes, their de novo synthesis remains a significant challenge that also limits their integration with modern high-throughput screening technologies. Herein, we describe a unique synthetic strategy that converts a wide range of acetaldehyde derivatives into iminosugars and imino-C-nucleoside analogues in two or three straightforward transformations. We also show that this strategy can be readily applied to the rapid production of indolizidine and pyrrolizidine iminosugars. The high levels of enantio- and diastereoselectivity, excellent overall yields, convenience and broad substrate scope make this an appealing process for diversity-oriented synthesis, and should enable drug discovery efforts. PMID:25903019

  16. Synthesis and antiviral activity of novel acyclic nucleoside analogues of 5-(1-azido-2-haloethyl)uracils.

    PubMed

    Kumar, R; Sharma, N; Nath, M; Saffran, H A; Tyrrell, D L

    2001-11-22

    We present the discovery of a novel category of 5-substituted acyclic pyrimidine nucleosides as potent antiviral agents. A series of 1-[(2-hydroxyethoxy)methyl] (5-7), 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl] (8-10), and 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl] (11-13) derivatives of 5-(1-azido-2-haloethyl)uracil were synthesized and evaluated for their biological activity in cell culture. 1-[4-Hydroxy-3-(hydroxymethyl)-1-butyl]-5-(1-azido-2-chloroethyl)uracil (12) was the most effective antiviral agent in the in vitro assays against DHBV (EC(50) = 0.31-1.55 microM) and HCMV (EC(50) = 3.1 microM). None of the compounds investigated showed any detectable toxicity to several stationary and proliferating host cells. PMID:11708924

  17. Effect of expression of adenine phosphoribosyltransferase on the in vivo anti-tumor activity of prodrugs activated by E. coli purine nucleoside phosphorylase.

    PubMed

    Parker, W B; Allan, P W; Waud, W R; Hong, J S; Sorscher, E J

    2011-06-01

    The use of E. coli purine nucleoside phosphorylase (PNP) to activate prodrugs has demonstrated excellent activity in the treatment of various human tumor xenografts in mice. E. coli PNP cleaves purine nucleoside analogs to generate toxic adenine analogs, which are activated by adenine phosphoribosyl transferase (APRT) to metabolites that inhibit RNA and protein synthesis. We created tumor cell lines that encode both E. coli PNP and excess levels of human APRT, and have used these new cell models to test the hypothesis that treatment of otherwise refractory human tumors could be enhanced by overexpression of APRT. In vivo studies with 6-methylpurine-2'-deoxyriboside (MeP-dR), 2-F-2'-deoxyadenosine (F-dAdo) or 9-β-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate (F-araAMP) indicated that increased APRT in human tumor cells coexpressing E. coli PNP did not enhance either the activation or the anti-tumor activity of any of the three prodrugs. Interestingly, expression of excess APRT in bystander cells improved the activity of MeP-dR, but diminished the activity of F-araAMP. In vitro studies indicated that increasing the expression of APRT in the cells did not significantly increase the activation of MeP. These results provide insight into the mechanism of bystander killing of the E. coli PNP strategy, and suggest ways to enhance the approach that are independent of APRT. PMID:21394111

  18. Adenosine A1 receptor activation modulates human equilibrative nucleoside transporter 1 (hENT1) activity via PKC-mediated phosphorylation of serine-281.

    PubMed

    Hughes, Scott J; Cravetchi, Xenia; Vilas, Gonzalo; Hammond, James R

    2015-05-01

    Equilibrative nucleoside transporter subtype 1 (ENT1) is critical for the regulation of the biological activities of endogenous nucleosides such as adenosine, and for the cellular uptake of chemotherapeutic nucleoside analogs. Previous studies have implicated protein kinase C (PKC) in the regulation of ENT1 expression/function. It was hypothesized that hENT1 activity at the plasma membrane is regulated by PKC-mediated phosphorylation of Ser281. WT (wild-type)-hENT1 or S281A-hENT1 was stably transfected into a PK15 cell variant that is deficient in nucleoside transport. Using [(3)H]nitrobenzylthioinosine (NBMPR) binding and [(3)H]2-chloroadenosine uptake analyses, it was determined that S281A-hENT1 exhibited functional characteristics similar to WT-hENT1. Direct activation of PKC with PMA or indirect activation with the adenosine A1 receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) led to significant increases in [(3)H]NBMPR binding and [(3)H]2-chloroadenosine uptake in WT-hENT1 transfected cells. The PKC inhibitor Gö6983 blocked these effects of both PMA and CCPA, and the CCPA-mediated increase was also blocked by the A1 adenosine receptor antagonist DPCPX. In contrast, neither PMA nor CCPA affected [(3)H]NBMPR binding or [(3)H]2-chloroadenosine uptake in cells transfected with S281A-hENT1. shRNAi silencing studies implicated PKCδ in this regulation of hENT1 activity. Immunocytochemical analysis and cell surface biotinylation assays showed that activation of PKC with PMA, but not CCPA, led to a significant increase in the plasma membrane localization of hENT1. These data suggest that phosphorylation of hENT1 by PKC has effects on both the function and subcellular trafficking of hENT1. This signaling pathway represents a feedback loop whereby adenosine receptor signaling can lead to increased adenosine reuptake into cells via hENT1. PMID:25725289

  19. NMR characterization of HIV-1 reverse transcriptase binding to various non-nucleoside reverse transcriptase inhibitors with different activities

    PubMed Central

    Thammaporn, Ratsupa; Yagi-Utsumi, Maho; Yamaguchi, Takumi; Boonsri, Pornthip; Saparpakorn, Patchreenart; Choowongkomon, Kiattawee; Techasakul, Supanna; Kato, Koichi; Hannongbua, Supa

    2015-01-01

    Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is an important target for antiviral therapy against acquired immunodeficiency syndrome. However, the efficiency of available drugs is impaired most typically by drug-resistance mutations in this enzyme. In this study, we applied a nuclear magnetic resonance (NMR) spectroscopic technique to the characterization of the binding of HIV-1 RT to various non-nucleoside reverse transcriptase inhibitors (NNRTIs) with different activities, i.e., nevirapine, delavirdine, efavirenz, dapivirine, etravirine, and rilpivirine. 1H-13C heteronuclear single-quantum coherence (HSQC) spectral data of HIV-1 RT, in which the methionine methyl groups of the p66 subunit were selectively labeled with 13C, were collected in the presence and absence of these NNRTIs. We found that the methyl 13C chemical shifts of the M230 resonance of HIV-1 RT bound to these drugs exhibited a high correlation with their anti-HIV-1 RT activities. This methionine residue is located in proximity to the NNRTI-binding pocket but not directly involved in drug interactions and serves as a conformational probe, indicating that the open conformation of HIV-1 RT was more populated with NNRTIs with higher inhibitory activities. Thus, the NMR approach offers a useful tool to screen for novel NNRTIs in developing anti-HIV drugs. PMID:26510386

  20. Synthesis and Anti-Influenza Activity of Pyridine, Pyridazine, and Pyrimidine C-Nucleosides as Favipiravir (T-705) Analogues.

    PubMed

    Wang, Guangyi; Wan, Jinqiao; Hu, Yujian; Wu, Xiangyang; Prhavc, Marija; Dyatkina, Natalia; Rajwanshi, Vivek K; Smith, David B; Jekle, Andreas; Kinkade, April; Symons, Julian A; Jin, Zhinan; Deval, Jerome; Zhang, Qingling; Tam, Yuen; Chanda, Sushmita; Blatt, Lawrence; Beigelman, Leonid

    2016-05-26

    Influenza viruses are responsible for seasonal epidemics and occasional pandemics which cause significant morbidity and mortality. Despite available vaccines, only partial protection is achieved. Currently, there are two classes of widely approved anti-influenza drugs: M2 ion channel blockers and neuraminidase inhibitors. However, the worldwide spread of drug-resistant influenza strains poses an urgent need for novel antiviral drugs, particularly with a different mechanism of action. Favipiravir (T-705), a broad-spectrum antiviral agent, has shown potent anti-influenza activity in cell-based assays, and its riboside (2) triphosphate inhibited influenza polymerase. In one of our approaches to treat influenza infection, we designed, prepared, and tested a series of C-nucleoside analogues, which have an analogy to 2 and were expected to act by a similar antiviral mechanism as favipiravir. Compound 3c of this report exhibited potent inhibition of influenza virus replication in MDCK cells, and its triphosphate was a substrate of and demonstrated inhibitory activity against influenza A polymerase. Metabolites of 3c are also presented. PMID:27120583

  1. Identification of Highly Promising Antioxidants/Neuroprotectants Based on Nucleoside 5'-Phosphorothioate Scaffold. Synthesis, Activity, and Mechanisms of Action.

    PubMed

    Azran, Sagit; Danino, Ortal; Förster, Daniel; Kenigsberg, Sarah; Reiser, Georg; Dixit, Mudit; Singh, Vijay; Major, Dan T; Fischer, Bilha

    2015-11-12

    With a view to identify novel and biocompatible neuroprotectants, we designed nucleoside 5'-thiophosphate analogues, 6-11. We identified 2-SMe-ADP(α-S), 7A, as a most promising neuroprotectant. 7A reduced ROS production in PC12 cells under oxidizing conditions, IC50 of 0.08 vs 21 μM for ADP. Furthermore, 7A rescued primary neurons subjected to oxidation, EC50 of 0.04 vs 19 μM for ADP. 7A is a most potent P2Y1-R agonist, EC50 of 0.0026 μM. Activity of 7A in cells involved P2Y1/12-R as indicated by blocking P2Y12-R or P2Y1-R. Compound 7A inhibited Fenton reaction better than EDTA, IC50 of 37 vs 54 μM, due to radical scavenging, IC50 of 12.5 vs 30 μM for ADP, and Fe(II)-chelation, IC50 of 80 vs >200 μM for ADP (ferrozine assay). In addition, 7A was stable in human blood serum, t1/2 of 15 vs 1.5 h for ADP, and resisted hydrolysis by NPP1/3, 2-fold vs ADP. Hence, we propose 7A as a highly promising neuroprotectant. PMID:26447940

  2. Label-free offline versus online activity methods for nucleoside diphosphate kinase b using high performance liquid chromatography.

    PubMed

    Lima, Juliana Maria; Salmazo Vieira, Plínio; Cavalcante de Oliveira, Arthur Henrique; Cardoso, Carmen Lúcia

    2016-08-01

    Nucleoside diphosphate kinase from Leishmania spp. (LmNDKb) has recently been described as a potential drug target to treat leishmaniasis disease. Therefore, screening of LmNDKb ligands requires methodologies that mimic the conditions under which LmNDKb acts in biological systems. Here, we compare two label-free methodologies that could help screen LmNDKb ligands and measure NDKb activity: an offline LC-UV assay for soluble LmNDKb and an online two-dimensional LC-UV system based on LmNDKb immobilised on a silica capillary. The target enzyme was immobilised on the silica capillary via Schiff base formation (to give LmNDKb-ICER-Schiff) or affinity attachment (to give LmNDKb-ICER-His). Several aspects of the ICERs resulting from these procedures were compared, namely kinetic parameters, stability, and procedure steps. Both the LmNDKb immobilisation routes minimised the conformational changes and preserved the substrate binding sites. However, considering the number of steps involved in the immobilisation procedure, the cost of reagents, and the stability of the immobilised enzyme, immobilisation via Schiff base formation proved to be the optimal procedure. PMID:27273166

  3. Synthesis of potent and broad genotypically active NS5B HCV non-nucleoside inhibitors binding to the thumb domain allosteric site 2 of the viral polymerase.

    PubMed

    Pierra Rouvière, Claire; Amador, Agnès; Badaroux, Eric; Convard, Thierry; Da Costa, Daniel; Dukhan, David; Griffe, Ludovic; Griffon, Jean-François; LaColla, Massimiliano; Leroy, Frédéric; Liuzzi, Michel; Loi, Anna Giulia; McCarville, Joe; Mascia, Valeria; Milhau, Julien; Onidi, Loredana; Paparin, Jean-Laurent; Rahali, Rachid; Sais, Efisio; Seifer, Maria; Surleraux, Dominique; Standring, David; Dousson, Cyril

    2016-09-15

    The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells and, as a consequence, is an attractive target for selective inhibition. This Letter describes the discovery of a new family of HCV NS5B non-nucleoside inhibitors, based on the bioisosterism between amide and phosphonamidate functions. As part of this program, SAR in this new series led to the identification of IDX17119, a potent non-nucleoside inhibitor, active on the genotypes 1b, 2a, 3a and 4a. The structure and binding domain of IDX17119 were confirmed by X-ray co-crystallization study. PMID:27520942

  4. Absence epileptic activity changing effects of non-adenosine nucleoside inosine, guanosine and uridine in Wistar Albino Glaxo Rijswijk rats.

    PubMed

    Kovács, Z; Kékesi, K A; Dobolyi, Á; Lakatos, R; Juhász, G

    2015-08-01

    Adenosine (Ado) and non-adenosine (non-Ado) nucleosides such as inosine (Ino), guanosine (Guo) and uridine (Urd) may have regionally different roles in the regulation of physiological and pathophysiological processes in the central nervous system (CNS) such as epilepsy. It was demonstrated previously that Ino and Guo decreased quinolinic acid (QA)-induced seizures and Urd reduced penicillin-, bicuculline- and pentylenetetrazole (PTZ)-induced seizures. It has also been demonstrated that Ino and Urd may exert their effects through GABAergic system by altering the function of GABA(A) type of gamma-aminobutyric acid receptors (GABAA receptors) whereas Guo decreases glutamate-induced excitability through glutamatergic system, which systems (GABAergic and glutamatergic) are involved in pathomechanisms of absence epilepsy. Thus, we hypothesized that Ino and Guo, similarly to the previously described effect of Urd, might also decrease absence epileptic activity. We investigated in the present study whether intraperitoneal (i.p.) application of Ino (500 and 1000mg/kg), Guo (20 and 50mg/kg), Urd (500 and 1000mg/kg), GABA(A) receptor agonist muscimol (1 and 3mg/kg), GABA(A) receptor antagonist bicuculline (2 and 4mg/kg), non-selective Ado receptor antagonist theophylline (5 and 10mg/kg) and non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo (a,d) cyclohepten-5,10-imine maleate (MK-801, 0.0625 and 0.1250mg/kg) alone and in combination have modulatory effects on absence epileptic activity in Wistar Albino Glaxo Rijswijk (WAG/Rij) rats. We found that Guo decreased the number of spike-wave discharges (SWDs) whereas Ino increased it dose-dependently. We strengthened that Urd can decrease absence epileptic activity. Our results suggest that Guo, Urd and their analogs could be potentially effective drugs for treatment of human absence epilepsy. PMID:26037802

  5. Mutational Analysis of Vaccinia Virus Nucleoside Triphosphate Phosphohydrolase I, a DNA-Dependent ATPase of the DExH Box Family

    PubMed Central

    Martins, Alexandra; Gross, Christian H.; Shuman, Stewart

    1999-01-01

    Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH-I) is a DNA-dependent ATPase that serves as a transcription termination factor during viral mRNA synthesis. NPH-I is a member of the DExH box family of nucleic acid-dependent nucleoside triphosphatases (NTPases), which is defined by the presence of several conserved sequence motifs. We have assessed the contributions of individual amino acids (underlined) in motifs I (GxGKT), II (DExHN), III (SAT), and VI (QxxGRxxR) to ATP hydrolysis by performing alanine scanning mutagenesis. Significant decrements in ATPase activity resulted from mutations at nine positions: Lys-61 and Thr-62 (motif I); Asp-141, Glu-142, His-144, and Asn-145 (motif II); and Gln-472, Arg-476, and Arg-479 (motif VI). Structure-function relationships at each of these positions were clarified by introducing conservative substitutions and by steady-state kinetic analysis of the mutant enzymes. Comparison of our findings for NPH-I with those of mutational studies of other DExH and DEAD box proteins underscores similarities as well as numerous disparities in structure-activity relationships. We conclude that the functions of the conserved amino acids of the NTPase motifs are context dependent. PMID:9882335

  6. Chlorella Virus Encoded Deoxyuridine triphosphatases Exhibit different Temperature Optima

    SciTech Connect

    Zhang,Y.; Moriyama, H.; Homma, K.; Van Etten, J.

    2005-01-01

    A putative deoxyuridine triphosphatase (dUTPase) gene from chlorella virus PBCV-1 was cloned, and the recombinant protein was expressed in Escherichia coli. The recombinant protein has dUTPase activity and requires Mg{sup 2+} for optimal activity, while it retains some activity in the presence of other divalent cations. Kinetic studies of the enzyme revealed a K{sub m} of 11.7 {mu}M, a turnover k{sub cat} of 6.8 s{sup -1}, and a catalytic efficiency of k{sub cat}/K{sub m} = 5.8 x 105 M{sup -1} s{sup -1}. dUTPase genes were cloned and expressed from two other chlorella viruses IL-3A and SH-6A. The two dUTPases have similar properties to PBCV-1 dUTPase except that IL-3A dUTPase has a lower temperature optimum (37{sup o}C) than PBCV-1 dUTPase (50{sup o}C). The IL-3A dUTPase differs from the PBCV-1 enzyme by nine amino acids, including two amino acid substitutions, Glu81{yields}Ser81 and Thr84{yields}Arg84, in the highly conserved motif III of the proteins. To investigate the difference in temperature optima between the two enzymes, homology modeling and docking simulations were conducted. The results of the simulation and comparisons of amino acid sequence suggest that adjacent amino acids are important in the temperature optima. To confirm this suggestion, three site-directed amino acid substitutions were made in the IL-3A enzyme: Thr84{yields}Arg84, Glu81{yields}Ser81, and Glu81{yields}Ser81 plus Thr84{yields}Arg84. The single substitutions affected the optimal temperature for enzyme activity. The temperature optimum increased from 37 to 55{sup o}C for the enzyme containing the two amino acid substitutions. We postulate that the change in temperature optimum is due to reduction in charge and balkiness in the active cavity that allows more movement of the ligand and protein before the enzyme and substrate complex is formed.

  7. Selective Non-nucleoside Inhibitors of Human DNA Methyltransferases Active in Cancer Including in Cancer Stem Cells

    PubMed Central

    2015-01-01

    DNA methyltransferases (DNMTs) are important enzymes involved in epigenetic control of gene expression and represent valuable targets in cancer chemotherapy. A number of nucleoside DNMT inhibitors (DNMTi) have been studied in cancer, including in cancer stem cells, and two of them (azacytidine and decitabine) have been approved for treatment of myelodysplastic syndromes. However, only a few non-nucleoside DNMTi have been identified so far, and even fewer have been validated in cancer. Through a process of hit-to-lead optimization, we report here the discovery of compound 5 as a potent non-nucleoside DNMTi that is also selective toward other AdoMet-dependent protein methyltransferases. Compound 5 was potent at single-digit micromolar concentrations against a panel of cancer cells and was less toxic in peripheral blood mononuclear cells than two other compounds tested. In mouse medulloblastoma stem cells, 5 inhibited cell growth, whereas related compound 2 showed high cell differentiation. To the best of our knowledge, 2 and 5 are the first non-nucleoside DNMTi tested in a cancer stem cell line. PMID:24387159

  8. Application of activated nucleoside analogs for the treatment of drug-resistant tumors by oral delivery of nanogel-drug conjugates

    PubMed Central

    Senanayake, Thulani H.; Warren, Galya; Wei, Xin; Vinogradov, Serguei V.

    2013-01-01

    A majority of nanoencapsulated drugs that have shown promise in cancer chemotherapy are administered intravenously. Development of effective oral nanoformulations presents a very challenging medical goal. Here, we describe successful applications of innovative polymeric nanogels in the form of conjugates with activated nucleoside analogs for oral administration in cancer chemotherapy. Previously, we reported the synthesis of amphiphilic polyvinyl alcohol and dextrin-based nanogel conjugates with the phosphorylated 5-FU nucleoside Floxuridine and demonstrated their enhanced activity against regular and drug-resistant cancers[1]. In this study, we synthesized and evaluated oral applications of nanogel conjugates of a protected Gemcitabine, the drug never used in oral therapies. These conjugates were able to quickly release an active form of the drug (Gemcitabine 5′-mono-, di- and triphosphates) by specific enzymatic activities, or slowly during hydrolysis. Gemcitabine conjugates demonstrated up to 127 times higher in vitro efficacy than the free drug against various cancer cells, including the lines resistant to nucleoside analogs. Surprisingly, these nanogel-drug conjugates were relatively stable in gastric conditions and able to actively penetrate through the gastrointestinal barrier based on permeability studies in Caco-2 cell model. In tumor xenograft models of several drug-resistant human cancers, we observed an efficient inhibition of tumor growth and extended the life-span of the animals by 4 times that of the control with orally treated Gemcitabine- or Floxuridine-nanogel conjugates. Thus, we have demonstrated a potential of therapeutic nanogel conjugates with the activated and stabilized Gemcitabine as a successful oral drug form against Gemcitabine-resistant and other drug-resistant tumors. PMID:23385032

  9. Persistent Hepatitis C Virus Infection Impairs Ribavirin Antiviral Activity through Clathrin-Mediated Trafficking of Equilibrative Nucleoside Transporter 1

    PubMed Central

    Panigrahi, Rajesh; Chandra, Partha K.; Ferraris, Pauline; Kurt, Ramazan; Song, Kyoungsub; Garry, Robert F.; Reiss, Krzysztof; Coe, Imogen R.; Furihata, Tomomi; Balart, Luis A.; Wu, Tong

    2014-01-01

    ABSTRACT Ribavirin (RBV) continues to be an important component of interferon-free hepatitis C treatment regimens, as RBV alone does not inhibit hepatitis C virus (HCV) replication effectively; the reason for this ineffectiveness has not been established. In this study, we investigated the RBV resistance mechanism using a persistently HCV-infected cell culture system. The antiviral activity of RBV against HCV was progressively impaired in the persistently infected culture, whereas interferon lambda 1 (IFN-λ1), a type III IFN, showed a strong antiviral response and induced viral clearance. We found that HCV replication in persistently infected cultures induces an autophagy response that impairs RBV uptake by preventing the expression of equilibrative nucleoside transporter 1 (ENT1). The Huh-7.5 cell line treated with an autophagy inducer, Torin 1, downregulated membrane expression of ENT1 and terminated RBV uptake. In contrast, the autophagy inhibitors hydroxychloroquine (HCQ), 3-methyladenine (3-MA), and bafilomycin A1 (BafA1) prevented ENT1 degradation and enhanced RBV antiviral activity. The HCV-induced autophagy response, as well as treatment with Torin 1, degrades clathrin heavy chain expression in a hepatoma cell line. Reduced expression of the clathrin heavy chain by HCV prevents ENT1 recycling to the plasma membrane and forces ENT1 to the lysosome for degradation. This study provides a potential mechanism for the impairment of RBV antiviral activity in persistently HCV-infected cell cultures and suggests that inhibition of the HCV-induced autophagy response could be used as a strategy for improving RBV antiviral activity against HCV infection. IMPORTANCE The results from this work will allow a review of the competing theories of antiviral therapy development in the field of HCV virology. Ribavirin (RBV) remains an important component of interferon-free hepatitis C treatment regimens. The reason why RBV alone does not inhibit HCV replication effectively has

  10. Design, Synthesis, and Evaluation of Anti-HBV Activity of Hybrid Molecules of Entecavir and Adefovir: Exomethylene Acycloguanine Nucleosides and Their Monophosphate Derivatives.

    PubMed

    Imoto, Shuhei; Kohgo, Satoru; Tokuda, Ryoh; Kumamoto, Hiroki; Aoki, Manabu; Amano, Masayuki; Kuwata-Higashi, Nobuyo; Mitsuya, Hiroaki; Haraguchi, Kazuhiro

    2015-01-01

    Exomethylene acycloguanine nucleosides 4, 6 and its monophosphate derivatives 5, 7, and 8 have been synthesized. Mitsunobu-type coupling of 2-N-acetyl-6-O-diphenylcarbamoylguanine (11) with primary alcohols proceeded regioselectively to furnish the desired N(9)-substituted products in moderate yield. Evaluation of 4-8 for anti-HBV activity in HepG2 cells revealed that the phosphonate derivative 8 was found to exhibit moderated activity (EC50 value of 0.29 μM), but cytotoxicity (CC50 value of 39 μM) against the host cells was also observed. PMID:26167667

  11. Influence of intermittent hypoxia and pyrimidinic nucleosides on cerebral enzymatic activities related to energy transduction.

    PubMed

    Dagani, F; Marzatico, F; Curti, D; Taglietti, M; Zanada, F; Benzi, G

    1984-08-01

    The effect of intermittent normobaric hypoxia and of biological pyrimidines (uridine and cytidine) on the specific activities of some enzymes related to cerebral energy metabolism were studied. Measurement were carried out on the following: homogenate in toto; purified mitochondrial fraction; crude synaptosomal fraction, in different areas of rat brain: cerebral cortex, hippocampus, corpus striatum, hypothalamus, cerebellum, and medulla oblongata. Intermittent normobaric hypoxia (12 hours daily for 5 days) caused modifications of the enzyme activities in the homogenate in toto (decrease of hexokinase in cerebellum; increase of pyruvate kinase in medulla oblongata), in the purified mitochondrial fraction (increase of succinate dehydrogenase in the corpus striatum) and in the crude synaptosomal fraction (decrease of cytochrome oxidase activity in cerebral cortex, hippocampus, and cerebellum; decrease of malate dehydrogenase in hippocampus and cerebellum; decrease of lactate dehydrogenase in cerebellum). Daily treatment with cytidine or uridine altered some enzyme activities either affected or unaffected by intermittent hypoxia. PMID:6493441

  12. Novel 5-vinyl pyrimidine nucleosides with potent anti-hepatitis B virus activity.

    PubMed

    Kumar, R; Tyrrell, D L

    2001-11-19

    Synthesis and antiviral activities of novel N-1 alkyl substituted pyrimidines, 1-[(2-hydroxyethoxy)methyl]-5-vinyluracil (5), 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]-5-vinyluracil (6), and 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl]-5-vinyluracil (7) are reported. Compounds 6 and 7 were potent inhibitors of DHBV in cell culture, in contrast, all of the compounds described were devoid of activity against TK(+) HSV-1 and TK(-) HSV-1. PMID:11677126

  13. Nucleoside Diphosphate Sugar-Starch Glucosyl Transferase Activity of wx Starch Granules 1

    PubMed Central

    Nelson, Oliver E.; Chourey, Prem S.; Chang, Ming Tu

    1978-01-01

    Starch granule preparations from the endosperm tissue of all waxy maize (Zea mays L.) mutants tested have low and approximately equal capability to incorporate glucose from adenosine diphosphate glucose into starch. As the substrate concentration is reduced, however, the activity of waxy preparations relative to nonmutant increases until, at the lowest substrate concentration utilized (0.1 μM), the activity of the waxy preparations is nearly equal to that of the nonmutant preparation. The apparent Km (adenosine diphosphate glucose) for starch granule preparations from wx-C/wx-C/wx-C endosperms was 7.1 × 10−5 M, which is compared to 3 × 10−3 M for preparations from nonwaxy endosperms. Starch granule preparations from three other waxy mutants of independent mutational origin have levels of enzymic activity approximately equal to wx-C at a given substrate concentration giving rise to similar apparent Km estimates. We conclude that there is in maize endosperm starch granules a second starch granule-bound glycosyl transferase, whose presence is revealed when mutation eliminates activity of the more active glucosyl transferase catalyzing the same reaction. PMID:16660522

  14. In vivo selection of active deoxyribonucleoside kinase by a mutagenic nucleoside analog.

    PubMed

    Kamiya, Hiroyuki; Ito, Mana; Nishi, Kosuke; Harashima, Hideyoshi

    2016-06-20

    A novel in vivo selection method for active deoxyribonucleoside kinase proteins is described here. A pool of randomly mutated genes for deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) was prepared and inserted into an expression vector. Enzymatically active mutants were selected by repeated cycles, including (i) introduction into Escherichia coli, (ii) treatment of the E. coli pool with a mutagenic deoxyribonucleoside (2-hydroxy-dA), and (iii) selection of antibiotic-resistant colonies resulting from mutations by phosphorylated 2-hydroxy-dA and the subsequent isolation of the plasmid DNAs. The ratio of the resistant colonies increased by two orders of magnitude from the first cycle to the fifth cycle, and then reached a plateau. Fifteen Dm-dNK mutants selected after the seventh and eighth evolution cycles were actually active in vivo. Moreover, one of the mutant proteins was as active as the wild-type protein in vitro. These results indicate that this novel in vivo evolution method was useful and that similar strategies would be applicable to other deoxyribonucleoside kinases. In addition, the distribution of mutated amino acids suggests important residues/regions in the Dm-dNK protein. PMID:27131895

  15. A kinetic description for sodium and potassium effects on (Na+ plus K+)-adenosine triphosphatase: a model for a two-nonequivalent site potassium activation and an analysis of multiequivalent site models for sodium activation.

    PubMed

    Lindenmayer, G E; Schwartz, A; Thompson, H K

    1974-01-01

    1. Dissociation constants for sodium and potassium of a site that modulates the rate of ouabain-(Na(+)+K(+))-ATPase interaction were applied to models for potassium activation of (Na(+)+K(+))-ATPase. The constants for potassium (0.213 mM) and for sodium (13.7 mM) were defined, respectively, as activation constant, K(a) and inhibitory constant, K(i).2. Tests of the one- and the two-equivalent site models, that describe sodium and potassium competition, revealed that neither model adequately predicts the activation effects of potassium in the presence of 100 or 200 mM sodium.3. The potassium-activation data, obtained at low potassium and high sodium, were explained by a two-nonequivalent site model where the dissociation constants of the first site are 0.213 mM for potassium and 13.7 mM for sodium. The second site was characterized by dissociation constants of 0.091 mM for potassium and 74.1 mM for sodium.4. The two-nonequivalent site model adequately predicted the responses to concentrations of potassium between 0.25 and 5 mM in the presence of 100-500 mM sodium. At lower sodium concentrations the predicted responses formed an upper limit for the function of observed activities. This limit was reached at lower concentrations of potassium and higher concentrations of sodium, which inferred saturation of the sodium-activation sites with sodium.5. Sodium-activation data were corrected for sodium interaction with potassium-activation sites by use of the two-nonequivalent site model for potassium activation. Tests of equivalent site models suggested that the corrected data for sodium activation may be most consistent with a model that has three-equivalent sites. Other multiequivalent site models (n = 2, 4, 5 or 6), however, cannot be statistically eliminated as possibilities. The three-equivalent site activation model was characterized by dissociation constants of 1.39 mM for sodium and 11.7 mM for potassium. The system theoretically would be half-maximally activated by

  16. Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4;#8201;Å

    SciTech Connect

    Johnson, Zachary Lee; Cheong, Cheom-Gil; Lee, Seok-Yong

    2012-07-11

    Nucleosides are required for DNA and RNA synthesis, and the nucleoside adenosine has a function in a variety of signalling processes. Transport of nucleosides across cell membranes provides the major source of nucleosides in many cell types and is also responsible for the termination of adenosine signalling. As a result of their hydrophilic nature, nucleosides require a specialized class of integral membrane proteins, known as nucleoside transporters (NTs), for specific transport across cell membranes. In addition to nucleosides, NTs are important determinants for the transport of nucleoside-derived drugs across cell membranes. A wide range of nucleoside-derived drugs, including anticancer drugs (such as Ara-C and gemcitabine) and antiviral drugs (such as zidovudine and ribavirin), have been shown to depend, at least in part, on NTs for transport across cell membranes. Concentrative nucleoside transporters, members of the solute carrier transporter superfamily SLC28, use an ion gradient in the active transport of both nucleosides and nucleoside-derived drugs against their chemical gradients. The structural basis for selective ion-coupled nucleoside transport by concentrative nucleoside transporters is unknown. Here we present the crystal structure of a concentrative nucleoside transporter from Vibrio cholerae in complex with uridine at 2.4 {angstrom}. Our functional data show that, like its human orthologues, the transporter uses a sodium-ion gradient for nucleoside transport. The structure reveals the overall architecture of this class of transporter, unravels the molecular determinants for nucleoside and sodium binding, and provides a framework for understanding the mechanism of nucleoside and nucleoside drug transport across cell membranes.

  17. Multicomponent reactions in nucleoside chemistry

    PubMed Central

    Buchowicz, Włodzimierz

    2014-01-01

    Summary This review covers sixty original publications dealing with the application of multicomponent reactions (MCRs) in the synthesis of novel nucleoside analogs. The reported approaches were employed for modifications of the parent nucleoside core or for de novo construction of a nucleoside scaffold from non-nucleoside substrates. The cited references are grouped according to the usually recognized types of the MCRs. Biochemical properties of the novel nucleoside analogs are also presented (if provided by the authors). PMID:25161730

  18. Large steric effect in the substitution reaction of amines with phosphoimidazolide-activated nucleosides

    NASA Technical Reports Server (NTRS)

    Kanavarioti, A.; Stronach, M. W.; Ketner, R. J.; Hurley, T. B.

    1995-01-01

    Aliphatic amines react with phosphoimidazolide-activated derivatives of guanosine and cytidine (ImpN) by replacing the imidazole group. The kinetics of reaction of guanosine 5'-phospho-2-methylimidazolide (2-MeImpG) with glycine ethyl ester, glycinamide, 2-methoxyethylamine, n-butylamine, morpholine, dimethylamine (Me2NH), ethylmethylamine (EtNHMe), diethylamine (Et2NH), pyrrolidine, and piperidine were determined in water at 37 degrees C. With primary amines, a plot of the logarithm of the rate constant for attack by the amine on the protonated substrate, log kSH(A), versus the pKa of the amine exhibits a good linear correlation with a Bronsted slope, beta nuc = 0.48. Most of the secondary amines tested react with slightly higher reactivity than primary amines of similar pKa. Interestingly, some secondary amines show substantially lower reactivity than might be expected: EtNHMe reacts about eight times, and Et2NH at least 100 times, more slowly than Me2NH although all three amines are of similar basicity. For comparison, the kinetics of reaction of guanosine 5'-phosphoimidazolide (ImpG) and cytidine 5'-phosphoimidazolide (ImpC) were determined with Me2NH, EtNHMe, and Et2NH, and similar results were obtained. These results establish that the increased steric hindrance observed with the successive addition of ethyl groups are not due to any special steric requirements imposed by the guanosine or the methyl on the 2-methylimidazole leaving group of 2-MeImpG. It is concluded that addition of ethyl and, perhaps, groups larger than ethyl dramatically increases the kinetic barrier for addition of aliphatic secondary amines to the P-N bond of ImpN. This study supports the observation that the primary amino groups on the natural polyamines are at least 2 orders of magnitude more reactive than the secondary amino groups in the reaction with ImpN.

  19. 1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

    PubMed

    Daluge, S M; Good, S S; Faletto, M B; Miller, W H; St Clair, M H; Boone, L R; Tisdale, M; Parry, N R; Reardon, J E; Dornsife, R E; Averett, D R; Krenitsky, T A

    1997-05-01

    1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human

  20. Carbocyclic pyrimidine nucleosides as inhibitors of S-adenosylhomocysteine hydrolase.

    PubMed

    Mosley, Sylvester L; Bakke, Brian A; Sadler, Joshua M; Sunkara, Naresh K; Dorgan, Kathleen M; Zhou, Zhaohui Sunny; Seley-Radtke, Katherine L

    2006-12-01

    The design, synthesis, and unexpected inhibitory activity against S-adenosyl-homocysteine (SAH) hydrolase (SAHase, EC 3.3.1.1) for a series of truncated carbocyclic pyrimidine nucleoside analogues is presented. Of the four nucleosides obtained, 10 was found to be active with a Ki value of 5.0 microM against SAHase. PMID:16904326

  1. Nucleoside transport in primary cultured rabbit tracheal epithelial cells.

    PubMed

    Mathias, Neil R; Wu, Sharon K; Kim, Kwang-Jin; Lee, Vincent H L

    2005-01-01

    The present study aimed at elucidating the mechanisms of nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) grown on a permeable filter support. Uptake of (3)H-uridine, the model nucleoside substrate, from the apical fluid of primary cultured RTEC was examined with respect to its dependence on Na(+), substrate concentration, temperature and its sensitivity to inhibitors, other nucleosides and antiviral nucleoside analogs. Apical (3)H-uridine uptake in primary cultured RTEC was strongly dependent on an inward Na(+) gradient and temperature. Ten micromolar nitro-benzyl-mercapto-purine-ribose (NBMPR) (an inhibitor of es-type nucleoside transport in the nanomolar range) did not further inhibit this process. (3)H-uridine uptake from apical fluid was inhibited by basolateral ouabain (10 microM) and apical phloridzin (100 microM), indicating that uptake may involve a secondary active transport process. Uridine uptake was saturable with a K(m) of 3.4 +/- 1.8 microM and the V(max) of 24.3 +/- 5.2 pmoles/mg protein/30 s. Inhibition studies indicated that nucleoside analogs that have a substitution on the nucleobase competed with uridine uptake from apical fluid, but those with modifications on the ribose sugar including acyclic analogs were ineffective. The pattern of inhibition of apical (3)H-uridine, (3)H-inosine and (3)H-thymidine uptake into RTEC cells by physiological nucleosides was consistent with multiple systems: A pyrimidine-selective transport system (CNT1); a broad nucleoside substrate transport system that excludes inosine (CNT4) and an equilibrative NBMPR-insensitive nucleoside transport system (ei type). These results indicate that the presence of apically located nucleoside transporters in the epithelial cells lining the upper respiratory tract can lead to a high accumulation of nucleosides in the trachea. At least one Na(+)-dependent, secondary, active transport process may mediate the apical absorption of nucleosides or

  2. Inhibition of renal Na+, K+-adenosine triphosphatase by gentamicin

    SciTech Connect

    Williams, P.D.; Trimble, M.E.; Crespo, L.; Holohan, P.D.; Freedman, J.C.; Ross, C.R.

    1984-11-01

    Inhibition of renal Na+,K+-adenosine triphosphatase is an early biochemical manifestation of gentamicin treatment in rats. Studies with isolated, perfused rat kidneys in filtering and nonfiltering modes indicate that gentamicin is transported across the brush border membrane before enzyme inhibition. The drug caused enzyme inhibition (42%) only in filtering kidneys, and this inhibition was blocked by spermine, an inhibitor of gentamicin binding. In purified rat renal basolateral membranes, bound (/sup 3/H)gentamicin was displaced 88% by unlabeled gentamicin. After in vivo exposure to (/sup 3/H)gentamicin, the radioactivity associated with the isolated basolateral membranes was displaced only 46% by unlabeled drug. These results suggest that inhibition of renal Na+,K+-adenosine triphosphatase by gentamicin is probably due to an interaction at the cytoplasmic face of the basolateral membrane. Scatchard plots of (/sup 3/H)gentamicin binding to basolateral and brush border membranes revealed a single class of noninteracting sites in each membrane. Gentamicin did not change the bulk membrane lipid fluidity, as estimated by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene.

  3. Nucleoside transporters and liver cell growth.

    PubMed

    Pastor-Anglada, M; Felipe, A; Casado, F J; del Santo, B; Mata, J F; Valdés, R

    1998-01-01

    Liver parenchymal cells show a wide variety of plasma membrane transporters that are tightly regulated by endocrine and nutritional factors. This review summarizes work performed in our laboratory on these transport systems, particularly nucleoside transporters, which are up-regulated in physiological situations associated with liver cell growth. Rat hepatocytes show a Na+-dependent nucleoside transport activity that is stimulated by pancreatic hormones. Indeed, this biological activity appears to be the result of the co-expression of at least two isoforms of nucleoside carriers, CNT1 and CNT2 (also called SPNT). These two transporters are up-regulated during the early phase of liver growth after partial hepatectomy, although to different extents, suggesting differential regulation of the two isoforms. The recent generation of isoform-specific antibodies allowed us to demonstrate that carrier expression may also have complex post-transcriptional regulation on the basis of the lack of correspondence between mRNA and protein levels. The analysis of nucleoside transport systems in hepatoma cells and the comparison with those in hepatocytes has also provided evidence that the differentiation status of liver parenchymal cells may determine the pattern of nucleoside transporters expressed. PMID:10353710

  4. Alpha-carboxy nucleoside phosphonates as universal nucleoside triphosphate mimics

    PubMed Central

    Balzarini, Jan; Das, Kalyan; Bernatchez, Jean A.; Martinez, Sergio E.; Ngure, Marianne; Keane, Sarah; Ford, Alan; Maguire, Nuala; Mullins, Niki; John, Jubi; Kim, Youngju; Dehaen, Wim; Vande Voorde, Johan; Liekens, Sandra; Naesens, Lieve; Götte, Matthias; Maguire, Anita R.; Arnold, Eddy

    2015-01-01

    Polymerases have a structurally highly conserved negatively charged amino acid motif that is strictly required for Mg2+ cation-dependent catalytic incorporation of (d)NTP nucleotides into nucleic acids. Based on these characteristics, a nucleoside monophosphonate scaffold, α-carboxy nucleoside phosphonate (α-CNP), was designed that is recognized by a variety of polymerases. Kinetic, biochemical, and crystallographic studies with HIV-1 reverse transcriptase revealed that α-CNPs mimic the dNTP binding through a carboxylate oxygen, two phosphonate oxygens, and base-pairing with the template. In particular, the carboxyl oxygen of the α-CNP acts as the potential equivalent of the α-phosphate oxygen of dNTPs and two oxygens of the phosphonate group of the α-CNP chelate Mg2+, mimicking the chelation by the β- and γ-phosphate oxygens of dNTPs. α-CNPs (i) do not require metabolic activation (phosphorylation), (ii) bind directly to the substrate-binding site, (iii) chelate one of the two active site Mg2+ ions, and (iv) reversibly inhibit the polymerase catalytic activity without being incorporated into nucleic acids. In addition, α-CNPs were also found to selectively interact with regulatory (i.e., allosteric) Mg2+-dNTP-binding sites of nucleos(t)ide-metabolizing enzymes susceptible to metabolic regulation. α-CNPs represent an entirely novel and broad technological platform for the development of specific substrate active- or regulatory-site inhibitors with therapeutic potential. PMID:25733891

  5. Basolateral Uptake of Nucleosides by Sertoli Cells Is Mediated Primarily by Equilibrative Nucleoside Transporter 1

    PubMed Central

    Klein, David M.; Evans, Kristen K.; Hardwick, Rhiannon N.; Dantzler, William H.; Wright, Stephen H.

    2013-01-01

    The blood-testis barrier (BTB) prevents the entry of many xenobiotic compounds into seminiferous tubules thereby protecting developing germ cells. Understanding drug transport across the BTB may improve drug delivery into the testis. Members of one class of drug, nucleoside reverse transcriptase inhibitors (NRTIs), do penetrate the BTB, presumably through interaction with physiologic nucleoside transporters. By investigating the mechanism of nucleoside transport, it may be possible to design other drugs to bypass the BTB in a similar manner. We present a novel ex vivo technique to study transport at the BTB that employs isolated, intact seminiferous tubules. Using this system, we found that over 80% of total uptake by seminiferous tubules of the model nucleoside uridine could be inhibited by 100 nM nitrobenzylmercaptopurine riboside (NBMPR, 6-S-[(4-nitrophenyl)methyl]-6-thioinosine), a concentration that selectively inhibits equilibrative nucleoside transporter 1 (ENT1) activity. In primary cultured rat Sertoli cells, 100 nM NBMPR inhibited all transepithelial transport and basolateral uptake of uridine. Immunohistochemical staining showed ENT1 to be located on the basolateral membrane of human and rat Sertoli cells, whereas ENT2 was located on the apical membrane of Sertoli cells. Transepithelial transport of uridine by rat Sertoli cells was partially inhibited by the NRTIs zidovudine, didanosine, and tenofovir disoproxil fumarate, consistent with an interaction between these drugs and ENT transporters. These data indicate that ENT1 is the primary route for basolateral nucleoside uptake into Sertoli cells and a possible mechanism for nucleosides and nucleoside-based drugs to undergo transepithelial transport. PMID:23639800

  6. Pestivirus NS3 (p80) protein possesses RNA helicase activity.

    PubMed Central

    Warrener, P; Collett, M S

    1995-01-01

    The pestivirus bovine viral diarrhea virus (BVDV) p80 protein (referred to here as the NS3 protein) contains amino acid sequence motifs predictive of three enzymatic activities: serine proteinase, nucleoside triphosphatase, and RNA helicase. We have previously demonstrated that the former two enzymatic activities are associated with this protein. Here, we show that a purified recombinant BVDV NS3 protein derived from baculovirus-infected insect cells possesses RNA helicase activity. BVDV NS3 RNA helicase activity was specifically inhibited by monoclonal antibodies to the p80 protein. The activity was dependent on the presence of nucleoside triphosphate and divalent cation, with a preference for ATP and Mn2+. Hydrolysis of the nucleoside triphosphate was necessary for strand displacement. The helicase activity required substrates with an un-base-paired region on the template strand 3' of the duplex region. As few as three un-base-paired nucleotides were sufficient for efficient oligonucleotide displacement. However, the enzyme did not act on substrates having a single-stranded region only to the 5' end of the duplex or on substrates lacking single-stranded regions altogether (blunt-ended duplex substrates), suggesting that the directionality of the BVDV RNA helicase was 3' to 5' with respect to the template strand. The BVDV helicase activity was able to displace both RNA and DNA oligonucleotides from RNA template strands but was unable to release oligonucleotides from DNA templates. The possible role of this activity in pestivirus replication is discussed. PMID:7853509

  7. Comparison of the binding of the therapeutically active nucleosides to DNA molecules with different level of lesions

    NASA Astrophysics Data System (ADS)

    Kruglova, E. B.; Gladkovskaya, N. A.

    2002-12-01

    Recently we have shown that DNA molecules extracted from epididymis of the Wistar male rats exposed to low doses of gamma radiation interact with some pyrimidine nucleosides. The bindign affinities of NUC to control DNA molecules are unessential. Comparing the UV melting curves for the various DNA sammples we show that observed differences are related to conformational chagnes in the DNA double helix. The samples of the damaged DNA have been obtained by partial denaturation of the calf thymus DNA in the salt-free aqueous solutions. The level of DNA damages in the model DNA smplase depends on the DNA concentration. It was shown that damages in the DNA molecules lead to changes of the melting curves of DNA-NUC mixtures that are similar to those for the DNA samples extracted from irradiated tissues. ALso it has been found that the binding mechanisms to cytosine arabinoside and 6-azacytosine to DNA molecuels having modifeid secondary structures are different.

  8. Polymeric nanogel formulations of nucleoside analogs

    PubMed Central

    Vinogradov, Serguei V

    2008-01-01

    Nanogels are colloidal microgel carriers that have been introduced recently as a prospective drug delivery system for nucleotide therapeutics. The crosslinked protonated polymer network of nanogels binds oppositely charged drug molecules, encapsulating them into submicron particles with a core-shell structure. The nanogel network also provides a suitable template for chemical engineering, surface modification and vectorisation. This review reveals recent attempts to develop novel drug formulations of nanogels with antiviral and antiproliferative nucleoside analogs in the active form of 5′-triphosphates; discusses structural approaches to the optimisation of nanogel properties, and; discusses the development of targeted nanogel drug formulations for systemic administration. Notably, nanogels can improve the CNS penetration of nucleoside analogs that are otherwise restricted from passing across the blood–brain barrier. The latest findings reviewed here demonstrate an efficient intracellular release of nucleoside analogs, encouraging further applications of nanogel carriers for targeted drug delivery. PMID:17184158

  9. Transcriptional activation of the nitrogenase promoter in vitro: adenosine nucleotides are required for inhibition of NIFA activity by NIFL.

    PubMed

    Eydmann, T; Söderbäck, E; Jones, T; Hill, S; Austin, S; Dixon, R

    1995-03-01

    The enhancer-binding protein NIFA is required for transcriptional activation of nif promoters by the alternative holoenzyme form of RNA polymerase, which contains the sigma factor sigma 54 (sigma N). NIFA hydrolyzes nucleoside triphosphates to catalyze the isomerization of closed promoter complexes to transcriptionally competent open complexes. The activity of NIFA is antagonized by the regulatory protein NIFL in response to oxygen and fixed nitrogen in vivo. We have investigated the requirement for nucleotides in the formation and stability of open promoter complexes by NIFA and inhibition of its activity by NIFL at the Klebsiella pneumoniae nifH promoter. Open complexes formed by sigma 54-containing RNA polymerase are considerably more stable to heparin challenge in the presence of GTP than in the presence of ATP. This differential stability is most probably a consequence of GTP being the initiating nucleotide at this promoter. Adenosine nucleosides are specifically required for Azotobacter vinelandii NIFL to inhibit open complex formation by native NIFA, and the nucleoside triphosphatase activity of NIFA is strongly inhibited by NIFL under these conditions. We propose a model in which NIFL modulates the activity of NIFA via an adenosine nucleotide switch. PMID:7868590

  10. Mycoplasmas and cancer: focus on nucleoside metabolism

    PubMed Central

    Vande Voorde, Johan; Balzarini, Jan; Liekens, Sandra

    2014-01-01

    The standard of care for patients suffering cancer often includes treatment with nucleoside analogues (NAs). NAs are internalized by cell-specific nucleobase/nucleoside transporters and, after enzymatic activation (often one or more phosphorylation steps), interfere with cellular nucleo(s)(t)ide metabolism and DNA/RNA synthesis. Therefore, their efficacy is highly dependent on the expression and activity of nucleo(s)(t)ide-metabolizing enzymes, and alterations thereof (e.g. by down/upregulated expression or mutations) may change the susceptibility to NA-based therapy and/or confer drug resistance. Apart from host cell factors, several other variables including microbial presence may determine the metabolome (i.e. metabolite concentrations) of human tissues. Studying the diversity of microorganisms that are associated with the human body has already provided new insights in several diseases (e.g. diabetes and inflammatory bowel disease) and the metabolic exchange between tissues and their specific microbiota was found to affect the bioavailability and toxicity of certain anticancer drugs, including NAs. Several studies report a preferential colonization of tumor tissues with some mycoplasma species (mostly Mycoplasma hyorhinis). These prokaryotes are also a common source of cell culture contamination and alter the cytostatic activity of some NAs in vitro due to the expression of nucleoside-catabolizing enzymes. Mycoplasma infection may therefore bias experimental work with NAs, and their presence in the tumor microenvironment could be of significance when optimizing nucleoside-based cancer treatment. PMID:26417262

  11. Oral Cyclosporin A Inhibits CD4 T cell P-glycoprotein Activity in HIV-Infected Adults Initiating Treatment with Nucleoside Reverse Transcriptase Inhibitors

    PubMed Central

    Hulgan, Todd; Donahue, John P.; Smeaton, Laura; Pu, Minya; Wang, Hongying; Lederman, Michael M.; Smith, Kimberly; Valdez, Hernan; Pilcher, Christopher; Haas, David W.

    2010-01-01

    Purpose P-glycoprotein limits tissue penetration of many antiretroviral drugs. We characterized effects of the P-glycoprotein substrate cyclosporin A on T cell P-glycoprotein activity in HIV-infected AIDS Clinical Trials Group study A5138 participants. Methods We studied P-glycoprotein activity on CD4 and CD8 T cells in 16 participants randomized to receive oral cyclosporin A (n=9) or not (n=7) during initiation antiretroviral therapy (ART) that did not include protease or non-nucleoside reverse transcriptase inhibitors. Results CD4 T cell P-glycoprotein activity decreased by a median of 8 percentage points with cyclosporin A/ART (difference between cyclosporin A/ART versus ART only P=0.001). Plasma trough cyclosporin A concentrations correlated with change in P-glycoprotein activity in several T cell subsets. Conclusions Oral cyclosporin A can inhibit peripheral blood CD4 T cell P-glycoprotein activity. Targeted P-glycoprotein inhibition might enhance delivery of ART to T cells. PMID:19779705

  12. Exploring the role of the α-carboxyphosphonate moiety in the HIV-RT activity of α-carboxy nucleoside phosphonates

    PubMed Central

    Mullins, Nicholas D.; Maguire, Nuala M.; Ford, Alan; Das, Kalyan; Arnold, Edward; Balzarini, Jan; Maguire, Anita R.

    2016-01-01

    As α-carboxy nucleoside phosphonates (α-CNPs) have demonstrated a novel mode of action of HIV-1 reverse transcriptase inhibition, structurally related derivatives were synthesized, namely the malonate 2, the unsaturated and saturated bisphosphonates 3 and 4, respectively and the amide 5. These compounds were evaluated for inhibition of HIV-1 reverse transcriptase in cell-free assays. The importance of the α-carboxy phosphonoacetic acid moiety for achieving reverse transcriptase inhibition, without the need for prior phosphorylation, was confirmed. The malonate derivative 2 was less active by two orders of magnitude than the original α-CNPs, while displaying the same pattern of kinetic behavior; interestingly the activity resides in the “L”-enantiomer of 2, as seen with the earlier series of α-CNPs. A crystal structure with an RT/DNA complex at 2.95 Å resolution revealed the binding of the “L”-enantiomer of 2, at the polymerase active site with a weaker metal ion chelation environment compared to 1a (T-α-CNP) which may explain the lower inhibitory activity of 2. PMID:26813581

  13. Arylthiopyrrole (AThP) derivatives as non-nucleoside HIV-1 reverse transcriptase inhibitors: synthesis, structure-activity relationships, and docking studies (part 2).

    PubMed

    Lavecchia, Antonio; Costi, Roberta; Artico, Marino; Miele, Gaetano; Novellino, Ettore; Bergamini, Alberto; Crespan, Emmanuele; Maga, Giovanni; Di Santo, Roberto

    2006-12-01

    Arylthio isopropyl pyridinylmethylpyrrolemethanols (AThPs) have been recently reported as a new class of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitors acting at the non-nucleoside binding site (NNBS) of this enzyme. Docking experiments of the potent inhibitors 4k (IC(50) = 0.24 microM, SI = 167) and 5e (IC(50) = 0.11 microM, SI > 1667) of wild-type RT prompted the synthesis and biological evaluation of novel AThP derivatives featuring a number of polar groups in position 3 of the pyrrole ring and larger and more hydrophobic alicyclic substituents in place of the isopropyl group at position 4. Among the compounds synthesized and tested in cell-based assays against HIV-1 infected cells, 19b was the most active, with EC(50) = 0.007 microM, CC(50) = 114.5 microm, and SI = 16357. This compound and its precursor 18b retained interesting activities against clinically relevant drug-resistant RT forms carrying K103N, Y181I, and L100I mutations. Docking calculations of 10, 14, 18b, and 19b were also performed to investigate their binding mode into the RT NNBS and to rationalize both structure-activity relationship and resistance data. PMID:17089434

  14. Fission yeast RNA triphosphatase reads an Spt5 CTD code.

    PubMed

    Doamekpor, Selom K; Schwer, Beate; Sanchez, Ana M; Shuman, Stewart; Lima, Christopher D

    2015-01-01

    mRNA capping enzymes are directed to nascent RNA polymerase II (Pol2) transcripts via interactions with the carboxy-terminal domains (CTDs) of Pol2 and transcription elongation factor Spt5. Fission yeast RNA triphosphatase binds to the Spt5 CTD, comprising a tandem repeat of nonapeptide motif TPAWNSGSK. Here we report the crystal structure of a Pct1·Spt5-CTD complex, which revealed two CTD docking sites on the Pct1 homodimer that engage TPAWN segments of the motif. Each Spt5 CTD interface, composed of elements from both subunits of the homodimer, is dominated by van der Waals contacts from Pct1 to the tryptophan of the CTD. The bound CTD adopts a distinctive conformation in which the peptide backbone makes a tight U-turn so that the proline stacks over the tryptophan. We show that Pct1 binding to Spt5 CTD is antagonized by threonine phosphorylation. Our results fortify an emerging concept of an "Spt5 CTD code" in which (i) the Spt5 CTD is structurally plastic and can adopt different conformations that are templated by particular cellular Spt5 CTD receptor proteins; and (ii) threonine phosphorylation of the Spt5 CTD repeat inscribes a binary on-off switch that is read by diverse CTD receptors, each in its own distinctive manner. PMID:25414009

  15. d- and l-2′,3′-Didehydro-2′,3′-Dideoxy-3′-Fluoro-Carbocyclic Nucleosides: Synthesis, Anti-HIV Activity and Mechanism of Resistance

    PubMed Central

    Wang, Jianing; Jin, Yunho; Rapp, Kimberly L.; Schinazi, Raymond F.; Chu, Chung K.

    2008-01-01

    Introducing 2′-fluoro substitution on the 2′,3′-double bond in carbocyclic nucleosides has provided biologically interesting compounds with potent anti-HIV activity. As an extension of our previous works in the discovery of anti-HIV agents, d- and l-2′,3′-unsaturated 3′-fluoro carbocyclic nucleosides were synthesized and evaluated against HIV-1 in human peripheral blood mononuclear (PBM) cells. Among the synthesized l-series nucleosides, compounds 18, 19, 26, 28 exhibited moderate antiviral activity (EC50 7.1 μM, 6.4 μM, 10.3 μM and 20.7 μM, respectively), while among the d-series, the guanosine analogue (35, d-3′-F-C-d4G) exhibited the most potent anti-HIV activity (EC50 0.4 μM, EC90 2.8 μM). However, the guanosine analogue 35 was cross-resistant to the lamivudine-resistant variants (HIV-1M184V). Molecular modeling studies suggest that hydrophobic interaction as well as hydrogen bonding stabilize the binding of compound 35 in the active site of wild type HIV reverse transcriptase (HIV-RT). In the case of l-nucleosides, these two effects are opposite which results in a loss of binding affinity. According to the molecular modeling studies, cross-resistance of d-3′-F-C-d4G (35) to M184V mutant may be caused by the realignment of the primer and template in the HIV-RTM184V interaction, which destabilizes the RT-inhibitor triphosphate complex, resulting in a significant reduction in anti-HIV activity of the d-guanine derivative 35. PMID:17373782

  16. Synthesis, structure-activity relationship and molecular docking of cyclohexenone based analogous as potent non-nucleoside reverse-transcriptase inhibitors

    NASA Astrophysics Data System (ADS)

    Nazar, Muhammad Faizan; Abdullah, Muhammad Imran; Badshah, Amir; Mahmood, Asif; Rana, Usman Ali; Khan, Salah Ud-Din

    2015-04-01

    The chalcones core in compounds is advantageously chosen effective synthons, which offer exciting perspectives in biological and pharmacological research. The present study reports the successful development of eight new cyclohexenone based anti-reverse transcriptase analogous using rational drug design synthesis principles. These new cyclohexenone derivatives (CDs) were synthesized by following a convenient route of Robinson annulation, and the molecular structure of these CDs were later confirmed by various analytical techniques such as 1H NMR, 13C NMR, FT-IR, UV-Vis spectroscopy and mass spectrometry. All the synthesized compounds were screened theoretically and experimentally against reverse transcriptase (RT) and found potentially active reverse transcriptase (RT) inhibitors. Of the compounds studied, the compound 2FC4 showed high interaction with RT at non-nucleoside binding site, contributing high free binding energy (ΔG -8.01 Kcal) and IC50 (0.207 μg/ml), respectively. Further results revealed that the compounds bearing more halogen groups, with additional hydrophobic character, offered superior anti-reverse transcriptase activity as compared to rest of compounds. It is anticipate that the present study would be very useful for the selection of potential reverse transcriptase inhibitors featuring inclusive pharmacological profiles.

  17. Membrane-permeable Triphosphate Prodrugs of Nucleoside Analogues.

    PubMed

    Gollnest, Tristan; Dinis de Oliveira, Thiago; Rath, Anna; Hauber, Ilona; Schols, Dominique; Balzarini, Jan; Meier, Chris

    2016-04-18

    The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate-limitations can be at the mono-, but also at the di- and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (TriPPPro-approach). In this approach, NTPs are masked by two bioreversible units at the γ-phosphate. Using a procedure involving H-phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme-triggered delivery of NTPs was demonstrated by pig liver esterase, in human T-lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro-compounds of some HIV-inactive nucleoside analogues showed marked anti-HIV activity. For cellular uptake studies, a fluorescent TriPPPro-compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells. PMID:27008042

  18. Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity

    SciTech Connect

    Wright, K.T.; Seabright, R.; Logan, A.; Lilly, A.J.; Khanim, F.; Bunce, C.M.; Johnson, W.E.B.

    2010-07-16

    Research highlights: {yields} Extracellular Nm23H1 stimulates nerve growth. {yields} Extracellular Nm23H1 provides pathfinding cues to growth cones. {yields} The neurotrophic activity of Nm23H1 is independent of NDP kinase activity. {yields} The neurotrophic activity of Nm23H1 is independent of NGF. -- Abstract: The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems.

  19. DNA 3' pp 5' G de-capping activity of aprataxin: effect of cap nucleoside analogs and structural basis for guanosine recognition

    DOE PAGESBeta

    Chauleau, Mathieu; Jacewicz, Agata; Shuman, Stewart

    2015-05-24

    DNA3' pp 5'G caps synthesized by the 3'-PO4/5'-OH ligase RtcB have a strong impact on enzymatic reactions at DNA 3'-OH ends. Aprataxin, an enzyme that repairs A5'pp5'DNA ends formed during abortive ligation by classic 3'-OH/5'-PO4 ligases, is also a DNA 3' de-capping enzyme, converting DNAppG to DNA3'p and GMP. By taking advantage of RtcB's ability to utilize certain GTP analogs to synthesize DNAppN caps, we show that aprataxin hydrolyzes inosine and 6-O-methylguanosine caps, but is not adept at removing a deoxyguanosine cap. We report a 1.5 Å crystal structure of aprataxin in a complex with GMP, which reveals that: (i)more » GMP binds at the same position and in the same anti nucleoside conformation as AMP; and (ii) aprataxin makes more extensive nucleobase contacts with guanine than with adenine, via a hydrogen bonding network to the guanine O6, N1, N2 base edge. Alanine mutations of catalytic residues His147 and His149 abolish DNAppG de-capping activity, suggesting that the 3' de-guanylylation and 5' de-adenylylation reactions follow the same pathway of nucleotidyl transfer through a covalent aprataxin-(His147)–NMP intermediate. Alanine mutation of Asp63, which coordinates the guanosine ribose hydroxyls, impairs DNAppG de-capping.« less

  20. Arylthiopyrrole (AThP) derivatives as non-nucleoside HIV-1 reverse transcriptase inhibitors: synthesis, structure-activity relationships, and docking studies (part 1).

    PubMed

    Di Santo, Roberto; Costi, Roberta; Artico, Marino; Miele, Gaetano; Lavecchia, Antonio; Novellino, Ettore; Bergamini, Alberto; Cancio, Reynel; Maga, Giovanni

    2006-12-01

    Novel arylthio isopropyl pyridinylmethylpyrrolemethanol (AThP) derivatives 3-5, which are related to capravirine (S-1153), were synthesized and tested for their ability to block the replication cycle of HIV-1 in infected cells. The newly synthesized AThPs are active in the concentration range of 0.008-53 microM. Even if compounds 3-5 are generally less potent than S-1153, their SI values are in some cases similar to that of the reference drug. In fact, the cytotoxicities of AThPs are generally lower than that of S-1153. Compound 4e was the most active derivative of this series in cell-based assays; its potency is similar to that of S-1153 (EC(50)=8 and 3 nM, respectively), as is its selectivity index (SI=6250 and 7000, respectively). AThP derivatives were proven to target HIV-1 RT. In fact, compounds 3-5 generally inhibited the viral enzyme at concentrations similar to those observed in cell-based assays. A selected number of AThPs (4k and 5a,e) were tested against clinically relevant drug-resistant forms of recombinant reverse transcriptase (rRT) carrying the K103N and Y181I mutations. Carbamate 5e showed an approximate 240-fold decrease in activity against Y181I, but only a 10-fold loss in potency against the K103N rRT form. Docking calculations were also performed to investigate the binding mode of compounds 2, 4e, 4j, 4k and 5e into the non-nucleoside binding site of HIV-1 RT and to rationalize some structure-activity relationships and resistance data. PMID:17089433

  1. Localization of GFP-tagged concentrative nucleoside transporters in a renal polarized epithelial cell line.

    PubMed

    Mangravite, L M; Lipschutz, J H; Mostov, K E; Giacomini, K M

    2001-05-01

    Many nucleosides undergo active reabsorption within the kidney, probably via nucleoside transporters. To date, two concentrative nucleoside transporters have been cloned, the sodium-dependent purine-selective nucleoside transporter (SPNT) and concentrative nucleoside transporter 1 (CNT1). We report the stable expression of green fluorescence protein (GFP)-tagged SPNT and CNT1 in Madin-Darby canine kidney (MDCK) cells, a polarized renal epithelial line. We demonstrate that the GFP tag does not alter the substrate selectivity and only modestly affects the kinetic activity of the transporters. By using confocal microscopy and functional studies, both SPNT and CNT1 are localized primarily to the apical membrane of MDCK and LLC-PK(1) cells. Apical localization of these transporters suggests a role in renal nucleoside reabsorption and regulation of tubular function via the adenosine pathway. PMID:11292631

  2. Nucleoside transporter expression and function in cultured mouse astrocytes.

    PubMed

    Peng, Liang; Huang, Rong; Yu, Albert C H; Fung, King Y; Rathbone, Michel P; Hertz, Leif

    2005-10-01

    Uptake of purine and pyrimidine nucleosides in astrocytes is important for several reasons: (1) uptake of nucleosides contributes to nucleic acid synthesis; (2) astrocytes synthesize AMP, ADP, and ATP from adenosine and GTP from guanosine; and (3) adenosine and guanosine function as neuromodulators, whose effects are partly terminated by cellular uptake. It has previously been shown that adenosine is rapidly accumulated by active uptake in astrocytes (Hertz and Matz, Neurochem Res 14:755-760, 1989), but the ratio between active uptake and metabolism-driven uptake of adenosine is unknown, as are uptake characteristics for guanosine. The present study therefore aims at providing detailed information of nucleoside transport and transporters in primary cultures of mouse astrocytes. Reverse transcription-polymerase chain reaction identified the two equilibrative nucleoside transporters, ENT1 and ENT2, together with the concentrative nucleoside transporter CNT2, whereas CNT3 was absent, and CNT1 expression could not be investigated. Uptake studies of tritiated thymidine, formycin B, guanosine, and adenosine (3-s uptakes at 1-4 degrees C to study diffusional uptake and 1-60-min uptakes at 37 degrees C to study concentrative uptake) demonstrated a fast diffusional uptake of all four nucleosides, a small, Na(+)-independent and probably metabolism-driven uptake of thymidine (consistent with DNA synthesis), larger metabolism-driven uptakes of guanosine (consistent with synthesis of DNA, RNA, and GTP) and especially of adenosine (consistent with rapid nucleotide synthesis), and Na(+)-dependent uptakes of adenosine (consistent with its concentrative uptake) and guanosine, rendering neuromodulator uptake independent of nucleoside metabolism. Astrocytes are accordingly well suited for both intense nucleoside metabolism and metabolism-independent uptake to terminate neuromodulator effects of adenosine and guanosine. PMID:15892125

  3. Novel triphosphate phosphohydrolase activity of Clostridium thermocellum TTM, a member of the triphosphate tunnel metalloenzyme superfamily.

    PubMed

    Keppetipola, Niroshika; Jain, Ruchi; Shuman, Stewart

    2007-04-20

    Triphosphate tunnel metalloenzymes (TTMs) are a newly recognized superfamily of phosphotransferases defined by a unique active site residing within an eight-stranded beta barrel. The prototypical members are the eukaryal metal-dependent RNA triphosphatases, which catalyze the initial step in mRNA capping. Little is known about the activities and substrate specificities of the scores of TTM homologs present in bacterial and archaeal proteomes, nearly all of which are annotated as adenylate cyclases. Here we have conducted a biochemical and structure-function analysis of a TTM protein (CthTTM) from the bacterium Clostridium thermocellum. CthTTM is a metal-dependent tripolyphosphatase and nucleoside triphosphatase; it is not an adenylate cyclase. We have identified 11 conserved amino acids in the tunnel that are critical for tripolyphosphatase and ATPase activity. The most salient findings are that (i) CthTTM is 150-fold more active in cleaving tripolyphosphate than ATP and (ii) the substrate specificity of CthTTM can be transformed by a single mutation (K8A) that abolishes tripolyphosphatase activity while strongly stimulating ATP hydrolysis. Our results underscore the plasticity of CthTTM substrate choice and suggest how novel specificities within the TTM superfamily might evolve through changes in the residues that line the tunnel walls. PMID:17303560

  4. Aqueous microwaves assisted cross-coupling reactions applied to unprotected nucleosides.

    NASA Astrophysics Data System (ADS)

    Len, Christophe; Hervé, Gwénaelle

    2015-02-01

    Nucleoside analogues have attracted much attention due to their potential biological activities. Amongst all synthetic nucleosides, C5-modified pyrimidines and C7- or C8-modified purines have mostly been prepared using palladium cross-coupling reactions and then studied as antitumoral and antiviral agents. Our objective is to focus this review on the Suzuki-Miyaura and on the Heck cross-couplings of nucleosides using microwave irradiations which are an alternative technology compatible with green chemistry and sustainable development.

  5. Polarization of the Vacuolar Adenosine Triphosphatase Delineates a Transition to High-Grade Pancreatic Intraepithelial Neoplasm Lesions

    PubMed Central

    Sreekumar, Bharath K.; Belinsky, Glenn S.; Einwachter, Henrik; Rhim, Andrew D.; Schmid, Roland; Chung, Chuhan

    2015-01-01

    Objectives A functional vacuolar adenosine triphosphatase (v-ATPase) complex regulates canonical Wnt/β-catenin signaling. The goal of this study was to identify the distribution of the v-ATPase in human and murine models of pancreatic intraepithelial neoplasms (PanINs) and assess its role in Wnt/β-catenin signaling. Methods We evaluated the immunolabeling pattern of the v-ATPase in human PanIN specimens and murine PanIN-1 and PanIN-2 lesions obtained from Ptf1aCre/+; LSL-KrasG12D mice. Wnt/β-catenin signaling was interrogated in primary PanIN cells by examining the phosphorylated levels of its surface coreceptor, low-density lipoprotein receptor-related protein-6 (LRP6), and its intracellular effector, nonphosphorylated β-catenin. The response of primary PanIN cells to epidermal growth factor (EGF) was assessed in the absence and presence of the v-ATPase inhibitor, concanamycin. Results In advanced (PanIN-2), but not early (PanIN-1), lesions, the v-ATPase assumed a polarized phenotype. Blocking the v-ATPase disrupted Wnt/β-catenin signaling in primary PanIN cells despite significantly higher levels of the total and activated Wnt cell surface coreceptor, LRP6. Vacuolar adenosine triphosphatase blockade significantly decreased the total and activated levels of EGF receptor, a determinant of PanIN progression. The activation of EGF receptor and its intracellular mediator, p44/42 mitogen-activated protein kinase, was also reduced by v-ATPase blockade. This led to diminished proliferation in response to EGF ligand. Conclusions The v-ATPase regulates Wnt/β-catenin and EGF receptor signaling in PanINs. PMID:25072283

  6. Flexibility as a Strategy in Nucleoside Antiviral Drug Design.

    PubMed

    Peters, H L; Ku, T C; Seley-Radtke, K L

    2015-01-01

    As far back as Melville Wolfrom's acyclic sugar synthesis in the 1960's, synthesis of flexible nucleoside analogues have been an area of interest. This concept, however, went against years of enzyme-substrate binding theory. Hence, acyclic methodology in antiviral drug design did not take off until the discovery and subsequent FDA approval of such analogues as Acyclovir and Tenofovir. More recently, the observation that flexible nucleosides could overcome drug resistance spawned a renewed interest in the field of nucleoside drug design. The next generation of flexible nucleosides shifted the focus from the sugar moiety to the nucleobase. With analogues such as Seley-Radtke "fleximers", and Herdewijn's C5 substituted 2'-deoxyuridines, the area of base flexibility has seen great expansion. More recently, the marriage of these methodologies with acyclic sugars has resulted in a series of acyclic flex-base nucleosides with a wide range of antiviral properties, including some of the first to exhibit anti-coronavirus activity. Various flexible nucleosides and their corresponding nucleobases will be compared in this review. PMID:26282942

  7. Microbial transformation of nucleosides

    NASA Technical Reports Server (NTRS)

    Lamba, S. S.

    1979-01-01

    A study involving the use of coulter counter in studying the effects of neomycin on E. coli, S. aureus and A. aerogenes was completed. The purpose of this was to establish proper technique for enumeration of cells per ml. It was found that inhibitory effects on growth of E. coli and A. aerogenes, both gram negative organisms, were directly related to the concentration of neomycin used. However, in case S. aureus, a gram positive organism, a decreased inhibition was noted at higher concentrations. A paper entitled, Use of Coulter Counter in Studying Effect of Drugs on Cells in Culture 1 - Effects of Neomycin on E. coli, S. aureus and A. aerogenes, is attached in the appendix. Laboratory procedures were also established to study the effects of nucleoside antibiotic cordycepin on He La cell grown in suspension cultures.

  8. [Purine nucleoside phosphorylase].

    PubMed

    Pogosian, L G; Akopian, Zh I

    2013-01-01

    Purine nucleoside phosphorylase (PNP) is one of the most important enzymes of the purine metabolism, wich promotes the recycling of purine bases. Nowadays is the actual to search for effective inhibitors of this enzyme which is necessary for creation T-cell immunodeficient status of the organism in the organs and tissues transplantation, and chemotherapy of a number pathologies as well. For their successful practical application necessary to conduct in-depth and comprehensive study of the enzyme, namely a structure, functions, and an affinity of the reaction mechanism. In the review the contemporary achievements in the study of PNP from various biological objects are presented. New data describing the structure of PNP are summarised and analysed. The physiological role of the enzyme is discussed. The enzyme basic reaction mechanisms and actions are considered. The studies on enzyme physicochemical, kinetic, and catalytic research are presented. PMID:24479338

  9. Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters

    PubMed Central

    Johnson, Zachary Lee; Lee, Jun-Ho; Lee, Kiyoun; Lee, Minhee; Kwon, Do-Yeon; Hong, Jiyong; Lee, Seok-Yong

    2014-01-01

    Concentrative nucleoside transporters (CNTs) are responsible for cellular entry of nucleosides, which serve as precursors to nucleic acids and act as signaling molecules. CNTs also play a crucial role in the uptake of nucleoside-derived drugs, including anticancer and antiviral agents. Understanding how CNTs recognize and import their substrates could not only lead to a better understanding of nucleoside-related biological processes but also the design of nucleoside-derived drugs that can better reach their targets. Here, we present a combination of X-ray crystallographic and equilibrium-binding studies probing the molecular origins of nucleoside and nucleoside drug selectivity of a CNT from Vibrio cholerae. We then used this information in chemically modifying an anticancer drug so that it is better transported by and selective for a single human CNT subtype. This work provides proof of principle for utilizing transporter structural and functional information for the design of compounds that enter cells more efficiently and selectively. DOI: http://dx.doi.org/10.7554/eLife.03604.001 PMID:25082345

  10. A comparison of enzymatic phosphorylation and phosphatidylation of beta-L- and beta-D-nucleosides.

    PubMed

    Birichevskaya, Larisa L; Kvach, Sergei V; Sivets, Grigorii G; Kalinichenko, Elena N; Zinchenko, Anatoly I; Mikhailopulo, Igor A

    2007-04-01

    Enzymatic 5'-monophosphorylation and 5'-phosphatidylation of a number of beta-L- and beta-D-nucleosides was investigated. The first reaction, catalyzed by nucleoside phosphotransferase (NPT) from Erwinia herbicola, consisted of the transfer of the phosphate residue from p-nitrophenylphosphate (p-NPP) to the 5'-hydroxyl group of nucleoside; the second was the phospholipase D (PLD)-catalyzed transphosphatidylation of L-alpha-lecithin with a series of beta-L- and beta-D-nucleosides as the phosphatidyl acceptor resulted in the formation of the respective phospholipid-nucleoside conjugates. Some beta-L-nucleosides displayed similar or even higher substrate activity compared to the beta-D-enantiomers. PMID:17206374

  11. A new class of acyclic nucleoside phosphonates: synthesis and biological activity of 9-[[(phosphonomethyl)aziridin-1-yl]methyl]guanine (PMAMG) and analogues.

    PubMed

    Abu Sheikha, Ghassan; La Colla, Paolo; Loi, Anna Giulia

    2002-10-01

    A new class of acyclic nucleoside phosphonates PMAMG, PMAMA, PMAMC, and PMAMT (compounds 1, 2, 3 and 4) have been synthesized and tested in vitro against a wide variety of viruses, fungi and bacteria. PMAMG (1) was synthesized by the alkylation reaction of acetylguanine with the phosphonate side-chain, diisopropyl [[2-(bromomethyl)aziridin-1-yl

  12. Challenges and solutions in the bioanalysis of BMS-986094 and its metabolites including a highly polar, active nucleoside triphosphate in plasma and tissues using LC-MS/MS.

    PubMed

    Liu, Ang; Lute, John; Gu, Huidong; Wang, Bonnie; Trouba, Kevin J; Arnold, Mark E; Aubry, Anne-Françoise; Wang, Jian

    2015-09-01

    BMS-986094, a nucleotide polymerase inhibitor of the hepatitis C virus, was withdrawn from clinical trials because of a serious safety issue. To investigate a potential association between drug/metabolite exposure and toxicity in evaluations conducted after the termination of the BMS-986094 development program, it was essential to determine the levels of BMS-986094 and its major metabolites INX-08032, INX-08144 and INX-09054 in circulation and the active nucleoside triphosphate INX-09114 in target and non-target tissues. However, there were many challenges in the bioanalysis of these compounds. The chromatography challenge for the extremely polar nucleoside triphosphate was solved by applying mixed-mode chromatography which combined anion exchange and reversed-phase interactions. The LC conditions provided adequate retention and good peak shape of the analyte and showed good robustness. A strategy using simultaneous extraction but separate LC analysis of the prodrug BMS-986094 and its major circulating metabolites was used to overcome a carryover issue of the hydrophobic prodrug while still achieving good chromatography of the polar metabolites. In addition, the nucleotide analytes were not stable in the presence of endogenous enzymes. Low pH and low temperature were required for blood collection and plasma sample processing. However, the use of phosphatase inhibitor and immediate homogenization and extraction were critical for the quantitative analysis of the active triphosphate, INX-09114, in tissue samples. To alleviate the bioanalytical complexity caused by multiple analytes, different matrices, and various species, a fit-for-purpose approach to assay validation was implemented based on the needs of drug safety assessment in non-clinical (GLP or non-GLP) studies. The assay for INX-08032 was fully validated in plasma of toxicology species. The lower limit of quantification was 1.00ng/mL and the linear curve range was 1.00-500.00ng/mL using a weighted (1/x(2

  13. DNA 3' pp 5' G de-capping activity of aprataxin: effect of cap nucleoside analogs and structural basis for guanosine recognition

    SciTech Connect

    Chauleau, Mathieu; Jacewicz, Agata; Shuman, Stewart

    2015-05-24

    DNA3' pp 5'G caps synthesized by the 3'-PO4/5'-OH ligase RtcB have a strong impact on enzymatic reactions at DNA 3'-OH ends. Aprataxin, an enzyme that repairs A5'pp5'DNA ends formed during abortive ligation by classic 3'-OH/5'-PO4 ligases, is also a DNA 3' de-capping enzyme, converting DNAppG to DNA3'p and GMP. By taking advantage of RtcB's ability to utilize certain GTP analogs to synthesize DNAppN caps, we show that aprataxin hydrolyzes inosine and 6-O-methylguanosine caps, but is not adept at removing a deoxyguanosine cap. We report a 1.5 Å crystal structure of aprataxin in a complex with GMP, which reveals that: (i) GMP binds at the same position and in the same anti nucleoside conformation as AMP; and (ii) aprataxin makes more extensive nucleobase contacts with guanine than with adenine, via a hydrogen bonding network to the guanine O6, N1, N2 base edge. Alanine mutations of catalytic residues His147 and His149 abolish DNAppG de-capping activity, suggesting that the 3' de-guanylylation and 5' de-adenylylation reactions follow the same pathway of nucleotidyl transfer through a covalent aprataxin-(His147)–NMP intermediate. Alanine mutation of Asp63, which coordinates the guanosine ribose hydroxyls, impairs DNAppG de-capping.

  14. Hypouricemic effects of novel concentrative nucleoside transporter 2 inhibitors through suppressing intestinal absorption of purine nucleosides.

    PubMed

    Hiratochi, Masahiro; Tatani, Kazuya; Shimizu, Kazuo; Kuramochi, Yu; Kikuchi, Norihiko; Kamada, Noboru; Itoh, Fumiaki; Isaji, Masayuki

    2012-09-01

    We have developed concentrative nucleoside transporter 2 (CNT2) inhibitors as a novel pharmacological approach for improving hyperuricemia by inhibiting intestinal absorption of purines. Dietary purine nucleosides are absorbed in the small intestines by CNTs expressed in the apical membrane. In humans, the absorbed purine nucleosides are rapidly degraded to their final end product, uric acid, by xanthine oxidase. Based on the expression profile of human CNTs in digestive tract tissues, we established a working hypothesis that mainly CNT2 contributes to the intestinal absorption of purine nucleosides. In order to confirm this possibility, we developed CNT2 inhibitors and found that (2R,3R,4S,5R)-2-(6-amino-8-{[3'-(3-aminopropoxy)-biphenyl-4-ylmethyl]-amino}-9H-purin-9-yl)-5-hydroxymethyl-tetrahydrofuran-3,4-diol (KGO-2142) and 1-[3-(5-{[1-((2R,3R,4S,5R)-3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-1H-benzimidazol-2-ylamino]-methyl}-2-ethoxyphenoxy)-propyl]-piperidine-4-carboxylic acid amide (KGO-2173) were inhibitory. These CNT2 inhibitors had potent inhibitory activity against inosine uptake via human CNT2, but they did not potently interfere with nucleoside uptake via human CNT1, CNT3 or equilibrative nucleoside transporters (ENTs) in vitro. After oral administration of KGO-2173 along with [(14)C]-inosine, KGO-2173 significantly decreased the urinary excretion of radioactivity at 6 and 24h in rats. Since dietary purine nucleosides are not utilized in the body and are excreted into the urine rapidly, this decrease in radioactivity in the urine represented the inhibitory activity of KGO-2173 toward the absorption of [(14)C]-inosine in the small intestines. KGO-2142 almost completely inhibited dietary RNA-induced hyperuricemia and the increase in urinary excretion of uric acid in cebus monkeys. These novel CNT2 inhibitors, KGO-2142 and KGO-2173, could be useful therapeutic options for the treatment of hyperuricemia. PMID:22709993

  15. A review of non-nucleoside anti-hepatitis B virus agents.

    PubMed

    Zhang, Fan; Wang, Gang

    2014-03-21

    Hepatitis B Virus is the most common cause of chronic liver disease worldwide. Currently approved agents of chronic HBV infection treatment include interferon and nucleoside analogues. However, the side effects of interferon and the viral resistance of nucleoside analogues make the current treatment far from satisfactory. Therefore, new drugs with novel structures and mechanisms are needed. Recently, a number of non-nucleoside HBV inhibitors have been obtained from natural sources or prepared by synthesis/semi-synthesis. Some of them exhibited potent anti-HBV activity with novel mechanisms. These compounds provide useful information for the medicinal chemist to develop novel non-nucleoside compounds as anti-HBV agents. PMID:24549242

  16. Purification and properties of the proton-translocating adenosine triphosphatase complex of bovine heart mitochondria.

    PubMed

    Serrano, R; Kanner, B I; Racker, E

    1976-04-25

    1. The proton-translocating adenosine triphosphatase (ATPase) of bovine heart mitochondria was highly purified by extraction of submitochondrial particles with cholate, fractionation with ammonium sulfate, and sucrose gradient centrifugation in the presence of methanol, deoxycholate, and lysolecithin. 2. The preparation had a very low content of phospholipids, respiratory components, and adenine nucleotide transporter. The ATPase activity (14 o 16 micromoles/min/mg at 30 degrees) was dependent on addition of phospholipids. The purified enzyme was reconstituted with phospholipids, coupling factor 1 (F1), and the oligomycin sensitivity-conferring protein (OSCP) yielding vesicles with highly active 32Pi-ATP exchange (up to 260 nanomoles/min/mg at 30 degrees), and a proton pump driven by ATP. Site III oxidative phosphorylation was reconstituted when purified cytochrome oxidase was included. 3. The 32Pi-ATP exchange of the reconstituted vesicles was sensitive to both rutamycin and dichylohexylcarbodiimide but the ATPase activity was sensitive to rutamycin and not to dicyclohexylcarbodiimide. 4. In sodium dodecyl sulfate-acrylamide gel scans of the complex, the subunits of F1, OSCP, and three other major bands with apparent molecular weights of 32,000, 23,000, and about 11,000 were noted. Three other minor bands with estimated molecular weights of 80,000, 70,000, and 52,000 were also detected. These bands apparently represent residual trace amounts of respiratory components. Quantitative assays of individual respiratory components revealed between 0 and 3% contamination. 5. We conclude that the rutamycin-sensitive ATPase complex functions as a reversible ATP-driven proton pump. PMID:177416

  17. Pyrrolobenzoxazepinone derivatives as non-nucleoside HIV-1 RT inhibitors: further structure-activity relationship studies and identification of more potent broad-spectrum HIV-1 RT inhibitors with antiviral activity.

    PubMed

    Campiani, G; Morelli, E; Fabbrini, M; Nacci, V; Greco, G; Novellino, E; Ramunno, A; Maga, G; Spadari, S; Caliendo, G; Bergamini, A; Faggioli, E; Uccella, I; Bolacchi, F; Marini, S; Coletta, M; Nacca, A; Caccia, S

    1999-10-21

    Pyrrolobenzoxazepinone (PBO) derivatives represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTs) whose prototype is (+/-)-6-ethyl-6-phenylpyrrolo[2,1-d][1,5]benzoxazepin-7(6H)- one (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis and biological evaluation of novel derivatives and analogues of 6 featuring a meta-substituted phenyl or a 2-thienyl ring at C-6 and a pyridine system in place of the fused-benzene ring to yield pyrrolopyridooxazepinones (PPOs). Compared with the lead 6 and nevirapine, several of the synthesized compounds (PBOs 13a-d and PPOs 13i-k) displayed higher inhibitory activity against wild-type RT and clinically relevant mutant RTs containing the single amino acid substitutions L100I, K103N, V106A, Y181I, and Y188L. The most potent inhibitors were further evaluated for in vitro antiviral activity on lymphocytes and monocyte-macrophages, for cytotoxicity on a panel of cell lines, and for potential synergistic antiviral activity with AZT. Pharmacokinetic studies performed on 13b, 13c, and 13i showed that these compounds achieve high concentrations in the brain. The results of the biological and pharmacokinetic experiments suggest a potential clinical utility of analogues such as 13b-d, 13i, and 13j, in combination with nucleoside RT inhibitors, against strains of HIV-1 bearing those mutations that confer resistance to known NNRTI. PMID:10543890

  18. Functionalization of 2H-1,2,3-Triazole C-Nucleoside Template via N(2) Selective Arylation.

    PubMed

    Lopes, Alexandra Basilio; Wagner, Patrick; de Souza, Rodrigo Octavio Mendonça Alves; Germain, Nadège Lubin; Uziel, Jacques; Bourguignon, Jean-Jacques; Schmitt, Martine; Miranda, Leandro S M

    2016-06-01

    C-Nucleosides are an underexplored and important class of nucleosides with antiviral and anticancer activity. In addition, triazole heterocycles are well employed as a strategy to modify nucleobase in nucleoside analogues, although rare examples were described for triazoyl C-nucleosides. N(2)-Aryl-1,2,3-triazole C-nucleoside compounds that could be obtained by selective 1,2,3-triazole heterocycle N(2) arylation in 1-β-d-ribofuranosyl-2H-1,2,3-triazole substrate were designed in this study. The optimized condition used AdBrettPhos/[PdCl(allyl)]2 as the catalyst system. This transformation was accomplished by aryl halides bearing an electron donor and withdrawing groups, as well as by heterocyclic halides in good to excellent yields. The transformation developed in this study represents a significant contribution to the nucleoside field, once it allows for the synthesis of unexplored scaffolds through selective functionalization of triazole nucleosides. PMID:27166644

  19. Genome segment 4 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes RNA triphosphatase and methyltransferases.

    PubMed

    Biswas, Poulomi; Kundu, Anirban; Ghosh, Ananta Kumar

    2015-01-01

    Cloning and sequencing of Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) genome segment S4 showed that it consists of 3410 nt with a single ORF of 1110 aa which could encode a protein of ~127 kDa (p127). Bioinformatics analysis showed the presence of a 5' RNA triphosphatase (RTPase) domain (LRDR), a S-adenosyl-l-methionine (SAM)-binding (GxGxG) motif and the KDKE tetrad of 2'-O-methyltransferase (MTase), which suggested that S4 may encode RTPase and MTase. The ORF of S4 was expressed in Escherichia coli as a His-tagged fusion protein and purified by nickel-nitrilotriacetic acid affinity chromatography. Biochemical analysis of recombinant p127 showed its RTPase as well as SAM-dependent guanine N(7)-and ribose 2'-O-MTase activities. A MTase assay using in vitro transcribed AmCPV S2 RNA having a 5' G*pppG end showed that guanine N(7) methylation occurred prior to the ribose 2'-O methylation to yield a m(7)GpppG/m(7)GpppGm RNA cap. Mutagenesis of the SAM-binding (GxGxG) motif (G831A) completely abolished N(7)- and 2'-O-MTase activities, indicating the importance of these residues for capping. From the kinetic analysis, the Km values of N(7)-MTase for SAM and RNA were calculated as 4.41 and 0.39 µM, respectively. These results suggested that AmCPV S4-encoded p127 catalyses RTPase and two cap methylation reactions for capping the 5' end of viral RNA. PMID:25228490

  20. Nucleoside transporters in the disposition and targeting of nucleoside analogs in the kidney.

    PubMed

    Mangravite, Lara M; Badagnani, Ilaria; Giacomini, Kathleen M

    2003-10-31

    Systemic disposition of nucleosides and nucleoside analogs is dependent on renal handling of these compounds. There are five known, functionally characterized nucleoside transporters with varying substrate specificities for nucleosides: concentrative nucleoside transporters (CNT1-CNT3; Solute Carrier (SLC) 28A1-28A3), which mediate the intracellular flux of nucleosides, and equilibrative nucleoside transporters (ENT1-ENT2; SLC29A1-SLC29A2), which mediate bi-directional facilitated diffusion of nucleosides. All five of these transporters are expressed in the kidney. Concentrative nucleoside transporters primarily localize to the apical membrane of renal epithelial cells while equilibrative nucleoside transporters primarily localize to the basolateral membrane. These transporters work in concert to mediate reabsorptive flux of naturally occurring nucleosides and nucleoside analogs. In addition, equilibrative transporters also participate in secretory flux of some nucleoside analogs. Nucleoside transporters also serve in the targeting of nucleoside analog therapies to renal tumors. This review examines the role that these transporters play in renal disposition of nucleosides and nucleoside analogs in both systemic and kidney-specific therapies. PMID:14612157

  1. Synthesis and anti-DNA viral activities in vitro of certain 2,4-disubstituted-7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo[2,3-d d pyrimidine nucleosides.

    PubMed

    Bhattacharya, B K; Ojwang, J O; Rando, R F; Huffman, J H; Revankar, G R

    1995-09-29

    Several novel 2,4-disubstituted-7-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl)pyrrolo[2,3-d]pyrimidines have been synthesized and evaluated for their anti-human cytomegalovirus (HCMV), anti-hepatitis B virus (HBV), and anti-herpes simplex virus (HSV) activities in vitro. These nucleosides were prepared starting from 2-amino-4-chloro-7-(2-deoxy-2-fluoro- 3,5-di-O-benzoyl-beta-D-arabinofuranosyl)pyrrolo[2,3-d]pyrimidine (3), which in turn was synthesized by direct glycosylation of the sodium salt of 2-amino-4-chloropyrrolo[2,3-d]pyrimidine (1) with 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha-D-arabinofuranosyl bromide (2). Displacement of the 4-chloro group of 3 with OH, NH2, NHOH, SH, and SeH nucleophiles furnished the corresponding nucleosides 6-8, 12, and 14, respectively. The 3'-deoxygenation of 2-amino-4-chloro-7- (2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo[2,3-d]pyrimidine (4) and subsequent amination gave 2,4-diamino-2',3'-dideoxy derivative 19. Catalytic hydrogenation of 3 followed by debenzoylation afforded 2-aminopyrrolo[2,3-d]pyrimidine nucleoside 23. Among the compounds evaluated for their ability to inhibit the growth of HCMV (strain AD169) in MRC-5 cells using a plaque reduction assay, only 7 was significantly active in vitro with a 50% inhibitory concentration (IC50) of 3.7 micrograms/mL (TI > 125), whereas the IC50 value of ganciclovir (DHPG) was 3.2 micrograms/mL. Strain D16 of HCMV was more resistant to 7 (IC50 11 micrograms/mL) than the AD169 strain. When 7 was tested in combination with DHPG, the resultant anti-HCMV activity was found to be moderately synergistic with no evidence of antagonism. Nucleoside 7 also reduced episomal HBV replication in human hepatoblastoma 2.2.15 cells with an IC50 of 0.7 micrograms/mL (TI > 143). Development of cells harboring HBV which had become resistant to the drug was not observed with 7. Compound 7 also exhibited significant activity against herpes simplex virus types 1 and 2 (IC50 of 4.1 and 6.3 micrograms

  2. Lack of stereospecificity of some cellular and viral enzymes involved in the synthesis of deoxyribonucleotides and DNA: molecular basis for the antiviral activity of unnatural L-beta-nucleosides.

    PubMed

    Spadari, S; Maga, G; Verri, A; Bendiscioli, A; Tondelli, L; Capobianco, M; Colonna, F; Garbesi, A; Focher, F

    1995-01-01

    Among enzymes involved in the synthesis of nucleotides and DNA, some exceptions have recently been found to the universal rule that enzymes act only on one enantiomer of a chiral substrate and that only one of the enantiomeric forms of chiral molecules may bind effectively at the catalytic site, displaying biological activity. The exceptions include: herpes virus thymidine kinases, cellular deoxycytidine kinase and deoxynucloside mono- and diphosphate kinases, cellular and viral DNA polymerases, such as DNA polymerase alpha, terminal transferase and HIV-1 reverse transcriptase. The ability of these enzymes to utilize unnatural L-beta-nucleosides or -nucleotides as substrate may be exploited from chemotherapeutic point of view. PMID:8824765

  3. The pivotal role of uridine-cytidine kinases in pyrimidine metabolism and activation of cytotoxic nucleoside analogues in neuroblastoma.

    PubMed

    van Kuilenburg, André B P; Meinsma, Rutger

    2016-09-01

    Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine as well as the pharmacological activation of several cytotoxic pyrimidine ribonucleoside analogues. In this study, we investigated the functional role of two isoforms of UCK in neuroblastoma cell lines. Analysis of mRNA coding for UCK1 and UCK2 showed that UCK2 is the most abundantly expressed UCK in a panel of neuroblastoma cell lines. Transient and stable overexpression of UCK2 in neuroblastoma cells increased the metabolism of uridine and cytidine as well as the cytotoxicity of 3-deazauridine. Knockdown of endogenous UCK2 as well as overexpression of UCK1 resulted in decreased metabolism of uridine and cytidine and protected the neuroblastoma cells from 3-deazauridine-induced toxicity. Subcellular localization studies showed that UCK1-GFP and UCK2-GFP were localized in the cell nucleus and cytosol, respectively. However, co-expression of UCK1 with UCK2 resulted in a nuclear localization of UCK2 instead of its normal cytosolic localization, thereby impairing its normal function. The physical association of UCK1 and UCK2 was further demonstrated through pull-down analysis using his-tagged UCK. The discovery that UCK2 is highly expressed in neuroblastoma opens the possibility for selectively targeting neuroblastoma cells using UCK2-dependent pyrimidine analogues, while sparing normal tissues. PMID:27239701

  4. Synthesis of carbocyclic nucleoside analogs with five-membered heterocyclic nucleobases

    PubMed Central

    Cho, Jong hyun; Coats, Steven J.; Schinazi, Raymond F.

    2015-01-01

    New carbocyclic nucleoside analogs with five-membered heterocyclic nucleobases were synthesized and evaluated as potential anti-HIV and anti-HCV agents. Among the synthesized carbocyclic nucleoside analogs, the pyrazole amide 15f exhibited modest selective anti-HIV-1 activity (EC50 = 24 µM). PMID:26028788

  5. Novel inhibitors of Mycobacterium tuberculosis growth based on modified pyrimidine nucleosides and their analogues

    NASA Astrophysics Data System (ADS)

    Shmalenyuk, E. R.; Kochetkov, S. N.; Alexandrova, L. A.

    2013-09-01

    The review summarizes data on the synthesis and antituberculosis activity of pyrimidine nucleoside derivatives and their analogues. Enzymes from M. tuberculosis as promising targets for prototypes of new-generation drugs are considered. Nucleosides as inhibitors of drug-resistant M. tuberculosis strains are characterized. The bibliography includes 101 references.

  6. Second-line protease inhibitor-based highly active antiretroviral therapy after failing non-nucleoside reverse transcriptase inhibitors-based regimens in Asian HIV-infected children

    PubMed Central

    Bunupuradah, Torsak; Puthanakit, Thanyawee; Fahey, Paul; Kariminia, Azar; Yusoff, Nik Khairulddin Nik; Khanh, Truong Huu; Sohn, Annette H.; Chokephaibulkit, Kulkanya; Lumbiganon, Pagakrong; Hansudewechakul, Rawiwan; Razali, Kamarul; Kurniati, Nia; Huy, Bui Vu; Sudjaritruk, Tavitiya; Kumarasamy, Nagalingeswaran; Fong, Siew Moy; Saphonn, Vonthanak; Ananworanich, Jintanat

    2013-01-01

    Background The WHO recommends boosted protease inhibitor (bPI)-based highly active antiretroviral therapy (HAART) after failing non-nucleoside reverse transcriptase inhibitor (NNRTI) treatment. We examined outcomes of this regimen in Asian HIV-infected children. Methods Children from five Asian countries in the TREAT Asia Pediatric HIV Observational Database (TApHOD) with ≥24 weeks of NNRTI-based HAART followed by ≥24 weeks of bPI-based HAART were eligible. Primary outcomes were the proportions with virologic suppression (HIV-RNA <400 copies/ml) and immune recovery (CD4% ≥25% if age <5 years and CD4 count ≥500 cells/mm3 if age ≥5 years) at 48 and 96 weeks. Results Of 3422 children, 153 were eligible; 52% were female. At switch, median age was 10 years, 26% were in WHO stage 4. Median weight-for-age z-score (WAZ) was −1.9 (n=121), CD4% was 12.5% (n=106), CD4 count was 237 (n=112) cells/mm3, and HIV-RNA was 4.6 log10copies/ml (n=61). The most common PI was lopinavir/ritonavir (83%). At 48 weeks, 61% (79/129) had immune recovery, 60% (26/43) had undetectable HIV-RNA and 73% (58/79) had fasting triglycerides ≥130mg/dl. By 96 weeks, 70% (57/82) achieved immune recovery, 65% (17/26) virologic suppression, and hypertriglyceridemia occurred in 66% (33/50). Predictors for virologic suppression at week 48 were longer duration of NNRTI-based HAART (p=0.006), younger age (p=0.007), higher WAZ (p=0.020), and HIV-RNA at switch <10,000 copies/ml (p=0.049). Conclusion In this regional cohort of Asian children on bPI-based second-line HAART, 60% of children tested had immune recovery by one year, and two-thirds had hyperlipidemia, highlighting difficulties in optimizing second-line HAART with limited drug options. PMID:23296119

  7. Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3*

    PubMed Central

    Slugoski, Melissa D.; Smith, Kyla M.; Ng, Amy M. L.; Yao, Sylvia Y. M.; Karpinski, Edward; Cass, Carol E.; Baldwin, Stephen A.; Young, James D.

    2009-01-01

    Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na+ and H+ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na+/nucleoside and H+/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H+ dependence (all mutants), shift in Na+:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na+-specific hCNT1, uncoupled Na+ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine. PMID:19380587

  8. Nucleoside transporter expression and adenosine uptake in the rat cochlea.

    PubMed

    Khan, Abdul F; Thorne, Peter R; Muñoz, David J B; Wang, Carol J H; Housley, Gary D; Vlajkovic, Srdjan M

    2007-02-12

    Even though extracellular adenosine plays multiple roles in the cochlea, the mechanisms that control extracellular adenosine concentrations in this organ are unclear. This study investigated the expression of nucleoside transporters and adenosine uptake in the rat cochlea. Reverse transcription-polymerase chain reaction revealed the expression of mRNA transcripts for two equilibrative (ENT1 and ENT2) and two concentrative (CNT1 and CNT2) nucleoside transporters. Exogenous adenosine perfused through the cochlear perilymphatic compartment was taken up by cells lining the compartment. Adenosine uptake was sensitive to changes in extracellular Na concentrations and inhibited by nitrobenzylthioinosine (an adenosine uptake blocker). The study suggests that the bi-directional nucleoside transport supports the uptake and recycling of purines and regulates the activation of adenosine receptors by altering adenosine concentrations in cochlear fluid spaces. PMID:17314663

  9. Brain transfer RNA. II. Analysis of modified nucleosides

    SciTech Connect

    Chaudhary, K.D.; Carrier-Malhotra, L.; Murthy, M.R.

    1982-01-01

    Transfer RNAs were isolated from rat and calf brains and their nucleosides were analysed by tritium derivative technique. Qualitative changes in the minor nucleoside components were compared on the fluorograms which showed differences in the intensities of spots. Cerebellar and cortical tRNAs were also compared, but revealed no significant quantitative differences in their methylated constituants despite 60% higher methyltransferase activity observed in cerebellum compared to cerebral cortex. An overall similarity was noticed between the relative proportions of the major and minor nucleosides of tRNAs derived from rat or calf brain, expressed as mol%. Brain tRNA was also analysed by two-dimensional polyacrylamide gel electrophoresis which showed qualitative and quantitative changes during postnatal development.

  10. The concentrative nucleoside transporter family, SLC28.

    PubMed

    Gray, Jennifer H; Owen, Ryan P; Giacomini, Kathleen M

    2004-02-01

    The SLC28 family consists of three subtypes of sodium-dependent, concentrative nucleoside transporters, CNT1, CNT2, and CNT3 (SLC28A1, SLC28A2, and SLC28A3, respectively), that transport both naturally occurring nucleosides and synthetic nucleoside analogs used in the treatment of various diseases. These subtypes differ in their substrate specificities: CNT1 is pyrimidine-nucleoside preferring, CNT2 is purine-nucleoside preferring, and CNT3 transports both pyrimidine and purine nucleosides. Recent studies have identified key amino acid residues that are determinants of pyrimidine and purine specificity of CNT1 and CNT2. The tissue distributions of the CNTs vary: CNT1 is localized primarily in epithelia, whereas CNT2 and CNT3 have more generalized distributions. Nucleoside transporters in the SLC28 and SLC29 families play critical roles in nucleoside salvage pathways where they mediate the first step of nucleotide biosynthesis. In addition, these transporters work in concert to terminate adenosine signaling. SLC28 family members are crucial determinants of response to a variety of anticancer and antiviral nucleoside analogs, as they modulate the entry of these analogs into target tissues. Further, this family is involved in the absorption and disposition of many nucleoside analogs. Several CNT single nucleoside polymorphisms (SNPs) have been identified, but have yet to be characterized. PMID:12856181

  11. Visualizing nucleic acid metabolism using non-natural nucleosides and nucleotide analogs.

    PubMed

    Choi, Jung-Suk; Berdis, Anthony J

    2016-01-01

    Nucleosides and their corresponding mono-, di-, and triphosphates play important roles in maintaining cellular homeostasis. In addition, perturbations in this homeostasis can result in dysfunctional cellular processes that cause pathological conditions such as cancer and autoimmune diseases. This review article discusses contemporary research areas applying nucleoside analogs to probe the mechanistic details underlying the complexities of nucleoside metabolism at the molecular and cellular levels. The first area describes classic and contemporary approaches used to quantify the activity of nucleoside transporters, an important class of membrane proteins that mediate the influx and efflux of nucleosides and nucleobases. A focal point of this section is describing how biophotonic nucleosides are replacing conventional assays employing radiolabeled substrates to study the mechanism of these proteins. The second section describes approaches to understand the utilization of nucleoside triphosphates by cellular DNA polymerases during DNA synthesis. Emphasis here is placed on describing how novel nucleoside analogs such as 5-ethynyl-2'-deoxyuridine are being used to quantify DNA synthesis during normal replication as well as during the replication of damaged DNA. In both sections, seminal research articles relevant to these areas are described to highlight how these novel probes are improving our understanding of these biological processes. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. PMID:26004088

  12. Versatile synthesis and biological evaluation of novel 3’-fluorinated purine nucleosides

    PubMed Central

    Ren, Hang; Hatala, Paul J; Stevens, William C; He, Baicheng

    2015-01-01

    Summary A unified synthetic strategy accessing novel 3'-fluorinated purine nucleoside derivatives and their biological evaluation were achieved. Novel 3’-fluorinated analogues were constructed from a common 3’-deoxy-3’-fluororibofuranose intermediate. Employing Suzuki and Stille cross-coupling reactions, fifteen 3’-fluororibose purine nucleosides 1–15 and eight 3’-fluororibose 2-chloro/2-aminopurine nucleosides 16–23 with various substituents at position 6 of the purine ring were efficiently synthesized. Furthermore, 3’-fluorine analogs of natural products nebularine and 6-methylpurine riboside were constructed via our convergent synthetic strategy. Synthesized nucleosides were tested against HT116 (colon cancer) and 143B (osteosarcoma cancer) tumor cell lines. We have demonstrated 3’-fluorine purine nucleoside analogues display potent tumor cell growth inhibition activity at sub- or low micromolar concentration. PMID:26734098

  13. 2011 Homer Smith Award: To serve and protect: classic and novel roles for Na+, K+ -adenosine triphosphatase.

    PubMed

    Aperia, Anita

    2012-08-01

    The ability of cells to maintain sharp ion gradients across their membranes is the foundation for the molecular transport and electrical excitability. Across animal species and cell types, Na(+),K(+)-adenosine triphosphatase (ATPase) is arguably the most powerful contributor to this phenomenon. By producing a steep concentration difference of sodium and potassium between the intracellular and extracellular milieu, Na(+),K(+)-ATPase in the tubules provides the driving force for renal sodium reabsorption. Pump activity is downregulated by natriuretic hormones, such as dopamine, and is upregulated by antinatriuretic hormones, such as angiotensin. In the past decade, studies have revealed a novel and surprising role: that Na(+),K(+)-ATPase is a transducer of signals from extracellular to intracellular compartments. The signaling function of Na(+),K(+)-ATPase is activated by ouabain, a mammalian steroid hormone, at far lower concentrations than those that inhibit pump activity. By promoting growth and inhibiting apoptosis, activation of Na(+),K(+)-ATPase exerts tissue-protective effects. Ouabain-stimulated Na(+),K(+)-ATPase signaling has recently shown clinical promise by protecting the malnourished embryonic kidney from adverse developmental programming. A deeper understanding of the tissue-protective role of Na(+),K(+)-ATPase signaling and the regulation of Na(+),K(+)-ATPase pumping activity is of fundamental importance for the understanding and treatment of kidney diseases and kidney-related hypertension. PMID:22745476

  14. Arabidopsis thaliana nucleosidase mutants provide new insights into nucleoside degradation

    PubMed Central

    Riegler, Heike; Geserick, Claudia; Zrenner, Rita

    2011-01-01

    A central step in nucleoside and nucleobase salvage pathways is the hydrolysis of nucleosides to their respective nucleobases. In plants this is solely accomplished by nucleosidases (EC 3.2.2.x). To elucidate the importance of nucleosidases for nucleoside degradation, general metabolism, and plant growth, thorough phenotypic and biochemical analyses were performed using Arabidopsis thaliana T-DNA insertion mutants lacking expression of the previously identified genes annotated as uridine ribohydrolases (URH1 and URH2). Comprehensive functional analyses of single and double mutants demonstrated that both isoforms are unimportant for seedling establishment and plant growth, while one participates in uridine degradation. Rather unexpectedly, nucleoside and nucleotide profiling and nucleosidase activity screening of soluble crude extracts revealed a deficiency of xanthosine and inosine hydrolysis in the single mutants, with substantial accumulation of xanthosine in one of them. Mixing of the two mutant extracts, and by in vitro activity reconstitution using a mixture of recombinant URH1 and URH2 proteins, both restored activity, thus providing biochemical evidence that at least these two isoforms are needed for inosine and xanthosine hydrolysis. This mutant study demonstrates the utility of in vivo systems for the examination of metabolic activities, with the discovery of the new substrate xanthosine and elucidation of a mechanism for expanding the nucleosidase substrate spectrum. PMID:21599668

  15. Theophylline inhibition of renal and cerebral nucleoside formation.

    PubMed Central

    Jensen, M. H.

    1981-01-01

    1 Theophylline inhibits the enzymatic formation of purine nucleosides, among these adenosine (dephosphorylated adenosine 5'-monophosphate), in kidney and brain of the rat. 2 Some pharmacological effects of theophylline on regional blood flow and electrophysiological activity of the nervous system may be caused by inhibition of the endogenous formation of adenosine. PMID:7236996

  16. (Biological applications of nucleosides and nucleotides)

    SciTech Connect

    Srivastava, P.C.

    1990-08-20

    The traveler was invited to visit The Meditech Group, VTT Technology, Inc., Reactor Laboratory, VTT Technical Research Center of Finland (VTT), Otakaari, Espoo, Finland. The Meditech Group commands a 70 percent market share of Finland's radiopharmaceutical business and plans to expand its activities to other Scandinavian countries as well as in the Leningrad area of USSR. Meditech has plans to separate itself from Technical Research Center of Finland and its subsidiary VTT Technology, Inc., to become a private radiopharmaceutical company in the near future. As a private company, Meditech could expand its activities to encompass radiopharmaceutical research and development and may require foreign technical experts to support its research endeavors. The traveler also attended the Ninth International Round Table Conference on Nucleosides, Nucleotides, and Their Biological Applications held at the Biomedical Center, University of Uppsala, Uppsala, Sweden. The meeting focused on the chemistry and biology of RNA and DNA and their building blocks, nucleosides and nucleotides. The traveler also presented an invited paper entitled Design, Synthesis and Tumor Specificity of Azomycin Ribo- and Acyclonucleosides,'' describing his recent work at Oak Ridge National Laboratory.

  17. Synthesis of small interfering RNAs containing acetal-type nucleoside analogs at their 3'-ends and analysis of their silencing activity and their ability to bind to the Argonaute2 PAZ domain.

    PubMed

    Inada, Natsumi; Nakamoto, Kosuke; Yokogawa, Takashi; Ueno, Yoshihito

    2015-10-20

    In this study, we aimed to create small interfering RNAs (siRNAs) with increased silencing activities and nuclease resistance properties. Therefore, we designed and synthesized five types of siRNA containing acetal-type nucleoside analogs at their 3'-dangling ends. We found that the siRNA containing 1-O-(2,2,2-trifluoroethyl)-β-D-ribofuranose at the 3'-dangling end was the most potent among the synthesized siRNAs and showed more resistance to nucleolytic degradation by a 3' exonuclease than a natural RNA did. Thus, modification of siRNAs by addition of 1-O-(2,2,2-trifluoroethyl)-β-D-ribofuranose may hold promise as a means of improving the silencing activity and nuclease resistance of siRNAs. PMID:26397394

  18. ELECTRON MICROSCOPE OBSERVATIONS ON THE SURFACE ADENOSINE TRIPHOSPHATASE-LIKE ENZYMES OF HELA CELLS INFECTED WITH HERPES VIRUS.

    PubMed

    EPSTEIN, M A; HOLT, S J

    1963-11-01

    HeLa cells infected with herpes simplex virus have been examined in thin sections by electron microscopy after cytochemical staining for the presence of surface enzymes splitting adenosine triphosphate. As with uninfected HeLa cultures (18), the opaque enzyme reaction product was localized at the plasma membranes of about half the cells, tending to be present where there were microvilli and absent on smooth surfaces. Where mature extracellular herpes particles were found in association with cell membranes showing the enzyme activity, they were invariably likewise stained, and conversely, those mature particles which lay close against cells without reaction product at the surface were themselves free of it. Particles found budding into cytoplasmic vacuoles were also always without opaque deposit since this was never seen at vacuolar membranes, even in cells having the activity at the surface. The enzyme reaction product thus provided a marker indicating the manner in which the particles escape from cells and mature by budding out through cellular membranes, carrying, in the process, a portion of the latter on to themselves to form the outer viral limiting membrane. In some instances, virus particles were observed with more opaque material covering them than was present at the cell membrane with which they were associated. This finding has been taken as evidence for a physiological waxing and waning of surface enzyme activity of adenosine triphosphatase type. The fine structure of the mature extracellular virus as prepared here, using glutaraldehyde fixation, is also recorded. The observations and interpretations are discussed in full. PMID:14086760

  19. The search for nucleoside/nucleotide analog inhibitors of dengue virus.

    PubMed

    Chen, Yen-Liang; Yokokawa, Fumiaki; Shi, Pei-Yong

    2015-10-01

    Nucleoside analogs represent the largest class of antiviral agents and have been actively pursued for potential therapy of dengue virus (DENV) infection. Early success in the treatment of human immunodeficiency virus (HIV) infection and the recent approval of sofosbuvir for chronic hepatitis C have provided proof of concept for this class of compounds in clinics. Here we review (i) nucleoside analogs with known anti-DENV activity; (ii) challenges of the nucleoside antiviral approach for dengue; and (iii) potential strategies to overcome these challenges. This article forms part of a symposium in Antiviral Research on flavivirus drug discovery. PMID:26241002

  20. Synthesis and anti-HIV activity of some [Nucleoside Reverse Transcriptase Inhibitor]-C5'-linker-[Integrase Inhibitor] heterodimers as inhibitors of HIV replication.

    PubMed

    Sugeac, Elena; Fossey, Christine; Ladurée, Daniel; Schmidt, Sylvie; Laumond, Geraldine; Aubertin, Anne-Marie

    2004-12-01

    Selected for their expected ability to inhibit HIV replication, a series of eight heterodimers containing a Nucleoside Reverse Transcriptase Inhibitor (NRTI) and an Integrase Inhibitor (INI), bound by a linker, were designed and synthesized. For the NRTIs, d4U, d2U and d4T were chosen. For the INIs, 4-[1-(4-fluorobenzyl)-1H-pyrrol-2-yl]-2,4-dioxobutyric acid (6) and 4-(3,5-dibenzyloxyphenyl)-2,4-dioxobutyric acid (9) (belonging to the beta-diketo acids class) were chosen. The conjugation of the two different inhibitors (NRTI and INI) was performed using an amino acid (glycine or beta-alanine) as a cleavable linker. PMID:15662954

  1. Marine Nucleosides: Structure, Bioactivity, Synthesis and Biosynthesis

    PubMed Central

    Huang, Ri-Ming; Chen, Yin-Ning; Zeng, Ziyu; Gao, Cheng-Hai; Su, Xiangdong; Peng, Yan

    2014-01-01

    Nucleosides are glycosylamines that structurally form part of nucleotide molecules, the building block of DNA and RNA. Both nucleosides and nucleotides are vital components of all living cells and involved in several key biological processes. Some of these nucleosides have been obtained from a variety of marine resources. Because of the biological importance of these compounds, this review covers 68 marine originated nucleosides and their synthetic analogs published up to June 2014. The review will focus on the structures, bioactivities, synthesis and biosynthetic processes of these compounds. PMID:25474189

  2. Synthesis of cycloalkyl substituted purine nucleosides via a metal-free radical route.

    PubMed

    Wang, Dong-Chao; Xia, Ran; Xie, Ming-Sheng; Qu, Gui-Rong; Guo, Hai-Ming

    2016-05-01

    An efficient route to synthesize cycloalkyl substituted purine nucleosides was developed. This metal-free C-H activation was accomplished by a tBuOOtBu initiated radical reaction. By adjusting the amount of tBuOOtBu and reaction time, the selective synthesis of C6-monocycloalkyl or C6,C8-dicycloalkyl substituted purine nucleosides could be realized. Furthermore, uracil and related nucleosides were also suitable substrates, giving the C5-cyclohexyl substituted uracil derivatives in good yields with excellent regioselectivities. PMID:27101306

  3. Protein sequence comparisons show that the 'pseudoproteases' encoded by poxviruses and certain retroviruses belong to the deoxyuridine triphosphatase family.

    PubMed Central

    McGeoch, D J

    1990-01-01

    Amino acid sequence comparisons show extensive similarities among the deoxyuridine triphosphatases (dUTPases) of Escherichia coli and of herpesviruses, and the 'protease-like' or 'pseudoprotease' sequences encoded by certain retroviruses in the oncovirus and lentivirus families and by poxviruses. These relationships suggest strongly that the 'pseudoproteases' actually are dUTPases, and have not arisen by duplication of an oncovirus protease gene as had been suggested. The herpesvirus dUTPase sequences differ from the others in that they are longer (about 370 residues, against around 140) and one conserved element ('Motif 3') is displaced relative to its position in the other sequences; a model involving internal duplication of the herpesvirus gene can account effectively for these observations. Sequences closely similar to Motif 3 are also found in phosphofructokinases, where they form part of the active site and fructose phosphate binding structure; thus these sequences may represent a class of structural element generally involved in phosphate transfer to and from glycosides. PMID:2165588

  4. Characterization of a Trifunctional Mimivirus mRNA Capping Enzyme and Crystal Structure of the RNA Triphosphatase Domain

    SciTech Connect

    Benarroch,D.; Smith, P.; Shuman, S.

    2008-01-01

    The RNA triphosphatase (RTPase) components of the mRNA capping apparatus are a bellwether of eukaryal taxonomy. Fungal and protozoal RTPases belong to the triphosphate tunnel metalloenzyme (TTM) family, exemplified by yeast Cet1. Several large DNA viruses encode metal-dependent RTPases unrelated to the cysteinyl-phosphatase RTPases of their metazoan host organisms. The origins of DNA virus RTPases are unclear because they are structurally uncharacterized. Mimivirus, a giant virus of amoeba, resembles poxviruses in having a trifunctional capping enzyme composed of a metal-dependent RTPase module fused to guanylyltransferase (GTase) and guanine-N7 methyltransferase domains. The crystal structure of mimivirus RTPase reveals a minimized tunnel fold and an active site strikingly similar to that of Cet1. Unlike homodimeric fungal RTPases, mimivirus RTPase is a monomer. The mimivirus TTM-type RTPase-GTase fusion resembles the capping enzymes of amoebae, providing evidence that the ancestral large DNA virus acquired its capping enzyme from a unicellular host.

  5. Adenosine Triphosphatase from Soybean Callus and Root Cells

    PubMed Central

    Hendrix, Donald L.; Kennedy, Ralph M.

    1977-01-01

    The ATPase activity of a membrane fraction from soybean (Glycine max L.) root and callus cells, presumed to be enriched in plasma membrane, has been characterized with respect to ion stimulation, pH requirement, and nucleotide specificity. The enzyme from both sources was activated by divalent cations (Mg2+ > Mn2+ > Zn2+ > Ca2+ > Sr2+) and further stimulated by monovalent salts. Preparations from root cells were stimulated by monovalent ions according to the sequence: K+ > Rb+ > Choline+ > Na+ > Li+ > NH4+ > Cs+ > tris+. Membrane preparations from callus cells showed similar stimulatory patterns except for a slight preference for Na+ over K+. No synergism between K+ and Na+ was found with preparations from either cell source. The pH optimum for ATP hydrolysis in the presence of 50 mm KCl and 3 mm MgSO4 was 6.5 for both preparations and slightly higher in the presence of 3 mm MgSO4 alone. The order of nucleotide preference was found to be: ATP ≫ ADP > GTP > CTP > UTP. Maximal glucan synthetase activity at high (1 mm), but not at low (1 μm), substrate was found to be coincident with the position of this fraction on the sucrose gradient. PMID:16659830

  6. Phospholipid association with the bovine cardiac mitochondrial adenosine triphosphatase.

    PubMed Central

    Brown, R E; Montgomery, R I; Spach, P I; Cunningham, C C

    1985-01-01

    The association of different phospholipids with a lipid-depleted oligomycin-sensitive ATPase from bovine cardiac mitochondria [Serrano, Kanner & Racker (1976) J. Biol. Chem. 251, 2453-2461] has been examined using three approaches. First, reconstitution of the ATPase with different synthetic diacyl phospholipids resulted in a 2-10-fold stimulation of ATPase specific activity depending upon the particular phospholipid employed. The phospholipid headgroup region displayed the following order of ATPase reactivation potential: dioleoylphosphatidylglycerol greater than dioleoylphosphatidic acid greater than dioleoylphosphatidylcholine. Furthermore, the ATPase showed higher levels of specific activity when reconstituted with dioleoyl phospholipid derivatives compared with dimyristoyl derivatives. Second, examination of the phospholipid remaining associated with the lipid-depleted ATPase upon purification showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were present. No relative enrichment of any of these phospholipids (compared with their distribution in submitochondrial particles) was noted. Therefore, no preferential association between the ATPase and any one phospholipid could be found in the mitochondrial ATPase. Third, the sodium cholate-mediated phospholipid exchange procedure was employed for studying the phospholipid requirements of the ATPase. Replacement of about 50% of the mitochondrial phospholipid remaining with the lipid-depleted ATPase could be achieved utilizing either synthetic phosphatidic acid or phosphatidylcholine. Examination of the displaced mitochondrial phospholipid showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were replaced with equal facility. Images Fig. 3. PMID:3156584

  7. A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation

    PubMed Central

    Motomura, Kei; Hirota, Ryuichi; Okada, Mai; Ikeda, Takeshi; Ishida, Takenori

    2014-01-01

    Inorganic polyphosphate (polyP) is a linear polymer of tens to hundreds of phosphate (Pi) residues linked by “high-energy” phosphoanhydride bonds as in ATP. PolyP kinases, responsible for the synthesis and utilization of polyP, are divided into two families (PPK1 and PPK2) due to differences in amino acid sequence and kinetic properties. PPK2 catalyzes preferentially polyP-driven nucleotide phosphorylation (utilization of polyP), which is important for the survival of microbial cells under conditions of stress or pathogenesis. Phylogenetic analysis suggested that the PPK2 family could be divided into three subfamilies (classes I, II, and III). Class I and II PPK2s catalyze nucleoside diphosphate and nucleoside monophosphate phosphorylation, respectively. Here, we demonstrated that class III PPK2 catalyzes both nucleoside monophosphate and nucleoside diphosphate phosphorylation, thereby enabling us to synthesize ATP from AMP by a single enzyme. Moreover, class III PPK2 showed broad substrate specificity over purine and pyrimidine bases. This is the first demonstration that class III PPK2 possesses both class I and II activities. PMID:24532069

  8. Modified nucleosides as biomarkers for early cancer diagnose in exposed populations.

    PubMed

    Seidel, Annerose; Seidel, Peter; Manuwald, Olaf; Herbarth, Olf

    2015-07-01

    There is increasing worldwide interest in developing of markers for tumor diagnosis and identification of individuals who are at high cancer risk. Cancer, like other diseases accompanied by metabolic disorders, causes characteristic effects on cell turnover rate, activity of modifying enzymes, and RNA/DNA modifications. This results in an increased excretion of modified nucleosides in cancer patients. Therefore, for many years modified nucleosides have been suggested as tumor markers. The aim of the study was to elucidate further the usefulness of urinary nucleosides as possible markers at early detection of cancer in persons which are exposed against tumor promoting influences during their working life. Uranium miners are exposed to many kinds of pollutants that can cause health damage even lead to carcinogenesis. We analyzed modified nucleosides in urine samples from 92 miners who are at high risk for lung cancer to assess the levels of nucleosides by a multilayer perceptron (MLP) classifier - a neural network model. Eighteen nucleosides/metabolites were detected with reversed-phase high-pressure liquid chromatography (RP-HPLC). A valid set of urinary metabolites were selected and multivariate statistical technique of multilayer perceptron neural network were applied. In a previous study, MLP shows a sensitivity and specificity of 97 and 85%, respectively. MLP classification including the most relevant markers/nucleosides clearly demonstrates the elevation of RNA metabolism in miners, which is associated with possible malignant disease. We found that there were 30 subjects with early health disorders among 92 uranium workers based on MLP technique using modified nucleosides. The combination of RP-HPLC analysis of modified nucleosides and subsequent MLP analyses represents a promising tool for the development of a non-invasive prediction system and may assist in developing management and surveillance procedures. PMID:24615900

  9. Lipophilic prodrugs of nucleoside triphosphates as biochemical probes and potential antivirals

    PubMed Central

    Gollnest, Tristan; de Oliveira, Thiago Dinis; Schols, Dominique; Balzarini, Jan; Meier, Chris

    2015-01-01

    The antiviral activity of nucleoside reverse transcriptase inhibitors is often limited by ineffective phosphorylation. We report on a nucleoside triphosphate (NTP) prodrug approach in which the γ-phosphate of NTPs is bioreversibly modified. A series of TriPPPro-compounds bearing two lipophilic masking units at the γ-phosphate and d4T as a nucleoside analogue are synthesized. Successful delivery of d4TTP is demonstrated in human CD4+ T-lymphocyte cell extracts by an enzyme-triggered mechanism with high selectivity. In antiviral assays, the compounds are potent inhibitors of HIV-1 and HIV-2 in CD4+ T-cell (CEM) cultures. Highly lipophilic acyl residues lead to higher membrane permeability that results in intracellular delivery of phosphorylated metabolites in thymidine kinase-deficient CEM/TK− cells with higher antiviral activity than the parent nucleoside. PMID:26503889

  10. Complex regulation of nucleoside transporter expression in epithelial and immune system cells.

    PubMed

    Pastor-Anglada, M; Casado, F J; Valdés, R; Mata, J; García-Manteiga, J; Molina, M

    2001-01-01

    Nucleoside transporters have a variety of functions in the cell, such as the provision of substrates for nucleic acid synthesis and the modulation of purine receptors by determining agonist availability. They also transport a wide range of nucleoside-derived antiviral and anticancer drugs. Most mammalian cells co-express several nucleoside transporter isoforms at the plasma membrane, which are differentially regulated. This paper reviews studies on nucleoside transporter regulation, which has been extensively characterized in the laboratory in several model systems: the hepatocyte, an epithelial cell type, and immune system cells, in particular B cells, which are non-polarized and highly specialized. The hepatocyte co-expresses at least two Na+-dependent nucleoside transporters, CNT1 and CNT2, which are up-regulated during cell proliferation but may undergo selective loss in certain experimental models of hepatocarcinomas. This feature is consistent with evidence that CNT expression also depends on the differentiation status of the hepatocyte. Moreover, substrate availability also modulates CNT expression in epithelial cells, as reported for hepatocytes and jejunum epithelia from rats fed nucleotide-deprived diets. In human B cell lines, CNT and ENT transporters are co-expressed but differentially regulated after B cell activation triggered by cytokines or phorbol esters, as described for murine bone marrow macrophages induced either to activate or to proliferate. The complex regulation of the expression and activity of nucleoside transporters hints at their relevance in cell physiology. PMID:11396615

  11. KP-1212/1461, a nucleoside designed for the treatment of HIV by viral mutagenesis.

    PubMed

    Harris, Kevin S; Brabant, William; Styrchak, Sheila; Gall, Alexander; Daifuku, Richard

    2005-07-01

    We report the activities of a novel nucleoside analog against HIV. This nucleoside (KP-1212) is not a chain terminator but exerts its antiviral effects via mutagenesis of the viral genome. Serial passaging of HIV in the presence of KP-1212 causes an increase in the mutation rate of the virus leading to viral ablation. HIV strains resistant to KP-1212 have not yet been isolated. Quite to the contrary, virus treated with KP-1212 exhibited an increased sensitivity not only to KP-1212 but also to another nucleoside reverse transcriptase inhibitor (NRTI), zidovudine. HIV strains resistant to other NRTIs (e.g. zidovudine, lamivudine, stavudine, abacavir, etc.) exhibited no cross-resistance towards KP-1212. Multiple assays confirmed that KP-1212 has a favorable (low) genotoxicity profile when compared to some approved antiviral nucleosides. In addition, KP-1212 is not toxic to mitochondria nor does it exhibit any inhibitory effects on mitochondrial DNA synthesis. PMID:15890415

  12. A method for determining the adenosine triphosphatase content of energy-transducing membranes. reaction of 4-chloro-7-nitrobenzofurazan with the adenosine triphosphatase of bovine heart submitochondrial particles.

    PubMed Central

    Ferguson, S J; Lloyd, W J; Radda, G K

    1976-01-01

    1. Modification of a single amino acid residue by introduction of the nitrobenzofurazan group inactivates mitochondrial ATPase (adenosine triphosphatase) when membrane-bound in submitochondrial particles. The similarity between the reactions of both membrane-bound and isolated ATPase with 4-chloro-7-nitrobenzofurazan indicates that the single essential tryosine residue identified in the isolated enzyme [Ferguson, Loyd, Lyons & Radda (1975) Eur. J. Biochem. 54, 117-126] Is also a feature of the membrane-bound ATPase. 2. A procedure is presented for estimating the ATPase content of the inner mitochondrial membrane. It is based on the specificity of the incorporation of the nitrobenzofurazan group, and the ready removal of this group by compounds that contain a thiol group. This method indicates that 8.5% of the membrane protein is ATPase. The procedure should be applicable to the titration of the energy-transducing ATPases of bacterial plasma membranes and of the thylakoid membranes of chloroplasts. 3. Combination of the data obtained on the ATPase content of the bovine heart inner mitochondrial membrane with a titration of the cytochrome bc1 complex with antimycin indicates that these two components of the membrane are present in approximately equal amounts. PMID:136962

  13. Aberrant Apoptotic Response of Colorectal Cancer Cells to Novel Nucleoside Analogues

    PubMed Central

    Harmse, Leonie; Dahan-Farkas, Nurit; Panayides, Jenny-Lee; van Otterlo, Willem; Penny, Clement

    2015-01-01

    Despite the increased understanding of colorectal cancer and the introduction of targeted drug therapy, the metastatic phase of the disease remains refractory to treatment. Since the deregulation of normal apoptosis contributes to the pathogenesis of colorectal cancer, novel nucleoside analogues were synthesized here and evaluated for their ability to induce apoptosis and cause cell death in two colorectal adeno-carcinoma cell lines, Caco-2 and HT-29. Three novel nucleoside analogues assessed here showed cytotoxic activity, as measured by the MTT assay against both cell lines: the IC50 values ranged between 3 and 37 μM, with Caco-2 cells being more sensitive than HT-29 cells. Compared to camptothecin, the positive control, the nucleoside analogues were significantly less toxic to normal unstimulated leukocytes (p>0.05). Moreover, the nucleosides were able to induce apoptosis as measured by an increase in caspase 8 and caspase 3 activity above that of the control. This was additionally supported by data derived from Annexin V-FITC assays. Despite marginal changes to the mitochondrial membrane potential, all three nucleosides caused a significant increase in cytosolic cytochrome c (p>0.05), with a corresponding decrease in mitochondrial cytochrome c. Morphological analysis of both cell lines showed the rapid appearance of vacuoles following exposure to two of the nucleosides, while a third caused cellular detachment, delayed cytoplasmic vacuolisation and nuclear abnormalities. Preliminary investigations, using the autophagic indicator monodansylcadaverine and chloroquine as positive control, showed that two of the nucleosides induced the formation of autophagic vacuoles. In summary, the novel nucleoside analogues showed selective cytotoxicity towards both cancer cell lines and are effective initiators of an unusual apoptotic response, demonstrating their potential to serve as structural scaffolds for more potent analogues. PMID:26390405

  14. Mast cell degranulation is negatively regulated by the Munc13-4-binding small-guanosine triphosphatase Rab37

    PubMed Central

    Higashio, Hironori; Satoh, Yoh-ichi; Saino, Tomoyuki

    2016-01-01

    Mast cell degranulation is regulated by the small guanosine triphosphatases (GTPases) Rab27a and Rab27b, which have distinct and opposing roles: Rab27b acts as a positive regulator through its effector protein Munc13-4, a non-neuronal isoform of the vesicle-priming Munc13 family of proteins, whereas Rab27a acts as a negative regulator through its effector protein melanophilin, by maintaining integrity of cortical filamentous actin (F-actin), a barrier to degranulation. Here we investigated the role of Rab37, one of the Rab GTPases assumed to be implicated in regulated secretion during mast cell degranulation. Using the RBL-2H3 mast cell line, we detected Rab37 on the secretory granules and found that antigen-induced degranulation was extensively increased by either knockdown of Rab37 or overexpression of a dominant-active Rab37 mutant. This hypersecretion phenotype in the Rab37-knockdown cells was suppressed by simultaneous knockdown of Rab27a and Rab27b or of Munc13-4, but not by disruption of cortical F-actin. We further found that Rab37 interacted with Munc13-4 in a GTP-independent manner and formed a Rab27-Munc13-4-Rab37 complex. These results suggest that Rab37 is a Munc13-4-binding protein that inhibits mast cell degranulation through its effector protein, by counteracting the vesicle-priming activity of the Rab27-Munc13-4 system. PMID:26931073

  15. Broad-spectrum non-nucleoside inhibitors of human herpesviruses

    PubMed Central

    McClain, Lora; Zhi, Yun; Cheng, Hoyee; Ghosh, Ayantika; Piazza, Paolo; Yee, Michael B.; Kumar, Santosh; Milosevic, Jadranka; Bloom, David C.; Arav-Boger, Ravit; Kinchington, Paul R.; Yolken, Robert; Nimgaonkar, Vishwajit; D’Aiuto, Leonardo

    2015-01-01

    Herpesvirus infections cause considerable morbidity and mortality through lifelong recurrent cycles of lytic and latent infection in several tissues, including the human nervous system. Acyclovir (ACV) and its prodrug, the current antivirals of choice for herpes simplex virus (HSV) and, to some extent, varicella zoster virus (VZV) infections are nucleoside analogues that inhibit viral DNA replication. Rising viral resistance and the need for more effective second-line drugs have motivated searches for additional antiviral agents, particularly non-nucleoside based agents. We evaluated the antiviral activity of five compounds with predicted lysosomotropic activity using conventional and human induced pluripotent stem cell-derived neuronal (iPSC-neurons) cultures. Their potency and toxicity were compared with ACV and the lysosomotropic agents chloroquine and bafilomycin A1. Out of five compounds tested, micromolar concentrations of 30N12, 16F19, and 4F17 showed antiviral activity comparable to ACV (50μM) during lytic herpes simplex virus type 1 (HSV-1) infections, reduced viral DNA copy number, and reduced selected HSV-1 protein levels. These compounds also inhibited the reactivation of ‘quiescent’ HSV-1 infection established in iPSC-neurons, but did not inhibit viral entry into host cells. The same compounds had greater potency than ACV against lytic VZV infection; they also inhibited replication of human cytomegalovirus. The anti-herpetic effects of these non-nucleoside agents merit further evaluation in vivo. PMID:26079681

  16. Broad-spectrum non-nucleoside inhibitors of human herpesviruses.

    PubMed

    McClain, Lora; Zhi, Yun; Cheng, Hoyee; Ghosh, Ayantika; Piazza, Paolo; Yee, Michael B; Kumar, Santosh; Milosevic, Jadranka; Bloom, David C; Arav-Boger, Ravit; Kinchington, Paul R; Yolken, Robert; Nimgaonkar, Vishwajit; D'Aiuto, Leonardo

    2015-09-01

    Herpesvirus infections cause considerable morbidity and mortality through lifelong recurrent cycles of lytic and latent infection in several tissues, including the human nervous system. Acyclovir (ACV) and its prodrug, the current antivirals of choice for herpes simplex virus (HSV) and, to some extent, varicella zoster virus (VZV) infections are nucleoside analogues that inhibit viral DNA replication. Rising viral resistance and the need for more effective second-line drugs have motivated searches for additional antiviral agents, particularly non-nucleoside based agents. We evaluated the antiviral activity of five compounds with predicted lysosomotropic activity using conventional and human induced pluripotent stem cell-derived neuronal (iPSC-neurons) cultures. Their potency and toxicity were compared with ACV and the lysosomotropic agents chloroquine and bafilomycin A1. Out of five compounds tested, micromolar concentrations of 30N12, 16F19, and 4F17 showed antiviral activity comparable to ACV (50μM) during lytic herpes simplex virus type 1 (HSV-1) infections, reduced viral DNA copy number, and reduced selected HSV-1 protein levels. These compounds also inhibited the reactivation of 'quiescent' HSV-1 infection established in iPSC-neurons, but did not inhibit viral entry into host cells. The same compounds had greater potency than ACV against lytic VZV infection; they also inhibited replication of human cytomegalovirus. The anti-herpetic effects of these non-nucleoside agents merit further evaluation in vivo. PMID:26079681

  17. Purine nucleoside phosphorylase as a cytosolic arsenate reductase.

    PubMed

    Gregus, Zoltán; Németi, Balázs

    2002-11-01

    The findings of the accompanying paper (Németi and Gregus, Toxicol: Sci. 70, 4-12) indicate that the arsenate (AsV) reductase activity of rat liver cytosol is due to an SH enzyme that uses phosphate (or its analogue, arsenate, AsV) and a purine nucleoside (guanosine or inosine) as substrates. Purine nucleoside phosphorylase (PNP) is such an enzyme. It catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides according to the following scheme: guanosine (or inosine) + phosphate <--> guanine (or hypoxanthine) + ribose-1-phosphate. Therefore, we have tested the hypothesis that PNP is responsible for the thiol- and purine nucleoside-dependent reduction of AsV to AsIII by rat liver cytosol. AsIII formed from AsV was quantified by HPLC-hydride generation-atomic fluorescence spectrometry analysis of the deproteinized incubates. The following findings support the conclusion that PNP reduces AsV to AsIII, using AsV instead of phosphate in the reaction above: (1) Specific PNP inhibitors (CI-1000, BCX-1777) at a concentration of 1 microM completely inhibited cytosolic AsV reductase activity. (2) During anion-exchange chromatography of cytosolic proteins, PNP activity perfectly coeluted with the AsV reductase activity, suggesting that both activities belong to the same protein. (3) PNP purified from calf spleen catalyzed reduction of AsV to AsIII in the presence of dithiothreitol (DTT) and a 6-oxopurine nucleoside (guanosine or inosine). (4) AsV reductase activity of purified PNP, like the cytosolic AsV reductase activity, was inhibited by phosphate (a substrate of PNP alternative to AsV), guanine and hypoxanthine (products of PNP favoring the reverse reaction), mercurial thiol reagents (nonspecific inhibitors of PNP), as well as CI-1000 and BCX-1777 (specific PNP inhibitors). Thus, PNP appears to be responsible for the AsV reductase activity of rat liver cytosol in the presence of DTT. Further research should clarify the mechanism and the in vivo significance of PNP

  18. Modified Nucleoside Triphosphates for In-vitro Selection Techniques

    PubMed Central

    Dellafiore, María A.; Montserrat, Javier M.; Iribarren, Adolfo M.

    2016-01-01

    The development of SELEX (Selective Enhancement of Ligands by Exponential Enrichment) provides a powerful tool for the search of functional oligonucleotides with the ability to bind ligands with high affinity and selectivity (aptamers) and for the discovery of nucleic acid sequences with diverse enzymatic activities (ribozymes and DNAzymes). This technique has been extensively applied to the selection of natural DNA or RNA molecules but, in order to improve chemical and structural diversity as well as for particular applications where further chemical or biological stability is necessary, the extension of this strategy to modified oligonucleotides is desirable. Taking into account these needs, this review intends to collect the research carried out during the past years, focusing mainly on the use of modified nucleotides in SELEX and the development of mutant enzymes for broadening nucleoside triphosphates acceptance. In addition, comments regarding the synthesis of modified nucleoside triphosphate will be briefly discussed. PMID:27200340

  19. A Novel and Fast Purification Method for Nucleoside Transporters.

    PubMed

    Hao, Zhenyu; Thomsen, Maren; Postis, Vincent L G; Lesiuk, Amelia; Sharples, David; Wang, Yingying; Bartlam, Mark; Goldman, Adrian

    2016-01-01

    Nucleoside transporters (NTs) play critical biological roles in humans, and to understand the molecular mechanism of nucleoside transport requires high-resolution structural information. However, the main bottleneck for structural analysis of NTs is the production of pure, stable, and high quality native protein for crystallization trials. Here we report a novel membrane protein expression and purification strategy, including construction of a high-yield membrane protein expression vector, and a new and fast purification protocol for NTs. The advantages of this strategy are the improved time efficiency, leading to high quality, active, stable membrane proteins, and the efficient use of reagents and consumables. Our strategy might serve as a useful point of reference for investigating NTs and other membrane proteins by clarifying the technical points of vector construction and improvements of membrane protein expression and purification. PMID:27376071

  20. A Novel and Fast Purification Method for Nucleoside Transporters

    PubMed Central

    Hao, Zhenyu; Thomsen, Maren; Postis, Vincent L. G.; Lesiuk, Amelia; Sharples, David; Wang, Yingying; Bartlam, Mark; Goldman, Adrian

    2016-01-01

    Nucleoside transporters (NTs) play critical biological roles in humans, and to understand the molecular mechanism of nucleoside transport requires high-resolution structural information. However, the main bottleneck for structural analysis of NTs is the production of pure, stable, and high quality native protein for crystallization trials. Here we report a novel membrane protein expression and purification strategy, including construction of a high-yield membrane protein expression vector, and a new and fast purification protocol for NTs. The advantages of this strategy are the improved time efficiency, leading to high quality, active, stable membrane proteins, and the efficient use of reagents and consumables. Our strategy might serve as a useful point of reference for investigating NTs and other membrane proteins by clarifying the technical points of vector construction and improvements of membrane protein expression and purification. PMID:27376071

  1. Synthetic strategies toward carbocyclic purine-pyrimidine hybrid nucleosides.

    PubMed

    Sadler, Joshua M; Mosley, Sylvester L; Dorgan, Kathleen M; Zhou, Zhaohui Sunny; Seley-Radtke, Katherine L

    2009-08-01

    The blending of key structural features from the purine and pyrimidine nucleobase scaffolds gives rise to a new class of hybrid nucleosides. The purine-pyrimidine hybrid nucleosides can be viewed as either N-3 ribosylated purines or 5,6-disubstituted pyrimidines, thus recognition by both purine- and pyrimidine-metabolizing enzymes is possible. Given the increasing reports of the development of resistance in many enzymatic systems, a drug that could be recognized by more than one enzyme could prove highly advantageous in overcoming resistance mechanisms related to binding site mutations. In that regard, the design, synthesis and results of preliminary biological activity for a series of carbocyclic uracil derivatives with either a fused imidazole or thiazole ring are presented herein. PMID:19592260

  2. Synthetic Strategies Toward Carbocyclic Purine-Pyrimidine Hybrid Nucleosides

    PubMed Central

    Sadler, Joshua M.; Mosley, Sylvester L.; Dorgan, Kathleen M.; Zhou, Zhaohui Sunny; Seley-Radtke, Katherine L.

    2009-01-01

    The blending of key structural features from the purine and pyrimidine nucleobase scaffolds gives rise to a new class of hybrid nucleosides. The purine-pyrimidine hybrid nucleosides can be viewed as either N-3 ribosylated purines or 5,6-disubstituted pyrimidines, thus recognition by both purine- and pyrimidine-metabolizing enzymes is possible. Given the increasing reports of the development of resistance in many enzymatic systems, a drug that could be recognized by more than one enzyme could prove highly advantageous in overcoming resistance mechanisms related to binding site mutations. In that regard, the design, synthesis and results of preliminary biological activity for a series of carbocyclic uracil derivatives with either a fused imidazole or thiazole ring are presented herein. PMID:19592260

  3. Modified Nucleoside Triphosphates for In-vitro Selection Techniques.

    PubMed

    Dellafiore, María A; Montserrat, Javier M; Iribarren, Adolfo M

    2016-01-01

    The development of SELEX (Selective Enhancement of Ligands by Exponential Enrichment) provides a powerful tool for the search of functional oligonucleotides with the ability to bind ligands with high affinity and selectivity (aptamers) and for the discovery of nucleic acid sequences with diverse enzymatic activities (ribozymes and DNAzymes). This technique has been extensively applied to the selection of natural DNA or RNA molecules but, in order to improve chemical and structural diversity as well as for particular applications where further chemical or biological stability is necessary, the extension of this strategy to modified oligonucleotides is desirable. Taking into account these needs, this review intends to collect the research carried out during the past years, focusing mainly on the use of modified nucleotides in SELEX and the development of mutant enzymes for broadening nucleoside triphosphates acceptance. In addition, comments regarding the synthesis of modified nucleoside triphosphate will be briefly discussed. PMID:27200340

  4. Overcoming nucleoside analog chemoresistance of pancreatic cancer: A therapeutic challenge

    PubMed Central

    Hung, Sau Wai; Mody, Hardik R.; Govindarajan, Rajgopal

    2013-01-01

    Clinical refractoriness to nucleoside analogs (e.g., gemcitabine, capecitabine) is a major scientific problem and is one of the main reasons underlying the extremely poor prognostic state of pancreatic cancer. The drugs’ effects are suboptimal partly due to cellular mechanisms limiting their transport, activation, and overall efficacy. Nonetheless, novel therapeutic approaches are presently under study to circumvent nucleoside analog resistance in pancreatic cancer. With these new approaches come additional challenges to be addressed. This review describes the determinants of chemoresistance in the gemcitabine cytotoxicity pathways, provides an overview of investigational approaches for overcoming chemoresistance, and discusses new challenges presented. Understanding the future directions of the field may assist in the successful development of novel treatment strategies for enhancing chemotherapeutic efficacy in pancreatic cancer. PMID:22425961

  5. Modified Nucleoside Triphosphates for in-vitro Selection Techniques

    NASA Astrophysics Data System (ADS)

    Iribarren, Adolfo; Dellafiore, María; Montserrat, Javier

    2016-05-01

    The development of SELEX (Selective Enhancement of Ligands by Exponential Enrichment) provides a powerful tool for the search of functional oligonucleotides with the ability to bind ligands with high affinity and selectivity (aptamers) and for the discovery of nucleic acid sequences with diverse enzymatic activities (ribozymes and DNAzymes). This technique has been extensively applied to the selection of natural DNA or RNA molecules but, in order to improve chemical and structural diversity as well as for particular applications where further chemical or biological stability is necessary, the extension of this strategy to modified oligonucleotides is desirable. Taking into account these needs, this review intends to collect the research carried out during the past years, focusing mainly on the use of modified nucleotides in SELEX and the development of mutant enzymes for broadening nucleoside triphosphates acceptance. In addition, comments regarding the synthesis of modified nucleoside triphosphate will be briefly discussed.

  6. The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.

    PubMed Central

    Zammit, V A; Newsholme, E A

    1976-01-01

    suggested that, under anaerobic conditions, muscles of marine invertebrates form lactate and/or octopine or succinate (or similar end product) according to the activities of the enzymes present in the muscles (see above). The muscles investigated possess low activities of cytosolic glycerol 3-phosphate dehydrogenase, which indicates that glycerol phosphate formation is quantitatively unimportant under anaerobic conditions, and low activities of mitochondrial glycerol phosphate dehydrogenase, which indicates that the glycerol phosphate cycle is unimportant in the re-oxidation of glycolytically produced NADH in these muscles under aerobic conditions. Conversely, high activities of glutamate-oxaloacetate transaminase are present in some muscles, which indicates that the malate-aspartate cycle may be important in oxidation of glycolytically produced NADH under aerobic conditions. 3. High activities of nucleoside diphosphate kinase were found in muscles that function for prolonged periods under anaerobic conditions (e.g... PMID:13783

  7. Synthesis, DNA Polymerase Incorporation, and Enzymatic Phosphate Hydrolysis of Formamidopyrimidine Nucleoside Triphosphates

    PubMed Central

    Imoto, Shuhei; Patro, Jennifer N.; Jiang, Yu Lin; Oka, Natsuhisa; Greenberg, Marc M.

    2007-01-01

    The nucleoside triphosphates of N6-(2-deoxy-α,β-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy·dGTP) and its C-nucleoside analogue (β-C-Fapy·dGTP) were synthesized. The lability of the formamide group required that nucleoside triphosphate formation be carried out using an umpolung strategy in which pyrophosphate was activated toward nucleophilic attack. The Klenow fragment of DNA polymerase I from Escherichia coli accepted Fapy·dGTP and β-C-Fapy·dGTP as substrates much less efficiently than it did dGTP. Subsequent extension of a primer containing either modified nucleotide was less affected compared to when the native nucleotide is present at the 3′-terminus. The specificity constants are sufficiently large that nucleoside triphosphate incorporation could account for the level of Fapy·dG observed in cells if 1% of the dGTP pool is converted to Fapy·dGTP. Similarly, polymerase-mediated introduction of β-C-Fapy·dG could be useful for incorporating useful amounts of this nonhydrolyzable analogue for use as an inhibitor of base excision repair. The kinetic viability of these processes is enhanced by inefficient hydrolysis of Fapy·dGTP and β-C-Fapy·dGTP by MutT, the E. coli enzyme that releases pyrophosphate and the corresponding nucleoside monophosphate upon reaction with structurally related nucleoside triphosphates. PMID:17090045

  8. Cloning, expression, and functional characterization of a Ca(2+)-dependent endoplasmic reticulum nucleoside diphosphatase.

    PubMed

    Failer, Bernd U; Braun, Norbert; Zimmermann, Herbert

    2002-10-01

    We have isolated and characterized the cDNA encoding a Ca(2+)-dependent nucleoside diphosphatase (EC ) related to two secreted ATP- and ADP-hydrolyzing apyrases of the bloodsucking insects, Cimex lectularius and Phlebotomus papatasi. The rat brain-derived cDNA has an open reading frame of 1209 bp encoding a protein of 403 amino acids and a calculated molecular mass of 45.7 kDa. The mRNA was expressed in all tissues investigated, revealing two major transcripts with varying preponderance. The immunohistochemical analysis of the Myc-His-tagged enzyme expressed in Chinese hamster ovary cells revealed its association with the endoplasmic reticulum and also with pre-Golgi intermediates. Ca(2+)-dependent nucleoside diphosphatase is a membrane protein with its catalytic site facing the organelle lumen. It hydrolyzes nucleoside 5'-diphosphates in the order UDP >GDP = IDP >CDP but not ADP. Nucleoside 5'-triphosphates were hydrolyzed to a minor extent, and no hydrolysis of nucleoside 5'-monophosphates was observed. The enzyme was strongly activated by Ca(2+), insensitive to Mg(2+), and had a K(m) for UDP of 216 microm. Ca(2+)-dependent nucleoside diphosphatase may support glycosylation reactions related to quality control in the endoplasmic reticulum. PMID:12167635

  9. Transport of physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs by recombinant Escherichia coli nucleoside-H(+) cotransporter (NupC) produced in Xenopus laevis oocytes.

    PubMed

    Loewen, Shaun K; Yao, Sylvia Y M; Slugoski, Melissa D; Mohabir, Nadira N; Turner, Raymond J; Mackey, John R; Weiner, Joel H; Gallagher, Maurice P; Henderson, Peter J F; Baldwin, Stephen A; Cass, Carol E; Young, James D

    2004-01-01

    The recently identified human and rodent plasma membrane proteins CNT1, CNT2 and CNT3 belong to a gene family (CNT) that also includes the bacterial nucleoside transport protein NupC. Heterologous expression in Xenopus oocytes has established that CNT1-3 correspond functionally to the three major concentrative nucleoside transport processes found in human and other mammalian cells (systems cit, cif and cib, respectively) and mediate Na(+) - linked uptake of both physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs. Here, one describes a complementary Xenopus oocyte transport study of Escherichia coli NupC using the plasmid vector pGEM-HE in which the coding region of NupC was flanked by 5'- and 3'-untranslated sequences from a Xenopus beta-globin gene. Recombinant NupC resembled human (h) and rat (r) CNT1 in nucleoside selectivity, including an ability to transport adenosine and the chemotherapeutic drugs 3'-azido-3'-deoxythymidine (AZT), 2',3'- dideoxycytidine (ddC) and 2'-deoxy-2',2'-difluorocytidine (gemcitabine), but also interacted with inosine and 2',3'- dideoxyinosine (ddl). Apparent affinities were higher than for hCNT1, with apparent K(m) values of 1.5-6.3 microM for adenosine, uridine and gemcitabine, and 112 and 130 microM, respectively, for AZT and ddC. Unlike the relatively low translocation capacity of hCNT1 and rCNT1 for adenosine, NupC exhibited broadly similar apparent V(max) values for adenosine, uridine and nucleoside drugs. NupC did not require Na(+) for activity and was H(+) - dependent. The kinetics of uridine transport measured as a function of external pH were consistent with an ordered transport model in which H(+) binds to the transporter first followed by the nucleoside. These experiments establish the NupC-pGEM-HE/oocyte system as a useful tool for characterization of NupC-mediated transport of physiological nucleosides and clinically relevant nucleoside therapeutic drugs. PMID:14668133

  10. Structural modifications of nucleosides in ionic liquids

    PubMed Central

    Kumar, Vineet; Parmar, Virinder S.; Malhotra, Sanjay V.

    2011-01-01

    Nucleoside chemistry represents an important research area for drug discovery, as many nucleoside analogs are prominent drugs and have been widely applied for cancer and viral chemotherapy. However, the synthesis of modified nucleosides presents a major challenge, which is further aggravated by poor solubility of these compounds in common organic solvents. Most of the currently available methods for nucleoside modification employ toxic high boiling solvents; require long reaction time and tedious workup methods. As such, there is constant effort to develop process chemistry in alternative medium to limit the use of organic solvents that are hazardous to the environment and can be deleterious to human health. One such approach is to use ionic liquids, which are ‘designer materials’ with unique and tunable physico-chemical properties. Studies have shown that methodologies using ionic liquids are highly efficient and convenient for the synthesis of nucleoside analogs, as demonstrated by the preparation of pharmaceutically important anti-viral drugs. This article summarizes recent efforts on nucleoside modification using ionic liquids. PMID:20178825

  11. Attenuation of nucleoside and anti-cancer nucleoside analog drug uptake in prostate cancer cells by Cimicifuga racemosa extract BNO-1055.

    PubMed

    Dueregger, Andrea; Guggenberger, Fabian; Barthelmes, Jan; Stecher, Günther; Schuh, Markus; Intelmann, Daniel; Abel, Gudrun; Haunschild, Jutta; Klocker, Helmut; Ramoner, Reinhold; Sampson, Natalie

    2013-11-15

    This study aimed to investigate the mechanisms underlying the anti-proliferative effects of the ethanolic Cimicifuga racemosa extract BNO-1055 on prostate cells and evaluate its therapeutic potential. BNO-1055 dose-dependently attenuated cellular uptake and incorporation of thymidine and BrdU and significantly inhibited cell growth after long-time exposure. Similar results were obtained using saponin-enriched sub-fractions of BNO-1055. These inhibitory effects of BNO-1055 could be mimicked using pharmacological inhibitors and isoform-specific siRNAs targeting the equilibrative nucleoside transporters ENT1 and ENT2. Moreover, BNO-1055 attenuated the uptake of clinically relevant nucleoside analogs, e.g. the anti-cancer drugs gemcitabine and fludarabine. Consistent with inhibition of the salvage nucleoside uptake pathway BNO-1055 potentiated the cytotoxicity of the de novo nucleotide synthesis inhibitor 5-FU without significantly altering its uptake. Collectively, these data show for the first time that the anti-proliferative effects of BNO-1055 result from hindered nucleoside uptake due to impaired ENT activity and demonstrate the potential therapeutic use of BNO-1055 for modulation of nucleoside transport. PMID:23972793

  12. Evaluation of anti-HIV-1 mutagenic nucleoside analogues.

    PubMed

    Vivet-Boudou, Valérie; Isel, Catherine; El Safadi, Yazan; Smyth, Redmond P; Laumond, Géraldine; Moog, Christiane; Paillart, Jean-Christophe; Marquet, Roland

    2015-01-01

    Because of their high mutation rates, RNA viruses and retroviruses replicate close to the threshold of viability. Their existence as quasi-species has pioneered the concept of "lethal mutagenesis" that prompted us to synthesize pyrimidine nucleoside analogues with antiviral activity in cell culture consistent with an accumulation of deleterious mutations in the HIV-1 genome. However, testing all potentially mutagenic compounds in cell-based assays is tedious and costly. Here, we describe two simple in vitro biophysical/biochemical assays that allow prediction of the mutagenic potential of deoxyribonucleoside analogues. The first assay compares the thermal stabilities of matched and mismatched base pairs in DNA duplexes containing or not the nucleoside analogues as follows. A promising candidate should display a small destabilization of the matched base pair compared with the natural nucleoside and the smallest gap possible between the stabilities of the matched and mismatched base pairs. From this assay, we predicted that two of our compounds, 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine, should be mutagenic. The second in vitro reverse transcription assay assesses DNA synthesis opposite nucleoside analogues inserted into a template strand and subsequent extension of the newly synthesized base pairs. Once again, only 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine are predicted to be efficient mutagens. The predictive potential of our fast and easy first line screens was confirmed by detailed analysis of the mutation spectrum induced by the compounds in cell culture because only compounds 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine were found to increase the mutation frequency by 3.1- and 3.4-fold, respectively. PMID:25398876

  13. Nucleoside derivatives from the marine-derived fungus Aspergillus versicolor.

    PubMed

    Chen, Min; Fu, Xiu-Mei; Kong, Chui-Jian; Wang, Chang-Yun

    2014-01-01

    Four nucleoside derivatives (1-4) were isolated from the fungus Aspergillus versicolor derived from the gorgonian Dichotella gemmacea collected in the South China Sea. Their structures were elucidated by comprehensive spectroscopic method of NMR and MS analysis. All isolated metabolites were evaluated for their cytotoxicity, antibacterial activity and lethality towards brine shrimp Artemia salina. Compounds 1/2 exhibited selective antibacterial activity against Staphylococcus epidermidis with an MIC value of 12.5 μM. It should be noted that 1 and 2, whose structures were listed in SciFinder Scholar, had no associated reference. This is the first report about their isolation, structure elucidation and biological activities. PMID:24670197

  14. Cladribine Analogues via O6-(Benzotriazolyl) Derivatives of Guanine Nucleosides

    PubMed Central

    Satishkumar, Sakilam; Vuram, Prasanna K.; Relangi, Siva Subrahmanyam; Gurram, Venkateshwarlu; Zhou, Hong; Kreitman, Robert J.; Montemayor, Michelle M. Martínez; Yang, Lijia; Kaliyaperumal, Muralidharan; Sharma, Somesh; Pottabathini, Narender; Lakshman, Mahesh K.

    2016-01-01

    Cladribine, 2-chloro-2′-deoxyadenosine, is a highly efficacious clinically used nucleoside for the treatment of hairy cell leukemia. It is also being evaluated against other lymphoid malignancies and has been a molecule of interest for well over half a century. In continuation of our interest on the amide bond-activation in purine nucleosides via the use of (benzotriazol-1yl-oxy)tris(dimethylamino)phosphonium hexafluorophosphate, we have evaluated the use of O6-(benzotriazol-1-yl)-2′-deoxyguanosine as a potential precursor to cladribine and its analogues. These compounds, after appropriate deprotection, were assessed for their biological activities and the data are presented herein. Against hairy cell leukemia (HCL), T-cell lymphoma (TCL), and chronic lymphocytic leukemia (CLL) cladribine was the most active against all. The bromo analogue of cladribine showed comparable activity to the ribose analogue of cladribine against HCL, but was more active against TCL and CLL. The bromo ribo analogue of cladribine possessed activity, but was least active among the C6-NH2-containing compounds. Substitution with alkyl groups at the exocyclic amino group appears detrimental to activity, and only the C6 piperidinyl cladribine analogue demonstrated any activity. Against adenocarcinoma MDA-MB-231 cells, only cladribine and its ribose analogue were most active. PMID:26556315

  15. Farnesides A and B, Sesquiterpenoid Nucleoside Ethers from a Marine-Derived Streptomyces sp., strain CNT-372 from Fiji

    PubMed Central

    Ilan, Ella Zafrir; Torres, Manuel R.; Prudhomme, Jacques; Le Roch, Karine; Jensen, Paul R.; Fenical, William

    2013-01-01

    Farnesides A and B (1, 2), linear sesquiterpenoids connected by ether links to a ribose dihydrouracil nucleoside, were isolated from a marine-derived Streptomyces sp., strain CNT-372, grown in saline liquid culture. The structures of the new compounds were assigned by comprehensive spectroscopic analysis primarily involving 1D and 2D NMR analysis and by comparison of spectroscopic data to the recently reported ribose nucleoside JBIR-68 (3). The farnesides are only the second example of this exceedingly rare class of microbial terpenoid nucleoside metabolites. Farneside A (1) was found to have modest antimalarial activity against the parasite Plasmodium falciparum. PMID:23987585

  16. Sequencing, functional expression and characterization of rat NTPDase6, a nucleoside diphosphatase and novel member of the ecto-nucleoside triphosphate diphosphohydrolase family.

    PubMed

    Braun, N; Fengler, S; Ebeling, C; Servos, J; Zimmermann, H

    2000-11-01

    We have isolated and characterized the cDNA encoding nucleoside triphosphate diphosphohydrolase 6 (NTPDase6), a novel member of the ecto-nucleoside triphosphate diphosphohydrolase family. The rat-brain-derived cDNA has an open reading frame of 1365 bp encoding a protein of 455 amino acid residues, a calculated molecular mass of 49971 Da and a predicted N-terminal hydrophobic sequence. It shares 86% sequence identity with the human CD39L2 sequence and 48% and 51% identity respectively with sequences of the two related human and murine nucleoside diphosphatases (CD39L4, NTPDase5/ER-UDPase). The mRNA was expressed in all tissues investigated, revealing two major transcripts with differing abundances. PCR analysis suggests a single open reading frame. A Myc-His-tagged NTPDase6 was expressed in Chinese hamster ovary (CHO) and PC12 cells for immunological analysis and protein isolation. The protein was contained in membrane fractions of transfected CHO cells and occurred in a soluble form in the cell culture supernatants. NTPDase6 preferentially hydrolysed nucleoside 5'-diphosphates. With different substrates the order of activity was GDP>IDP>UDP,CDP>ADP. Nucleoside 5'-triphosphates were hydrolysed only to a minor extent and no hydrolysis of nucleoside 5'-monophosphates was observed. The enzyme was strongly and equally activated by Ca(2+) and Mg(2+) and had a K(m) for GDP of 211 microM. The immunohistochemical analysis of transfected CHO and PC12 cells suggests that NTPDase6 is associated with the Golgi apparatus and to a small extent also with the plasma membrane. The enzyme might support glycosylation reactions in the Golgi apparatus and, when released from cells, might catalyse the hydrolysis of extracellular nucleotides. PMID:11042118

  17. Basal Expression of Nucleoside Transporter mRNA Differs Among Small Intestinal Epithelia of Beef Steers and is Differentially Altered by Ruminal or Abomasal Infusion of Starch Hydrolysate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In ruminants, microbial-derived nucleic acids are a major source of N and are absorbed as nucleosides by small intestinal epithelia. Although the biochemical activities of 2 nucleoside transport systems have been described for cattle, little is known regarding the regulation of their gene expression...

  18. Nucleoside transporter subtype expression: effects on potency of adenosine kinase inhibitors

    PubMed Central

    Sinclair, C J D; Powell, A E; Xiong, W; LaRivière, C G; Baldwin, S A; Cass, C E; Young, J D; Parkinson, F E

    2001-01-01

    Adenosine kinase (AK) inhibitors can enhance adenosine levels and potentiate adenosine receptor activation. As the AK inhibitors 5′ iodotubercidin (ITU) and 5-amino-5′-deoxyadenosine (NH2dAdo) are nucleoside analogues, we hypothesized that nucleoside transporter subtype expression can affect the potency of these inhibitors in intact cells.Three nucleoside transporter subtypes that mediate adenosine permeation of rat cells have been characterized and cloned: equilibrative transporters rENT1 and rENT2 and concentrative transporter rCNT2. We stably transfected rat C6 glioma cells, which express rENT2 nucleoside transporters, with rENT1 (rENT1-C6 cells) or rCNT2 (rCNT2-C6 cells) nucleoside transporters.We tested the effects of ITU and NH2dAdo on [3H]-adenosine uptake and conversion to [3H]-adenine nucleotides in the three cell types. NH2dAdo did not show any cell type selectivity. In contrast, ITU showed significant inhibition of [3H]-adenosine uptake and [3H]-adenine nucleotide formation at concentrations ⩽100 nM in rENT1-C6 cells, while concentrations ⩾3 μM were required for C6 or rCNT2-C6 cells.Nitrobenzylthioinosine (NBMPR; 100 nM), a selective inhibitor of rENT1, abolished the effects of nanomolar concentrations of ITU in rENT1-C6 cells.This study demonstrates that the effects of ITU, but not NH2dAdo, in whole cell assays are dependent upon nucleoside transporter subtype expression. Thus, cellular and tissue differences in expression of nucleoside transporter subtypes may affect the pharmacological actions of some AK inhibitors. PMID:11682452

  19. Purine nucleoside modulation of functions of human lymphocytes.

    PubMed

    Priebe, T; Platsoucas, C D; Seki, H; Fox, F E; Nelson, J A

    1990-09-01

    The accumulation of endogenous substrates in patients with adenosine deaminase deficiency or purine nucleoside phosphorylase deficiency is believed to be responsible for the immunodeficiency observed in these patients. To identify the lymphocyte populations that are most susceptible to these substrates, we investigated the effect of their nucleoside analogs on a number of T and B cell functions of human lymphocytes. We found that tubercidin (Tub), 2-chloro 2'deoxyadenosine (2CldA), 2-fluoro adenine arabinoside-5'phosphate (FaraAMP), and 9-beta-D-arabinosyl guanine (AraGua) inhibited the proliferative responses of human peripheral blood mononuclear cells (PBMC) to polyclonal activators (PHA, OKT3 mab) or to allogeneic PBMC in mixed lymphocyte cultures (MLC). Addition of recombinant IL-2 from the beginning of the culture did not alter the inhibition by Tub of the proliferative responses of PBMC. These purine nucleoside analogs also inhibited the proliferative responses of purified human peripheral blood CD4+ and CD8+ T cells to PHA and of purified B cells to SAC. The concentrations of these nucleosides required to achieve a given degree of inhibition of proliferative responses of T lymphocyte subpopulations or B cells was similar, suggesting that these analogs do not exhibit any selectivity for these purified lymphocyte populations. Tub and FaraAMP, respectively, inhibited and enhanced, at the effector phase, both NK cytotoxicity and specific T cell-mediated cytotoxicity. In contrast to these findings, LAK cytotoxicity at the effector phase was not significantly inhibited by Tub, and was not enhanced by FaraAMP. Both analogs inhibited rIL-2-induced proliferative responses of PBMC, but did not affect the generation of LAK cytotoxicity (induction phase) against the K562 targets when added at the beginning of the culture. This suggests that DNA synthesis is not required for LAK cell induction. Both Tub and FaraAMP inhibited immunoglobulin production (IgG and IgM) by

  20. Synthesis and biological activity of novel 5'-arylamino-nucleosides by microwave-assisted one-pot tandem Staudinger/aza-Wittig/reduction.

    PubMed

    Chen, Hua; Zhao, Jianpeng; Li, Yanan; Shen, Fengjuan; Li, Xiaoliu; Yin, Qingmei; Qin, Zhanbin; Yan, Xinhao; Wang, Yanfei; Zhang, Pingzhu; Zhang, Jinchao

    2011-01-01

    Novel pseudonucleosides with benzylamino group on 5'-position (4) were synthesized by using the microwave-assisted one-pot tandem Staudinger/aza-Wittig/reduction reaction in good yields of 55.2-71.7%. The deacetylation of 4 afforded compounds 5. HIV-1 reverse transcriptase (RT) inhibitory and antitumor activities were preliminarily evaluated with 5. The results showed that the new pseudonucleosides (5) could effectively inhibit HIV-1 RT activity, but no antitumor activity. PMID:21095125

  1. Crystallization and Crystal-Packing Studies of Chlorella Virus Deoxyuridine Triphosphatase

    SciTech Connect

    Homma, K.; Moriyama, H

    2009-01-01

    The 141-amino-acid deoxyuridine triphosphatase (dUTPase) from the algal Chlorella virus IL-3A and its Glu81Ser/Thr84Arg-mutant derivative Mu-22 were crystallized using the hanging-drop vapor-diffusion method at 298 K with polyethylene glycol as the precipitant. An apo IL-3A dUTPase with an amino-terminal T7 epitope tag and a carboxy-terminal histidine tag yielded cubic P2{sub 1}3 crystals with unit-cell parameter a = 106.65 {angstrom}. In the presence of dUDP, the enzyme produced thin stacked orthorhombic P222 crystals with unit-cell parameters a = 81.0, b = 96.2, c = 132.8 {angstrom}. T7-histidine-tagged Mu-22 dUTPase formed thin stacked rectangular crystals. Amino-terminal histidine-tagged dUTPases did not crystallize but formed aggregates. Glycyl-seryl-tagged dUTPases yielded cubic P2{sub 1}3 IL-3A crystals with unit-cell parameter a = 105.68 {angstrom} and hexagonal P6{sub 3} Mu-22 crystals with unit-cell parameters a = 132.07, c = 53.45 {angstrom}, {gamma} = 120{sup o}. Owing to the Thr84Arg mutation, Mu-22 dUTPase had different monomer-to-monomer interactions to those of IL-3A dUTPase.

  2. Prebiotic phosphorylation of nucleosides in formamide

    NASA Technical Reports Server (NTRS)

    Schoffstall, A. M.

    1976-01-01

    Results are presented for an experimental study intended to assess phosphorylation under neither aqueous nor dry thermal conditions. Instead, phosphorylations were attempted in possible nonaqueous prebiotic solvents. Formamide appeared to be the most obvious candidate for phosphorylation studies. Three main classes of phosphorylated products were formed in formamide solution: adenosine monophosphates, cyclic adenosine phosphate, and adenosine diphosphates. Experiments were designed to investigate the extent of phosphorylation of nucleosides in formamide, the relative amounts of nucleoside monophosphate, diphosphates and cyclic phosphate formed and the relative effectiveness of different sources of phosphate as phosphorylating agents in formamide. Reaction variables were temperature, nature of the phosphate or condensed phosphate, nucleoside, concentration of reactants and possible effects of additives. Product identification was based on qualitative and quantitative thin layer chromatography.

  3. Membrane bound pyrophosphatase and P-type adenosine triphosphatase of Leishmania donovani as possible chemotherapeutic targets: similarities and differences in inhibitor sensitivities.

    PubMed

    Sen, S S; Bhuyan, N R; Lakshman, K; Roy, A K; Chakraborty, B; Bera, T

    2009-12-01

    The activities of inorganic pyrophosphatase (PPase) and adenosine triphosphatase (ATPase) were studied in the plasma membrane of Leishmania donovani promastigotes and amastigotes. It was shown that the specific activity of PPase was greater than that of ATPase in the promastigote plasma membrane. We characterized H+-PPase present in the plasma membrane of L. donovani and investigated its possible role in the survival of promastigote and amastigote. PPase activity was stimulated by K+ and sodium orthovanadate and inhibited by pyrophosphate analogs (imidodiphosphate and alendronate), KF, N,N'-dicyclohexylcarbodiimide (DCCD), thiol reagents (p-chloromercuribenzenesulfonate (PCMBS), N-ethylmaleimide (NEM), and phenylarsine oxide (PAO)), the ABC superfamily transport modulator verapamil, and also by the F(1)F(o)-ATPase inhibitor quercetin. ATPase activity was stimulated by K+ and verapamil, inhibited by DCCD, PCMBS, NEM, sodium azide, sodium orthovanadate, and quercetin, and was unaffected by PAO. We conclude that there are significant differences within promastigote, amastigote, and mammalian host in cytosolic pH homeostasis to merit the inclusion of PPase transporter as a putative target for rational drug design. PMID:19961421

  4. Synthesis of Nucleoside Triphosphates from 2'-3'-Protected Nucleosides Using Trimetaphosphate.

    PubMed

    Mohamady, Samy; Taylor, Scott D

    2016-02-01

    Chemists have been attempting to triphosphorylate nucleosides and other alcohols using trimetaphosphate (TriMP) since the 1960s. However, this route appears to have been abandoned due to poor yields. The first practical syntheses of nucleoside triphosphates (NTPs) are reported using TriMP as the key reagent. This was achieved by reacting the tetrabutylammonium salt of TriMP with mesitylenesulfonyl chloride in the presence of DABCO in pyridine followed by the addition of an appropriately protected nucleoside and phthalimide. Quenching the reaction with aqueous buffer followed by hydrolysis of the OH protecting groups gave the NTPs in good yield. PMID:26759914

  5. Synthesis of phosphonate analogues of the antiviral cyclopropane nucleoside A-5021.

    PubMed

    Onishi, Tomoyuki; Sekiyama, Takaaki; Tsuji, Takashi

    2005-01-01

    A series of phosphonate analogues of the antiviral cyclopropane nucleoside A-5021 were synthesized from (1S*, 7R*)-3,5-dioxa-4,4-diphenylbicyclo[5. 1.0]octane-l-methanol by a 10-step process. In contrast to the potent antiherpetic activity of A-5021, they were all devoid of antiviral activity. PMID:16270661

  6. Cloning and functional expression of a complementary DNA encoding a mammalian nucleoside transport protein.

    PubMed

    Huang, Q Q; Yao, S Y; Ritzel, M W; Paterson, A R; Cass, C E; Young, J D

    1994-07-01

    Expression screening in Xenopus oocytes was used to isolate a cDNA from rat jejunal epithelium encoding a Na(+)-dependent nucleoside transport protein (named cNT1). The cDNA sequence of cNT1 predicts a protein of 648 amino acids (relative molecular mass 71,000) with 14 potential transmembrane domains. Data base searches indicate significant sequence similarity to the NUPC proton/nucleoside symporter of Escherichia coli. There is no sequence similarity between cNT1 and proteins of mammalian origin. Functionally, cNT1 exhibited the transport characteristics of the nucleoside transport system cit (selective for pyrimidine nucleosides and adenosine) and accepted both 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC) as permeants (Km = 0.49 and 0.51 mM, respectively). The demonstration of transport of AZT by cNT1 expressed in Xenopus oocytes provides the first direct evidence that AZT enters cells by transporter-mediated processes, as well as by passive diffusion. Consistent with the tissue distribution of system cit transport activity, transcripts for cNT1 were detected in kidney as well as jejunum. cNT1 therefore belongs to a potential new gene family and may be involved in the intestinal absorption and renal handling of pyrimidine nucleoside analogs used to treat acquired immunodeficiency syndrome (AIDS). PMID:8027026

  7. Molecular Modeling, Synthesis, and Anti-HIV Activity of Novel Isoindolinedione Analogues as Potent Non-nucleoside Reverse Transcriptase Inhibitors.

    PubMed

    Kumari, Garima; Singh, Ramendra K

    2016-02-01

    Different isoindolinedione derivatives bearing imine, amide, thioamide, and sulfonamide linkages have been designed in silico using discovery studio software (BIOVIA, San Diego, CA, USA), synthesized, and evaluated for their anti-HIV activity. SAR studies revealed that the linkages in these molecules did affect their anti-HIV activity and the molecules having sulfonamide linkages were the most potent HIV-RT inhibitors as the S=O bonds of the sulfonamide moiety interacted with Lys103 (NH or carbonyl or both) and Pro236; the NH part of the sulfonamide linkage formed bond with carbonyl of Lys101. blood-brain barrier (BBB) plots were also studied, and it was found that all the designed molecules have potential to cross BBB, a very vital criteria for anti-HIV drugs. In vitro screening was performed using HIV-1 strain IIIB in MT-4 cells using the MTT assay, and it was seen that some of these molecules were effective inhibitors of HIV-1 replication at nanomolar concentration with selectivity indices ranging from 33.75 to 73.33 under in vitro conditions. Some of these molecules have shown good anti-HIV activity at 3-4 nm concentrations. These derivatives have potential to be developed as lead molecules effective against HIV-1. Novel isoindolinedione derivatives as probable NNRTIs have been synthesized and characterized. Some of these molecules have shown good anti-HIV activity at 3-4 nm concentrations. PMID:26212217

  8. Inhibition of Siderophore Biosynthesis in Mycobacterium tuberculosis with Nucleoside Bisubstrate Analogues: Structure–Activity Relationships of the Nucleobase Domain of 5′-O-[N-(Salicyl)sulfamoyl]adenosine

    PubMed Central

    Neres, João; Labello, Nicholas P.; Somu, Ravindranadh V.; Boshoff, Helena I.; Wilson, Daniel J.; Vannada, Jagadeshwar; Chen, Liqiang; Barry, Clifton E.; Bennett, Eric M.; Aldrich, Courtney C.

    2009-01-01

    5′-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS) is a prototype for a new class of antitubercular agents that inhibit the aryl acid adenylating enzyme (AAAE) known as MbtA involved in biosynthesis of the mycobactins. Herein, we report the structure-based design, synthesis, biochemical, and biological evaluation of a comprehensive and systematic series of analogues, exploring the structure–activity relationship of the purine nucleobase domain of Sal-AMS. Significantly, 2-phenyl-Sal-AMS derivative 26 exhibited exceptionally potent antitubercular activity with an MIC99 under iron-deficient conditions of 0.049 µM while the N-6-cyclopropyl-Sal-AMS 16 led to improved potency and to a 64-enhancement in activity under iron-deficient conditions relative to iron-replete conditions, a phenotype concordant with the designed mechanism of action. The most potent MbtA inhibitors disclosed here display in vitro antitubercular activity superior to most current first line TB drugs, and these compounds are also expected to be useful against a wide range of pathogens that require aryl-capped siderphores for virulence. PMID:18690677

  9. Site specific replacements of a single loop nucleoside with a dibenzyl linker may switch the activity of TBA from anticoagulant to antiproliferative

    PubMed Central

    Scuotto, Maria; Rivieccio, Elisa; Varone, Alessia; Corda, Daniela; Bucci, Mariarosaria; Vellecco, Valentina; Cirino, Giuseppe; Virgilio, Antonella; Esposito, Veronica; Galeone, Aldo; Borbone, Nicola; Varra, Michela; Mayol, Luciano

    2015-01-01

    Many antiproliferative G-quadruplexes (G4s) arise from the folding of GT-rich strands. Among these, the Thrombin Binding Aptamer (TBA), as a rare example, adopts a monomolecular well-defined G4 structure. Nevertheless, the potential anticancer properties of TBA are severely hampered by its anticoagulant action and, consequently, no related studies have appeared so far in the literature. We wish to report here that suitable chemical modifications in the TBA sequence can preserve its antiproliferative over anticoagulant activity. Particularly, we replaced one residue of the TT or TGT loops with a dibenzyl linker to develop seven new quadruplex-forming TBA based sequences (TBA-bs), which were studied for their structural (CD, CD melting, 1D NMR) and biological (fibrinogen, PT and MTT assays) properties. The three-dimensional structures of the TBA-bs modified at T13 (TBA-bs13) or T12 (TBA-bs12), the former endowed with selective antiproliferative activity, and the latter acting as potently as TBA in both coagulation and MTT assays, were further studied by 2D NMR restrained molecular mechanics. The comparative structural analyses indicated that neither the stability, nor the topology of the G4s, but the different localization of the two benzene rings of the linker was responsible for the loss of the antithrombin activity for TBA-bs13. PMID:26250112

  10. Synthesis and antiviral activity of novel 5-(1-cyanamido-2-haloethyl) and 5-(1-hydroxy(or methoxy)-2-azidoethyl) analogues of uracil nucleosides.

    PubMed

    Kumar, R; Rai, D; Sharma, S K; Saffran, H A; Blush, R; Tyrrell, D L

    2001-10-11

    A new class of 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) and arabinouridines (7, 8) were synthesized by the regiospecific addition of halogenocyanamides (X-NHCN) to the 5-vinyl substituent of the respective 5-vinyl-2'-deoxyuridine (2) and 2'-arabinouridine (3). Reaction of 2 with sodium azide, ceric ammonium nitrate, and acetonitrile-methanol or water afforded the 5-(1-hydroxy-2-azidoethyl)-(10) and 5-(1-methoxy-2-azidoethyl)-2'-deoxyuridines (11). In vitro antiviral activities against HSV-1-TK(+) (KOS and E-377), HSV-1-TK(-), HSV-2, VZV, HCMV, and DHBV were determined. Of the newly synthesized compounds, 5-(1-cyanamido-2-iodoethyl)-2'-deoxyuridine (6) exhibited the most potent anti-HSV-1 activity, which was equipotent to acyclovir and superior to 5-ethyl-2'-deoxyuridine (EDU). In addition, it was significantly inhibitory for thymidine kinase deficient strain of HSV-1 (EC(50) = 2.3-15.3 microM). The 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) all were approximately equipotent against HSV-2 and were approximately 1.5- and 15-fold less inhibitory for HSV-2 than EDU and acyclovir, respectively. Compounds 4-6 were all inactive against HCMV but exhibited appreciable antiviral activity against VZV. Their anti-VZV activity was similar or higher to that of EDU and approximately 5-12-fold lower than that of acyclovir. The 5-(1-cyanamido-2-haloethyl)-(7,8) analogues of arabinouridine were moderately inhibitory for VZV and HSV-1 (strain KOS), whereas compounds 10 and 11 were inactive against herpes viruses. Compounds 5 and 6 also demonstrated modest anti-hepatitis B virus activity against DHBV (EC(50) = 19.9-23.6 microM). Interestingly, the related 5-(1-azido-2-bromoethyl)-2'-deoxyuridine (1n) analogue proved to be markedly inhibitory to DHBV replication (EC(50) = 2.6-6.6 microM). All compounds investigated exhibited low host cell toxicity to several stationary and proliferating host cell lines as well as mitogen-stimulated proliferating human T lymphocytes

  11. Nucleoside inhibitors of tick-borne encephalitis virus.

    PubMed

    Eyer, Luděk; Valdés, James J; Gil, Victor A; Nencka, Radim; Hřebabecký, Hubert; Šála, Michal; Salát, Jiří; Černý, Jiří; Palus, Martin; De Clercq, Erik; Růžek, Daniel

    2015-09-01

    Tick-borne encephalitis virus (TBEV) is a leading cause of human neuroinfections in Europe and Northeast Asia. There are no antiviral therapies for treating TBEV infection. A series of nucleoside analogues was tested for the ability to inhibit the replication of TBEV in porcine kidney cells and human neuroblastoma cells. The interactions of three nucleoside analogues with viral polymerase were simulated using advanced computational methods. The nucleoside analogues 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), 2'-C-methyladenosine (2'-CMA), and 2'-C-methylcytidine (2'-CMC) inhibited TBEV replication. These compounds showed dose-dependent inhibition of TBEV-induced cytopathic effects, TBEV replication (50% effective concentrations [EC50]of 5.1 ± 0.4 μM for 7-deaza-2'-CMA, 7.1 ± 1.2 μM for 2'-CMA, and 14.2 ± 1.9 μM for 2'-CMC) and viral antigen production. Notably, 2'-CMC was relatively cytotoxic to porcine kidney cells (50% cytotoxic concentration [CC50] of ∼50 μM). The anti-TBEV effect of 2'-CMA in cell culture diminished gradually after day 3 posttreatment. 7-Deaza-2'-CMA showed no detectable cellular toxicity (CC50 > 50 μM), and the antiviral effect in culture was stable for >6 days posttreatment. Computational molecular analyses revealed that compared to the other two compounds, 7-deaza-2'-CMA formed a large cluster near the active site of the TBEV polymerase. High antiviral activity and low cytotoxicity suggest that 7-deaza-2'-CMA is a promising candidate for further investigation as a potential therapeutic agent in treating TBEV infection. PMID:26124166

  12. Nucleoside Inhibitors of Tick-Borne Encephalitis Virus

    PubMed Central

    Eyer, Luděk; Valdés, James J.; Gil, Victor A.; Nencka, Radim; Hřebabecký, Hubert; Šála, Michal; Salát, Jiří; Černý, Jiří; Palus, Martin; De Clercq, Erik

    2015-01-01

    Tick-borne encephalitis virus (TBEV) is a leading cause of human neuroinfections in Europe and Northeast Asia. There are no antiviral therapies for treating TBEV infection. A series of nucleoside analogues was tested for the ability to inhibit the replication of TBEV in porcine kidney cells and human neuroblastoma cells. The interactions of three nucleoside analogues with viral polymerase were simulated using advanced computational methods. The nucleoside analogues 7-deaza-2′-C-methyladenosine (7-deaza-2′-CMA), 2′-C-methyladenosine (2′-CMA), and 2′-C-methylcytidine (2′-CMC) inhibited TBEV replication. These compounds showed dose-dependent inhibition of TBEV-induced cytopathic effects, TBEV replication (50% effective concentrations [EC50]of 5.1 ± 0.4 μM for 7-deaza-2′-CMA, 7.1 ± 1.2 μM for 2′-CMA, and 14.2 ± 1.9 μM for 2′-CMC) and viral antigen production. Notably, 2′-CMC was relatively cytotoxic to porcine kidney cells (50% cytotoxic concentration [CC50] of ∼50 μM). The anti-TBEV effect of 2′-CMA in cell culture diminished gradually after day 3 posttreatment. 7-Deaza-2′-CMA showed no detectable cellular toxicity (CC50 > 50 μM), and the antiviral effect in culture was stable for >6 days posttreatment. Computational molecular analyses revealed that compared to the other two compounds, 7-deaza-2′-CMA formed a large cluster near the active site of the TBEV polymerase. High antiviral activity and low cytotoxicity suggest that 7-deaza-2′-CMA is a promising candidate for further investigation as a potential therapeutic agent in treating TBEV infection. PMID:26124166

  13. The concentrative nucleoside transporter family (SLC28): new roles beyond salvage?

    PubMed

    Aymerich, I; Duflot, S; Fernández-Veledo, S; Guillén-Gómez, E; Huber-Ruano, I; Casado, F J; Pastor-Anglada, M

    2005-02-01

    The concentrative nucleoside transporter (CNT) family (SLC28) has three members: SLC28A1 (CNT1), SLC28A2 (CNT2) and SLC28A3 (CNT3). The CNT1 and CNT2 transporters are co-expressed in liver parenchymal cells and macrophages, two suitable models in which to study cell cycle progression. Despite initial observations suggesting that these transporter proteins might contribute to nucleoside salvage during proliferation, their subcellular localization and regulatory properties suggest alternative roles in cell physiology. In particular, CNT2 is a suitable candidate for modulation of purinergic responses, since it is under the control of the adenosine 1 receptor. Increasing evidence also suggests a role for CNT2 in energy metabolism, since its activation relies on the opening of ATP-sensitive K(+) channels. Animal and cell models genetically modified to alter nucleoside transporter expression levels may help to elucidate the particular roles of CNT proteins in cell physiology. PMID:15667311

  14. Conformational analysis of a nucleoside of 1,4-dihydro-4-oxoquinoline-3-carboxylic acid analogue

    NASA Astrophysics Data System (ADS)

    Zaccur Leal, Kátia; Rudolf Seidl, Peter; Diniz Yoneda, Julliane; Santos, CarlaV. B.; Marques, Isakelly P.; Souza, Maria Cecília B. V.; Francisco Ferreira, Vitor

    2005-06-01

    The synthesis of new ribonucleosides is an essential research area in the investigation of new therapeutically useful agents, particularly those used in the treatment of HIV infection. The conformation of these nucleosides may have direct implications for their ability to bind to receptor targets. We have prepared the 7-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic acid derivatives and used the ensemble of low-energy minima to develop conformational profiles of quinolonic nucleosides and verify their accuracy in different calculations of structural parameters. Results are compared with experimental data obtained by X-ray and NMR analysis. Finally, we intend to test the applicability of these methods to conformational analysis of other nucleosides and verify if the preferential conformation is the one which gives the best anti-HIV or antiviral activity.

  15. Development of a Diastereoselective Phosphorylation of a Complex Nucleoside via Dynamic Kinetic Resolution.

    PubMed

    Tran, Kristy; Beutner, Gregory L; Schmidt, Michael; Janey, Jacob; Chen, Ke; Rosso, Victor; Eastgate, Martin D

    2015-05-15

    The development of a diastereoselective nucleoside phosphorylation is described, which produces a single isomer of a complex nucleoside monophosphate pro-drug. A stable phosphoramidic acid derivative is coupled to the nucleoside, in a process mediated by HATU and quinine, to deliver the coupled product in high chemical yield and good diastereoselectivity. This unusual process was shown to proceed through a dynamic kinetic resolution of a 1:1 mixture of activated phosphonate ester diastereoisomers. The optimized conditions afforded the product with a combined [S,S(P)] and [S,R(P)] in-process yield of 89% and a ∼7:1 [S,S(P):S,R(P)] diastereomeric ratio. Isolation of the major isomer was facilitated by single crystallization from anisole, where the product was obtained in 57% isolated yield, excellent purity (>95%), and a high diastereomeric ratio (>50:1). PMID:25840459

  16. Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism.

    PubMed Central

    Lu, Q; Inouye, M

    1996-01-01

    Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8650159

  17. The Ugi reaction in the generation of new nucleosides as potential antiviral and antileishmanial agents.

    PubMed

    Fan, Xuesen; Zhang, Xinying; Bories, Christian; Loiseau, Philippe M; Torrence, Paul F

    2007-04-01

    5-Formyl-2'-deoxyuridine-3',5'-diacetate was converted to a small library of 5-substituted pyrimidine nucleoside N-acylamino acid amides by means of a Ugi multicomponent reaction. The reaction allowed introduction of various substituents at the acyl moiety, at the amino acid alpha-amide group, and at the amino acid carboxyl function. Evaluation of these novel 5-substituted nucleosides against vaccinia virus and cowpox virus provided one compound with discernable activity against cowpox virus but five- to eightfold less active than the Cidofovir standard. More promising activity was seen for the inhibition of Leishmania donovani promastigotes. Several synthetic products showed antileishmanial activity in the 10(-5)M range. When compared to earlier studies demonstrating anti-orthopoxviral and antileishmanial activity of 5-substituted pyrimidine nucleosides, these results imply that the 5-(N-acylamino acid amide)-derivatized pyrimidine nucleosides may possess more steric bulk, greater hydrophobicity, and more flexibility than is compatible with these particular biological activities. PMID:16996561

  18. Inosine Triphosphatase Genetic Variants are Protective Against Anemia During Antiviral Therapy for HCV2/3 But Do Not Decrease Dose Reductions of RBV Or Increase SVR

    PubMed Central

    Thompson, Alexander J.; Santoro, Rosanna; Piazzolla, Valeria; Clark, Paul J.; Naggie, Susanna; Tillmann, Hans L.; Patel, Keyur; Muir, Andrew J.; Shianna, Kevin V.; Mottola, Leonardo; Petruzzellis, Daniela; Romano, Mario; Sogari, Fernando; Facciorusso, Domenico; Goldstein, David B.; McHutchison, John G.; Mangia, Alessandra

    2016-01-01

    Two functional variants in the inosine triphosphatase (ITPA) gene causing inosine triphos-phatase (ITPase) deficiency protect against ribavirin (RBV)-induced hemolytic anemia and the need for RBV dose reduction in patients with genotype 1 hepatitis C virus (HCV). No data are available for genotype 2/3 HCV. We evaluated the association between the casual ITPA variants and on-treatment anemia in a well-characterized cohort of genotype 2/3 patients treated with variable-duration pegylated interferon alfa-2b (PEG-IFN-α2b) and RBV. Two hundred thirty-eight Caucasian patients were included in this retrospective study [185 (78%) with genotype 2 and 53 (22%) with genotype 3]. Patients were treated with PEG-IFN-α2b plus weight-based RBV (1000/1200 mg) for 12 (n = 109) or 24 weeks (n = 129). The ITPA polymorphisms rs1127354 and rs7270101 were genotyped, and an ITPase deficiency variable was defined that combined both ITPA variants according to their effect on ITPase activity. The primary endpoint was hemoglobin (Hb) reduction in week 4. We also considered Hb reduction over the course of therapy, the need for RBV dose modification, and the rate of sustained virological response (SVR). The ITPA variants were strongly and independently associated with protection from week 4 anemia (P = 10−6 for rs1127354 and P = 10−7 for rs7270101). Combining the variants into the ITPase deficiency variable increased the strength of association (P = 10−11). ITPase deficiency protected against anemia throughout treatment. ITPase deficiency was associated with a delayed time to an Hb level < 10 g/dL (hazard ratio = 0.25, 95% confidence interval = 0.08–0.84, P = 0.025) but not with the rate of RBV dose modification (required per protocol at Hb < 9.5 g/dL). There was no association between the ITPA variants and SVR. Conclusion Two ITPA variants were strongly associated with protection against treatment-related anemia in patients with genotype 2/3 HCV, but they did not decrease the need

  19. Nucleoside phosphorylation by the mineral schreibersite

    PubMed Central

    Gull, Maheen; Mojica, Mike A.; Fernández, Facundo M.; Gaul, David A.; Orlando, Thomas M.; Liotta, Charles L.; Pasek, Matthew A.

    2015-01-01

    Phosphorylation of the nucleosides adenosine and uridine by the simple mixing and mild heating of aqueous solutions of the organic compounds with synthetic analogs of the meteoritic mineral schreibersite, (Fe,Ni)3P under slightly basic conditions (pH ~9) is reported. These results suggest a potential role for meteoritic phosphorus in the origin and development of early life. PMID:26606901

  20. X-ray structure and activities of an essential Mononegavirales L-protein domain.

    PubMed

    Paesen, Guido C; Collet, Axelle; Sallamand, Corinne; Debart, Françoise; Vasseur, Jean-Jacques; Canard, Bruno; Decroly, Etienne; Grimes, Jonathan M

    2015-01-01

    The L protein of mononegaviruses harbours all catalytic activities for genome replication and transcription. It contains six conserved domains (CR-I to -VI; Fig. 1a). CR-III has been linked to polymerase and polyadenylation activity, CR-V to mRNA capping and CR-VI to cap methylation. However, how these activities are choreographed is poorly understood. Here we present the 2.2-Å X-ray structure and activities of CR-VI+, a portion of human Metapneumovirus L consisting of CR-VI and the poorly conserved region at its C terminus, the +domain. The CR-VI domain has a methyltransferase fold, which besides the typical S-adenosylmethionine-binding site ((SAM)P) also contains a novel pocket ((NS)P) that can accommodate a nucleoside. CR-VI lacks an obvious cap-binding site, and the (SAM)P-adjoining site holding the nucleotides undergoing methylation ((SUB)P) is unusually narrow because of the overhanging +domain. CR-VI+ sequentially methylates caps at their 2'O and N7 positions, and also displays nucleotide triphosphatase activity. PMID:26549102

  1. X-ray structure and activities of an essential Mononegavirales L-protein domain

    PubMed Central

    Paesen, Guido C.; Collet, Axelle; Sallamand, Corinne; Debart, Françoise; Vasseur, Jean-Jacques; Canard, Bruno; Decroly, Etienne; Grimes, Jonathan M.

    2015-01-01

    The L protein of mononegaviruses harbours all catalytic activities for genome replication and transcription. It contains six conserved domains (CR-I to -VI; Fig. 1a). CR-III has been linked to polymerase and polyadenylation activity, CR-V to mRNA capping and CR-VI to cap methylation. However, how these activities are choreographed is poorly understood. Here we present the 2.2-Å X-ray structure and activities of CR-VI+, a portion of human Metapneumovirus L consisting of CR-VI and the poorly conserved region at its C terminus, the +domain. The CR-VI domain has a methyltransferase fold, which besides the typical S-adenosylmethionine-binding site (SAMP) also contains a novel pocket (NSP) that can accommodate a nucleoside. CR-VI lacks an obvious cap-binding site, and the SAMP-adjoining site holding the nucleotides undergoing methylation (SUBP) is unusually narrow because of the overhanging +domain. CR-VI+ sequentially methylates caps at their 2′O and N7 positions, and also displays nucleotide triphosphatase activity. PMID:26549102

  2. Acyclic nucleoside/nucleotide analogues with an imidazole ring skeleton.

    PubMed

    Chen, H M; Hosmane, R S

    2001-08-01

    Syntheses of a few acyclic nucleoside and acyclic nucleoside phosphonate analogues containing an imidazole ring have been reported. These analogues include methyl 1-(2-hydroxyethoxymethyl)imidazole-4, 5-dicarbo-xylate (1), 4,5-dicarbamoyl-1-(2-hydroxyethoxymethyl)imidazole (2), 4,5-dicyano-1-(2-hydroxyethoxymethyl)imidazole (4), Methyl 1-(2-bromoethoxymethyl)imidazole-4,5-dicarboxylate (7), 4,5-dicyano-(2-bromoethoxymethyl)imidazole (8), and Methyl 1-(2-phosphonomethoxyethyl)imidazole (10). Also reported are a few potential prodrugs of the above compounds, including the acetyl derivatives 5 and 6 (of 1 and 4, respectively), and the diethyl phosphonate ester 9 (of 10). In addition, the corresponding benzyl-protected precursors 11 and 12 (of 1 and 4, respectively), along with their common hydrolysis product, 1-(2-benzyloxy-ethoxymethyl)-4,5-imidazoledicarboxylic acid (3), are reported. Another potential prodrug included in the list is 1-(2-acetoxyethyl)-4,5-dicyanoimidazole (15). The compounds were screened for in vitro antiviral activity against a wide variety of herpes and respiratory viruses. The most active compound was the phosphonate analogue 9 which exhibited an anti-measles virus activity with an EC50 of <2.5 microg/mL and an SI value of > 176. PMID:11554548

  3. Novel anticancer compound [trifluoromethyl-substituted pyrazole N-nucleoside] inhibits FLT3 activity to induce differentiation in acute myeloid leukemia cells.

    PubMed

    Saleh, Ayman M; Taha, Mutasem O; Aziz, Mohammad A; Al-Qudah, Mahmoud A; AbuTayeh, Reem F; Rizvi, Syed A

    2016-06-01

    Anticancer properties of chemically synthesized compounds have continuously been optimized for better efficacy and selectivity. Derivatives of heterocyclic compounds are well known to have selective antiproliferative effect against many types of cancer. In this study, we investigated the ability of an indigenously synthesized anticancer molecule, G-11 [1-(2",3",4",6"-Tetra-O-acetyl-β-D-glucopyranosyl)-4-(3'-trifluoromethylphenylhydrazono)-3-trifluoromethyl-1,4-dihydropyrazol-5-one], to cause selective cytotoxicity and induce differentiation in the acute myeloid leukemia HL-60 cells. G-11 was able to exert cytotoxic effect on hematological (Jurkat, U937, K562, HL-60, CCRF-SB) and solid tumor (MCF-7, HepG2, HeLa, Caco-2) cell lines, with IC50 values significantly lower than noncancerous cells (HEK-293, BJ and Vero) and normal peripheral blood mononuclear cells. G-11 induced differentiation of HL-60 cells to granulocytes and monocytes/macrophages by inhibiting the activation of FLT3 (CD135 tyrosine kinase). ITD-FLT3 mutation found in many acute myeloid leukemia patients could also be targeted by G-11 as exhibited by its inhibitory effect on MOLM-13 and MV4-11 cell lines. Molecular docking studies suggest the involvement of Leu616, Asp698, Cys694 and Cys828 residues in binding of G-11 to FLT3. The ability of G-11 to cause selective cytotoxicity and induce differentiation in cancer cells could be clinically relevant for therapeutic gains. PMID:26916980

  4. Nucleolipids of Canonical Purine ß‐d‐Ribo‐Nucleosides: Synthesis and Cytostatic/Cytotoxic Activities Toward Human and Rat Glioblastoma Cells

    PubMed Central

    Knies, Christine; Hammerbacher, Katharina; Kinscherf, Ralf

    2015-01-01

    Abstract We report on the synthesis of two series of canonical purine ß‐d‐ribonucleoside nucleolipids derived from inosine and adenosine, which have been characterized by elemental analyses, electrospray ionization mass spectrometry (ESI MS) as well as by 1H and 13C NMR, and pH‐dependent UV/Vis spectroscopy. A selection of the novel nucleolipids with different lipophilic moieties were first tested on their cytotoxic effect toward human macrophages. Compounds without a significant inhibitory effect on the viability of the macrophages were tested on their cytostatic/cytotoxic effect toward human astrocytoma/oligodendroglioma GOS‐3 cells as well as against the rat malignant neuroectodermal BT4Ca cell line. In order to additionally investigate the potential molecular mechanisms involved in the cytotoxic effects of the derivatives on GOS‐3 or BT4Ca cells, we evaluated the induction of apoptosis and observed the particular activity of the nucleolipid ethyl 3‐{4‐hydroxymethyl‐2‐methyl‐6‐[6‐oxo‐1‐(3,7,11‐trimethyl‐dodeca‐2,6,10‐trienyl)‐1,6‐dihydro‐purin‐9‐yl]‐tetrahydro‐furo[3,4‐d][1,3]dioxol‐2‐yl}propionate (8 c) toward both human and rat glioblastoma cell lines in vitro. PMID:27308225

  5. An efficient antigene activity and antiproliferative effect by targeting the Bcl-2 or survivin gene with triplex forming oligonucleotides containing a W-shaped nucleoside analogue (WNA-βT).

    PubMed

    Taniguchi, Yosuke; Sasaki, Shigeki

    2012-10-01

    Triplex forming oligonucleotides (TFOs) are some of the most promising tools in the antigene strategy for the development of gene targeting therapeutics. However, the stable triplex formation is restricted to the homopurine sequences consisting of purine nucleosides, dG and dA. Therefore, the T or dC nucleoside in the homopurine strand inhibits the stable triplex formation. We have developed W-shaped nucleoside analogues (WNAs) for the formation of the unnatural type triplex DNA, with sequences containing the interrupting site in an antiparallel triplex formation. In the present study, we tested the antigene effect of TFOs having WNA-βT, which increased the stability of the triplex formation with a target sequence including the TA interrupting site. We designed the GU TFO (WNA) and GU TFO (natural) for targeting sequences of the Bcl-2 or survivin oncogene. The gel shift assay showed that the TFO (WNA) formed more stable triplexes than the natural TFO. Remarkably, the Bcl-2- or survivin-targeted TFO (WNA) inhibited the cell proliferation and induced a caspase-dependent apoptosis. It was confirmed that the survivin-targeted TFO (WNA) more effectively decreased the number of survivin products in the A549 cell than the natural TFOs. PMID:22987068

  6. Binding of nucleotides to nucleoside diphosphate kinase: a calorimetric study.

    PubMed

    Cervoni, L; Lascu, I; Xu, Y; Gonin, P; Morr, M; Merouani, M; Janin, J; Giartosio, A

    2001-04-17

    The source of affinity for substrates of human nucleoside diphosphate (NDP) kinases is particularly important in that its knowledge could be used to design more effective antiviral nucleoside drugs (e.g., AZT). We carried out a microcalorimetric study of the binding of enzymes from two organisms to various nucleotides. Isothermal titration calorimetry has been used to characterize the binding in terms of Delta G degrees, Delta H degrees and Delta S degrees. Thermodynamic parameters of the interaction of ADP with the hexameric NDP kinase from Dictyostelium discoideum and with the tetrameric enzyme from Myxococcus xanthus, at 20 degrees C, were similar and, in both cases, binding was enthalpy-driven. The interactions of ADP, 2'deoxyADP, GDP, and IDP with the eukaryotic enzyme differed in enthalpic and entropic terms, whereas the Delta G degrees values obtained were similar due to enthalpy--entropy compensation. The binding of the enzyme to nonphysiological nucleotides, such as AMP--PNP, 3'deoxyADP, and 3'-deoxy-3'-amino-ADP, appears to differ in several respects. Crystallography of the protein bound to 3'-deoxy-3'-amino-ADP showed that the drug was in a distorted position, and was unable to interact correctly with active site side chains. The interaction of pyrimidine nucleoside diphosphates with the hexameric enzyme is characterized by a lower affinity than that with purine nucleotides. Titration showed the stoichiometry of the interaction to be abnormal, with 9--12 binding sites/hexamer. The presence of supplementary binding sites might have physiological implications. PMID:11294625

  7. Novel regulation of equlibrative nucleoside transporter 1 (ENT1) by receptor-stimulated Ca2+-dependent calmodulin binding.

    PubMed

    Bicket, Alex; Mehrabi, Pedram; Naydenova, Zlatina; Wong, Victoria; Donaldson, Logan; Stagljar, Igor; Coe, Imogen R

    2016-05-15

    Equilibrative nucleoside transporters (ENTs) facilitate the flux of nucleosides, such as adenosine, and nucleoside analog (NA) drugs across cell membranes. A correlation between adenosine flux and calcium-dependent signaling has been previously reported; however, the mechanistic basis of these observations is not known. Here we report the identification of the calcium signaling transducer calmodulin (CaM) as an ENT1-interacting protein, via a conserved classic 1-5-10 motif in ENT1. Calcium-dependent human ENT1-CaM protein interactions were confirmed in human cell lines (HEK293, RT4, U-87 MG) using biochemical assays (HEK293) and the functional assays (HEK293, RT4), which confirmed modified nucleoside uptake that occurred in the presence of pharmacological manipulations of calcium levels and CaM function. Nucleoside and NA drug uptake was significantly decreased (∼12% and ∼39%, respectively) by chelating calcium (EGTA, 50 μM; BAPTA-AM, 25 μM), whereas increasing intracellular calcium (thapsigargin, 1.5 μM) led to increased nucleoside uptake (∼26%). Activation of N-methyl-d-aspartate (NMDA) receptors (in U-87 MG) by glutamate (1 mM) and glycine (100 μM) significantly increased nucleoside uptake (∼38%) except in the presence of the NMDA receptor antagonist, MK-801 (50 μM), or CaM antagonist, W7 (50 μM). These data support the existence of a previously unidentified novel receptor-dependent regulatory mechanism, whereby intracellular calcium modulates nucleoside and NA drug uptake via CaM-dependent interaction of ENT1. These findings suggest that ENT1 is regulated via receptor-dependent calcium-linked pathways resulting in an alteration of purine flux, which may modulate purinergic signaling and influence NA drug efficacy. PMID:27009875

  8. Carbocyclic nucleoside analogues: classification, target enzymes, mechanisms of action and synthesis

    NASA Astrophysics Data System (ADS)

    Matyugina, E. S.; Khandazhinskaya, A. P.; Kochetkov, Sergei N.

    2012-08-01

    Key biological targets (S-adenosyl-L-homocysteine hydrolase, telomerase, human immunodeficiency virus reverse transcriptase, herpes virus DNA polymerase and hepatitis B virus DNA polymerase) and the mechanisms of action of carbocyclic nucleoside analogues are considered. Structural types of analogues are discussed. Methods of synthesis for the most promising compounds and the spectrum of their biological activities are described. The bibliography includes 126 references.

  9. Effects of nucleosides on glia - neuron interactions open up new vistas in the development of more effective antiepileptic drugs.

    PubMed

    Kovacs, Zsolt; Kardos, Julianna; Kekesi, Katalin A; Juhasz, Gabor; Lakatos, Renata; Heja, Laszlo

    2015-01-01

    One-third of epileptic patients are drug refractory due to the limited efficacy of antiepileptic therapy. Thus, there is an immense need to find more effective, safer and well-tolerated antiepileptic drugs. A great deal of results suggests that adenosine (Ado), guanosine (Guo), inosine (Ino) or uridine (Urd) are endogenous antiepileptogenic modulators. Furthermore, nucleosides and their derivatives may be safe and effective potential drugs in the treatment of epilepsy. Conversely, nucleosidergic modulatory system implying nucleoside levels, metabolism, receptors and transporters may also be involved in seizure pathomechanisms. Application of Ado receptor agonists as well as antagonists, elevation of nucleoside levels (e.g., by nucleoside metabolism inhibitors, and Adoreleasing implants) or utilization of non-Ado nucleosides may also turn to be useful approaches to decrease epileptic activity. However, all drugs exerting their effects on the nucleosidergic modulatory system may affect the fine regulation of glia-neuron interactions that are intimately governed by various nucleosidergic processes. Perturbation of the complex, bidirectional communication between neurons and astrocytes through these nucleosidergic modulatory mechanisms may lead to pathological changes in the central nervous system (CNS) and therefore may cause significant side effects. Thus, a deeper understanding of the nucleosidergic modulatory control over glia-neuron interactions is essential in order to develop more effective and safe nucleoside-based antiepileptic drugs. In this review article we focus on the role of Ado and Urd in glia-neuron interactions, placing emphasis on their implications for the treatment of epilepsy. PMID:25666791

  10. Involvement of concentrative nucleoside transporter 1 in intestinal absorption of trifluorothymidine, a novel antitumor nucleoside, in rats.

    PubMed

    Okayama, Takashige; Yoshisue, Kunihiro; Kuwata, Keizo; Komuro, Masahito; Ohta, Shigeru; Nagayama, Sekio

    2012-02-01

    ααα-Trifluorothymidine (TFT), an anticancer nucleoside analog, is a potent thymidylate synthase inhibitor. TFT exerts its antitumor activity primarily by inducing DNA fragmentation after incorporation of the triphosphate form of TFT into the DNA. Although an oral combination of TFT and a thymidine phosphorylase inhibitor has been clinically developed, there is little information regarding TFT absorption. Therefore, we investigated TFT absorption in the rat small intestine. After oral administration of TFT in rats, more than 75% of the TFT was absorbed. To identify the uptake transport system, uptake studies were conducted by using everted sacs prepared from rat small intestines. TFT uptake was saturable, significantly reduced under Na(+)-free conditions, and strongly inhibited by the addition of an endogenous pyrimidine nucleoside. From these results, we suggested the involvement of concentrative nucleoside transporters (CNTs) in TFT absorption into rat small intestine. In rat small intestines, the mRNAs coding for rat CNT1 (rCNT1) and rCNT2, but not for rCNT3, were predominantly expressed. To investigate the roles of rCNT1 and rCNT2 in TFT uptake, we conducted uptake assays by using Xenopus laevis oocytes injected with rCNT1 complementary RNA (cRNA) and rCNT2 cRNA. TFT uptake by X. laevis oocytes injected with rCNT1 cRNA, and not rCNT2 cRNA, was significantly greater than that by water-injected oocytes. In addition, in situ single-pass perfusion experiments performed using rat jejunum regions showed that thymidine, a substrate for CNT1, strongly inhibited TFT uptake. In conclusion, TFT is absorbed via rCNT1 in the intestinal lumen in rats. PMID:22076553

  11. 5-(1-Substituted) alkyl pyrimidine nucleosides as antiviral (herpes) agents.

    PubMed

    Kumar, Rakesh

    2004-10-01

    The treatment of viral diseases remains one of the major challenges to modern medicine. During the past two decades there has been increased recognition of the consequences of serious viral illnesses that are not controlled by vaccination. These illnesses include human immunodeficiency virus, human herpes viruses, and viruses that cause hepatitis. There are now eight pathogens recognized in the herpes virus family that cause infections in humans. Infections by the herpes viruses are opportunistic and often life-threatening, leading to significant morbidity and mortality in the increasing number of chronically immune compromised individuals such as AIDS patients, cancer patients and transplant recipients on immunosuppressive therapy. Nearly all individuals with AIDS are infected with one or more of the herpes viruses. Antiviral therapy with guanosine nucleoside analogs acyclovir and ganciclovir has had a major impact on diseases caused by herpes simplex virus type-1 and type-2 (HSV-1, HSV-2), Varicella zoster virus (VZV), and human cytomegalovirus (HCMV) but development of resistant virus strains and the absence of any effective treatment for other members of the herpes family provide a stimulus for increased search of new agents effective against various herpes viruses. Pyrimidine nucleosides have taken up an important role in the therapy of virus infection. Significant progress in the study of anti-herpes nucleosides has been made by the advent of 5-substituted pyrimidine nucleosides such as 5-iodo-, 5-ethyl-, 5-(2-chloroethyl)-, and (E)-5-(2-bromovinyl)- derivatives of 2'-deoxyuridine. These are highly specific inhibitors of HSV-1, HSV-2, and/or VZV infections. However, Epstein Barr virus (EBV) and HCMV are much less sensitive to these agents. In 5-substituted pyrimidine nucleosides the nature of substituents, particularly at the C-5 position, has been found to be an important determinant of anti-herpes activity. Structural requirements at the C-2 carbon of the 5

  12. Characterization of nucleoside transport systems in cultured rat epididymal epithelium.

    PubMed

    Leung, G P; Ward, J L; Wong, P Y; Tse, C M

    2001-05-01

    The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na(+) dependent, while 30% was Na(+) independent. The Na(+)-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na(+)-dependent [(3)H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na(+)-dependent [(3)H]guanosine uptake was inhibited by thymidine by 20% and Na(+)-dependent [(3)H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na(+)-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma. PMID:11287319

  13. Synthesis of purine and 7-deazapurine nucleoside analogues of 6-N-(4-Nitrobenzyl)adenosine; inhibition of nucleoside transport and proliferation of cancer cells.

    PubMed

    Rayala, Ramanjaneyulu; Theard, Patricia; Ortiz, Heysell; Yao, Sylvia; Young, James D; Balzarini, Jan; Robins, Morris J; Wnuk, Stanislaw F

    2014-09-01

    Human equilibrative nucleoside transporter 1 (hENT1) is a prototypical nucleoside transporter protein ubiquitously expressed on the cell surface of almost all human tissue. Given the role of hENT1 in the transport of nucleoside drugs, an important class of therapeutics in the treatment of various cancers and viral infections, efforts have been made to better understand the mechanisms by which hENT1 modulates nucleoside transport. To that end, we report here the design and synthesis of novel tool compounds for the further study of hENT1. The 7-deazapurine nucleoside antibiotic tubercidin was converted into its 4-N-benzyl and 4-N-(4-nitrobenzyl) derivatives by alkylation at N3 followed by a Dimroth rearrangement to the 4-N-isomer or by fluoro-diazotization followed by SN Ar displacement of the 4-fluoro group by a benzylamine. The 4-N-(4-nitrobenzyl) derivatives of sangivamycin and toyocamycin antibiotics were prepared by the alkylation approach. Cross-membrane transport of labeled uridine by hENT1 was inhibited to a weaker extent by the 4-nitrobenzylated tubercidin and sangivamycin analogues than was observed with 6-N-(4-nitrobenzyl)adenosine. Type-specific inhibition of cancer cell proliferation was observed at micromolar concentrations with the 4-N-(4-nitrobenzyl) derivatives of sangivamycin and toyocamycin, and also with 4-N-benzyltubercidin. Treatment of 2',3',5'-O-acetyladenosine with aryl isocyanates gave the 6-ureido derivatives but none of them exhibited inhibitory activity against cancer cell proliferation or hENT1. PMID:24788480

  14. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA.

    PubMed

    Karikó, Katalin; Buckstein, Michael; Ni, Houping; Weissman, Drew

    2005-08-01

    DNA and RNA stimulate the mammalian innate immune system through activation of Toll-like receptors (TLRs). DNA containing methylated CpG motifs, however, is not stimulatory. Selected nucleosides in naturally occurring RNA are also methylated or otherwise modified, but the immunomodulatory effects of these alterations remain untested. We show that RNA signals through human TLR3, TLR7, and TLR8, but incorporation of modified nucleosides m5C, m6A, m5U, s2U, or pseudouridine ablates activity. Dendritic cells (DCs) exposed to such modified RNA express significantly less cytokines and activation markers than those treated with unmodified RNA. DCs and TLR-expressing cells are potently activated by bacterial and mitochondrial RNA, but not by mammalian total RNA, which is abundant in modified nucleosides. We conclude that nucleoside modifications suppress the potential of RNA to activate DCs. The innate immune system may therefore detect RNA lacking nucleoside modification as a means of selectively responding to bacteria or necrotic tissue. PMID:16111635

  15. Equilibrium of Phosphointermediates of Sodium and Potassium Ion Transport Adenosine Triphosphatase

    PubMed Central

    Suzuki, Kuniaki; Post, Robert L.

    1997-01-01

    Sodium and potassium ion transport adenosine triphosphatase accepts and donates a phosphate group in the course of its reaction sequence. The phosphorylated enzyme has two principal reactive states, E1P and E2P. E1P is formed reversibly from ATP in the presence of Na+ and is precursor to E2P, which equilibrates with Pi in the presence of K+. We studied equilibrium between these states at 4°C and the effect of Na+ on it. To optimize the reaction system we used a Hofmeister effect, replacing the usual anion, chloride, with a chaotropic anion, usually nitrate. We phosphorylated enzyme from canine kidney with [32P]ATP. We estimated interconversion rate constants for the reaction E1P ⇌ E2P and their ratio. To estimate rate constants we terminated phosphorylation and observed decay kinetics. We observed E1P or E2P selectively by adding K+ or ADP respectively. K+ dephosphorylates E2P leaving E1P as observable species; ADP dephosphorylates E1P leaving E2P as observable species. We fitted a 2-pool model comprising two reactive species or a twin 2-pool model, comprising a pair of independent 2-pool models, to the data and obtained interconversion and hydrolysis rate constants for each state. Replacing Na+ with Tris+ or lysine+ did not change the ratio of interconversion rate constants between E1P and E2P. Thus Na+ binds about equally strongly to E1P and E2P. This conclusion is consistent with a model of Pedemonte (1988. J. Theor. Biol. 134:165–182.). We found that Na+ affected another equilibrium, that of transphosphorylation between ATP·dephosphoenzyme and ADP·E1P. We used the reactions and model of Pickart and Jencks (1982. J. Biol. Chem. 257:5319–5322.) to generate and fit data. Decreasing the concentration of Na+ 10-fold shifted the equilibrium constant 10-fold favoring ADP·E1P over ATP·dephosphoenzyme. Thus Na+ can dissociate from E1P·Na3. Furthermore, we found two characteristics of Hofmeister effects on this enzyme. PMID:9154903

  16. Kinetic and biochemical characterization of Trypanosoma evansi nucleoside triphosphate diphosphohydrolase.

    PubMed

    Weiss, Paulo Henrique Exterchoter; Batista, Franciane; Wagner, Glauber; Magalhães, Maria de Lourdes Borba; Miletti, Luiz Claudio

    2015-06-01

    Nucleoside triphosphate diphospho-hydrolases (NTPDases) catalyze the hydrolysis of several nucleosides tri and diphosphate playing major roles in eukaryotes including purinergic signaling, inflammation, hemostasis, purine salvage and host-pathogen interactions. These enzymes have been recently described in parasites where several evidences indicated their involvement in virulence and infection. Here, we have investigated the presence of NTPDase in the genome of Trypanosoma evansi. Based on the genomic sequence from Trypanosoma brucei, we have amplified an 1812 gene fragment corresponding to the T. evansi NTPDase gene. The protein was expressed in the soluble form and purified to homogeneity and enzymatic assays were performed confirming the enzyme identity. Kinetic parameters and substrate specificity were determined. The dependence of cations on enzymatic activity was investigated indicating the enzyme is stimulated by divalent cations and carbohydrates but inhibited by sodium. Bioinformatic analysis indicates the enzyme is a membrane bound protein facing the extracellular side of the cell with 98% identity to the T. brucei homologous NTPDase gene. PMID:25819299

  17. Summary: the modified nucleosides of RNA.

    PubMed Central

    Limbach, P A; Crain, P F; McCloskey, J A

    1994-01-01

    A comprehensive listing is made of posttranscriptionally modified nucleosides from RNA reported in the literature through mid-1994. Included are chemical structures, common names, symbols, Chemical Abstracts registry numbers (for ribonucleoside and corresponding base), Chemical Abstracts Index Name, phylogenetic sources, and initial literature citations for structural characterization or occurrence, and for chemical synthesis. The listing is categorized by type of RNA: tRNA, rRNA, mRNA, snRNA, and other RNAs. A total of 93 different modified nucleosides have been reported in RNA, with the largest number and greatest structural diversity in tRNA, 79; and 28 in rRNA, 12 in mRNA, 11 in snRNA and 3 in other small RNAs. PMID:7518580

  18. Nucleoside diphosphate kinase of Saccharomyces cerevisiae, Ynk1p: localization to the mitochondrial intermembrane space.

    PubMed Central

    Amutha, Boominathan; Pain, Debkumar

    2003-01-01

    Nucleoside diphosphate kinase (NDPK) is a highly conserved multifunctional enzyme. It catalyses the transfer of gamma phosphates from nucleoside triphosphates to nucleoside diphosphates by a mechanism that involves formation of an autophosphorylated enzyme intermediate. The phosphate is usually supplied by ATP. NDPK activity in different subcellular compartments may regulate the crucial balance between ATP and GTP or other nucleoside triphosphates. NDPKs are homo-oligomeric proteins and are predominantly localized in the cytosol. In this paper, we demonstrate that in Saccharomyces cerevisiae a small fraction of total NDPK activity encoded by YNK1 is present in the intermembrane space (IMS) of mitochondria, and the corresponding protein Ynk1p in the IMS represents approx. 0.005% of total mitochondrial proteins. Ynk1p, synthesized as a single gene product, must therefore be partitioned between cytoplasm and mitochondrial IMS fractions. A mechanism for this partitioning is suggested by our observations that interaction with a 40 kDa protein of the translocase of outer mitochondrial membrane (Tom40p), occurs preferentially with unfolded, unphosphorylated forms of Ynk1p. A population of newly translated, but not yet folded or autophosphorylated, Ynk1p intermediates may be imported into the IMS of mitochondria and trapped there by subsequent folding and oligomerization. Within the small volume of the IMS, Ynk1p may be more concentrated and may be required to supply GTP to several important proteins in this compartment. PMID:12472466

  19. 2'-modified nucleosides for site-specific labeling of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Krider, Elizabeth S.; Miller, Jeremiah E.; Meade, Thomas J.

    2002-01-01

    We report the synthesis of 2'-modified nucleosides designed specifically for incorporating labels into oligonucleotides. Conversion of these nucleosides to phosphoramidite and solid support-bound derivatives proceeds in good yield. Large-scale synthesis of 11-mer oligonucleotides possessing the 2'-modified nucleosides is achieved using these derivatives. Thermal denaturation studies indicate that the presence of 2'-modified nucleosides in 11-mer duplexes has minimal destabilizing effects on the duplex structure when the nucleosides are placed at the duplex termini. The powerful combination of phosphoramidite and support-bound derivatives of 2'-modified nucleosides affords the large-scale preparation of an entirely new class of oligonucleotides. The ability to synthesize oligonucleotides containing label attachment sites at 3', intervening, and 5' locations of a duplex is a significant advance in the development of oligonucleotide conjugates.

  20. Stable expression of a recombinant sodium-dependent, pyrimidine-selective nucleoside transporter (CNT1) in a transport-deficient mouse leukemia cell line.

    PubMed

    Crawford, C R; Cass, C E; Young, J D; Belt, J A

    1998-01-01

    Previous studies of nucleoside transport in mammalian cells have identified two types of activities: the equilibrative nucleoside transporters and concentrative, Na+-nucleoside cotransporters. Characterization of the concentrative nucleoside transporters has been hampered by the presence in most cells and tissues of multiple transporters with overlapping permeant specificities. With the recent cloning of cDNAs encoding rat and human members of the concentrative nucleoside transporter (CNT) family, it is now possible to study the concentrative transporters in isolation by use of functional expression systems. We report here the isolation of a nucleoside transport-deficient subline of L1210 mouse leukemia (L1210/DNC3) that is a suitable recipient for stable expression of cloned nucleoside transporter cDNAs. We have used L1210/DNC3 as the recipient in gene transfer studies to develop a stable cell line (L1210/DU5) that produces the recombinant concentrative nucleoside transporter with selectivity for pyrimidine nucleosides (CNT1) that was initially identified in rat intestine (Q.Q. Huang, S.Y. Yao, M.W. Ritzel, A.R.P. Paterson, C.E. Cass, and J.D. Young. 1994. J. Biol. Chem. 269: 17,757-17,760). L1210/DU5 was used to examine the permeant selectivity of recombinant rat CNT1 by comparing a series of nucleoside analogs with respect to (i) inhibition of inward fluxes of [3H]thymidine, (ii) initial rates of transport of 3H-analog, and (iii) cytotoxicity to L1210/DU5 versus the parental transport-deficient cell line. By all three criteria, recombinant CNT1 transported 5-fluoro-2'-deoxyuridine and 5-fluorouridine well and cytosine arabinoside poorly. Although some purine nucleosides (2'-deoxyadenosinedeoxyadeno-2'-deoxyadenosine, 7-deazaadenosine) were potent inhibitors of CNT1, they were poor permeants when uptake was measured directly by analysis of isotopic fluxes or indirectly by comparison of cytotoxicity ratios. We conclude that comparison of analog cytotoxicity to L

  1. Na+-dependent nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells.

    PubMed

    Felipe, A; Valdes, R; Santo, B; Lloberas, J; Casado, J; Pastor-Anglada, M

    1998-03-01

    Hepatocytes show a Na+-dependent nucleoside transport activity that is kinetically heterogeneous and consistent with the expression of at least two independent concentrative Na+-coupled nucleoside transport systems (Mercader et al. Biochem. J. 317, 835-842, 1996). So far, only a single nucleoside carrier-related cDNA (SPNT) has been isolated from liver cells (Che et al. J. Biol. Chem. 270, 13596-13599, 1995). This cDNA presumably encodes a plasma membrane protein responsible for Na+-dependent purine nucleoside transport activity. Thus, the liver must express, at least, a second nucleoside transporter which should be pyrimidine-preferring. Homology cloning using RT-PCR revealed that a second isoform is indeed present in liver. This second isoform turned out to be identical to the 'epithelial-specific isoform' called cNT1, which shows in fact high specificity for pyrimidine nucleosides. Although cNT1 mRNA is present at lower amounts than SPNT mRNA, the amounts of cNT1 protein, when measured using isoform-specific polyclonal antibodies, were even higher than the SPNT protein levels. Moreover, partially purified basolateral plasma membrane vesicles from liver were enriched in the SPNT but not in the cNT1 protein, which suggests that the subcellular localization of these carrier proteins is different. SPNT and cNT1 protein amounts in crude membrane extracts from 6 h-regenerating rat livers are higher than in the preparations from sham-operated controls (3.5- and 2-fold, respectively). These results suggest that liver parenchymal cells express at least two different isoforms of concentrative nucleoside carriers, the cNT1 and SPNT proteins, which show differential regulation and subcellular localization. PMID:9480921

  2. Na+-dependent nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells.

    PubMed Central

    Felipe, A; Valdes, R; Santo, B; Lloberas, J; Casado, J; Pastor-Anglada, M

    1998-01-01

    Hepatocytes show a Na+-dependent nucleoside transport activity that is kinetically heterogeneous and consistent with the expression of at least two independent concentrative Na+-coupled nucleoside transport systems (Mercader et al. Biochem. J. 317, 835-842, 1996). So far, only a single nucleoside carrier-related cDNA (SPNT) has been isolated from liver cells (Che et al. J. Biol. Chem. 270, 13596-13599, 1995). This cDNA presumably encodes a plasma membrane protein responsible for Na+-dependent purine nucleoside transport activity. Thus, the liver must express, at least, a second nucleoside transporter which should be pyrimidine-preferring. Homology cloning using RT-PCR revealed that a second isoform is indeed present in liver. This second isoform turned out to be identical to the 'epithelial-specific isoform' called cNT1, which shows in fact high specificity for pyrimidine nucleosides. Although cNT1 mRNA is present at lower amounts than SPNT mRNA, the amounts of cNT1 protein, when measured using isoform-specific polyclonal antibodies, were even higher than the SPNT protein levels. Moreover, partially purified basolateral plasma membrane vesicles from liver were enriched in the SPNT but not in the cNT1 protein, which suggests that the subcellular localization of these carrier proteins is different. SPNT and cNT1 protein amounts in crude membrane extracts from 6 h-regenerating rat livers are higher than in the preparations from sham-operated controls (3.5- and 2-fold, respectively). These results suggest that liver parenchymal cells express at least two different isoforms of concentrative nucleoside carriers, the cNT1 and SPNT proteins, which show differential regulation and subcellular localization. PMID:9480921

  3. L-Aspartic and l-glutamic acid ester-based ProTides of anticancer nucleosides: Synthesis and antitumoral evaluation.

    PubMed

    Gao, Ling-Jie; De Jonghe, Steven; Daelemans, Dirk; Herdewijn, Piet

    2016-05-01

    A series of novel aryloxyphosphoramidate nucleoside prodrugs based on l-aspartic acid and l-glutamic acid as amino acid motif has been synthesized and evaluated for antitumoral activity. Depending on the cancer cell line studied and on the nature of the parent nucleoside compound (gemcitabine, 5-iodo-2'-deoxy-uridine, floxuridine or brivudin), the corresponding ProTides are endowed with an improved or decreased cytotoxic activity. PMID:27032331

  4. Isolation of Enzymatically Active Replication Complexes from Feline Calicivirus-Infected Cells

    PubMed Central

    Green, Kim Y.; Mory, Aaron; Fogg, Mark H.; Weisberg, Andrea; Belliot, Gaël; Wagner, Mariam; Mitra, Tanaji; Ehrenfeld, Ellie; Cameron, Craig E.; Sosnovtsev, Stanislav V.

    2002-01-01

    A membranous fraction that could synthesize viral RNA in vitro in the presence of magnesium salt, ribonucleotides, and an ATP-regenerating system was isolated from feline calicivirus (FCV)-infected cells. The enzymatically active component of this fraction was designated FCV replication complexes (RCs), by analogy to other positive-strand RNA viruses. The newly synthesized RNA was characterized by Northern blot analysis, which demonstrated the production of both full-length (8.0-kb) and subgenomic-length (2.5-kb) RNA molecules similar to those synthesized in FCV-infected cells. The identity of the viral proteins associated with the fraction was investigated. The 60-kDa VP1 major capsid protein was the most abundant viral protein detected. VP2, a minor structural protein encoded by open reading frame 3 (ORF3), was also present. Nonstructural proteins associated with the fraction included the precursor polypeptides Pro-Pol (76 kDa) and p30-VPg (43 kDa), as well as the mature nonstructural proteins p32 (derived from the N-terminal region of the ORF1 polyprotein), p30 (the putative “3A-like” protein), and p39 (the putative nucleoside triphosphatase). The isolation of enzymatically active RCs containing both viral and cellular proteins should facilitate efforts to dissect the contributions of the virus and the host to FCV RNA replication. PMID:12163578

  5. Radiolabeled nucleoside analogs in cancer diagnosis and therapy.

    PubMed

    Kassis, A I; Adelstein, S J; Mariani, G

    1996-09-01

    Radiolabeled nucleosides, specifically 5-iodo-2'-deoxyuridine (IUdR) radioiodinated with the Auger-electronemitting 123I or 125I, have been shown to produce extensive DNA damage in mammalian cell systems in vitro. Such nucleosides are cycle-dependent agents that are taken up by mitotically dividing cells in the S phase of the cell cycle. The degree of damage that occurs is related to the fact that these nucleosides bind covalently to DNA bringing the decaying Augerelectron-emitting radionuclide in close proximity to the genome. The use of these radiohalogenated nucleosides in vivo is associated with several problems. The first relates to their extremely short biologic half-life in blood (T1/2 of minutes in humans). The second involves achieving therapeutic ratios in tumor cells in the face of efficient hepatic dehalogenation. The third concerns the uptake of these radiopharmaceuticals by actively proliferating normal cell renewal systems, thus potentially causing toxic side effects. The fourth, one shared with other cycle-dependent drugs, relates to the matter of labeling the whole tumor cell population. To facilitate targeting to tumors, investigators have been examining the direct introduction of these agents into the targeted area or into an arterial blood supply that immediately precedes the target. For example, radiopharmaceutical administration could be intracavitary (bladder, spinal fluid, peritoneum), intralesional (brain tumor, breast mass) or intra-arterial (liver, pancreas). In all these situations, the following conditions must be met: (a) once within the vicinity of the tumor the agent can freely diffuse through the tissues and is selectively taken up by cancerous cells; (b) once the agent has left the target area it is converted quickly into a nontoxic form and/or excreted from the body; and finally, (c) the biologic behavior of the agent is not altered by repeated injections. We report herein our experience and that of others with [123I/125I/131I

  6. Purification and characterization of purine nucleoside phosphorylase from developing embryos of Hyalomma dromedarii.

    PubMed

    Kamel, M Y; Fahmy, A S; Ghazy, A H; Mohamed, M A

    1991-04-01

    Purine nucleoside phosphorylase from Hyalomma dromedarii, the camel tick, was purified to apparent homogeneity. A molecular weight of 56,000 - 58,000 was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. Unlike purine nucleoside phosphorylase preparations from other tissues, the H. dromedarii enzyme was unstable in the presence of beta-mercaptoethanol. The enzyme had a sharp pH optimum at pH 6.5. It catalyzed the phosphorolysis and arsenolysis of ribo- and deoxyribo-nucleosides of hypoxanthine and guanine, but not of adenine or pyrimidine nucleosides. The Km values of the enzyme at the optimal pH for inosine, deoxyinosine, guanosine, and deoxyguanosine were 0.31, 0.67, 0.55, and 0.33 mM, respectively. Inactivation and kinetic studies suggested that histidine and cysteine residues were essential for activity. The pKa values determined for catalytic ionizable groups were 6-7 and 8-9. The enzyme was completely inactivated by thiol reagents and reactivated by excess beta-mercaptoethanol. The enzyme was also susceptible to pH-dependent photooxidation in the presence of methylene blue, implicating histidine. Initial velocity studies showed an intersecting pattern of double-reciprocal plots of the data, consistent with a sequential mechanism. PMID:1905141

  7. Base release in nucleosides induced by low-energy electrons: a DFT study.

    PubMed

    Li, Xifeng; Sanche, Léon; Sevilla, Michael D

    2006-06-01

    Low-energy electrons are known to induce strand breaks and base damage in DNA and RNA through fragmentation of molecular bonding. Recently the glycosidic bond cleavage of nucleosides by low-energy electrons has been reported. These experimental results call for a theoretical investigation of the strength of the C(1)'-N link in nucleosides (dA, dC and dT) between the base and deoxyribose before and after electron attachment. Through density functional theory (DFT) calculations, we compare the C(1)'-N bond strength, i.e., the bond dissociation energy of the neutral and its anionic radical, and find that an excess electron effectively weakens the C(1)'- N bond strength in nucleosides by 61-75 kcal/mol in the gas phase and 76-83 kcal/mol in the solvated environment. As a result, electron-induced fragmentation of the C(1)'-N bond in the gas phase is exergonic for dA (DeltaG=-14 kcal/mol) and for dT (DeltaG=-6 kcal/mol) and is endergonic (DeltaG=+1 kcal/ mol) only for dC. In the gas phase all the anionic nucleosides are found to be in valence states. Solvation is found to increase the exergonic nature by an additional 20 kcal, making the fragmentation both exothermic and exergonic for all nucleoside anion radicals. Thus C(1)'-N bond breaking in nucleoside anion radicals is found to be thermodynamically favorable both in the gas phase and under solvation. The activation barrier for the C(1)'-N bond breaking process was found to be about 20 kcal/mol in every case examined, suggesting that a 1 eV electron would induce spontaneous cleavage of the bond and that stabilized anion radicals on the DNA strand would undergo base release at only a modest rate at room temperature. These results suggest that base release from nucleosides and DNA is an expected consequence of low-energy electron-induced damage but that the high barrier would inhibit this process in the stable anion radicals. PMID:16802873

  8. Expression of nucleoside transporter in freshly isolated neurons and astrocytes from mouse brain.

    PubMed

    Li, B; Gu, L; Hertz, L; Peng, L

    2013-11-01

    Nucleoside transporters comprise equilibrative ENT1-4 and concentrative CNT1-3. CNTs transport against an intracellular/extracellular gradient and are essential for transmitter removal, independently of metabolic need. ENT1-4 mediate transport until intracellular/extracellular equilibrium of the transported compound, but are very efficient, when the accumulated nucleoside or nucleobase is rapidly eliminated by metabolism. Most nucleoside transporters are membrane-bound, but ENT3 is mainly intracellular. This study uses freshly isolated neurons and astrocytes from two adult mouse strains. In one transgenic strain the neuronal marker Thy1 was associated with a compound fluorescing at one wavelength, and in the other the astrocytic marker GFAP was associated with a compound fluorescent at a different wavelength. Highly purified astrocytic and neuronal populations (as determined by presence/absence of cell-specific genes) were obtained from these mice by fluorescence-activated cell sorting. In each population mRNA analysis was performed by reverse-transcription polymerase chain reaction. CNT1 was absent in both cell types; all other nucleoside transporters were expressed to at least a similar degree (in relation to applied amount of RNA and to a house-keeping gene) in astrocytes as in neurons. Astrocytic ENT3 enrichment was dramatic, but it was not up-regulated after fluoxetine-mediated increase in DNA synthesis. A comparison with results obtained in cultured astrocytes shows that the latter are generally compatible with the present findings and suggests that many observations obtained in intact tissue, mainly by in situ hybridization (which also determines mRNA expression) may underestimate astrocytic nucleoside transporter expression. PMID:24026568

  9. Reduced Ribavirin Antiviral Efficacy via Nucleoside Transporter-Mediated Drug Resistance▿

    PubMed Central

    Ibarra, Kristie D.; Pfeiffer, Julie K.

    2009-01-01

    Treatment for hepatitis C virus infection currently consists of pegylated interferon and ribavirin (RBV), a nucleoside analog. Although RBV clearly plays a role in aiding the treatment response, its antiviral mechanism is unclear. Regardless of the specific mechanism of RBV, we hypothesize that differences in levels of cellular uptake of RBV may affect antiviral efficacy and treatment success and that cells may become RBV resistant through reduced uptake. We monitored RBV uptake in various cell lines and determined the effect of uptake capacity on viral replication. RBV-resistant cells demonstrated reduced RBV uptake and increased growth of a model RNA virus, poliovirus, in the presence of RBV. Overexpression of equilibrative nucleoside transporter 1 (ENT1) or concentrative nucleoside transporter 3 (CNT3) increased RBV uptake in RBV-sensitive cell lines and restored the uptake defect in most RBV-resistant cell lines. However, CNT3 is not expressed in Huh-7 liver cells, and inhibition of concentrative transport did not affect RBV uptake. Blocking equilibrative transport using the inhibitor nitrobenzylmercaptopurine riboside recapitulated the RBV-resistant phenotype in RBV-sensitive cell lines, with a reduction in RBV uptake and increased poliovirus growth. Taken together, these results indicate that RBV uptake is restricted primarily to ENT1 in the cell lines examined. Interestingly, some RBV-resistant cell lines may compensate for reduced ENT1-mediated nucleoside uptake by increasing the activity of an alternative nucleoside transporter, ENT2. It is possible that RBV uptake affects the antiviral treatment response, either through natural differences in patients or through acquired resistance. PMID:19244331

  10. Hybridization accompanying FRET event in labeled natural nucleoside-unnatural nucleoside containing chimeric DNA duplexes.

    PubMed

    Bag, Subhendu Sekhar; Das, Suman K; Pradhan, Manoj Kumar; Jana, Subhashis

    2016-09-01

    Förster resonance energy transfer (FRET) is a highly efficient strategy in illuminating the structures, structural changes and dynamics of DNA, proteins and other biomolecules and thus is being widely utilized in studying such phenomena, in designing molecular/biomolecular probes for monitoring the hybridization event of two single stranded DNA to form duplex, in gene detection and in many other sensory applications in chemistry, biology and material sciences. Moreover, FRET can give information about the positional status of chromophores within the associated biomolecules with much more accuracy than other methods can yield. Toward this end, we want to report here the ability of fluorescent unnatural nucleoside, triazolylphenanthrene ((TPhen)BDo) to show FRET interaction upon hybridization with fluorescently labeled natural nucleosides, (Per)U or (OxoPy)U or (Per)U, forming two stable chimeric DNA duplexes. The pairing selectivity and the thermal duplex stability of the chimeric duplexes are higher than any of the duplexes with natural nucleoside formed. The hybridization results in a Förster resonance energy transfer (FRET) from donor triazolylphenanthrene of (TPhen)BDo to acceptor oxopyrene of (OxoPy)U and/or to perylene chromophore of (Per)U, respectively, in two chimeric DNA duplexes. Therefore, we have established the FRET process in two chimeric DNA duplexes wherein a fluorescently labeled natural nucleoside ((OxoPy)U or (Per)U) paired against an unnatural nucleoside ((TPhen)BDo) without sacrificing the duplex stability and B-DNA conformation. The hybridization accompanying FRET event in these classes of interacting fluorophores is new. Moreover, there is no report of such designed system of chimeric DNA duplex. Our observed phenomenon and the design can potentially be exploited in designing more of such efficient FRET pairs for useful application in the detection and analysis of biomolecular interactions and in material science application. PMID:27498231

  11. Multistep virtual screening for rapid and efficient identification of non-nucleoside bacterial thymidine kinase inhibitors.

    PubMed

    Zander, Johannes; Hartenfeller, Markus; Hähnke, Volker; Proschak, Ewgenij; Besier, Silke; Wichelhaus, Thomas A; Schneider, Gisbert

    2010-08-16

    Antimicrobial activity of trimethoprim/sulfamethoxazole (SXT) against Staphylococcus aureus (S. aureus) is antagonized by thymidine, which is abundant in infected or inflamed human tissue. To restore the antimicrobial activity of SXT in the presence of thymidine, we screened for small-molecule inhibitors of S. aureus thymidine kinase with non-nucleoside scaffolds. We present the successful application of an adaptive virtual screening protocol for novel antibiotics using a combination of ligand- and structure-based approaches. Two consecutive rounds of virtual screening and in vitro testing were performed that resulted in several non-nucleoside hits. The most potent compound exhibits substantial antimicrobial activity against both methicillin-resistant S. aureus strain ATCC 700699 and nonresistant strain ATCC 29213, when combined with SXT in the presence of thymidine. This study demonstrates how virtual screening can be used to guide hit finding in antibacterial screening campaigns with minimal experimental effort. PMID:20648496

  12. Inosine triphosphatase allele frequency and association with ribavirin-induced anaemia in Brazilian patients receiving antiviral therapy for chronic hepatitis C

    PubMed Central

    Delvaux, Nathália; da Costa, Vanessa Duarte; da Costa, Maristella Matos; Villar, Livia Melo; Coelho, Henrique Sérgio Moraes; Esberard, Eliane Bordalo Cathalá; Flores, Priscila Pollo; Brandão-Mello, Carlos Eduardo; Villela-Nogueira, Cristiane Alves; de Almeida, Adilson José; Lampe, Elisabeth

    2015-01-01

    Inosine triphosphatase (ITPA) single nucleotide polymorphisms (SNPs) are strongly associated with protection against ribavirin (RBV)-induced anaemia in European, American and Asian patients; however, there is a paucity of data for Brazilian patients. The aim of this study was to evaluate the ITPA SNP (rs7270101/rs1127354) frequency in healthy and hepatitis C virus (HCV)-infected patients from Brazil and the association with the development of severe anaemia during antiviral therapy. ITPA SNPs were determined in 200 HCV infected patients and 100 healthy individuals by sequencing. Biochemical parameters and haemoglobin (Hb) levels were analysed in 97 patients who underwent antiviral therapy. A combination of AArs7270101+CCrs1127354 (100% ITPase activity) was observed in 236/300 individuals. Anaemia was observed in 87.5% and 86.2% of treated patients with AA (rs7270101) and CC genotypes (rs1127354), respectively. Men with AA (rs7270101) showed a considerable reduction in Hb at week 12 compared to those with AC/CC (p = 0.1475). In women, there was no influence of genotype (p = 0.5295). For rs1127354, men with the CC genotype also showed a sudden reduction in Hb compared to those with AC. Allelic distribution of rs7270101 and rs1127354 shows high rates of the genotypes AA and CC, respectively, suggesting that the study population had a great propensity for developing RBV-induced anaemia. A progressive Hb reduction during treatment was observed; however, this reduction was greater in men at week 12 than in women. PMID:26154744

  13. The halo-substituent effect on Pseudomonas cepacia lipase-mediated regioselective acylation of nucleosides: A comparative investigation.

    PubMed

    Wang, Zhao-Yu; Bi, Yan-Hong; Yang, Rong-Ling; Duan, Zhang-Qun; Nie, Ling-Hong; Li, Xiang-Qian; Zong, Min-Hua; Wu, Jie

    2015-10-20

    In this work, comparative experiments were explored to investigate the substrate specificity of Pseudomonas cepacia lipase in regioselective acylation of nucleosides carrying various substituents (such as the H, F, Cl, Br, I) at 2'- and 5-positions. Experimental data indicated that the catalytic performance of the enzyme depended very much on the halo-substituents in nucleosides. The increased bulk of 2'-substituents in ribose moiety of the nucleoside might contribute to the improved 3'-regioselectivity (90-98%, nucleosides a-d) in enzymatic decanoylation, while the enhancement of regioselectivity (93-99%) in 3'-O-acylated nucleosides e-h could be attributable to the increasing hydrophobicity of the halogen atoms at 5-positions. With regard to the chain-length selectivity, P. cepacia lipase displayed the highest 3'-regioselectivity toward the longer chain (C14) as compared to shorter (C6 and C10) ones. The position, orientation and property of the substituent, specific structure of the lipase's active site, and acyl structure could account for the diverse results. PMID:26325198

  14. FUN26 (function unknown now 26) protein from saccharomyces cerevisiae is a broad selectivity, high affinity, nucleoside and nucleobase transporter.

    PubMed

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Duggan, Kelli D; Roe-Žurž, Zygy; Schmitz, Hannah; Burleson, Carter; Hays, Franklin A

    2014-08-29

    Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2')- and C(5')-positions on the ribose sugar and is not stimulated by a membrane pH differential. [(3)H]Adenine nucleobase transport efficiency is increased ∼4-fold relative to nucleosides tested with no observed [(3)H]adenosine or [(3)H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system. PMID:25035431

  15. FUN26 (Function Unknown Now 26) Protein from Saccharomyces cerevisiae Is a Broad Selectivity, High Affinity, Nucleoside and Nucleobase Transporter*

    PubMed Central

    Boswell-Casteel, Rebba C.; Johnson, Jennifer M.; Duggan, Kelli D.; Roe-Žurž, Zygy; Schmitz, Hannah; Burleson, Carter; Hays, Franklin A.

    2014-01-01

    Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2′)- and C(5′)-positions on the ribose sugar and is not stimulated by a membrane pH differential. [3H]Adenine nucleobase transport efficiency is increased ∼4-fold relative to nucleosides tested with no observed [3H]adenosine or [3H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system. PMID:25035431

  16. Nonenantioselectivity Property of Human Deoxycytidine Kinase Explained by Structures of the Enzyme in Complex with [subscript L]- and [subscript D]-Nucleosides

    SciTech Connect

    Sabini, Elisabetta; Hazra, Saugata; Konrad, Manfred; Lavie, Arnon

    2008-07-31

    Biological molecules are predominantly enantioselective. Yet currently two nucleoside analogue prodrugs (3TC and FTC) with opposite chirality compared to physiological nucleosides are clinically approved for the treatment of HIV infections. These prodrugs require conversion to their triphosphorylated forms to achieve pharmacological activity. The first step in the activation of these agents is catalyzed by human deoxycytidine kinase (dCK). This enzyme possesses the ability to phosphorylate nucleosides of the unnatural L-chirality. To understand the molecular basis for the nonenantioselectivity of dCK, we solved the crystal structures of the enzyme in complex with the L-enantiomer and of its physiological substrate deoxycytidine and with the L-nucleoside analogue FTC. These were compared to a structure solved with D-dC. Our results highlight structural adjustments imposed on the L-nucleosides and properties of the enzyme endowing it with the ability to phosphorylate substrates with nonphysiological chirality. This work reveals the molecular basis for the activation of L-nucleosides by dCK.

  17. Selective Phosphorylation of South and North-Cytidine and Adenosine Methanocarba-Nucleosides by Human Nucleoside and Nucleotide Kinases Correlates with Their Growth Inhibitory Effects on Cultured Cells.

    PubMed

    Sjuvarsson, Elena; Marquez, Victor E; Eriksson, Staffan

    2015-01-01

    Here bicyclo[3.1.0]hexane locked deoxycytidine (S-MCdC, N-MCdC), and deoxyadenosine analogs (S-MCdA and N-MCdA) were examined as substrates for purified preparations of human deoxynucleoside kinases: dCK, dGK, TK2, TK1, the ribonucleoside kinase UCK2, two NMP kinases (CMPK1, TMPK) and a NDP kinase. dCK can be important for the first step of phosphorylation of S-MCdC in cells, but S-MCdCMP was not a substrate for CMPK1, TMPK, or NDPK. dCK and dGK had a preference for the S-MCdA whereas N-MCdA was not a substrate for dCK, TK1, UCK2, TK2, dGK nucleoside kinases. The cell growth experiments suggested that N-MCdC and S-MCdA could be activated in cells by cellular kinases so that a triphosphate metabolite was formed. List of abbreviations: ddC, 2', 3'-didioxycytosine, Zalcitabine; 3TC, β-L-(-)-2',3'-dideoxy-3'-thiacytidine, Lamivudine; CdA, 2-cloro-2'-deoxyadenosine, Cladribine; AraA, 9-β-D-arabinofuranosyladenine; hCNT 1-3, human Concentrative Nucleoside Transporter type 1, 2 and 3; hENT 1-4, human Equilibrative Nucleoside Transporter type 1, 2, 3, and 4. PMID:26167664

  18. An improved method for the enzymatic transformation of nucleosides into 5'-monophosphates.

    PubMed

    Barai, Vladimir N; Kvach, Sergei V; Zinchenko, Anatoli I; Mikhailopulo, Igor A

    2004-12-01

    An improved method to transform nucleosides into 5'-monophosphates using nucleoside phosphotransferase from Erwinia herbicola is reported. The method is based on the shift in the equilibrium state of the reaction to the formation of desired product due to its precipitation by Zn2+. Under optimal conditions, the extent of nucleoside transformations into nucleoside-5'-monophosphates were 41-91% (mol). PMID:15672226

  19. Natural and engineered biosynthesis of nucleoside antibiotics in Actinomycetes.

    PubMed

    Chen, Wenqing; Qi, Jianzhao; Wu, Pan; Wan, Dan; Liu, Jin; Feng, Xuan; Deng, Zixin

    2016-03-01

    Nucleoside antibiotics constitute an important family of microbial natural products bearing diverse bioactivities and unusual structural features. Their biosynthetic logics are unique with involvement of complex multi-enzymatic reactions leading to the intricate molecules from simple building blocks. Understanding how nature builds this family of antibiotics in post-genomic era sets the stage for rational enhancement of their production, and also paves the way for targeted persuasion of the cell factories to make artificial designer nucleoside drugs and leads via synthetic biology approaches. In this review, we discuss the recent progress and perspectives on the natural and engineered biosynthesis of nucleoside antibiotics. PMID:26153500

  20. Lights and shadows in the challenge of binding acyclovir, a synthetic purine-like nucleoside with antiviral activity, at an apical-distal coordination site in copper(II)-polyamine chelates.

    PubMed

    Pérez-Toro, Inmaculada; Domínguez-Martín, Alicia; Choquesillo-Lazarte, Duane; Vílchez-Rodríguez, Esther; González-Pérez, Josefa María; Castiñeiras, Alfonso; Niclós-Gutiérrez, Juan

    2015-07-01

    Several nucleic acid components and their metal complexes are known to be involved in crucial metabolic steps. Therefore the study of metal-nucleic acid interactions becomes essential to understand these biological processes. In this work, the synthetic purine-like nucleoside acyclovir (acv) has been used as a model of guanosine recognition with copper(II)-polyamine chelates. The chemical stability of the N9-acyclic arm in acv offers the possibility to use this antiviral drug to deepen the knowledge of metal-nucleoside interactions. Cu(II) chelates with cyclam, cyclen and trien were used as suitable receptors. All these copper(II) tetraamine chelates have in common the potential ability to yield a Cu-N7(apical) bond assisted by an appropriate (amine)N-H⋯O6(acv) intra-molecular interligand interaction. A series of synthesis afforded the following compounds: [Cu(cyclam)(ClO4)2] (1), {[Cu(cyclam)(μ2-NO3)](NO3)}n (2), {[Cu(cyclam)(μ2-SO4)]·MeOH}n (3), {[Cu(cyclam)(μ2-SO4)]·5H2O}n (4), [Cu(cyclen)(H2O)]SO4·2H2O (5), [Cu(cyclen)(H2O)]SO4·3H2O (6), [Cu(trien)(acv)](NO3)2·acv (7) and [Cu(trien)(acv)]SO4·0.71H2O (8). All these compounds have been characterized by X-ray crystallography and FT-IR spectroscopy. Our results reveal that the macrochelates Cu(cyclen)(2+) and Cu(cyclam)(2+) are unable to bind acv at an apical site. In contrast, the Cu(trien)(2+) complex has proved to be an efficient receptor for acv in compounds (7) and (8). In the ternary complex [Cu(trien)(acv)](2+), the metal binding pattern of acv consists of an apical Cu-N7 bond assisted by an intra-molecular (primary amino)N-H⋯O6(acv) interligand interaction. Structural comparisons reveal that this unprecedented apical role of acv is due to the acyclic nature of trien together with the ability of the Cu(trien)(2+) chelate to generate five-coordinated (type 4+1) copper(II) complexes. PMID:25863571

  1. Inhibitory Effect of Bridged Nucleosides on Thermus aquaticus DNA Polymerase and Insight into the Binding Interactions

    PubMed Central

    Kim, Sung-Kun; Castro, Aaron; Kim, Edward S.; Dinkel, Austin P.; Liu, Xiaoyun; Castro, Miguel

    2016-01-01

    Modified nucleosides have the potential to inhibit DNA polymerases for the treatment of viral infections and cancer. With the hope of developing potent drug candidates by the modification of the 2’,4’-position of the ribose with the inclusion of a bridge, efforts were focused on the inhibition of Taq DNA polymerase using quantitative real time PCR, and the results revealed the significant inhibitory effects of 2’,4’-bridged thymidine nucleoside on the polymerase. Study on the mode of inhibition revealed the competitive mechanism with which the 2’,4’-bridged thymidine operates. With a Ki value of 9.7 ± 1.1 μM, the 2’,4’-bridged thymidine proved to be a very promising inhibitor. Additionally, docking analysis showed that all the nucleosides including 2’,4’-bridged thymidine were able to dock in the active site, indicating that the substrate analogs reflect a structural complementarity to the enzyme active site. The analysis also provided evidence that Asp610 was a key binding site for 2’,4’-bridged thymidine. Molecular dynamics (MD) simulations were performed to further understand the conformational variations of the binding. The root-mean-square deviation (RMSD) values for the peptide backbone of the enzyme and the nitrogenous base of the inhibitor stabilized within 0.8 and 0.2 ns, respectively. Furthermore, the MD analysis indicates substantial conformational change in the ligand (inhibitor) as the nitrogenous base rotated anticlockwise with respect to the sugar moiety, complemented by the formation of several new hydrogen bonds where Arg587 served as a pivot axis for binding formation. In conclusion, the active site inhibition of Taq DNA polymerase by 2’,4’-bridged thymidine suggests the potential of bridged nucleosides as drug candidates. PMID:26820310

  2. TAOK3 Phosphorylates the Methylenecyclopropane Nucleoside MBX 2168 to its Monophosphate

    PubMed Central

    Komazin-Meredith, Gloria; Cardinale, Steven C.; Comeau, Katelyn; Magalhaes, Kevin J.; Hartline, Caroll B.; Williams, John D.; Opperman, Timothy J.; Prichard, Mark N.; Bowlin, Terry L.

    2015-01-01

    Monohydroxymethyl methylenecyclopropane nucleosides (MCPNs) with ether or thioether substituents at the 6-position show promise as broad-spectrum herpes virus inhibitors. Their proposed mechanism of action involves sequential phosphorylation to a triphosphate, which can then inhibit viral DNA polymerase. The inhibition of herpes simplex virus (HSV) by these compounds is not dependent on the viral thymidine kinase (TK), which is known to phosphorylate acyclovir (ACV), a standard treatment for HSV infections. Previous studies on the mechanism of action of these compounds against human cytomegalovirus (HCMV) implicated a host kinase in addition to HCMV UL97 kinase in performing the initial phosphorylation. After first eliminating other candidate HSV-1 encoded kinases (UL13 and US3) as well as potential host nucleoside kinases, using activity-based fractionation, we have now identified the host serine-threonine protein kinase TAOK3 as the kinase responsible for transforming the representative monohydroxymethyl MCPN analog MBX 2168 to its monophosphate. PMID:25857706

  3. Rapid and Liquid-Based Selection of Genetic Switches Using Nucleoside Kinase Fused with Aminoglycoside Phosphotransferase

    PubMed Central

    Kawai-Noma, Shigeko; Saito, Kyoichi; Umeno, Daisuke

    2015-01-01

    The evolutionary design of genetic switches and circuits requires iterative rounds of positive (ON-) and negative (OFF-) selection. We previously reported a rapid OFF selection system based on the kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the artificial mutator nucleoside dP. By fusing hsvTK with the kanamycin resistance marker aminoglycoside-(3’)-phosphotransferase (APH), we established a novel selector system for genetic switches. Due to the bactericidal nature of kanamycin and nucleoside-based lethal mutagenesis, both positive and negative selection could be completed within several hours. Using this new selector system, we isolated a series of homoserine lactone-inducible genetic switches with different expression efficiencies from libraries of the Vibrio fischeri lux promoter in two days, using only liquid handling. PMID:25790096

  4. Crystal structures of HIV-1 reverse transcriptase complexes with thiocarbamate non-nucleoside inhibitors

    SciTech Connect

    Spallarossa, Andrea Cesarini, Sara; Ranise, Angelo; Ponassi, Marco; Unge, Torsten; Bolognesi, Martino

    2008-01-25

    O-Phthalimidoethyl-N-arylthiocarbamates (TCs) have been recently identified as a new class of potent HIV-1 reverse transcriptase (RT) non-nucleoside inhibitors (NNRTIs), by means of computer-aided drug design techniques [Ranise A. Spallarossa, S. Cesarini, F. Bondavalli, S. Schenone, O. Bruno, G. Menozzi, P. Fossa, L. Mosti, M. La Colla, et al., Structure-based design, parallel synthesis, structure-activity relationship, and molecular modeling studies of thiocarbamates, new potent non-nucleoside HIV-1 reverse transcriptase inhibitor isosteres of phenethylthiazolylthiourea derivatives, J. Med. Chem. 48 (2005) 3858-3873]. To elucidate the atomic details of RT/TC interaction and validate an earlier TC docking model, the structures of three RT/TC complexes were determined at 2.8-3.0 A resolution by X-ray crystallography. The conformations adopted by the enzyme-bound TCs were analyzed and compared with those of bioisosterically related NNRTIs.

  5. Purification, crystallization, and preliminary X-ray diffraction study of purine nucleoside phosphorylase from E. coli

    SciTech Connect

    Abramchik, Yu. A. Timofeev, V. I. Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2015-07-15

    Crystals of E. coli purine nucleoside phosphorylase were grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one crystal at the Spring-8 synchrotron facility to 0.99 Å resolution. The crystals belong to sp. gr. P2{sub 1} and have the following unit-cell parameters: a = 74.1 Å, b = 110.2 Å, c = 88.2 Å, α = γ = 90°, β = 111.08°. The crystal contains six subunits of the enzyme comprising a hexamer per asymmetric unit. The hexamer is the biological active form of E. coli. purine nucleoside phosphorylase.

  6. TAOK3 phosphorylates the methylenecyclopropane nucleoside MBX 2168 to its monophosphate.

    PubMed

    Komazin-Meredith, Gloria; Cardinale, Steven C; Comeau, Katelyn; Magalhaes, Kevin J; Hartline, Caroll B; Williams, John D; Opperman, Timothy J; Prichard, Mark N; Bowlin, Terry L

    2015-07-01

    Monohydroxymethyl methylenecyclopropane nucleosides (MCPNs) with ether or thioether substituents at the 6-position show promise as broad-spectrum herpes virus inhibitors. Their proposed mechanism of action involves sequential phosphorylation to a triphosphate, which can then inhibit viral DNA polymerase. The inhibition of herpes simplex virus (HSV) by these compounds is not dependent on the viral thymidine kinase (TK), which is known to phosphorylate acyclovir (ACV), a standard treatment for HSV infections. Previous studies on the mechanism of action of these compounds against human cytomegalovirus (HCMV) implicated a host kinase in addition to HCMV UL97 kinase in performing the initial phosphorylation. After first eliminating other candidate HSV-1 encoded kinases (UL13 and US3) as well as potential host nucleoside kinases, using activity-based fractionation, we have now identified the host serine-threonine protein kinase TAOK3 as the kinase responsible for transforming the representative monohydroxymethyl MCPN analog MBX 2168 to its monophosphate. PMID:25857706

  7. Hepatotoxicity of nucleoside reverse transcriptase inhibitors.

    PubMed

    Montessori, Valentina; Harris, Marianne; Montaner, Julio S G

    2003-05-01

    Hepatotoxicity is an adverse effect of all available classes of antiretrovirals, including nucleoside reverse transcriptase inhibitors (NRTI). A syndrome of hepatic steatosis and lactic acidosis has been recognized as a rare, potentially fatal complication since the advent of NRTI monotherapy in the early 1990s. Today, NRTI remain the backbone of antiretroviral combination regimens, and, with the success of current treatment strategies, exposure to two or more of these agents may occur over a number of years. Hepatic steatosis and lactic acidosis are accordingly being observed more frequently, along with a more recently recognized syndrome of chronic hyperlactatemia. These as well as other adverse effects of NRTI are mediated by inhibition of human DNA polymerase gamma, resulting in mitochondrial dysfunction in the liver and other tissues. Early recognition and intervention are essential to avert serious outcomes. PMID:12800069

  8. Functional non-nucleoside adenylyl cyclase inhibitors.

    PubMed

    Lelle, Marco; Hameed, Abdul; Ackermann, Lisa-Maria; Kaloyanova, Stefka; Wagner, Manfred; Berisha, Filip; Nikolaev, Viacheslav O; Peneva, Kalina

    2015-05-01

    In this study, we describe the synthesis of novel functional non-nucleoside adenylyl cyclase inhibitors, which can be easily modified with thiol containing biomolecules such as tumour targeting structures. The linkage between inhibitor and biomolecule contains cleavable bonds to enable efficient intracellular delivery in the reductive milieu of the cytosol as well as in the acidic environment within endosomes and lysosomes. The suitability of this synthetic approach was shown by the successful bioconjugation of a poor cell-permeable inhibitor with a cell-penetrating peptide. Additionally, we have demonstrated the excellent inhibitory effect of the compounds presented here in a live-cell Förster resonance energy transfer-based assay in human embryonic kidney cells. PMID:25319071

  9. Effect of dietary nucleosides on growth and maturation of the developing gut in the rat.

    PubMed

    Uauy, R; Stringel, G; Thomas, R; Quan, R

    1990-05-01

    Dietary nucleoside (DN) as a precursor for nucleic acid synthesis may be important for rapidly dividing cells, since gut epithelial cells have limited capacity for de novo purine and pyrimidine synthesis. We evaluated in a controlled blinded study the effect of added nucleosides, 0.8% by weight, given for 2 weeks, on gut growth and maturation in 20 weanling rats. Mucosal protein and DNA in the proximal intestinal segment were 50% and 77% higher, respectively, in the DN-supplemented group (n = 10; p less than 0.05). Villus height based on cell count was 25% greater in the DN group (p less than 0.05). Maltase activity was significantly greater in proximal, middle, and distal intestinal segments, and the largest increase, 87%, was seen in the proximal gut mucosa. The maltase/lactase ratio was also higher in this segment. Increases in sucrase were less prominent. Lactase was minimally affected. The pattern of change in disaccharidase activity suggests that DN may enhance gut growth and maturation of the intestine in the weanling rat, the effects being more pronounced in the proximal segment. Diets free of nucleosides and nitrogenous bases may have adverse effects on the gut. PMID:2358983

  10. Design, synthesis and biological evaluation of phosphorodiamidate prodrugs of antiviral and anticancer nucleosides

    PubMed Central

    McGuigan, Christopher; Bourdin, Claire; Derudas, Marco; Hamon, Nadège; Hinsinger, Karen; Kandil, Sahar; Madela, Karolina; Meneghesso, Silvia; Pertusati, Fabrizio; Serpi, Michaela; Slusarczyk, Magdalena; Chamberlain, Stanley; Kolykhalov, Alexander; Vernachio, John; Vanpouille, Christophe; Introini, Andrea; Margolis, Leonid; Balzarini, Jan

    2014-01-01

    We herein report the application of the phosphorodiamidate phosphate prodrug approach to a series of thirteen nucleoside analogs with antiviral or anticancer activity. Twenty-five symmetrical phosphorodiamidates were synthesized, bearing esterified l-Alanine (and in one case d-alanine) in the prodrug moiety, each as single stereoisomer. The presence of an achiral phosphorus represents a potential advantage over the phosphoramidate ProTide approach, where diastereoisomeric mixtures are routinely obtained, and different biological profiles may be expected from the diastereoisomers. Optimization of the synthetic pathway allowed us to identify two general methods depending on the particular nucleoside analogs. All the compounds were biologically evaluated in antiviral and anticancer assays and several showed improvement of activity compared to their parent nucleosides, as in the case of ddA, d4T, abacavir and acyclovir against HIV-1 and/or HIV-2. The biological results were supported by metabolism studies with carboxypeptidase Y monitored by 31P NMR to investigate their bioactivation. This work further validates the phosphorodiamidate approach as a monophosphate prodrug motif with broad application in the antiviral and anticancer fields. PMID:24177359

  11. Poliovirus protein 2C has ATPase and GTPase activities.

    PubMed

    Rodríguez, P L; Carrasco, L

    1993-04-15

    Poliovirus protein 2C belongs to an expanding group of proteins containing a nucleotide binding motif in their sequence. We present evidence that poliovirus 2C has nucleoside triphosphatase (NTPase) activity and binds to RNA. Poliovirus 2C was expressed in Escherichia coli cells as a fusion protein with the maltose binding protein (MBP). The fusion protein MBP-2C is efficiently cut by protease Xa within the 2C region. Thus, the fusion protein as such was used to assay for the putative activities of poliovirus 2C. Deletion mutants were constructed which lacked different portions of the 2C carboxyl terminus: mutant 2C delta 1 lacked the last 169 amino acids, whereas mutant 2C delta 2 had the last 74 amino acids deleted. The fusion proteins MBP-2C, MBP-2BC, and the mutant MBP-2C delta 2 that contained the first 255 amino acids of 2C had NTPase activity. Both ATPase and GTPase activities are inhibited by antibodies directed against the MBP-2C protein. Analysis of the ability of the different proteins to bind to labeled RNA indicates that MBP-2C and MBP-2BC form a complex, whereas none of the mutants interacted with RNA, indicating that the RNA binding domain lies beyond amino acid 255. None of the fusion proteins had detectable helicase activity. We suggest that poliovirus protein 2C shows similarities to the GTPases group involved in vesicular traffic and transports the viral RNA replication complexes. These results provide the first experimental evidence that poliovirus protein 2C is an NTPase and that this protein has affinity for nucleic acids. PMID:8385138

  12. Targeted Delivery of Deoxycytidine Kinase to Her2-Positive Cells Enhances the Efficacy of the Nucleoside Analog Fludarabine

    PubMed Central

    Kay, Brian K.; Lavie, Arnon

    2016-01-01

    Cytotoxic drugs, such as nucleoside analogs and toxins, commonly suffer from off-target effects. One approach to mitigate this problem is to deliver the cytotoxic drug selectively to the intended site. While for toxins this can be achieved by conjugating the cell-killing moiety to a targeting moiety, it is not an option for nucleoside analogs, which rely on intracellular enzymes to convert them to their active triphosphorylated form. To overcome this limitation, and achieve site-targeted activation of nucleoside analogs, we fused the coding region of a prodrug-activating enzyme, deoxycytidine kinase (dCK), to affinity reagents that bind to the Her2 cell surface protein. We evaluated dCK fusions to an anti-Her2 affibody and Designed Ankyrin Repeat Protein (DARPin) for their ability to kill cancer cells by promoting the activation of the nucleoside analog fludarabine. Cell staining and flow cytometry experiments with three Her2 positive cancer cell lines (BT-474-JB, JIMT-1 and SK-OV-3) indicate dCK fusions binding and cellular internalization. In contrast, these reagents bind only weakly to the Her2 negative cell line, MCF-7. Cell proliferation assays indicate that SK-OV-3 and BT-474-JB cell lines exhibit significantly reduced proliferation rates when treated with targeting-module fused dCK and fludarabine, compared to fludarabine alone. These findings demonstrate that we have succeeded in delivering active dCK into the Her2-positive cells, thereby increasing the activation of fludarabine, which ultimately reduces the dose of nucleoside analog needed for cell killing. This strategy may help establish the therapeutic index required to differentiate between healthy tissues and cancer cells. PMID:27280468

  13. Base-Modified Nucleosides as Chemotherapeutic Agents: Past and Future.

    PubMed

    Burke, Matthew P; Borland, Kayla M; Litosh, Vladislav A

    2016-01-01

    Nucleoside and nucleobase antimetabolites have substantially impacted treatment of cancer and infections. Their close resemblance to natural analogs gives them the power to interfere with a variety of intracellular targets, which on one hand gives them high potency, but on the other hand incurs severe side effects, especially of the chemotherapeutics used against malignancies. Therefore, the development of novel nucleoside analogs with widened therapeutic windows represents an attractive target to synthetic organic and medicinal chemists. This review discusses the current antimetabolite drugs: 5- fluorouracil, 6-mercaptopurine, 6-thioguanine, Cladribine, Vidaza, Decitabine, Emtricitabine, Abacavir, Sorivudine, Clofarabine, Fludarabine, and Nelarabine; gives insight into the nucleoside drug candidates that are being developed; and outlines the approaches to nucleobase modifications that may help discover novel bioactive nucleoside analogs with the mechanism of action focused on termination of DNA synthesis, which is expected to diminish the off-target toxicity in non-proliferating human cells. PMID:26369814

  14. Medicinal chemistry of fluorinated cyclic and acyclic nucleoside phosphonates.

    PubMed

    Baszczyňski, Ondřej; Janeba, Zlatko

    2013-11-01

    The fluorine atom plays an important role in medicinal chemistry because fluorine substitution has a strong impact on the physical, chemical, and biological properties of bioactive compounds. Such fluorine modifications have also been extensively studied among the pharmaceutically important class of nucleoside phosphonates, nucleotide analogues in which the phosphate group is replaced by the enzymatically and chemically stable phosphonate moiety. The fluorinated nucleoside phosphonates abound with antiviral, antiparasitic, and anticancer properties because they are able to act as inhibitors of important enzymes of nucleoside/nucleotide metabolism. In this paper, we review the biological properties of cyclic and acyclic nucleoside phosphonates modified by the attachment of one or more fluorine atoms to various parts of the molecule, namely to nucleobases, alkylphosphonate groups, cyclic or acyclic linkers, or to prodrug moieties. PMID:23893552

  15. A novel bis(pinacolato)diboron-mediated N-O bond deoxygenative route to C6 benzotriazolyl purine nucleoside derivatives.

    PubMed

    Basava, Vikram; Yang, Lijia; Pradhan, Padmanava; Lakshman, Mahesh K

    2016-08-01

    Reaction of amide bonds in t-butyldimethylsilyl-protected inosine, 2'-deoxyinosine, guanosine, 2'-deoxyguanosine, and 2-phenylinosine with commercially available peptide-coupling agents (benzotriazol-1H-yloxy)tris(dimethylaminophosphonium) hexafluorophosphate (BOP), (6-chloro-benzotriazol-1H-yloxy)trispyrrolidinophosphonium hexafluorophosphate (PyClocK), and (7-azabenzotriazol-1H-yloxy)trispyrrolidinophosphonium hexafluorophospate (PyAOP) gave the corresponding O(6)-(benzotriazol-1-yl) nucleoside analogues containing a C-O-N bond. Upon exposure to bis(pinacolato)diboron and base, the O(6)-(benzotriazol-1-yl) and O(6)-(6-chlorobenzotriazol-1-yl) purine nucleoside derivatives obtained from BOP and PyClocK, respectively, underwent N-O bond reduction and C-N bond formation, leading to the corresponding C6 benzotriazolyl purine nucleoside analogues. In contrast, the 7-azabenzotriazolyloxy purine nucleoside derivatives did not undergo efficient deoxygenation, but gave unsymmetrical nucleoside dimers instead. This is consistent with a prior report on the slow reduction of 1-hydroxy-1H-4-aza and 1-hydroxy-1H-7-azabenzotriazoles. Because of the limited number of commercial benzotriazole-based peptide coupling agents, and to show the applicability of the method when such coupling agents are unavailable, 1-hydroxy-1H-5,6-dichlorobenzotriazole was synthesized. Using this compound, silyl-protected inosine and 2'-deoxyinosine were converted to the O(6)-(5,6-dichlorobenzotriazol-1-yl) derivatives via in situ amide activation with PyBroP. The O(6)-(5,6-dichlorobenzotriazol-1-yl) purine nucleosides so obtained also underwent smooth reduction to afford the corresponding C6 5,6-dichlorobenzotriazolyl purine nucleoside derivatives. A total of 13 examples were studied with successful reactions occurring in 11 cases (the azabenzotriazole derivatives, mentioned above, being the only unreactive entities). To understand whether these reactions are intra or intermolecular processes, a

  16. Human cytomegalovirus resistance to deoxyribosylindole nucleosides maps to a transversion mutation in the terminase subunit-encoding gene UL89.

    PubMed

    Gentry, Brian G; Phan, Quang; Hall, Ellie D; Breitenbach, Julie M; Borysko, Katherine Z; Kamil, Jeremy P; Townsend, Leroy B; Drach, John C

    2015-01-01

    Human cytomegalovirus (HCMV) infection can cause severe illnesses, including encephalopathy and mental retardation, in immunocompromised and immunologically immature patients. Current pharmacotherapies for treating systemic HCMV infections include ganciclovir, cidofovir, and foscarnet. However, long-term administration of these agents can result in serious adverse effects (myelosuppression and/or nephrotoxicity) and the development of viral strains with reduced susceptibility to drugs. The deoxyribosylindole (indole) nucleosides demonstrate a 20-fold greater activity in vitro (the drug concentration at which 50% of the number of plaques was reduced with the presence of drug compared to the number in the absence of drug [EC50] = 0.34 μM) than ganciclovir (EC50 = 7.4 μM) without any observed increase in cytotoxicity. Based on structural similarity to the benzimidazole nucleosides, we hypothesize that the indole nucleosides target the HCMV terminase, an enzyme responsible for packaging viral DNA into capsids and cleaving the DNA into genome-length units. To test this hypothesis, an indole nucleoside-resistant HCMV strain was isolated, the open reading frames of the genes that encode the viral terminase were sequenced, and a G766C mutation in exon 1 of UL89 was identified; this mutation resulted in an E256Q change in the amino acid sequence of the corresponding protein. An HCMV wild-type strain, engineered with this mutation to confirm resistance, demonstrated an 18-fold decrease in susceptibility to the indole nucleosides (EC50 = 3.1 ± 0.7 μM) compared to that of wild-type virus (EC50 = 0.17 ± 0.04 μM). Interestingly, this mutation did not confer resistance to the benzimidazole nucleosides (EC50 for wild-type HCMV = 0.25 ± 0.04 μM, EC50 for HCMV pUL89 E256Q = 0.23 ± 0.04 μM). We conclude, therefore, that the G766C mutation that results in the E256Q substitution is unique for indole nucleoside resistance and distinct from previously discovered substitutions

  17. Synthesis and Evaluation of 2'-Deoxy-2'-Spirodiflurocyclopropyl Nucleoside Analogs.

    PubMed

    Liu, Xiao; Xia, Xueliang; Sun, Chenghai; Lin, Cai; Zhou, Yiqian; Hussain, Muzammal; Tang, Fei; Liu, Lu; Li, Xue; Zhang, Jiancun

    2016-09-01

    The preparation of 2'-deoxy-2'-siprodifluorocyclopropany-lnucleoside analogs has been achieved from α-d-glucose in several steps. The key step in the synthesis was the introduction of the difluorocyclopropane through a difluorocarbene type reaction at the 2'-position. Then, a series of novel 2'-deoxy-2'-spirodifluorocyclopropanyl nucleoside analogs were synthesized using the Vorbrüggen method. All the synthesized nucleosides were characterized and subsequently evaluated against hepatitis C and influenza A virus strains in vitro. PMID:27556785

  18. Distribution of nucleosides in populations of Cordyceps cicadae.

    PubMed

    Zeng, Wen-Bo; Yu, Hong; Ge, Feng; Yang, Jun-Yuan; Chen, Zi-Hong; Wang, Yuan-Bing; Dai, Yong-Dong; Adams, Alison

    2014-01-01

    A rapid HPLC method had been developed and used for the simultaneous determination of 10 nucleosides (uracil, uridine, 2'-deoxyuridine, inosine, guanosine, thymidine, adenine, adenosine, 2'-deoxyadenosine and cordycepin) in 10 populations of Cordyceps cicadae, in order to compare four populations of Ophicordyceps sinensis and one population of Cordyceps militaris. Statistical analysis system (SAS) 8.1 was used to analyze the nucleoside data. The pattern of nucleoside distribution was analyzed in the sampled populations of C. cicadae, O. sinensis and C. militaris, using descriptive statistical analysis, nested analysis and Q cluster analysis. The total amount of the 10 nucleosides in coremium was 1,463.89-5,678.21 µg/g in 10 populations of C. cicadae, 1,369.80-3,941.64 µg/g in sclerotium. The average contents of the 10 analytes were 4,392.37 µg/g and 3,016.06 µg/g in coremium and sclerotium, respectively. The coefficient of variation (CV) of nucleosides ranged from 8.36% to 112.36% in coremium of C. cicadae, and from 10.77% to 155.87% in sclerotium of C. cicadae. The CV of the nucleosides was wide within C. cicadae populations. The nested variation analysis by the nine nucleosides' distribution indicated that about 42.29% of the nucleoside variability in coremium was attributable to the differentiation among populations, and the remaining 57.71% resided in the populations. It was also shown that about 28.94% of the variation in sclerotium was expressed between populations, while most of the variation (71.06%) corresponded to the populations. PMID:24830714

  19. Transport characteristics of mouse concentrative nucleoside transporter 1.

    PubMed

    Niitani, Miho; Nishida, Kentaro; Okuda, Hiroto; Nagai, Katsuhito; Fujimoto, Sadaki; Nagasawa, Kazuki

    2010-03-30

    Concentrative nucleoside transporter 1 (CNT1, SLC28A1) is a key molecule for determining the pharmacokinetic/pharmacodynamic profile of a candidate compound derived from a pyrimidine nucleoside, but there is no available information on the differences in the functional profile of this ortholog between man and mouse. Here, using a clone of mouse CNT1 (mCNT1), we investigated its transport characteristics and substrate specificity for synthetic nucleoside analogues, and compared them with those of human CNT1 (hCNT1). In mCNT1-transfected Cos-7 cells, pyrimidine, but not purine, nucleosides showed sodium- and concentration-dependent uptake, and uridine uptake was competitively inhibited by uridine analogues, the rank order of the inhibitory effects being 5-bromouridine>3'-deoxyuridine>2'-deoxyuridine. cis- and trans-Inhibition studies involving synthetic nucleoside drugs revealed that gemcitabine and zidovudine greatly inhibited [(3)H]uridine uptake mediated by mCNT1 in the both cases, while cytarabine and zalcitabine showed small cis-inhibitory effect, and no trans-inhibitory effect on the uptake. These results demonstrate that the transport characteristics of mCNT1 are almost the same as those of hCNT1, suggesting that mice may be a good animal model in evaluation of pyrimidine nucleoside analogues as to their applicability in human therapy. PMID:20060452

  20. Expedient and generic synthesis of imidazole nucleosides by enzymatic transglycosylation.

    PubMed

    Vichier-Guerre, S; Dugué, L; Bonhomme, F; Pochet, S

    2016-04-14

    A straightforward route to original imidazole-based nucleosides that makes use of an enzymatic N-transglycosylation step is reported in both the ribo- and deoxyribo-series. To illustrate the scope of this approach, a diverse set of 4-aryl and 4-heteroaryl-1H-imidazoles featuring variable sizes and hydrogen-bonding patterns was prepared using a microwave-assisted Suzuki-Miyaura cross-coupling reaction. These imidazole derivatives were examined as possible substrates for the nucleoside 2'-deoxyribosyltransferase from L. leichmannii and the purine nucleoside phosphorylase from E. coli. The optimum transglycosylation conditions, including the use of co-adjuvants to address solubility issues, were defined. Enzymatic conversion of 4-(hetero)arylimidazoles to 2'-deoxyribo- or ribo-nucleosides proceeded in good to high conversion yields, except bulky hydrophobic imidazole derivatives. Nucleoside deoxyribosyltransferase of class II was found to convert the widest range of functionalized imidazoles into 2'-deoxyribonucleosides and was even capable of bis-glycosylating certain heterocyclic substrates. Our findings should enable chemoenzymatic access to a large diversity of flexible nucleoside analogues as molecular probes, drug candidates and original building blocks for synthetic biology. PMID:26986701

  1. Insecticidal properties of essential oils against Tribolium castaneum (Herbst) and their inhibitory effects on acetylcholinesterase and adenosine triphosphatases.

    PubMed

    Abou-Taleb, Hamdy K; Mohamed, Magdy I E; Shawir, Mohamed S; Abdelgaleil, Samir A M

    2016-01-01

    Essential oils from 20 Egyptian plants were obtained by using hydrodistillation. The chemical composition of the isolated oils was identified by gas chromatograph/mass spectrometer. Fumigant and contact toxicities of the essential oils were evaluated against the adults of Tribolium castaneum. In fumigation assays, the oil of Origanum vulgare (LC50 = 9.97 mg/L air) displayed the highest toxicity towards the adults of T. castaneum. In contact assays, the oils of Artemisia monosperma (LC50 = 0.07 mg/cm(2)) and O. vulgare (LC50 = 0.07 mg/cm(2)) were the most potent toxicants against the adults of T. castaneum. Biochemical studies showed that the tested oils caused pronounced inhibition of acetylcholinesterase (AChE) and adenosine triphosphatases (ATPases) isolated from the larvae of T. castaneum. The oil Cupressus macrocarpa (IC50 = 12.3 mg/L) was the most potent inhibitor of AChE, while the oil of Calistemon viminals (IC50 = 4.4 mg/L) was the most potent inhibitor of ATPases. PMID:25978134

  2. Inhibition of nucleoside transport by a new series of compounds related to lidoflazine and mioflazine.

    PubMed

    Ijzerman, A P; Thedinga, K H; Custers, A F; Hoos, B; Van Belle, H

    1989-08-15

    A new series of compounds related to the nucleoside transport inhibitors, lidoflazine and mioflazine, is introduced. The influence of these derivatives on nucleoside-specific transport proteins was studied in two ways. First, a rapid, non-radioactive assay was developed for the screening of this type of material for actual transport inhibition in human erythrocytes. The method is based on the dose-dependent reversal of the inhibition of inorganic phosphate release induced by inosine when human erythrocytes are suspended in a phosphate-free medium. It enables the estimation of the potency and specificity of this new series of nucleoside transport inhibitors, most of which are highly active (EC50 values as low as 13 nM). Second, the displacement of a radiolabeled transport inhibitor, [3H]nitrobenzylthioinosine, was examined. All compounds were capable of displacing specific [3H]nitrobenzylthioinosine binding to crude and solubilized plasma membranes of calf lung tissue, displaying affinities in the nanomolar range. Pseudo-Hill coefficients derived from the shape of the displacement curves were significantly greater than unity for most derivatives, in contrast to values of approximately unity obtained for dipyridamole and analogs. These findings were incorporated in a mathematical model describing the interaction of mioflazine analogs with the transport protein, suggesting that one molecule of mioflazine is capable of displacing two or more molecules of [3H]nitrobenzylthioinosine at a time. The consequences of this model regarding the nature of the transport protein are discussed. PMID:2776845

  3. An unusual UMP C-5 methylase in nucleoside antibiotic polyoxin biosynthesis.

    PubMed

    Chen, Wenqing; Li, Yan; Li, Jie; Wu, Lian; Li, Yan; Wang, Renxiao; Deng, Zixin; Zhou, Jiahai

    2016-09-01

    Polyoxin is a group of structurally-related peptidyl nucleoside antibiotics bearing C-5 modifications on the nucleoside skeleton. Although the structural diversity and bioactivity preference of polyoxin are, to some extent, affected by such modifications, the biosynthetic logic for their occurence remains obscure. Here we report the identification of PolB in polyoxin pathway as an unusual UMP C-5 methylase with thymidylate synthase activity which is responsible for the C-5 methylation of the nucleoside skeleton. To probe its molecular mechanism, we determined the crystal structures of PolB alone and in complexes with 5-Br UMP and 5-Br dUMP at 2.15 Å, 1.76 Å and 2.28 Å resolutions, respectively. Loop 1 (residues 117-131), Loop 2 (residues 192-201) and the substrate recognition peptide (residues 94-102) of PolB exhibit considerable conformational flexibility and adopt distinct structures upon binding to different substrate analogs. Consistent with the structural findings, a PolB homolog that harbors an identical function from Streptomyces viridochromogenes DSM 40736 was identified. The discovery of UMP C5-methylase opens the way to rational pathway engineering for polyoxin component optimization, and will also enrich the toolbox for natural nucleotide chemistry. PMID:27412636

  4. A novel nucleoside hydrolase from Lactobacillus buchneri LBK78 catalyzing hydrolysis of 2'-O-methylribonucleosides.

    PubMed

    Mitsukawa, Yuuki; Hibi, Makoto; Matsutani, Narihiro; Horinouchi, Nobuyuki; Takahashi, Satomi; Ogawa, Jun

    2016-08-01

    2'-O-Methylribonucleosides (2'-OMe-NRs) are promising raw materials for nucleic acid drugs because of their high thermal stability and nuclease tolerance. In the course of microbial screening for metabolic activity toward 2'-OMe-NRs, Lactobacillus buchneri LBK78 was found to decompose 2'-O-methyluridine (2'-OMe-UR). The enzyme responsible was partially purified from L. buchneri LBK78 cells by a four-step purification procedure, and identified as a novel nucleoside hydrolase. This enzyme, LbNH, belongs to the nucleoside hydrolase superfamily, and formed a homotetrameric structure composed of subunits with a molecular mass around 34 kDa. LbNH hydrolyzed 2'-OMe-UR to 2'-O-methylribose and uracil, and the kinetic constants were Km of 0.040 mM, kcat of 0.49 s(-1), and kcat/Km of 12 mM(-1) s(-1). In a substrate specificity analysis, LbNH preferred ribonucleosides and 2'-OMe-NRs as its hydrolytic substrates, but reacted weakly with 2'-deoxyribonucleosides. In a phylogenetic analysis, LbNH showed a close relationship with purine-specific nucleoside hydrolases from trypanosomes. PMID:27180876

  5. Vaccinia virus lacking the deoxyuridine triphosphatase gene (F2L) replicates well in vitro and in vivo, but is hypersensitive to the antiviral drug (N)-methanocarbathymidine

    PubMed Central

    Prichard, Mark N; Kern, Earl R; Quenelle, Debra C; Keith, Kathy A; Moyer, Richard W; Turner, Peter C

    2008-01-01

    Background The vaccinia virus (VV) F2L gene encodes a functional deoxyuridine triphosphatase (dUTPase) that catalyzes the conversion of dUTP to dUMP and is thought to minimize the incorporation of deoxyuridine residues into the viral genome. Previous studies with with a complex, multigene deletion in this virus suggested that the gene was not required for viral replication, but the impact of deleting this gene alone has not been determined in vitro or in vivo. Although the crystal structure for this enzyme has been determined, its potential as a target for antiviral therapy is unclear. Results The F2L gene was replaced with GFP in the WR strain of VV to assess its effect on viral replication. The resulting virus replicated well in cell culture and its replication kinetics were almost indistinguishable from those of the wt virus and attained similar titers. The virus also appeared to be as pathogenic as the WR strain suggesting that it also replicated well in mice. Cells infected with the dUTPase mutant would be predicted to affect pyrimidine deoxynucleotide pools and might be expected to exhibit altered susceptibility to pyrimidine analogs. The antiviral activity of cidofovir and four thymidine analogs were evaluated both in the mutant and the parent strain of this virus. The dUTPase knockout remained fully susceptible to cidofovir and idoxuridine, but was hypersensitive to the drug (N)-methanocarbathymidine, suggesting that pyrimidine metabolism was altered in cells infected with the mutant virus. The absence of dUTPase should reduce cellular dUMP pools and may result in a reduced conversion to dTMP by thymidylate synthetase or an increased reliance on the salvage of thymidine by the viral thymidine kinase. Conclusion We confirmed that F2L was not required for replication in cell culture and determined that it does not play a significant role on virulence of the virus in intranasally infected mice. The recombinant virus is hypersensitive to (N

  6. The reactivity of the thiol groups of the adenosine triphosphatase of sarcoplasmic reticulum and their location on tryptic fragments of the molecule

    PubMed Central

    Thorley-Lawson, David A.; Green, N. Michael

    1977-01-01

    The ATPase (adenosine triphosphatase) from sarcoplasmic reticulum contains 20 thiol groups/115000 daltons, measured by using either N-ethyl[14C]maleimide or 5,5′-dithiobis-(2-nitrobenzoate) in sodium dodecyl sulphate. After reduction there were 26 thiol groups, in good agreement with 26.5 residues of cysteic acid found by amino acid analysis. The difference between this and the 20 residues measured before reduction implies the presence of three disulphide residues. The same number of disulphide residues was found by direct measurement. Three to six fewer thiol groups were found in preparations made in the absence of dithiothreitol. The missing residues were accounted for as cysteic acid. The distribution of disulphide bonds and of exposed and buried thiol groups among the tryptic fragments of the molecule was measured after labelling with N-ethyl[14C]-maleimide. The disulphides were confined to fragment B (mol.wt. 55000), whereas several thiol groups were present on each of the fragments (A, B, A1 and A2). The kinetics of the reaction of the ATPase with 5,5′-dithiobis-(2-nitrobenzoate) showed that four or five of the thiol groups were unreactive in the absence of detergent and that 13 of the remainder reacted with a single first-order rate constant. In the presence of ATP and Ca2+ the reaction rate of all but two groups of this class was uniformly decreased. In the presence or absence of ATP and Ca2+ the rate constant for inactivation was close to the rate constant for this class, but was not identical with it. No selective protection of a specific active-site-thiol group was observed. Parallel experiments with sarcoplasmic reticulum gave similar results, except that the reaction rates were a little lower and there were two more buried groups. Solution of ATPase of sarcoplasmic reticulum in detergent greatly increased the reactivity of all thiol groups. The effects of low concentrations of deoxycholate were reversible. EGTA or low concentrations (0.02mm) of Ca2

  7. Triple nucleoside reverse transcriptase inhibitor therapy in children.

    PubMed

    Handforth, Jennifer; Sharland, Mike

    2004-01-01

    Much of the success attributed to HIV therapy in the last few years has resulted from improved ways of using existing drugs in combination therapy regimens. The availability of new, more potent drugs such as protease inhibitors and more accurate viral load tests to aid decisions to start or change treatment has also contributed to the success. Published recommendations for pediatric HIV therapy, generated by a panel of experts and specialists, are readily available and regularly updated. Preferred regimens of 'potent' therapy (referred to as highly active antiretroviral therapy, or HAART) currently consist of two nucleoside reverse transcriptase inhibitors (NRTIs) combined with either a non-nucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor. More intense four-drug regimens using an NNRTI or a second protease inhibitor as a fourth drug are being evaluated. Problems with HAART include: unpalatable drug formulations and adverse effects, coupled with lack of data on the pharmacokinetics, efficacy, and safety of various drug combinations. Adherence is a major factor influencing the efficacy and outcome of antiretroviral therapy. Many children cannot adhere to complex multidrug regimens, which cause virologic failure, despite excellent CD4+ cell count responses. This means a rapid progression through the limited number of treatment regimens available. Simpler regimens such as those containing three NRTIs have been proposed as a method of treatment that will allow suppression of the virus, yet circumvent many of the problems previously mentioned. An additional benefit would be the preservation of antiretroviral drugs from other classes for future treatment options if required. The major advantages of triple NRTI regimens are the simplicity of the regimen, good tolerability, few drug-drug interactions, and infrequent adverse effects coupled with a low pill burden. However, abacavir hypersensitivity remains a major problem. Up to 3% of patients may

  8. Functional characterization of nucleoside transporter gene replacements in Leishmania donovani.

    PubMed

    Liu, Wei; Boitz, Jan M; Galazka, Jon; Arendt, Cassandra S; Carter, Nicola S; Ullman, Buddy

    2006-12-01

    Leishmania donovani express two nucleoside transporters of non-overlapping ligand selectivity. To evaluate the physiological role of nucleoside transporters in L. donovani, homozygous null mutants of the genes encoding the LdNT1 adenosine-pyrimidine nucleoside transporter and the LdNT2 inosine-guanosine transporter were created singly and in combination by single targeted gene replacement followed by selection for loss-of-heterozygosity. The mutant alleles were verified by Southern blotting, and the effects of gene replacement on transport phenotype were evaluated by rapid sampling transport measurements and by drug resistance profiles. The Deltaldnt1, Deltaldnt2, and Deltaldnt1/Deltaldnt2 mutants were all capable of proliferation in defined culture medium supplemented with any of a spectrum of purine nucleobases or nucleosides, except that a Deltaldnt2 lesion conferred an inability to efficiently salvage exogenous xanthosine, a newly discovered ligand of LdNT2. Each of the three knockout strains was viable as promastigotes and axenic amastigotes and capable of maintaining an infection in J774 and bone marrow-derived murine macrophages. These genetic studies demonstrate: (1) that L. donovani promastigotes, axenic amastigotes, and tissue amastigotes are viable in the absence of nucleoside transport; (2) that nucleoside transporters are not essential for sustaining an infection in mammalian host cells; (3) that the phagolysosome of macrophages is likely to contain purines that are not LdNT1 or LdNT2 ligands, i.e., nucleobases. Furthermore, the Deltaldnt1, Deltaldnt2, and Deltaldnt1/Deltaldnt2 knockouts offer a unique genetically defined null background for the biochemical and genetic characterization of nucleoside transporter genes and cDNAs from phylogenetically diverse species and of genetically manipulated LdNT1 and LdNT2 constructs. PMID:17050001

  9. Impaired Ranvier node sodium-potassium adenosine triphosphatase may induce facial palsy.

    PubMed

    Kanoh, N; Sakagami, M

    1996-09-01

    To clarify the part of the neuron essential for myelinated nerve conduction, the cytochemical localization of potassium ion (K+)-dependent p-nitrophenylphosphatase (K-NPPase) activity was investigated in the normal and reserpine-treated facial nerve of guinea pigs. In the normal animals, K-NPPase activity was localized to the internodal axolemma and Schmidt-Lanterman incisures. In the Ranvier nodes, enzyme activity was observed along the paranodal and nodal axolemma. In reserpinized nerves, K-NPPase activity was absent along the internodal axolemma and Schmidt-Lanterman incisures. In the Ranvier nodes, however, enzyme activity was detectable only in the nodal axolemma. The reserpinized animals demonstrated no evidence of facial palsy. Because K-NPPase is essential for nerve conduction, these results indicate that the location of enzyme activity in reserpinized animals, namely the nodal axolemma, may be of prime importance in saltatory nerve conduction. PMID:8822727

  10. Role of the transporter regulator protein (RS1) in the modulation of concentrative nucleoside transporters (CNTs) in epithelia.

    PubMed

    Errasti-Murugarren, Ekaitz; Fernández-Calotti, Paula; Veyhl-Wichmann, Mayke; Diepold, Maximilian; Pinilla-Macua, Itziar; Pérez-Torras, Sandra; Kipp, Helmut; Koepsell, Hermann; Pastor-Anglada, Marçal

    2012-07-01

    SLC28 genes encode three plasma membrane transporter proteins, human concentrative nucleoside transporter (CNT)1, CNT2, and CNT3, all of which are implicated in the uptake of natural nucleosides and a variety of nucleoside analogs used in the chemotherapy of cancer and viral and inflammatory diseases. Mechanisms determining their trafficking toward the plasma membrane are not well known, although this might eventually become a target for therapeutic intervention. The transporter regulator RS1, which was initially identified as a short-term, post-transcriptional regulator of the high-affinity, Na(+)-coupled, glucose transporter sodium-dependent glucose cotransporter 1, was evaluated in this study as a candidate for coordinate regulation of membrane insertion of human CNT-type proteins. With a combination of studies with mammalian cells, Xenopus laevis oocytes, and RS1-null mice, evidence that RS1 down-regulates the localization and activity at the plasma membrane of the three members of this protein family (CNT1, CNT2, and CNT3) is provided, which indicates the biochemical basis for coordinate regulation of nucleoside uptake ability in epithelia and probably in other RS1-expressing cell types. PMID:22492015

  11. Synthesis of novel fluorocarbocyclic nucleosides and nucleotides as potential inhibitors of human immunodeficiency virus

    SciTech Connect

    Hilpert, H.

    1989-01-01

    3[prime]-Azido-3[prime]-deoxythymidine (AZT) and 2[prime], 3[prime]-dideoxycytidine (DDC) are potent in vivo inhibitors of human immunodeficiency virus. Due to their short half-life in the body and undesired side-effects compounds with improved bioavailability were designed. A feature of these analogues was the replacement of the heterocyclic oxygen atom by an isosteric CHF-group thus stabilizing the labile glycosidic bond against metabolic breakdown. A versatile and short synthesis, starting from ketone, serves to construct the highly functionalized and protected key intermediates. These ([alpha]- and [beta]-fluoro epimeric) intermediates were elaborated to eight fluorocarbocyclic nucleoside analogues linked with a thymine base, an adenine base, and a guanine base. An attempt was made to prepare analogues of the potent HIV inhibitor carbovir c. The unexpected oxidation of the double bond of compound d, instead of the desired Baeyer-Villiger ring-expansion, meant that the synthetic scheme was redundant. A second total synthesis involves the preparation of the three fluorocarbocyclic phosphonates. These analogues possess additionally a P-C linkage which should markedly enhance the stability of the side chain. To perform enzyme inhibition tests, three analogues were chemically activated to the biologically active triphosphates. Inhibition tests on HIV associated reverse transcriptase confirmed the high activity of one of the AZT triphosphates. The fluorocarbocyclic counterpart was two orders of magnitude less active. A fluorocarbocyclic phosphonate was twice as active as the AZT triphosphate. Neither the eight nucleoside analogues nor the three phosphonates displayed significant activity against HIV infected cells. Crystallographic data of two fluorocarbocyclic nucleosides, two potent HIV inhibitors, and some 20 examples of 2[prime]-deoxyribonucleosides have been compared.

  12. Evidence for the occurrence of an ecto-(adenosine triphosphatase) in rat epididymal spermatozoa.

    PubMed Central

    Majumder, G C; Biswas, R

    1979-01-01

    Intact spermatozoa from rat cauda epididymis possess a Mg2+-dependent ATPase activity that hydrolyses externally added [gamma-32P]ATP. The ATPase reaction was linear with time for approx. 6 min and there was no detectable uptake of ATP by these cells. The ATPase activity of the whole spermatozoa was not due to leakage of the intracellular enzymic activity, contamination of the broken cells or any possible cell damage during incubation and isolation of spermatozoa. The activity of the enzyme was strongly inhibited (approx. 85%) by p-chloromercuribenzenesulphonic acid (50 microM) or the diazonium salt of sulphanilic acid (50 microM), which are believed not to enter the cells, whereas ouabain (0.5 mM), NaF (10 mM), NaN3 (2.5 mM) and oligomycin (5 microM) had no appreciable effect on the activity of the spermatozoal APTase. There was little loss of ATPase activity from the cells when washed with 0.5 mM-EDTA and an iso-osmotic or hyperosmotic medium. These data are consistent with the view that the observed ATPase activity is located on the external surface of spermatozoa. The sperm ecto-ATPase activity is resistant to the action of proteinases (50 micrograms/ml), namely trypsin, chymotrypsin and Pronase. Studies with various unlabelled phosphate esters indicate that the sperm ecto-ATPase is not a non-specific phosphatase and it has high degree of substrate specificity for ATP. PMID:231971

  13. Ethenoguanines undergo glycosylation by nucleoside 2'-deoxyribosyltransferases at non-natural sites.

    PubMed

    Ye, Wenjie; Paul, Debamita; Gao, Lina; Seckute, Jolita; Sangaiah, Ramiah; Jayaraj, Karupiah; Zhang, Zhenfa; Kaminski, P Alexandre; Ealick, Steven E; Gold, Avram; Ball, Louise M

    2014-01-01

    Deoxyribosyl transferases and functionally related purine nucleoside phosphorylases are used extensively for synthesis of non-natural deoxynucleosides as pharmaceuticals or standards for characterizing and quantitating DNA adducts. Hence exploring the conformational tolerance of the active sites of these enzymes is of considerable practical interest. We have determined the crystal structure at 2.1 Å resolution of Lactobacillus helveticus purine deoxyribosyl transferase (PDT) with the tricyclic purine 8,9-dihydro-9-oxoimidazo[2,1-b]purine (N2,3-ethenoguanine) at the active site. The active site electron density map was compatible with four orientations, two consistent with sites for deoxyribosylation and two appearing to be unproductive. In accord with the crystal structure, Lactobacillus helveticus PDT glycosylates the 8,9-dihydro-9-oxoimidazo[2,1-b]purine at N7 and N1, with a marked preference for N7. The activity of Lactobacillus helveticus PDT was compared with that of the nucleoside 2'-deoxyribosyltransferase enzymes (DRT Type II) from Lactobacillus leichmannii and Lactobacillus fermentum, which were somewhat more effective in the deoxyribosylation than Lactobacillus helveticus PDT, glycosylating the substrate with product profiles dependent on the pH of the incubation. The purine nucleoside phosphorylase of Escherichia coli, also commonly used in ribosylation of non-natural bases, was an order of magnitude less efficient than the transferase enzymes. Modeling based on published active-site structures as templates suggests that in all cases, an active site Phe is critical in orienting the molecular plane of the purine derivative. Adventitious hydrogen bonding with additional active site residues appears to result in presentation of multiple nucleophilic sites on the periphery of the acceptor base for ribosylation to give a distribution of nucleosides. Chemical glycosylation of O9-benzylated 8,9-dihydro-9-oxoimidazo[2,1-b]purine also resulted in N7 and N1

  14. Ethenoguanines Undergo Glycosylation by Nucleoside 2′-Deoxyribosyltransferases at Non-Natural Sites

    PubMed Central

    Ye, Wenjie; Paul, Debamita; Gao, Lina; Seckute, Jolita; Jayaraj, Karupiah; Zhang, Zhenfa; Kaminski, P. Alexandre

    2014-01-01

    Deoxyribosyl transferases and functionally related purine nucleoside phosphorylases are used extensively for synthesis of non-natural deoxynucleosides as pharmaceuticals or standards for characterizing and quantitating DNA adducts. Hence exploring the conformational tolerance of the active sites of these enzymes is of considerable practical interest. We have determined the crystal structure at 2.1 Å resolution of Lactobacillus helveticus purine deoxyribosyl transferase (PDT) with the tricyclic purine 8,9-dihydro-9-oxoimidazo[2,1-b]purine (N2,3-ethenoguanine) at the active site. The active site electron density map was compatible with four orientations, two consistent with sites for deoxyribosylation and two appearing to be unproductive. In accord with the crystal structure, Lactobacillus helveticus PDT glycosylates the 8,9-dihydro-9-oxoimidazo[2,1-b]purine at N7 and N1, with a marked preference for N7. The activity of Lactobacillus helveticus PDT was compared with that of the nucleoside 2′-deoxyribosyltransferase enzymes (DRT Type II) from Lactobacillus leichmannii and Lactobacillus fermentum, which were somewhat more effective in the deoxyribosylation than Lactobacillus helveticus PDT, glycosylating the substrate with product profiles dependent on the pH of the incubation. The purine nucleoside phosphorylase of Escherichia coli, also commonly used in ribosylation of non-natural bases, was an order of magnitude less efficient than the transferase enzymes. Modeling based on published active-site structures as templates suggests that in all cases, an active site Phe is critical in orienting the molecular plane of the purine derivative. Adventitious hydrogen bonding with additional active site residues appears to result in presentation of multiple nucleophilic sites on the periphery of the acceptor base for ribosylation to give a distribution of nucleosides. Chemical glycosylation of O9-benzylated 8,9-dihydro-9-oxoimidazo[2,1-b]purine also resulted in N7 and N1

  15. Nucleoside 2'-deoxyribosyltransferase from psychrophilic bacterium Bacillus psychrosaccharolyticus--preparation of an immobilized biocatalyst for the enzymatic synthesis of therapeutic nucleosides.

    PubMed

    Fresco-Taboada, Alba; Serra, Immacolata; Fernández-Lucas, Jesús; Acebal, Carmen; Arroyo, Miguel; Terreni, Marco; de la Mata, Isabel

    2014-01-01

    Nucleoside 2'-deoxyribosyltransferase (NDT) from the psychrophilic bacterium Bacillus psychrosaccharolyticus CECT 4074 has been cloned and produced for the first time. A preliminary characterization of the recombinant protein indicates that the enzyme is an NDT type II since it catalyzes the transfer of 2'-deoxyribose between purines and pyrimidines. The enzyme (BpNDT) displays a high activity and stability in a broad range of pH and temperature. In addition, different approaches for the immobilization of BpNDT onto several supports have been studied in order to prepare a suitable biocatalyst for the one-step industrial enzymatic synthesis of different therapeutic nucleosides. Best results were obtained by adsorbing the enzyme on PEI-functionalized agarose and subsequent cross-linking with aldehyde-dextran (20 kDa and 70% oxidation degree). The immobilized enzyme could be recycled for at least 30 consecutive cycles in the synthesis of 2'-deoxyadenosine from 2'-deoxyuridine and adenine at 37 °C and pH 8.0, with a 25% loss of activity. High conversion yield of trifluridine (64.4%) was achieved in 2 h when 20 mM of 2'-deoxyuridine and 10 mM 5-trifluorothymine were employed in the transglycosylation reaction catalyzed by immobilized BpNDT at 37 °C and pH 7.5. PMID:25090115

  16. Effect of Ethanol on Branchial Adenosine Triphosphatases in Oreochromis mossambicus (Peters)

    PubMed Central

    Bhanu, Smitha V.; Philip, Babu

    2011-01-01

    The aim of this work was to determine the toxicity of ethanol in an aquatic system by means of bioassays with Oreochromis mossambicus (Peters) as a test organism. The study revealed changes in the gill ATPase activities. The results obtained indicated that ethanol brought about a decrease in the body weight, followed by significant inhibition on total ATPase, Na+/K+ ATPase, Ca2+ ATPase and Mg2+ ATPase activities. The studies also indicated that these can be employed as suitable biomarkers in ethanol related toxicity studies. PMID:21430917

  17. Expression and hepatobiliary transport characteristics of the concentrative and equilibrative nucleoside transporters in sandwich-cultured human hepatocytes

    PubMed Central

    Govindarajan, Rajgopal; Endres, Christopher J.; Whittington, Dale; LeCluyse, Edward; Pastor-Anglada, Marçal; Tse, Chung-Ming; Unadkat, Jashvant D.

    2008-01-01

    We previously reported that both the concentrative (hCNT) and equilibrative (hENT) nucleoside transporters are expressed in the human liver (21). Here we report a study that investigated the expression of these transporters (transcripts and proteins) and their role in the hepatobiliary transport of nucleosides/nucleoside drugs using sandwich-cultured human hepatocytes. In the hepatic tissue, the rank order of the mRNA expression of the transporters was hCNT1 ≈ hENT1 > hENT2 ≈ hCNT2 > hCNT3. In sandwich-cultured hepatocytes, the mRNA expression of hCNT2 and hENT2 was comparable to that in hepatic tissue, whereas the expression of corresponding transporters in the two-dimensional hepatocyte cultures was lower. Colocalization studies demonstrated predominant localization of these transporters at the sinusoidal membrane and of hENT1, hCNT1, and hCNT2 at the canalicular membrane. In the sandwich-cultured hepatocytes, ENTs were the major contributors to the transport of thymidine (hENT1, 63%; hENT2, 23%) or guanosine (hENT1, 53%; hENT2, 24%) into the hepatocytes followed by hCNT1 (10%) for thymidine or hCNT2 (23%) for guanosine. Although ribavirin was predominately transported (89%) into the hepatocytes by hENT1, fialuridine (FIAU) was transported by both hENT1 (30%) and hCNTs (61%). The extensively metabolized natural nucleosides were not effluxed into the bile, whereas significant biliary-efflux was observed of FIAU (19%), ribavirin (30%), and formycin B (35%). We conclude that the hepatic activity of hENT1 and hCNT1/2 transporters will determine the in vivo hepatic distribution and therefore the efficacy and/or toxicity of nucleoside drugs used to treat hepatic diseases. PMID:18635603

  18. Assays To Detect the Formation of Triphosphates of Unnatural Nucleotides: Application to Escherichia coli Nucleoside Diphosphate Kinase.

    PubMed

    Matsuura, Mariko F; Shaw, Ryan W; Moses, Jennifer D; Kim, Hyo-Joong; Kim, Myong-Jung; Kim, Myong-Sang; Hoshika, Shuichi; Karalkar, Nilesh; Benner, Steven A

    2016-03-18

    One frontier in synthetic biology seeks to move artificially expanded genetic information systems (AEGIS) into natural living cells and to arrange the metabolism of those cells to allow them to replicate plasmids built from these unnatural genetic systems. In addition to requiring polymerases that replicate AEGIS oligonucleotides, such cells require metabolic pathways that biosynthesize the triphosphates of AEGIS nucleosides, the substrates for those polymerases. Such pathways generally require nucleoside and nucleotide kinases to phosphorylate AEGIS nucleosides and nucleotides on the path to these triphosphates. Thus, constructing such pathways focuses on engineering natural nucleoside and nucleotide kinases, which often do not accept the unnatural AEGIS biosynthetic intermediates. This, in turn, requires assays that allow the enzyme engineer to follow the kinase reaction, assays that are easily confused by ATPase and other spurious activities that might arise through "site-directed damage" of the natural kinases being engineered. This article introduces three assays that can detect the formation of both natural and unnatural deoxyribonucleoside triphosphates, assessing their value as polymerase substrates at the same time as monitoring the progress of kinase engineering. Here, we focus on two complementary AEGIS nucleoside diphosphates, 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one. These assays provide new ways to detect the formation of unnatural deoxyribonucleoside triphosphates in vitro and to confirm their incorporation into DNA. Thus, these assays can be used with other unnatural nucleotides. PMID:26829203

  19. The Crystal Structure of Streptococcus pyogenes Uridine Phosphorylase Reveals a Distinct Subfamily of Nucleoside Phosphorylases

    SciTech Connect

    Tran, Timothy H.; Christoffersen, S.; Allan, Paula W.; Parker, William B.; Piskur, Jure; Serra, I.; Terreni, M.; Ealick, Steven E.

    2011-09-20

    Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 {angstrom} resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an ?/? monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is {approx}7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.

  20. Supramolecular gels made from nucleobase, nucleoside and nucleotide analogs.

    PubMed

    Peters, Gretchen Marie; Davis, Jeffery T

    2016-06-01

    Supramolecular or molecular gels are attractive for various applications, including diagnostics, tissue scaffolding and targeted drug release. Gelators derived from natural products are of particular interest for biomedical purposes, as they are generally biocompatible and stimuli-responsive. The building blocks of nucleic acids (i.e. nucleobases, nucleosides, and nucleotides) are desirable candidates for supramolecular gelation as they readily engage in reversible, noncovalent interactions. In this review, we describe a number of organo- and hydrogels formed through the assembly of nucleosides, nucleotides, and their derivatives. While natural nucleosides and nucleotides generally require derivatization to induce gelation, guanosine and its corresponding nucleotides are well known gelators. This unique gelating ability is due to propensity of the guanine nucleobase to self-associate into stable higher-order assemblies, such as G-ribbons, G4-quartets, and G-quadruplexes. PMID:27146863

  1. Flow cytomeric measurement of DNA and incorporated nucleoside analogs

    DOEpatents

    Dolbeare, Frank A.; Gray, Joe W.

    1989-01-01

    A method is provided for simultaneously measuring total cellular DNA and incorporated nucleoside analog. The method entails altering the cellular DNA of cells grown in the presence of a nucleoside analog so that single stranded and double stranded portions are present. Separate stains are used against the two portions. An immunochemical stain is used against the single stranded portion to provide a measure of incorporated nucleoside analog, and a double strand DNA-specific stain is used against the double stranded portion to simultaneously provide a measure of total cellular DNA. The method permits rapid flow cytometric analysis of cell populations, rapid identification of cycling and noncycling subpopulations, and determination of the efficacy of S phase cytotoxic anticancer agents.

  2. Chemoselective N-Deacetylation of Protected Nucleosides and Nucleotides Promoted by Schwartz's Reagent

    PubMed Central

    Ferrari, Valentina; Serpi, Michaela; McGuigan, Christopher; Pertusati, Fabrizio

    2015-01-01

    Protection and deprotection strategies involving the N-acetyl group are widely utilized in nucleoside and nucleotide chemistry. Herein, we present a mild and selective N-deacetylation methodology, applicable to purine and pyrimidine nucleosides, by means of Schwartz's reagent, compatible with most of the common protecting groups used in nucleoside chemistry. PMID:26492555

  3. Mechanism of inhibition of rat brain adenosine triphosphatase by mercuric chloride

    SciTech Connect

    Chetty, C.S.; Rajanna, B.; Rajanna, S. )

    1989-02-09

    Mercuric Chloride (Hg), a neurotoxic compound inhibited ATPase system of rat brain microsomes. Membrane bound enzymes, Na{sup +}-K{sup +} ATPase (IC{sub 50} = 2.35 {times} 10{sup {minus}7M}) and K-paranitrophenyl phosphatase (K-PNPPase) (IC{sub 50} = 2.7 {times} 10{sup {minus}7M}) and {sup 3}H-Ouabain binding (IC{sub 50} = 3.3 {times} 10{sup {minus}7M}) were inhibited by Hg at micromolar concentrations in a dose dependent manner. Hydrolysis of ATP was linear with time with or without Hg in the reaction mixtures. Altered pH or temperature versus enzyme activity showed higher inhibition by Hg at basic pH (8.0-9.0) and at lower temperatures (17-32{degree}C). Activation energy ({Delta}E) values were increased at 27-37{degree}C in the presence of Hg. Kinetic studies of cationic-substrate activation of Na{sup +}-K{sup +} ATPase and K-PNPPase in the presence of Hg showed significant changes in kinetic constant (K{sub m} and V{sub max}). Inhibition of Na{sup +}-K{sup +} ATPase was partially restored by repeated washings of microsomes. Preincubation with sulfhydryl agents protected Na{sup +}-K{sup +} ATPase from Hg inhibition. Cumulative inhibition studies with Hg and ouabain indicated possible interaction between the two inhibitors of Na{sup +}-K{sup +} ATPase by interacting at Na{sup +} and K{sup +} sites.

  4. Testing nucleoside analogues as inhibitors of Bacillus anthracis spore germination in vitro and in macrophage cell culture.

    PubMed

    Alvarez, Zadkiel; Lee, Kyungae; Abel-Santos, Ernesto

    2010-12-01

    Bacillus anthracis, the etiological agent of anthrax, has a dormant stage in its life cycle known as the endospore. When conditions become favorable, spores germinate and transform into vegetative bacteria. In inhalational anthrax, the most fatal manifestation of the disease, spores enter the organism through the respiratory tract and germinate in phagosomes of alveolar macrophages. Germinated cells can then produce toxins and establish infection. Thus, germination is a crucial step for the initiation of pathogenesis. B. anthracis spore germination is activated by a wide variety of amino acids and purine nucleosides. Inosine and l-alanine are the two most potent nutrient germinants in vitro. Recent studies have shown that germination can be hindered by isomers or structural analogues of germinants. 6-Thioguanosine (6-TG), a guanosine analogue, is able to inhibit germination and prevent B. anthracis toxin-mediated necrosis in murine macrophages. In this study, we screened 46 different nucleoside analogues as activators or inhibitors of B. anthracis spore germination in vitro. These compounds were also tested for their ability to protect the macrophage cell line J774a.1 from B. anthracis cytotoxicity. Structure-activity relationship analysis of activators and inhibitors clarified the binding mechanisms of nucleosides to B. anthracis spores. In contrast, no structure-activity relationships were apparent for compounds that protected macrophages from B. anthracis-mediated killing. However, multiple inhibitors additively protected macrophages from B. anthracis. PMID:20921305

  5. Syntheses of Isoxazoline-Carbocyclic Nucleosides and Their Antiviral Evaluation: A Standard Protocol

    PubMed Central

    Quadrelli, Paolo; Vazquez Martinez, Naiara; Scrocchi, Roberto; Corsaro, Antonino; Pistarà, Venerando

    2014-01-01

    The current synthesis of racemic purine and pyrimidine isoxazoline-carbocyclic nucleosides is reported, detailing the key-steps for standard and reliable preparations. Improved yields were obtained by the proper tuning of the single synthetic steps, opening the way for the preparation of a variety of novel compounds. Some of the obtained compounds were also evaluated against a wide variety of DNA and RNA viruses including HIV. No specific antiviral activity was observed in the cases at hand. Novel compounds were prepared for future biological tests. PMID:25544956

  6. Substituted indoles as HIV-1 non-nucleoside reverse transcriptase inhibitors: a patent evaluation (WO2015044928).

    PubMed

    Li, Xiao; Gao, Ping; Zhan, Peng; Liu, Xinyong

    2016-05-01

    The invention described in this patent (WO2015044928) is related to compounds based on the substituted indole scaffold, their synthetic process and application to inhibit HIV-1 replication as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Some of the newly claimed compounds presented improved potency against wild-type (WT) HIV-1 strain in comparison to previously disclosed indole-based NNRTIs and were also shown to be effective against common resistant HIV-1 strains. In light of their novel structural characteristics, simple synthetic route and improved anti-HIV activity, these compounds deserve further study as promising NNRTIs. PMID:26742549

  7. Formation of ATP by the adenosine triphosphatase complex from spinach chloroplasts reconstituted together with bacteriorhodopsin.

    PubMed

    Winget, G D; Kanner, N; Racker, E

    1977-06-01

    The energy-linked ATPase complex has been isolated from spinach chloroplasts. This protein complex contained all the subunits of the chloroplast coupling factor (CF1) as well as several hydrophobic compoenents. When the activated complex was reconstituted with added soybean phospholipids, it catalyzed the exchange of radioactive inorganic phosphate with ATP. Sonication of the complex into proteoliposomes together with bacteriorhodopsin yield vesicles that catalyzed light-dependent ATP formation. Both the 32Pi-ATP exchange reactions and ATP formation were sensitive to uncouplers such as 3-tert-butyl-5,2'-dichloro-4'-nitrosalicylanilide, bis-(hexafluoroacetonyl)acetone and carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone, that act to dissipate a proton gradient. The energy transfer inhibitors dicyclohexylcarbodiimide, triphenyltin chloride and 2-beta-D-glucopyranosyl-4,6'-dihydroxydihydrochalcone were also effective inhibitors of both reactions. PMID:141938

  8. A highly stable adenosine triphosphatase from a thermophillie bacterium. Purification, properties, and reconstitution.

    PubMed

    Yoshida, M; Sone, N; Hirata, H; Kagawa, Y

    1975-10-10

    1. A highly stable ATPase (TF1) was purified to a monodispersed state from the membranes of a thermophilic bacterium PS3. Its molecular weight was 380,000, and it was composed of five subunits alpha, beta, gamma, sigma', and sigma with molecular weights of 56,000, 53,000, 32,000, 15,500, and 11,000, respectively. 2. TF1 was stable against dissociating agents such as 5.5 M urea and 4.0 M LiCl, organic solvents, such as 60% acetone, heavy metals, and detergents. Low concentrations of all these agents stimulated its activity at 60 degrees. 3. TF1 was not cold-labile and showed a maximal activity at 70 degrees. Its CD spectrum revealed that its conformation changed between 81 and 96 degrees, and that its contents of alpha helices and beta structures were 27.3 and 12.8%, respectively, at 75 degrees. 4. TF1 was completely dissociated by treatment with dodecyl sulfate at 60 degrees and then with 7.1 M urea. The dissociated TF1 was reconstituted by treatment with Dowex 1-X2, and then dialysis. 5. [3H]Acetyl-TF1 bound to TF1-depleted membranes. TF1 only catalyzed 32Pi-ATP exchange and showed sensitivity to inhibitors of energy transfer when bound to the membranes. 6. A hydrophobic membrance component (TFo) was isolated which rendered TF1 sensitive to inhibitors of energy transfer. It was composed of three subunits (with molecular weights of 19,000, 13,500, and 5,400) and P-lipids. PMID:240842

  9. Inhibition of nucleoside diphosphate kinase in rat liver mitochondria by added 3'-azido-3'-deoxythymidine.

    PubMed

    Valenti, D; Barile, M; Quagliariello, E; Passarella, S

    1999-02-12

    The effect of 3'-azido-3'-deoxythymidine on nucleoside diphosphate kinase of isolated rat liver mitochondria has been studied. This is done by monitoring the increase in the rate of oxygen uptake by nucleoside diphosphate (TDP, UDP, CDP or GDP) addition to mitochondria in state 4. It is shown that 3'-azido-3'-deoxythymidine inhibits the mitochondrial nucleoside diphosphate kinase in a competitive manner, with a Ki value of about 10 microM as measured for each tested nucleoside diphosphate. It is also shown that high concentrations of GDP prevent 3'-azido-3'-deoxythymidine inhibition of the nucleoside diphosphate kinase. PMID:10050777

  10. Hepatocyte nuclear factor-4alpha and bile acids regulate human concentrative nucleoside transporter-1 gene expression.

    PubMed

    Klein, Kerstin; Kullak-Ublick, Gerd A; Wagner, Martin; Trauner, Michael; Eloranta, Jyrki J

    2009-04-01

    The concentrative nucleoside transporter-1 (CNT1) is a member of the solute carrier 28 (SLC28) gene family and is expressed in the liver, intestine, and kidneys. CNT1 mediates the uptake of naturally occurring pyrimidine nucleosides, but also nucleoside analogs used in anticancer and antiviral therapy. Thus expression levels of CNT1 may affect the pharmacokinetics of these drugs and the outcome of drug therapy. Because little is known about the transcriptional regulation of human CNT1 gene expression, we have characterized the CNT1 promoter with respect to DNA response elements and their binding factors. The transcriptional start site of the CNT1 gene was determined by 5'-RACE. In silico analysis revealed the existence of three putative binding sites for the nuclear receptor hepatocyte nuclear factor-4alpha (HNF-4alpha) within the CNT1 promoter. A luciferase reporter gene construct containing the CNT1 promoter region was transactivated by HNF-4alpha in human cell lines derived from the liver, intestine, and kidneys. Consistent with this, we showed in electromobility shift assays that HNF-4alpha specifically binds to two conserved direct repeat-1 motifs within the proximal CNT1 promoter. In cotransfection experiments, the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha further increased, whereas the bile acid-inducible corepressor small heterodimer partner reduced, HNF-4alpha-dependent CNT1 promoter activity. Consistent with the latter phenomenon, CNT1 mRNA expression levels were suppressed in primary human hepatocytes upon bile acid treatment. Supporting the physiological relevance and species conservation of this effect, ileal Cnt1 mRNA expression was decreased upon bile acid feeding and increased upon bile duct ligation in mice. PMID:19228884

  11. Structural determinants of the 5'-methylthioinosine specificity of Plasmodium purine nucleoside phosphorylase.

    PubMed

    Donaldson, Teraya M; Ting, Li-Min; Zhan, Chenyang; Shi, Wuxian; Zheng, Renjian; Almo, Steven C; Kim, Kami

    2014-01-01

    Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5'-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5'-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5'-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5'-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket. PMID:24416224

  12. Concentrative nucleoside transporter (rCNT1) is targeted to the apical membrane through the hepatic transcytotic pathway.

    PubMed

    Duflot, Sylvie; Calvo, Maria; Casado, F Javier; Enrich, Carlos; Pastor-Anglada, Marçal

    2002-11-15

    The Na+-dependent nucleoside transporter CNT1 has been identified in a caveolin-enriched plasma membrane fraction (CEF), in transcytotic endosomes, and in canalicular membranes isolated from quiescent rat liver in which the transporter appears to be biologically active. CNT1 was also detected, albeit in small amounts, in the early/sorting endosomes. Plasma membrane preparations enriched in basolateral markers showed Na+-dependent nucleoside transport activity that is mostly, if not exclusively, accounted for by CNT2, a transporter protein which was not detected in CEF nor in the endosomal fractions. These data are consistent with different localization and trafficking pathways of the two isoforms in hepatocytes. CNT1 is the first transporter which is reported to follow the transcytotic pathway to be inserted on the apical side of liver parenchymal cells. PMID:12441131

  13. The GABA transaminase, ABAT, is essential for mitochondrial nucleoside metabolism

    PubMed Central

    Besse, Arnaud; Wu, Ping; Bruni, Francesco; Donti, Taraka; Graham, Brett H.; Craigen, William J.; McFarland, Robert; Moretti, Paolo; Lalani, Seema; Scott, Kenneth L.; Taylor, Robert W.; Bonnen, Penelope E.

    2015-01-01

    Summary ABAT is a key enzyme responsible for catabolism of principal inhibitory neurotransmitter gamma-aminobutyric acid (GABA). We report an essential role for ABAT in a seemingly unrelated pathway, mitochondrial nucleoside salvage, and demonstrate that mutations in this enzyme cause an autosomal recessive neurometabolic disorder and mtDNA depletion syndrome (MDS). We describe a family with encephalomyopathic MDS caused by a homozygous missense mutation in ABAT that results in elevated GABA in subjects’ brains as well as decreased mtDNA levels in subjects’ fibroblasts. Nucleoside rescue and co-IP experiments pinpoint that ABAT functions in the mitochondrial nucleoside salvage pathway to facilitate conversion of dNDPs to dNTPs. Pharmacological inhibition of ABAT through the irreversible inhibitor Vigabatrin caused depletion of mtDNA in photoreceptor cells that was prevented through addition of dNTPs in cell culture media. This work reveals ABAT as a connection between GABA metabolism and nucleoside metabolism and defines a neurometabolic disorder that includes MDS. PMID:25738457

  14. Nucleoside-Based Diarylethene Photoswitches: Synthesis and Photochromic Properties.

    PubMed

    Wang, Hai-Xia; Xi, Dan-Dan; Xie, Ming-Sheng; Wang, Hui-Xuan; Qu, Gui-Rong; Guo, Hai-Ming

    2016-07-01

    Diarylethene photoswitches based on the natural nucleoside deoxyadenosine were designed and synthesized. In aqueous solution, some of them exhibited good photochromic properties, including clear changes in color upon irradiation at 365 nm, red-shifts of the absorption wavelength, with good fatigue resistance, thermal stability, conversion efficiency, and base-pairing properties. PMID:27124421

  15. Cross-linked polymeric nanogel formulations of 5'-triphosphates of nucleoside analogues: role of the cellular membrane in drug release.

    PubMed

    Vinogradov, Serguei V; Kohli, Ekta; Zeman, Arin D

    2005-01-01

    Activation of cytotoxic nucleoside analogues in vivo depends primarily on their cell-specific phosphorylation. Anticancer chemotherapy using nucleoside analogues may be significantly enhanced by intracellular administration of active phosphorylated drugs. However, the cellular transport of anionic compounds is very ineffective and restricted by many drug efflux transporters. Recently developed cationic nanogel carriers can encapsulate large amounts of nucleoside 5'-triphosphates that form polyionic complexes with protonated amino groups on the polyethylenimine backbone of the nanogels. In this paper, the 5'-triphosphate of an antiviral nucleoside analogue, 3'-azido-2',3'-dideoxythymidine (AZT), was efficiently synthesized and its complexes with nanogels were obtained and evaluated as potential cytotoxic drug formulations for treatment of human breast carcinoma cells. A selective phosphorylating reagent, tris-imidazolylphosphate, was used to convert AZT into the nucleoside analogue 5'-triphosphate using a one-pot procedure. The corresponding 3'-azido-2',3'-dideoxythymidine 5'-triphosphate (AZTTP) was isolated with high yield (75%). Nanogels encapsulated up to 30% of AZTTP by weight by mixing solutions of the carrier and the drug. The AZTTP/nanogel formulation showed enhanced cytotoxicity in two breast cancer cell lines, MCF-7 and MDA-MB-231, demonstrating IC50 values 130-200 times lower than those values for AZT alone. The exact mechanism of drug release from nanogels remains unclear. One mechanism could involve interaction with negatively charged counterions. A high affinity of nanogels to isolated cellular membranes has been observed, especially for nanogels made of amphiphilic block copolymer, Pluronic P85. Cellular trafficking of nanogel particles, contrasted by polyethylenimine-coordinated copper(II) ions, was studied by transmission electron microscopy (TEM), which revealed membranotropic properties of nanogels. A substantial release of encapsulated drug was

  16. Novel reactivity of Fhit proteins: catalysts for fluorolysis of nucleoside 5'-phosphoramidates and nucleoside 5'-phosphosulfates to generate nucleoside 5'-phosphorofluoridates.

    PubMed

    Wojdyła-Mamoń, Anna M; Zimny, Jarosław; Romanowska, Joanna; Kraszewski, Adam; Stawinski, Jacek; Bieganowski, Paweł; Guranowski, Andrzej

    2015-06-01

    Fragile histidine triad (HIT) proteins (Fhits) occur in all eukaryotes but their function is largely unknown. Human Fhit is presumed to function as a tumour suppressor. Previously, we demonstrated that Fhits catalyse hydrolysis of not only dinucleoside triphosphates but also natural adenosine 5'-phosphoramidate (NH2-pA) and adenosine 5'-phosphosulfate (SO4-pA) as well as synthetic adenosine 5'-phosphorofluoridate (F-pA). In the present study, we describe an Fhit-catalysed displacement of the amino group of nucleoside 5'-phosphoramidates (NH2-pNs) or the sulfate moiety of nucleoside 5'-phosphosulfates (SO4-pNs) by fluoride anion. This results in transient accumulation of the corresponding nucleoside 5'-phosphorofluoridates (F-pNs). Substrate specificity and kinetic characterization of the fluorolytic reactions catalysed by the human Fhit and other examples of involvement of fluoride in the biochemistry of nucleotides are described. Among other HIT proteins, human histidine triad nucleotide-binding protein (Hint1) catalysed fluorolysis of NH2-pA 20 times and human Hint2 40 times more slowly than human Fhit. PMID:25826698

  17. Selective loss of nucleoside carrier expression in rat hepatocarcinomas.

    PubMed

    Dragan, Y; Valdés, R; Gomez-Angelats, M; Felipe, A; Javier Casado, F; Pitot, H; Pastor-Anglada, M

    2000-08-01

    Evidence that hepatoma cell lines show differential expression of concentrative nucleoside transporters (CNT1 and CNT2) prompted us to study the transporter proteins in 2 models of hepatocarcinogenesis, the chemically induced Solt and Farber model and the albumin-SV40 large T antigen (Alb-SV40) transgenic rat. CNT1 expression was lower in tumor biopsy specimens from Alb-SV40 rat livers than in normal tissue. Immunocytochemistry revealed that the CNT1 protein was indeed absent in the tumor lesions. CNT1 was also absent in a cell line, L25, derived from the Alb-SV40 transgenic rat liver tumors, whereas another cell line, L37, derived from the normal-appearing parenchyma, retained the expression of both carrier isoforms. The protein expression correlated with the nucleoside transport properties of these cell lines. Moreover, although CNT2 expression was highly dependent on the growth characteristics of the 2 cell lines, as was CNT1 (albeit to a lower extent) in L37 cells, it was not expressed in L25 cells at any stage of cell growth. In contrast to the transgenic model of hepatocarcinogenesis, in the chemically induced tumors the expression of CNT2 was lower, although still detectable. In summary, these data indicate that hepatocarcinogenesis leads to a selective loss or diminished expression of nucleoside carrier isoforms, a feature that may be relevant to our understanding of the molecular basis of the bioavailability of those drugs that are nucleoside derivatives and may be substrates of these carriers. The transport properties and isoform-expression profile of the L25 and L37 cell lines make them suitable hepatocyte culture models with which to study nucleoside transport processes and drug sensitivity. PMID:10915730

  18. Sensing Metal Ions with DNA Building Blocks: Fluorescent Pyridobenzimidazole Nucleosides

    PubMed Central

    Kim, Su Jeong; Kool, Eric T.

    2008-01-01

    We describe novel fluorescent N-deoxyribosides (1 and 2) having 2-pyrido-2-benzimidazole and 2-quino-2-benzimidazole as aglycones. The compounds were prepared from the previously unknown heterocyclic precursors and Hoffer’s chlorosugar, yielding alpha anomers as the chief products. X-ray crystal structures confirmed the geometry, and showed that the pyridine and benzimidazole ring systems deviated from coplanarity in the solid state by 154° and 140°, respectively. In methanol the compounds 1 and 2 had absorption maxima at 360 and 370 nm respectively, and emission maxima at 494 and 539 nm. Experiments revealed varied fluorescence responses of the nucleosides to a panel of seventeen monovalent, divalent and trivalent metal ions in methanol. One or both of the nucleosides showed significant changes with ten of the metal ions. The most pronounced spectral changes for ligand-nucleoside 1 included red shifts in fluorescence (Au+, Au3+), strong quenching (Cu2+, Ni2+, Pt2+), and in substantial enhancements in emission intensity coupled with redshifts (Ag+, Cd2+, Zn2+). The greatest spectral changes for ligand-nucleoside 2 included a redshift in fluorescence (Ag+), a blueshift (Cd2+), strong quenching (Pd2+, Pt2+), and in substantial enhancements in emission intensity coupled with a blueshift (Zn2+). The compounds could be readily incorporated into oligodeoxynucleotides, where an initial study revealed that they retained sensitivity to metal ions in aqueous solution, and demonstrated possible cooperative sensing behavior with several ions. The two free nucleosides alone can act as differential sensors for at multiple metal ions, and they are potentially useful monomers for contributing metal ion sensing capability to DNAs. PMID:16669686

  19. Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1.

    PubMed

    Rehan, Shahid; Ashok, Yashwanth; Nanekar, Rahul; Jaakola, Veli-Pekka

    2015-12-15

    Many nucleoside transport inhibitors are in clinical use as anti-cancer, vasodilator and cardioprotective drugs. However, little is known about the binding energetics of these inhibitors to nucleoside transporters (NTs) due to their low endogenous expression levels and difficulties in the biophysical characterization of purified protein with ligands. Here, we present kinetics and thermodynamic analyses of inhibitor binding to the human equilibrative nucleoside transporter-1 (hENT1), also known as SLC29A1. Using a radioligand binding assay, we obtained equilibrium binding and kinetic rate constants of well-known NT inhibitors--[(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR), dilazep, and dipyridamole--and the native permeant, adenosine, to hENT1. We observed that the equilibrium binding affinities for all inhibitors decreased whereas, the kinetic rate constants increased with increasing temperature. Furthermore, we found that binding is enthalpy driven and thus, an exothermic reaction, implying that the transporter does not discriminate between its inhibitors and substrates thermodynamically. This predominantly enthalpy-driven binding by four chemically distinct ligands suggests that the transporter may not tolerate diversity in the type of interactions that lead to high affinity binding. Consistent with this, the measured activation energy of [(3)H]NBMPR association was relatively large (20 kcal mol(-1)) suggesting a conformational change upon inhibitor binding. For all three inhibitors the enthalpy (ΔH°) and entropy (ΔS°) contributions to the reaction energetics were determined by van't Hoff analysis to be roughly similar (25-75% ΔG°). Gains in enthalpy with increasing polar surface area of inhibitors suggest that the binding is favored by electrostatic or polar interactions between the ligands and the transporter. PMID:26428002

  20. Human UMP-CMP kinase 2, a novel nucleoside monophosphate kinase localized in mitochondria.

    PubMed

    Xu, Yunjian; Johansson, Magnus; Karlsson, Anna

    2008-01-18

    Enzyme deficiency in the salvage pathway of deoxyribonucleotide synthesis in mitochondria can cause mtDNA depletion syndromes. We have identified a human mitochondrial UMP-CMP kinase (UMP-CMPK, cytidylate kinase; EC 2.7.4.14), designated as UMP-CMP kinase 2 (UMP-CMPK2). The C-terminal domain of this 449-amino acid protein contains all consensus motifs of a nucleoside monophosphate kinase. Phylogenetic analysis showed that UMP-CMPK2 belonged to a novel nucleoside monophosphate kinase family, which was closer to thymidylate kinase than to cytosolic UMP-CMP kinase. Subcellular localization with green fluorescent protein fusion proteins illustrated that UMP-CMPK2 was localized in the mitochondria of HeLa cells and that the mitochondrial targeting signal was included in the N-terminal 22 amino acids. The enzyme was able to phosphorylate dUMP, dCMP, CMP, and UMP with ATP as phosphate donor, but the kinetic properties were different compared with the cytosolic UMP-CMPK. Its efficacy to convert dUMP was highest, followed by dCMP, whereas CMP and UMP were the poorest substrates. It also phosphorylated the monophosphate forms of the nucleoside analogs ddC, dFdC, araC, BVDU, and FdUrd, which suggests that UMP-CMPK2 may be involved in mtDNA depletion caused by long term treatment with ddC or other pyrimidine analogs. UMP-CMPK2 mRNA expression was exclusively detected in chronic myelogenous leukemia K-562 and lymphoblastic leukemia MOLT-4 among eight studied cancer cell lines. Particular high expression in leukemia cells, dominant expression in bone marrow, and tight correlation with macrophage activation and inflammatory response suggest that UMP-CMPK2 may have other functions in addition to the supply of substrates for mtDNA synthesis. PMID:17999954

  1. An intersubunit disulfide bridge stabilizes the tetrameric nucleoside diphosphate kinase of Aquifex aeolicus.

    PubMed

    Boissier, Fanny; Georgescauld, Florian; Moynié, Lucile; Dupuy, Jean-William; Sarger, Claude; Podar, Mircea; Lascu, Ioan; Giraud, Marie-France; Dautant, Alain

    2012-06-01

    The nucleoside diphosphate kinase (Ndk) catalyzes the reversible transfer of the γ-phosphate from nucleoside triphosphate to nucleoside diphosphate. Ndks form hexamers or two types of tetramers made of the same building block, namely, the common dimer. The secondary interfaces of the Type I tetramer found in Myxococcus xanthus Ndk and of the Type II found in Escherichia coli Ndk involve the opposite sides of subunits. Up to now, the few available structures of Ndk from thermophiles were hexameric. Here, we determined the X-ray structures of four crystal forms of the Ndk from the hyperthermophilic bacterium Aquifex aeolicus (Aa-Ndk). Aa-Ndk displays numerous features of thermostable proteins and is made of the common dimer but it is a tetramer of Type I. Indeed, the insertion of three residues in a surface-exposed spiral loop, named the Kpn-loop, leads to the formation of a two-turn α-helix that prevents both hexamer and Type II tetramer assembly. Moreover, the side chain of the cysteine at position 133, which is not present in other Ndk sequences, adopts two alternate conformations. Through the secondary interface, each one forms a disulfide bridge with the equivalent Cys133 from the neighboring subunit. This disulfide bridge was progressively broken during X-ray data collection by radiation damage. Such crosslinks counterbalance the weakness of the common-dimer interface. A 40% decrease of the kinase activity at 60°C after reduction and alkylation of the protein corroborates the structural relevance of the disulfide bridge on the tetramer assembly and enzymatic function. PMID:22467275

  2. Identification of a nucleoside triphosphate binding site on calf thymus RNA polymerase II

    SciTech Connect

    Freund, E.; McGuire, P.M.

    1986-01-14

    A nucleoside triphosphate binding site on calf thymus RNA polymerase II was identified by using photoaffinity analogues of adenosine 5'-triphosphate and guanosine 5'-triphosphate. Both radiolabeled 8-azidoadenosine 5'-triphosphate (8-N3ATP) and radiolabeled 8-azidoguanosine 5'-triphosphate (8-N3GTP) bound to a single polypeptide of this enzyme. This polypeptide has a molecular mass of 37 kilodaltons and an isoelectric point of 5.4. Ultraviolet (UV) irradiation was necessary for photolabeling to occur. In addition, no labeling occurred when the probe was prephotolyzed or when the enzyme was inactivated. Furthermore, photolabeling of the enzyme could be decreased by preincubation with natural substrates. To provide evidence that the radiolabeled polypeptide forms a part of the domain of the nucleoside triphosphate binding site, experiments were performed using unlabeled 8-N3ATP. Although this unlabeled analogue was not a substrate for RNA polymerase II, it photoinactivated the enzyme in the presence of UV irradiation, and it inhibited transcription elongation by the enzyme in a competitive manner in the absence of UV irradiation. As in the case with photolabeling, photoinactivation by 8-N3ATP could be decreased by natural substrates; in both cases, purine ribonucleoside triphosphates were more efficient than pyrimidine nucleoside triphosphates. Furthermore, photoinactivation was saturable at about the same concentration as the inhibition constant for 8-N3ATP. Collectively, these results provide evidence that the radiolabeled polypeptide in calf thymus RNA polymerase II is an essential component for activity and suggest that this polypeptide may be part of this enzyme's purine ribonucleoside triphosphate binding site.

  3. Cellular Uptake of Decitabine by Equilibrative Nucleoside Transporters in HCT116 Cells.

    PubMed

    Ueda, Kumiko; Hosokawa, Mika; Iwakawa, Seigo

    2015-01-01

    DNA hypermethylation, an epigenetic change that silences gene expression without altering nucleotide sequences, plays a critical role in the formation and progression of colorectal cancers as well as in the acquisition of drug resistance. Decitabine (DAC), a DNA methyltransferase 1 inhibitor of nucleoside analogues, has been shown to restore gene expression silenced by hypermethylation. In the present study, the mechanisms underlying both uridine and DAC uptake were examined in the human colon cancer cell line HCT116. Real-time polymerase chain reaction analysis revealed that ENT1 mRNA was the most abundant among the nucleoside transporters examined in HCT116 cells. The ENT1 protein was detected in the membrane fraction, as determined by Western blotting. The uptake of uridine or DAC was time- and concentration-dependent, but also Na(+)-independent. The uptake of these agents was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), an inhibitor of equilibrative nucleoside transporters (ENTs), and was also decreased in cells treated with ENT1 small interfering RNA. The uptake of both uridine and DAC was inhibited by uridine, cytidine, adenosine, or inosine, while that of DAC was also inhibited by thymidine. The expression of MAGEA1 mRNA, the DNA of which was methylated in HCT116 cells, was increased by DAC treatment, and this increment was attenuated by concomitant treatment with NBMPR. The IC50 value of DAC was also increased in the presence of NBMPR. These results suggest that DAC is mainly taken up by ENT1 and that this uptake is one of the key determinants of the activity of DAC in HCT116 cells. PMID:26235575

  4. The chemoenzymatic synthesis of clofarabine and related 2'-deoxyfluoroarabinosyl nucleosides: the electronic and stereochemical factors determining substrate recognition by E. coli nucleoside phosphorylases.

    PubMed

    Fateev, Ilja V; Antonov, Konstantin V; Konstantinova, Irina D; Muravyova, Tatyana I; Seela, Frank; Esipov, Roman S; Miroshnikov, Anatoly I; Mikhailopulo, Igor A

    2014-01-01

    Two approaches to the synthesis of 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine (1, clofarabine) were studied. The first approach consists in the chemical synthesis of 2-deoxy-2-fluoro-α-D-arabinofuranose-1-phosphate (12a, (2F)Ara-1P) via three step conversion of 1,3,5-tri-O-benzoyl-2-deoxy-2-fluoro-α-D-arabinofuranose (9) into the phosphate 12a without isolation of intermediary products. Condensation of 12a with 2-chloroadenine catalyzed by the recombinant E. coli purine nucleoside phosphorylase (PNP) resulted in the formation of clofarabine in 67% yield. The reaction was also studied with a number of purine bases (2-aminoadenine and hypoxanthine), their analogues (5-aza-7-deazaguanine and 8-aza-7-deazahypoxanthine) and thymine. The results were compared with those of a similar reaction with α-D-arabinofuranose-1-phosphate (13a, Ara-1P). Differences of the reactivity of various substrates were analyzed by ab initio calculations in terms of the electronic structure (natural purines vs analogues) and stereochemical features ((2F)Ara-1P vs Ara-1P) of the studied compounds to determine the substrate recognition by E. coli nucleoside phosphorylases. The second approach starts with the cascade one-pot enzymatic transformation of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a, followed by its condensation with 2-chloroadenine thereby affording clofarabine in ca. 48% yield in 24 h. The following recombinant E. coli enzymes catalyze the sequential conversion of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a: ribokinase (2-deoxy-2-fluoro-D-arabinofuranose-5-phosphate), phosphopentomutase (PPN; no 1,6-diphosphates of D-hexoses as co-factors required) (12a), and finally PNP. The substrate activities of D-arabinose, D-ribose and D-xylose in the similar cascade syntheses of the relevant 2-chloroadenine nucleosides were studied and compared with the activities of 2-deoxy-2-fluoro-D-arabinose. As expected, D-ribose exhibited the best substrate activity

  5. Structure of nucleoside diphosphate kinase from pacific shrimp (Litopenaeus vannamei) in binary complexes with purine and pyrimidine nucleoside diphosphates

    PubMed Central

    López-Zavala, Alonso A.; Quintero-Reyes, Idania E.; Carrasco-Miranda, Jesús S.; Stojanoff, Vivian; Weichsel, Andrzej; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R.

    2014-01-01

    Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012 ▶), J. Bioenerg. Biomembr. 44, 325–331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2′-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism. PMID:25195883

  6. Structure of nucleoside diphosphate kinase from pacific shrimp (Litopenaeus vannamei) in binary complexes with purine and pyrimidine nucleoside diphosphates.

    PubMed

    López-Zavala, Alonso A; Quintero-Reyes, Idania E; Carrasco-Miranda, Jesús S; Stojanoff, Vivian; Weichsel, Andrzej; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R

    2014-09-01

    Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012), J. Bioenerg. Biomembr. 44, 325-331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2'-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism. PMID:25195883

  7. Nucleoside transporters, bcl-2 and apoptosis in CLL cells exposed to nucleoside analogues in vitro.

    PubMed

    Petersen, A J; Brown, R D; Gibson, J; Pope, B; Luo, X F; Schutz, L; Wiley, J S; Joshua, D E

    1996-04-01

    The purine nucleoside analogues fludarabine (F1) and chlorodeoxyadenosine (2-CdA) are considered to be cell cycle specific agents which require DNA synthesis for cytotoxicity. However, their efficacy in the treatment of CLL, an indolent lymphoid malignancy suggests additional mechanisms of action. Like cytosine arabinoside (AraC), F1 and 2-CdA gain access to the cell via a specific nucleoside transporter (NST) protein. To investigate the mode of action of these drugs in CLL, we used a fluorescent ligand for the NST (5'-(SAENTA- x8)-fluorescein) and 3-colour flow cytometry to determine NST expression on CD5+/CD19+ B-cells from the peripheral blood (PB) of patients with CLL. NST levels on these cells was found to be not significantly different from normal control lymphocytes (mean = 485 +/- 425) vs. (mean = 553 +/- 178). Exposure to varying concentrations (0, 3 microM and 30 microM) of F1 and 2-CdA, however, resulted in an upregulation of NST (mean = 1552 +/- 775 with 30 microM FL; mean = 3392 +/- 2197 with 30 microM 2-CdA) after 48. "Large" lymphoid cells (not present in normal PB) were found to express significantly more NST (mean = 2540 +/- 2861) and have a higher proliferative capacity than "small" cells (mean = 357 +/- 517 NST/cell). Incubation of CLL cells with F1 (n = 6) and 2-CdA (n = 8) in vitro over 48 h also resulted in an increase in the proportion of cells in S-phase (0 microM = 0.2 + 2 - 0.1; 30 microM FL = 2.4 +/- 2.0; 30 microM 2-CdA = 3.3 +/- 1.3) and a significant increase in morphologically identifiable apoptosis. Apoptosis was confirmed by flow cytometric DNA analysis (0 microM = 13 +/- 8%; 30 microM FL = 40 +/- 20%; 30 microM 2-CdA = 48 +/- 11%). In situ hybridization using a biotinylated cDNA bcl-2 probe demonstrated that bcl-2 mRNA expression was markedly decreased in treated cells after 24 h. These studies have demonstrated that: (1) NST expression on CLL lymphocytes is low; (2) in vitro exposure to the analogues increases both the level of

  8. Detection of RNA nucleoside modifications with the uridine-specific ribonuclease MC1 from Momordica charantia.

    PubMed

    Addepalli, Balasubrahmanym; Lesner, Nicholas P; Limbach, Patrick A

    2015-10-01

    A codon-optimized recombinant ribonuclease, MC1 is characterized for its uridine-specific cleavage ability to map nucleoside modifications in RNA. The published MC1 amino acid sequence, as noted in a previous study, was used as a template to construct a synthetic gene with a natural codon bias favoring expression in Escherichia coli. Following optimization of various expression conditions, the active recombinant ribonuclease was successfully purified as a C-terminal His-tag fusion protein from E. coli [Rosetta 2(DE3)] cells. The isolated protein was tested for its ribonuclease activity against oligoribonucleotides and commercially available E. coli tRNA(Tyr I). Analysis of MC1 digestion products by ion-pairing reverse phase liquid-chromatography coupled with mass spectrometry (IP-RP-LC-MS) revealed enzymatic cleavage of RNA at the 5'-termini of uridine and pseudouridine, but cleavage was absent if the uridine was chemically modified or preceded by a nucleoside with a bulky modification. Furthermore, the utility of this enzyme to generate complementary digestion products to other common endonucleases, such as RNase T1, which enables the unambiguous mapping of modified residues in RNA is demonstrated. PMID:26221047

  9. Synthesis of Conformationally North-Locked Pyrimidine Nucleosides Built on an Oxa-bicyclo[3.1.0]hexane Scaffold

    PubMed Central

    Ludek, Olaf R.; Marquez, Victor E.

    2011-01-01

    Beginning with a known 3-oxabicyclo[3.1.0]hexane scaffold (I), the relocation of the fused cyclopropane ring bond and the shifting of the oxygen atom to an alternative location engendered a new 2-oxabicyclo[3.1.0]hexane template (II) that mimics more closely the tetrahydrofuran ring of conventional nucleosides. The synthesis of this new class of locked nucleosides involved a novel approach that required the isocyanate II (B = NCO) with a hydroxyl-protected scaffold as a pivotal intermediate that was obtained in eleven steps from a known dihydrofuran precursor. The completion of the nucleobases was successfully achieved by quenching the isocyanate with the lithium salts of the corresponding acrylic amides that led to the uracil and thymidine precursors in a single step. Ring closure of these intermediates led to the target, locked nucleosides. The anti-HIV activity of 29 (uridine analogue), 31 (thymidine analogue), and 34 (cytidine analogue) was explored in human osteosarcoma (HOS) cells or modified HOS cells (HOS-313) expressing the herpes simplex virus 1 thymidine kinase (HSV-1 TK). Only the cytidine analogue showed moderate activity in HOS-313 cells, which means that the compounds are not good substrates for the cellular kinases. PMID:22026578

  10. The SLC28 (CNT) and SLC29 (ENT) nucleoside transporter families: a 30-year collaborative odyssey.

    PubMed

    Young, James D

    2016-06-15

    Specialized nucleoside transporter (NT) proteins are required for passage of nucleosides and hydrophilic nucleoside analogues across biological membranes. Physiologic nucleosides serve as central salvage metabolites in nucleotide biosynthesis, and nucleoside analogues are used as chemotherapeutic agents in the treatment of cancer and antiviral diseases. The nucleoside adenosine modulates numerous cellular events via purino-receptor cell signalling pathways. Human NTs are divided into two structurally unrelated protein families: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family. Human CNTs are inwardly directed Na(+)-dependent nucleoside transporters found predominantly in intestinal and renal epithelial and other specialized cell types. Human ENTs mediate bidirectional fluxes of purine and pyrimidine nucleosides down their concentration gradients and are ubiquitously found in most, possibly all, cell types. Both protein families are evolutionarily old: CNTs are present in both eukaryotes and prokaryotes; ENTs are widely distributed in mammalian, lower vertebrate and other eukaryote species. This mini-review describes a 30-year collaboration with Professor Stephen Baldwin to identify and understand the structures and functions of these physiologically and clinically important transport proteins. PMID:27284054

  11. 4'-Substituted pyrimidine nucleosides lacking 5'-hydroxyl function as potential anti-HCV agents.

    PubMed

    Shakya, Neeraj; Vedi, Satish; Liang, Chao; Yang, Fang; Agrawal, Babita; Kumar, Rakesh

    2014-03-01

    Hepatitis C virus (HCV) infection is one of the major health problems worldwide. If left untreated, it leads to liver cirrhosis, liver cancer and death. Herein, we report synthesis and anti-HCV activity of a new class of pyrimidine nucleosides possessing a 4'-carboxymethyl (9-16, 21 and 23) or 4'-carboxamide function (17-19 and 24). Among these, 10-12 (EC50=33.1-42.4 μM), 14 and 21 (EC50=43.4-59.5 μM) exhibited potent activity in HCV-1a replicon cells without any toxicity to parent Huh-7 cells (CC50=>829-1055 μM). The anti-HCV activities demonstrated by this unusual class of compounds were superior to that of ribavirin (EC50=81.9 μM). Further, the most active analog, 12, was found to interact synergistically with ribavirin to inhibit HCV RNA replication. PMID:24485784

  12. The role of human equilibrative nucleoside transporter 1 on the cellular transport of the DNA methyltransferase inhibitors 5-azacytidine and CP-4200 in human leukemia cells.

    PubMed

    Hummel-Eisenbeiss, Johanna; Hascher, Antje; Hals, Petter-Arnt; Sandvold, Marit Liland; Müller-Tidow, Carsten; Lyko, Frank; Rius, Maria

    2013-09-01

    The nucleoside analog 5-azacytidine is an archetypical drug for epigenetic cancer therapy, and its clinical effectiveness has been demonstrated in the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). However, therapy resistance in patients with MDS/AML remains a challenging issue. Membrane proteins that are involved in drug uptake are potential mediators of drug resistance. The responsible proteins for the transport of 5-azacytidine into MDS/AML cells are unknown. We have now systematically analyzed the expression and activity of various nucleoside transporters. We identified the human equilibrative nucleoside transporter 1 (hENT1) as the most abundant nucleoside transporter in leukemia cell lines and in AML patient samples. Transport assays using [¹⁴C]5-azacytidine demonstrated Na⁺-independent uptake of the drug into the cells, which was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a hENT1 inhibitor. The cellular toxicity of 5-azacytidine and its DNA demethylating activity were strongly reduced after hENT1 inhibition. In contrast, the cellular activity of the 5-azacytidine derivative 5-azacytidine-5'-elaidate (CP-4200), a nucleoside transporter-independent drug, persisted after hENT1 inhibition. A strong dependence of 5-azacytidine-induced DNA demethylation on hENT1 activity was also confirmed by array-based DNA methylation profiling, which uncovered hundreds of loci that became demethylated only when hENT1-mediated transport was active. Our data establish hENT1 as a key transporter for the cellular uptake of 5-azacytidine in leukemia cells and raise the possibility that hENT1 expression might be a useful biomarker to predict the efficiency of 5-azacytidine treatments. Furthermore, our data suggest that CP-4200 may represent a valuable compound for the modulation of transporter-related 5-azacytidine resistances. PMID:23814180

  13. Selective non-nucleoside HIV-1 reverse transcriptase inhibitors. New 2,3-dihydrothiazolo[2,3-a]isoindol-5(9bH)-ones and related compounds with anti-HIV-1 activity.

    PubMed

    Mertens, A; Zilch, H; König, B; Schäfer, W; Poll, T; Kampe, W; Seidel, H; Leser, U; Leinert, H

    1993-08-20

    A series of substituted 2,3-dihydrothiazolo[2,3-a]isoindol-5(9bH)-ones and related compounds 1-73 were synthesized and evaluated for their ability to inhibit reverse transcriptase (RT) of the human immune deficiency virus 1 (HIV-1) and replication of HIV-1 in MT2 cells. The antiviral activity of these compounds depends on the stereoselective configuration of the substituent in position 9b. Structure-activity studies were done within these series of compounds to determine the optimum substituents for antiviral activity. The most potent inhibitors were found in the class of 2,3-dihydrothiazolo[2,3-a]isoindol-5(9bH)-ones bearing a phenyl ring system in position 9b optionally substituted with one or two methyl groups or a chlorine atom in position 8. The most active analogues (R)-(+)-1, (R)-(+)-6, (R)-(+)-13, (R)-(+)-26, and (R)-(+)-53 inhibit the HIV-1 RT with an IC50 between 16 and 300 nM and an IC50 between 10 and 392 nM in MT2 cells, respectively. PMID:7689109

  14. Synthesis and biological investigations of 5-substituted pyrimidine nucleosides coupled to a dihydropyridine/pyridinium salt redox chemical delivery system.

    PubMed

    Kumar, R; Wang, L; Wiebe, L I; Knaus, E E

    2001-11-01

    The syntheses, antiviral activities, and partition coefficients (P) of 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-coupled nucleosides are described. These novel compounds were designed in an effort to enhance the lipophilicity, and thereby the delivery to the CNS, without compromising the anti-HSV-1 activity of the parental nucleosides. We have previously reported the synthesis of 3'-O-(1-methyl-1,4-dihydropyridyl-3- carbonyl) analogs of 5-iodo-(5), 5-vinyl-(6), and (E)-5-(2-iodovinyl)-2'-deoxyuridines (7). We now report the synthesis of 5-iodo-3'-O-(1-methyl-1,4-dihydropyridyl-3- carbonyl)-5'-O-acetyl-2'-deoxyuridine (15) and 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2'-deoxyuridine (17). Quarternization of the 3'-O-(3-pyridylcarbonyl) compounds (10,12) using iodomethane afforded the corresponding 1-methyl pyridinium salts (13,14) which were reduced with sodium dithionite to yield the corresponding 3'-O-1-methyl-1,4-dihydropyridyl-3-carbonyl compounds (15,16). The deprotection of 3'-O-(1-methyl-1,4-dihydropyridyl- 3-carbonyl)-5'-O-t-butyldimethylsilyl-2'-deoxyuridine (16) with Bu4N+F- afforded 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2'-deoxyuridine (17). Compounds 5-7 and 15 were evaluated for their antiviral activity in vitro against HSV-1, HSV-2, HCMV, and VZV, and were found to retain anti-HSV-1, HSV-2 and VZV activity as compared to their parental nucleosides (1-3). In addition, the cellular toxicity of 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-coupled compounds (5-7 and 15) was found to be lower than the parent nucleosides. The lipophilicity of compounds (5-7,15,17) are enhanced substantially, compared to the parent nucleosides, as indicated by an increase in corresponding P values (1-octanol-water) upon replacement of the C-3' hydroxyl by 1-methyl-1,4-dihydropyridyl-3-carbonyl moiety. PMID:11822172

  15. Docking and small angle X-ray scattering studies of purine nucleoside phosphorylase.

    PubMed

    Filgueira de Azevedo, Walter; dos Santos, Giovanni César; dos Santos, Denis Marangoni; Olivieri, Johnny Rizzieri; Canduri, Fernanda; Silva, Rafael Guimarães; Basso, Luiz Augusto; Renard, Gaby; da Fonseca, Isabel Osório; Mendes, Maria Anita; Palma, Mário Sérgio; Santos, Diógenes Santiago

    2003-10-01

    Docking simulations have been used to assess protein complexes with some success. Small angle X-ray scattering (SAXS) is a well-established technique to investigate protein spatial configuration. This work describes the integration of geometric docking with SAXS to investigate the quaternary structure of recombinant human purine nucleoside phosphorylase (PNP). This enzyme catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for PNP causes gradual decrease in T-cell immunity. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant rejection, rheumatoid arthritis, lupus, and T-cell lymphomas. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. The present analysis confirms the trimeric structure observed in the crystal. The potential application of the present procedure to other systems is discussed. PMID:13679062

  16. Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides.

    PubMed

    Yin, Junfa; Xu, Tian; Zhang, Ning; Wang, Hailin

    2016-08-01

    Structure-based DNA modification analysis provides accurate and important information on genomic DNA changes from epigenetic modifications to various DNA lesions. However, genomic DNA strands are often required to be efficiently digested into single nucleosides. It is an arduous task because of the involvement of multiple enzymes with different catalytic acitivities. Here we constructed a three-enzyme cascade capillary monolithic bioreactor that consists of immobilized deoxyribonuclease I (DNase I), snake venom phosphodiesterase (SVP), and alkaline phosphatase (ALPase). By the use of this cascade capillary bioreactor, genomic DNA can be efficiently digested into single nucleosides with an increasing rate of ∼20 folds. The improvement is mainly attributed to dramatically increase enzymatic capacity and activity. With a designed macro-porous structure, genomic DNA of 5-30 Kb (∼1.6-10 million Daltons) can be directly passed through the bioreactor simply by hand pushing or a low-pressure microinjection pump. By coupling with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we further developed a sensitive assay for detection of an oxidative stress biomarker 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in DNA. The proposed three-enzyme cascade bioreactor is also potentially applicable for fast identification and quantitative detection of other lesions and modifications in genomic DNA. PMID:27416319

  17. Structural optimization of non-nucleoside DNA methyltransferase inhibitor as anti-cancer agent.

    PubMed

    Zhong, Bo; Vatolin, Sergei; Idippily, Nethrie D; Lama, Rati; Alhadad, Laila A; Reu, Frederic J; Su, Bin

    2016-02-15

    Inhibition of DNA methyltransferase 1 (DNMT1) can reverse the malignant behavior of cancer cells by restoring expression of aberrantly silenced genes that are required for differentiation, senescence, and apoptosis. Clinically used DNMT1 inhibitors decitabine and azacitidine inhibit their target by covalent trapping after incorporation into DNA as azacytidine analogs. These nucleoside compounds are prone to rapid enzymatic inactivation in blood, posing challenges to the development of purely epigenetic dosing schedules. Non-nucleoside compounds that suppress expression or function of DNMT1 may overcome this problem. Using a high-throughput PCR-based site specific chromatin condensation assay, we identified a compound that reactivated Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) in myeloma cells and suppressed expression of DNMT1 from a library of 5120 chemically diverse small molecules. Lead optimization was performed to generate 26 new analogs with lung cancer proliferation and DNMT1 expression as activity readout. Two of the new derivatives showed 2 fold improvement of growth inhibiting potency and also decreased DNMT1 protein levels in lung cancer cells. PMID:26774653

  18. Non-nucleoside reverse transcriptase inhibitors: a review on pharmacokinetics, pharmacodynamics, safety and tolerability

    PubMed Central

    Usach, Iris; Melis, Virginia; Peris, José-Esteban

    2013-01-01

    Introduction Human immunodeficiency virus (HIV) type-1 non-nucleoside and nucleoside reverse transcriptase inhibitors (NNRTIs) are key drugs of highly active antiretroviral therapy (HAART) in the clinical management of acquired immune deficiency syndrome (AIDS)/HIV infection. Discussion First-generation NNRTIs, nevirapine (NVP), delavirdine (DLV) and efavirenz (EFV) are drugs with a low genetic barrier and poor resistance profile, which has led to the development of new generations of NNRTIs. Second-generation NNRTIs, etravirine (ETR) and rilpivirine (RPV) have been approved by the Food and Drug Administration and European Union, and the next generation of drugs is currently being clinically developed. This review describes recent clinical data, pharmacokinetics, metabolism, pharmacodynamics, safety and tolerability of commercialized NNRTIs, including the effects of sex, race and age differences on pharmacokinetics and safety. Moreover, it summarizes the characteristics of next-generation NNRTIs: lersivirine, GSK 2248761, RDEA806, BILR 355 BS, calanolide A, MK-4965, MK-1439 and MK-6186. Conclusions This review presents a wide description of NNRTIs, providing useful information for researchers interested in this field, both in clinical use and in research. PMID:24008177

  19. The type 1 equilibrative nucleoside transporter regulates anxiety-like behavior in mice

    PubMed Central

    Chen, J.; Rinaldo, L.; Lim, S.-J.; Young, H.; Messing, R. O.; Choi, D.-S.

    2010-01-01

    Activation of adenosine receptors in the brain reduces anxiety-like behavior in animals and humans. Because nucleoside transporters regulate adenosine levels, we used mice lacking the type 1 equilibrative nucleoside transporter (ENT1) to investigate whether ENT1 contributes to anxiety-like behavior. The ENT1 null mice spent more time in the center of an open field compared with wild-type littermates. In the elevated plus maze, ENT1 null mice entered more frequently into and spent more time exploring the open arms. The ENT1 null mice also spent more time exploring the light side of a light–dark box compared with wild-type mice. Microinjection of an ENT1-specific antagonist, nitrobenzylthioinosine (nitrobenzylmercaptopurine riboside), into the amygdala of C57BL/6J mice reduced anxiety-like behavior in the open field and elevated plus maze. These findings show that amygdala ENT1 modulates anxiety-like behavior. The ENT1 may be a drug target for the treatment of anxiety disorders. PMID:17376149

  20. pH-Cleavable Nucleoside Lipids: A New Paradigm for Controlling the Stability of Lipid-Based Delivery Systems.

    PubMed

    Oumzil, Khalid; Benizri, Sébastien; Tonelli, Giovanni; Staedel, Cathy; Appavoo, Ananda; Chaffanet, Max; Navailles, Laurence; Barthélémy, Philippe

    2015-11-01

    Lipid-based delivery systems are an established technology with considerable clinical acceptance and several applications in human. Herein, we report the design, synthesis and evaluation of novel orthoester nucleoside lipids (ONLs) for the modulation of liposome stability. The ONLs contain head groups with 3'-orthoester nucleoside derivatives featuring positive or negative charges. The insertion of the orthoester function in the NL structures allows the formation of pH-sensitive liposomes. ONL-based liposomes can be hydrolyzed to provide nontoxic products, including nucleoside derivatives and hexadecanol. To allow the release to be tunable at different hydrolysis rates, the charge of the polar head structure is modulated, and the head group can be released at a biologically relevant pH. Crucially, when ONLs are mixed with natural phosphocholine lipids (PC), the resultant liposome evolves toward the formation of a hexadecanol/PC lamellar system. Biological evaluation shows that stable nucleic acid lipid particles (SNALPs) formulated with ONLs and siRNAs can effectively enter into tumor cells and release their nucleic acid payload in response to an intracellular acidic environment. This results in a much higher antitumor activity than conventional SNALPs. The ability to use pH-cleavable nucleolipids to control the stability of lipid-based delivery systems represents a promising approach for the intracellular delivery of drug cargos. PMID:26381578

  1. Compositions containing nucleosides and manganese and their uses

    SciTech Connect

    Daly, Michael J.; Gaidamakova, Elena K.; Matrosova, Vera Y.; Levine, Rodney L.; Wehr, Nancy B.

    2015-11-17

    This invention encompasses methods of preserving protein function by contacting a protein with a composition comprising one or more purine or pyrimidine nucleosides (such as e.g., adenosine or uridine) and an antioxidant (such as e.g., manganese). In addition, the invention encompasses methods of treating and/or preventing a side effect of radiation exposure and methods of preventing a side effect of radiotherapy comprising administration of a pharmaceutically effective amount of a composition comprising one or more purine or pyrimidine nucleosides (such as e.g., adenosine or uridine) and an antioxidant (such as e.g., manganese) to a subject in need thereof. The compositions may comprise D. radiodurans extracts.

  2. Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals. Quantitative basis for Urh1 and purine nucleoside phosphorylase function in NAD+ metabolism.

    PubMed

    Belenky, Peter; Christensen, Kathryn C; Gazzaniga, Francesca; Pletnev, Alexandre A; Brenner, Charles

    2009-01-01

    NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification. PMID:19001417

  3. Synthesis of oligodeoxyribonucleotides using N-benzyloxycarbonyl-blocked nucleosides

    SciTech Connect

    Watkins, B.E.; Kiely, J.S.; Rapoport, H.

    1982-10-20

    The exo amino groups of 2'-deoxyadenosine and 2'-deoxycytidine have been blocked as the benzyl carbamates, and 2'-deoxyguanosine has been blocked as its 2-N-(benzyloxycarbonyl)carbamate and 6-O-benzyl ether. These blocked nucleosides have been incorporated into an efficient oligodeoxyribonucleotide synthetic scheme, and the resulting oligomer has been successfully deblocked by using transfer hydrogenation. The deblocking conditions result in no reduction of the pyrimidine bases.

  4. Membrane events and ionic processes involved in dopamine release from tuberoinfundibular neurons. I. Effect of the inhibition of the Na+,K+-adenosine triphosphatase pump by ouabain

    SciTech Connect

    Taglialatela, M.; Amoroso, S.; Kaparos, G.; Maurano, F.; Di Renzo, G.F.; Annunziato, L.

    1988-08-01

    In the present study we investigated the membrane events and the ionic processes which mediate the stimulatory effect of ouabain on the release of endogenous dopamine (DA) and previously taken-up (3H)DA release from rat hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons. Ouabain (0.1-1 mM) dose-dependently stimulated endogenous DA and newly taken-up (3H)DA release. This effect was counteracted partially by nomifensine (10 microM). Removal of Ca++ ions from the extracellular space in the presence of the Ca++-chelator ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid prevented completely ouabain-elicited (3H)DA release. Lanthanum (1 mM) and cobalt (2 mM), two inorganic Ca++-entry blockers, were able to inhibit this stimulatory effect, whereas verapamil (10 microM) and nitrendipine (50 microM), two organic antagonists of the voltage-operated channel for Ca++ ions, failed to affect ouabain-induced (3H)DA release. By contrast, adriamycin (100-300 microM), a putative inhibitor of cardiac Na+-Ca++ antiporter, dose-dependently prevented ouabain-induced (3H)DA release from TIDA neurons. Finally, tetrodotoxin reduced digitalis-stimulated (3H)DA release. In conclusion, these results seem to be compatible with the idea that the inhibition of Na+,K+-adenosine triphosphatase by ouabain stimulates the release of (3H)DA from a central neuronal system like the TIDA tract and that this effect is critically dependent on the entrance of Ca++ ions into the nerve terminals of these neurons. In addition the Na+-Ca++ exchange antiporter appears to be the membrane system which transports Ca++ ions into the neuronal cytoplasm during Na+,K+-adenosine triphosphatase inhibition. The enhanced intracellular Ca++ availability triggers DA release which could occur partially through a carrier-dependent process.

  5. Structural requirements for potent anti-human immunodeficiency virus (HIV) and sperm-immobilizing activities of cyclohexenyl thiourea and urea non-nucleoside inhibitors of HIV-1 reverse transcriptase.

    PubMed

    D'Cruz, Osmond J; Venkatachalam, Taracad K; Mao, Chen; Qazi, Sanjive; Uckun, Fatih M

    2002-12-01

    The current pandemic of sexually transmitted human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) has created an urgent need for a new type of microbicide, one that is both a spermicide and a virucide. In a systematic effort to identify a non-detergent-type antiviral spermicide, we have rationally designed and synthesized a series of cyclohexenyl thiourea (CHET) nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase (RT) with sperm-immobilizing activity (SIA). To gain further insight into the structural requirements for the optimal activity of these dual-function NNIs, we compared the effects of thiazolyl, benzothiazolyl, and pyridyl ring substitutions and functionalization with electron-donating and electron-withdrawing groups as well as the importance of thiourea and urea moieties of 15 heterocyclic ring-substituted NNIs. RT activity and p24 antigen production in HIV-infected peripheral blood mononuclear cells were used as markers of viral replication. Computer-assisted sperm analysis was used for evaluating SIA of CHET compounds. The rabbit model was used for evaluation of in vivo mucosal toxicity and contraceptive activity of the lead NNIs. Three CHET-NNIs with a bromo, chloro, or methyl substitution at the 5 position of the pyridyl ring exhibited potent anti-HIV activity at nanomolar concentrations (IC(50) = 3-5 nM) and SIA at micromolar concentrations (EC(50) = 45-96 micro M). The dual-function CHET-NNIs were potent inhibitors of drug-resistant HIV-1 strains with genotypic and phenotypic NNI resistance. Upon substitution of the sulfur atom of the thiourea moiety with an oxygen atom, the most striking difference noted was a 38-fold reduction in time required for 50% sperm immobilization (T(1/2)). A quantitative structure-activity relationship (QSAR) analysis was used in deriving regression equations between 20 physicochemical properties and SIA of NNIs. QSAR analysis showed that the T(1/2) values positively correlated with

  6. All-trans-retinoic Acid Promotes Trafficking of Human Concentrative Nucleoside Transporter-3 (hCNT3) to the Plasma Membrane by a TGF-β1-mediated Mechanism*

    PubMed Central

    Fernández-Calotti, Paula; Pastor-Anglada, Marçal

    2010-01-01

    Human concentrative nucleoside transporter-3 (hCNT3) is a sodium-coupled nucleoside transporter that exhibits high affinity and broad substrate selectivity, making it the most suitable candidate for mediating the uptake and cytotoxic action of most nucleoside-derived drugs. The drug of this class most commonly used in the treatment of chronic lymphocytic leukemia (CLL) is the pro-apoptotic nucleoside analog fludarabine (Flu), which enters CLL cells primarily through human equilibrative nucleoside transporters (hENTs). Although CLL cells lack hCNT3 activity, they do express this transporter protein, which is located mostly in the cytosol. The aim of our study was to identify agents and mechanisms capable of promoting hCNT3 trafficking to the plasma membrane. Here, we report that all-trans-retinoic acid (ATRA), currently used in the treatment of acute promyelocytic leukemia (APL), increases hCNT3-related activity through a mechanism that involves trafficking of pre-existing hCNT3 proteins to the plasma membrane. This effect is mediated by the autocrine action of transforming growth factor (TGF)-β1, which is transcriptionally activated by ATRA in a p38-dependent manner. TGF-β1 acts through activation of ERK1/2 and the small GTPase RhoA to promote plasma membrane trafficking of the hCNT3 protein. PMID:20172853

  7. Nucleobase and nucleoside transport and integration into plant metabolism

    PubMed Central

    Girke, Christopher; Daumann, Manuel; Niopek-Witz, Sandra; Möhlmann, Torsten

    2014-01-01

    Nucleotide metabolism is an essential process in all living organisms. Besides newly synthesized nucleotides, the recycling (salvage) of partially degraded nucleotides, i.e., nucleosides and nucleobases serves to keep the homeostasis of the nucleotide pool. Both types of metabolites are substrates of at least six families of transport proteins in Arabidopsis thaliana (Arabidopsis) with a total of 49 members. In the last years several members of such transport proteins have been analyzed allowing to present a more detailed picture of nucleoside and nucleobase transport and the physiological function of these processes. Besides functioning in nucleotide metabolism it turned out that individual members of the before named transporters exhibit the capacity to transport a wide range of different substrates including vitamins and phytohormones. The aim of this review is to summarize the current knowledge on nucleobase and nucleoside transport processes in plants and integrate this into nucleotide metabolism in general. Thereby, we will focus on those proteins which have been characterized at the biochemical level. PMID:25250038

  8. Effects of halides on reaction of nucleosides with ozone.

    PubMed

    Suzuki, Toshinori; Kaya, Eriko; Inukai, Michiyo

    2012-01-01

    Ozone (O(3)), a major component of photochemical oxidants, is used recently as a deodorizer in living spaces. It has been reported that O(3) can directly react with DNA, causing mutagenesis in human cells and carcinogenesis in mice. However, little is known about the effects of coexistent ions in the reaction of O(3). In the present study, we analyzed the effects of halides on the reaction of O(3) with nucleosides using reversed-phase high-performance liquid chromatography with ultraviolet detection. When aqueous O(3) solution was added to a nucleoside mixture in potassium phosphate buffer (pH 7.3), the nucleosides were consumed with the following decreasing order of importance: dGuo > Thd > dCyd > dAdo. The effects of addition of fluoride and chloride in the system were slight. Bromide suppressed the reactions of dGuo, Thd, and dAdo but enhanced the reaction of dCyd. The major products were 5-hydroxy-2'-deoxycytidine, 5-bromo-2'-deoxycytidine, and 8-bromo-2'-deoxyguanosine. The time course and pH dependence of the product yield indicated formation of hypobromous acid as the reactive agent. Iodide suppressed all the reactions effectively. The results suggest that bromide may alter the mutation spectrum by O(3) in humans. PMID:22646086

  9. Structure-catalytic activity relationships of dicyclohexylcarboxamidine analogs in phosphorylation and alkylation of nucleosides by a two-step phosphorylating agent, 2-methylthio-4H-1,3,2-benzodioxaphosphorin 2-oxide (MTBO).

    PubMed

    Eto, M; Kawasaki, S

    1986-01-01

    Adenosine borate complex was phosphorylated and o-hydroxybenzylated by 2-methylthio-4H-1,3,2-benzodioxaphosphorin 2-oxide (MTBO) in the presence of 4-morpholine-N,N'-dicyclohexylcarboxamidine (MDC) at first to give 1-(o-hydroxybenzyl)adenosine derivative followed by the rearrangement of the benzyl group to the N-6 amino group to give N6-(o-hydroxybenzyl)adenosine 5'-S-methyl phosphorothiolate. More than 20 analogs of MDC were examined for their catalytic activity in phosphorylation and o-hydroxybenzylation of ribonucleoside by MTBO. Dicyclohexylformamidine (DCF) and n-alkylamino analogs of MDC had no effect on the o-hydroxybenzylation of ribonucleoside by MTBO, but had great effect on the phosphorylation. Dialkylamino and cyclic imino analogs of MDC had high catalytic activities to the both reaction. The dicyclohexylcarboxamidine structure of MDC gave the catalytic ability for phosphorylation by MTBO, while the morpholine moiety had great effect on the selectivity of o-hydroxybenzylation by MTBO. PMID:3562278

  10. Nucleoside transport at the blood-testis barrier studied with primary-cultured sertoli cells.

    PubMed

    Kato, Ryo; Maeda, Tomoji; Akaike, Toshihiro; Tamai, Ikumi

    2005-02-01

    Nucleosides are essential for nucleotide synthesis in testicular spermatogenesis. In the present study, the mechanism of the supply of nucleosides to the testicular system across the blood-testis barrier was studied using primary-cultured Sertoli cells from rats and TM4 cells from mice. Uptake of uridine by these cells was time- and concentration-dependent. Uridine uptake was decreased under Na(+)-free conditions, and the system was presumed to be high affinity, indicating an Na(+)-dependent concentrative nucleoside transporter (CNT) is involved. On the other hand, nitrobenzylthioinosine, a potent inhibitor of Na(+)-independent equilibrative nucleoside transporters (ENTs), inhibited uridine uptake by the Sertoli cells in a concentration-dependent manner. Expression of nucleoside transporters ENT1, ENT2, ENT3, CNT1, CNT2, and CNT3 was detected in Sertoli cells by reverse transcriptase-polymerase chain reaction analysis. Inhibition studies of the uptake of uridine by various nucleosides both in the presence and absence of Na(+) indicated that the most of those expressed nucleoside transporters, ENTs and CNTs, are involved functionally. These results demonstrated that Sertoli cells are equipped with multiple nucleoside transport systems, including ENT1, ENT2, and CNTs, to provide nucleosides for spermatogenesis. PMID:15547112

  11. Nucleosides of 4-methylthio-1,2,3-triazol-5-yl-carboxylic acid derivatives

    SciTech Connect

    Shingarova, I.D.; Yartseva, I.V.; Preobrazhenskaya, M.N.

    1987-08-01

    2-..beta..-D-Ribofuranosyl-4-methylthio-5-methoxycarbonyl-1,2,3-triazole was obtained by fusing 4-methylthio-5-methoxycarbonyl-1,2,3-triazole together with tetraacyl-D-ribofuranose, followed by deacylation, and its amide and hydrazide were prepared. The structures of the new nucleosides were established by converting them into known 2-nucleosides of 1,2,3-triazol-4-yl-carboxylic acid derivatives. By comparing PMR spectra with previously reported PMR spectra for the isomeric 1- and 2-nucleosides of 1,2,3-triazol-4-yl-carboxylic acid derivatives, the synthesized nucleosides could be assigned to 2-substituted triazoles.

  12. Chemical Logic and Enzymatic Machinery for Biological Assembly of Peptidyl Nucleoside Antibiotics

    PubMed Central

    Walsh, Christopher T.; Zhang, Wenjun

    2011-01-01

    Peptidyl nucleoside antibiotics are a group of natural products targeting MraY, a bacterial translocase involved in the lipid-linked cycle in peptidoglycan biosynthesis. In this Perspective, we explore how Nature builds complex peptidyl nucleoside antibiotics scaffolds from simple nucleoside and amino acid building blocks. We discuss the current stage of research on biosynthetic pathways for peptidyl nucleoside antibiotics, primarily focusing on chemical logic and enzymatic machinery for uridine transformation and coupling to peptides. We further survey the nonribosomal biosynthetic paradigm for a subgroup of uridyl peptide antibiotics represented by pacidamycins, concluded by diversification opportunities for antibiotic optimization. PMID:21851099

  13. Identification of 8-Aminoadenosine Derivatives as a New Class of Human Concentrative Nucleoside Transporter 2 Inhibitors

    PubMed Central

    2015-01-01

    Purine-rich foods have long been suspected as a major cause of hyperuricemia. We hypothesized that inhibition of human concentrative nucleoside transporter 2 (hCNT2) would suppress increases in serum urate levels derived from dietary purines. To test this hypothesis, the development of potent hCNT2 inhibitors was required. By modifying adenosine, an hCNT2 substrate, we successfully identified 8-aminoadenosine derivatives as a new class of hCNT2 inhibitors. Compound 12 moderately inhibited hCNT2 (IC50 = 52 ± 3.8 μM), and subsequent structure–activity relationship studies led to the discovery of compound 48 (IC50 = 0.64 ± 0.19 μM). Here we describe significant findings about structural requirements of 8-aminoadenosine derivatives for exhibiting potent hCNT2 inhibitory activity. PMID:25815140

  14. Discovery, characterization, and lead optimization of 7-azaindole non-nucleoside HIV-1 reverse transcriptase inhibitors.

    PubMed

    Stanton, Richard A; Lu, Xiao; Detorio, Mervi; Montero, Catherine; Hammond, Emily T; Ehteshami, Maryam; Domaoal, Robert A; Nettles, James H; Feraud, Michel; Schinazi, Raymond F

    2016-08-15

    A library of 585 compounds built off a 7-azaindole core was evaluated for anti-HIV-1 activity, and ten hits emerged with submicromolar potency and therapeutic index >100. Of these, three were identified as non-nucleoside reverse transcriptase (RT) inhibitors and were assayed against relevant resistant mutants. Lead compound 8 inhibited RT with submicromolar potency (IC50=0.73μM) and also maintained some activity against the clinically important RT mutants K103N and Y181C (IC50=9.2, 3.5μM) in cell-free assays. Free energy perturbation guided lead optimization resulted in the development of a compound with a two-fold increase in potency against RT (IC50=0.36μM). These data highlight the discovery of a unique scaffold with the potential to move forward as next-generation anti-HIV-1 agents. PMID:27390064

  15. Stimulation of phospholipase D in rabbit platelet membranes by nucleoside triphosphates and by phosphocreatine: roles of membrane-bound GDP, nucleoside diphosphate kinase and creatine kinase.

    PubMed Central

    Fan, X T; Sherwood, J L; Haslam, R J

    1994-01-01

    Previous work has shown that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and GTP stimulate phospholipase D (PLD) in rabbit platelet membranes and that these effects are greatly enhanced by pretreatment of platelets with phorbol esters that activate protein kinase C [Van der Meulen and Haslam (1990), Biochem. J. 271, 693-700]. In the present study, the effects of Mg2+, various nucleoside triphosphates and phosphocreatine (PCr) were investigated. Platelet membranes containing phospholipids labelled with [3H]glycerol were assayed for PLD in the presence of an optimal Mg2+ concentration (10 mM) by measuring [3H]phosphatidylethanol formation in incubations that included 300 mM ethanol. In membranes from phorbolester-treated platelets, the same maximal increases in PLD activity (5-fold) were seen with 1 microM GTP[S]), and 100 microM GTP. Addition of adenosine 5'-[gamma-thio]triphosphate (ATP[S]), ITP, XTP, UTP and CTP had similar stimulatory effects, but only at > or = 1 mM. In contrast, ATP had a biphasic action, causing a maximal (2-fold) stimulation at 10 microM and smaller effects at higher concentrations; the inhibitory component of the action of ATP was blocked by 2 microM staurosporine. Guanosine 5'-[beta-thio]diphosphate decreased the stimulatory effects of ATP and ATP[S]. UDP, which can inhibit nucleoside diphosphate kinase (NDPK), decreased the activation of PLD by ATP[S], ATP, XTP, CTP and to a lesser extent ITP, but had no effect on the actions of GTP[S] and GTP. Rabbit platelet membranes contained NDPK and addition of [gamma-32P]ATP led to the formation of [32P]GTP in amounts sufficient to explain most or all of the activation of PLD; UDP prevented GTP formation. PCr (0.04-1 mM) also stimulated membrane PLD activity, an effect that was dependent on endogenous membrane-bound creatine kinase (CK). UDP and guanosine 5'-[beta-thio]diphosphate each inhibited this effect of PCr. The results show that in rabbit platelet membranes, CK, NDPK and the GTP

  16. Kinetico-mechanistic Studies on the Substitution Reactivity on the {Ru(II)(bpy)2} Core with Nucleosides and Nucleotides at Physiological pH.

    PubMed

    Vázquez, Marta; Martínez, Manuel

    2016-07-01

    The kinetico-mechanistic study of the substitution reactions of the aquo ligands in cis-[Ru(bpy)2(H2O)2](2+) by different nucleotides and nucleosides has been conducted at pH close to the physiological value. The concentration dependence and thermal and pressure activation parameters have been measured to ascertain the activation via which reactions take place. Substitution processes are found associatively activated for nitrogen-bonded nucleosides or nucleotides, with outer-sphere hydrogen-bonded aggregates being determinant. For reactions leading to oxygen-bonded nucleotides, the process is clearly dissociatively activated. A selectively induced lability of the inert {Ru(II)(bpy)2} core is observed on the formation of nitrogen(amide)-bonded complexes at relatively low pH values, which might be relevant for the effective intercalation of designed, ruthenium(II)-bonded, aromatic rings. PMID:27327488

  17. An alternative route to the arylvinyltriazole nucleosides.

    PubMed

    Chudinov, Mikhail V; Prutkov, Alexander N; Matveev, Andrey V; Grebenkina, Lyubov E; Konstantinova, Irina D; Berezovskaya, Yulia V

    2016-07-15

    A new pathway to synthesis of arylvinyl ribavirin analogues is developed which makes it possible to obtain not only trans- but also cis-isomers at vinyl bond. By this route eight ribavirin 5-arylvinyl analogues are synthesized and their antiviral activity is evaluated. PMID:27262598

  18. Carboranyl Nucleosides & Oligonucleotides for Neutron Capture Therapy Final Report

    SciTech Connect

    Schinazi, Raymond F.

    2004-12-01

    This proposal enabled us to synthesize and develop boron-rich nucleosides and oligonucleotide analogues for boron neutron capture therapy (BNCT) and the treatment of various malignancies. First, we determined the relationship between structure, cellular accumulation and tissue distribution of 5-o-carboranyl-2'-deoxyuridine (D-CDU) and its derivatives D-ribo-CU and 5-o-carboranyluracil (CU), to potentially target brain and other solid tumors for neutron capture therapy. Synthesized carborane containing nucleoside derivatives of CDU, D- and L-enantiomers of CDU, D-ribo-CU and CU were used. We measured tissue disposition in xenografted mice bearing 9479 human prostate tumors xenografts and in rats bearing 9L gliosarcoma isografts in their flanks and intracranially. The accumulation of D-CDU, 1-({beta}-L-arabinosyl)-5-o-carboranyluracil, D-ribo-CU, and CU were also studied in LnCap human prostate tumor cells and their retention was measured in male nude mice bearing LnCap and 9479 human prostate tumor xenografts. D-CDU, D-ribo-CU and CU levels were measured after administration in mice bearing 9479 human prostate tumors in their flanks. D-CDU achieved high cellular concentrations in LnCap cells and up to 2.5% of the total cellular compound was recovered in the 5'-monophosphorylated form. D-CDU cellular concentrations were similar in LnCap and 9479 tumor xenografts. Studies in tumor bearing animals indicated that increasing the number of hydroxyl moieties in the sugar constituent of the carboranyl nucleosides lead to increased rate and extent of renal elimination, a decrease in serum half-lives and an increased tissue specificity. Tumor/brain ratios were greatest for CDU and D-ribo-CU, while tumor/prostate ratios were greatest with CU. CDU and D-ribo-CU have potential for BNCT of brain malignancies, while CU may be further developed for prostate cancer. A method was developed for the solid phase synthesis of oligonucleotides containing (ocarboran-1-yl

  19. Origin, Utilization, and Recycling of Nucleosides in the Central Nervous System

    ERIC Educational Resources Information Center

    Ipata, Piero Luigi

    2011-01-01

    The brain relies on the salvage of preformed purine and pyrimidine rings, mainly in the form of nucleosides, to maintain its nucleotide pool in the proper qualitative and quantitative balance. The transport of nucleosides from blood into neurons and glia is considered to be an essential prerequisite to enter their metabolic utilization in the…

  20. Analysis of Nucleosides in Municipal Wastewater by Large-Volume Liquid Chromatography Tandem Mass Spectrometry

    PubMed Central

    Brewer, Alex J.; Lunte, Craig

    2015-01-01

    Nucleosides are components of both DNA and RNA, and contain either a ribose (RNA) or 2deoxyribose (DNA) sugar and a purine or pyrimidine base. In addition to DNA and RNA turnover, modified nucleosides found in urine have been correlated to a diminished health status associated with AIDS, cancers, oxidative stress and age. Nucleosides found in municipal wastewater influent are potentially useful markers of community health status, and as of now, remain uninvestigated. A method was developed to quantify nucleosides in municipal wastewater using large-volume injection, liquid chromatography, and mass spectrometry. Method accuracy ranged from 92 to 139% when quantified by using isotopically labeled internal standards. Precision ranged from 6.1 to 19% of the relative standard deviation. The method’s utility was demonstrated by the analysis of twenty-four hour composite wastewater influent samples that were collected over a week to investigate community nucleoside excretion. Nucleosides originating from RNA were more abundant that DNA over the study period, with total loads of nucleosides ranging from 2 to 25 kg/day. Given this relatively high amount of nucleosides found over the study period they present an attractive analyte for the investigation of community health. PMID:26322136

  1. A procedure for the preparation and isolation of nucleoside-5’-diphosphates

    PubMed Central

    Korhonen, Heidi J; Bolt, Hannah L

    2015-01-01

    Summary Tris[bis(triphenylphosphoranylidene)ammonium] pyrophosphate (PPN pyrophosphate) was used in the SN2 displacements of the tosylate ion from 5’-tosylnucleosides to afford nucleoside-5’-diphosphates. Selective precipitation permitted the direct isolation of nucleoside-5’-diphosphates from crude reaction mixtures. PMID:25977720

  2. Mass Modulation of Protein Dynamics Associated with Barrier Crossing in Purine Nucleoside Phosphorylase

    PubMed Central

    Antoniou, Dimitri; Ge, Xiaoxia; Schramm, Vern L.; Schwartz, Steven D.

    2012-01-01

    The role of protein dynamics on different time scales in enzyme catalysis remains an area of active debate. The connection between enzyme dynamics on the femtosecond time scale and transition state formation has been demonstrated in human purine nucleoside phosphorylase (PNP) through the study of a mass-altered enzyme. Isotopic substitution in human PNP (heavy PNP) decreased the rate of on-enzyme chemistry but did not alter either the transition state structure or steady-state kinetic parameters. Here we investigate the underlying atomic motions associated with altered barrier crossing probability for heavy PNP. Transition path sampling was employed to illuminate the molecular differences between barrier crossing in light and heavy enzymes. The mass effect is apparent in promoting vibrations that polarize the N-ribosidic bond, and that promote the stability of the purine leaving group. These motions facilitate barrier crossing. PMID:24496053

  3. Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies.

    PubMed

    de Moraes, Marcela Cristina; Temporini, Caterina; Calleri, Enrica; Bruni, Giovanna; Ducati, Rodrigo Gay; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra; Massolini, Gabriella

    2014-04-18

    The aim of this work was to optimize the preparation of a capillary human purine nucleoside phosphorylase (HsPNP) immobilized enzyme reactor (IMER) for characterization and affinity screening studies of new inhibitors by frontal affinity chromatography coupled to mass spectrometry (FAC-MS). For this purpose two monolithic supports, a Chromolith Speed Rod (0.1mm I.D.×5cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25mm I.D.×5cm) with epoxy reactive groups were considered and compared to an IMER previously developed using an open fused silica capillary. Each HsPNP-IMER was characterized in terms of catalytic activity using Inosine as standard substrate. Furthermore, they were also explored for affinity ranking experiments. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed. PMID:24630982

  4. Structural basis for selective inhibition of purine nucleoside phosphorylase from Schistosoma mansoni: kinetic and structural studies.

    PubMed

    Castilho, Marcelo S; Postigo, Matheus P; Pereira, Humberto M; Oliva, Glaucius; Andricopulo, Adriano D

    2010-02-15

    Selectivity plays a crucial role in the design of enzyme inhibitors as novel antiparasitic agents, particularly in cases where the target enzyme is also present in the human host. Purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive target for the discovery of potential antischistosomal agents. In the present work, kinetic studies were carried out in order to determine the inhibitory potency, mode of action and enzyme selectivity of a series of inhibitors of SmPNP. In addition, crystallographic studies provided important structural insights for rational inhibitor design, revealing consistent structural differences in the binding mode of the inhibitors in the active sites of the SmPNP and human PNP (HsPNP) structures. The molecular information gathered in this work should be useful for future medicinal chemistry efforts in the design of new inhibitors of SmPNP having increased affinity and selectivity. PMID:20129792

  5. Anti-Mycobacterial Nucleoside Antibiotics from a Marine-Derived Streptomyces sp. TPU1236A

    PubMed Central

    Bu, Ying-Yue; Yamazaki, Hiroyuki; Ukai, Kazuyo; Namikoshi, Michio

    2014-01-01

    Five new nucleoside antibiotics, named streptcytosines A–E (1–5), and six known compounds, de-amosaminyl-cytosamine (6), plicacetin (7), bamicetin (8), amicetin (9), collismycin B (10), and SF2738 C (11), were isolated from a culture broth of Streptomyces sp. TPU1236A collected in Okinawa, Japan. The structures of new compounds were elucidated on the basis of their spectroscopic data (HRFABMS, IR, UV, and 2D NMR experiments including 1H-1H COSY, HMQC, HMBC, and NOESY spectra). Streptcytosine A (1) belonged to the amicetin group antibiotics, and streptcytosines B–E (2–5) were derivatives of de-amosaminyl-cytosamine (6), 2,3,6-trideoxyglucopyranosyl cytosine. Compound 1 inhibited the growth of Mycobacterium smegmatis (MIC = 32 µg/mL), while compounds 2–5 were not active at 50 µg/disc. Bamicetin (8) and amicetin (9) showed the MICs of 16 and 8 µg/mL, respectively. PMID:25522318

  6. Complete inactivation of HIV-1 using photo-labeled non-nucleoside reverse transcriptase inhibitors.

    PubMed

    Rios, Adan; Quesada, Jorge; Anderson, Dallas; Goldstein, Allan; Fossum, Theresa; Colby-Germinario, Susan; Wainberg, Mark A

    2011-01-01

    We demonstrate that a photo-labeled derivative of the non-nucleoside reverse transcriptase inhibitor (NNRTI) dapivirine termed DAPY, when used together with exposure to ultraviolet light, was able to completely and irreversibly inactivate both HIV-1 RT activity as well as infectiousness in each of a T cell line and peripheral blood mononuclear cells. Control experiments using various concentrations of DAPY revealed that a combination of exposure to ultraviolet light together with use of the specific, high affinity photo-labeled compound was necessary for complete inactivation to occur. This method of HIV RT inactivation may have applicability toward preservation of an intact viral structure and warrants further investigation in regard to the potential of this approach to elicit a durable, broad protective immune response. PMID:20937333

  7. Computer-generated Model of Purine Nucleoside Phosphorylase (PNP)

    NASA Technical Reports Server (NTRS)

    1987-01-01

    Purine Nucleoside Phosphorylase (PNP) is an important target enzyme for the design of anti-cancer and immunosuppressive drugs. Bacterial PNP, which is slightly different from the human enzyme, is used to synthesize chemotherapuautic agents. Knowledge of the three-dimensional structure of the bacterial PNP molecule is useful in efforts to engineer different types of PNP enzymes, that can be used to produce new chemotherapeutic agents. This picture shows a computer model of bacterial PNP, which looks a lot like a display of colorful ribbons. Principal Investigator was Charles Bugg.

  8. Polymerization of the cyclic pyrophosphates of nucleosides and their analogues

    NASA Technical Reports Server (NTRS)

    Tohidi, Mahrokh; Orgel, Leslie E.

    1990-01-01

    When 2-prime-deoxythymidine 3-prime, 5-prime-cyclic diphosphate, or the cyclic pyrophosphates of the acyclic nucleoside analogs II and IV are heated to 65-85 C in the presence of imidazole, oligomers with lengths up to 20-30 are formed in excellent yield. This reaction provides a useful source of oligomers for use as templates in aqueous condensation reactions. In the absence of evidence to the contrary, it is assumed that the oligomers are atactic. The potential significance of this reaction in prebiotic chemistry is discussed.

  9. Oligodeoxyribonucleotides containing 1,3-propanediol as nucleoside substitute.

    PubMed Central

    Seela, F; Kaiser, K

    1987-01-01

    1,3-Propanediol was protected with one dimethoxytrityl residue and converted into the methoxy- and cyanoethoxyphosphoramidites 2a and 2b, respectively. Solid-phase oligonucleotide synthesis, employing the phosphoramidite 2a resulted in the dodecamers d(CGCGAATTCGCG) (6-9), in which dA or dT residues were replaced by 1,3-propanediol. These oligomers showed a high tendency to form hairpins. Their phosphodiester bonds between the 3'-position of a nucleoside and the propanediol moiety was not cleaved by snake venom phosphodiesterase. PMID:3562246

  10. Novel nucleoside-based antimalarial compounds.

    PubMed

    Zheng, Zhaoyan; Tran, Huu-Anh; Manivannan, Srinivasan; Wen, Xianghui; Kaiser, Marcel; Brun, Reto; Snyder, Floyd F; Back, Thomas G

    2016-06-15

    The malaria-causing parasite Plasmodium falciparum employs a salvage pathway for the biosynthesis of nucleotides, in contrast to de novo biosynthesis that is utilized by the human host. A series of twenty-two 2-, 6- and 5'-modified adenosine ribonucleosides was synthesized, with the expectation that these compounds would generate toxic metabolites instead of active nucleotides by the pathogen, while remaining inert in host cells. Bioassays with P. falciparum (K1 strain) indicated IC50 values as low as 110nM and a selectivity index with respect to cytotoxicity toward an L6 rat myoblast cell line of >1000 for the most potent analogue. PMID:27156774

  11. Synthesis of 5-Fluoroalkylated Pyrimidine Nucleosides via Negishi Cross-Coupling

    PubMed Central

    Chacko, Ann-Marie; Qu, Wenchao

    2014-01-01

    5-fluoroalkylated pyrimidine nucleosides (1) have potential as therapeutic agents and molecular imaging agents targeting HSV1-tk suicide gene therapy. Thus, straightforward preparation of 5-fluoroalkylated nucleoside derivatives has been developed. Reported herein are the first examples of Pd-catalyzed Negishi cross-coupling of 3-N-benzoyl-3′,5′-di-O-benzoyl-5-iodo-2′-deoxyuridine (2a) and 3-N-benzoyl-3′,5′-di-O-benzoyl-5-iodo-2′-deoxy-2′-fluoroarabinouridine (2b) with unactivated Csp3 fluoroalkylzinc bromides. This paper demonstrates the first synthesis of six 5-fluoroalkyl-2′-deoxy pyrimidine nucleoside derivatives with three to five methylene-chain lengths (5). Furthermore, this methodology has been extended to create a series of thirteen 5-alkyl substituted nucleosides, including the target nucleosides 5 and 5-silyloxypropyl and 5-cyanobutyl derivatives. PMID:18522415

  12. Theoretical understanding of two-photon-induced fluorescence of isomorphic nucleoside analogs.

    PubMed

    Samanta, Pralok K; Pati, Swapan K

    2015-04-21

    We use ab initio Density Functional Theory (DFT) and Time-dependent DFT (TDDFT) calculations for a detailed understanding of one-photon absorption (1PA) and two-photon absorption (2PA) cross sections of eight different nucleoside analogs. The results are compared and contrasted with the available experimental data. Our calculated results show that the low energy peaks in the absorption spectra mainly arise because of the π-π* electronic transition of the nucleoside analogs. The emission spectra of the nucleoside analogs are also calculated using TDDFT methods. The calculated absorption and emission spectra in the presence of a solvent follow the same trend as those found experimentally. Our results demonstrate that the nucleoside analogs show significantly different electronic and optical properties, although their bonding aspects towards Watson-Crick base pairing remain the same. We also derive the microscopic details of the origin of nonlinear optical properties of the nucleoside analogs. PMID:25785569

  13. A high-yielding, strictly regioselective prebiotic purine nucleoside formation pathway.

    PubMed

    Becker, Sidney; Thoma, Ines; Deutsch, Amrei; Gehrke, Tim; Mayer, Peter; Zipse, Hendrik; Carell, Thomas

    2016-05-13

    The origin of life is believed to have started with prebiotic molecules reacting along unidentified pathways to produce key molecules such as nucleosides. To date, a single prebiotic pathway to purine nucleosides had been proposed. It is considered to be inefficient due to missing regioselectivity and low yields. We report that the condensation of formamidopyrimidines (FaPys) with sugars provides the natural N-9 nucleosides with extreme regioselectivity and in good yields (60%). The FaPys are available from formic acid and aminopyrimidines, which are in turn available from prebiotic molecules that were also detected during the Rosetta comet mission. This nucleoside formation pathway can be fused to sugar-forming reactions to produce pentosides, providing a plausible scenario of how purine nucleosides may have formed under prebiotic conditions. PMID:27174989

  14. Aquaporin 3 (AQP3) participates in the cytotoxic response to nucleoside-derived drugs

    PubMed Central

    2012-01-01

    Background Nucleoside analogs used in the chemotherapy of solid tumors, such as the capecitabine catabolite 5′-deoxy-5-fluorouridine (5′-DFUR) trigger a transcriptomic response that involves the aquaglyceroporin aquaporin 3 along with other p53-dependent genes. Here, we examined whether up-regulation of aquaporin 3 (AQP3) mRNA in cancer cells treated with 5′-DFUR represents a collateral transcriptomic effect of the drug, or conversely, AQP3 participates in the activity of genotoxic agents. Methods The role of AQP3 in cell volume increase, cytotoxicity and cell cycle arrest was analyzed using loss-of-function approaches. Results 5′-DFUR and gemcitabine, but not cisplatin, stimulated AQP3 expression and cell volume, which was partially and significantly blocked by knockdown of AQP3. Moreover, AQP3 siRNA significantly blocked other effects of nucleoside analogs, including G1/S cell cycle arrest, p21 and FAS up-regulation, and cell growth inhibition. Short incubations with 5-fluorouracil (5-FU) also induced AQP3 expression and increased cell volume, and the inhibition of AQP3 expression significantly blocked growth inhibition triggered by this drug. To further establish whether AQP3 induction is related to cell cycle arrest and apoptosis, cells were exposed to long incubations with escalating doses of 5-FU. AQP3 was highly up-regulated at doses associated with cell cycle arrest, whereas at doses promoting apoptosis induction of AQP3 mRNA expression was reduced. Conclusions Based on the results, we propose that the aquaglyceroporin AQP3 is required for cytotoxic activity of 5’-DFUR and gemcitabine in the breast cancer cell line MCF7 and the colon adenocarcinoma cell line HT29, and is implicated in cell volume increase and cell cycle arrest. PMID:23017148

  15. Targeting the Plasmodium vivax equilibrative nucleoside transporter 1 (PvENT1) for antimalarial drug development.

    PubMed

    Deniskin, Roman; Frame, I J; Sosa, Yvett; Akabas, Myles H

    2016-04-01

    Infection with Plasmodium falciparum and vivax cause most cases of malaria. Emerging resistance to current antimalarial medications makes new drug development imperative. Ideally a new antimalarial drug should treat both falciparum and vivax malaria. Because malaria parasites are purine auxotrophic, they rely on purines imported from the host erythrocyte via Equilibrative Nucleoside Transporters (ENTs). Thus, the purine import transporters represent a potential target for antimalarial drug development. For falciparum parasites the primary purine transporter is the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Recently we identified potent PfENT1 inhibitors with nanomolar IC50 values using a robust, yeast-based high throughput screening assay. In the current work we characterized the Plasmodium vivax ENT1 (PvENT1) homologue and its sensitivity to the PfENT1 inhibitors. We expressed a yeast codon-optimized PvENT1 gene in Saccharomyces cerevisiae. PvENT1-expressing yeast imported both purines ([(3)H]adenosine) and pyrimidines ([(3)H]uridine), whereas wild type (fui1Δ) yeast did not. Based on radiolabel substrate uptake inhibition experiments, inosine had the lowest IC50 (3.8 μM), compared to guanosine (14.9 μM) and adenosine (142 μM). For pyrimidines, thymidine had an IC50 of 183 μM (vs. cytidine and uridine; mM range). IC50 values were higher for nucleobases compared to the corresponding nucleosides; hypoxanthine had a 25-fold higher IC50 than inosine. The archetypal human ENT1 inhibitor 4-nitrobenzylthioinosine (NBMPR) had no effect on PvENT1, whereas dipyridamole inhibited PvENT1, albeit with a 40 μM IC50, a 1000-fold less sensitive than human ENT1 (hENT1). The PfENT1 inhibitors blocked transport activity of PvENT1 and the five known naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) with similar IC50 values. Thus, the PfENT1 inhibitors also target PvENT1. This implies that development of novel antimalarial

  16. Targeting the Plasmodium vivax equilibrative nucleoside transporter 1 (PvENT1) for antimalarial drug development

    PubMed Central

    Deniskin, Roman; Frame, I.J.; Sosa, Yvett; Akabas, Myles H.

    2015-01-01

    Infection with Plasmodium falciparum and vivax cause most cases of malaria. Emerging resistance to current antimalarial medications makes new drug development imperative. Ideally a new antimalarial drug should treat both falciparum and vivax malaria. Because malaria parasites are purine auxotrophic, they rely on purines imported from the host erythrocyte via Equilibrative Nucleoside Transporters (ENTs). Thus, the purine import transporters represent a potential target for antimalarial drug development. For falciparum parasites the primary purine transporter is the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Recently we identified potent PfENT1 inhibitors with nanomolar IC50 values using a robust, yeast-based high throughput screening assay. In the current work we characterized the Plasmodium vivax ENT1 (PvENT1) homologue and its sensitivity to the PfENT1 inhibitors. We expressed a yeast codon-optimized PvENT1 gene in Saccharomyces cerevisiae. PvENT1-expressing yeast imported both purines ([3H]adenosine) and pyrimidines ([3H]uridine), whereas wild type (fui1Δ) yeast did not. Based on radiolabel substrate uptake inhibition experiments, inosine had the lowest IC50 (3.8 μM), compared to guanosine (14.9 μM) and adenosine (142 μM). For pyrimidines, thymidine had an IC50 of 183 μM (vs. cytidine and uridine; mM range). IC50 values were higher for nucleobases compared to the corresponding nucleosides; hypoxanthine had a 25-fold higher IC50 than inosine. The archetypal human ENT1 inhibitor 4-nitrobenzylthioinosine (NBMPR) had no effect on PvENT1, whereas dipyridamole inhibited PvENT1, albeit with a 40 μM IC50, a 1000-fold less sensitive than human ENT1 (hENT1). The PfENT1 inhibitors blocked transport activity of PvENT1 and the five known naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) with similar IC50 values. Thus, the PfENT1 inhibitors also target PvENT1. This implies that development of novel antimalarial drugs

  17. Na+ reabsorption in cultured rat epididymal epithelium via the Na+/nucleoside cotransporter.

    PubMed

    Leung, G P; Cheung, K H; Tse, C M; Wong, P Y

    2001-03-01

    The effect of nucleoside on Na+ reabsorption via Na+/nucleoside cotransporter in cultured rat epididymal epithelia was studied by short-circuit current (Isc) technique. Guanosine added apically stimulated Isc in a dose-dependent manner, with a median effective concentration (EC50) of 7 +/- 2 microM (mean +/- SEM). Removal of Na+ from the apical bathing solution or pretreatment with a nonspecific Na+/nucleoside cotransporter inhibitor, phloridzin, completely blocked the Isc response to guanosine. Moreover, the guanosine response was abolished by pretreatment of the tissue with ouabain, a Na+/K+-ATPase inhibitor, suggesting the involvement of Na+/nucleoside cotransporter on the apical side and Na+/K+-ATPase on the basolateral side in Na+ reabsorption. In contrast, the Isc response to guanosine was not affected after desensitization of purinoceptors by ATP. Addition of the Na+/K+/2Cl- symport inhibitor bumetanide to the basolateral side or the nonspecific Cl- channel blocker diphenylamine-2-carboxylate to the apical side showed no effect on the Isc response to guanosine, excluding stimulation of Cl- secretion by guanosine as the cause of the guanosine-induced Isc. The Isc response to purine nucleoside (guanosine and inosine) was much higher than that to pyrimidine nucleoside (thymidine and cytidine). Consistent with substrate specificity, results of reverse transcription-polymerase chain reaction revealed mRNA for concentrative nucleoside transporter (CNT2), which is a purine nucleoside-selective Na+/nucleoside cotransporter in the epididymis, but not for CNT1. It is suggested that the Na+/nucleoside cotransporter (i.e., CNT2) may be one of the elements involved in Na+ and fluid reabsorption in the epididymis, thereby providing an optimal microenvironment for the maturation and storage of spermatozoa. PMID:11207189

  18. Muraymycin nucleoside-peptide antibiotics: uridine-derived natural products as lead structures for the development of novel antibacterial agents

    PubMed Central

    Wirth, Marius; Niro, Giuliana; Leyerer, Kristin

    2016-01-01

    Summary Muraymycins are a promising class of antimicrobial natural products. These uridine-derived nucleoside-peptide antibiotics inhibit the bacterial membrane protein translocase I (MraY), a key enzyme in the intracellular part of peptidoglycan biosynthesis. This review describes the structures of naturally occurring muraymycins, their mode of action, synthetic access to muraymycins and their analogues, some structure–activity relationship (SAR) studies and first insights into muraymycin biosynthesis. It therefore provides an overview on the current state of research, as well as an outlook on possible future developments in this field. PMID:27340469

  19. Non-nucleoside inhibitors of HIV-1 reverse transcriptase: molecular modeling and X-ray structure investigations.

    PubMed

    Schäfer, W; Friebe, W G; Leinert, H; Mertens, A; Poll, T; von der Saal, W; Zilch, H; Nuber, B; Ziegler, M L

    1993-03-19

    The structural features of a new class of non-nucleoside HIV-1 reverse transcriptase inhibitors (3) are presented. Comparison of the structural and electronic properties with those of TIBO (1) and Nevirapine (2) yields a common three-dimensional model. This model permits the improvement of the lead compound 3 by chemical modification (5,6). Additionally, two new types of inhibitors (4, 7) with similar biological activity can be derived from this model. The structure of the new compounds, including their absolute configuration, are determined by X-ray crystallography. PMID:7681480

  20. Lethal Mutagenesis of HIV with Mutagenic Nucleoside Analogs

    NASA Astrophysics Data System (ADS)

    Loeb, Lawrence A.; Essigmann, John M.; Kazazi, Farhad; Zhang, Jue; Rose, Karl D.; Mullins, James I.

    1999-02-01

    The human immunodeficiency virus (HIV) replicates its genome and mutates at exceptionally high rates. As a result, the virus is able to evade immunological and chemical antiviral agents. We tested the hypothesis that a further increase in the mutation rate by promutagenic nucleoside analogs would abolish viral replication. We evaluated deoxynucleoside analogs for lack of toxicity to human cells, incorporation by HIV reverse transcriptase, resistance to repair when incorporated into the DNA strand of an RNA\\cdot DNA hybrid, and mispairing at high frequency. Among the candidates tested, 5-hydroxydeoxycytidine (5-OH-dC) fulfilled these criteria. In seven of nine experiments, the presence of this analog resulted in the loss of viral replicative potential after 9-24 sequential passages of HIV in human CEM cells. In contrast, loss of viral replication was not observed in 28 control cultures passaged in the absence of the nucleoside analog, nor with other analogs tested. Sequence analysis of a portion of the HIV reverse transcriptase gene demonstrated a disproportionate increase in G -> A substitutions, mutations predicted to result from misincorporation of 5-OH-dC into the cDNA during reverse transcription. Thus, "lethal mutagenesis" driven by the class of deoxynucleoside analogs represented by 5-OH-dC could provide a new approach to treating HIV infections and, potentially, other viral infections.

  1. Chutes and Ladders in Hepatitis C Nucleoside Drug Development§

    PubMed Central

    Coats, Steven J.; Garnier-Amblard, Ethel C.; Amblard, Franck; Ehteshami, Maryam; Amiralaei, Sheida; Zhang, Hongwang; Zhou, Longhu; Boucle, Sebastien R. L.; Lu, Xiao; Bondada, Lavanya; Shelton, Jadd R.; Li, Hao; Liu, Peng; Li, Chengwei; Cho, Jong Hyun; Chavre, Satish N.; Zhou, Shaoman; Mathew, Judy; Schinazi, Raymond F.

    2014-01-01

    Chutes and Ladders is an exciting up-and-down-again game in which players race to be the first to the top of the board. Along the way, they will find ladders to help them advance, and chutes that will cause them to move backwards. The development of nucleoside analogs for clinical treatment of hepatitis C presents a similar scenario in which taking shortcuts may help quickly advance a program, but there is always a tremendous risk of being sent backwards as one competes for the finish line. In recent years the treatment options for chronic hepatitis C virus (HCV) infection have expand due to the development of a replicon based in vitro evaluation system, allowing for the identification of multiple drugable viral targets along with a concerted and substantial drug discovery effort. Three major drug targets have reached clinical study for chronic HCV infection: the NS3/4A serine protease, the large phosphoprotein NS5A, and the NS5B RNA-dependent RNA polymerase. Recently, two oral HCV protease inhibitors were approved by the FDA and were the first direct acting anti-HCV agents to result from the substantial research in this area. There are currently many new chemical entities from several different target classes that are being evaluated worldwide in clinical trials for their effectiveness at achieving a sustained virologic response (SVR) (Pham et al., 2004; Radkowski et al., 2005). Clearly the goal is to develop therapies leading to a cure that are safe, widely accessible and available, and effective against all HCV genotypes (GT), and all stages of the disease. Nucleoside analogs that target the HCV NS5B polymerase that have reached human clinical trials is the focus of this review as they have demonstrated significant advantages in the clinic with broader activity against the various HCV GT and a higher barrier to the development of resistant viruses when compared to all other classes of HCV inhibitors. PMID:24275341

  2. Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells.

    PubMed

    Rehan, Shahid; Jaakola, Veli-Pekka

    2015-10-01

    Human equilibrative nucleoside transporter-1 (hENT1) is the major plasma membrane transporter involved in transportation of natural nucleosides as well as nucleoside analog drugs, used in anti-cancer and anti-viral therapies. Despite extensive biochemical and pharmacological studies, little is known about the structure-function relationship of this protein. The major obstacles to purification include a low endogenous expression level, the lack of an efficient expression and purification protocol, and the hydrophobic nature of the protein. Here, we report protein expression, purification and functional characterization of hENT1 from Sf9 insect cells. hENT1 expressed by Sf9 cells is functionally active as demonstrated by saturation binding with a Kd of 1.2±0.2nM and Bmax of 110±5pmol/mg for [(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR). We also demonstrate purification of hENT1 using FLAG antibody affinity resin in lauryl maltose neopentyl glycol detergent with a Kd of 4.3±0.7nM. The yield of hENT1 from Sf9 cells was ∼0.5mg active transporter per liter of culture. The purified protein is functionally active, stable, homogenous and appropriate for further biophysical and structural studies. PMID:26162242

  3. Synthesis of α-l-Threofuranosyl Nucleoside Triphosphates (tNTPs)

    PubMed Central

    Zou, Keyong; Horhota, Allen; Yu, Biao; Szostak, Jack W.

    2005-01-01

    The α-l-threofuranosyl nucleoside triphosphates of T, G, and D (tTTP, tGTP, and tDTP) were synthesized from the described 2‘-O-DMT-protected derivatives using the Eckstein method, while the corresponding C derivative (tCTP) was prepared from the 2‘-O-acetyl derivative. The prepared α-l-threofuranosyl nucleoside triphosphates, despite being one carbon shorter than the native 2‘-deoxyfuranosyl nucleoside triphosphates, are effective substrates for selected DNA polymerases. PMID:15816733

  4. Identification of a novel compound that inhibits osteoclastogenesis by suppressing nucleoside transporters.

    PubMed

    Katsuyama, Shun; Sugino, Kumi; Sasazawa, Yukiko; Nakano, Yoshihiko; Aono, Harumi; Morishita, Keisuke; Kawatani, Makoto; Umezawa, Kazuo; Osada, Hiroyuki; Simizu, Siro

    2016-04-01

    We screened small-molecule compounds that inhibit osteoclast differentiation to find new anti-osteoporosis agents and found that a novel compound, SUKU-1, suppressed osteoclastogenesis. We also synthesized 38 derivatives of SUKU-1 and discovered that nine of them had inhibitory effects on osteoclastogenesis and that SUKU-33 was the most potent inhibitor. Next, we investigated the mechanisms by which SUKU-33 suppressed osteoclast differentiation. By measuring the uptake of [(3) H]-uridine in cells, we found that SUKU-33 suppressed both equilibrative nucleoside transporters and concentrative nucleoside transporters. These results suggest that SUKU-33 inhibits osteoclast differentiation by suppressing nucleoside transporters. PMID:27001232

  5. Application of crystallographic and modeling methods in the design of purine nucleoside phosphorylase inhibitors.

    PubMed Central

    Ealick, S E; Babu, Y S; Bugg, C E; Erion, M D; Guida, W C; Montgomery, J A; Secrist, J A

    1991-01-01

    Competitive inhibitors of the salvage pathway enzyme purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) have been designed by using the three-dimensional structure of the enzyme as determined by x-ray crystallography. The process was an iterative one that utilized interactive computer graphics, Monte Carlo-based conformational searching, energy minimization, and x-ray crystallography. The proposed compounds were synthesized and tested by an in vitro assay. Among the compounds designed and synthesized are the most potent competitive inhibitors of purine nucleoside phosphorylase thus far reported. Images PMID:1763067

  6. An Efficient Protection-Free One-Pot Chemical Synthesis of Modified Nucleoside-5'-Triphosphates.

    PubMed

    Shanmugasundaram, Muthian; Senthilvelan, Annamalai; Xiao, Zejun; Kore, Anilkumar R

    2016-07-01

    A simple, reliable, and an efficient "one-pot, three step" chemical method for the synthesis of modified nucleoside triphosphates such as 5-methylcytidine-5'-triphosphate (5-MeCTP), pseudouridine-5'-triphosphate (pseudoUTP) and N(1)-methylpseudouridine-5'-triphosphate (N(1)-methylpseudoUTP) starting from the corresponding nucleoside is described. The overall reaction involves the monophosphorylation of nucleoside, followed by the reaction with pyrophosphate and subsequent hydrolysis of the cyclic intermediate to furnish the corresponding NTP in moderate yields with high purity (>99.5%). PMID:27159048

  7. Synthesis of α-L-threose nucleoside phosphonates via regioselective sugar protection.

    PubMed

    Dumbre, Shrinivas G; Jang, Mi-Yeon; Herdewijn, Piet

    2013-07-19

    A new synthesis route to α-L-threose nucleoside phosphonates via 2-O and 3-O selectively protected L-threose is developed. The key intermediates 2-O-benzoyl-L-threonolactone and 1-O-acetyl-2-O-benzoyl-3-O-t-butyldiphenylsilyl-L-threofuranose were functionalized to synthesize 2'-deoxy-2'-fluoro- and 3'-C-ethynyl L-threose 3'-O-phosphonate nucleosides. The key intermediates developed are important intermediates for the synthesis of new L-threose-based nucleoside analogues, TNA phosphoramidites, and TNA triphosphates. PMID:23822647

  8. Pyrimidine nucleoside phosphorylation in developing seeds and germinating seedlings of wheat

    SciTech Connect

    Rowe, M.L.

    1988-01-01

    Uridine- and thymidine-phosphorylating enzymes were measured in developing and germinating seeds of Triticum aestivum v. Arthur and T. aestivum v. Lemhi. Because crude extracts were to be used in the developmental study, characteristics of unpurified nucleoside phosphotransferase (NPTase) were examined. In the developmental study with two varieties of wheat, NPTase activity was found to be very low in all of the true seed tissues during seed maturation. Uridine-phosphorylating activity was due to primarily to uridine kinase. Thymidine phosphorylation was very low in all tissues throughout seed maturation, with a brief appearance by thymidine kinase in the developing embryo. In germinating seeds, uridine-phosphorylating activity was present from earliest stages of germination but showed a decrease in activity followed by a recovery after 48 hours inbibition. Experiments using ({alpha}-{sup 32}P)ATP indicated that uridine kinase was present during early germination but had disappeared by 96 hours. Uridine phosphorylation at later stages of germination was accomplished by NTPase. Thymidine phosphorylation did not begin until after 36 hours of germination and was the result of NPTase activity.

  9. Synthesis of Purine and 7-Deazapurine Nucleoside Analogues of 6-N-(4-Nitrobenzyl)adenosine; Inhibition of Nucleoside Transport and Proliferation of Cancer Cells

    PubMed Central

    Rayala, Ramanjaneyulu; Theard, Patricia; Ortiz, Heysell; Yao, Sylvia; Young, James D.; Balzarini, Jan; Robins, Morris J.

    2014-01-01

    The 7-deazapurine nucleoside antibiotic tubercidin was converted into its 4-N-benzyl and 4-N-(4-nitrobenzyl) derivatives by alkylation at N3 followed by Dimroth rearrangement to the 4-N- isomer or by fluoro-diazotization followed by SNAr displacement of the 4-fluoro group by a benzylamine. The 4-N-(4-nitrobenzyl) derivatives of sangivamycin and toyocamycin antibiotics were prepared by the alkylation approach. Cross-membrane transport of labeled uridine by hENT1 was inhibited to a weaker extent by the 4-nitrobenzylated tubercidin and sangivamycin analogues than was observed with 6-N-(4-nitrobenzyl)adenosine. Type-specific inhibition of cancer cell proliferation was observed at μM concentrations with the 4-N-(4-nitrobenzyl) derivatives of sangivamycin and toyocamycin, and also with 4-N-benzyltubercidin. Treatment of 2′,3′,5′-O-acetyladenosine with aryl isocyanates gave the 6-ureido derivatives but none of them exhibited inhibitory activity against cancer cell proliferation or hENT1. PMID:24788480

  10. [Study of the calmodulin-dependent regulation of calcium adenosine triphosphatase of erythrocyte membranes in patients with ischemic heart disease].

    PubMed

    Malaia, L T; Petruniaka, V V; Rudyk, Iu S

    1991-01-01

    The inhibitor calmodulin (R 24571) was examined for effects on the activity of red blood cell Ca-ATPases in patients with coronary heart disease during the treatment with nitrates, beta-blockers and calcium antagonists. The maximum activity of Ca-ATPase was measured in the erythrocytes perforated with saponine in the presence of endogenous regulators at a concentration of Ca2+ of 3-5 microM. Patients with high and low Ca-ATPase activity were identified. In the control group R24571 failed to affect Ca-ATPase activity. In patients, the calmodulin inhibitor caused both Ca-ATPase activation and inhibition. The effects of R 24571 correlated with the severity of the patients' condition. In effective therapy, the action of the calmodulin inhibitor became lower on Ca-ATPase activity. It was concluded that there was Ca-ATPase regulation imbalance in patients with coronary heart diseases. PMID:1838226

  11. Lipopolysaccharide-induced apoptosis of macrophages determines the up-regulation of concentrative nucleoside transporters Cnt1 and Cnt2 through tumor necrosis factor-alpha-dependent and -independent mechanisms.

    PubMed

    Soler, C; Valdés, R; Garcia-Manteiga, J; Xaus, J; Comalada, M; Casado, F J; Modolell, M; Nicholson, B; MacLeod, C; Felipe, A; Celada, A; Pastor-Anglada, M

    2001-08-10

    In murine bone marrow macrophages, lipopolysaccharide (LPS) induces apoptosis through the autocrine production of tumor necrosis factor-alpha (TNF-alpha), as demonstrated by the fact that macrophages from TNF-alpha receptor I knock-out mice did not undergo early apoptosis. In these conditions LPS up-regulated the two concentrative high affinity nucleoside transporters here shown to be expressed in murine bone marrow macrophages, concentrative nucleoside transporter (CNT) 1 and 2, in a rapid manner that is nevertheless consistent with the de novo synthesis of carrier proteins. This effect was not dependent on the presence of macrophage colony-stimulating factor, although LPS blocked the macrophage colony-stimulating factor-mediated up-regulation of the equilibrative nucleoside transport system es. TNF-alpha mimicked the regulatory response of nucleoside transporters triggered by LPS, but macrophages isolated from TNF-alpha receptor I knock-out mice similarly up-regulated nucleoside transport after LPS treatment. Although NO is produced by macrophages after LPS treatment, NO is not involved in these regulatory responses because LPS up-regulated CNT1 and CNT2 transport activity and expression in macrophages from inducible nitric oxide synthase and cationic amino acid transporter (CAT) 2 knock-out mice, both of which lack inducible nitric oxide synthesis. These data indicate that the early proapoptotic responses of macrophages, involving the up-regulation of CNT transporters, follow redundant regulatory pathways in which TNF-alpha-dependent- and -independent mechanisms are involved. These observations also support a role for CNT transporters in determining extracellular nucleoside availability and modulating macrophage apoptosis. PMID:11346649

  12. Synthesis of Methylenecyclopropane Analogues of Antiviral Nucleoside Phosphonates

    PubMed Central

    Yan, Zhaohua; Zhou, Shaoman; Kern, Earl R.; Zemlicka, Jiri

    2006-01-01

    Synthesis of methylenecyclopropane analogues of nucleoside phosphonates 6a, 6b, 7a and 7b is described. Cyclopropyl phosphonate 8 was transformed in four steps to methylenecyclopropane phosphonate 16. The latter intermediate was converted in seven steps to the key Z- and E-methylenecyclopropane alcohols 23 and 24 separated by chromatography. Selenoxide eliminations (15 → 16 and 22 → 23 + 24) were instrumental in the synthesis. The Z- and E-isomers 23 and 24 were transformed to bromides 25a and 25b which were used for alkylation of adenine and 2-amino-6-chloropurine to give intermediates 26a, 26b, 26c and 26d. Acid hydrolysis provided the adenine and guanine analogues 6a, 6b, 7a and 7b. Phosphonates 6b and 7b are potent inhibitors of replication of Epstein-Barr virus (EBV). PMID:16758001

  13. RIBOSE MODIFIED NUCLEOSIDES AND NUCLEOTIDES AS LIGANDS FOR PURINE RECEPTORS

    PubMed Central

    Ravi, R. G.; Nandanan, E.; Kim, H. S.; Moro, S.; Kim, Y. C.; Lee, K.; Barak, D.; Marquez, V. E.; Ji, X. D.

    2016-01-01

    Molecular modeling of receptors for adenosine and nucleotide (P2) receptors with docked ligand, based on mutagenesis, was carried out. Adenosine 3′,5′-bisphosphate derivatives act as selective P2Y1 antagonists/partial agonists. The ribose moiety was replaced with carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics, producing compounds that retained receptor affinity. Conformational constraints were built into the ribose rings of nucleoside and nucleotide ligands using the methanocarba approach, i.e. fused cyclopropane and cyclopentane rings in place of ribose, suggesting a preference for the Northern (N) conformation among ligands for P2Y1 and A1 and A3ARs. PMID:11563046

  14. Pan-Pathway Based Interaction Profiling of FDA-Approved Nucleoside and Nucleobase Analogs with Enzymes of the Human Nucleotide Metabolism

    PubMed Central

    Egeblad, Louise; Welin, Martin; Flodin, Susanne; Gräslund, Susanne; Wang, Liya; Balzarini, Jan; Eriksson, Staffan; Nordlund, Pär

    2012-01-01

    To identify interactions a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs was screened against 23 enzymes of the human nucleotide metabolism using a thermal shift assay. The method was validated with deoxycytidine kinase; eight interactions known from the literature were detected and five additional interactions were revealed after the addition of ATP, the second substrate. The NAL screening gave relatively few significant hits, supporting a low rate of “off target effects.” However, unexpected ligands were identified for two catabolic enzymes guanine deaminase (GDA) and uridine phosphorylase 1 (UPP1). An acyclic guanosine prodrug analog, valaciclovir, was shown to stabilize GDA to the same degree as the natural substrate, guanine, with a ΔTagg around 7°C. Aciclovir, penciclovir, ganciclovir, thioguanine and mercaptopurine were also identified as ligands for GDA. The crystal structure of GDA with valaciclovir bound in the active site was determined, revealing the binding of the long unbranched chain of valaciclovir in the active site of the enzyme. Several ligands were identified for UPP1: vidarabine, an antiviral nucleoside analog, as well as trifluridine, idoxuridine, floxuridine, zidovudine, telbivudine, fluorouracil and thioguanine caused concentration-dependent stabilization of UPP1. A kinetic study of UPP1 with vidarabine revealed that vidarabine was a mixed-type competitive inhibitor with the natural substrate uridine. The unexpected ligands identified for UPP1 and GDA imply further metabolic consequences for these nucleoside analogs, which could also serve as a starting point for future drug design. PMID:22662200

  15. Novel indole-3-sulfonamides as potent HIV non-nucleoside reverse transcriptase inhibitors (NNRTIs)

    SciTech Connect

    Zhao, Zhijian; Wolkenberg, Scott E.; Lu, Meiqing; Munshi, Vandna; Moyer, Gregory; Feng, Meizhen; Carella, Anthony V.; Ecto, Linda T.; Gabryelski, Lori J.; Lai, Ming-Tain; Prasad, Sridar G.; Yan, Youwei; McGaughey, Georgia B.; Miller, Michael D.; Lindsley, Craig W.; Hartman, George D.; Vacca, Joseph P.; Williams, Theresa M.

    2008-09-29

    This Letter describes the design, synthesis, and biological evaluation of novel 3-indole sulfonamides as potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) with balanced profiles against common HIV RT mutants K103N and Y181C.

  16. Comparison of nucleoside concentrations in blood of fish with and without tumors

    SciTech Connect

    Kuehl, D.W.; Johnson, R.D. ); Eisenschenk, L.; Naumann, S. ); Regal, R.; Barnidge, P. ); McKim, J. Jr. )

    1991-05-01

    The objective of this study was to develop and use HPLC based analytical methodology to characterize nucleosides in blood plasma and serum from fish with and without tumors, with a goal of determining if fish blood nucleoside concentrations could similarly be used as a bioindicator of tumor development in fish. The approach was to develop analytical methodology and quality assurance criteria for the analysis of nucleosides in fish blood, and to characterize nucleoside concentrations in blood of fish for which both healthy and tumor-bearing samples were available. Data would then be used to establish parameters with which tumor-bearing fish could be distinguished from healthy fish. Blood samples used to establish the diagnostic parameters were from control rainbow trout (Oncorhynchus mykiss) and those with tumors developed after exposure to aflatoxins. A second set of blood samples was from field collected black bullheads (Ictalurus melas).

  17. The nonspecificity of the lead method for the histochemical demonstration of adenosine triphosphatases in human skeletal muscle fibres.

    PubMed

    Erzen, I; Sirca, A

    1985-01-01

    The lead method for the histochemical demonstration of presumptive mitochondrial adenosinetriphosphatase was applied to biopsy and autopsy samples of the human vastus lateralis muscle. The effect of p-chloromercuribenzoate and of Triton X-100 was tested microdensitometrically and the activity of 'mitochondrial' ATPase was compared to the activity of enzymes of the oxidative metabolism succinic dehydrogenase and NAD-tetrazolium reductase. It is concluded that the ATPase activity displayed is not mainly mitochondrial. In autopsy material, it seems to be predominantly myofibrillar. PMID:2933378

  18. The nonspecificity of the lead method for the histochemical demonstration of adenosine triphosphatases in human skeletal muscle fibres.

    PubMed Central

    Erzen, I; Sirca, A

    1985-01-01

    The lead method for the histochemical demonstration of presumptive mitochondrial adenosinetriphosphatase was applied to biopsy and autopsy samples of the human vastus lateralis muscle. The effect of p-chloromercuribenzoate and of Triton X-100 was tested microdensitometrically and the activity of 'mitochondrial' ATPase was compared to the activity of enzymes of the oxidative metabolism succinic dehydrogenase and NAD-tetrazolium reductase. It is concluded that the ATPase activity displayed is not mainly mitochondrial. In autopsy material, it seems to be predominantly myofibrillar. Images Fig. 1 PMID:2933378

  19. a Nucleoside Under Observation in the Gas Phase: a Rotational Study of Uridine

    NASA Astrophysics Data System (ADS)

    Peña, Isabel; Alonso, José L.

    2014-06-01

    The nucleoside of uridine has been placed in the gas phase by laser ablation and the most stable C2{'}-anti conformation characterized by broadband chirped pulse (CP-FTMW) and narrowband molecular beam Fourier transform microwave (LA-MB-FTMW) spectroscopies. The quadrupole hyperfine structure, originated by two 14N nuclei, has been completely resolved. Intramolecular hydrogen bonds involving uracil and ribose moieties have been found to play an important role in the stabilization of the nucleoside.

  20. Enantioselective Intermolecular Cyclopropanations for the Synthesis of Chiral Pyrimidine Carbocyclic Nucleosides.

    PubMed

    Xie, Ming-Sheng; Zhou, Peng; Niu, Hong-Ying; Qu, Gui-Rong; Guo, Hai-Ming

    2016-09-01

    A direct route to chiral cyclopropylpyrimidine carbocyclic nucleoside analogues has been reported via highly enantioselective intermolecular cyclopropanation reactions of N1-vinylpyrimidines with α-diazoesters. With chiral ruthenium(II)-phenyloxazoline complex (2 mol %) as the catalyst, cyclopropyl pyrimidine nucleoside analogues could be obtained in good yields (71-96% yields) with high levels of diastereo- and enantioselectivities (10:1 to >20:1 dr and 96-99% ee) in 1 min. PMID:27526779

  1. Mild, efficient, selective and "green" benzoylation of nucleosides using benzoyl cyanide in ionic liquid.

    PubMed

    Prasad, Ashok K; Kumar, Vineet; Maity, Jyotirmoy; Sanghvi, Yogesh S; Ravikumar, Vasulinga T; Parmar, Virinder S

    2005-01-01

    Use of benzoyl cyanide (BzCN) for benzoylation of nucleosides has been studied, both in pyridine and in ionic liquid BzCN in 1-methoxyethyl-3-methylimidazolium methanesulfonate as ionic liquid has been found to be a "green "alternative compared to the pyridine-BzCN system. An efficient and selective benzoylation of nucleosides of both, the 2'-deoxy- and the ribo-series at ambient temperature was accomplished. PMID:16248029

  2. Permeation of aldopentoses and nucleosides through fatty acid and phospholipid membranes: implications to the origins of life.

    PubMed

    Wei, Chenyu; Pohorille, Andrew

    2013-02-01

    Permeation of aldopentoses and nucleosides through fatty acid and phospholipid membranes was investigated by way of molecular dynamics simulations. Calculated permeability coefficients of membranes to aldopentoses, which exist predominantly in the pyranose form, are in a very good agreement with experimental results. The unexpected preferential permeation of ribose, compared to its diastereomers, found by Sacerdote and Szostak, is explained in terms of inter- and intramolecular interactions involving hydroxyl groups. In aqueous solution, these groups favor the formation of intermolecular hydrogen bonds with neighboring water molecules. Inside the membrane, however, they form intramolecular hydrogen bonds, which in ribose are arranged in a chain. In its diastereomers this chain is broken, which yields higher free energy barrier to transfer through membranes. Faster permeation of ribose would lead to its preferential accumulation inside cells if sugars were converted sufficiently quickly to nonpermeable derivatives. An estimate for the rate of such reaction was derived. Preferential accumulation of ribose would increase the probability of correct monomers' incorporation during synthesis of nucleic acids inside protocells. The same mechanism does not apply to nucleosides or their activated derivatives because sugars are locked in the furanose form, which contains fewer exocyclic hydroxyl groups than does pyranose. The results of this study underscore concerted early evolution of membranes and the biochemical processes that they encapsulated. PMID:23397957

  3. Nucleoside uptake in macrophages from various murine strains: a short-time and a two-step stimulation model

    SciTech Connect

    Busolo, F.; Conventi, L.; Grigolon, M.; Palu, G. )

    1991-06-28

    Kinetics of (3H)-uridine uptake by murine peritoneal macrophages (pM phi) is early altered after exposure to a variety of stimuli. Alterations caused by Candida albicans, lipopolysaccharide (LPS) and recombinant interferon-gamma (rIFN-gamma) were similar in SAVO, C57BL/6, C3H/HeN and C3H/HeJ mice, and were not correlated with an activation process as shown by the amount of tumor necrosis factor-alpha (TNF-alpha) being released. Short-time exposure to all stimuli resulted in an increased nucleoside uptake by SAVO pM phi, suggesting that the tumoricidal function of this cell either depends from the type of stimulus or the time when the specific interaction with the cell receptor is taking place. Experiments with priming and triggering signals confirmed the above findings, indicating that the increase or the decrease of nucleoside uptake into the cell depends essentially on the chemical nature of the priming stimulus. The triggering stimulus, on the other hand, is only able to amplify the primary response.

  4. Enzymatic regioselective acylation of nucleosides in biomass-derived 2-methyltetrahydrofuran: kinetic study and enzyme substrate recognition.

    PubMed

    Gao, Wen-Li; Li, Ning; Zong, Min-Hua

    2013-03-10

    Enzymatic regioselective acylation of pyrimidine nucleosides was mediated by immobilized lipase from Penicillium expansum in 2-methyltetrahydrofuran (MeTHF), a bio-solvent derived from biomass. Despite of the moderate dissolution ability of MeTHF toward nucleosides, the initial enzymatic reaction rate was much higher in this eco-friendly solvent than in other commonly used organic solvents. This could be explained by the lower apparent activation energy of the enzymatic reaction (24.5 vs. 43.3-57.1kJ/mol) and the higher catalytic efficiency of the enzyme (Vmax/Km, 5.8 vs. 1.1-2.9h(-1)) in MeTHF. The enzymatic acylation of a group of ribonucleosides afforded the desirable 5'-esters with the conversions of 96-99% and 5'-regioselectivities of 96 to >99%. In enzymatic acylation of 2'-deoxynucleosides, however, 5'-regioselectivities showed a clear dependence on the 5-substituents present in the base moiety although the substrate conversions reached >98% within 1-3h. In the cases of 2',3'-dideoxynucleoside analogs, the reaction rate decreased markedly due to the lack of 3'-hydroxyl. PMID:23337886

  5. An adenosine kinase inhibitor, ABT-702, inhibits spinal nociceptive transmission by adenosine release via equilibrative nucleoside transporters in rat.

    PubMed

    Otsuguro, Ken-ichi; Tomonari, Yuki; Otsuka, Saori; Yamaguchi, Soichiro; Kon, Yasuhiro; Ito, Shigeo

    2015-10-01

    Adenosine kinase (AK) inhibitor is a potential candidate for controlling pain, but some AK inhibitors have problems of adverse effects such as motor impairment. ABT-702, a non-nucleoside AK inhibitor, shows analgesic effect in animal models of pain. Here, we investigated the effects of ABT-702 on synaptic transmission via nociceptive and motor reflex pathways in the isolated spinal cord of neonatal rats. The release of adenosine from the spinal cord was measured by HPLC. ABT-702 inhibited slow ventral root potentials (sVRPs) in the nociceptive pathway more potently than monosynaptic reflex potentials (MSRs) in the motor reflex pathway. The inhibitory effects of ABT-702 were mimicked by exogenously applied adenosine, blocked by 8CPT (8-cyclopentyl-1,3-dipropylxanthine), an adenosine A1 receptor antagonist, and augmented by EHNA (erythro-9-(2-hydroxy-3-nonyl) adenine), an adenosine deaminase (ADA) inhibitor. Equilibrative nucleoside transporter (ENT) inhibitors reversed the effects of ABT-702, but not those of adenosine. ABT-702 released adenosine from the spinal cord, an effect that was also reversed by ENT inhibitors. The ABT-702-facilitated release of adenosine by way of ENTs inhibits nociceptive pathways more potently than motor reflex pathways in the spinal cord via activation of A1 receptors. This feature is expected to lead to good analgesic effects, but, caution may be required for the use of AK inhibitors in the case of ADA dysfunction or a combination with ENT inhibitors. PMID:26066576

  6. The HCV non-nucleoside inhibitor Tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase function.

    PubMed

    Hebner, Christy M; Han, Bin; Brendza, Katherine M; Nash, Michelle; Sulfab, Maisoun; Tian, Yang; Hung, Magdeleine; Fung, Wanchi; Vivian, Randall W; Trenkle, James; Taylor, James; Bjornson, Kyla; Bondy, Steven; Liu, Xiaohong; Link, John; Neyts, Johan; Sakowicz, Roman; Zhong, Weidong; Tang, Hengli; Schmitz, Uli

    2012-01-01

    Tegobuvir (TGV) is a novel non-nucleoside inhibitor (NNI) of HCV RNA replication with demonstrated antiviral activity in patients with genotype 1 chronic HCV infection. The mechanism of action of TGV has not been clearly defined despite the identification of resistance mutations mapping to the NS5B polymerase region. TGV does not inhibit NS5B enzymatic activity in biochemical assays in vitro, suggesting a more complex antiviral mechanism with cellular components. Here, we demonstrate that TGV exerts anti-HCV activity utilizing a unique chemical activation and subsequent direct interaction with the NS5B protein. Treatment of HCV subgenomic replicon cells with TGV results in a modified form of NS5B with a distinctly altered mobility on a SDS-PAGE gel. Further analysis reveals that the aberrantly migrating NS5B species contains the inhibitor molecule. Formation of this complex does not require the presence of any other HCV proteins. The intensity of the aberrantly migrating NS5B species is strongly dependent on cellular glutathione levels as well as CYP 1A activity. Furthermore analysis of NS5B protein purified from a heterologous expression system treated with TGV by mass spectrometry suggests that TGV undergoes a CYP- mediated intracellular activation step and the resulting metabolite, after forming a glutathione conjugate, directly and specifically interacts with NS5B. Taken together, these data demonstrate that upon metabolic activation TGV is a specific, covalent inhibitor of the HCV NS5B polymerase and is mechanistically distinct from other classes of the non-nucleoside inhibitors (NNI) of the viral polymerase. PMID:22720059

  7. Post-Translational Phosphorylation of Serine 74 of Human Deoxycytidine Kinase Favors the Enzyme Adopting the Open Conformation Making It Competent for Nucleoside Binding and Release

    SciTech Connect

    Hazra, Saugata; Szewczak, Andrzej; Ort, Stephan; Konrad, Manfred; Lavie, Arnon

    2012-03-26

    Deoxycytidine kinase (dCK) uses either ATP or UTP as a phosphoryl donor to catalyze the phosphorylation of nucleoside acceptors. The kinetic properties of human dCK are modulated in vivo by phosphorylation of serine 74. This residue is a part of the insert region and is distant from the active site. Replacing the serine with a glutamic acid (S74E variant) can mimic phosphorylation of Ser74. To understand how phosphorylation affects the catalytic properties of dCK, we examined the S74E variant of dCK both structurally and kinetically. We observe that the presence of a glutamic acid at position 74 favors the adoption by the enzyme of the open conformation. Glu74 stabilizes the open conformation by directly interacting with the indole side chain of Trp58, a residue that is in the proximity of the base of the nucleoside substrate. The open dCK conformation is competent for the binding of nucleoside but not for phosphoryl transfer. In contrast, the closed conformation is competent for phosphoryl transfer but not for product release. Thus, dCK must make the transition between the open and closed states during the catalytic cycle. We propose a reaction scheme for dCK that incorporates the transition between the open and closed states, and this serves to rationalize the observed kinetic differences between wild-type dCK and the S74E variant.

  8. Synthesis and In Vitro Evaluation of 5-[18F]Fluoroalkyl Pyrimidine Nucleosides for Molecular Imaging of Herpes Simplex Virus Type-1 Thymidine Kinase Reporter Gene Expression

    PubMed Central

    Chacko, Ann-Marie; Qu, Wenchao; Kung, Hank F.

    2014-01-01

    Two novel series of 5-fluoroalkyl-2′-deoxyuridines (FPrDU, FBuDU, FPeDU) and 2′-fluoro-2′-deoxy-5-fluoroalkylarabinouridines (FFPrAU, FFBuAU, FFPeAU), having three, four or five methylene units (propyl, butyl, or pentyl) at C-5, were prepared and tested as reporter probes for imaging HSV1-tk gene expression. The Negishi coupling methodology was employed to efficiently synthesize the radiolabeling precursors. All six 5-[18F]fluoroalkyl pyrimidines were prepared readily from 3-N-benzoyl-3′,5′-di-O-benzoyl-protected 5-O-mesylate precursors in 17–35% radiochemical yield (decay-corrected). In vitro studies highlighted that all six [18F]labeled nucleosides selectively accumulated in cells expressing the HSV1-TK protein, with negligible uptake in control cells. [18F]FPrDU, [18F]FBuDU, [18F]FPeDU, and [18F]FFBuAU had the best uptake profiles. Despite selective accumulation in HSV1-tk expressing cells, all 5-fluoroalkyl pyrimidine nucleosides had low to negligible cytotoxic activity (CC50>1000–209 μM). Ultimately, results demonstrated that 5-[18F]fluoropropyl, [18F]fluorobutyl, and [18F]fluoropentyl pyrimidine nucleosides have potential as in vivo HSV1-TK PET reporter probes over a dynamic range of reporter gene expression levels. PMID:18800764

  9. Expression and purification of an engineered, yeast-expressed Leishmania donovani nucleoside hydrolase with immunogenic properties.

    PubMed

    Hudspeth, Elissa M; Wang, Qian; Seid, Christopher A; Hammond, Molly; Wei, Junfei; Liu, Zhuyun; Zhan, Bin; Pollet, Jeroen; Heffernan, Michael J; McAtee, C Patrick; Engler, David A; Matsunami, Risë K; Strych, Ulrich; Asojo, Oluwatoyin A; Hotez, Peter J; Bottazzi, Maria Elena

    2016-07-01

    Leishmania donovani is the major cause of visceral leishmaniasis (kala-azar), now recognized as the parasitic disease with the highest level of mortality second only to malaria. No human vaccine is currently available. A 36 kDa L. donovani nucleoside hydrolase (LdNH36) surface protein has been previously identified as a potential vaccine candidate antigen. Here we present data on the expression of LdNH36 in Pichia pastoris and its purification at the 20 L scale to establish suitability for future pilot scale manufacturing. To improve efficiency of process development and ensure reproducibility, 4 N-linked glycosylation sites shown to contribute to heterogeneous high-mannose glycosylation were mutated to glutamine residues. The mutant LdNH36 (LdNH36-dg2) was expressed and purified to homogeneity. Size exclusion chromatography and light scattering demonstrated that LdNH36-dg2 existed as a tetramer in solution, similar to the wild-type recombinant L. major nucleoside hydrolase. The amino acid mutations do not affect the tetrameric interface as confirmed by theoretical modeling, and the mutated amino acids are located outside the major immunogenic domain. Immunogenic properties of the LdNH36-dg2 recombinant protein were evaluated in BALB/c mice using formulations that included a synthetic CpG oligodeoxynucleotide, together with a microparticle delivery platform (poly(lactic-co-glycolic acid)). Mice exhibited high levels of IgG1, IgG2a, and IgG2b antibodies that were reactive to both LdNH36-dg2 and LdNH36 wild-type. While the point mutations did affect the hydrolase activity of the enzyme, the IgG antibodies elicited by LdNH36-dg2 were shown to inhibit the hydrolase activity of the wild-type LdNH36. The results indicate that LdNH36-dg2 as expressed in and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing in support of future first-in-humans phase 1 clinical trials. PMID:26839079

  10. Kinetics and mechanism of the acid-catalyzed hydrolysis of a hypermodified nucleoside wyosine and its 5'-monophosphate.

    PubMed Central

    Golankiewicz, B; Zielonacka-Lis, E; Folkman, W

    1985-01-01

    The rates of acid-catalyzed hydrolysis of a hypermodified nucleoside, wyosine and its 5'-monophosphate were determined at various pH, temperature and buffer concentrations. The results show that despite distinct differences in structure and the glycosyl bond stability, the hydrolysis of wyosine proceeds via cleavage of the C-N bond by A-1 mechanism, analogously to simple nucleosides. Unlike majority of other monophosphates studied so far, wyosine 5'-monophosphate is not more stable than respective nucleoside. PMID:4000960

  11. Purine nucleoside metabolism in the erythrocytes of patients with adenosine deaminase deficiency and severe combined immunodeficiency.

    PubMed Central

    Agarwal, R P; Crabtree, G W; Parks, R E; Nelson, J A; Keightley, R; Parkman, R; Rosen, F S; Stern, R C; Polmar, S H

    1976-01-01

    Deficiency of erythrocytic and lymphocytic adenosine deaminase (ADA) occurs in some patients with severe combined immunodeficiency disease (SCID). SCID with ADA deficiency is inherited as an autosomal recessive trait. ADA is markedly reduced or undetectable in affected patients (homozygotes), and approximately one-half normal levels are found in individuals heterozygous for ADA deficiency. The metabolism of purine nucleosides was studied in erythrocytes from normal individuals, four ADA-deficiency patients, and two heterozygous individuals. ADA deficiency in intake erythrocytes was confirmed by a very sensitive ammonia-liberation technique. Erythrocytic ADA activity in three heterozygous individuals (0.07,0.08, and 0.14 mumolar units/ml of packed cells) was between that of the four normal controls (0.20-0.37 mumol/ml) and the ADA-deficient patients (no activity). In vitro, adenosine was incorporated principally into IMP in the heterozygous and normal individuals but into the adenosine nucleotides in the ADa-deficient patients. Coformycin (3-beta-D-ribofuranosyl-6,7,8-trihydroimidazo[4,5-4] [1,3] diazepin-8 (R)-ol), a potent inhibitor of ADA, made possible incorporation of adenosine nucleotides in the ADA-deficient patients... PMID:947948

  12. Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of Mycobacterium tuberculosis.

    PubMed

    Georgescauld, Florian; Moynié, Lucile; Habersetzer, Johann; Cervoni, Laura; Mocan, Iulia; Borza, Tudor; Harris, Pernile; Dautant, Alain; Lascu, Ioan

    2013-01-01

    Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg(80)-Asp(93) as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved. PMID:23526954

  13. Intersubunit Ionic Interactions Stabilize the Nucleoside Diphosphate Kinase of Mycobacterium tuberculosis

    PubMed Central

    Georgescauld, Florian; Moynié, Lucile; Habersetzer, Johann; Cervoni, Laura; Mocan, Iulia; Borza, Tudor; Harris, Pernile; Dautant, Alain; Lascu, Ioan

    2013-01-01

    Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg80-Asp93 as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved. PMID:23526954

  14. Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies.

    PubMed

    de Moraes, Marcela Cristina; Cardoso, Carmen L; Cass, Quezia B

    2013-05-01

    The parasite Schistosoma mansoni (Sm) depends exclusively on the salvage pathway for its purine requirements. The enzyme purine nucleoside phosphorylase (PNP) is, therefore, a promising target for development of antischistosomal agents and an assay for screening of inhibitors. To enable this, immobilized SmPNP reactors were produced. By quantification of hypoxanthine by liquid chromatography, kinetic constants (K M) for the substrate inosine were determined for the free and immobilized enzyme as 110 ± 6.90 μmol L (-1) and 164 ± 13.4 μmol L (-1), respectively, indicating that immobilization did not affect enzyme activity. Furthermore, the enzyme retained 25 % of its activity after four months. Non-Michaelis kinetics for the phosphate substrate, and capacity for Pi-independent hydrolysis were also demonstrated, despite the low rate of enzymatic catalysis. Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated with a small library of 9-deazaguanine analogues. The method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives. PMID:23535739

  15. Increase of salt dependence of halophilic nucleoside diphosphate kinase caused by a single amino acid substitution.

    PubMed

    Ishibashi, Matsujiro; Hayashi, Tomoe; Yoshida, Chiho; Tokunaga, Masao

    2013-07-01

    Nucleoside diphosphate kinase (HsNDK) from an extremely halophilic archaea, Halobacterium salinarum, is composed of a homo hexamer, assembled as a trimer of basic dimeric units. It requires >2 M NaCl for refolding, although it does not require NaCl for stability or enzymatic activity below 30 °C. A HisN111L mutant with an N-terminal extension sequence containing hexa-His tag, in which Asn111 was replaced with Leu, was designed to be less stable between basic dimeric units. This mutant can lose between 6 and 12 hydrogen bonds between basic dimeric units in the hexamer structure. The HisN111L mutant had enhanced salt requirements for enzymatic activity and refolding even though the secondary structure of the HisN111L mutant was confirmed to be similar to the control, HisNDK, in low and high salt solutions using circular dichroism. We reported previously that G114R and D148C mutants, which had enhanced interactions between basic dimeric units, showed facilitated refolding and stabilization in low salt solution. The results of this study help to elucidate the process for engineering industrial enzymes by controlling subunit-subunit interactions through mutations. PMID:23609188

  16. Searching for novel scaffold of triazole non-nucleoside inhibitors of HIV-1 reverse transcriptase.

    PubMed

    Frączek, Tomasz; Paneth, Agata; Kamiński, Rafał; Krakowiak, Agnieszka; Paneth, Piotr

    2016-06-01

    Azoles are a promising class of the new generation of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). From thousands of reported compounds, many possess the same basic structure of an aryl substituted azole ring linked by a thioglycolamide chain with another aromatic ring. In order to find novel extensions for this basic scaffold, we explored the 5-position substitution pattern of triazole NNRTIs using molecular docking followed by the synthesis of selected compounds. We found that heterocyclic substituents in the 5-position of the triazole ring are detrimental to the inhibitory activity of compounds with four-membered thioglycolamide linker and this substitution seems to be viable only for compounds with shorter two-membered linker. Promising compound, N-(4-carboxy-2-chlorophenyl)-2-((4-benzyl-5-methyl-4H-1,2,4-triazol-3-yl)sulfanyl)acetamide, with potent inhibitory activity and acceptable aqueous solubility has been identified in this study that could serve as lead scaffold for the development of novel water-soluble salts of triazole NNRTIs. PMID:25942362

  17. Rigid Adenine Nucleoside Derivatives as Novel Modulators of the Human Sodium Symporters for Dopamine and Norepinephrine.

    PubMed

    Janowsky, Aaron; Tosh, Dilip K; Eshleman, Amy J; Jacobson, Kenneth A

    2016-04-01

    Thirty-two congeneric rigid adenine nucleoside derivatives containing a North (N)-methanocarba ribose substitution and a 2-arylethynyl group either enhanced (up to 760% of control) or inhibited [(125)I] methyl (1R,2S,3S)-3-(4-iodophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate (RTI-55) binding at the human dopamine (DA) transporter (DAT) and inhibited DA uptake. Several nucleosides also enhanced [(3)H]mazindol [(±)-5-(4-chlorophenyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol] binding to the DAT. The combination of binding enhancement and functional inhibition suggests possible allosteric interaction with the tropanes. The structure-activity relationship of this novel class of DAT ligands was explored: small N(6)-substition (methyl or ethyl) was favored, while the N1 of the adenine ring was essential. Effective terminal aryl groups include thien-2-yl (compounds 9 and 16), with EC50 values of 35.1 and 9.1 nM, respectively, in [(125)I]RTI-55 binding enhancement, and 3,4-difluorophenyl as in the most potent DA uptake inhibitor (compound 6) with an IC50 value of 92 nM (3-fold more potent than cocaine), but not nitrogen heterocycles. Several compounds inhibited or enhanced binding at the norepinephrine transporter (NET) and serotonin transporter (SERT) and inhibited function in the micromolar range; truncation at the 4'-position in compound 23 allowed for weak inhibition of the SERT. We have not yet eliminated adenosine receptor affinity from this class of DAT modulators, but we identified modifications that remove DAT inhibition as an off-target effect of potent adenosine receptor agonists. Thus, we have identified a new class of allosteric DAT ligands, rigidified adenosine derivatives, and explored their initial structural requirements. They display a very atypical pharmacological profile, i.e., either enhancement by increasing affinity or inhibition of radioligand binding at the DAT, and in some cases the NET and SERT, and inhibition of neurotransmitter

  18. Inhibition of Mycoplasma pneumoniae growth by FDA-approved anticancer and antiviral nucleoside and nucleobase analogs

    PubMed Central

    2013-01-01

    Background Mycoplasma pneumoniae (Mpn) is a human pathogen that causes acute and chronic respiratory diseases and has been linked to many extrapulmonary diseases. Due to the lack of cell wall, Mpn is resistant to antibiotics targeting cell wall synthesis such as penicillin. During the last 10 years macrolide-resistant Mpn strains have been frequently reported in Asian countries and have been spreading to Europe and the United States. Therefore, new antibiotics are needed. In this study, 30 FDA-approved anticancer or antiviral drugs were screened for inhibitory effects on Mpn growth and selected analogs were further characterized by inhibition of target enzymes and metabolism of radiolabeled substrates. Results Sixteen drugs showed varying inhibitory effects and seven showed strong inhibition of Mpn growth. The anticancer drug 6-thioguanine had a MIC (minimum inhibitory concentration required to cause 90% of growth inhibition) value of 0.20 μg ml-1, whereas trifluorothymidine, gemcitabine and dipyridamole had MIC values of approximately 2 μg ml-1. In wild type Mpn culture the presence of 6-thioguanine and dipyridamole strongly inhibited the uptake and metabolism of hypoxanthine and guanine while gemcitabine inhibited the uptake and metabolism of all nucleobases and thymidine. Trifluorothymidine and 5-fluorodeoxyuridine, however, stimulated the uptake and incorporation of radiolabeled thymidine and this stimulation was due to induction of thymidine kinase activity. Furthermore, Mpn hypoxanthine guanine phosphoribosyl transferase (HPRT) was cloned, expressed, and characterized. The 6-thioguanine, but not other purine analogs, strongly inhibited HPRT, which may in part explain the observed growth inhibition. Trifluorothymidine and 5-fluorodeoxyuridine were shown to be good substrates and inhibitors for thymidine kinase from human and Mycoplasma sources. Conclusion We have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent

  19. Rigid Adenine Nucleoside Derivatives as Novel Modulators of the Human Sodium Symporters for Dopamine and Norepinephrine

    PubMed Central

    Tosh, Dilip K.; Eshleman, Amy J.; Jacobson, Kenneth A.

    2016-01-01

    Thirty-two congeneric rigid adenine nucleoside derivatives containing a North (N)-methanocarba ribose substitution and a 2-arylethynyl group either enhanced (up to 760% of control) or inhibited [125I] methyl (1R,2S,3S)-3-(4-iodophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate (RTI-55) binding at the human dopamine (DA) transporter (DAT) and inhibited DA uptake. Several nucleosides also enhanced [3H]mazindol [(±)-5-(4-chlorophenyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol] binding to the DAT. The combination of binding enhancement and functional inhibition suggests possible allosteric interaction with the tropanes. The structure-activity relationship of this novel class of DAT ligands was explored: small N6-substition (methyl or ethyl) was favored, while the N1 of the adenine ring was essential. Effective terminal aryl groups include thien-2-yl (compounds 9 and 16), with EC50 values of 35.1 and 9.1 nM, respectively, in [125I]RTI-55 binding enhancement, and 3,4-difluorophenyl as in the most potent DA uptake inhibitor (compound 6) with an IC50 value of 92 nM (3-fold more potent than cocaine), but not nitrogen heterocycles. Several compounds inhibited or enhanced binding at the norepinephrine transporter (NET) and serotonin transporter (SERT) and inhibited function in the micromolar range; truncation at the 4′-position in compound 23 allowed for weak inhibition of the SERT. We have not yet eliminated adenosine receptor affinity from this class of DAT modulators, but we identified modifications that remove DAT inhibition as an off-target effect of potent adenosine receptor agonists. Thus, we have identified a new class of allosteric DAT ligands, rigidified adenosine derivatives, and explored their initial structural requirements. They display a very atypical pharmacological profile, i.e., either enhancement by increasing affinity or inhibition of radioligand binding at the DAT, and in some cases the NET and SERT, and inhibition of neurotransmitter uptake

  20. Identification of an alternative nucleoside triphosphate: 5'-deoxyadenosylcobinamide phosphate nucleotidyltransferase in Methanobacterium thermoautotrophicum delta H.

    PubMed

    Thomas, M G; Escalante-Semerena, J C

    2000-08-01

    Computer analysis of the archaeal genome databases failed to identify orthologues of all of the bacterial cobamide biosynthetic enzymes. Of particular interest was the lack of an orthologue of the bifunctional nucleoside triphosphate (NTP):5'-deoxyadenosylcobinamide kinase/GTP:adenosylcobinamide-phosphate guanylyltransferase enzyme (CobU in Salmonella enterica). This paper reports the identification of an archaeal gene encoding a new nucleotidyltransferase, which is proposed to be the nonorthologous replacement of the S. enterica cobU gene. The gene encoding this nucleotidyltransferase was identified using comparative genome analysis of the sequenced archaeal genomes. Orthologues of the gene encoding this activity are limited at present to members of the domain Archaea. The corresponding ORF open reading frame from Methanobacterium thermoautotrophicum Delta H (MTH1152; referred to as cobY) was amplified and cloned, and the CobY protein was expressed and purified from Escherichia coli as a hexahistidine-tagged fusion protein. This enzyme had GTP:adenosylcobinamide-phosphate guanylyltransferase activity but did not have the NTP:AdoCbi kinase activity associated with the CobU enzyme of S. enterica. NTP:adenosylcobinamide kinase activity was not detected in M. thermoautotrophicum Delta H cell extract, suggesting that this organism may not have this activity. The cobY gene complemented a cobU mutant of S. enterica grown under anaerobic conditions where growth of the cell depended on de novo adenosylcobalamin biosynthesis. cobY, however, failed to restore adenosylcobalamin biosynthesis in cobU mutants grown under aerobic conditions where de novo synthesis of this coenzyme was blocked, and growth of the cell depended on the assimilation of exogenous cobinamide. These data strongly support the proposal that the relevant cobinamide intermediates during de novo adenosylcobalamin biosynthesis are adenosylcobinamide-phosphate and adenosylcobinamide-GDP, not adenosylcobinamide

  1. A Computational Study of Expanded Heterocyclic Nucleosides in DNA

    PubMed Central

    O'Daniel, Peter I.; Jefferson, Malcolm; Wiest, Olaf; Seley-Radtke, Katherine L.

    2008-01-01

    The first molecular dynamics study of a series of heterospacer-expanded tricyclic bases in DNA using modified force field parameters in AMBER is detailed. The expanded purine nucleoside monomers have been designed to probe the effects of a heteroaromatic spacer ring on the structure, function, and dynamics of the DNA helix. The heterobase scaffold has been expanded with a furan, pyrrole, or thiophene spacer ring. This structural modification increases the polarizability of the bases and provides an additional hydrogen bond donor with the amine hydrogen of the pyrrole ring or hydrogen bond acceptor with the furan or thiophene ring free electron pairs. The polarizability of the expanded bases were determined by AM1 calculations and the results of the MD simulations of 20-mers predict that the modified curvature of the expanded base leads to a much larger major groove, while the effect on the minor groove is negligible. Overall, the structure resembles A-DNA. MD simulations of 10-mers suggest that the balance between base pairing vs. base stacking and intercalation can be shifted towards the latter due to the increased surface area and polariz-ability of the expanded bases. PMID:18808194

  2. Interaction of purine bases and nucleosides with serum albumin

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Michnik, A.

    1997-06-01

    The proton NMR spectra of alkyl derivatives of adenine and adenosine have been studied. High-resolution (400 MHz) proton spectra were recorded at 300 K at increasing concentrations of serum albumin. The dependence of the chemical shifts and the line width of the individual spectral lines on the protein concentration provides some detailed information about the nature of the complexes between the purine derivatives and albumin. Comparison of data for the methylated and non-methylated purine bases and nucleosides indicates the formation of non-specific complexes with serum albumin. However, the presence of the ethyl group in 8-ethyl-9 N-methyladenine means that in the adenine derivative-serum albumin complex the ethyl chain preserves its dominant role in binding. An advantage of our model is that the π-π interaction between the adenine ring and the amino acids of the protein can be replaced by hydrophobic interaction in the case of complexation of the ethyl adenine derivative.

  3. DNA nucleoside composition and methylation in several species of microalgae

    SciTech Connect

    Jarvis, E.E.; Dunahay, T.G.; Brown, L.M. )

    1992-06-01

    Total DNA was isolated from 10 species of microalgae, including representatives of the Chlorophyceae (Chlorella ellipsoidea, Chlamydomonas reinhardtii, and Monoraphidium minutum), Bacillariophyceae (Cyclotella cryptica, Navicula saprophila, Nitzschia pusilla, and Phaeodactylum tricornutum), Charophyceae (Stichococcus sp.), Dinophyceae (Crypthecodinium cohnii), and Prasinophyceae (Tetraselmis suecica). Control samples of Escherichia coli and calf thymus DNA were also analyzed. The nucleoside base composition of each DNA sample was determined by reversed-phase high performance liquid chromatography. All samples contained 5-methyldeoxycytidine, although at widely varying levels. In M. minutum, about one-third of the cytidine residues were methylated. Restriction analysis supported this high degree of methylation in M. minutum and suggested that methylation is biased toward 5[prime]-CG dinucleotides. The guanosine + cytosine (GC) contents of the green algae were, with the exception of Stichococcus sp., consistently higher than those of the diatoms. Monoraphidium minutum exhibited an extremely high GC content of 71%. Such a value is rare among eukaryotic organisms and might indicate an unusual codon usage. This work is important for developing strategies for transformation and gene cloning in these algae. 46 refs., 1 fig., 2 tabs.

  4. Fluorescent pyrimidopyrimidoindole nucleosides: control of photophysical characterizations by substituent effects.

    PubMed

    Mizuta, Masahiro; Seio, Kohji; Miyata, Kenichi; Sekine, Mitsuo

    2007-07-01

    10-(2-Deoxy-beta-D-ribofuranosyl)pyrimido[4',5':4,5]pyrimido[1,6-a]indole-6,9(7H)-dione (dCPPI) and its derivatives were synthesized via the Suzuki-Miyaura coupling reaction of 5-iododeoxycytidine with 5-substituted N-Boc-indole-2-borates and characterized by UV-vis and fluorescence spectroscopy. The new fluorescent nucleosides showed rather large Stokes shifts (116-139 nm) in an aqueous buffer. The fluorescent intensities were dependent on the nature of the substituents on the indole rings. The electron-withdrawing groups increased the fluorescent intensity while the electron-donating groups having lone pairs decreased it. Among the substituted dCPPI derivatives tested, the trimethylammonium derivative of dCPPI was found to emit the brightest fluorescent light. The solvatochromism of dCPPI and its derivatives was also studied. Some of the dCPPI derivatives showed interesting solvent-dependent fluorescence enhancement and could be useful as new fluorescent structural probes for nucleic acids. The Lippert-Mataga analyses of the Stokes shift were also carried out to obtain estimated values of the dipole moment of the excited states of some of the derivatives. PMID:17555352

  5. Allelic isoforms of the H+/nucleoside co-transporter (CaCNT) from Candida albicans reveal separate high- and low-affinity transport systems for nucleosides.

    PubMed

    Slugoski, Melissa D; Loewen, Shaun K; Ng, Amy M L; Baldwin, Stephen A; Cass, Carol E; Young, James D

    2004-11-01

    Contigs 19-10196 and 19-20196 of the Stanford Candida albicans genome sequence databank encode two putative allelic isoforms of C. albicans CaCNT, a recently characterized 608 amino acid residue H+-coupled fungal member of the CNT family of concentrative nucleoside transport proteins. The single Ser/Gly difference between CaCNT/19-20196 and CaCNT occurs at position 328 in putative TM 7, and corresponds to a Ser/Gly substitution previously shown to contribute to the contrasting pyrimidine and purine nucleoside selectivities of human (h) and rat (r) Na+-dependent CNT1 and CNT2. CaCNT/19-10196 differs from CaCNT by four amino acids, but has Gly at position 328. These new proteins were recreated by site-directed mutagenesis of CaCNT and characterized functionally by heterologous expression in Xenopus laevis oocytes. In marked contrast to h/rCNT1/2, both CaCNT/19-10196 and CaCNT/19-20196 exhibited permeant selectivities for purine nucleosides (adenosine, guanosine and inosine) and uridine similar to that of CaCNT. However, although H+-coupled, CaCNT/19-20196 exhibited a approximately 10-fold higher apparent Km for uridine than either CaCNT or CaCNT/19-10196. CaCNT/19-20196 also exhibited a low apparent affinity for inosine. We conclude that the three proteins correspond to high-affinity (CaCNT, CaCNT/19-10196) and low-affinity (CaCNT/19-20196) allelic isoforms of the C. albicans CNT nucleoside transporter. This is the first example of a single amino acid residue substitution altering a CNT protein's overall apparent affinity for nucleosides. PMID:15543539

  6. Mass Spectrometric Characterization of HIV-1 Reverse Transcriptase Interactions with Non-nucleoside Reverse Transcriptase Inhibitors.

    PubMed

    Thammaporn, Ratsupa; Ishii, Kentaro; Yagi-Utsumi, Maho; Uchiyama, Susumu; Hannongbua, Supa; Kato, Koichi

    2016-01-01

    Non-nucleoside reverse transcriptase inhibitors (NNRTIs) of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) have been developed for the treatment of acquired immunodeficiency syndrome. HIV-1 RT binding to NNRTIs has been characterized by various biophysical techniques. However, these techniques are often hampered by the low water solubility of the inhibitors, such as the current promising diarylpyrimidine-based inhibitors rilpivirine and etravirine. Hence, a conventional and rapid method that requires small sample amounts is desirable for studying NNRTIs with low water solubility. Here we successfully applied a recently developed mass spectrometric technique under non-denaturing conditions to characterize the interactions between the heterodimeric HIV-1 RT enzyme and NNRTIs with different inhibitory activities. Our data demonstrate that mass spectrometry serves as a semi-quantitative indicator of NNRTI binding affinity for HIV-1 RT using low and small amounts of samples, offering a new high-throughput screening tool for identifying novel RT inhibitors as anti-HIV drugs. PMID:26934936

  7. Transgene Expression of Drosophila melanogaster Nucleoside Kinase Reverses Mitochondrial Thymidine Kinase 2 Deficiency*♦

    PubMed Central

    Krishnan, Shuba; Zhou, Xiaoshan; Paredes, João A.; Kuiper, Raoul V.; Curbo, Sophie; Karlsson, Anna

    2013-01-01

    A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK+/− transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK+/−TK2−/− mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK+/−TK2−/− mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency. PMID:23288848

  8. Synthesis of nucleoside 3'-(S-alkyl phosphorothioates) and their use as substrates for nucleases.

    PubMed

    Saba, D; Dekker, C A

    1981-09-15

    The synthesis of cytidine, uridine, guanosine, and adenosine 3'-(S-methyl phosphorothioates) by treatment of the 2',5'-di-O-(4-methoxytetrahydropyran-4-yl)ribonucleosides with 2-(methylthio 4H-1,3,2-benzodioxaphosphorin 2-oxide is described. These nucleotide analogues are stable compounds both in the solid state and the neutral aqueous solution. All four of these compounds are degraded by RNase T2 to the parent nucleotides and methanethiol. In addition, cytidine and uridine 3'-(S-methyl phosphorothioates) are substrates for bovine pancreatic ribonuclease and guanosine 3'-(S-methyl phosphorothioate) is a substrate for RNase T1 and RNase U1. When used in conjunction with a chromophore-producing reagent, nucleoside 3'-(S-methyl phosphorothioates) provide a means for direct kinetic measurement of ribonuclease activity over a wide pH range (pH 2-9). The reactivities of these substrates with ribonucleases are compared to the reactivities of other synthetic substrates as well as a number of natural substrates. The utility of ribonucleoside 3'-(S-methyl phosphorothioates) as substrates for the assay of ribonucleases is discussed. PMID:6271188

  9. Nucleoside diphosphate regulation of overall rates of protein biosynthesis acting at the level of initiation.

    PubMed

    Hucul, J A; Henshaw, E C; Young, D A

    1985-12-15

    A sensitive assay method developed to examine the effects of subtle, physiologically relevant, changes in the levels of adenine and guanine mono-, di-, and triphosphorylated nucleotides specifically on the initiation of protein synthesis is described. Initiation rates are quantified by measuring the amount of protein synthesis resulting from the run-off of ribosomes which have initiated during defined intervals in a modified in vitro protein-synthesizing system developed from Ehrlich ascites tumor cell lysates (Henshaw, E.C., and Panniers, R. (1983) Methods Enzymol. 101, 616-629). The modifications include the attenuation of the ATP-regenerating system so that the relative nucleotide levels more nearly reflect actual intracellular conditions. With this system the rate of initiation is highly sensitive to changes in the ADP:ATP and GDP:GTP ratios, but indifferent to the absolute levels of either diphosphate. While the tight coupling of these two ratios by endogenous nucleoside diphosphate kinase activity prevents the independent manipulation of either ratio, the data do eliminate both AMP and GMP per se as inhibitory species. The close agreement of our data calculated in terms of energy charge to previously published results on overall rates of protein synthesis in rat thymocytes (Mendelsohn, S.K., Nordeen, S.K., and Young, D.A. (1977) Biochem. Biophys. Res. Commun. 79, 53-60) continues to suggest a physiologically relevant regulatory influence of subtle changes in nucleotides acting at the level of the initiation reaction. PMID:2999123

  10. Highly ordered graphene-isolated silver nanodot arrays as SERS substrate for detection of urinary nucleosides

    NASA Astrophysics Data System (ADS)

    Xu, Shicai; Jiang, Shouzhen; Hu, Guodong; Wei, Jie; Wang, Li; Zhang, Junye; Li, Qiuju

    2015-11-01

    An efficient surface enhanced Raman scattering (SERS) substrate has been developed based on highly ordered arrays of graphene-isolated Ag nanodot (G/AgND) arrays. By combining the plasmonic activity of AgND arrays and unique physical/chemical properties of graphene, the G/AgND arrays show high performance in terms of sensitivity, signal-to-noise ratio and reproducibility. The SERS signals of R6G are clearly detected even at very low concentration of 10-11M. The maximum deviations of SERS intensities from 20 positions of the SERS substrate are less than 5%. The G/AgND arrays were employed for detection of human urinary nucleosides. The diagnostic algorithms based on principal component analysis achieved a diagnostic sensitivity of 91.1% and specificity of 93.3% for separating cancer from normal samples. This work demonstrates that G/AgND arrays used in Raman spectroscopy could be developed as a smart and promising system for detection and screening of cancers.

  11. Mechanistic Insights into the Rate-Limiting Step in Purine-Specific Nucleoside Hydrolase.

    PubMed

    Chen, Nanhao; Zhao, Yuan; Lu, Jianing; Wu, Ruibo; Cao, Zexing

    2015-07-14

    A full enzymatic catalysis cycle in the inosine-adenosine-guanosine specific nucleoside hydrolase (IAG-NH) was assumed to be comprised of four steps: substrate binding, chemical reaction, base release, and ribose release. Nevertheless, the mechanistic details for the rate-limiting step of the entire enzymatic reaction are still unknown, even though the ribose release was likely to be the most difficult stage. Based on state-of-the-art quantum mechanics and molecular mechanics (QM/MM) molecular dynamics (MD) simulations, the ribose release process can be divided into two steps: "ribose dissociation" and "ribose release". The "ribose dissociation" includes "cleavage" and "exchange" stages, in which a metastable 6-fold intermediate will recover to an 8-fold coordination shell of Ca(2+) as observed in apo- IAG-NH. Extensive random acceleration molecular dynamics and MD simulations have been employed to verify plausible release channels, and the estimated barrier for the rate-determining step of the entire reaction is 13.0 kcal/mol, which is comparable to the experimental value of 16.7 kcal/mol. Moreover, the gating mechanism arising from loop1 and loop2, as well as key residues around the active pocket, has been found to play an important role in manipulating the ribose release. PMID:26575755

  12. Sinefungin, a Natural Nucleoside Analogue of S-Adenosylmethionine, Inhibits Streptococcus pneumoniae Biofilm Growth

    PubMed Central

    Park, Seok-Won; Chae, Sung-Won

    2014-01-01

    Pneumococcal colonization and disease is often associated with biofilm formation, in which the bacteria exhibit elevated resistance both to antibiotics and to host defense systems, often resulting in infections that are persistent and difficult to treat. We evaluated the effect of sinefungin, a nucleoside analogue of S-adenosylmethionine, on pneumococcal in vitro biofilm formation and in vivo colonization. Sinefungin is bacteriostatic to pneumococci and significantly decreased biofilm growth and inhibited proliferation and structure of actively growing biofilms but did not alter growth or the matrix structure of established biofilms. Sinefungin significantly reduced pneumococcal colonization in rat middle ear. The quorum sensing molecule (autoinducer-2) production was significantly reduced by 92% in sinefungin treated samples. The luxS, pfs, and speE genes were downregulated in biofilms grown in the presence of sinefungin. This study shows that sinefungin inhibits pneumococcal biofilm growth in vitro and colonization in vivo, decreases AI-2 production, and downregulates luxS, pfs, and speE gene expressions. Therefore, the S-adenosylmethionine (SAM) inhibitors could be used as lead compounds for the development of novel antibiofilm agents against pneumococci. PMID:25050323

  13. The Dipeptide Monoester Prodrugs of Floxuridine and Gemcitabine—Feasibility of Orally Administrable Nucleoside Analogs

    PubMed Central

    Tsume, Yasuhiro; Bermejo, Blanca Borras; Amidon, Gordon L.

    2014-01-01

    Dipeptide monoester prodrugs of floxuridine and gemcitabine were synthesized. Their chemical stability in buffers, enzymatic stability in cell homogenates, permeability in mouse intestinal membrane along with drug concentration in mouse plasma, and anti-proliferative activity in cancer cells were determined and compared to their parent drugs. Floxuridine prodrug was more enzymatically stable than floxuridine and the degradation from prodrug to parent drug works as the rate-limiting step. On the other hand, gemcitabine prodrug was less enzymatically stable than gemcitabine. Those dipeptide monoester prodrugs exhibited 2.4- to 48.7-fold higher uptake than their parent drugs in Caco-2, Panc-1, and AsPC-1 cells. Floxuridine and gemcitabine prodrugs showed superior permeability in mouse jejunum to their parent drugs and exhibited the higher drug concentration in plasma after in situ mouse perfusion. Cell proliferation assays in ductal pancreatic cancer cells, AsPC-1 and Panc-1, indicated that dipeptide prodrugs of floxuridine and gemcitabine were more potent than their parent drugs. The enhanced potency of nucleoside analogs was attributed to their improved membrane permeability. The prodrug forms of 5′-l-phenylalanyl-l-tyrosyl-floxuridine and 5′-l-phenylalanyl-l-tyrosyl-gemcitabine appeared in mouse plasma after the permeation of intestinal membrane and the first-pass effect, suggesting their potential for the development of oral dosage form for anti-cancer agents. PMID:24473270

  14. Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

    SciTech Connect

    Mastrangelo, Eloise; Bollati, Michela; Milani, Mario; Lamballeire, Xavier de; Brisbare, Nadege; Dalle, Karen; Lantez, Violaine; Egloff, Marie-Pierre; Coutard, Bruno; Canard, Bruno; Gould, Ernest; Forrester, Naomi; Bolognesi, Martino

    2006-08-01

    Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed, purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup.

  15. Capillary bioreactors based on human purine nucleoside phosphorylase: a new approach for ligands identification and characterization.

    PubMed

    de Moraes, Marcela Cristina; Ducati, Rodrigo Gay; Donato, Augusto José; Basso, Luiz Augusto; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra

    2012-04-01

    The enzyme purine nucleoside phosphorylase (PNP) is a target for the discovery of new lead compounds employed on the treatment severe T-cell mediated disorders. Within this context, the development of new, direct, and reliable methods for ligands screening is an important task. This paper describes the preparation of fused silica capillaries human PNP (HsPNP) immobilized enzyme reactor (IMER). The activity of the obtained IMER is monitored on line in a multidimensional liquid chromatography system, by the quantification of the product formed throughout the enzymatic reaction. The K(M) value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255 ± 29.2 μM and 133 ± 14.9 μM, respectively). A new fourth-generation immucillin derivative (DI4G; IC(50)=40.6 ± 0.36 nM), previously identified and characterized in HsPNP free enzyme assays, was used to validate the IMER as a screening method for HsPNP ligands. The validated method was also used for mechanistic studies with this inhibitor. This new approach is a valuable tool to PNP ligand screening, since it directly measures the hypoxanthine released by inosine phosphorolysis, thus furnishing more reliable results than those one used in a coupled enzymatic spectrophotometric assay. PMID:22099222

  16. SwoHp, a Nucleoside Diphosphate Kinase, Is Essential in Aspergillus nidulans

    PubMed Central

    Lin, Xiaorong; Momany, Cory; Momany, Michelle

    2003-01-01

    The temperature-sensitive swoH1 mutant of Aspergillus nidulans was previously identified in a screen for mutants with defects in polar growth. In the present work, we found that the swoH1 mutant swelled, lysed, and did not produce conidia during extended incubation at the restrictive temperature. When shifted from the permissive to the restrictive temperature, swoH1 showed the temperature-sensitive swelling phenotype only after 8 h at the higher temperature. The swoH gene was mapped to chromosome II and cloned by complementation of the temperature-sensitive phenotype. The sequence showed that swoH encodes a homologue of nucleoside diphosphate kinases (NDKs) from other organisms. Deletion experiments showed that the swoH gene is essential. A hemagglutinin-SwoHp fusion complemented the mutant phenotype, and the purified fusion protein possessed phosphate transferase activity in thin-layer chromatography assays. Sequencing of the mutant allele showed a predicted V83F change. Structural modeling suggested that the swoH1 mutation would lead to perturbation of the NDK active site. Crude cell extracts from the swoH1 mutant grown at the permissive temperature had ∼20% of the NDK activity seen in the wild type and did not show any decrease in activity when assayed at higher temperatures. Though the data are not conclusive, the lack of temperature-sensitive NDK activity in the swoH1 mutant raises the intriguing possibility that the SwoH NDK is required for growth at elevated temperatures rather than for polarity maintenance. PMID:14665452

  17. Binding Strength of Nucleobases and Nucleosides on Silver Nanoparticles Probed by a Colorimetric Method.

    PubMed

    Yu, Lu; Li, Na

    2016-06-01

    Because of their unique and tunable properties, oligonucleotide-functionalized noble metal nanoparticles have provided a versatile platform for various engineering and biomedical applications. The vast majority of such applications were demonstrated with gold nanoparticles (AuNPs) while only a few were demonstrated with sliver nanoparticles (AgNPs). This is largely due to the lack of robust protocols to functionalize AgNPs with thiol-modified oligonucleotides. Previous studies have revealed strong interactions between nucleobases and AgNPs. This could enable an alternative way to functionalize AgNPs with non-thiolated oligonucleotides. However, there is no quantitative study on the interaction strengths between AgNPs and oligonucleotides. Several methods have been used for quantitative evaluation of the interaction strengths between AuNPs and oligonucleotides. These methods often require specialized equipment that might not be widely accessible or rely on labor-intensive procedures to obtain the adsorption isotherms. Herein, we developed a colorimetric method, as a simple and high-throughput alternative of existing methods, to quantify the binding strength between AgNPs and nucleobases/nucleosides. In this colorimetric method, concentration-dependent destabilizing effects of nucleobase/nucleoside adsorption on AgNPs are utilized to indirectly quantify the amount of nucleobases/nucleosides adsorbed on AgNPs, thus deriving the binding strength between AgNPs and nucleobases/nucleosides. First, the concentration-dependent AgNP aggregation kinetics in the presence of nucleobases/nucleosides were systematically investigated. Then, this colorimetric method was used to determine the binding strengths between AgNPs and various DNA/RNA nucleobases/nucleosides. It was found that the ranking of interaction strengths between AgNPs and DNA/RNA nucleosides (dC < dT < dA, rC < rU < rA) is generally agreed with that between AgNPs and corresponding nucleobases (C < T < U < A). This

  18. Saturable Hepatic Extraction of Gemcitabine Involves Biphasic Uptake Mediated by Nucleoside Transporters Equilibrative Nucleoside Transporter 1 and 2.

    PubMed

    Shimada, Takuya; Nakanishi, Takeo; Tajima, Hidehiro; Yamazaki, Maiko; Yokono, Rina; Takabayashi, Makiko; Shimada, Tsutomu; Sawamoto, Kazuki; Miyamoto, Ken-Ichi; Kitagawa, Hirohisa; Ohta, Tetsuo; Tamai, Ikumi; Sai, Yoshimichi

    2015-09-01

    Hepatic arterial infusion (HAI) chemotherapy with gemcitabine (GEM) is expected to be more effective and safer method to treat hepatic metastasis of pancreatic cancer compared with intravenous administration, because it affords higher tumor exposure with lower systemic exposure. Thus, a key issue for dose selection is the saturability of hepatic uptake of GEM. Therefore, we investigated GEM uptake in rat and human isolated hepatocytes. Hepatic GEM uptake involved high- and low-affinity saturable components with Km values of micromolar and millimolar order, respectively. The uptake was inhibited concentration dependently by S-(4-nitrobenzyl)-6-thioinosine (NBMPR) and was sodium-ion-independent, suggesting a contribution of equilibrative nucleoside transporters (ENTs). The concentration dependence of uptake in the presence of 0.1 μM NBMPR showed a single low-affinity binding site. Therefore, the high- and low-affinity sites correspond to ENT1 and ENT2, respectively. Our results indicate hepatic extraction of GEM is predominantly mediated by the low-affinity site (hENT2), and at clinically relevant hepatic concentrations of GEM, hENT2-mediated uptake would not be completely saturated. This is critical for HAI, because saturation of hepatic uptake would result in a marked increase of GEM concentration in the peripheral circulation, abrogating the advantage of HAI over intravenous administration in terms of severe adverse events. PMID:26037416

  19. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers

    DOE PAGESBeta

    Wang, Jun; Bonnesen, Peter V; Rangel, E.; Vallejo, E.; Sanchez-Castillo, Ariadna; Cleaves, II, H. James; Baddorf, Arthur P; Sumpter, Bobby G; Pan, Minghu; Maksymovych, Petro; et al

    2016-01-04

    The self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N9-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two ormore » more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. The resulting characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Moreover, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers.« less

  20. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers

    NASA Astrophysics Data System (ADS)

    Wang, Jun; Bonnesen, Peter V.; Rangel, E.; Vallejo, E.; Sanchez-Castillo, Ariadna; James Cleaves, H., II; Baddorf, Arthur P.; Sumpter, Bobby G.; Pan, Minghu; Maksymovych, Petro; Fuentes-Cabrera, Miguel

    2016-01-01

    Self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N9-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two or more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. These characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Further, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers.

  1. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers

    PubMed Central

    Wang, Jun; Bonnesen, Peter V.; Rangel, E.; Vallejo, E.; Sanchez-Castillo, Ariadna; James Cleaves II, H.; Baddorf, Arthur P.; Sumpter, Bobby G.; Pan, Minghu; Maksymovych, Petro; Fuentes-Cabrera, Miguel

    2016-01-01

    Self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N9-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two or more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. These characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Further, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers. PMID:26725380

  2. Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC-UV-MS.

    PubMed

    Russell, Susan P; Limbach, Patrick A

    2013-04-01

    Post-transcriptional chemical covalent modification of adenosine, guanosine, uridine and cytidine occurs frequently in all types of ribonucleic acids (RNAs). In ribosomal RNA (rRNA) and transfer RNA (tRNA) these modifications make important contributions to RNA structure and stability and to the accuracy and efficiency of protein translation. The functional dynamics, synergistic nature and regulatory roles of these posttranscriptional nucleoside modifications within the cell are not well characterized. These modifications are present at very low levels and isolation of individual nucleosides for analysis requires a complex multi-step approach. The focus of this study is to characterize the reproducibility of a liquid chromatography method used to isolate and quantitatively characterize modified nucleosides in tRNA and rRNA when nucleoside detection is performed using ultraviolet and mass spectrometric detection (UV and MS, respectively). Despite the analytical challenges of sample isolation and dynamic range, quantitative profiling of modified nucleosides obtained from bacterial tRNAs and rRNAs is feasible at relative standard deviations of 5% RSD or less. PMID:23500350

  3. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers.

    PubMed

    Wang, Jun; Bonnesen, Peter V; Rangel, E; Vallejo, E; Sanchez-Castillo, Ariadna; James Cleaves Ii, H; Baddorf, Arthur P; Sumpter, Bobby G; Pan, Minghu; Maksymovych, Petro; Fuentes-Cabrera, Miguel

    2016-01-01

    Self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N(9)-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two or more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. These characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Further, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers. PMID:26725380

  4. Apoplastic Nucleoside Accumulation in Arabidopsis Leads to Reduced Photosynthetic Performance and Increased Susceptibility Against Botrytis cinerea

    PubMed Central

    Daumann, Manuel; Fischer, Marietta; Niopek-Witz, Sandra; Girke, Christopher; Möhlmann, Torsten

    2015-01-01

    Interactions between plant and pathogen often occur in the extracellular space and especially nucleotides like ATP and NAD have been identified as key players in this scenario. Arabidopsis mutants accumulating nucleosides in the extracellular space were generated and studied with respect to susceptibility against Botrytis cinerea infection and general plant fitness determined as photosynthetic performance. The mutants used are deficient in the main nucleoside uptake system ENT3 and the extracellular nucleoside hydrolase NSH3. When grown on soil but not in hydroponic culture, these plants markedly accumulate adenosine and uridine in leaves. This nucleoside accumulation was accompanied by reduced photosystem II efficiency and altered expression of photosynthesis related genes. Moreover, a higher susceptibility toward Botrytis cinerea infection and a reduced induction of pathogen related genes PR1 and WRKY33 was observed. All these effects did not occur in hydroponically grown plants substantiating a contribution of extracellular nucleosides to these effects. Whether reduced general plant fitness, altered pathogen response capability or more direct interactions with the pathogen are responsible for these observations is discussed. PMID:26779190

  5. Short Communication: Transplacental Nucleoside Analogue Exposure and Mitochondrial Parameters in HIV-Uninfected Children

    PubMed Central

    Brogly, Susan B.; DiMauro, Salvatore; Van Dyke, Russell B.; Williams, Paige L.; Naini, Ali; Libutti, Daniel E.; Choi, Julia; Chung, Michelle

    2011-01-01

    Abstract Transplacental nucleoside analogue exposure can affect infant mitochondrial DNA (mtDNA). We evaluated mitochondria in peripheral blood mononuclear cells of children with and without clinical signs of mitochondrial dysfunction (MD) and antiretroviral (ARV) exposure. We previously identified 20 children with signs of MD (cases) among 1037 HIV-uninfected children born to HIV-infected women. We measured mtDNA copies/cell and oxidative phosphorylation (OXPHOS) NADH dehydrogenase (complex I) and cytochrome c oxidase (complex IV) protein levels and enzyme activities, determined mtDNA haplogroups and deletions in 18 of 20 cases with stored samples and in sex- and age-matched HIV-uninfected children, both ARV exposed and unexposed, (1) within 18 months of birth and (2) at the time of presentation of signs of MD. In specimens drawn within 18 months of birth, mtDNA levels were higher and OXPHOS protein levels and enzyme activities lower in cases than controls. In contrast, at the time of MD presentation, cases and ARV-exposed controls had lower mtDNA levels, 214 and 215 copies/cell, respectively, than ARV-unexposed controls, 254 copies/cell. OXPHOS protein levels and enzyme activities were lower in cases than exposed controls, and higher in cases than unexposed controls, except for complex IV activity, which was higher in cases. Haplotype H was less frequent among cases (6%) than controls (31%). No deletions were found. The long-term significance of these small but potentially important alterations should continue to be studied as these children enter adolescence and adulthood. PMID:21142587

  6. Probing the TRAP-RNA interaction with nucleoside analogs.

    PubMed

    Elliott, M B; Gottlieb, P A; Gollnick, P

    1999-10-01

    The trp RNA-binding Attenuation Protein (TRAP) from Bacillus subtilis binds a series of GAG and UAG repeats separated by 2-3 nonconserved spacer nucleotides in trp leader mRNA. To identify chemical groups on the RNA required for stability of the TRAP-RNA complex, we introduced several different nucleoside analogs into each pentamer of the RNA sequence 5'-(UAGCC)-3' repeated 11 times and measured their effect on the TRAP-RNA interaction. Deoxyribonucleoside substitutions revealed that a 2'-hydroxyl group (2'-OH) is required only on the guanosine occupying the third residue of the RNA triplets for high-affinity binding to TRAP. The remaining hydroxyl groups are dispensable. Base analog substitutions identified all of the exocyclic functional groups and N1 nitrogens of adenine and guanine in the second and third nucleotides, respectively, of the triplets as being involved in binding TRAP. In contrast, none of the substitutions made in the first residue caused any detectable changes in affinity, indicating that elements of these bases are not necessary for complex formation and stability. Studies using abasic nucleotides in the first residue of the triplets and in the two spacer residues confirmed that the majority of the specificity and stability of the TRAP-RNA complex is provided by the AG dinucleotide of the triplet repeats. In addition to direct effects on binding, we demonstrate that the N7-nitrogen of adenosine and guanosine in UAG triplet and the 2'-OHs of (UAGCC)11 RNA are involved in the formation of an as yet undetermined structure that interferes with TRAP binding. PMID:10573119

  7. Resistance issues with new nucleoside/nucleotide backbone options.

    PubMed

    Wainberg, Mark A; Turner, Dan

    2004-09-01

    The nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs) remain an enduring feature of combination therapy. As NRTI/NtRTI options continue to expand, questions arise about how best to combine these agents to create effective dual NRTI/NtRTI backbones in antiretroviral regimens while avoiding treatment-emergent drug resistance. Clinicians must consider how NRTIs/NtRTIs such as tenofovir DF (TDF), abacavir (ABC), and emtricitabine (FTC), as well as new once-daily and coformulated NRTIs/NtRTIs, interact with older agents when combined in novel regimens and how sequencing the new NRTIs can preserve future treatment options. Resistance data from clinical trials have revealed important information on the patterns, prevalence, and effects of resistance seen among patients experiencing virologic failure. In recent years, the prevalence of some mutations such as M184V and Q151M has remained relatively constant, while the L74V mutation, the 69 insertions, and thymidine analogue mutations have decreased in prevalence. Other mutations such as K65R and Y115F, while still relatively uncommon, are increasing in prevalence. This increase may be due to the use of new treatment combinations that select for these mutations at a higher rate. Clinical trials suggest that new regimens containing TDF or ABC select for K65R and that this mutation is observed more frequently with TDF; in contrast, L74V is observed more frequently in ABC-containing regimens but is not commonly selected by TDF-containing regimens. Several lines of evidence are converging to suggest that the presence of zidovudine may decrease the risk of L74V and K65R in ABC- or TDF-containing regimens. This review summarizes the clinical implications of resistance profiles associated with new NRTI/NtRTI regimens in current use and in advanced clinical studies. PMID:15319668

  8. NMR-spectroscopic screening of spider venom reveals sulfated nucleosides as major components for the brown recluse and related species.

    PubMed

    Schroeder, Frank C; Taggi, Andrew E; Gronquist, Matthew; Malik, Rabia U; Grant, Jacqualine B; Eisner, Thomas; Meinwald, Jerrold

    2008-09-23

    Extensive chemical analyses of spider venoms from many species have revealed complex mixtures of biologically active compounds, of which several have provided important leads for drug development. We have recently shown that NMR spectroscopy can be used advantageously for a direct structural characterization of the small-molecule content of such complex mixtures. Here, we report the application of this strategy to a larger-scale analysis of a collection of spider venoms representing >70 species, which, in combination with mass spectrometric analyses, allowed the identification of a wide range of known, and several previously undescribed, small molecules. These include polyamines, common neurotransmitters, and amino acid derivatives as well as two additional members of a recently discovered family of natural products, the sulfated nucleosides. In the case of the well studied brown recluse spider, Loxosceles reclusa, sulfated guanosine derivatives were found to comprise the major small-molecule components of the venom. PMID:18794518

  9. NMR-spectroscopic screening of spider venom reveals sulfated nucleosides as major components for the brown recluse and related species

    PubMed Central

    Schroeder, Frank C.; Taggi, Andrew E.; Gronquist, Matthew; Malik, Rabia U.; Grant, Jacqualine B.; Eisner, Thomas; Meinwald, Jerrold

    2008-01-01

    Extensive chemical analyses of spider venoms from many species have revealed complex mixtures of biologically active compounds, of which several have provided important leads for drug development. We have recently shown that NMR spectroscopy can be used advantageously for a direct structural characterization of the small-molecule content of such complex mixtures. Here, we report the application of this strategy to a larger-scale analysis of a collection of spider venoms representing >70 species, which, in combination with mass spectrometric analyses, allowed the identification of a wide range of known, and several previously undescribed, small molecules. These include polyamines, common neurotransmitters, and amino acid derivatives as well as two additional members of a recently discovered family of natural products, the sulfated nucleosides. In the case of the well studied brown recluse spider, Loxosceles reclusa, sulfated guanosine derivatives were found to comprise the major small-molecule components of the venom. PMID:18794518

  10. B cell hyperactivity and abnormalities in T cell markers and immunoregulatory function in a patient with nucleoside phosphorylase deficiency.

    PubMed Central

    Zabay, J M; De La Concha, E G; Ludeña, C; Lozano, C; Pascual-Salcedo, D; Bootello, A; Gonzalezporqué, P

    1982-01-01

    We describe a 2 year old girl with nucleoside phosphorylase (PNP) deficiency, who had low blood T cell numbers and T lymphocyte blastogenic response to mitogens, hypergammaglobulinaemia, high titres of antibodies to many common antigens, various autoantibodies, a monoclonal IgM-kappa protein, an increased frequency of mature Ig containing blood B cells and a high production of Ig in vitro in unstimulated cultures. E rosetting cells showed faint or no immunofluorescence staining with monoclonal antibodies directed against T cell membrane antigens. In vitro Ig production in response to pokeweed mitogen was defective, and no T cell helper or suppressor activity was observed. It is suggested that the immunoregulatory deficiency might have caused the B cell hyperactivity. PMID:6819909

  11. Synthesis and Biological Evaluation of 2-Oxo/Thioxoquinoxaline and 2-Oxo/Thioxoquinoxaline-Based Nucleoside Analogues.

    PubMed

    El-Sayed, Hassan A; Said, Said A; Moustafa, Ahmed H; Baraka, Mohamed M; Abdel-Kader, Rimaa T

    2016-01-01

    Several O- and S-quinoxaline glycosides have been prepared by glycosidation of 3-methyl-2-oxo(thioxo)-1,2-dihydroquinoxalines 1a,b with α-D-glucopyranosyl, α-D-galactopyranosyl, and α-D-lactosyl bromide in the presence of K2CO3 followed by deacetylation with Et3N/H2O. Furthermore, alkylation of 1a,b with 4-bromobutyl acetate, 2-acetoxyethoxymethyl bromide, and 3-chloropropanol afforded the corresponding O- and S-acycloquinoxaline nucleosides. Reaction of 1b with chloroacetic acid followed by condensation with sulfacetamide and sulfadiazine in the presence of Et3N/THF and ethyl chloroformate gave the corresponding sulfonamide derivatives 14 and 15, respectively. The structures of new compounds were confirmed by using IR, (1)H, (13)C NMR spectra and microanalysis. Some of these compounds were screened in vitro for antitumor and antifungal activities. PMID:26810144

  12. Transition State Analogues of Purine Nucleoside Phosphorylase: the Work of Vernon L. Schramm

    PubMed Central

    Kresge, Nicole; Simoni, Robert D.; Hill, Robert L.

    2010-01-01

    Transition State Analogue Inhibitors of Purine Nucleoside Phosphorylase from Plasmodium falciparum (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Kim, K., and Schramm, V. L. (2002) J. Biol. Chem. 277, 3219–3225) Purine-less Death in Plasmodium falciparum Induced by Immucillin-H, a Transition State Analogue of Purine Nucleoside Phosphorylase (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Schramm, V. L., and Kim, K. (2002) J. Biol. Chem. 277, 3226–3231) Achieving the Ultimate Physiological Goal in Transition State Analogue Inhibitors for Purine Nucleoside Phosphorylase (Lewandowicz, A., Tyler, P. C., Evans, G. B., Furneaux, R. H., and Schramm, V. L. (2003) J. Biol. Chem. 278, 31465–31468)

  13. N-Branched acyclic nucleoside phosphonates as monomers for the synthesis of modified oligonucleotides.

    PubMed

    Hocková, Dana; Rosenbergová, Šárka; Ménová, Petra; Páv, Ondřej; Pohl, Radek; Novák, Pavel; Rosenberg, Ivan

    2015-04-21

    Protected N-branched nucleoside phosphonates containing adenine and thymine bases were prepared as the monomers for the introduction of aza-acyclic nucleotide units into modified oligonucleotides. The phosphotriester and phosphoramidite methods were used for the incorporation of modified and natural units, respectively. The solid phase synthesis of a series of nonamers containing one central modified unit was successfully performed in both 3'→5' and 5'→3' directions. Hybridization properties of the prepared oligoribonucleotides and oligodeoxyribonucleotides were evaluated. The measurement of thermal characteristics of the complexes of modified nonamers with the complementary strand revealed a considerable destabilizing effect of the introduced units. We also examined the substrate/inhibitory properties of aza-acyclic nucleoside phosphono-diphosphate derivatives (analogues of nucleoside triphosphates) but neither inhibition of human and bacterial DNA polymerases nor polymerase-mediated incorporation of these triphosphate analogues into short DNA was observed. PMID:25766752

  14. Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus

    PubMed Central

    Duong-ly, Krisna C.; Schoeffield, Andrew J.; Pizarro-Dupuy, Mario A.; Zarr, Melissa; Pineiro, Silvia A.; Amzel, L. Mario; Gabelli, Sandra B.

    2015-01-01

    Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 –- a Nudix hydrolase from Bdellovibrio bacteriovorus–that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases. PMID:26524597

  15. Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis

    SciTech Connect

    Grenha, Rosa; Levdikov, Vladimir M.; Fogg, Mark J.; Blagova, Elena V.; Brannigan, James A. Wilkinson, Anthony J.; Wilson, Keith S.

    2005-05-01

    The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis was solved by X-ray crystallography using molecular replacement and refined at a resolution of 2.24 Å. Protein structures from the causative agent of anthrax (Bacillus anthracis) are being determined as part of a structural genomics programme. Amongst initial candidates for crystallographic analysis are enzymes involved in nucleotide biosynthesis, since these are recognized as potential targets in antibacterial therapy. Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway. The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis has been solved by molecular replacement at 2.24 Å resolution and refined to an R factor of 18.4%. This is the first report of a DeoD structure from a Gram-positive bacterium.

  16. Functional and genetic diversity in the concentrative nucleoside transporter, CNT1, in human populations.

    PubMed

    Gray, Jennifer H; Mangravite, Lara M; Owen, Ryan P; Urban, Thomas J; Chan, Wendy; Carlson, Elaine J; Huang, Conrad C; Kawamoto, Michiko; Johns, Susan J; Stryke, Douglas; Ferrin, Thomas E; Giacomini, Kathleen M

    2004-03-01

    The concentrative nucleoside transporter, CNT1 (SLC28A1), mediates the cellular uptake of naturally occurring pyrimidine nucleosides and many structurally diverse anticancer and antiviral nucleoside analogs. As a first step toward understanding whether genetic variation in CNT1 contributes to variation in the uptake and disposition of clinically used nucleoside analogs, we determined the haplotype structure and functionally analyzed all coding region variants of CNT1 identified in ethnically diverse populations (100 African Americans, 100 European Americans, 30 Asians, 10 Mexican Americans, and 7 Pacific Islanders) (Leabman et al., 2003). A total of 58 coding region haplotypes were identified using PHASE analysis, 44 of which contained at least one amino acid variant. More than half of the coding region haplotypes were population-specific. Using site-directed mutagenesis, 15 protein-altering CNT1 variants, including one amino acid insertion and one base pair (bp) deletion, were constructed and expressed in Xenopus laevis oocytes. All variant transporters took up [3H]thymidine with the exception of CNT1-Ser546Pro, a rare variant, and CNT1-1153del, a single bp deletion found at a frequency of 3% in the African American population. The bp deletion results in a frame-shift followed by a stop-codon. The anticancer nucleoside analog gemcitabine had a reduced affinity for CNT1-Val189Ile (a common CNT1 variant found at a frequency of 26%) compared with reference CNT1 (IC50=13.8 +/- 0.60 microM for CNT1-reference and 23.3 +/- 1.5 microM for CNT1-Val189Ile, p<0.05). These data suggest that common genetic variants of CNT1 may contribute to variation in systemic and intracellular levels of anti-cancer nucleoside analogs. PMID:14978229

  17. Characterization of intestinal absorption of mizoribine mediated by concentrative nucleoside transporters in rats.

    PubMed

    Mori, Nobuhiro; Yokooji, Tomoharu; Kamio, Yoshihiro; Murakami, Teruo

    2008-05-31

    Mizoribine, an imidazole nucleoside, is an inhibitor of purine synthesis and has been used as an orally available immunosuppressive agent in human renal transplantation. In the present study, the intestinal absorption of mizoribine was characterized by examining the contribution of concentrative nucleoside transporters (CNT1, CNT2) in rats. When mizoribine was administered orally in conscious rats, the bioavailability of mizoribine estimated by urinary excretion percentage of unchanged mizoribine was a dose dependent: 53.1+/-6.0% at 5 mg/kg and 24.0+/-5.1% at 20 mg/kg. In in-situ loop studies, the disappearance rate, or absorption rate, of mizoribine from the intestinal lumen was comparable between 1 and 5 mg/kg, but significantly lower at 25 mg/kg. Coadministration of adenosine (a substrate of both CNT1 and CNT2), thymidine (a CNT1 substrate) and inosine (a CNT2 substrate) significantly suppressed the intestinal mizoribine absorption, depending on the nucleoside concentrations coadministered. Gemcitabine (a pyrimidine nucleoside analogue, a CNT1 substrate) and ribavirin (a purine nucleoside analog, a CNT2 substrate) also significantly suppressed the mizoribine intestinal absorption. Bile salts such as sodium cholate and sodium glycocholate (10 mM) also significantly suppressed the intestinal mizoribine absorption, but not ribavirin absorption. Mizoribine is an amphoteric compound, however, the suppression of intestinal absorption by bile salts was not ascribed to the electrostatic interaction or micellar formation between mizoribine and bile salts. In conclusion, the intestinal absorption of mizoribine is mediated by CNT1 and CNT2, and nucleoside-derived drugs such as gemcitabine and ribavirin can suppress the intestinal absorption of mizoribine. Bile salts such as sodium glycocholate were also found to cause interaction with mizoribine. PMID:18371949

  18. Nucleoside transporter proteins as biomarkers of drug responsiveness and drug targets

    PubMed Central

    Pastor-Anglada, Marçal; Pérez-Torras, Sandra

    2015-01-01

    Nucleoside and nucleobase analogs are currently used in the treatment of solid tumors, lymphoproliferative diseases, viral infections such as hepatitis and AIDS, and some inflammatory diseases such as Crohn. Two gene families are implicated in the uptake of nucleosides and nucleoside analogs into cells, SCL28 and SLC29. The former encodes hCNT1, hCNT2, and hCNT3 proteins. They translocate nucleosides in a Na+ coupled manner with high affinity and some substrate selectivity, being hCNT1 and hCNT2 pyrimidine- and purine-preferring, respectively, and hCNT3 a broad selectivity transporter. SLC29 genes encode four members, being hENT1 and hENT2 the only two which are unequivocally implicated in the translocation of nucleosides and nucleobases (the latter mostly via hENT2) at the cell plasma membrane. Some nucleoside-derived drugs can also interact with and be translocated by members of the SLC22 gene family, particularly hOCT and hOAT proteins. Inter-individual differences in transporter function and perhaps, more importantly, altered expression associated with the disease itself might modulate the transporter profile of target cells, thereby determining drug bioavailability and action. Drug transporter pharmacology has been periodically reviewed. Thus, with this contribution we aim at providing a state-of-the-art overview of the clinical evidence generated so far supporting the concept that these membrane proteins can indeed be biomarkers suitable for diagnosis and/or prognosis. Last but not least, some of these transporter proteins can also be envisaged as drug targets, as long as they can show “transceptor” functions, in some cases related to their role as modulators of extracellular adenosine levels, thereby providing a functional link between P1 receptors and transporters. PMID:25713533

  19. Isolation and functional characterization of the PfNT1 nucleoside transporter gene from Plasmodium falciparum.

    PubMed

    Carter, N S; Ben Mamoun, C; Liu, W; Silva, E O; Landfear, S M; Goldberg, D E; Ullman, B

    2000-04-01

    Plasmodium falciparum, the causative agent of the most lethal form of human malaria, is incapable of de novo purine synthesis, and thus, purine acquisition from the host is an indispensable nutritional requirement. This purine salvage process is initiated by the transport of preformed purines into the parasite. We have identified a gene encoding a nucleoside transporter from P. falciparum, PfNT1, and analyzed its function and expression during intraerythrocytic parasite development. PfNT1 predicts a polypeptide of 422 amino acids with 11 transmembrane domains that is homologous to other members of the equilibrative nucleoside transporter family. Southern analysis and BLAST searching of The Institute for Genomic Research (TIGR) malaria data base indicate that PfNT1 is a single copy gene located on chromosome 14. Northern analysis of RNA from intraerythrocytic stages of the parasite demonstrates that PfNT1 is expressed throughout the asexual life cycle but is significantly elevated during the early trophozoite stage. Functional expression of PfNT1 in Xenopus laevis oocytes significantly increases their ability to take up naturally occurring D-adenosine (K(m) = 13.2 microM) and D-inosine (K(m) = 253 microM). Significantly, PfNT1, unlike the mammalian nucleoside transporters, also has the capacity to transport the stereoisomer L-adenosine (K(m) > 500 microM). Inhibition studies with a battery of purine and pyrimidine nucleosides and bases as well as their analogs indicate that PfNT1 exhibits a broad substrate specificity for purine and pyrimidine nucleosides. These data provide compelling evidence that PfNT1 encodes a functional purine/pyrimidine nucleoside transporter whose expression is strongly developmentally regulated in the asexual stages of the P. falciparum life cycle. Moreover, the unusual ability to transport L-adenosine and the vital contribution of purine transport to parasite survival makes PfNT1 an attractive target for therapeutic evaluation. PMID

  20. Evidence for incorporation of intact dietary pyrimidine (but not purine) nucleosides into hepatic RNA.

    PubMed Central

    Berthold, H K; Crain, P F; Gouni, I; Reeds, P J; Klein, P D

    1995-01-01

    The absorption and metabolism of dietary nucleic acids have received less attention than those of other organic nutrients, largely because of methodological difficulties. We supplemented the rations of poultry and mice with the edible alga Spirulina platensis, which had been uniformly labeled with 13C by hydroponic culture in 13CO2. The rations were ingested by a hen for 4 wk and by four mice for 6 days; two mice were fed a normal diet and two were fed a nucleic acid-deficient diet. The animals were killed and nucleosides were isolated from hepatic RNA. The isotopic enrichment of all mass isotopomers of the nucleosides was analyzed by selected ion monitoring of the negative chemical ionization mass spectrum and the labeling pattern was deconvoluted by reference to the enrichment pattern of the tracer material. We found a distinct difference in the 13C enrichment pattern between pyrimidine and purine nucleosides; the isotopic enrichment of uniformly labeled [M + 9] isotopomers of pyrimidines exceeded that of purines [M + 10] by > 2 orders of magnitude in the avian nucleic acids and by 7- and 14-fold in the murine nucleic acids. The purines were more enriched in lower mass isotopomers, those less than [M + 3], than the pyrimidines. Our results suggest that large quantities of dietary pyrimidine nucleosides and almost no dietary purine nucleosides are incorporated into hepatic nucleic acids without hydrolytic removal of the ribose moiety. In addition, our results support a potential nutritional role for nucleosides and suggest that pyrimidines are conditionally essential organic nutrients. PMID:7479738

  1. A low Km nucleoside 3'(2'),5-bisphosphate 3'(2')-phosphohydrolase from rat liver.

    PubMed

    Meireles Ribeiro, J; Ruiz, A; Sillero, A; Günther Sillero, M A

    1990-04-01

    In the course of an investigation on the occurrence in rat liver of a specific hydrolytic activity on adenosine 2',5'-bisphosphate, a nucleoside 3'(2'),5'-bisphosphate 3'(2')-phosphohydrolase was purified following standard procedures. The enzyme hydrolyzes the phosphate group joined to the 3' or the 2' position of the following nucleotides (relative velocities indicated in brackets): PAdoP (100), PCydP (95), PGuoP (80), PAdo2'P (40), PdAdoP (4), SPAdoP (18). Other nucleotides were not substrates of the reaction: NADP+, PAdoPP, PPGuoP, AdoP, PAdo, GuoP, PGuo, ADP, ATP, cAMP, adenosine(3')phospho(5')adenosine. The Km values determined for PAdoP and PAdo2'P were 10 and 7 microM, respectively. Two isoforms were separated by chromatography on a Mono Q column. Both isoforms were kinetically indistinguishable, presenting a pI value of 5.35, a molecular mass of 38 kDa, pH optimum of 8.0, and strictly required Mg2+ or Mn2+. An enzymatic activity similar to the one described here has already been reported in guinea pig liver [5]. These authors however only obtained 1 enzymatic form with Km values of 3.1 and 1.8 mM for PAdoP and PAdo2'P, respectively. The potential physiological role of this enzyme in the metabolism of sulphate is also considered. The previously registered number EC 3.1.3.7 could be applied to this activity. PMID:2166593

  2. Predictive markers of capecitabine sensitivity identified from the expression profile of pyrimidine nucleoside-metabolizing enzymes.

    PubMed

    Yasuno, Hideyuki; Kurasawa, Mitsue; Yanagisawa, Mieko; Sato, Yasuko; Harada, Naoki; Mori, Kazushige

    2013-02-01

    Molecular markers predicting sensitivity to anticancer drugs are important and useful not only for selecting potential responders but also for developing new combinations. In the present study, we analyzed the difference in the sensitivity of xenograft models to capecitabine (Xeloda®), 5'-deoxy-5-fluorouridine (5'-DFUR, doxifluridine, Furtulon®) and 5-FU by comparing the mRNA levels of 12 pyrimidine nucleoside-metabolizing enzymes. Amounts of mRNA in the tumor tissues of 80 xenograft models were determined by real-time RT-PCR and mutual correlations were examined. A clustering analysis revealed that the 12 enzymes were divided into two groups; one group consisted of 8 enzymes, including orotate phosphoribosyl transferase (OPRT), TMP kinase (TMPK) and UMP kinase (UMPK), and was related to the de novo synthesis pathway for nucleotides, with mRNA expression levels showing significant mutual correlation. In the other group, 4 enzymes, including thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD), were involved in the salvage/degradation pathway of the nucleotides, and the mRNA levels of this group were dispersed more widely than that of the de novo group. Antitumor activity was assessed in 24 xenograft models for each drug. The antitumor activity of capecitabine and 5'-DFUR correlated significantly with the mRNA levels of TP and with the TP/DPD ratio, whereas the activity of 5-FU correlated significantly with OPRT, TMPK, UMPK and CD. In a stepwise regression analysis, TP and DPD were found to be independent predictive factors of sensitivity to capecitabine and 5'-DFUR, and UMPK was predictive of sensitivity to 5-FU. These results indicate that the predictive factors for sensitivity to capecitabine and 5'-DFUR in xenograft models may be different from those for 5-FU, suggesting that these drugs may have different responders in clinical usage. PMID:23229803

  3. Correlation of nucleoside and nucleobase transporter gene expression with antimetabolite drug cytotoxicity.

    PubMed

    Lu, Xin; Gong, Shimei; Monks, Anne; Zaharevitz, Daniel; Moscow, Jeffrey A

    2002-01-01

    Antimetabolite drugs that inhibit nucleic acid metabolism are widely used in cancer chemotherapy. Nucleoside and nucleobase transporters are important for the cellular uptake of nucleic acids and their corresponding anticancer analogue drugs. Thus, these transporters may play a role both in antimetabolite drug sensitivity, by mediating the uptake of nucleoside analogues, and in antimetabolite drug resistance, by mediating the uptake of endogenous nucleosides that may rescue cells from toxicity. Therefore, we examined the relation of the expression of nucleoside and nucleobase transporters to antimetabolite cytotoxicity. We measured the RNA levels of all eight known nucleoside and nucleobase transporters in 50 cell lines included in the National Cancer Institute's Anticancer Drug Screen panel. RNA levels of concentrative nucleoside transporters (CNTs), equilibrative nucleoside transporters (ENTs) and nucleobase transporters (NCBTs) were determined by quantitative RT-PCR using real-time fluorescence acquisition. This method was validated by measuring the expression of the MDR1 gene, and correlating our results with independently determined measurements of MDR1 RNA levels and protein function in these cell lines. We then correlated the pattern of RNA levels to the pattern of cytotoxicity of anticancer drugs in the NCI drug screen database using the COMPARE analysis. Several hypothesized relations between transporter gene expression and cytotoxicity, based upon known interactions between certain nucleoside analogues and transporter proteins, were not observed, suggesting that expression of individual transporters may not be a significant determinant of the cytotoxicity of these drugs. The most closely correlated drug cytotoxicity patterns to transporter gene expression patterns (where increased expression corresponds to increase sensitivity) included those between CNT1 and O6-methylguanine and between ENT2 and hydroxyurea. We also observed that p53 status influenced

  4. Distribution of the modified nucleoside Q and its derivatives in animal and plant transfer RNA's.

    PubMed Central

    Kasai, H; Kuchino, Y; Nihei, K; Nishimura, S

    1975-01-01

    The modified nucleoside, 7-(4,5-cis-dihydroxy-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanosine, designated as Q, and its derivative, Q*, were found in tRNA's from various organisms, including several mammalian tissues, other animals such as starfish, lingula and hagfish, and wheat germ. Q isolated from rat liver tRNA was found to be identical with E. coli Q by mass spectrometry and thin-layer chromatography. Thus the rare modified nucleoside Q originally isolated from E. coli tRNA, is widely distributed in various organisms. Analysis of the mass spectrum of Q* suggested that it has a different side chain from Q. PMID:1187350

  5. Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis

    PubMed Central

    Grenha, Rosa; Levdikov, Vladimir M.; Fogg, Mark J.; Blagova, Elena V.; Brannigan, James A.; Wilkinson, Anthony J.; Wilson, Keith S.

    2005-01-01

    Protein structures from the causative agent of anthrax (Bacillus anthracis) are being determined as part of a structural genomics programme. Amongst initial candidates for crystallographic analysis are enzymes involved in nucleotide biosynthesis, since these are recognized as potential targets in antibacterial therapy. Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway. The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis has been solved by molecular replacement at 2.24 Å resolution and refined to an R factor of 18.4%. This is the first report of a DeoD structure from a Gram-positive bacterium. PMID:16511068

  6. Equilibrative nucleoside transporter 3 depletion in β-cells impairs mitochondrial function and promotes apoptosis: Relationship to pigmented hypertrichotic dermatosis with insulin-dependent diabetes.

    PubMed

    Liu, B; Czajka, A; Malik, A N; Hussain, K; Jones, P M; Persaud, S J

    2015-10-01

    Loss of function recessive mutations in the SLC29A3 gene that encodes human equilibrative nucleoside transporter 3 (ENT3) have been identified in patients with pigmented hypertrichotic dermatosis with insulin-dependent diabetes (PHID). ENT3 is a member of the equilibrative nucleoside transporter (ENT) family whose primary function is mediating transport of nucleosides and nucleobases. The aims of this study were to characterise ENT3 expression in islet β-cells and identify the effects of its depletion on β-cell mitochondrial activity and apoptosis. RT-PCR amplification identified ENT3 expression in human and mouse islets and exocrine pancreas, and in MIN6 β-cells. Immunohistochemistry using human and mouse pancreas sections exhibited extensive ENT3 immunostaining of β-cells, which was confirmed by co-staining with an anti-insulin antibody. In addition, exposure of dispersed human islet cells and MIN6 β-cells to MitoTracker and an ENT3 antibody showed co-localisation of ENT3 to β-cell mitochondria. Consistent with this, Western blot analysis confirmed enhanced ENT3 immunoreactivity in β-cell mitochondria-enriched fractions. Furthermore, ENT3 depletion in β-cells increased mitochondrial DNA content and promoted an energy crisis characterised by enhanced ATP-linked respiration and proton leak. Finally, inhibition of ENT3 activity by dypridamole and depletion of ENT3 by siRNA-induced knockdown resulted in increased caspase 3/7 activities in β-cells. These observations demonstrate that ENT3 is predominantly expressed by islet β-cells where it co-localises with mitochondria. Depletion of ENT3 causes mitochondrial dysfunction which is associated with enhanced β-cell apoptosis. Thus, apoptotic loss of islet β-cells may contribute to the occurrence of autoantibody-negative insulin-dependent diabetes in individuals with non-functional ENT3 mutations. PMID:26163994

  7. The long-term outcomes of antiretroviral treatment initiated with mono or dual nucleoside reverse transcriptase inhibitors in HIV-1-infected children: an Asian observational study

    PubMed Central

    Wittawatmongkol, Orasri; Mohamed, Thahira J; Le, Thoa PK; Ung, Vibol; Maleesatharn, Alan; Hansudewechakul, Rawiwan; Nguyen, Lam V; Kumarasamy, Nagalingeswaran; Lumbiganon, Pagakrong; Sudjaritruk, Tavitiya; Bunupuradah, Torsak; Yusoff, Nik KN; Kurniati, Nia; Fong, Moy S.; Nallusamy, Revathy; Kariminia, Azar; Sohn, Annette H.; Chokephaibulkit, Kulkanya

    2016-01-01

    After a median of 115.9 months of follow-up, 90% of 206 HIV-1-infected children in a cohort in Asia who initiated antiretroviral treatment (ART) with mono or dual nucleoside reverse transcriptase inhibitors were alive and had comparable immunological and virological outcomes as compared to the 1,915 children who had started with highly active antiretroviral regimens. However, these children had higher rates of treatment-related adverse events, opportunistic infections, and cumulative mortality, and were more likely to require protease inhibitor-containing regimens or other more novel ART-based regimens. PMID:27076917

  8. O2 Protonation Controls Threshold Behavior for N-Glycosidic Bond Cleavage of Protonated Cytosine Nucleosides.

    PubMed

    Wu, R R; Rodgers, M T

    2016-06-01

    IRMPD action spectroscopy studies of protonated 2'-deoxycytidine and cytidine, [dCyd+H](+) and [Cyd+H](+), have established that both N3 and O2 protonated conformers coexist in the gas phase. Threshold collision-induced dissociation (CID) of [dCyd+H](+) and [Cyd+H](+) is investigated here using guided ion beam tandem mass spectrometry techniques to elucidate the mechanisms and energetics for N-glycosidic bond cleavage. N-Glycosidic bond cleavage is observed as the major dissociation pathways resulting in competitive elimination of either protonated or neutral cytosine for both protonated cytosine nucleosides. Electronic structure calculations are performed to map the potential energy surfaces (PESs) for both N-glycosidic bond cleavage pathways observed. The molecular parameters derived from theoretical calculations are employed for thermochemical analysis of the energy-dependent CID data to determine the minimum energies required to cleave the N-glycosidic bond along each pathway. B3LYP and MP2(full) computed activation energies for N-glycosidic bond cleavage associated with elimination of protonated and neutral cytosine, respectively, are compared to measured values to evaluate the efficacy of these theoretical methods in describing the dissociation mechanisms and PESs for N-glycosidic bond cleavage. The 2'-hydroxyl of [Cyd+H](+) is found to enhance the stability of the N-glycosidic bond vs that of [dCyd+H](+). O2 protonation is found to control the threshold energies for N-glycosidic bond cleavage as loss of neutral cytosine from the O2 protonated conformers is found to require ∼25 kJ/mol less energy than the N3 protonated analogues, and the activation energies and reaction enthalpies computed using B3LYP exhibit excellent agreement with the measured thresholds for the O2 protonated conformers. PMID:27159774

  9. Disrupted sleep-wake regulation in type 1 equilibrative nucleoside transporter knockout mice.

    PubMed

    Kim, T; Ramesh, V; Dworak, M; Choi, D-S; McCarley, R W; Kalinchuk, A V; Basheer, R

    2015-09-10

    The type 1 equilibrative nucleoside transporter (ENT1) is implicated in regulating levels of extracellular adenosine ([AD]ex). In the basal forebrain (BF) levels of [AD]ex increase during wakefulness and closely correspond to the increases in the electroencephalogram (EEG) delta (0.75-4.5Hz) activity (NRδ) during subsequent non-rapid eye movement sleep (NREMS). Thus in the BF, [AD]ex serves as a biochemical marker of sleep homeostasis. Waking EEG activity in theta range (5-9Hz, Wθ) is also described as a marker of sleep homeostasis. An hour-by-hour temporal relationship between the Wθ and NRδ is unclear. In this study we examined the relationship between these EEG markers of sleep homeostasis during spontaneous sleep-wakefulness and during sleep deprivation (SD) and recovery sleep in the ENT1 gene knockout (ENT1KO) mouse. We observed that baseline NREMS amount was decreased during the light period in ENT1KO mice, accompanied by a weak correlation between Wθ of each hour and NRδ of its subsequent hour when compared to their wild-type (WT) littermates. Perfusion of low dose of adenosine into BF not only strengthened the Wθ-NRδ relationship, but also increased NREMS to match with the WT littermates suggesting decreased [AD]ex in ENT1KO mice. However, the SD-induced [AD]ex increase in the BF and the linear correlation between the EEG markers of sleep homeostasis were unaffected in ENT1KO mice suggesting that during SD, sources other than ENT1 contribute to increase in [AD]ex. Our data provide evidence for a differential regulation of wakefulness-associated [AD]ex during spontaneous vs prolonged waking. PMID:26143012

  10. Synthesis and pharmacological evaluation of nucleoside prodrugs designed to target siderophore biosynthesis in Mycobacterium tuberculosis.

    PubMed

    Dawadi, Surendra; Kawamura, Shuhei; Rubenstein, Anja; Remmel, Rory; Aldrich, Courtney C

    2016-03-15

    The nucleoside antibiotic, 5'-O-[N-(salicyl)sulfamoyl]adenosine (1), possesses potent whole-cell activity against Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). This compound is also active in vivo, but suffers from poor drug disposition properties that result in poor bioavailability and rapid clearance. The synthesis and evaluation of a systematic series of lipophilic ester prodrugs containing linear and α-branched alkanoyl groups from two to twelve carbons at the 3'-position of a 2'-fluorinated analog of 1 is reported with the goal to improve oral bioavailability. The prodrugs were stable in simulated gastric fluid (pH 1.2) and under physiological conditions (pH 7.4). The prodrugs were also remarkably stable in mouse, rat, and human serum (relative serum stability: human∼rat≫mouse) displaying a parabolic trend in the SAR with hydrolysis rates increasing with chain length up to eight carbons (t1/2=1.6 h for octanoyl prodrug 7 in mouse serum) and then decreasing again with higher chain lengths. The permeability of the prodrugs was also assessed in a Caco-2 cell transwell model. All of the prodrugs were found to have reduced permeation in the apical-to-basolateral direction and enhanced permeation in the basolateral-to-apical direction relative to the parent compound 2, resulting in efflux ratios 5-28 times greater than 2. Additionally, Caco-2 cells were found to hydrolyze the prodrugs with SAR mirroring the serum stability results and a preference for hydrolysis on the apical side. Taken together, these results suggest that the described prodrug strategy will lead to lower than expected oral bioavailability of 2 and highlight the contribution of intestinal esterases for prodrug hydrolysis. PMID:26875934

  11. Non-nucleoside reverse transcriptase inhibitor efavirenz increases monolayer permeability of human coronary artery endothelial cells.

    PubMed

    Jamaluddin, Md Saha; Lin, Peter H; Yao, Qizhi; Chen, Changyi

    2010-01-01

    Highly active antiretroviral therapy (HAART) is often associated with endothelial dysfunction and cardiovascular complications. In this study, we determined whether HIV non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) could increase endothelial permeability. Human coronary artery endothelial cells (HCAECs) were treated with EFV (1, 5 and 10 microg/ml) and endothelial permeability was determined by a transwell system with a fluorescence-labeled dextran tracer. HCAECs treated with EFV showed a significant increase of endothelial permeability in a concentration-dependent manner. With real time PCR analysis, EFV significantly reduced the mRNA levels of tight junction proteins claudin-1, occludin, zonula occluden-1 and junctional adhesion molecule-1 compared with controls (P<0.05). Protein levels of these tight junction molecules were also reduced substantially in the EFV-treated cells by western blot and flow cytometry analyses. In addition, EFV also increased superoxide anion production with dihydroethidium and cellular glutathione assays, while it decreased mitochondrial membrane potential with JC-staining. Antioxidants (ginkgolide B and MnTBAP) effectively blocked EFV-induced endothelial permeability and mitochondrial dysfunction. Furthermore, EFV increased the phosphorylation of MAPK JNK and IkappaBalpha, thereby increasing NFkappaB translocation to the nucleus. Chemical JNK inhibitor and dominant negative mutant JNK and IkappaBalpha adenoviruses effectively blocked the effects of EFV on HCAECs. Thus, EFV increases endothelial permeability which may be due to the decrease of tight junction proteins and the increase of superoxide anion. JNK and NFkappaB activation may be directly involved in the signal transduction pathway of EFV action in HCAECs. PMID:19674747

  12. Anopheles gambiae Purine Nucleoside Phosphorylase: Catalysis, Structure, and Inhibition

    SciTech Connect

    Taylor,E.; Rinaldo-Matthis, A.; Li, L.; Ghanem, M.; Hazleton, K.; Cassera, M.; Almo, S.; Schramm, V.

    2007-01-01

    The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been identified in genome searches on the basis of sequence homology with characterized enzymes. Purine nucleoside phosphorylase (PNP) is a target for the development of therapeutic agents in humans and purine auxotrophs, including malarial parasites. The PNP from Anopheles gambiae (AgPNP) was expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP has kcat values of 54 and 41 s-1 for 2'-deoxyinosine and inosine, its preferred substrates, and 1.0 s-1 for guanosine. However, the chemical step is fast for AgPNP at 226 s-1 for guanosine in pre-steady-state studies. 5'-Deaza-1'-aza-2'-deoxy-1'-(9-methylene)-Immucillin-H (DADMe-ImmH) is a transition-state mimic for a 2'-deoxyinosine ribocation with a fully dissociated N-ribosidic bond and is a slow-onset, tight-binding inhibitor with a dissociation constant of 3.5 pM. This is the tightest-binding inhibitor known for any PNP, with a remarkable Km/Ki* of 5.4 x 107, and is consistent with enzymatic transition state predictions of enhanced transition-state analogue binding in enzymes with enhanced catalytic efficiency. Deoxyguanosine is a weaker substrate than deoxyinosine, and DADMe-Immucillin-G is less tightly bound than DADMe-ImmH, with a dissociation constant of 23 pM for AgPNP as compared to 7 pM for HsPNP. The crystal structure of AgPNP was determined in complex with DADMe-ImmH and phosphate to a resolution of 2.2 Angstroms to reveal the differences in substrate and inhibitor specificity. The distance from the N1' cation to the phosphate O4 anion is shorter in the AgPNP{center_dot}DADMe-ImmH{center_dot}PO4 complex than in HsPNP{center_dot}DADMe-ImmH{center_dot}SO4, offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP.

  13. Hypoxanthine enters human vascular endothelial cells (ECV 304) via the nitrobenzylthioinosine-insensitive equilibrative nucleoside transporter.

    PubMed Central

    Osses, N; Pearson, J D; Yudilevich, D L; Jarvis, S M

    1996-01-01

    The transport properties of the nucleobase hypoxanthine were examined in the human umbilical vein endothelial cell line ECV 304. Initial rates of hypoxanthine influx were independent of extracellular cations: replacement of Na+ with Li+, Rb+, N-methyl-D-glucamine or choline had no significant effect on hypoxanthine uptake by ECV 304 cells. Kinetic analysis demonstrated the presence of a single saturable system for the transport of hypoxanthine in ECV 304 cells with an apparent K(m) of 320 +/- 10 microM and a Vmax of 5.6 +/- 0.9 pmol/10(6) cells per s. Hypoxanthine uptake was inhibited by the nucleosides adenosine, uridine and thymidine (apparent Ki 41 +/- 6, 240 +/- 27 and 59 +/- 8 microM respectively) and the nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole (apparent Ki 2.5 +/- 0.3, 11 +/- 3 and 0.16 +/- 0.006 microM respectively), whereas the nucleobases adenine, guanine and thymine had little effect (50% inhibition at > 1 mM). ECV 304 cells were also shown to transport adenosine via both the NBMPR-sensitive and -insensitive nucleoside carriers. Hypoxanthine specifically inhibited adenosine transport via the NBMPR-insensitive system in a competitive manner (apparent Ki 290 +/- 14 microM). These results indicate that hypoxanthine entry into ECV 304 endothelial cells is mediated by the NBMPR-insensitive nucleoside carrier present in these cells. PMID:8760371

  14. Release of nucleosides from canine and human hearts as an index of prior ischemia.

    PubMed

    Fox, A C; Reed, G E; Meilman, H; Silk, B B

    1979-01-01

    During ischemia, myocardial adenosine triphosphate is degraded to adenosine, inosine and hypoxanthine. These nucleosides are released into coronary venous blood and may provide an index of ischemia; adenosine may also participate in the autoregulation of coronary flow. In dogs, the temporal relations between reactive hyperemic flow and nucleoside concentrations in regional venous blood were correlated after brief occlusions of a segmental coronary artery. Reactive hyperemia and adenosine release peaked together in 10 seconds, persisted for 10 to 30 seconds and then decreased in a pattern consistent with the hypothesis that they are related. During initial reflow after 45 seconds of ischemia, mean concentrations of adenosine, inosine and hypoxanthine increased, respectively, to 52, 67 and 114 nmol/100 ml plasma; after 5 minutes of ischemia, the respective levels increased to 58, 1,570 and 1,134 nmol and fell quickly. In nine patients there was a similar release of nucleosides into coronary sinus blood during reperfusion after 59 to 80 minutes of ischemic arrest during cardiac surgery. With initial reflow, adenosine, inosine and hypoxanthine levels reached 65, 655 and 917 nmol/100 ml of blood, respectively. Inosine and hypoxanthine concentrations remained high for 5 to 10 minutes after cardiac beating resumed, often when production of lactate had decreased. The results indicate that postischemic release of nucleosides reaches significant levels in man as well as animals, is parallel with the duration of ischemia, is temporary and may be a useful supplement to measurement of lactate as an index of prior myocardial ischemia. PMID:758770

  15. Pseudobond parameters for QM/MM studies involving nucleosides, nucleotides, and their analogs

    NASA Astrophysics Data System (ADS)

    Chaudret, Robin; Parks, Jerry M.; Yang, Weitao

    2013-01-01

    In biological systems involving nucleosides, nucleotides, or their respective analogs, the ribose sugar moiety is the most common reaction site, for example, during DNA replication and repair. However, nucleic bases, which comprise a sizable portion of nucleotide molecules, are usually unreactive during such processes. In quantum mechanical/molecular simulations of nucleic acid reactivity, it may therefore be advantageous to describe specific ribosyl or ribosyl phosphate groups quantum mechanically and their respective nucleic bases with a molecular mechanics potential function. Here, we have extended the pseudobond approach to enable quantum mechanical/molecular mechanical simulations involving nucleotides, nucleosides, and their analogs in which the interface between the two subsystems is located between the sugar and the base, namely, the C(sp3)-N(sp2) bond. The pseudobond parameters were optimized on a training set of 10 molecules representing several nucleotide and nucleoside bases and analogs, and they were then tested on a larger test set of 20 diverse molecules. Particular emphasis was placed on providing accurate geometries and electrostatic properties, including electrostatic potential, natural bond orbital (NBO) and atoms in molecules (AIM) charges and AIM first moments. We also tested the optimized parameters on five nucleotide and nucleoside analogues of pharmaceutical relevance and a small polypeptide (triglycine). Accuracy was maintained for these systems, which highlights the generality and transferability of the pseudobond approach.

  16. Donor/acceptor chromophores-decorated triazolyl unnatural nucleosides: synthesis, photophysical properties and study of interaction with BSA.

    PubMed

    Bag, Subhendu Sekhar; Talukdar, Sangita; Das, Suman Kalyan; Pradhan, Manoj Kumar; Mukherjee, Soumen

    2016-06-14

    Much effort has been put forth to develop unnatural, stable, hydrophobic base pairs with orthogonal recognition properties and study their effect on DNA duplex stabilisation. Our continuous efforts on the design of fluorescent unnatural biomolecular building blocks lead us to the synthesis of some triazolyl donor/acceptor unnatural nucleosides via an azide-alkyne 1,3-dipolar cycloaddition reaction as a key step, which we want to report herein. We have studied their photophysical properties and found interesting solvatochromic fluorescence for two of the nucleosides. Photophysical interactions among two donor-acceptor β-nucleosides as well as a pair of α/β-nucleosides have also been evaluated. Furthermore, we have exploited one of the fluorescent nucleosides in studying its interaction with BSA with the help of UV-visible and steady state fluorescence techniques. Our design concept is based on the hypothesis that a pair of such donor/acceptor nucleosides might be involved in π-stacking as well as in photophysical interactions, leading to stabilization of the DNA duplex if such nucleosides can be incorporated into short oligonucleotide sequences. Therefore, the designed bases may find application in biophysical studies in the context of DNA. PMID:27181694

  17. Regulation by equilibrative nucleoside transporter of adenosine outward currents in adult rat spinal dorsal horn neurons.

    PubMed

    Liu, Tao; Fujita, Tsugumi; Kawasaki, Yasuhiko; Kumamoto, Eiichi

    2004-07-30

    A current response induced by superfusing adenosine was examined in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. In 78% of the neurons examined, adenosine induced an outward current at -70 mV [18.8 +/- 1.1 pA (n = 98) at 1mM] in a dose-dependent manner (EC(50) = 177 microM). A similar current was induced by A(1) agonist N(6)-cyclopentyladenosine (1 microM), whereas A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 microM) reversed the adenosine action. The adenosine current reversed its polarity at a potential being close to the equilibrium potential for K(+), and was attenuated by Ba(2+) (100 microM) and 4-aminopyridine (5mM) but not tetraethylammonium (5mM). The adenosine current was enhanced in duration by equilibrative nucleoside-transport (rENT1) inhibitor S-(4-nitrobenzyl)-6-thioinosine (1 microM) and adenosine deaminase (ADA) inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (1 microM), and slowed in falling phase by adenosine kinase (AK) inhibitor iodotubercidine (1 microM). We conclude that a Ba(2+)- and 4-aminopyridine-sensitive K(+) channel in SG neurons is opened via the activation of A(1) receptors by adenosine whose level is possibly regulated by rENT1, adenosine deaminase and adenosine kinase. Considering that intrathecally-administered adenosine analogues produce antinociception, the regulatory systems of adenosine may serve as targets for antinociceptive drugs. PMID:15275960

  18. Use of Nucleoside Reverse Transcriptase Inhibitor Only Regimens in HIV-infected Children and Adolescents

    PubMed Central

    Neely, Michael; Rutstein, Richard; Del Bianco, Gabriela; Heresi, Gloria; Barton, Theresa; Wiznia, Andrew; Wiegand, Ryan; Wheeling, Travis; Bohannon, Beverly; Dominguez, Kenneth

    2013-01-01

    In adults, nucleoside reverse transcriptase inhibitor (NRTI)-only antiretroviral regimens (NOARs) with ≥ three NRTIs are less potent than highly active antiretroviral therapy (HAART). However published pediatric experience with NOARs is limited. Methods We analyzed data from NOAR-treated participants in LEGACY, a multicenter observational cohort study of HIV-infected children and adolescents. NOAR-treated case-participantswere matched to participantswithout prior NOAR who initiated HAART during the same year for comparison. Results Of 575 participants with data from time of HIV diagnosis through 2006, 67 (12%) received NOARs for at least 24 weeks; most (46%) received the fixed dose combination of zidovudine/lamivudine/abacavir. NOAR use peaked in 2001-2002. NOAR-treated participants were significantly older and more treatment-experienced than HAART-treated participants. Virologic outcomes, including the percentage of participants with a plasma HIV RNA viral load <400 copies/mL at week 24 (47% vs. 34%) and the mean 24-week change in log10 plasma HIV RNA viral load from baseline (−0.63 vs. −1.02) were similar between NOAR- and HAART-treated participants, but virologic rebound was more likely in NOAR-treated participants (77% vs. 54%, P = 0.02). Increase in CD4 percentage points from baseline to 24 weeks was negligible in NOAR-treated participants compared with HAART-treated participants (0.95% vs. 10.1%, P <0.001). Anemia and leukopenia were more commonly reported with NOARs than HAART. Discussion Week 24 virologic outcomes were similar between NOAR- and HAART-treated participants, but NOAR durability was poorer and their use was associated with less immunologic reconstitution. NOARs should play a limited role in pediatric and adolescent ART. PMID:24008749

  19. Four Generations of Transition State Analogues for Human Purine Nucleoside Phosphorylase

    SciTech Connect

    Ho, M.; Shi, W; Rinaldo-Mathis, A; Tyler, P; Evans, G; Almo, S; Schramm, V

    2010-01-01

    Inhibition of human purine nucleoside phosphorylase (PNP) stops growth of activated T-cells and the formation of 6-oxypurine bases, making it a target for leukemia, autoimmune disorders, and gout. Four generations of ribocation transition-state mimics bound to PNP are structurally characterized. Immucillin-H (K*{sub i} = 58 pM, first-generation) contains an iminoribitol cation with four asymmetric carbons. DADMe-Immucillin-H (K*{sub i} = 9 pM, second-generation), uses a methylene-bridged dihydroxypyrrolidine cation with two asymmetric centers. DATMe-Immucillin-H (K*{sub i} = 9 pM, third-generation) contains an open-chain amino alcohol cation with two asymmetric carbons. SerMe-ImmH (K*{sub i} = 5 pM, fourth-generation) uses achiral dihydroxyaminoalcohol seramide as the ribocation mimic. Crystal structures of PNPs establish features of tight binding to be; (1) ion-pair formation between bound phosphate (or its mimic) and inhibitor cation, (2) leaving-group interactions to N1, O6, and N7 of 9-deazahypoxanthine, (3) interaction between phosphate and inhibitor hydroxyl groups, and (4) His257 interacting with the 5{prime}-hydroxyl group. The first generation analogue is an imperfect fit to the catalytic site with a long ion pair distance between the iminoribitol and bound phosphate and weaker interactions to the leaving group. Increasing the ribocation to leaving-group distance in the second- to fourth-generation analogues provides powerful binding interactions and a facile synthetic route to powerful inhibitors. Despite chemical diversity in the four generations of transition-state analogues, the catalytic site geometry is almost the same for all analogues. Multiple solutions in transition-state analogue design are available to convert the energy of catalytic rate enhancement to binding energy in human PNP.

  20. Cysteine Cross-linking Defines the Extracellular Gate for the Leishmania donovani Nucleoside Transporter 1.1 (LdNT1.1)*

    PubMed Central

    Valdés, Raquel; Shinde, Ujwal; Landfear, Scott M.

    2012-01-01

    Equilibrative nucleoside transporters are a unique family of proteins that enable uptake of nucleosides/nucleobases into a wide range of eukaryotes and internalize a myriad of drugs used in the treatment of cancer, heart disease, AIDs, and parasitic infections. In previous work we generated a structural model for such a transporter, the LdNT1.1 nucleoside permease from the parasitic protozoan Leishmania donovani, using ab initio computation. The model suggested that aromatic residues present in transmembrane helices 1, 2, and 7 interact to form an extracellular gate that closes the permeation pathway in the inward-open conformation. Mutation of residues Phe-48TM1 and Trp-75TM2 abrogated transport activity, consistent with such prediction. In this study cysteine mutagenesis and oxidative cross-linking were combined to analyze proximity relationships of helices 1, 2, and 7 in LdNT1.1. Disulfide bond formation between introduced paired cysteines at the interface of such helices (A61CTM1/F74CTM2, A61CTM1/G350CTM7, and F74CTM2/G350CTM7) was analyzed by transport measurement and gel mobility shifts upon oxidation with Cu (II)-(1,10-phenanthroline)3. In all cases cross-linking inhibited transport. However, if LdNT1.1 ligands were included during cross-linking, inhibition of transport was reduced, suggesting that ligands moved the three gating helices apart. Moreover, all paired cysteine mutants exhibited a mobility shift upon oxidation, corroborating the formation of a disulfide bond. These data support the notion that helices 1, 2, and 7 constitute the extracellular gate of LdNT1.1, thus further validating the computational model and the previously demonstrated importance of F48TM1 and Trp-75TM2 in tethering together helices that are part of the gate. PMID:23150661

  1. Synthesis of nucleoside and nucleotide conjugates of bile acids, and polymerase construction of bile acid-functionalized DNA.

    PubMed

    Ikonen, Satu; Macícková-Cahová, Hana; Pohl, Radek; Sanda, Miloslav; Hocek, Michal

    2010-03-01

    Aqueous Sonogashira cross-coupling reactions of 5-iodopyrimidine or 7-iodo-7-deazaadenine nucleosides with bile acid-derived terminal acetylenes linked via an ester or amide tether gave the corresponding bile acid-nucleoside conjugates. Analogous reactions of halogenated nucleoside triphosphates gave directly bile acid-modified dNTPs. Enzymatic incorporation of these modified nucleotides to DNA was successfully performed using Phusion polymerase for primer extension. One of the dNTPs (dCTP bearing cholic acid) was also efficient for PCR amplification. PMID:20165813

  2. Design and synthesis of novel 5-substituted acyclic pyrimidine nucleosides as potent and selective inhibitors of hepatitis B virus.

    PubMed

    Kumar, Rakesh; Nath, Mahendra; Tyrrell, D Lorne J

    2002-05-01

    A novel class of 5-substituted acyclic pyrimidine nucleosides, 1-[(2-hydroxyethoxy)methyl]-5-(1-azidovinyl)uracil (9a), 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]-5-(1-azidovinyl)uracil (9b), and 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl]-5-(1-azidovinyl)uracil (9c), were synthesized by regiospecific addition of bromine azide to the 5-vinyl substituent of the respective 5-vinyluracils (2a-c) followed by treatment of the obtained 5-(1-azido-2-bromoethyl) compounds (3a-c) with t-BuOK, to affect the base-catalyzed elimination of HBr. Thermal decomposition of 9b and 9c at 110 degrees C in dioxane yielded corresponding 5-[2-(1-azirinyl)]uracil analogues (10b,c). The 5-(1-azidovinyl)uracil derivatives 9a-c were found to exhibit potent and selective in vitro anti-HBV activity against duck hepatitis B virus (DHBV) infected primary duck hepatocytes at low concentrations (EC(50) = 0.01-0.1 microg/mL range). The most active anti-DHBV agent (9c), possessing a [4-hydroxy-3-(hydroxymethyl)-1-butyl] substituent at N-1, exhibited an activity (EC(50) of 0.01-0.05 microg/mL) comparable to that of reference compound (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (3-TC) (EC(50) = 0.01-0.05 microg/mL). In contrast, related 5-[2-(1-azirinyl)]uracil analogues (10b,c) were devoid of anti-DHBV activity, indicating that an acyclic side chain at C-5 position of the pyrimidine ring is essential for anti-HBV activity. The pyrimidine nucleosides (9a-c, 10b,c) exhibited no cytotoxic activity against a panel of 60 human cancer cell lines. All of the compounds investigated did not show any detectable toxicity to several stationary and proliferating host cell lines or to mitogen stimulated proliferating human T lymphocytes, up to the highest concentration tested. PMID:11985471

  3. Nucleotides as nucleophiles - Reactions of nucleotides with phosphoimidazolide activated guanosine

    NASA Technical Reports Server (NTRS)

    Kanavarioti, Anastassia; Rosenbach, Morgan T.; Hurley, T. B.

    1992-01-01

    On the basis of recently discovered RNAs with catalytic capabilities resembling those of enzymes, it is postulated that an 'RNA world' may have played a determining role in prebiotic chemistry and led evolution from prebiological to biological systems. The advent of the RNA world thus postulated, however, entails the preexistence of ribomononucleotides, and presumes that their reactions resulted in templatelike oligonucleotides. Attention is presently given to the reaction of nucleoside monophosphates with the phosphoimidazolide-activated nucleosides that (1) have successfully been used in place of the natural nucleoside triphosphates and (2) for whose prebiotic existence there is now some evidence.

  4. Basal expression of nucleoside transporter mRNA differs among small intestinal epithelia of beef steers and is differentially altered by ruminal or abomasal infusion of starch hydrolysate.

    PubMed

    Liao, S F; Alman, M J; Vanzant, E S; Miles, E D; Harmon, D L; McLeod, K R; Boling, J A; Matthews, J C

    2008-04-01

    In ruminants, microbial-derived nucleic acids are a major source of N and are absorbed as nucleosides by small intestinal epithelia. Although the biochemical activities of 2 nucleoside transport systems have been described for cattle, little is known regarding the regulation of their gene expression. This study was conducted to test 2 hypotheses: (1) the small intestinal epithelia of beef cattle differentially express mRNA for 3 concentrative (CNT1, 2, 3) and 2 equilibrative (ENT1, 2) nucleoside transporters (NT), and (2) expression of these NT is responsive to small intestine luminal supply of rumen-derived microbes (hence, nucleosides), energy (cornstarch hydrolysate, SH), or both. Eighteen ruminally and abomasally catheterized Angus steers (260 +/- 17 kg of BW) were fed an alfalfa cube-based diet at 1.33x NE(m) requirement. Six steers in each of 3 periods were blocked by BW (heavy vs. light). Within each block, 3 steers were randomly assigned to 3 treatments (n = 6): ruminal and abomasal water infusion (control), ruminal SH infusion/abomasal water infusion, or ruminal water infusion/abomasal SH infusion. The dosage of SH infusion amounted to 20% of ME intake. After a 14-or 16-d infusion period, steers were slaughtered, and duodenal, jejunal, and ileal epithelia were harvested for total RNA extraction and the relative amounts of mRNA expressed were determined using real-time RT-PCR quantification methodologies. All 5 NT mRNA were found expressed by each epithelium, but their abundance differed among epithelia. Specifically, jejunal expression of all 5 NT mRNA was higher than that by the ileum, whereas jejunal expression of CNT1, CNT3, and ENT1 mRNA was higher, or tended to be higher, than duodenal expression. Duodenal expression of CNT2, CNT3, and ENT2 mRNA was higher than ileal expression. With regard to SH infusion treatments, ruminal infusion increased duodenal expression of CNT3 (67%), ENT1 (51%), and ENT2 (39%) mRNA and ileal expression of CNT3 (210%) and

  5. YZGD from Paenibacillus thiaminolyticus, a pyridoxal phosphatase of the HAD (haloacid dehalogenase) superfamily and a versatile member of the Nudix (nucleoside diphosphate x) hydrolase superfamily.

    PubMed

    Tirrell, Isaac M; Wall, Jennifer L; Daley, Christopher J; Denial, Sarah J; Tennis, Frances G; Galens, Kevin G; O'Handley, Suzanne F

    2006-03-15

    YZGD from Paenibacillus thiaminolyticus is a novel bifunctional enzyme with both PLPase (pyridoxal phosphatase) and Nudix (nucleoside diphosphate x) hydrolase activities. The PLPase activity is catalysed by the HAD (haloacid dehalogenase) superfamily motif of the enzyme, and the Nudix hydrolase activity is catalysed by the conserved Nudix signature sequence within a separate portion of the enzyme, as confirmed by site-directed mutagenesis. YZGD's phosphatase activity is very specific, with pyridoxal phosphate being the only natural substrate, while YZGD's Nudix activity is just the opposite, with YZGD being the most versatile Nudix hydrolase characterized to date. YZGD's Nudix substrates include the CDP-alcohols (CDP-ethanol, CDP-choline and CDP-glycerol), the ADP-coenzymes (NADH, NAD and FAD), ADP-sugars, TDP-glucose and, to a lesser extent, UDP- and GDP-sugars. Regardless of the Nudix substrate, one of the products is always a nucleoside monophosphate, suggesting a role in nucleotide salvage. Both the PLPase and Nudix hydrolase activities require a bivalent metal cation, but while PLPase activity is supported by Co2+, Mg2+, Zn2+ and Mn2+, the Nudix hydrolase activity is Mn2+-specific. YZGD's phosphatase activity is optimal at an acidic pH (pH 5), while YZGD's Nudix activities are optimal at an alkaline pH (pH 8.5). YZGD is the first enzyme reported to be a member of both the HAD and Nudix hydrolase superfamilies, the first PLPase to be recognized as a member of the HAD superfamily and the first Nudix hydrolase capable of hydrolysing ADP-x, CDP-x and TDP-x substrates with comparable substrate specificity. PMID:16336194

  6. YZGD from Paenibacillus thiaminolyticus, a pyridoxal phosphatase of the HAD (haloacid dehalogenase) superfamily and a versatile member of the Nudix (nucleoside diphosphate x) hydrolase superfamily

    PubMed Central

    Tirrell, Isaac M.; Wall, Jennifer L.; Daley, Christopher J.; Denial, Sarah J.; Tennis, Frances G.; Galens, Kevin G.; O'Handley, Suzanne F.

    2005-01-01

    YZGD from Paenibacillus thiaminolyticus is a novel bifunctional enzyme with both PLPase (pyridoxal phosphatase) and Nudix (nucleoside diphosphate x) hydrolase activities. The PLPase activity is catalysed by the HAD (haloacid dehalogenase) superfamily motif of the enzyme, and the Nudix hydrolase activity is catalysed by the conserved Nudix signature sequence within a separate portion of the enzyme, as confirmed by site-directed mutagenesis. YZGD's phosphatase activity is very specific, with pyridoxal phosphate being the only natural substrate, while YZGD's Nudix activity is just the opposite, with YZGD being the most versatile Nudix hydrolase characterized to date. YZGD's Nudix substrates include the CDP-alcohols (CDP-ethanol, CDP-choline and CDP-glycerol), the ADP-coenzymes (NADH, NAD and FAD), ADP-sugars, TDP-glucose and, to a lesser extent, UDP- and GDP-sugars. Regardless of the Nudix substrate, one of the products is always a nucleoside monophosphate, suggesting a role in nucleotide salvage. Both the PLPase and Nudix hydrolase activities require a bivalent metal cation, but while PLPase activity is supported by Co2+, Mg2+, Zn2+ and Mn2+, the Nudix hydrolase activity is Mn2+-specific. YZGD's phosphatase activity is optimal at an acidic pH (pH 5), while YZGD's Nudix activities are optimal at an alkaline pH (pH 8.5). YZGD is the first enzyme reported to be a member of both the HAD and Nudix hydrolase superfamilies, the first PLPase to be recognized as a member of the HAD superfamily and the first Nudix hydrolase capable of hydrolysing ADP-x, CDP-x and TDP-x substrates with comparable substrate specificity. PMID:16336194

  7. Antiviral activities of isometric dideoxynucleosides of D- and L-related stereochemistry.

    PubMed Central

    Nair, V; Jahnke, T S

    1995-01-01

    In summary, many isomeric analogs of ddNs of both D-related and L-related absolute stereochemistries have been synthesized and evaluated in vitro for their antiviral activities. A few of these compounds exhibit potent antiviral activity and, interestingly, belong to both the D and L families. The synthetic methodologies developed will allow accessibility to many more novel modified nucleosides. While some structure-activity relationships are emerging from this work, it is clear that these chiral isomeric nucleosides have opened a new chapter in the field of antiviral nucleosides. PMID:7625783

  8. Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening.