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Sample records for apyrase nucleoside triphosphate-diphosphohydrolase

  1. Leishmania infantum ecto-nucleoside triphosphate diphosphohydrolase-2 is an apyrase involved in macrophage infection and expressed in infected dogs.

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    Raphael De Souza Vasconcellos

    2014-11-01

    Full Text Available Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (∼70 kDa and ∼45 kDa have been found in the L. infantum genome. Here, we studied the ∼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection.We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP  =  UDP> ADP> UTP  =  ATP in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis.In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in naturally infected dogs. Additionally, the blockade of

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

    Science.gov (United States)

    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

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

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    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

  4. Leishmania (Viannia) braziliensis nucleoside triphosphate diphosphohydrolase (NTPDase 1): localization and in vitro inhibition of promastigotes growth by polyclonal antibodies.

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    Porcino, Gabriane Nascimento; Carvalho-Campos, Cristiane; Maia, Ana Carolina Ribeiro Gomes; Detoni, Michelle Lima; Faria-Pinto, Priscila; Coimbra, Elaine Soares; Marques, Marcos José; Juliano, Maria Aparecida; Juliano, Luiz; Diniz, Vanessa Álvaro; Corte-Real, Suzana; Vasconcelos, Eveline Gomes

    2012-10-01

    Nucleoside triphosphate diphosphohydrolase (NTPDase) activity was recently characterized in Leishmania (Viannia) braziliensis promastigotes (Lb), and an antigenic conserved domain (r82-121) from the specific NTPDase 1 isoform was identified. In this work, mouse polyclonal antibodies produced against two synthetic peptides derived from this domain (LbB1LJ, r82-103; LbB2LJ, r102-121) were used. The anti-LbB1LJ or anti-LbB2LJ antibodies were immobilized on protein A-sepharose and immunoprecipitated the NTPDase 1 of 48 kDa and depleted approximately 40% of the phosphohydrolytic activity from detergent-homogenized Lb preparation. Ultrastructural immunocytochemical microscopy identified the NTPDase 1 on the parasite surface and in its subcellular cytoplasmic vesicles, mitochondria, kinetoplast and nucleus. The ATPase and ADPase activities of detergent-homogenized Lb preparation were partially inhibited by anti-LbB1LJ antibody (43-79%), which was more effective than that inhibition (18-47%) by anti-LbB2LJ antibody. In addition, the immune serum anti-LbB1LJ (67%) or anti-LbB2LJ (33%) was cytotoxic, significantly reducing the promastigotes growth in vitro. The results appoint the conserved domain from the L. braziliensis NTPDase as an important target for inhibitor design and the potential application of these biomolecules in experimental protocols of disease control. PMID:22921497

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

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    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

  6. The biochemical properties of the Arabidopsis ecto-nucleoside triphosphate diphosphohydrolase AtAPY1 contradict a direct role in purinergic signaling.

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    Carolin Massalski

    Full Text Available The Arabidopsis E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase AtAPY1 was previously shown to be involved in growth and development, pollen germination and stress responses. It was proposed to perform these functions through regulation of extracellular ATP signals. However, a GFP-tagged version was localized exclusively in the Golgi and did not hydrolyze ATP. In this study, AtAPY1 without the bulky GFP-tag was biochemically characterized with regard to its suggested role in purinergic signaling. Both the full-length protein and a soluble form without the transmembrane domain near the N-terminus were produced in HEK293 cells. Of the twelve nucleotide substrates tested, only three--GDP, IDP and UDP--were hydrolyzed, confirming that ATP was not a substrate of AtAPY1. In addition, the effects of pH, divalent metal ions, known E-NTPDase inhibitors and calmodulin on AtAPY1 activity were analyzed. AtAPY1-GFP extracted from transgenic Arabidopsis seedlings was included in the analyses. All three AtAPY1 versions exhibited very similar biochemical properties. Activity was detectable in a broad pH range, and Ca(2+, Mg(2+ and Mn(2+ were the three most efficient cofactors. Of the inhibitors tested, vanadate was the most potent one. Surprisingly, sulfonamide-based inhibitors shown to inhibit other E-NTPDases and presumed to inhibit AtAPY1 as well were not effective. Calmodulin stimulated the activity of the GFP-tagless membranous and soluble AtAPY1 forms about five-fold, but did not alter their substrate specificities. The apparent Km values obtained with AtAPY1-GFP indicate that AtAPY1 is primarily a GDPase. A putative three-dimensional structural model of the ecto-domain is presented, explaining the potent inhibitory potential of vanadate and predicting the binding mode of GDP. The found substrate specificity classifies AtAPY1 as a nucleoside diphosphatase typical of N-terminally anchored Golgi E-NTPDases and negates a direct function in

  7. Crystal Structure of a Legionella pneumophila Ecto -Triphosphate Diphosphohydrolase, A Structural and Functional Homolog of the Eukaryotic NTPDases

    Energy Technology Data Exchange (ETDEWEB)

    Vivian, Julian P.; Riedmaier, Patrice; Ge, Honghua; Le Nours, Jérôme; Sansom, Fiona M.; Wilce, Matthew C.J.; Byres, Emma; Dias, Manisha; Schmidberger, Jason W.; Cowan, Peter J.; d' Apice, Anthony J.F.; Hartland, Elizabeth L.; Rossjohn, Jamie; Beddoe, Travis (Monash); (Melbourne)

    2010-04-19

    Many pathogenic bacteria have sophisticated mechanisms to interfere with the mammalian immune response. These include the disruption of host extracellular ATP levels that, in humans, is tightly regulated by the nucleoside triphosphate diphosphohydrolase family (NTPDases). NTPDases are found almost exclusively in eukaryotes, the notable exception being their presence in some pathogenic prokaryotes. To address the function of bacterial NTPDases, we describe the structures of an NTPDase from the pathogen Legionella pneumophila (Lpg1905/Lp1NTPDase) in its apo state and in complex with the ATP analog AMPPNP and the subtype-specific NTPDase inhibitor ARL 67156. Lp1NTPDase is structurally and catalytically related to eukaryotic NTPDases and the structure provides a basis for NTPDase-specific inhibition. Furthermore, we demonstrate that the activity of Lp1NTPDase correlates directly with intracellular replication of Legionella within macrophages. Collectively, these findings provide insight into the mechanism of this enzyme and highlight its role in host-pathogen interactions.

  8. Extracellular ATP signaling and homeostasis in plant cells

    OpenAIRE

    Sun, Jian; Zhang, Chunlan; Zhang, Xuan; Deng, Shurong; Zhao, Rui; Shen, Xin; Chen, Shaoliang

    2012-01-01

    Extracellular ATP (eATP) is now recognized as an important signaling agent in plant growth and defense response to environmental stimuli. eATP has dual functions in plant cell signaling, which is largely dependent on its concentration in the extracellular matrix (ECM). A lethal level of eATP (extremely low or high) causes cell death, whereas a moderate level of eATP benefits plant growth and development. Ecto-apyrases (Nucleoside Triphosphate-Diphosphohydrolase) help control the eATP concentr...

  9. YND1, a homologue of GDA1, encodes membrane-bound apyrase required for Golgi N- and O-glycosylation in Saccharomyces cerevisiae.

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    Gao, X D; Kaigorodov, V; Jigami, Y

    1999-07-23

    The gene for the open reading frame YER005w that is homologous to yeast Golgi GDPase encoded by the GDA1 gene was cloned and named YND1. It encodes a 630-amino acid protein that contains a single transmembrane region near the carboxyl terminus. The overexpression of the YND1 gene in the gda1 null mutant caused a significant increase in microsomal membrane-bound nucleoside phosphatase activity with a luminal orientation. The activity was equally high toward ADP/ATP, GDP/GTP, and UDP/UTP and approximately 50% less toward CDP/CTP and thiamine pyrophosphate, but there was no activity toward GMP, indicating that the Ynd1 protein belongs to the apyrase family. This substrate specificity is different from that of yeast GDPase, but similar to that of human Golgi UDPase. The Deltaynd1 mutant cells were defective in O- and N-linked glycosylation in the Golgi compartments. The overexpression of the YND1 gene complemented some glycosylation defects in Deltagda1 disruptants, suggesting a partially redundant function of yeast apyrase and GDPase. From these results and the phenotype of the Deltaynd1Deltagda1 double deletion showing a synthetic effect, we conclude that yeast apyrase is required for Golgi glycosylation and cell wall integrity, providing the first direct evidence for the in vivo function of intracellular apyrase in eukaryotic cells. PMID:10409709

  10. Nucleoside Sensing

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    Thomas Schrader; Frank-Gerrit Klärner; Michael Fokkens

    2006-01-01

    A rigid molecular clip comprising bisphosphonate binding sites and aromatic sidewalls forming an electron-rich cavity is able to distinguish between nucleosides and nucleotides in aqueous solution. Neutral nucleosides as well as antibiotics derived thereof are drawn into the unpolar interior of the cleft and lead to substantial upfield-shifts in the 1H NMR spectrum. Nucleoside drugs can therefore be detected with high selectivity in the presence of their phosphorylated pendants or nucleic acids.

  11. Alteration in apyrase enzyme attenuated virulence of Shigella flexneri.

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    BangaSingh, Kirnpal Kaur; Nisha, Mehru; Lau, Hut Yee; Ravichandran, Manickam; Salleh, Mohd Zaki

    2016-02-01

    Virulence of Shigella is attributed to the genes presence in chromosome or in the megaplasmid. The apy gene which is located in the megaplasmid of Shigella species encodes for apyrase enzyme, a pathogenesis-associated enzyme causing mitochondrial damage and host cell death. In this study we constructed an apy mutant of Shigella flexneri by insertional activation using a kanamycin resistant gene cassette. The wild type apy gene of S. flexneri 2a was PCR amplified, cloned and mutated with insertion of kanamycin resistant gene cassette (aphA). The mutated construct (apy: aphA) was subcloned into a conjugative suicidal vector (pWM91) at the unique Sma1 and Sac1 sites. The mutation of the wild apy gene in the construct was confirmed by DNA sequencing. The mutated construct was introduced into wild type S. flexneri 2a by conjugation with Escherichia coli. After undergoing homologous recombination, the wild apy gene was deleted from the construct using the sucrose selection method. Non-functional activity of the apyrase enzyme in the constructed strain by colorimetric test indicated the successful mutation of the apyrase enzyme. This strain with mutated apy gene was evaluated for its protective efficacy using the guinea pig keratoconjunctivitis model. The strain was Sereny negative and it elicited a significant protection following challenge with wild S. flexneri strain. This apy mutant strain will form a base for the development of a vaccine target for shigellosis. PMID:26706344

  12. Analysis on the Interaction Domain of VirG and Apyrase by Pull-Down Assay

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    Yu Wang

    2014-11-01

    Full Text Available VirG is outer membrane protein of Shigella and affects the spread of Shigella. Recently it has been reported that apyrase influences the location of VirG, although the underlying mechanism remains poorly understood. The site of interaction between apyrase and VirG is the focus of our research. First we constructed recombinant plasmid pHIS-phoN2 and pS-(v1–1102, v53–758, v759–1102, v53–319, v320–507, v507–758 by denaturation-renaturation, the phoN2:kan mutant of Shigella flexneri 5a M90T by a modified version of the lambda red recombination protocol originally described by Datsenko and Wanner and the complemented strain M90TΔphoN2/pET24a(PhisphoN2. Second, the recombinant plasmid pHIS-phoN2 and the pS-(v1–1102, v53–758, v759–1102, v53–319, v320–507, v507–758 were transformed into E. coli BL21 (DE3 and induced to express the fusion proteins. Third, the fusion proteins were purified and the interaction of VirG and apyrase was identified by pull-down. Fourth, VirG was divided and the interaction site of apyrase and VirG was determined. Finally, how apyrase affects the function of VirG was analyzed by immunofluorescence. Accordingly, the results provided the data supporting the fact that apyrase combines with the α-domain of VirG to influence the function of VirG.

  13. Histochemical Detection of Ischemia-Like Alterations Induced in Kidney Tissue in vitro - Different Sensitivity to Oxidant Stress of Glomerular ENTPD1 versus E5NT

    NARCIS (Netherlands)

    Vlaar, Alexander P. J.; van Son, Willem J.; Bakker, Winston W.

    2009-01-01

    The expression of ENTPD1 (ecto-nucleoside triphosphate diphosphohydrolase) along the glomerular microvasculature of the kidney is downregulated in ischemic conditions, in contrast to E5NT (ecto-5'-nucleotidase), which may explain the increased tendency for intraglomerular microthrombus formation in

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

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    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

  15. In vivo glioblastoma growth is reduced by apyrase activity in a rat glioma model

    International Nuclear Information System (INIS)

    ATP is an important signalling molecule in the peripheral and central nervous system. Both glioma growth and tumor resection induces cell death, thus liberating nucleotides to the extracellular medium. Nucleotides are hydrolyzed very slowly by gliomas when compared with astrocytes and induce neuronal cell death and glioma proliferation. The objective of the present study was to test the involvement of extracellular ATP in glioblastoma growth in a rat glioma model. To deplete the extracellular ATP, the enzyme apyrase was tested on the treatment of gliomas implanted in the rats CNS. One million glioma C6 cells in 3 microliters of DMEM/FCS were injected in the right striata of male Wistar rats, 250–270 g. After 20 days, the rats were decapitated and the brain sectioning and stained with hematoxylin and eosine. We performed immunohistochemical experiments with Ki67, CD31 and VEGF. Total RNA was isolated from cultured glioma C6 cells and the cDNA was analyzed by Real Time-PCR with primers for the NTPDase family. C6 glioma cells effectively have a low expression of all NTPDases investigated, in comparison with normal astrocytes. The implanted glioma co-injected with apyrase had a significant reduction in the tumor size (p < 0.05) when compared with the rats injected only with gliomas or with gliomas plus inactivated apyrase. According to the pathological analysis, the malignant gliomas induced by C6 injection and co-injected with apyrase presented a significant reduction in the mitotic index and other histological characteristics that indicate a less invasive/proliferative tumor. Reduction of proliferation induced by apyrase co-injection was confirmed by counting the percentage of Ki67 positive glioma cell nuclei. According to counts with CD31, vessel density and neoformation was higher in the C6 group 20 days after implantation. Confirming this observation, rats treated with apyrase presented less VEGF staining in comparison to the control group. These results

  16. Ticlopidine in Its Prodrug Form Is a Selective Inhibitor of Human NTPDase1

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    Joanna Lecka

    2014-01-01

    Full Text Available Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, like other ectonucleotidases, controls extracellular nucleotide levels and consequently their (pathophysiological responses such as in thrombosis, inflammation, and cancer. Selective NTPDase1 inhibitors would therefore be very useful. We previously observed that ticlopidine in its prodrug form, which does not affect P2 receptor activity, inhibited the recombinant form of human NTPDase1 (Ki=14 μM. Here we tested whether ticlopidine can be used as a selective inhibitor of NTPDase1. We confirmed that ticlopidine inhibits NTPDase1 in different forms and in different assays. The ADPase activity of intact HUVEC as well as of COS-7 cells transfected with human NTPDase1 was strongly inhibited by 100 µM ticlopidine, 99 and 86%, respectively. Ticlopidine (100 µM completely inhibited the ATPase activity of NTPDase1 in situ as shown by enzyme histochemistry with human liver and pancreas sections. Ticlopidine also inhibited the activity of rat and mouse NTPDase1 and of potato apyrase. At 100 µM ticlopidine did not affect the activity of human NTPDase2, NTPDase3, and NTPDase8, nor of NPP1 and NPP3. Weak inhibition (10–20% of NTPDase3 and -8 was observed at 1 mM ticlopidine. These results show that ticlopidine is a specific inhibitor of NTPDase1 that can be used in enzymatic and histochemistry assays.

  17. Piperidine nucleosides and nucleotides

    Czech Academy of Sciences Publication Activity Database

    Kovačková, Soňa; Dračínský, Martin; Rosenberg, Ivan; Rejman, Dominik

    Lyon : Université de Lyon, 2010, s. 315-316. [International Roundtable on Nucleosides, Nucleotides and Nucleic Acids. IRT 2010. Lyon (FR), 29.08.2010-03.09.2010] Institutional research plan: CEZ:AV0Z40550506 Keywords : phosphonate analogs * piperidine nucleosides * piperidine nucleotides Subject RIV: CC - Organic Chemistry http://irt2010.univ-lyon1.fr

  18. NTPDase2 and Purinergic Signaling Control Progenitor Cell Proliferation in Neurogenic Niches of the Adult Mouse Brain

    OpenAIRE

    Gampe, Kristine; Stefani, Jennifer; Hammer, Klaus; Brendel, Peter; Pötzsch, Alexandra; Enikolopov, Grigori; Enjyoji, Keiichi; Acker-Palmer, Amparo; Robson, Simon C.; Zimmermann, Herbert

    2015-01-01

    Nerve cells are continuously generated from stem cells in the adult mammalian subventricular zone (SVZ) and hippocampal dentate gyrus. We have previously noted that stem/progenitor cells in the SVZ and the subgranular layer (SGL) of the dentate gyrus express high levels of plasma membrane-bound nucleoside triphosphate diphosphohydrolase 2 (NTPDase2), an ectoenzyme that hydrolyzes extracellular nucleoside di- and triphosphates. We inferred that deletion of NTPDase2 would increase local extrace...

  19. Golgi-Located NTPDase1 of Leishmania major Is Required for Lipophosphoglycan Elongation and Normal Lesion Development whereas Secreted NTPDase2 Is Dispensable for Virulence

    OpenAIRE

    Sansom, Fiona M.; Julie E Ralton; M Fleur Sernee; Cohen, Alice M.; Hooker, David J.; Hartland, Elizabeth L.; Thomas Naderer; McConville, Malcolm J.

    2014-01-01

    Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri- and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence an...

  20. A calcium-dependent, structural homologue of a novel salivary apyrase from blood-feeding arthropods has been identified in Ostertagia third stage larvae

    Science.gov (United States)

    Apyrases (ATP diphosphohydrolase) comprise a ubiquitous class of glycosylated nucleotidases that hydrolyze extracellular ATP and ADP to orthophosphate and AMP. Most apyrases have been structurally linked to the heat shock70/sugar kinase/actin superfamily which is associated with motility, adhesion, ...

  1. Functional characterization of a salivary apyrase from the sand fly, Phlebotomus duboscqi, a vector of Leishmania major.

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    Hamasaki, Ryoichi; Kato, Hirotomo; Terayama, Yoshimi; Iwata, Hiroyuki; Valenzuela, Jesus G

    2009-11-01

    Two transcripts coding for proteins homologous to apyrases were identified by massive sequencing of a Phlebotomus (P.) duboscqi salivary gland cDNA library. The sequence analysis revealed that the amino acids important for enzymatic activity including nucleotidase activity and the binding of calcium and nucleotides were well conserved in these molecules. A recombinant P. duboscqi salivary apyrase was expressed in Escherichia coli and purified. The resulting protein efficiently hydrolyzed ADP and ATP, but not AMP, GDP, CDP or UDP, in a calcium-dependent manner. Further, the recombinant protein inhibited ADP- and collagen-induced platelet aggregation. The results indicated that this salivary protein plays an important role in the blood-feeding process in P. duboscqi. Its unique enzymatic activity makes the salivary apyrase an attractive candidate as a therapeutic agent for the treatment of thrombotic pathologies as well as a reagent for a wide variety of research purposes. PMID:19651132

  2. Extracellular Nucleotides and Apyrases Regulate Stomatal Aperture in Arabidopsis1[W][OA

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    Clark, Greg; Fraley, Devin; Steinebrunner, Iris; Cervantes, Andrew; Onyirimba, James; Liu, Angela; Torres, Jonathan; Tang, Wenqiang; Kim, Joshua; Roux, Stanley J.

    2011-01-01

    This study investigates the role of extracellular nucleotides and apyrase enzymes in regulating stomatal aperture. Prior data indicate that the expression of two apyrases in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, is strongly correlated with cell growth and secretory activity. Both are expressed strongly in guard cell protoplasts, as determined by reverse transcription-polymerase chain reaction and immunoblot analyses. Promoter activity assays for APY1 and APY2 show that expression of both apyrases correlates with conditions that favor stomatal opening. Correspondingly, immunoblot data indicate that APY expression in guard cell protoplasts rises quickly when these cells are moved from darkness into light. Both short-term inhibition of ectoapyrase activity by polyclonal antibodies and long-term suppression of APY1 and APY2 transcript levels significantly disrupt normal stomatal behavior in light. Stomatal aperture shows a biphasic response to applied adenosine 5′-[γ-thio]triphosphate (ATPγS) or adenosine 5′-[β-thio] diphosphate, with lower concentrations inducing stomatal opening and higher concentrations inducing closure. Equivalent concentrations of adenosine 5′-O-thiomonophosphate have no effect on aperture. Two mammalian purinoceptor inhibitors block ATPγS- and adenosine 5′-[β-thio] diphosphate-induced opening and closing and also partially block the ability of abscisic acid to induce stomatal closure and of light to induce stomatal opening. Treatment of epidermal peels with ATPγS induces increased levels of nitric oxide and reactive oxygen species, and genetically suppressing the synthesis of these agents blocks the effects of nucleotides on stomatal aperture. A luciferase assay indicates that treatments that induce either the closing or opening of stomates also induce the release of ATP from guard cells. These data favor the novel conclusion that ectoapyrases and extracellular nucleotides play key roles in regulating stomatal functions

  3. Detection of IgG1 and IgG4 subtypes reactive against potato apyrase in schistosomiasis patients

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    Priscila de Faria-Pinto

    2010-07-01

    Full Text Available In this paper, we showed for the first time that the conserved domains within Schistosoma mansoni ATP diphosphohydrolase isoforms, shared with potato apyrase, possess epitopes for the IgG1 and IgG4 subtypes, as 24 (80% of the 30 schistosomiasis patients were seropositive for this vegetable protein. The analyses for each patient cured (n = 14 after treatment (AT with praziquantel revealed variable IgG1 and IgG4 reactivity against potato apyrase. Different antigenic epitopes shared between the vegetable and parasite proteins could be involved in susceptibility or resistance to S. mansoni AT with praziquantel and these possibilities should be explored.

  4. Piperidine nucleosides and nucleotides

    Czech Academy of Sciences Publication Activity Database

    Kovačková, Soňa; Pačes, Ondřej; Dračínský, Martin; Rosenberg, Ivan; Rejman, Dominik

    -, č. 52 (2008), s. 587-587. ISSN 0261-3166. [Joint Symposium of the International Roundtable on Nucleosides, Nucleotides and Nucleic Acids /18./ and the International Symposium on Nucleic Acid Chemistry /35./. Kyoto, 08.09.2008-12.09.2008] R&D Projects: GA MŠk(CZ) LC06077; GA MŠk 2B06065; GA MŠk LC512 Grant ostatní: GA MZd(CZ) NR9138 Institutional research plan: CEZ:AV0Z40550506 Keywords : piperidine * nucleosidation * phosphonate Subject RIV: CC - Organic Chemistry

  5. Acyclic Nucleoside Phosphonates

    Czech Academy of Sciences Publication Activity Database

    Holý, Antonín; Jansa, Petr

    Beijing: -, 2009. s. 119-119. [BIT's Annual World Summit of Antivirals 2009 /2./. 18.07.2009-20.07.2009, Beijing] R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * tenofovir * adefovir Subject RIV: CC - Organic Chemistry

  6. Piperidine nucleosides and nucleotides

    Czech Academy of Sciences Publication Activity Database

    Kovačková, Soňa; Pačes, Ondřej; Dračínský, Martin; Rosenberg, Ivan; Rejman, Dominik

    Manchester : University of Manchester, 2009. s. 50-50. [Nucleic Acids at the Chemistry - Biology Interface. 07.09.2009-08.09.2009, Manchester] R&D Projects: GA MZd NR9138; GA MŠk 2B06065 Institutional research plan: CEZ:AV0Z40550506 Keywords : phosphonate * piperidine nucleosides * nucleotides Subject RIV: CC - Organic Chemistry

  7. Piperidine nucleoside phosphonic acid derivatives

    Czech Academy of Sciences Publication Activity Database

    Kovačková, Soňa; Dračínský, Martin; Rejman, Dominik

    Praha : Institute of Organic Chemistry and Biochemistry AS CR, v. v. i., 2011 - (Hocek, M.), s. 372-374 ISBN 978-80-86241-37-1. - (Collection Symposium Series. 12). [Chemistry of Nucleic Acid Components /15./. Český Krumlov (CZ), 05.06.2011-10.06.2011] Institutional research plan: CEZ:AV0Z40550506 Keywords : piperidine nucleoside * nucleoside phosphonic acids * nucleoside diphosphate analogs * nucleotide analogs Subject RIV: CC - Organic Chemistry

  8. Nucleoside Inhibitors of Zika Virus.

    Science.gov (United States)

    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

  9. The Arabidopsis apyrase AtAPY1 is localized in the Golgi instead of the extracellular space

    OpenAIRE

    Schiller Madlen; Massalski Carolin; Kurth Thomas; Steinebrunner Iris

    2012-01-01

    Abstract Background The two highly similar Arabidopsis apyrases AtAPY1 and AtAPY2 were previously shown to be involved in plant growth and development, evidently by regulating extracellular ATP signals. The subcellular localization of AtAPY1 was investigated to corroborate an extracellular function. Results Transgenic Arabidopsis lines expressing AtAPY1 fused to the SNAP-(O6-alkylguanine-DNA alkyltransferase)-tag were used for indirect immunofluorescence and AtAPY1 was detected in punctate st...

  10. [Purine nucleoside phosphorylase].

    Science.gov (United States)

    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

  11. Studies on the sodium borohydride reduction of unsaturated keto nucleosides. Novel route to deoxy nucleosides

    International Nuclear Information System (INIS)

    Reduction of α,β-unsaturated (ketohexosyl)purines, which constitute the first examples of unsaturated keto nucleosides, with sodium borohydride gave the corresponding deoxy nucleosides. Contrary to the recently reported (4',6'-dideoxy-β-L-glycero-hex3'-enopyranosulosyl)purines, which were obtained by acetylation of the corresponding keto nucleosides, the α anomer, subsequently described, was prepared by oxidation of the partially protected deoxyhexosylpurine. The mechanisms of these reductions were established by a study of the NMR spectra of the deoxy nucleosides using NaBH4 in deuterated solvents and sodium borodeuteride in light solvents. 1,2 addition of the hydride was shown to be the mode of reduction of all the studied α- and β-unsaturated keto nucleosides. The ready availability of these unsaturated keto nucleosides provides extremely useful synthetic intermediate nucleosides especially for the preparation of nucleosides containing rare deoxy sugars

  12. The Arabidopsis apyrase AtAPY1 is localized in the Golgi instead of the extracellular space

    Directory of Open Access Journals (Sweden)

    Schiller Madlen

    2012-07-01

    Full Text Available Abstract Background The two highly similar Arabidopsis apyrases AtAPY1 and AtAPY2 were previously shown to be involved in plant growth and development, evidently by regulating extracellular ATP signals. The subcellular localization of AtAPY1 was investigated to corroborate an extracellular function. Results Transgenic Arabidopsis lines expressing AtAPY1 fused to the SNAP-(O6-alkylguanine-DNA alkyltransferase-tag were used for indirect immunofluorescence and AtAPY1 was detected in punctate structures within the cell. The same signal pattern was found in seedlings stably overexpressing AtAPY1-GFP by indirect immunofluorescence and live imaging. In order to identify the nature of the AtAPY1-positive structures, AtAPY1-GFP expressing seedlings were treated with the endocytic marker stain FM4-64 (N-(3-triethylammoniumpropyl-4-(p-diethylaminophenyl-hexatrienyl-pyridinium dibromide and crossed with a transgenic line expressing the trans-Golgi marker Rab E1d. Neither FM4-64 nor Rab E1d co-localized with AtAPY1. However, live imaging of transgenic Arabidopsis lines expressing AtAPY1-GFP and either the fluorescent protein-tagged Golgi marker Membrin 12, Syntaxin of plants 32 or Golgi transport 1 protein homolog showed co-localization. The Golgi localization was confirmed by immunogold labeling of AtAPY1-GFP. There was no indication of extracellular AtAPY1 by indirect immunofluorescence using antibodies against SNAP and GFP, live imaging of AtAPY1-GFP and immunogold labeling of AtAPY1-GFP. Activity assays with AtAPY1-GFP revealed GDP, UDP and IDP as substrates, but neither ATP nor ADP. To determine if AtAPY1 is a soluble or membrane protein, microsomal membranes were isolated and treated with various solubilizing agents. Only SDS and urea (not alkaline or high salt conditions were able to release the AtAPY1 protein from microsomal membranes. Conclusions AtAPY1 is an integral Golgi protein with the substrate specificity typical for Golgi apyrases. It is

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

    Directory of Open Access Journals (Sweden)

    Ri-Ming Huang

    2014-12-01

    Full Text Available 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.

  14. Base-Modified Nucleosides: Etheno Derivatives

    Science.gov (United States)

    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

  15. ATP release, generation and hydrolysis in exocrine pancreatic duct cells

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena; Yegutkin, G.G.; Novak, Ivana

    2015-01-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our...... dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting...

  16. Synthetic approaches to novel acyclic nucleoside phosphonates

    Czech Academy of Sciences Publication Activity Database

    Janeba, Zlatko

    Riga: -, 2013. s. 31-31. [Conference on Heterocycles in Bio-organic Chemistry /15./. 27.05.2013-30.05.2013, Riga] Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * antiviral * anticancer Subject RIV: CC - Organic Chemistry

  17. Prebiotic phosphorylation of nucleosides in formamide

    Science.gov (United States)

    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.

  18. Pyrrolidine analogues of nucleosides and nucleotides

    Czech Academy of Sciences Publication Activity Database

    Rejman, Dominik; Pohl, Radek; Kovačková, Soňa; Kočalka, Petr; Švenková, Alžběta; Šanderová, Hana; Krásný, Libor; Rosenberg, Ivan

    -, č. 52 (2008), s. 577-578. ISSN 0261-3166. [Joint Symposium of the International Roundtable on Nucleosides, Nucleotides and Nucleic Acids /18./ and the International Symposium on Nucleic Acid Chemistry /35./. Kyoto, 08.09.2008-12.09.2008] R&D Projects: GA MŠk(CZ) LC06077; GA MŠk 2B06065; GA MZd NR9138 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50200510; CEZ:AV0Z50520514 Keywords : pyrrolidine * nucleoside * nucleotide Subject RIV: CC - Organic Chemistry

  19. Structure-activity-relationships (SAR) in pyrimidine nucleoside transport

    International Nuclear Information System (INIS)

    Several series of pyrimidine nucleosides were evaluated as part of a larger program to develop non-invasive brain imaging agents. The interaction of these antitumor/antiviral nucleosides with an NBMPR-sensitive murine erythroctye nucleoside transporter was evaluated by determining their inhibitory effect (Ki) on zero-trans influx of thymidine. Within each series of compounds, which had F, Cl, Br or I as halogen substituents, an increase in size of the halogen atom or a decrease in electronegativity decreased affinity for the transporter. Partition coefficients (P) of these pyrimidine nucleosides were measured to determine their potential to diffuse across the blood-brain-barrier (BBB). Most of the pyrimidine nucleosides had lower P values (log P i for nucleosides with a particular sugar moiety. Within a nucleoside series with a given sugar component, the binding affinity for the transporter was inversely proportional to lipophilicity. 25 refs., 2 tabs., 4 figs

  20. Novel modular approach to C-nucleosides

    Czech Academy of Sciences Publication Activity Database

    Hocek, Michal; Urban, Milan; Joubert, Nicolas

    Kyoto : The Kinki Chemical Society, 2006. s. 183. [International Kyoto Conference on New Aspects of Organic Chemistry /10./. 13.11.2006-17.11.2006, Kyoto] R&D Projects: GA MŠk(CZ) LC512 Institutional research plan: CEZ:AV0Z40550506 Keywords : purines * cross-couplings * nucleosides Subject RIV: CC - Organic Chemistry

  1. Tetrofuranose nucleoside phosphonic acids: Synthesis and properties

    Czech Academy of Sciences Publication Activity Database

    Poláková, Ivana; Buděšínský, Miloš; Točík, Zdeněk; Rosenberg, Ivan

    2011-01-01

    Roč. 76, č. 5 (2011), s. 503-536. ISSN 0010-0765 R&D Projects: GA AV ČR KAN200520801; GA ČR GA203/09/0820; GA MŠk(CZ) LC06061; GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z40550506 Keywords : tetrofuranosyl phosphonate * nucleotide analogues * phosphonomethoxy nucleosides * sugar hydroxyphosphonates Subject RIV: CC - Organic Chemistry Impact factor: 1.283, year: 2011

  2. Synthesis of 1,4-Diazepine Nucleosides

    OpenAIRE

    M Zia-Ul-Haq; Hameed, Shahid

    2002-01-01

    The phenolic b-diketones I prepared by modified Baker-Venkataraman rearrangement were converted to the flavones II in acidic medium which on treatment with aqueous ethylenediamine/propylenediamine gave diazepine derivatives III. After coupling with acetobromo sugars in the presence of mercuric cyanide and nitromethane, deacetylation in methanolic ammonia yielded nucleosides V. The structures of all the intermediates and final products were confirmed with the help of modern spectrosc...

  3. Distribution of Nucleosides in Populations of Cordyceps cicadae

    OpenAIRE

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

    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...

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

    Science.gov (United States)

    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

  5. Carbocyclic nucleosides with norbornane pseudosugar moiety locked in North conformation

    Czech Academy of Sciences Publication Activity Database

    Dejmek, Milan; Hřebabecký, Hubert; Šála, Michal; Nencka, Radim

    Crete : University of Crete, 2011. P2.121-P2.121. [ESOC 2011. European Symposium on Organic Chemistry /17./. 10.07.2011-15.07.2011, Crete] R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : norbornane * conformationally lock ed nucleosides * carbocyclic nucleosides Subject RIV: CC - Organic Chemistry

  6. An Efficient and Facile Methodology for Bromination of Pyrimidine and Purine Nucleosides with Sodium Monobromoisocyanurate (SMBI

    Directory of Open Access Journals (Sweden)

    Roger Stromberg

    2013-10-01

    Full Text Available An efficient and facile strategy has been developed for bromination of nucleosides using sodium monobromoisocyanurate (SMBI. Our methodology demonstrates bromination at the C-5 position of pyrimidine nucleosides and the C-8 position of purine nucleosides. Unprotected and also several protected nucleosides were brominated in moderate to high yields following this procedure.

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

    Science.gov (United States)

    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

  8. Functional non-nucleoside adenylyl cyclase inhibitors.

    Science.gov (United States)

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

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, Zachary Lee; Cheong, Cheom-Gil; Lee, Seok-Yong (Duke)

    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.

  10. Meteorite-catalyzed synthesis of nucleosides and other prebiotic compounds

    Czech Academy of Sciences Publication Activity Database

    Ferus, Martin; Knížek, Antonín; Civiš, Svatopluk

    2015-01-01

    Roč. 112, č. 23 (2015), s. 7109-7110. ISSN 0027-8424 Institutional support: RVO:61388955 Keywords : meteorite-catalzzed synthesis * nucleosides * prebiotic compounds Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 9.674, year: 2014

  11. Medicinal Chemistry of Fluorinated Cyclic and Acyclic Nucleoside Phosphonates

    Czech Academy of Sciences Publication Activity Database

    Baszczyňski, Ondřej; Janeba, Zlatko

    2013-01-01

    Roč. 33, č. 6 (2013), s. 1304-1344. ISSN 0198-6325 Institutional support: RVO:61388963 Keywords : nucleoside phosphosphonates * fluorine * antiviral * anticancer * antiparasitic Subject RIV: CC - Organic Chemistry Impact factor: 8.131, year: 2013

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

    Science.gov (United States)

    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

  13. Distribution of nucleosides in populations of Cordyceps cicadae.

    Science.gov (United States)

    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

  14. Synthesis of coumarin or ferrocene labeled nucleosides via Staudinger ligation

    Directory of Open Access Journals (Sweden)

    Kois Pavol

    2006-11-01

    Full Text Available Abstract Background Reaction of azides with triaryl phosphines under mild conditions gives iminophosphoranes which can react with almost any kind of electrophilic reagent, e.g. aldehydes/ketones to form imines or esters to form amides. This so-called Staudinger ligation has been employed in a wide range of applications as a general tool for bioconjugation including specific labeling of nucleic acids. Results A new approach for the preparation of labeled nucleosides via intermolecular Staudinger ligation is described. Reaction of azidonucleosides with triphenylphosphine lead to iminophosphorane intermediates, which react subsequently with derivatives of coumarin or ferrocene to form coumarin or ferrocene labeled nucleosides. Fluorescent properties of coumarin labeled nucleosides are determined. Conclusion New coumarin and ferrocene labeled nucleosides were prepared via intermolecular Staudinger ligation. This reaction joins the fluorescent coumarin and biospecific nucleoside to the new molecule with promising fluorescent and electrochemical properties. The isolated yields of products depend on the structure of azidonucleoside and carboxylic acids. A detailed study of the kinetics of the Staudinger ligation with nucleoside substrates is in progress.

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

    Science.gov (United States)

    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

  16. Distribution of Nucleosides in Populations of Cordyceps cicadae

    Directory of Open Access Journals (Sweden)

    Wen-Bo Zeng

    2014-05-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

  18. Synergistic suppression of dengue virus replication using a combination of nucleoside analogs and nucleoside synthesis inhibitors.

    Science.gov (United States)

    Yeo, Kim Long; Chen, Yen-Liang; Xu, Hao Ying; Dong, Hongping; Wang, Qing-Yin; Yokokawa, Fumiaki; Shi, Pei-Yong

    2015-04-01

    Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Currently, there is no clinically approved vaccine or antiviral for DENV. Combination therapy is a common practice in antiviral treatment and a potential approach to search for new treatments for infectious pathogens. In this study, we performed a combination treatment in cell culture by using three distinct classes of inhibitors, including ribavirin (a guanosine analog with several antiviral mechanisms), brequinar (a pyrimidine biosynthesis inhibitor), and INX-08189 (a guanosine analog). The compound pairs were evaluated for antiviral activity by use of a DENV-2 luciferase replicon assay. Our result indicated that the combination of ribavirin and INX-08189 exhibited strong antiviral synergy. This result suggests that synergy can be achieved with compound pairs in which one compound suppresses the synthesis of the nucleoside for which the other compound is a corresponding nucleoside analog. In addition, we found that treatment of cells with brequinar alone could activate interferon-stimulated response elements (ISREs); furthermore, brequinar and NITD-982 (another pyrimidine biosynthesis inhibitor) potentiated interferon-induced ISRE activation. Compared to treatment with brequinar, treatment of cells with ribavirin alone could also induce ISRE activation, but to a lesser extent; however, when cells were cotreated with ribavirin and beta interferon, ribavirin did not augment the interferon-induced ISRE activation. PMID:25624323

  19. Rat pancreas secretes particulate ecto-nucleotidase CD39

    DEFF Research Database (Denmark)

    Sørensen, Christiane Elisabeth; Amstrup, Jan; Rasmussen, Hans N;

    2003-01-01

    In exocrine pancreas, acini release ATP and the excurrent ducts express several types of purinergic P2 receptors. Thereby, ATP, or its hydrolytic products, might play a role as a paracrine regulator between acini and ducts. The aim of the present study was to elucidate whether this acinar......-ductal signalling is regulated by nucleotidase(s), and to characterize and localize one of the nucleotidases within the rat pancreas. Using RT-PCR and Western blotting we show that pancreas expresses the full length ecto-nucleoside triphosphate diphosphohydrolase, CD39. Immunofluorescence shows CD39 localization...... relocalizes in clusters towards the lumen and is secreted. As a result, pancreatic juice collected from intact pancreas stimulated with CCK-8 contained nucleotidase activity, including that of CD39, and no detectable amounts of ATP. Anti-CD39 antibodies detected the full length (78 kDa) CD39 in pancreatic...

  20. New insights into the synergism of nucleoside analogs with radiotherapy

    International Nuclear Information System (INIS)

    Nucleoside analogs have been frequently used in combination with radiotherapy in the clinical setting, as it has long been understood that inhibition of DNA repair pathways is an important means by which many nucleoside analogs synergize. Recent advances in our understanding of the structure and function of deoxycytidine kinase (dCK), a critical enzyme required for the anti-tumor activity for many nucleoside analogs, have clarified the mechanistic role this kinase plays in chemo- and radio-sensitization. A heretofore unrecognized role of dCK in the DNA damage response and cell cycle machinery has helped explain the synergistic effect of these agents with radiotherapy. Since most currently employed nucleoside analogs are primarily activated by dCK, these findings lend fresh impetus to efforts focused on profiling and modulating dCK expression and activity in tumors. In this review we will briefly review the pharmacology and biochemistry of the major nucleoside analogs in clinical use that are activated by dCK. This will be followed by discussions of recent advances in our understanding of dCK activation via post-translational modifications in response to radiation and current strategies aimed at enhancing this activity in cancer cells

  1. Metabolism of pyrimidine bases and nucleosides in the coryneform bacteria Brevibacterium ammoniagenes and Micrococcus luteus.

    OpenAIRE

    Auling, G; Moss, B

    1984-01-01

    The metabolism of exogenous pyrimidine bases and nucleosides was investigated in Brevibacterium ammoniagenes and Micrococcus luteus with fluorinated analogs and radioactive precursors. Salvage of thymine and thymidine was found in M. luteus, but not in B. ammoniagenes. Exogenous uracil or uracil nucleosides, but not cytosine or cytosine nucleosides, were nucleic acid precursors for both bacteria. By examining the possible nucleoside-metabolizing enzymes, it can be suggested that the pyrimidin...

  2. Synthesis and Antiviral Activity of 3-Aminoindole Nucleosides of 2-Acetamido-2-deoxy-D-glucose

    Energy Technology Data Exchange (ETDEWEB)

    Abdelrahman, Adel A. H.; Elessawy, Farag A.; Barakat, Yousif A. [Menoufia Univ., Shebin El-Koam (Egypt); Ellatif, Mona M. Abd [The British Univ. in Egypt, Cairo (Egypt)

    2012-10-15

    A new method for the construction of 3-aminoindole nucleosides of 2-acetamido-2-deoxy-D-glucose based is presented. Nitration and acetylation of the indole nucleosides by acetic anhydride-nitric acid mixture followed by reduction using silver catalyst (SNSM) impregnated on silica gel, afforded the corresponding amino indole nucleosides. The nucleosides were tested for antiviral activity against hepatitis B virus (HBV) to show different degrees of antiviral activities or inhibitory actions.

  3. Prolinol-based nucleoside phosphonic acids: Synthesis and properties

    Czech Academy of Sciences Publication Activity Database

    Vaněk, Václav; Buděšínský, Miloš; Liboska, Radek; Hurychová, Vladimíra; Rosenberg, Ivan

    -, č. 52 (2008), s. 537-538. ISSN 0261-3166. [Joint Symposium of the International Roundtable on Nucleosides, Nucleotides and Nucleic Acids /18./ and the International Symposium on Nucleic Acid Chemistry /35./. Kyoto, 08.09.2008-12.09.2008] R&D Projects: GA MŠk(CZ) LC06061; GA MŠk(CZ) LC06077; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506 Keywords : oligonucleotide phosphonate * nucleoside analogue * pyrrolidine * prolinol Subject RIV: CC - Organic Chemistry

  4. Deoxyribonucleoside kinases activate nucleoside antibiotics in severely pathogenic bacteria

    DEFF Research Database (Denmark)

    Sandrini, Michael; Shannon, O.; Clausen, A.R.;

    2007-01-01

    kinase-deficient Escherichia coli strains become highly susceptible to nucleoside analogs when they express recombinant kinases from Staphylococcus aureus or Streptococcus pyogenes. We further demonstrate that recombinant S. aureus deoxyadenosine kinase efficiently phosphorylates the anticancer drug...... gemcitabine in vitro and is therefore the key enzyme in the activation pathway. When adult mice were infected intraperitoneally with a fatal dose of S. pyogenes strain AP1 and afterwards received gemcitabine, they failed to develop a systemic infection. Nucleoside analogs may therefore represent a promising...

  5. Synthesis of some novel hydrazono acyclic nucleoside analogues

    Directory of Open Access Journals (Sweden)

    Mohammad N. Soltani Rad

    2010-05-01

    Full Text Available The syntheses of novel hydrazono acyclic nucleosides similar to miconazole scaffolds are described. In this series of acyclic nucleosides, pyrimidine as well as purine and other azole derivatives replaced the imidazole function in miconazole and the ether group was replaced with a hydrazone moiety using phenylhydrazine. To interpret the dominant formation of (E-hydrazone derivatives rather than (Z-isomers, PM3 semiempirical quantum mechanic calculations were carried out which indicated that the (E-isomers had the lower heats of formation.

  6. Synthesis and Conformation of Novel 4'-Fluorinated 5'-Deoxythreosyl Phosphonic Acid Nucleosides as Antiviral Agents

    International Nuclear Information System (INIS)

    Efficient synthetic route to novel 4'-fluorinated 5'-deoxythreosyl phosphonic acid nucleosides was described from glyceraldehyde using Horner-Emmons reaction in the presence of triethyl α-fluorophosphonoacetate. Glycosylation reaction of nucleosidic bases with glycosly donor 14 gave the nucleosides which were further phosphonated and hydrolyzed to reach desired nucleoside analogues. Synthesized nucleoside analogues 18, 21, 25 and 28 were tested for anti-HIV activity as well as cytotoxicity. Adenine derivatives 18 and 21 showed significant anti-HIV activity up to 100 μM

  7. Novel types of acyclic nucleoside phosphonates: Chemistry and biology

    Czech Academy of Sciences Publication Activity Database

    Janeba, Zlatko

    La Habana: Cuban Chemical Society, 2015. LO009. [Quimicuba 2015. Congress of Chemical Sciences, Technology and Innovation /9./. 13.10.2015-16.10.2015, La Habana] R&D Projects: GA ČR GAP207/11/0108; GA MV VG20102015046 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * malaria * tuberculosis * inhibitors * 6-oxopurine phosphoribosyltransferases Subject RIV: CC - Organic Chemistry

  8. Synthesis of (Purin-6-yl)methylphosphonate Bases and Nucleosides

    Czech Academy of Sciences Publication Activity Database

    Hasník, Zbyněk; Pohl, Radek; Hocek, Michal

    2010-01-01

    Roč. 51, č. 18 (2010), s. 2464-2466. ISSN 0040-4039 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : purines * nucleosides * phosphonates * cross-coupling Subject RIV: CC - Organic Chemistry Impact factor: 2.618, year: 2010

  9. Novel oligonucleotides modified with acyclic nucleoside phosphonate units

    Czech Academy of Sciences Publication Activity Database

    Janeba, Zlatko; Hocková, Dana; Kaiser, Martin Maxmilian; Ménová, Petra; Novák, Pavel; Rosenbergová, Šárka; Páv, Ondřej; Pohl, Radek; Poštová Slavětínská, Lenka; Rosenberg, Ivan

    Newport : Gordon Research Conference, 2015. [Nucleosides, Nucleotides & Oligonucleotides. Gordon Research Conference 2015. 28.06.2015-03.07.2015, Newport] R&D Projects: GA ČR GA13-26526S Institutional support: RVO:61388963 Keywords : nucleotide analogues * modified oligonucleotides Subject RIV: CC - Organic Chemistry

  10. Conformation analysis of Se and Te containing nucleoside analogues

    Czech Academy of Sciences Publication Activity Database

    Poštová Slavětínská, Lenka; Rejman, Dominik; Kleinpeter, E.; Pohl, Radek

    Santiago de Compostela : -, 2013. s. 221-221. [SMASH 2013. 22.9.2013-25.9.2013, Santiago de Compostela] R&D Projects: GA ČR GA13-24880S Institutional support: RVO:61388963 Keywords : low-temperature NMR * DFT * nucleoside analogues Subject RIV: CC - Organic Chemistry

  11. Protecting group-free strategies for the synthesis of nucleosides

    Czech Academy of Sciences Publication Activity Database

    Downey, Alan Michael; Hocek, Michal

    Praha: Czech Chemical Society, 2015. s. 37. [Liblice 2015. Advances in Organic , Bioorganic and Pharmaceutical Chemistry /50./. 06.11.2015-08.11.2015, Olomouc] R&D Projects: GA ČR GAP207/11/0344 Institutional support: RVO:61388963 Keywords : nucleosides * cytostatics Subject RIV: CC - Organic Chemistry

  12. Novel modified nucleobases, nucleosides and nucleotides: biological activity and applications

    Czech Academy of Sciences Publication Activity Database

    Hocek, Michal

    Louvain-La Neuve : -, 2007. PL4. [ Organic Chemistry , Present and Future. 10.04.2007-13.04.2007, Louvain-La-Neuve] R&D Projects: GA MŠk 1M0508; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleobases * nucleosides * nucleotides Subject RIV: CC - Organic Chemistry

  13. Novel reactivity of Fhit proteins: catalysts for fluorolysis of nucleoside 5'-phosphoramidates and nucleoside 5'-phosphosulfates to generate nucleoside 5'-phosphorofluoridates.

    Science.gov (United States)

    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

  14. Synthesis, Spectroscopic Properties and Biochemical Evaluation of 6-Chloro-2-aminopurine Nucleoside Triphosphate%6-氯-2-氨基嘌呤核苷酸合成、光谱性质及生化评价

    Institute of Scientific and Technical Information of China (English)

    陈长宝; 吴春辉; 周杰

    2009-01-01

    A purine nucleotide derivative, 6-chloro-2-aminopurine nucleoside triphosphate, was synthesized in four steps and characterized with ~1H NMR, ~(31)P NMR, ~(13)C NMR, EA and IR. Its ultraviolet properties at various pH values were studied. The pK_a value of the compound was determined by the curves of UV abso-rption dependency on pH, which was 1.44. Hydrolysis of the nucleotide derivative under the catalysis of potato apyrase was studied. The competion of the ATP analogue with ATP for potato apyrase′ active site was proved to be mixed prohibition mechanism.%经过4步反应合成了嘌呤核苷酸衍生物6-氯-2-氨基嘌呤核苷三磷酸钠盐,使用~1H NMR、~(13)C NMR、~(31)P NMR、IR和元素分析测试技术表征了其化学结构. 在不同pH值的溶液中研究了它的紫外光谱性质,并通过pH光度微量滴定法测定了一个质子化物种的pK_a=1.44. 最后,在马铃薯三磷酸腺苷双磷酸酶的催化下研究了该化合物的水解特性,该核苷酸衍生物与ATP竞争马铃薯三磷酸腺苷双磷酸酶的活性位点的机理被证明为混合型抑制机理.

  15. The SLC28 (CNT) and SLC29 (ENT) nucleoside transporter families: a 30-year collaborative odyssey.

    Science.gov (United States)

    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

  16. Modified Nucleoside Triphosphates for in-vitro Selection Techniques

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  18. A Novel and Fast Purification Method for Nucleoside Transporters

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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. Compositions containing nucleosides and manganese and their uses

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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

  2. Locked and unlocked nucleosides in functional nucleic acids

    DEFF Research Database (Denmark)

    Doessing, Holger; Vester, Birte

    2011-01-01

    Nucleic acids are able to adopt a plethora of structures, many of which are of interest in therapeutics, bio- or nanotechnology. However, structural and biochemical stability is a major concern which has been addressed by incorporating a range of modifications and nucleoside derivatives. This rev...... review summarizes the use of locked nucleic acid (LNA) and un-locked nucleic acid (UNA) monomers in functional nucleic acids such as aptamers, ribozymes, and DNAzymes....

  3. Tyrosine-based prodrugs of acyclic nucleoside phosphonates

    Czech Academy of Sciences Publication Activity Database

    Tichý, Tomáš; Pomeisl, Karel; Krečmerová, Marcela; McKenna, Ch. E.

    Praha : Institute of Organic Chemistry and Biochemistry AS CR, v. v. i, 2014 - (Hocek, M.), s. 126-128 ISBN 978-80-86241-50-0. - (Collection Symposium Series. 14). [Symposium on Chemistry of Nucleic Acid Components /16./. Český Krumlov (CZ), 08.06.2014-13.06.2014] R&D Projects: GA ČR(CZ) GA14-00522S Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * prodrug * tyrosine Subject RIV: CC - Organic Chemistry

  4. Synthesis of chiral open-ring acyclic nucleoside bisphosphonates

    Czech Academy of Sciences Publication Activity Database

    Doláková, Petra; Masojídková, Milena; Dračínský, Martin; Holý, Antonín

    Napoli : University of Napoli, 2006. s. 73. [International Symposium on Applied Bioinorganic Chemistry /9./. 02.12.2006-05.12.2006, Napoli] R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Grant ostatní: René Descartes Prize(XE) HPAW-2002-100096 Institutional research plan: CEZ:AV0Z40550506 Keywords : purine * acyclic nucleoside bisphosphonates Subject RIV: CC - Organic Chemistry

  5. Acyclic Nucleoside Phosphonates as Potential Anti-Malarial Agents

    Czech Academy of Sciences Publication Activity Database

    Janeba, Zlatko; Hocková, Dana; Holý, Antonín; Česnek, Michal; Baszczyňski, Ondřej; Tichý, Tomáš; Krečmerová, Marcela; Naesens, L.; Skinner-Adams, T. S.; Edstein, M.; Keough, D. T.; de Jersey, J.; Guddat, L. W.

    Riviera Maya: -, 2011. s. 21-21. [Zing Conferences 2011. Drug Discovery - Tropical Diseases Conference. 11.12.2011-15.12.2011, Riviera Maya] R&D Projects: GA ČR GAP207/11/0108; GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * HGXPRT * malaria * Plasmodium Subject RIV: CC - Organic Chemistry

  6. Trends and challenges in chemistry of acyclic nucleoside phosphonates

    Czech Academy of Sciences Publication Activity Database

    Krečmerová, Marcela

    Rehovot : European Federation for Medicinal Chemistry, 2015. s. 164. [International Symposium on Advances in Synthetic and Medicinal Chemistry (EFMC-ASMC'15) /6./ and Annual Meeting of the Medicinal Chemistry Section of the Israel Chemical Society /13./. 15.11.2015-18.11.2015, Rehovot] R&D Projects: GA MPO(CZ) FR-TI4/625 Institutional support: RVO:61388963 Keywords : 5-azacytosine * PME-azaC * acyclic nucleoside phosphonate * antivirals * prodrug Subject RIV: CC - Organic Chemistry

  7. Oligonucleotides modified with acyclic nucleoside phosphonate (HPEP) units

    Czech Academy of Sciences Publication Activity Database

    Kaiser, Martin Maxmilian; Novák, Pavel; Rosenbergová, Šárka; Poštová Slavětínská, Lenka; Rosenberg, Ivan; Janeba, Zlatko

    Praha: Institute of Organic Chemistry and Biochemistry AS CR, v. v. i, 2014 - (Hocek, M.), s. 293-294. (Collection Symposium Series. 14). ISBN 978-80-86241-50-0. [Symposium on Chemistry of Nucleic Acid Components /16./. Český Krumlov (CZ), 08.06.2014-13.06.2014] R&D Projects: GA TA ČR TA03010598 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonate * HPEP * oligonucleotides Subject RIV: CC - Organic Chemistry

  8. C-Nucleosides: Synthetic Strategies and Biological Applications

    Czech Academy of Sciences Publication Activity Database

    Štambaský, J.; Hocek, Michal; Kočovský, P.

    2009-01-01

    Roč. 109, č. 12 (2009), s. 6729-6764. ISSN 0009-2665 R&D Projects: GA MŠk LC512; GA AV ČR IAA400550902 Grant ostatní: NIH(US) 1R03TW007372-01 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleosides * nucleobases * biological activity * extension of genetic alphabet Subject RIV: CC - Organic Chemistry Impact factor: 35.957, year: 2009

  9. Effects of halides on reaction of nucleosides with ozone.

    Science.gov (United States)

    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

  10. ABC Transporters and their Role in Nucleoside and Nucleotide Drug Resistance

    OpenAIRE

    Fukuda, Yu; Schuetz, John D.

    2012-01-01

    ATP-binding cassette (ABC) transporters confer drug resistance against a wide range of chemotherapeutic agents, including nucleoside and nucleotide based drugs. While nucleoside based drugs have been used for many years in the treatment of solid and hematological malignancies as well as viral and autoimmune diseases, the potential contribution of ABC transporters has only recently been recognized. This neglect is likely because activation of nucleoside derivatives require an initial carrier-m...

  11. Nucleoside phosphonic acids as selective inhibitors of human pyrimidine- specific 5'-nucleotidases

    Czech Academy of Sciences Publication Activity Database

    Šimák, Ondřej (ed.); Buděšínský, Miloš; Petrová, Magdalena; Zborníková, Eva; Pachl, Petr; Brynda, Jiří; Rosenberg, Ivan

    Montreal : International Society of Nucleosides, Nucleotides and Nucleic Acids, 2012. s. 49-49. [International Round Table on Nucleosides Nucleotides and Nucleic Acids /20./. 05.08.2012-09.08.2012, Montreal] R&D Projects: GA AV ČR KAN200520801; GA ČR GA203/09/0820 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : nucleoside phosphonic acids Subject RIV: CC - Organic Chemistry

  12. Nucleoside and Nucleotide Analogues for the Treatment of Herpesvirus Infections: Current Stage and New Prospects in the Field of Acyclic Nucleoside Phosphonates

    Czech Academy of Sciences Publication Activity Database

    Krečmerová, Marcela

    Rijeka : InTech, 2012 - (Magel, G.; Tyring, S.), s. 245-270 ISBN 978-953-51-0186-4 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * acyclic nucleoside analogue * antimetabolite * DHPA * 5-azacytosine * cidofovir * HPMPDAP * prodrugs * animal herpesviruses Subject RIV: CC - Organic Chemistry http://www.intechopen.com/books/herpesviridae-a-look-into-this-unique-family-of-viruses/nucleoside-and-nucleotide-analogues-for-the-treatment-of-herpesvirus-infections-current-stage-and-ne

  13. Carboranyl Nucleosides & Oligonucleotides for Neutron Capture Therapy Final Report

    Energy Technology Data Exchange (ETDEWEB)

    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

  14. An enzymatic glycosylation of nucleoside analogues using beta-galactosidase from Escherichia coli

    Czech Academy of Sciences Publication Activity Database

    Blažek, Jiří; Jansa, Petr; Baszczyňski, Ondřej; Kaiser, Martin Maxmilian; Otmar, Miroslav; Krečmerová, Marcela; Dračínský, Martin; Holý, Antonín; Králová, B.

    2012-01-01

    Roč. 20, č. 9 (2012), s. 3111-3118. ISSN 0968-0896 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : glycosylation * galactosylation * beta-galactosidase * enzymatic synthesis * nucleoside * acyclic nucleoside analogues Subject RIV: CC - Organic Chemistry Impact factor: 2.903, year: 2012

  15. A procedure for the preparation and isolation of nucleoside-5’-diphosphates

    Directory of Open Access Journals (Sweden)

    Heidi J. Korhonen

    2015-04-01

    Full Text Available Tris[bis(triphenylphosphoranylideneammonium] 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.

  16. A procedure for the preparation and isolation of nucleoside-5’-diphosphates

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  19. Synthesis of nucleoside 5'-S-methylphosphonates and related compounds

    Czech Academy of Sciences Publication Activity Database

    Kóšiová, Ivana; Rosenberg, Ivan

    -, č. 52 (2008), s. 569-570. ISSN 0261-3166. [Joint Symposium of the International Roundtable on Nucleosides, Nucleotides and Nucleic Acids /18./ and the International Symposium on Nucleic Acid Chemistry /35./. Kyoto, 08.09.2008-12.09.2008] R&D Projects: GA MŠk(CZ) LC06061; GA MŠk(CZ) LC06077 Grant ostatní: EMIL-FP6(XE) 503569 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleotide * S'-methylphosphonate analog Subject RIV: CC - Organic Chemistry

  20. Nucleoside phosphonic acids in thymidine phosphorylase inhibition: Structure - activity relationship

    Czech Academy of Sciences Publication Activity Database

    Panova, Natalya; Kóšiová, Ivana; Petrová, Magdalena; Vaněk, Václav; Liboska, Radek; Kovačková, Soňa; Kočalka, Petr; Králíková, Šárka; Točík, Zdeněk; Páv, Ondřej; Pačes, Ondřej; Rejman, Dominik; Rosenberg, Ivan

    -, č. 52 (2008), s. 665-666. ISSN 0261-3166. [Joint Symposium of the International Roundtable on Nucleosides, Nucleotides and Nucleic Acids /18./ and the International Symposium on Nucleic Acid Chemistry /35./. Kyoto, 08.09.2008-12.09.2008] R&D Projects: GA MŠk(CZ) LC06061; GA MŠk(CZ) LC06077; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506 Keywords : thymidine phosphorylase * inhibitors * phosphonic acids Subject RIV: CC - Organic Chemistry

  1. Polymerization of the cyclic pyrophosphates of nucleosides and their analogues

    Science.gov (United States)

    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.

  2. A high-yielding, strictly regioselective prebiotic purine nucleoside formation pathway.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  4. Construcción de un Inmunosensor Amperométrico utilizando Apirasa de Solanum tuberosum para la Detección de Esquistosomiasis Construction of an Amperometric Immunosensor using Solanum tuberosum potato Apyrase for the Detection of Schistosomiasis

    Directory of Open Access Journals (Sweden)

    Ninoska I Bojorge Ramírez

    2009-01-01

    Full Text Available Este trabajo presenta el desarrollo de un inmunosensor amperométrico composito a base de una proteína de origen vegetal para la determinación de antí-anticuerpo de S. mansoni. El inmunosensor consiste en una matriz electródica rígida construida con grafito y resina epoxi en la que se inmoviliza covalentemente la Apirasa. La estrategia de inmovilización se basa en silanización de la matriz compósita con 3-aminopropilsilano (3-APTES y su activación mediante glutaraldehído. La inmovilización fue monitoreada mediante voltaamperometría cíclica en Fe(CN6(4-/Fe(CN6³, obteniéndose una respuesta cuasi-reversible del inmunosensor y muy estable en el tiempo. La inmovilización también fue caracterizada mediante ángulos de contactos y microscopía electrónica de barrido. Estas cualidades son aprovechables para la determinación clínica de esquistosomiasis dada la rapidez y sensibilidad de respuesta.This paper presents the development of an amperometric composites inmunosensor based on a vegetable protein for the determination of anti-Ac S. mansoni. The inmunosensor consists of a rigid matrix built with graphite and epoxy resin in which immobilizes covalently the Apyrase. The strategy of immobilization is based in silanization of the matrix composites with 3-Aminopropyltriethoxysilane (3-APTES and its activation by glutaraldehyde. The detection was monitored by cyclic voltammetry Fe(CN6(4-/ Fe(CN6³, obtaining a response quasi-reversible of the immunosensor and very stable over time. The immobilization was also characterized by angles contacts and scanning electron microscopy. These qualities are useful for determination clinical of schistosomiasis given the speed of response and sensitivity.

  5. Alkylsulfanylphenyl derivatives of cytosine and 7-deazaadenine nucleosides, nucleotides and nucleoside triphosphates. Synthesis, polymerase incorporation to DNA and electrochemical study

    Czech Academy of Sciences Publication Activity Database

    Macíčková-Cahová, Hana; Pohl, Radek; Horáková Brázdilová, Petra; Havran, Luděk; Špaček, Jan; Fojta, Miroslav; Hocek, Michal

    2011-01-01

    Roč. 17, č. 21 (2011), s. 5833-5841. ISSN 0947-6539 R&D Projects: GA MŠk(CZ) LC06035; GA MŠk LC512; GA ČR GA203/09/0317; GA AV ČR(CZ) IAA400040901 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : DNA polymerases * electrochemistry * nucleosides * nucleotides * organosulfur compounds Subject RIV: CC - Organic Chemistry Impact factor: 5.925, year: 2011

  6. Synthesis of nucleosides and nucleoside triphosphates bearing anthraquinone substituents as redox probes and their enzymatic incorporation to DNA

    Czech Academy of Sciences Publication Activity Database

    Balintová, Jana; Havran, Luděk; Fojta, Miroslav; Hocek, Michal

    Praha : Institute of Organic Chemistry and Biochemistry AS CR, v. v. i., 2011 - (Hocek, M.), s. 297-299 ISBN 978-80-86241-37-1. - (Collection Symposium Series. 12). [Chemistry of Nucleic Acid Components /15./. Český Krumlov (CZ), 05.06.2011-10.06.2011] R&D Projects: GA MŠk LC512; GA ČR GA203/09/0317; GA MŠk 1M0508; GA AV ČR(CZ) IAA400040901 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleosides * anthraquinone * DNA Subject RIV: CC - Organic Chemistry

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

    Directory of Open Access Journals (Sweden)

    Sakilam Satishkumar

    2015-10-01

    Full Text Available 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 in the amide bond-activation in purine nucleosides via the use of (benzotriazol-1yl-oxytris(dimethylaminophosphonium 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 ribose analogue of cladribine showed activity, but was the 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, cladribine and its ribose analogue were most active.

  8. Nucleoside analogs and tuberculosis: new weapons against an old enemy.

    Science.gov (United States)

    Ferrari, Valentina; Serpi, Michaela

    2015-01-01

    Purine and pyrimidine nucleoside and nucleotide analogs have been extensively studied as anticancer and antiviral agents. In addition to this, they have recently shown great potential against Mycobacterium Tuberculosis, the causative agent of TB. TB ranks as the tenth most common cause of death in the world. The current treatment for TB infection is limited by side effects and cost of the drugs and most importantly by the development of resistance to the therapy. Therefore the development of novel drugs, capable of overcoming the drawbacks of the existing treatments, has become the focus of many research programs. In parallel to that, a tremendous effort has been made to elucidate the unique metabolism of this pathogen with the aim to identify new possible targets. This review presents the state of the art in nucleoside and nucleotide analogs in the treatment of TB. In particular, we report on the inhibitory activity of this class of compounds, both in enzymatic and whole-cell assays, providing a brief insight to which reported target these novel compounds are hitting. PMID:25826361

  9. Synthesis of tritium labelled nucleoside triphosphates by enzymatic phosphorylation

    International Nuclear Information System (INIS)

    [5-3H]UMP, [5-3H]CMP, [8-3H]AMP and [8-3H]GMP were prepared from 5BrUMP 5BrCMP 8BrAMP and 8BrGMP by catalytic halogentritium replacement at the same time. [5-3H]UTP, [5-3H]CTP, [8-3H]ATP and [8-3H]GTP were subsequently synthesized from [5-3H]UMP, [5-3H]CMP, [8-3H]AMP and [8-3H]GMP by enzymatic phosphorylation with the crude enzyme prepared from brewer's yeasts and purified by paper chromatography simultaneously. In addition, four kinds of tritium labelled nucleoside monophosphates and four kinds of tritium labelled nucleoside diphosphates were obtained as the by-products. The specific activity of these products is between 14-19 Ci/mmol and the radiochemical purity is more than 98%

  10. Synthesis and Conformation of Novel 4'-Fluorinated 5'-Deoxythreosyl Phosphonic Acid Nucleosides as Antiviral Agents

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Lien; Kim, Eunae; Choi, Eun Joo; Yoo, Jin Cheol; Lee, Wonjae; Hong, Joon Hee [Chosun Univ., Kwangju (Korea, Republic of)

    2012-12-15

    Efficient synthetic route to novel 4'-fluorinated 5'-deoxythreosyl phosphonic acid nucleosides was described from glyceraldehyde using Horner-Emmons reaction in the presence of triethyl α-fluorophosphonoacetate. Glycosylation reaction of nucleosidic bases with glycosly donor 14 gave the nucleosides which were further phosphonated and hydrolyzed to reach desired nucleoside analogues. Synthesized nucleoside analogues 18, 21, 25 and 28 were tested for anti-HIV activity as well as cytotoxicity. Adenine derivatives 18 and 21 showed significant anti-HIV activity up to 100 μM.

  11. Radiolabeled nucleoside analogs in cancer diagnosis and therapy.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  14. Red Fluorescent Protein pH Biosensor to Detect Concentrative Nucleoside Transport*

    OpenAIRE

    Johnson, Danielle E.; Ai, Hui-wang; Wong, Peter; Young, James D.; Campbell, Robert E.; Casey, Joseph R

    2009-01-01

    Human concentrative nucleoside transporter, hCNT3, mediates Na+/nucleoside and H+/nucleoside co-transport. We describe a new approach to monitor H+/uridine co-transport in cultured mammalian cells, using a pH-sensitive monomeric red fluorescent protein variant, mNectarine, whose development and characterization are also reported here. A chimeric protein, mNectarine fused to the N terminus of hCNT3 (mNect.hCNT3), enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluor...

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

    Science.gov (United States)

    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

  16. Synthesis and conformation of novel 3'-branched threo syl-5'-deoxy phosphonic acid nucleoside analogues

    International Nuclear Information System (INIS)

    The discovery that threo syl phosphonate nucleoside (PMDTA, EC50 = 2.53 μM) is a potent anti-HIV agent has led to the synthesis and biological evaluation of 5'-deoxy versions of threo syl phosphonate nucleosides. In the present study, (E)-3'-phosphono alkenyl and 3'-phosphono alkyl nucleoside analogues 13, 16, 20 and 23 were synthesized from acetol and tested for anti-HIV activity and cytotoxicity. The adenine analogue 16 was found to exhibit moderate in vitro anti-HIV-1 activity (EC50 = 22.2 μM)

  17. Interaction between N-Phospho-Amino Acids and Nucleoside in Aqueous Medium

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Nucleosides were phosphorylated with different N- (O, O-diisopropyl) phosphoryl amino acids to give nucleoside mono phosphates in aqueous solution. 2', 3', and 5'-isomers had been confirmed by comparison with authentic samples on the basis of HPLC analysis. The conversion percentage of nucleoside indicated that N- (O, O-diisopropyl) phosphoryl aspartic acid reacted with adenosine and guanosine at a much higher rate than other kinds of N- phosphoryl amino acids, while phosphorylation of cytidine and uridine was relatively easy by using N- (O, O-diisopropyl) phosphoryl threonine. The result could give some clue to the prebiotic code origin of nucleic acid and protein.

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

    Science.gov (United States)

    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

  19. Modification of Purine and Pyrimidine Nucleosides by Direct C-H Bond Activation

    Directory of Open Access Journals (Sweden)

    Yong Liang

    2015-03-01

    Full Text Available Transition metal-catalyzed modifications of the activated heterocyclic bases of nucleosides as well as DNA or RNA fragments employing traditional cross-coupling methods have been well-established in nucleic acid chemistry. This review covers advances in the area of cross-coupling reactions in which nucleosides are functionalized via direct activation of the C8-H bond in purine and the C5-H or C6-H bond in uracil bases. The review focuses on Pd/Cu-catalyzed couplings between unactivated nucleoside bases with aryl halides. It also discusses cross-dehydrogenative arylations and alkenylations as well as other reactions used for modification of nucleoside bases that avoid the use of organometallic precursors and involve direct C-H bond activation in at least one substrate. The scope and efficiency of these coupling reactions along with some mechanistic considerations are discussed.

  20. Preclinical development of biodegradable polymer foils for intracerebral delivery of cytotoxic nucleosides.

    Science.gov (United States)

    Grieb, Paweł; Ryba, Mirosław; Janisz, Monika; Walski, Michał

    2003-01-01

    Intracerebral implantation of biodegradable polymers loaded with cytotoxic or radiosensitising nucleoside analogues is a promising treatment strategy for malignant gliomas, which are currently intractable. The aim of the study was to develop biodegradable polymers containing nucleosides which could be implanted intracerebrally. Methods of synthesis were developed for the copolymers composed of D,L-lactide, glycolide and caprolactone in different proportions, as well as a novel method of introducing nucleosides to these copolymers at the polymerisation step. Upon degradation in an aqueous medium some of these copolymers emit nucleosides in micromolar concentration over several months. Their in situ degradation and biocompatibility with brain tissues was assessed by means of scanning and transmission electron microscopy. At the ultrastructural level tissue responses to the copolymer implantation closely resembled the responses to mechanical trauma. PMID:12899199

  1. Novel indole-3-sulfonamides as potent HIV non-nucleoside reverse transcriptase inhibitors (NNRTIs)

    Energy Technology Data Exchange (ETDEWEB)

    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. (Merck)

    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.

  2. Activity of different classes of nucleoside and nucleotide analogues against adenovirus in cell culture

    Czech Academy of Sciences Publication Activity Database

    Naesens, L.; Lenaerts, L.; Holý, Antonín; Balzarini, J.; De Clercq, E.

    Elsevier. Roč. 62, č. 2 (2004), s. A74. ISSN 0166-3542. [International Conference on Antiviral Research /17./. 02.05.2004-06.05.2004, Tucson] Keywords : nucleosides * nucleotides Subject RIV: CC - Organic Chemistry

  3. The Effect of Stereochemistry on Sodium Ion Complexation in Nucleoside-Metallacarborane Conjugates

    OpenAIRE

    Jan Milecki; Grzegorz Schroeder; Olejniczak, Agnieszka B.

    2010-01-01

    Conjugates of purine and pyrimidine nucleosides: thymidine and 2  -deoxyguanosine with cobalt-metallacarborane were studied for their sodium ion complexing properties. Formation of stable complexes of 1 : 1 stoichiometry was proved by ESI MS spectroscopy and 2 3 N a NMR. Equilibrium constants and energies of complex formation were calculated. Complexation of alkali-metals by nucleoside-metallacarborane conjugates may affect the physicochemical and biological properties of the conjugates and ...

  4. Preparation of tetrofuranosylnucleoside phosphonic acids as shortened analogues of 5´-nucleoside phosphates

    Czech Academy of Sciences Publication Activity Database

    Točík, Zdeněk; Poláková, Ivana; Buděšínský, Miloš; Rosenberg, Ivan

    Lyon : Université de Lyon, 2010, s. 345-346. [International Roundtable on Nucleosides, Nucleotides and Nucleic Acids. IRT 2010. Lyon (FR), 29.08.2010-03.09.2010] R&D Projects: GA MŠk(CZ) LC06077; GA ČR GA203/09/0820 Institutional research plan: CEZ:AV0Z40550506 Keywords : phosphonate nucleoside * nucleotide analog * tetrofuranosylnucleoside phosphonate Subject RIV: CC - Organic Chemistry http://irt2010.univ-lyon1.fr

  5. Small scale synthesis of nucleoside mono-, di-, and triphosphates on Controlled Pore Glass (CPG)

    Czech Academy of Sciences Publication Activity Database

    Liboska, Radek; Zborníková, Eva; Rosenberg, Ivan

    Lyon : Université de Lyon, 2010, s. 335-335. [International Roundtable on Nucleosides, Nucleotides and Nucleic Acids. IRT 2010. Lyon (FR), 29.08.2010-03.09.2010] R&D Projects: GA AV ČR KAN200520801; GA MŠk(CZ) LC06061 Institutional research plan: CEZ:AV0Z40550506 Keywords : triphosphate synthesis * nucleoside triphosphate * solid phase synthesis Subject RIV: CC - Organic Chemistry http://irt2010.univ-lyon1.fr

  6. Determination of nucleosides in Cordyceps sinensis and Ganoderma lucidum by high performance liquid chromatography method

    OpenAIRE

    Masood Shah Khan; Rabea Parveen; Kshipra Mishra; Rajkumar Tulsawani; Sayeed Ahmad

    2015-01-01

    Background: Nucleosides are supportive in the regulation and modulation of various physiological processes in body, they acts as precursors in nucleic acid synthesis, enhance immune response, help in absorption of iron and influence the metabolism of fatty acids. Cordyceps sinensis and Ganoderma lucidum are well-known for its use in traditional medicine of China, Nepal and India. They are rich in nucleosides such as adenine, adenosine, cordycepin, etc. Hence, a simple, economic and accurate h...

  7. Aqueous microwave-assisted cross-coupling reactions applied to unprotected nucleosides

    Science.gov (United States)

    Hervé, Gwénaëlle; Len, Christophe

    2015-01-01

    Metal catalyzed cross-coupling reactions have been the preferred tools to access to modified nucleosides (on the C5-position of pyrimidines and on the C7- or C8-positions of purines). Our objective is to focus this mini-review on the Suzuki-Miyaura and on the Heck cross-couplings of nucleosides using microwave irradiations which is an alternative technology compatible with green chemistry and sustainable development PMID:25741506

  8. Cytotoxic purine nucleoside analogues bind to A1, A2A and A3 adenosine receptors

    OpenAIRE

    Jensen, Kyle; Johnson, L’Aurelle A.; Jacobson, Pamala A.; Kachler, Sonja; Kirstein, Mark N.; Lamba, Jatinder; Klotz, Karl-Norbert

    2012-01-01

    Fludarabine, clofarabine and cladribine are anti-cancer agents which are analogues of the purine nucleoside adenosine. These agents have been associated with cardiac and neurological toxicities. Because these agents are analogues of adenosine, they may act through adenosine receptors to elicit their toxic effects. The objective of this study was to evaluate the ability of cytotoxic nucleoside analogues to bind and activate adenosine receptor subtypes (A1, A2A, A2B, and A3). Radioligand bindin...

  9. Nucleoside-nucleotide free diet protects rat colonic mucosa from damage induced by trinitrobenzene sulphonic acid.

    OpenAIRE

    Adjei, A A; Morioka, T.; Ameho, C K; Yamauchi, K.; Kulkarni, A. D.; Al-Mansouri, H M; Kawajiri, A; Yamamoto, S.

    1996-01-01

    BACKGROUND: Growing evidence suggests that intestinal recovery from injury induced by radiation, endotoxin, and protein deficiency is improved by the ingestion of nucleosides and nucleotides. AIM: This study examined the effect of dietary nucleosides and nucleotides supplementation on trinitrobenzene sulphonic acid induced colonic damage in experimental colitis. METHODS: Sprague-Dawley rats were randomised into two groups and fed nucleic acid free 20% casein diet (control) or this diet supple...

  10. Synthesis of Novel 2'-Spirocyclopropanoid 4'-Deoxythreosyl Phosphonic Acid Nucleoside Analogues

    International Nuclear Information System (INIS)

    Efficient synthetic route to novel 2'-spirocyclopropanoid 4'-deoxythreosyl phosphonic acid nucleosides was described from 1,4-dihydroxy-2-butene. Cyclopropane moiety was prepared via ester enolate alkylation using (2-chloroethyl)dimethylsulfonium iodide. Synthesized nucleoside analogues 16, 19, 23 and 26 were tested for anti-HIV activity as well as cytotoxicity. However, none of them showed any anti-HIV activity or cytotoxicity up to 100 μM

  11. Synthesis of Novel 2'-Spirocyclopropanoid 4'-Deoxythreosyl Phosphonic Acid Nucleoside Analogues

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Guang Huan; Hong, Joon Hee [Chosun Univ., Kwangju (Korea, Republic of)

    2013-03-15

    Efficient synthetic route to novel 2'-spirocyclopropanoid 4'-deoxythreosyl phosphonic acid nucleosides was described from 1,4-dihydroxy-2-butene. Cyclopropane moiety was prepared via ester enolate alkylation using (2-chloroethyl)dimethylsulfonium iodide. Synthesized nucleoside analogues 16, 19, 23 and 26 were tested for anti-HIV activity as well as cytotoxicity. However, none of them showed any anti-HIV activity or cytotoxicity up to 100 μM.

  12. 2,6-Diazidopurine Deoxyribo-Nucleoside as Substrate for the Click Reaction

    OpenAIRE

    Cīrule, D; Ozols, K; Bizdēna, Ē; Novosjolova, I

    2014-01-01

    The chemistry of triazolyl-substituted purine nucleosides is attractive since some of them exhibit antiviral, anticancer or fluorescent properties. Our group has reported the synthesis of 2,6-bistriazolyl purine arabino- and ribo-nucleosides using Cu(I) catalysed azide - alkyne 1,3-dipolar cycloaddition reaction. Various nucleophilic substitution reactions with the obtained products were carried out. In addition to our previous work, herein we report the synthesis of the novel...

  13. Formation of Mixed-Ligand Complexes of Pd2+ with Nucleoside 5'-Monophosphates and Some Metal-Ion-Binding Nucleoside Surrogates

    Directory of Open Access Journals (Sweden)

    Oleg Golubev

    2014-10-01

    Full Text Available Formation of mixed-ligand Pd2+ complexes between canonical nucleoside 5'-monophosphates and five metal-ion-binding nucleoside analogs has been studied by 1H-NMR spectroscopy to test the ability of these nucleoside surrogates to discriminate between unmodified nucleobases by Pd2+-mediated base pairing. The nucleoside analogs studied included 2,6-bis(3,5-dimethylpyrazol-1-yl-, 2,6-bis(1-methylhydrazinyl- and 6-(3,5-dimethylpyrazol-1-yl-substituted 9-(β-d-ribofuranosylpurines 1–3, and 2,4-bis(3,5-dimethylpyrazol-1-yl- and 2,4-bis(1-methylhydrazinyl-substituted 5-(β-d-ribofuranosyl-pyrimidines 4–5. Among these, the purine derivatives 1-3 bound Pd2+ much more tightly than the pyrimidine derivatives 4, 5 despite apparently similar structures of the potential coordination sites. Compounds 1 and 2 formed markedly stable mixed-ligand Pd2+ complexes with UMP and GMP, UMP binding favored by 1 and GMP by 2. With 3, formation of mixed-ligand complexes was retarded by binding of two molecules of 3 to Pd2+.

  14. Binding Strength of Nucleobases and Nucleosides on Silver Nanoparticles Probed by a Colorimetric Method.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  16. Resistance issues with new nucleoside/nucleotide backbone options.

    Science.gov (United States)

    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

  17. Two purine nucleoside phosphorylases in Bacillus subtilis. Purification and some properties of the adenosine-specific phosphorylase

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank

    1978-01-01

    Two purine nucleoside phosphorylases (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) were purified from vegetative Bacillus subtilis cells. One enzyme, inosine-guanosine phosphorylase, showed great similarity to the homologous enzyme of Bacillus cereus. It appeared to be a...

  18. Synthesis of nucleoside and nucleotide conjugates of bile acids, and polymerase construction of bile acid-functionalized DNA

    Czech Academy of Sciences Publication Activity Database

    Ikonen, Satu; Macíčková-Cahová, Hana; Pohl, Radek; Šanda, Miloslav; Hocek, Michal

    2010-01-01

    Roč. 8, č. 5 (2010), s. 1194-1201. ISSN 1477-0520 R&D Projects: GA MŠk LC512; GA ČR GA203/09/0317 Institutional research plan: CEZ:AV0Z40550506 Keywords : steroids * nucleosides * nucleoside triphosphates * DNA polymerase Subject RIV: CC - Organic Chemistry Impact factor: 3.451, year: 2010

  19. Synthesis of 4'α-C Phenyl-Branched Carbocyclic Nucleoside Using Ring-Closing Metathesis

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Joon Hee; Ko, Ok Hyun [Chosun University, Gwangju(Korea, Republic of)

    2003-09-15

    An efficient synthetic route for preparing novel 4'α-C phenyl branched carbocyclic nucleoside is described. The installation of phenyl group at the 4'-position of carbocyclic nucleoside was successfully accomplished via a sequential [3,3]-sigmatropic rearrangement and ring-closing metathesis (RCM) beginning from simple ketone such as 2-hydroxy acetophenone.

  20. Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis

    International Nuclear Information System (INIS)

    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

  1. Synthesis of Novel Homo-N-Nucleoside Analogs Composed of a Homo-1,4-Dioxane Sugar Analog and Substituted 1,3,5-Triazine Base Equivalents

    Directory of Open Access Journals (Sweden)

    Qiang Yu

    2008-12-01

    Full Text Available Enantioselective syntheses from dimethyl tartrate of 1,3,5-triazine homo-N-nucleoside analogs, containing a 1,4-dioxane moiety replacing the sugar unit in natural nucleosides, were accomplished. The triazine heterocycle in the nucleoside analogs was further substituted with combinations of NH2, OH and Cl in the 2,4-triazine positions.

  2. Normal And Modified Urinary Nucleosides As Novel Bio makers For Colorectal Cancer

    International Nuclear Information System (INIS)

    Background: Up till now, there is still no ideal tumour marker for early diagnosis and effective monitoring, especially for patients who undergo surgical resection of colorectal cancer (CRC). Aim of the study: To evaluate the clinical utility of normal and modified urinary nucleosides as diagnostic bio markers to be used for the purpose of screening for colorectal cancer, in addition to assessment of the correlation between their preoperative levels, tumour size and modified Duke's staging, as well as their role in monitoring of surgery, as compared to CEA, the routinely used serum marker. Subjects and methods: This study was conducted on 30 patients with CRC (Group I), 30 patients with benign colorectal pathological conditions (Group II) and 30 apparently healthy subjects (Group III). Morning urine and serum samples were collected before surgery and on day 7 postoperative, for the assay of urinary nucleosides (adenosine, cytidine, guanosine, uridine, 1-methyladenosine, 7- methylguanosine and N4-acetyl cytidine) by reversed phase high-performance liquid chromatography, and serum CEA by chemiluminescent sequential immuno metric assay Results: The levels of the measured urinary nucleosides in group I were significantly higher than those of group II or group III. Moreover, the elevated levels of the urinary nucleosides significantly decreased after curative resection of CRC. A significant positive correlation was found between the preoperative levels of some nucleosides and the tumour size, as well as the modified Duke's staging of CRC. Conclusion: These findings indicate that urinary nucleosides are satisfactory diagnostic bio markers of CRC. Moreover, they are apparently of value in the postoperative monitoring of CRC patients

  3. Isolation and functional characterization of the PfNT1 nucleoside transporter gene from Plasmodium falciparum.

    Science.gov (United States)

    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

  4. Versatile synthesis of amino acid functionalized nucleosides via a domino carboxamidation reaction

    Directory of Open Access Journals (Sweden)

    Vicky Gheerardijn

    2014-11-01

    Full Text Available Functionalized oligonucleotides have recently gained increased attention for incorporation in modified nucleic acid structures both for the design of aptamers with enhanced binding properties as well as the construction of catalytic DNA and RNA. As a shortcut alternative to the incorporation of multiple modified residues, each bearing one extra functional group, we present here a straightforward method for direct linking of functionalized amino acids to the nucleoside base, thus equipping the nucleoside with two extra functionalities at once. As a proof of principle, we have introduced three amino acids with functional groups frequently used as key-intermediates in DNA- and RNAzymes via an efficient and straightforward domino carboxamidation reaction.

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

    Science.gov (United States)

    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

  6. Use of nucleoside (tide) analogues in patients with hepatitis B-related acute liver failure

    DEFF Research Database (Denmark)

    Dao, Doan Y; Seremba, Emmanuel; Ajmera, Veeral;

    2012-01-01

    The efficacy of nucleoside(tide) analogues (NA) in the treatment of acute liver failure due to hepatitis B virus (HBV-ALF) remains controversial. We determined retrospectively the impact of NAs in a large cohort of patients with HBV-ALF.......The efficacy of nucleoside(tide) analogues (NA) in the treatment of acute liver failure due to hepatitis B virus (HBV-ALF) remains controversial. We determined retrospectively the impact of NAs in a large cohort of patients with HBV-ALF....

  7. 5-(1-Substituted) alkyl pyrimidine nucleosides as antiviral (herpes) agents.

    Science.gov (United States)

    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

  8. Aminopropanedinitrile (aminomalononitrile, AMN) in the synthesis of C-nucleosides and exocyclic amino thiazole N-nucleosides. Formation and reactions of 2-substituted-5-amino-4-oxazolecarbonitriles

    Energy Technology Data Exchange (ETDEWEB)

    Scheuerman, R.A.

    1992-01-01

    Aminopropanedinitrile (aminomalononitrile, AMN) reacts with a wide variety of alkyl, aryl, or heteroaryl acid chlorides in the presence of 1-methyl-2-pyrrolidinone to give N-(dicyanomethyl)carboxamides which are easily cyclized in situ or after isolation to 2-substituted-5-amino-4-oxazolecarbonitriles in good to excellent yields. Electron attracting or electron releasing groups on the phenyl rings do not appear to greatly influence the yields of oxazoles and steric factors do not appear to be important in the aliphatic series. The reaction of 2, 5-anhydro-3, 4, 6-tri-O-benzoyl-[beta]-D-allonyl chloride with aminopropane-dinitrile gives 2, 5-anhydro-N-(dicyanomethyl)-[beta]-D-allonamide-3, 4, 6-tribenzoate which is converted to 5-amino-2-(2, 3, 5-tri-O-benzoyl-[beta]-D-ribofuranosyl)-4-oxazolecarbonitrile, which is used to prepare other C-nucleosides including 2-([beta]-D-ribofuranosyl)oxazole-4-carboxamide (oxazofurin), an analogue of the antitumor and antiviral C-nucleoside tiazofurin. Attempted deblocking of several benzoyl protected C-nucleosides with sodium methoxide led to double elimination reactions and the formation of furan derivatives. The 2-substituted-5-amino-4-oxazolecarbonitriles react with ortho esters to give imidates which are cyclized to axazolo[5,4-d]pyrimidines. Reactions of 2-substituted-5-amino-4-oxazolecarbonitriles include acylation of the 5-amino group, dediazotization of the 5-amino group, nucleophilic attack and ring opening of the oxazole, and acid catalyzed ring opening of the oxazole. Sugar isothiocyanates are prepared and react with aminopropane-dinitrile (aminomalononitrile, AMN) in the presence of 1-methyl-2-pyrrolidinone to afford exocyclic amino thiazole N-nucleosides.

  9. Anopheles gambiae Purine Nucleoside Phosphorylase: Catalysis, Structure, and Inhibition

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. Synthesis of side-chain gem-difluorinated acyclic nucleoside phosphonates

    Czech Academy of Sciences Publication Activity Database

    Pomeisl, Karel; Beier, Petr; Pohl, Radek; Krečmerová, Marcela

    London: Elsevier, 2014. P1.68. [Tetrahedron Symposium. Challenges in Bioorganic and Organic Medicinal Chemistry /15./. 24.06.2014-27.06.2014, London] Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * difluoromethylation * FPMPA * diethyldifluoromethylphosphonate Subject RIV: CC - Organic Chemistry

  11. Pyrimidine acyclic nucleoside phosphonates and their phosphorylated analogs as potential multisubstrate inhibitors of thymidine phosphorylase

    Czech Academy of Sciences Publication Activity Database

    Pomeisl, Karel; Votruba, Ivan; Holý, Antonín; Pohl, Radek; Blažek, Jiří; Krečmerová, Marcela

    Ottawa : -, 2012. -. [Ottawa 2012 International Symposium On Biochemistry and Biophysics. 24.10.2012-25.10.2012, Ottawa] R&D Projects: GA MPO FR-TI4/625 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * thymidine phosphorylase * phosphorylation * pyrimidines Subject RIV: CC - Organic Chemistry

  12. Modular and practical synthesis of 6-substituted pyridin-3-yl C-nucleosides

    Czech Academy of Sciences Publication Activity Database

    Joubert, Nicolas; Pohl, Radek; Klepetářová, Blanka; Hocek, Michal

    2007-01-01

    Roč. 72, - (2007), s. 6797-6805. ISSN 0022-3263 R&D Projects: GA MŠk LC512 Grant ostatní: NIH(US) 1R03TW007372-01 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleosides * pyridines * cross-coupling Subject RIV: CC - Organic Chemistry Impact factor: 3.959, year: 2007

  13. Inhibition of hypoxanthine-guanine phosphoribosyltransferase by acyclic nucleoside phosphonates: A new class of antimalarial therapeutics

    Czech Academy of Sciences Publication Activity Database

    Keough, D. T.; Hocková, Dana; Holý, Antonín; Naesens, L.; Skinner-Adams, T. S.; de Jersey, J.; Guddat, L. W.

    2009-01-01

    Roč. 52, č. 14 (2009), s. 4391-4399. ISSN 0022-2623 R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * phosphoribosyltransferase * enzyme inhibitors * Plasmodium falciparum Subject RIV: CC - Organic Chemistry Impact factor: 4.802, year: 2009

  14. Inhibition of the Escherichia coli 6-oxopurine phosphoribosyltransferases by nucleoside phosphonates: potential for new antibacterial agents

    Czech Academy of Sciences Publication Activity Database

    Keough, D. T.; Hocková, Dana; Rejman, Dominik; Špaček, Petr; Vrbková, Silvie; Krečmerová, Marcela; Eng, W. S.; Jans, H.; West, N. P.; Naesens, L. M. J.; de Jersey, J.; Guddat, L. W.

    2013-01-01

    Roč. 56, č. 17 (2013), s. 6967-6984. ISSN 0022-2623 R&D Projects: GA ČR GAP207/11/0108 Institutional support: RVO:61388963 Keywords : nucleoside phosphonates * antibacterial agents * hypoxanthine-guanine phosphoribosyltransferase * state analog inhibitor * antimalarial chemotherapy Subject RIV: CC - Organic Chemistry Impact factor: 5.480, year: 2013

  15. Structural studies of nucleoside analog and feedback inhibitor binding to Drosophila melanogaster multisubstrate deoxyribonucleoside kinase

    DEFF Research Database (Denmark)

    Mikkelsen, Niels Egil; Munch-Petersen, Birgitte; Eklund, Hans

    2008-01-01

    relate them to the binding of substrate and feedback inhibitors. dCTP and dGTP binds similarly as the feedback inhibitor dTTP with the base in the substrate site. All investigated nucleoside analogs bind similarly as the pyrimidine substrates with many interactions in common. In contrast, the base of d...

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

    Science.gov (United States)

    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

  17. Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Georgescauld, Florian; Moynie, Lucile; Habersetzer, Johann; Cervoni, Laura; Mocan, Iulia; Borza, Tudor; Harris, Pernille Hanne; 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...

  18. Secretion of antiretroviral chemokines by human cells cultured with acyclic nucleoside phosphonates

    Czech Academy of Sciences Publication Activity Database

    Zídek, Zdeněk; Kmoníčková, Eva; Holý, Antonín

    2007-01-01

    Roč. 574, - (2007), s. 77-84. ISSN 0014-2999 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z40550506 Keywords : Acyclic nucleoside phosphonate * Chemokine * Cytokine Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.376, year: 2007

  19. Dietary nucleotide and nucleoside exposure in infancy and atopic dermatitis, recurrent wheeze, and allergic sensitization

    NARCIS (Netherlands)

    Timmermans, M.J.C.; Dagnelie, P.C.; Theunisz, E.H.; Ewalds, D.; Thijs, C.; Mommers, M.; Arts, I.C.

    2015-01-01

    We hypothesized that early life exposure to nucleotides and nucleosides lowers the risk of recurrent wheeze, atopic dermatitis, and allergic sensitization among n = 429 children. Concentrations in breast milk were established by high-performance liquid chromatography; concentrations in formula milks

  20. Cu(I)-catalyzed efficient synthesis of 2′-Triazolo-nucleoside conjugates

    DEFF Research Database (Denmark)

    Mathur, D.; Rana, N.; Olsen, Carl Erik; Parmar, V. S.; Prasad, A. K.

    2015-01-01

    -nucleoside conjugates, which can be evaluated for different biological activity for suitable drug development, were unambiguously identified on the basis of 1H NMR, 13C NMR, IR, and HRMS data analysis. These compounds have been synthesized for the first time and have not been reported in the literature earlier....

  1. Determination of the nucleosidic structural parameters by means of DNA vibrational markers

    Science.gov (United States)

    Ghomi, M.; Letellier, R.; Taillandier, E.

    1990-10-01

    Normal mode calculations based on the GF-Wilson method and a reliable force field allow the vibrational markers arising from the deoxyadenosine (dA) and deoxyguanosine (dG) residues in DNA double helical chains (right- and left-handed conformations) to be reproduced. To do this, a fast, optimized code running on a CRAY-2 computer has been performed. The normal modes of these nucleosides have been calculated as a function of their structural parameters, on the basis of a non-redundant set of internal coordinates. This study permits a better understanding of the behaviour of the main nucleosidic markers used experimentally to determine the conformation of an oligonucleotide or polynucleotide in the crystalline phase and in solution. Taking account of the calculated data, we propose a reliable set of values for the nucleosidic structural parameters which are in good agreement with those estimated by other techniques such as X-ray diffraction or nuclear magnetic resonance (NMR) spectroscopy. We have extended this study to follow the evolution of the nucleosidic vibrational markers as a function of the sugar conformation and the glycosidic torsion angle.

  2. Novel class of acyclic nucleoside phosphonates: (S)-3-hydroxy-2-(phosphonoethoxy)propyl analogues

    Czech Academy of Sciences Publication Activity Database

    Kaiser, Martin Maxmilian; Dračínský, Martin; Poštová Slavětínská, Lenka; Hocková, Dana; Keough, D. T.; Guddat, L. W.; Janeba, Zlatko

    Riga: -, 2013. s. 180-180. [Conference on Heterocycles in Bio-organic Chemistry /15./. 27.05.2013-30.05.2013, Riga] R&D Projects: GA ČR GAP207/11/0108 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * antiparasitic * HPEP * Plasmodium Subject RIV: CC - Organic Chemistry

  3. Norbornane-based nucleoside and nucleotide analogues locked in North conformation

    Czech Academy of Sciences Publication Activity Database

    Dejmek, Milan; Šála, Michal; Hřebabecký, Hubert; Dračínský, Martin; Procházková, Eliška; Chalupská, Dominika; Klíma, Martin; Plačková, Pavla; Hájek, Miroslav; Andrei, G.; Naesens, L.; Leyssen, P.; Neyts, J.; Balzarini, J.; Bouřa, Evžen; Nencka, Radim

    2015-01-01

    Roč. 23, č. 1 (2015), s. 184-191. ISSN 0968-0896 R&D Projects: GA ČR GPP207/12/P625; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : carbocyclic nucleosides * purines * norbornane * antiviral * PI4KIIalpha Subject RIV: CC - Organic Chemistry Impact factor: 2.793, year: 2014

  4. Activities of Different Classes of Acyclic Nucleoside Phosphonates against BK Virus in Primary Human Renal Cells

    Czech Academy of Sciences Publication Activity Database

    Topalis, D.; Lebeau, I.; Krečmerová, Marcela; Andrei, G.; Snoeck, R.

    2011-01-01

    Roč. 55, č. 5 (2011), s. 1961-1967. ISSN 0066-4804 Institutional research plan: CEZ:AV0Z40550506 Keywords : polyomavirus * BK virus * nephropathy * acyclic nucleoside phosphonates * HPMP-5-azaC Subject RIV: CC - Organic Chemistry Impact factor: 4.841, year: 2011

  5. N-Branched acyclic nucleoside phosphonates as monomers for the synthesis of modified oligonucleotides

    Czech Academy of Sciences Publication Activity Database

    Hocková, Dana; Rosenbergová, Šárka; Ménová, Petra; Páv, Ondřej; Pohl, Radek; Novák, Pavel; Rosenberg, Ivan

    2015-01-01

    Roč. 13, č. 15 (2015), s. 4449-4458. ISSN 1477-0520 R&D Projects: GA ČR GAP207/11/0108; GA ČR GA13-26526S Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * oligonucleotides * solid phase synthesis Subject RIV: CC - Organic Chemistry Impact factor: 3.562, year: 2014

  6. Synthesis and properties of novel types of chiral open-ring acyclic nucleoside phosphonates

    Czech Academy of Sciences Publication Activity Database

    Holý, Antonín; Doláková, Petra

    2005-01-01

    Roč. 65, č. 3 (2005), A31. ISSN 0166-3542. [International Conference on Antiviral Research /8./. Barcelona, 11.04.2005-14.04.2005] Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonate * pyrimidine * antiviral activity Subject RIV: CC - Organic Chemistry Impact factor: 3.406, year: 2005

  7. Synthesis of nucleoside and nucleotide conjugates of bile acids, and polymerase construction of bile acid-functionalized DNA.

    Science.gov (United States)

    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

  8. Synthesis and conformation of novel 3'-branched threo syl-5'-deoxy phosphonic acid nucleoside analogues

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Guang Huan; Kang, Li En; Kim, Eun Ae; Lee, Won Jae; Hong, Joon Hee [Chosun Univ., Kwangju (Korea, Republic of)

    2012-04-15

    The discovery that threo syl phosphonate nucleoside (PMDTA, EC{sub 50} = 2.53 μM) is a potent anti-HIV agent has led to the synthesis and biological evaluation of 5'-deoxy versions of threo syl phosphonate nucleosides. In the present study, (E)-3'-phosphono alkenyl and 3'-phosphono alkyl nucleoside analogues 13, 16, 20 and 23 were synthesized from acetol and tested for anti-HIV activity and cytotoxicity. The adenine analogue 16 was found to exhibit moderate in vitro anti-HIV-1 activity (EC{sub 50} = 22.2 μM)

  9. Purification, crystallization and preliminary X-ray diffraction study on pyrimidine nucleoside phosphorylase TTHA1771 from Thermus thermophilus HB8

    OpenAIRE

    Shimizu, Katsumi; Kunishima, Naoki

    2007-01-01

    The pyrimidine nucleoside phosphorylase TTHA1771 from T. thermophilus HB8 has been overexpressed, purified and crystallized. The crystals diffract X-rays to 1.8 Å resolution using synchrotron radiation.

  10. Activity of novel acyclic pyrimidine nucleoside phosphonates against different DNA polymerase mutants of herpes simplex virus type 1 (HSV-1)

    Czech Academy of Sciences Publication Activity Database

    Andrei, G.; Holý, Antonín; Fiten, P.; Opdenakker, G.; De Clercq, E.; Snoeck, R.

    Chicago, 2003. s. 490. [Interscience Conference on Antimicrobial Agents and Chemotherapy /43./. 14.09.2003-17.09.2003, Chicago] Institutional research plan: CEZ:AV0Z4055905 Keywords : pyrimidine nucleoside phosphonates Subject RIV: CC - Organic Chemistry

  11. Acyclic nucleoside phosphonates as inhibitors of mycobacterium tuberculosis hypoxanthine-guanine phosphoribosyltransferase: Crystal structures and antituberculosis activity

    Czech Academy of Sciences Publication Activity Database

    Hocková, Dana; Eng, W. S.; Špaček, Petr; Janeba, Zlatko; West, N. P.; Woods, K.; Naesens, L. M. J.; Keough, D. T.; Guddat, L. W.

    Newport: Gordon Research Conference, 2015. [Nucleosides, Nucleotides & Oligonucleotides. Gordon Research Conference 2015. 28.06.2015-03.07.2015, Newport] Institutional support: RVO:61388963 Keywords : nucleotide analogues * enzyme inhibitors * tuberculosis Subject RIV: CC - Organic Chemistry

  12. Acyclic nucleoside phosphonates as inhibitors of Mycobacterium tuberculosis hypoxanthine-guanine phosphoribosyltransferase: Crystal structures and antituberculosis activity

    Czech Academy of Sciences Publication Activity Database

    Hocková, Dana; Eng, W. S.; Špaček, Petr; Janeba, Zlatko; West, N. P.; Woods, K.; Naesens, L. M. J.; Keough, D. T.; Guddat, L. W.

    Praha: Czech Chemical Society, 2015. s. 74. [Liblice 2015. Advances in Organic, Bioorganic and Pharmaceutical Chemistry /50./. 06.11.2015-08.11.2015, Olomouc] Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * phosphoribosyltransferase * prodrugs Subject RIV: CC - Organic Chemistry

  13. N-Phosphonocarbonylpyrrolidine Derivatives of Guanine: A New Class of Bi-Substrate Inhibitors of Human Purine Nucleoside Phosphorylase

    Czech Academy of Sciences Publication Activity Database

    Rejman, Dominik; Panova, Natalya; Klener, P.; Maswabi, B.; Pohl, Radek; Rosenberg, Ivan

    2012-01-01

    Roč. 55, č. 4 (2012), s. 1612-1621. ISSN 0022-2623 R&D Projects: GA MŠk 2B06065; GA MŠk(CZ) LC06077; GA AV ČR KAN200520801 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleoside * phosphonic acid * pyrrolidine * purine nucleoside Subject RIV: CC - Organic Chemistry Impact factor: 5.614, year: 2012

  14. Synergistic antiviral effects of ribavirin and the C-nucleoside analogs tiazofurin and selenazofurin against togaviruses, bunyaviruses, and arenaviruses.

    OpenAIRE

    Huggins, J W; Robins, R K; Canonico, P G

    1984-01-01

    Binary combinations of the N-nucleoside ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) and the C-nucleoside analog selenazofurin (2-beta-D-ribofuranosylselenazole-4-carboxamide) or tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) were tested in vitro for activity against Venezuelan equine encephalomyelitis, Japanese encephalitis, yellow fever, Rift Valley fever, Korean hemorrhagic fever, and Pichinde viruses. The 50% effective dose for each compound alone or in a ser...

  15. Amalgamation of Nucleosides and Amino Acids in Antibiotic Biosynthesis: Discovery of an L-Threonine:Uridine-5'-Aldehyde Transaldolase

    OpenAIRE

    Barnard-Britson, Sandra; Chi, Xiuling; Nonaka, Koichi; Spork, Anatol P.; Tibrewal, Nidhi; Goswami, Anwesha; Pahari, Pallab; Ducho, Christian; Rohr, Jurgen; Van Lanen, Steven G

    2012-01-01

    The lipopeptidyl nucleoside antibiotics reperesented by A-90289, caprazamycin, and muraymycin, are structurally highlighted by a nucleoside core that contains a nonproteinogenic β-hydroxy-α-amino acid named 5′-C-glycyluridine (GlyU). Bioinformatic analysis of the biosynthetic gene clusters revealed a shared open reading frame encoding a protein with sequence similarity to serine hydroxymethyltransferases, resulting in the proposal that this shared enzyme catalyzes an aldol-type condensation w...

  16. Novel Type of Acyclic Nucleoside Phosphonates as Inhibitors of 6-Oxopurine Phosphoribosyltransferases: New Potential Antimalarial Chemotherapy Leads

    Czech Academy of Sciences Publication Activity Database

    Hocková, Dana; Keough, D. T.; Janeba, Zlatko; Wang, T.; de Jersey, J.; Guddat, L. W.

    Montreal: International Society of Nucleosides, Nucleotides and Nucleic Acids, 2012. -. [International Round Table on Nucleosides Nucleotides and Nucleic Acids /20./. 05.08.2012-09.08.2012, Montreal] R&D Projects: GA ČR GAP207/11/0108 Grant ostatní: National Health and Medical Research Council(AU) NHMRC 1030353 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleotide analogues * enzyme inhibitors * Plasmodium Subject RIV: CC - Organic Chemistry

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

    Science.gov (United States)

    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

  18. The chemoenzymatic synthesis of clofarabine and related 2′-deoxyfluoroarabinosyl nucleosides: the electronic and stereochemical factors determining substrate recognition by E. coli nucleoside phosphorylases

    OpenAIRE

    Fateev, Ilja V; Konstantin V. Antonov; Konstantinova, Irina D; Tatyana I. Muravyova; Seela, Frank; Esipov, Roman S; Anatoly I. Miroshnikov; 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, 2FAra-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 nucleosid...

  19. Altered E-NTPDase/E-ADA activities and CD39 expression in platelets of sickle cell anemia patients.

    Science.gov (United States)

    Castilhos, Lívia G; Doleski, Pedro H; Adefegha, Stephen A; Becker, Lara V; Ruchel, Jader B; Leal, Daniela B R

    2016-04-01

    Sickle cell anemia (SCA) is a hemoglobinopathy characterized by hemolysis and vaso-occlusions caused by rigidly distorted red blood cells. Sickle cell crisis is associated with extracellular release of nucleotides and platelets, which are critical mediators of hemostasis participating actively in purinergic thromboregulatory enzymes system.This study aimed to investigate the activities of purinergic system ecto-enzymes present on the platelet surface as well as CD39 and CD73 expressions on platelets of SCA treated patients. Fifteen SCA treated patients and 30 health subjects (control group) were selected. Ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-5'-nucleotidase (E-5'-NT) and ecto-adenosine deaminase (E-ADA) activities were measured in platelets isolated from these individuals. Results demonstrated an increase of 41 % in the E-NTPDase for ATP hydrolysis, 52% for ADP hydrolysis and 60 % in the E-ADA activity in SCA patients (P<0.05); however, a two folds decrease in the CD39 expression in platelets was observed in the same group (P<0.01). The increased E-NTPDase activity could be a compensatory mechanism associated with the low expression of CD39 in platelets. Besides, alteration of these enzymes activities suggests that the purinergic system could be involved in the thromboregulatory process in SCA patients. PMID:27044834

  20. The essential oil from Lippia alba induces biochemical stress in the silver catfish (Rhamdia quelen after transportation

    Directory of Open Access Journals (Sweden)

    Joseânia Salbego

    2014-12-01

    Full Text Available This study investigated the effects of the essential oil (EO from Lippia alba on biochemical parameters related to oxidative stress in the brain and liver of silver catfish (Rhamdia quelen after six hours of transport. Fish were transported in plastic bags and divided into three treatments groups: control, 30 µL L- 1 EO from L.alba and 40 µL L-1 EO from L.alba. Prior to transport, the fish were treated with the EO from L. alba (200 µL L -1 for three minutes, except for the control group. Fish transported in bags containing the EO did not have any alterations in acetylcholinesterase, ecto -nucleoside triphosphate diphosphohydrolase and 5'nucleotidase activity in the brain or superoxide dismutase activity in the liver. The hepatic catalase (CAT, glutathione-S-transferase (GST, glutathione peroxidase (GPx, nonprotein thiol and ascorbic acid levels were significantly lower compared to the control group. However, the hepatic thiobarbituric acid- reactive substances, protein oxidation levels and the lipid peroxidation/catalase+glutathione peroxidase (LPO/CAT+GPx ratio were significantly higher in fish transported with both concentrations of the EO, indicating oxidative stress in the liver. In conclusion, considering the hepatic oxidative stress parameters analyzed in the present experiment, the transport of previously sedated silver catfish in water containing 30 or 40 µL L-1 of EO from L. alba is less effective than the use of lower concentrations.

  1. NTPDase2+ cells generate lingual epithelia and papillae

    Directory of Open Access Journals (Sweden)

    Li eFeng

    2012-11-01

    Full Text Available The tongue epithelium is one of the most rapidly self-renewing tissues in adult mammals. Multiple stem cell populations are currently believed to exist in tongue epithelia. Keratin 14 (K14 positive cells differentiate into either lingual epithelia or lingual papillae, while ecto-nucleoside triphosphate diphosphohydrolase 2 (NTPDase2 is associated with neural stem cells and astrocyte-like cells ensheathing the migrating neuroblasts. Here, using a transgenic mouse expressing rtTA from the mouse NTPDase2 promoter, we generated an inducible model by treatment with Doxycycline. By immunohistochemical analysis and in situ hybridization, we found exclusive expression of NTPDase2 in lingual epithelia and lingual papillae. Using inducible genetic cell fate mapping, we further showed that the NTPDase2+ cells generated lingual papillae and epithelia in the adult tongue. Finally, building on our previously proposed paradigm of cell migration stream, a model is further described here for lingual epithelia cell genesis. In short, the current results not only extend our understanding of the cell migration stream in lingual epithelia and lingual papillae, but they also support the concept of multiple stem cell populations in lingual epithelia and papillae.

  2. Dereplication of Known Nucleobase and Nucleoside Compounds in Natural Product Extracts by Capillary Electrophoresis-High Resolution Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Junhui Chen

    2015-03-01

    Full Text Available Nucleobase and nucleoside compounds exist widely in various organisms. An often occurring problem in the discovery of new bioactive compounds from natural products is reisolation of known nucleobase and nucleoside compounds. To resolve this problem, a capillary electrophoresis-high resolution mass spectrometry (CE-HR-MS method providing both rapid separation and accurate mass full-scan MS data was developed for the first time to screen and dereplicate known nucleobase and nucleoside compounds in crude extracts of natural products. Instrumental parameters were optimized to obtain optimum conditions for CE separation and electrospray ionization-time-of-flight mass spectrometry (ESI-TOF/MS detection. The proposed method was verified to be precise, reproducible, and sensitive. Using this method, known nucleobase and nucleoside compounds in different marine medicinal organisms including Syngnathus acus Linnaeus; Hippocampus japonicus Kaup and Anthopleura lanthogrammica Berkly were successfully observed and identified. This work demonstrates that CE-HR-MS combined with an accurate mass database may be used as a powerful tool for dereplicating known nucleobase and nucleoside compounds in different types of natural products. Rapid dereplication of known nucleobase and nucleoside compounds allows researchers to focus on other leads with greater potential to yield new substances.

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

    Science.gov (United States)

    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

  4. Electronic Signatures of all Four DNA Nucleosides in a Tunneling Gap

    Science.gov (United States)

    Chang, Shuai

    2011-03-01

    New approaches to DNA sequencing are required to reduce costs and increase the availability of personalized genomics. Using Scanning Tunneling Microscope as a tool, we report measurements of the current signals generated as free nucleosides diffuse into a tunnel junction in which both electrodes are functionalized with a reagent that presents a hydrogen bond donor and acceptor to the nucleosides. This functionalization serves to both limit the range of molecular orientations in the tunnel gap and reduce the contact resistance, increasing the selectivity of the tunneling signal, so that a direct readout may be possible with a few repeated reads. This work was supported by a grant from the Sequencing Technology Program of the National Human Genome Research Institute (HG004378).

  5. Three-dimensional structure of E. Coli purine nucleoside phosphorylase at 0.99 Å resolution

    Science.gov (United States)

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

    2016-03-01

    Purine nucleoside phosphorylases (PNPs) catalyze the reversible phosphorolysis of nucleosides and are key enzymes involved in nucleotide metabolism. They are essential for normal cell function and can catalyze the transglycosylation. Crystals of E. coli PNP were grown in microgravity by the capillary counterdiffusion method through a gel layer. The three-dimensional structure of the enzyme was determined by the molecular-replacement method at 0.99 Å resolution. The structural features are considered, and the structure of E. coli PNP is compared with the structures of the free enzyme and its complexes with purine base derivatives established earlier. A comparison of the environment of the purine base in the complex of PNP with formycin A and of the pyrimidine base in the complex of uridine phosphorylase with thymidine revealed the main structural features of the base-binding sites. Coordinates of the atomic model determined with high accuracy were deposited in the Protein Data Bank (PDB_ID: 4RJ2).

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

    Energy Technology Data Exchange (ETDEWEB)

    Abramchik, Yu. A., E-mail: inna@ns.crys.ras.ru; Timofeev, V. I., E-mail: espiov@ibch.ru; Zhukhlistova, N. E., E-mail: tostars@mail.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Muravieva, T. I.; Esipov, R. S. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Kuranova, I. P. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    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.

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

    International Nuclear Information System (INIS)

    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. P21 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

  8. Using conformationally locked nucleosides to calibrate the anomeric effect: Implications for glycosyl bond stability

    Science.gov (United States)

    Moon, Hyung Ryong; Siddiqui, Maqbool A.; Sun, Guangyu; Filippov, Igor V.; Landsman, Nicholas A.; Lee, Yi-Chien; Adams, Kristie M.; Barchi, Joseph J.; Deschamps, Jeffrey R.; Nicklaus, Marc C.; Kelley, James A.

    2010-01-01

    Steric and electronic parameters such as the anomeric effect (AE) and gauche effect play significant roles in steering the North ⇆ South equilibrium of nucleosides in solution. Two isomeric oxa-bicyclo[3.1.0]hexane nucleosides that are conformationally locked in either the North or the South conformation of the pseudorotational cycle were designed to study the consequences of having the AE operational or not, independent of other parameters. The rigidity of the system allowed the orientation of the orbitals involved to be set in “fixed” relationships, either antiperiplanar where the AE is permanently “on”, or gauche where the AE is impaired. The consequences of these two alternatives were subject to high-level calculations and measured experimentally by x-ray crystallography, hydrolytic stability of the glycosyl bond, and pKa values. PMID:21052524

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

    International Nuclear Information System (INIS)

    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

  10. Modular synthesis of 6-substituted pyridin-3-yl C-nucleosides

    Czech Academy of Sciences Publication Activity Database

    Joubert, Nicolas; Hocek, Michal

    Dublin : University Cillege Dublin, 2007. s. 214. [European Symposium on Organic Chemistry /15./. 08.07.2007-13.07.2007, Dublin] R&D Projects: GA MŠk LC512 Grant ostatní: NIH(US) 1R03TW007372-01 Institutional research plan: CEZ:AV0Z40550506 Keywords : C-nucleosides * modular synthesis Subject RIV: CC - Organic Chemistry

  11. Mechanisms by Which Human DNA Primase Chooses To Polymerize a Nucleoside Triphosphate

    Czech Academy of Sciences Publication Activity Database

    Urban, M.; Joubert, Nicolas; Purse, B. W.; Hocek, Michal; Kuchta, R. D.

    2010-01-01

    Roč. 49, č. 4 (2010), s. 727-735. ISSN 0006-2960 R&D Projects: GA MŠk LC512; GA AV ČR IAA400550902 Grant ostatní: NIH(US) GM54194 Institutional research plan: CEZ:AV0Z40550506 Keywords : C-nucleosides * DNA polymerase * primase * mechanism Subject RIV: CC - Organic Chemistry Impact factor: 3.226, year: 2010

  12. Tunnel Conductance of Watson-Crick Nucleoside-Base Pairs from Telegraph Noise

    OpenAIRE

    Chang, Shuai; He, Jin; Lin, Lisha; Zhang, Peiming; Liang, Feng; Young, Michael; Huang, Shuo; Lindsay, Stuart

    2009-01-01

    The use of tunneling signals to sequence DNA is presently hampered by the small tunnel conductance of a junction spanning an entire DNA molecule. The design of a readout system that uses a shorter tunneling path requires knowledge of the absolute conductance across base-pairs. We have exploited the stochastic switching of hydrogen-bonded DNA base-nucleoside pairs trapped in a tunnel junction to determine the conductance of individual molecular pairs. This conductance is found to be sensitive ...

  13. Comparison of Clostridium difficile detection by monolayer and by inhibition of nucleoside uptake

    International Nuclear Information System (INIS)

    Detection and identification of Clostridium difficile toxin by traditional monolayer assay were compared with results obtained by a new procedure based on toxin-dependent inhibition of target cell uptake of a radioactive nucleoside. A high degree of correlation was noted between the two determinations. Although the new procedure was quantitative and objective, its value is seen at present as a rapid screen that may support results obtained in monolayers and as a potential assay for other, currently unidentified, toxins

  14. Synthesis and properties of chiral open-ring acyclic nucleoside bisphosphonates

    Czech Academy of Sciences Publication Activity Database

    Doláková, Petra; Dračínský, Martin; Holý, Antonín

    Elsevier. Roč. 74, č. 3 (2007), A72. ISSN 0166-3542. [Conference on Antiviral Research. 29.04.2007-03.05.2007, Palm Springs] R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Grant ostatní: René Descartes Prize-2001(XE) HPAW-2002-100096 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonate * bisphosphonate * pyrimidine Subject RIV: CC - Organic Chemistry

  15. PC-1 Nucleoside Triphosphate Pyrophosphohydrolase Deficiency in Idiopathic Infantile Arterial Calcification

    OpenAIRE

    Rutsch, Frank; Vaingankar, Sucheta; Johnson, Kristen; Goldfine, Ira; Maddux, Betty; Schauerte, Petra; Kalhoff, Hermann; Sano, Kimihiko; Boisvert, William A.; Superti-Furga, Andrea; Terkeltaub, Robert

    2001-01-01

    Inogranic pyrophosphate (PPi) inhibits hydroxyapatite deposition, and mice deficient in the PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) Plasma cell membrane glycoprotein-1 (PC-1) develop peri-articular and arterial calcification in early life. In idiopathic infantile arterial calcification (IIAC), hydroxyapatite deposition and smooth muscle cell (SMC) proliferation occur, sometimes associated with peri-articular calcification. Thus, we assessed PC-1 expression and PPi...

  16. Enhanced Topical and Transdermal Delivery of Antineoplastic and Antiviral Acyclic Nucleoside Phosphonate cPr-PMEDAP

    Czech Academy of Sciences Publication Activity Database

    Vávrová, K.; Kovaříková, P.; Školová, B.; Líbalová, M.; Roh, J.; Čáp, R.; Holý, Antonín; Hrabálek, A.

    2011-01-01

    Roč. 28, č. 12 (2011), s. 3105-3115. ISSN 0724-8741 R&D Projects: GA MŠk 1M0508 Grant ostatní: GA ČR(CZ) GAP207/11/0365 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * antivirals * antineoplastics * permeation enhancer * topical skin application * transdermal delivery Subject RIV: CC - Organic Chemistry Impact factor: 4.093, year: 2011

  17. Enantioselective Synthesis of Homo-N-Nucleosides Containing a 1,4-Dioxane Sugar Analog

    Directory of Open Access Journals (Sweden)

    Qiang Yu

    2008-12-01

    Full Text Available A dioxane homo-sugar analog, (2S,5S-and (2R,5S-5-[(4S-2,2-dimethyl-1,3-dioxolan-4-yl]-2-iodomethyl-1,4-dioxane was prepared from (2R,3R-dimethyl tartrate, and further elaborated into the corresponding homo-N-nucleoside analogs by its reactions with uracil and adenine, respectively.

  18. Synthesis of some nucleosides derivatives from L- rhamnose with expected biological activity

    OpenAIRE

    Ghoneim Amira

    2011-01-01

    Abstract Practical procedures for production of variously blocked compounds from L-rhamnose have been developed. These compounds are highly useful as indirect β-L-rhamnosyl donors. This approach represents a new method for the synthesis of aromatic nucleoside analogues and the synthesis of (3S, 4S, 5S, 6R) 3, 4, 5-triacetoxy-2-methyl-7,9-diaza-1-oxa-spiro [4,5]decane-10-one-8-thione (7).

  19. Current Status of Older and New Purine Nucleoside Analogues in the Treatment of Lymphoproliferative Diseases

    OpenAIRE

    Pawel Robak; Ewa Lech-Maranda; Tadeusz Robak; Anna Korycka

    2009-01-01

    For the past few years more and more new cytotoxic agents active in the treatment of hematological malignancies have been synthesized and become available for either in vitro studies or clinical trials. Among them the class of antineoplastic drugs belonging to the purine nucleoside analogues group (PNAs) plays an important role. Three of them: pentostatin (DCF), cladribine (2-CdA) and fludarabine (FA) were approved by Food and Drug Administration (FDA) for the treatment of hematological malig...

  20. Synergistic cytotoxicity of the DNA alkylating agent busulfan, nucleoside analogs and SAHA in lymphoma cell lines

    OpenAIRE

    Valdez, Benigno C.; Murray, David; Nieto, Yago; Li, Yang; Wang, Guiyun; Champlin, Richard E.; Andersson, Borje S.

    2011-01-01

    Hematopoietic stem cell transplantation (HSCT) is a promising treatment for lymphomas. Its success depends on effective pre-transplant conditioning regimens. We previously reported on the efficacy of DNA alkylating agent-nucleoside analog (NA) combinations for conditioning in AML. We hypothesized that a similar combinatory approach can be used for lymphomas. A combination of busulfan (Bu) with two NAs – clofarabine (Clo), fludarabine (Flu) or gemcitabine (Gem) – resulted in synergistic cytoto...

  1. Conformation analysis of nucleoside analogues containing selenium and tellurium in five-membered pseudosugar ring

    Czech Academy of Sciences Publication Activity Database

    Poštová Slavětínská, Lenka; Pohl, Radek; Rejman, Dominik

    Praha : Institute of Organic Chemistry and Biochemistry AS CR, v. v. i, 2014 - (Hocek, M.), s. 354-355 ISBN 978-80-86241-50-0. - (Collection Symposium Series. 14). [Symposium on Chemistry of Nucleic Acid Components /16./. Český Krumlov (CZ), 08.06.2014-13.06.2014] R&D Projects: GA ČR GA13-24880S Institutional support: RVO:61388963 Keywords : nucleoside analogues * five-membered pseudosugar ring * NMR Subject RIV: CC - Organic Chemistry

  2. Differential effects of acyclic nucleoside phosphonates on nitric oxide and cytokines in rat hepatocytes and macrophages

    Czech Academy of Sciences Publication Activity Database

    Kostecká, Petra; Holý, Antonín; Farghali, H.; Zídek, Zdeněk; Kmoníčková, Eva

    2012-01-01

    Roč. 12, č. 2 (2012), s. 342-349. ISSN 1567-5769 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * cytokines * nitric oxide Subject RIV: FR - Pharmacology ; Medidal Chemistry; CC - Organic Chemistry (UOCHB-X) Impact factor: 2.417, year: 2012

  3. Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus

    OpenAIRE

    de la Peña, Andres H.; Suarez, Allison; 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...

  4. The Hypolipidemic Activity of Boronated Nucleosides in Male Mice and Rats

    OpenAIRE

    Burnham, Bruce S.; Sood, Anup; Tomasz, Jeno; Powell, W J; Spielvogel, Bernard F.; Chen, S. Y.; Hall, Iris H.

    1996-01-01

    The boronated nucleosides with varying bases and sugar moieties were shown to be potent hypolipidemic agents in rodents. The 3′– aminocynaoborane dideoxythymidine derivative caused reductions in serum cholesterol and triglyceride levels, tissue lipids, VLDL and LDL cholesterol levels while elevating HDL cholesterol levels in rodents. The agents suppressed rat hepatic acetyl CoA synthetase, HMG-CoA reductase, acyl-CoA cholesterol acyl transferase, phosphatidylate phosphohydrolase and lipoprote...

  5. Synthesis of conformationally locked carbocyclic nucleosides with norbornane as pseudosugar moiety

    Czech Academy of Sciences Publication Activity Database

    Dejmek, Milan; Hřebabecký, Hubert; Šála, Michal; Dračínský, Martin; Nencka, Radim

    Praha : Institute of Organic Chemistry and Biochemistry AS CR, v. v. i., 2011 - (Hocek, M.), s. 208-215 ISBN 978-80-86241-37-1. - (Collection Symposium Series. 12). [Chemistry of Nucleic Acid Components /15./. Český Krumlov (CZ), 05.06.2011-10.06.2011] R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : norbornane * conformationally locked cyrbocyclic nucleosides Subject RIV: CC - Organic Chemistry

  6. Synthesis and properties of triplex-forming oligonucleotides containing 2'-modified nucleoside analogues

    OpenAIRE

    Lou, Chenguang

    2011-01-01

    Triplex-forming oligonucleotides (TFOs) bind to the major groove of the DNA duplex via the Hoogsteen interactions to generate triple helices. Potential applications of triplex technology are in regulation of gene expression, site-directed gene-knockout, mutation correction and as tools in molecular biotechnology. The presence of 2’-modified nucleosides in therapeutic oligonucleotides inhibits enzymatic degradation in vivo. Therefore such sugar modifications have the potential to improve the b...

  7. The antiretroviral nucleoside analogue Abacavir reduces cell growth and promotes differentiation of human medulloblastoma cells

    OpenAIRE

    De Rossi, Alessandra; Russo, Giuseppe; Puca, Andrew; La Montagna, Raffaele; Caputo, Mariella; Mattioli, Eliseo; Lopez, Massimo; Giordano, Antonio; Pentimalli, Francesca

    2009-01-01

    Abacavir is one of the most efficacious nucleoside analogues, with a well-characterized inhibitory activity on reverse transcriptase enzymes of retroviral origin, and has been clinically approved for the treatment of AIDS. Recently, Abacavir has been shown to inhibit also the human telomerase activity. Telomerase activity seems to be required in essentially all tumours for the immortalization of a subset of cells, including cancer stem cells. In fact, many cancer cells are dependent on telome...

  8. Nuclear quadrupole resonance of 14N and 2H in pyrimidines, purines, and their nucleosides

    Science.gov (United States)

    Rabbani, S. R.; Edmonds, D. T.; Gosling, P.

    Using nuclear quadrupole double-resonance techniques, nitrogen-14 and deuterium nuclear quadrupole coupling constants and asymmetry parameters have been measured in uracil, 5-bromouracil, cytosine, adenine, xanthine, hypoxanthine, their nucleosides, 2-aminopyrimidine, and benzimidazole. Zeeman studies and the detection of the simultaneous transitions of neighboring nuclei allowed in many cases a complete assignment of the observed spectral lines to particular 14N and 2D sites.

  9. Affinity capillary electrophoresis applied to investigation of complexes of acyclic nucleoside phosphonates with beta-cyclodextrin

    Czech Academy of Sciences Publication Activity Database

    Šolínová, Veronika; Mikysková, Hana; Kaiser, Martin Maxmilian; Janeba, Zlatko; Holý, Antonín; Kašička, Václav

    Ljubljana: National Institute of Chemistry, 2015 - (Vovk, I.; Glavnik, V.; Albreht, A.). s. 127 ISBN 978-961-6104-28-9. [ISSS 2015. International Symposium on Separation Sciences /21./. 30.06.2015-03.07.2015, Ljubljana] R&D Projects: GA ČR(CZ) GA13-17224S; GA MV VG20102015046 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * complexes * capillary electrophoresis Subject RIV: CB - Analytical Chemistry, Separation

  10. Determination of apparent binding constants of complexes of acyclic nucleoside phosphonates with cyclodextrins by capillary electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Šolínová, Veronika; Kaiser, Martin Maxmilian; Lukáč, Miloš; Janeba, Zlatko; Kašička, Václav

    Salzburg: Society of Analytical Chemistry, 2014. P510. [ISC 2014. International Symposium on Chromatography /30./. 14.09.2014-18.09.2014, Salzburg] R&D Projects: GA ČR(CZ) GAP206/12/0453; GA ČR(CZ) GA13-17224S; GA MV VG20102015046 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * capillary electrophoresis * binding constant Subject RIV: CB - Analytical Chemistry, Separation

  11. Conformational analysis and enzymatic decomposition of a locked nucleoside phosphoramidates using NMR spectroscopy

    Czech Academy of Sciences Publication Activity Database

    Procházková, Eliška; Hřebabecký, Hubert; Nencka, Radim; Dračínský, Martin

    Brno : Masaryk University, 2014 - (Novotný, J.; Foroutan -Nejad, C.; Marek, R.). C26 ISBN 978-80-86441-45-0. [NMR Valtice. Central European NMR Meeting /29./. 27.4.-30.4.2014, Valtice] R&D Projects: GA ČR GA13-24880S Institutional support: RVO:61388963 Keywords : enzymatic decomposition * locked nucleosides * conformation * NMR spectroscopy * DFT calculations Subject RIV: CC - Organic Chemistry

  12. Modification of guanine bases by nucleoside phosphoramidite reagents during the solid phase synthesis of oligonucleotides.

    OpenAIRE

    Pon, R T; Damha, M J; Ogilvie, K K

    1985-01-01

    Nucleoside 3'-phosphoramidite and chlorophosphite reagents have been found to react with the lactam function of guanine. This reaction caused unsatisfactory results when oligodeoxyribonucleotides containing a large number of guanine bases were prepared in an automated solid phase synthesizer. The guanine modification is unstable, and leads to depurination and chain cleavage. This side reaction can be eliminated by protecting the O6-position. A new O6-p-nitrophenylethyldeoxyguanosine phosphora...

  13. Detergent inhibited, heat labile nucleoside triphosphatase in cores of avian myeloblastosis virus

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank

    1978-01-01

    Endogenous DNA synthesis was studied in isolated core particles of avian myeloblastosis virus. It was found that cores contained an enzymatic activity which rapidly converted the added nucleoside triphosphates to diphosphates (but not further) at 0 degrees C, thus inhibiting DNA synthesis. This t...... triphosphatase probably originates from the viral membranes. In the cores the enzyme is completely inactivated by low concentrations (0.02%) of Nonident P-40. Also, the enzyme is very thermolabile and denatures rapidly at 38 degrees C....

  14. Altered Enthalpy-Entropy Compensation in Picomolar Transition State Analogues of Human Purine Nucleoside Phosphorylase†

    OpenAIRE

    Edwards, Achelle A.; Mason, Jennifer M.; Clinch, Keith; Tyler, Peter C.; Evans, Gary B.; Schramm, Vern L.

    2009-01-01

    Human purine nucleoside phosphorylase (PNP) belongs to the trimeric class of PNPs and is essential for catabolism of deoxyguanosine. Genetic deficiency of PNP in humans causes a specific T-cell immune deficiency and transition state analogue inhibitors of PNP are in development for treatment of T-cell cancers and autoimmune disorders. Four generations of Immucillins have been developed, each of which contains inhibitors binding with picomolar affinity to human PNP. Full inhibition of PNP occu...

  15. Sinefungin, a Natural Nucleoside Analogue of S-Adenosylmethionine, Inhibits Streptococcus pneumoniae Biofilm Growth

    OpenAIRE

    Mukesh Kumar Yadav; Seok-Won Park; Sung-Won Chae; Jae-Jun Song

    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 ...

  16. Comparison of Clostridium difficile detection by monolayer and by inhibition of nucleoside uptake

    Energy Technology Data Exchange (ETDEWEB)

    Fuhr, J.E.; Trent, D.J.; Collmann, I.R.

    1987-02-01

    Detection and identification of Clostridium difficile toxin by traditional monolayer assay were compared with results obtained by a new procedure based on toxin-dependent inhibition of target cell uptake of a radioactive nucleoside. A high degree of correlation was noted between the two determinations. Although the new procedure was quantitative and objective, its value is seen at present as a rapid screen that may support results obtained in monolayers and as a potential assay for other, currently unidentified, toxins.

  17. Synthesis and properties of chiral open-ring acyclic nucleoside bisphosphonates

    Czech Academy of Sciences Publication Activity Database

    Doláková, Petra; Masojídková, Milena; Holý, Antonín

    Praha : ÚOCHB AV ČR, 2005 - (Hocek, M.), s. 395-396 ISBN 80-86241-25-4. - (Collection Symposium Series. 7). [Chemistry of Nucleic Acid Components /13./. Špindlerův Mlýn (CZ), 03.09.2005-09.09.2005] R&D Projects: GA MŠk(CZ) 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonate * pyrimidine * antiviral activity Subject RIV: CC - Organic Chemistry

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

    Science.gov (United States)

    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

  19. Preliminary crystallographic studies of purine nucleoside phosphorylase from the cariogenic pathogen Streptococcus mutans

    International Nuclear Information System (INIS)

    Purine nucleoside phosphorylase (PNP), which is a pivotal enzyme in the nucleotide-salvage pathway, has been expressed in Escherichia coli strain BL21 (DE3) in a soluble form at a high level. After purification of the PNP enzyme, the protein was crystallized using the sitting-drop vapour-diffusion technique. The punA gene of the cariogenic pathogen Streptococcus mutans encodes purine nucleoside phosphorylase (PNP), which is a pivotal enzyme in the nucleotide-salvage pathway, catalyzing the phosphorolysis of purine nucleosides to generate purine bases and α-ribose 1-phosphate. In the present work, the PNP protein was expressed in Escherichia coli strain BL21 (DE3) in a soluble form at a high level. After purification of the PNP enzyme, the protein was crystallized using the sitting-drop vapour-diffusion technique; the crystals diffracted to 1.6 Å resolution at best. The crystals belonged to space group H3, with unit-cell parameters a = b = 113.0, c = 60.1 Å

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

    Science.gov (United States)

    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

  1. The chemoenzymatic synthesis of clofarabine and related 2'-deoxyfluoroarabinosyl nucleosides: the electronic and stereochemical factors determining substrate recognition by E. coli nucleoside phosphorylases.

    Science.gov (United States)

    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

  2. The chemoenzymatic synthesis of clofarabine and related 2′-deoxyfluoroarabinosyl nucleosides: the electronic and stereochemical factors determining substrate recognition by E. coli nucleoside phosphorylases

    Directory of Open Access Journals (Sweden)

    Ilja V. Fateev

    2014-07-01

    Full Text Available Two approaches to the synthesis of 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyladenine (1, clofarabine were studied. The first approach consists in the chemical synthesis of 2-deoxy-2-fluoro-α-D-arabinofuranose-1-phosphate (12a, 2FAra-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 (2FAra-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

  3. An intersubunit disulfide bridge stabilizes the tetrameric nucleoside diphosphate kinase of Aquifex aeolicus.

    Science.gov (United States)

    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

  4. Enantioselective Supramolecular Carriers for Nucleoside Drugs. A Thermodynamic and Kinetic Gas Phase Investigation

    Science.gov (United States)

    Fraschetti, Caterina; Filippi, Antonello; Crestoni, Maria Elisa; Villani, Claudio; Roselli, Graziella; Mortera, Stefano Levi; Speranza, Maurizio

    2012-10-01

    The enantioselective interactions between chiral tetra-amidic receptors and nucleosides have been investigated by the ESI-IT-MS and ESI-FT-ICR-MS methodologies. Configurational effects on the CID fragmentation of diastereomeric [ M H 2 •H•A] + aggregates (A = 2'-deoxycytidine dC, citarabine ( ara-C) were found to be mostly offset by isotope effect in [ S X 2 •H•A] + (X = H, D) differently from the results obtained on the analogues (A = cytidine C and gemcitabine G). This result points the involvement of two different nucleoside/tetraamide isoforms. The structural differences of the [ M H 2 •H•A] + (A = C and G) complexes vs. the [ M H 2 •H•A] + ( dC and ara-C) ones is fully confirmed by the kinetics of their uptake of the 2-aminobutane enantiomers, measured by FT-ICR mass spectrometry. Indeed, uptake of the 2-aminobutane enantiomers by [ M H n •H•A] + (n = 1,2; A = dC and ara-C) complexes is reversible, while that by [ M H n •H•A] + (n = 1,2; A = C and G) is not. The most encouraging result concerning the measured fragmentation and kinetic differences between C and ara-C, that are just epimers, indicates the possibility to subtly modulate the non-covalent drug/receptor interactions, through the electronic properties of the 2'-substituent on the nucleoside furanose ring, and furthermore on its three-dimensional position.

  5. Analysis of the Main Nucleosides in Cordyceps Sinensis by LC/ESI-MS

    Directory of Open Access Journals (Sweden)

    Yun-Biao He

    2010-01-01

    Full Text Available A sensitive, selective and reliable liquid chromatography-mass spectrometry coupled with electrospray ionization interface method for simultaneous separation and determination of thymine, adenine, adenosine and cordycepin in Cordyceps sinensis has been established. The optimum separation for these analytes was achieved using a gradient elution system and a 2.0 × 150 mm Shimadzu VP-ODS column. 2-Chloroadenosine was used as internal standard for this assay. [M+H]+ions at m/z 127, 136, 268, 252 and 302 were chosen and selective ion monitoring (SIM mode was used for quantitative analysis of the four main nucleosides. The regression equations were linear in the range of 1.0–117.5 μg·mL-1 for thymine, 1.8-127.0 μg·mL-1 for adenine, 0.6-114.0 μg·mL-1 for adenosine and 0.5-107.5 μg·mL-1 for cordycepin. The limits of quantitation (LOQ and detection (LOD were 1.0 and 0.2 μg·mL-1 for thymine, 1.8 and 0.6 μg·mL-1 for adenine, 0.6 and 0.1 μg·mL-1 for adenosine and 0.5 and 0.1 μg·mL-1 for cordycepin, respectively. The recoveries of the four nucleosides ranged from 98.47 to 99.32%. The developed method was successfully used to determine nucleosides in Cordyceps sinensis from different sources.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tran, Timothy H.; Christoffersen, S.; Allan, Paula W.; Parker, William B.; Piskur, Jure; Serra, I.; Terreni, M.; Ealick, Steven E. (Cornell); (Pavia); (Lund); (Southern Research)

    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.

  8. Screening of new non-nucleoside reverse transcriptase inhibitors of HIV-1 based on traditional Chinese medicines database

    Institute of Scientific and Technical Information of China (English)

    Tao Liu; Ai Xiu Li; You Pan Miao; Ke Zhu Wu; Yi Ma

    2009-01-01

    HIV-1 RT is an important target for the treatment of AIDS. There are two major classes of antiviral agents that inhibit HIV-1 RT have been identified, nucleoside RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). In this report, a noval class of non-nucleoside compound with potential RT inhibitory activity were found from the traditional Chinese medicines database (TCMD) using a combination of virtual screening, docking, molecular dynamic simulations, where results were ranked by scoring function of the docking tool. The result indicates that M4753 (a compound derived from TCMD) has not only the lowest bonding energy but also the best match in geometric conformation with the forthcoming NNRTIs. Accordingly M4753 might possibly become a promising lead compound of NNRTIs for AIDS therapy.

  9. Focus on Chirality of HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors

    Directory of Open Access Journals (Sweden)

    Valeria Famiglini

    2016-02-01

    Full Text Available Chiral HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs are of great interest since one enantiomer is often more potent than the corresponding counterpart against the HIV-1 wild type (WT and the HIV-1 drug resistant mutant strains. This review exemplifies the various studies made to investigate the effect of chirality on the antiretroviral activity of top HIV-1 NNRTI compounds, such as nevirapine (NVP, efavirenz (EFV, alkynyl- and alkenylquinazolinone DuPont compounds (DPC, diarylpyrimidine (DAPY, dihydroalkyloxybenzyloxopyrimidine (DABO, phenethylthiazolylthiourea (PETT, indolylarylsulfone (IAS, arylphosphoindole (API and trifluoromethylated indole (TFMI The chiral separation, the enantiosynthesis, along with the biological properties of these HIV-1 NNRTIs, are discussed.

  10. Biophysical Insights into the Inhibitory Mechanism of Non-Nucleoside HIV-1 Reverse Transcriptase Inhibitors

    Directory of Open Access Journals (Sweden)

    Nicolas Sluis-Cremer

    2013-11-01

    Full Text Available HIV-1 reverse transcriptase (RT plays a central role in HIV infection. Current United States Federal Drug Administration (USFDA-approved antiretroviral therapies can include one of five approved non-nucleoside RT inhibitors (NNRTIs, which are potent inhibitors of RT activity. Despite their crucial clinical role in treating and preventing HIV-1 infection, their mechanism of action remains elusive. In this review, we introduce RT and highlight major advances from experimental and computational biophysical experiments toward an understanding of RT function and the inhibitory mechanism(s of NNRTIs.

  11. Metabolic Abnormalities Associated with the Use of Protease Inhibitors and Non-nucleoside Reverse Transcriptase Inhibitors

    Directory of Open Access Journals (Sweden)

    Madhu N. Rao

    2006-01-01

    Full Text Available The use of protease inhibitors and non-nucleoside reverse transcriptase inhibitors for the treatment of HIV infection and AIDS has been associated with multiple abnormalities in glucose and lipid metabolism. Specifically, these abnormalities include insulin resistance, increased triglycerides and increased LDL cholesterol levels. The metabolic disturbances are due to a combination of factors, including the direct effect of medications, restoration to health and HIV disease, as well as individual genetic predisposition. Of the available anti-retroviral medications, indinavir has been associated with causing the most insulin resistance and ritonavir with causing the most hypertriglyceridemia.

  12. Acyclic nucleoside phosphonates with 5-azacytosine moiety: A new class of antiviral agents

    Czech Academy of Sciences Publication Activity Database

    Krečmerová, Marcela; Holý, Antonín; Pískala, Alois; Masojídková, Milena; Andrei, G.; Snoeck, R.; Naesens, L.; Balzarini, J.; Neyts, J.; De Clercq, E.

    Bern : DCB University of Bern, 2006. s. 340. [IRT-International Roundtable on Nucleosides, Nucleotides and Nucleic Acids /17./. 03.09.2006-07.09.2006, Bern] R&D Projects: GA MŠk(CZ) 1M0508; GA AV ČR(CZ) 1QS400550501 Grant ostatní: Descartes Prize(XE) HPAW-2002-100096; NIAID/NIH(US) UC1AI062540-01 Institutional research plan: CEZ:AV0Z40550506 Keywords : HPMP-5-azaC * 5-azacytosine * antiviral activity Subject RIV: CC - Organic Chemistry

  13. Conformationally constrained nucleoside phosphonic acids - potent inhibitors of human mitochondrial and cytosolic 5'(3')-nucleotidases

    Czech Academy of Sciences Publication Activity Database

    Šimák, Ondřej; Pachl, Petr; Fábry, Milan; Buděšínský, Miloš; Jandušík, T.; Hnízda, Aleš; Skleničková, Radka; Petrová, Magdalena; Veverka, Václav; Řezáčová, Pavlína; Brynda, Jiří; Rosenberg, Ivan

    2014-01-01

    Roč. 12, č. 40 (2014), s. 7971-7982. ISSN 1477-0520 R&D Projects: GA ČR GA203/09/0820; GA ČR GA13-24880S; GA ČR GA13-26526S; GA MŠk(CZ) LK11205; GA AV ČR KAN200520801 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : 5'(3')-nucleotidase * structure * inhibition * cdN * mdN * nucleoside * SAR * phosphonic acid Subject RIV: CC - Organic Chemistry Impact factor: 3.562, year: 2014

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

    Directory of Open Access Journals (Sweden)

    Wenjie Ye

    Full Text Available 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

  15. Substituted indoles as HIV-1 non-nucleoside reverse transcriptase inhibitors: a patent evaluation (WO2015044928).

    Science.gov (United States)

    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

  16. Development of a general and modular approach to C-nucleosides

    Czech Academy of Sciences Publication Activity Database

    Kubelka, Tomáš; Štefko, Martin; Bárta, Jan; Joubert, Nicolas; Urban, Milan; Chapuis, Hubert Jean; Hocek, Michal

    Praha : Institute of Organic Chemistry and Biochemistry AS CR, v. v. i., 2011 - (Hocek, M.), s. 180-183 ISBN 978-80-86241-37-1. - (Collection Symposium Series. 12). [Chemistry of Nucleic Acid Components /15./. Český Krumlov (CZ), 05.06.2011-10.06.2011] R&D Projects: GA MŠk LC512; GA AV ČR IAA400550902 Institutional research plan: CEZ:AV0Z40550506 Keywords : C-nucleosides * cross-coupling * palladium Subject RIV: CC - Organic Chemistry

  17. Modular synthesis of 5-substituted thiophene and furan C-nucleosides and their analogues

    Czech Academy of Sciences Publication Activity Database

    Bárta, Jan; Hocek, Michal

    Praha : Institute of Organic Chemistry and Biochemistry AS CR, v. v. i., 2011 - (Hocek, M.), s. 300-301 ISBN 978-80-86241-37-1. - (Collection Symposium Series. 12). [Chemistry of Nucleic Acid Components /15./. Český Krumlov (CZ), 05.06.2011-10.06.2011] R&D Projects: GA MŠk LC512; GA AV ČR IAA400550902 Institutional research plan: CEZ:AV0Z40550506 Keywords : C-nucleosides * furan * thiophene Subject RIV: CC - Organic Chemistry

  18. Facile syntheses of pyrimidine acyclic nucleoside phosphonates and their potential evaluation for biomedical applications

    Czech Academy of Sciences Publication Activity Database

    Pomeisl, Karel; Holý, Antonín; Votruba, Ivan; Nencka, Radim; Pohl, Radek

    Praha : Institute of Organic Chemistry and Biochemistry ASCR, 2008 - (Hocek, M.), s. 201-205 ISBN 978-80-86241-29-6. - (Collection Symposium Series. 10). [Symposium on Chemistry of Nucleic Acid Components /14./. Český Krumlov (CZ), 08.06.2008-13.06.2008] R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * thymidine phosphorylase * dUTPpase Subject RIV: CC - Organic Chemistry

  19. Evaluation of localized bacterial infection using radioisotope-labeled nucleosides imaging modality

    International Nuclear Information System (INIS)

    Conventional diagnostic methods for infections are difficult to distinguish localized bacterial infections from sites of sterile inflammation. For this reason, the importance of developing methods to image bacterial infections is widely recognized. In this study to acquire bacterial infection imaging with radiolabeled nucleosides, in vitro bacterial thymidine kinase (tk) activities of Salmonella typhimurium with [18F]FLT and [125I]IVDU were measured and localized infections model in BALB/c mice was imaged with [18F]FLT or [125I]FIAU

  20. Inhibition of human thymidine phosphorylase by phosphonate analogues of pyrimidine nucleoside 2’(3’)-phosphates

    Czech Academy of Sciences Publication Activity Database

    Panova, Natalya; Kóšiová, Ivana; Šimák, Ondřej; Petrová, Magdalena; Liboska, Radek; Buděšínský, Miloš; Rejman, Dominik; Rosenberg, Ivan

    Lyon : Université de Lyon, 2010, s. 336-337. [International Roundtable on Nucleosides, Nucleotides and Nucleic Acids. IRT 2010. Lyon (FR), 29.08.2010-03.09.2010] R&D Projects: GA MŠk(CZ) LC06077; GA AV ČR KAN200520801; GA MŠk(CZ) LC06061 Institutional research plan: CEZ:AV0Z40550506 Keywords : human thymidine phosphorylase * phosphorylase inhibition * phosphonate nucleosides Subject RIV: CC - Organic Chemistry http://irt2010.univ-lyon1.fr

  1. N2-(1-Methoxycarbonylethyl)guanosine, a new nucleoside coupled with an amino acid derivative from Amanita exitialis

    Institute of Scientific and Technical Information of China (English)

    Yu Lang Chi; Hui Ye Zhang; Jing Hua Xue; Jing Hao; Mei Fang Liu; Xiao Yi Wei

    2009-01-01

    A new purine nucleoside coupled with an amino acid derivative, N2-(1-methoxycarbonylethyl)guanosine 1, along with βearboline and russulaceramide was isolated from the fruiting bodies ofAmanita exitialis, a newly described poisonous mushroom. Its structure was elucidated by spectroscopic methods. This is the first report of naturally occurring nucleosides in which an α-amino acid derivative is bonded through its a-amino nitrogen to a nucleobase aglycone by a C-N bond. The new compound was found to be toxic in brine shrimp lethality test (BST).

  2. Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.

    Science.gov (United States)

    Menezes, Camila Braz; Durgante, Juliano; de Oliveira, Rafael Rodrigues; Dos Santos, Victor Hugo Jacks Mendes; Rodrigues, Luiz Frederico; Garcia, Solange Cristina; Dos Santos, Odelta; Tasca, Tiana

    2016-05-01

    Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final

  3. Non-nucleoside reverse transcriptase inhibitors: a review on pharmacokinetics, pharmacodynamics, safety and tolerability

    Directory of Open Access Journals (Sweden)

    Iris Usach

    2013-09-01

    Full Text Available 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.

  4. Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1

    International Nuclear Information System (INIS)

    T. cruzi TcNDPK1 was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl2, 20% PEG 3350. Data were collected to 3.5 Å resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 Å. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease

  5. Parameterization of AZT-A widely used nucleoside inhibitor of HIV-1 reverse transcriptase

    Science.gov (United States)

    Carvalho, Alexandra T. P.; Fernandes, Pedro A.; Ramos, Maria J.

    Seven nucleoside reverse transcriptase (RT) inhibitors are currently used in the clinical treatment of acquired immunodeficiency syndrome (AIDS). These substrate analogues block DNA synthesis by the viral enzyme RT. However, the emergence of resistant variants of RT allied to their long-term toxicity requires the design of new and better RT inhibitors, with long-term in vivo efficacy. In this work we used density functional theory (DFT) calculations to develop a set of molecular mechanics (MM) parameters committed to the AMBER force field for one of the most used in the clinic nucleoside reverse transcriptase inhibitors (NRTIs): zidovudine (AZT). These parameters were tested by comparing the optimized geometries of AZT at both the DFT and MM levels of theory. The ability of the new parameters to reproduce the torsional energy of the azide group was also verified by scanning the surface in MM with the new parameters and comparing the results with the same potential energy surface (PES) at the DFT level. Finally, the parameters were validated through classical MD simulations of AZT in aqueous environment.

  6. Proteomic of the nucleoside diphosphate kinase from T. cruzi: preliminary structural studies, expression and purification

    International Nuclear Information System (INIS)

    Full text: The enzyme nucleoside diphosphate kinase (NDPK) is a major component of the pathway for the synthesis of nucleosides triphosphates other than ATP. The mechanism of reaction involves the formation of a phospho-histidine intermediate. All known NDPK are oligomers made of small polypeptides of about 150 residues (17kDa) with a high degree of sequence similarity. T. cruzi NDPK (TcNDPK) has been characterized and could be involved in flagellar movement and therefore in the pathology of the parasite. T. cruzi NDPK (TcNDPK) has been cloned in E. coli DH5α strain. The gene has been cloned into a pRSET A plasmid and expressed in E. coli BL21-DE3 strain as a fusion protein containing TcNDPK preceded by an hexa-His-tag. The fusion protein has been purified with a HiTrap chelating column loaded with 0,1M NiSO4 in different buffer conditions and its purification level visualized in SDS-PAGE. The conformational state of the purified TcNDPK has been observed by different methods such as native PAGE. We have obtained large quantities of TcNDPK to be used for crystallization and interaction assays. The objective of this work is the resolution of the three-dimensional structure of TcNDPK by X-ray crystallography as a first step for rational drug design based on the structure. (author)

  7. Non-nucleoside reverse transcriptase inhibitors (NNRTIs): past, present, and future.

    Science.gov (United States)

    De Clercq, Erik

    2004-01-01

    Non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) have become an inherent ingredient of the drug combination schemes that are currently used in the treatment of human immunodeficiency virus type 1 (HIV-1) infections. Starting from the 1-[(2-hydroxyethoxy)methyl]-6-(phenylsulfanyl)thymine (HEPT) and 4,5,6,7-tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO) derivatives, numerous classes of compounds have been described as NNRTIs. Only three compounds have so far been approved for clinical use: nevirapine, delavirdine, and efavirenz. NNRTIs are notorious for rapidly leading to virus-drug resistance development, primarily based on the emergence of the K103N and Y181C mutations in the HIV-1 RT. Newer NNRTIs, such as capravirine, dapivirine (TMC 125), and DPC 083, are resilient to these 'NNRTI' mutations, and, therefore, offer considerable promise as future anti-HIV-1 drugs. NNRTIs are targeted at a specific 'pocket' binding site within the HIV-1 RT, that is distinct from, but both spatially and functionally related to, the catalytic site, where the nucleoside RT inhibitors (NRTIs) and nucleotide RT inhibitors (NtRTIs) interact. NNRTIs have acquired a definitive position, as part of a combination regimen with NRTIs and NtRTIs, in the first-line treatment of HIV-1 infections. PMID:17191775

  8. Crystallization and preliminary X ray analysis of nucleoside diphosphate kinase 1 from T. cruzi

    International Nuclear Information System (INIS)

    Introduction: Trypanosoma cruzi is the etiologic agent of Chagas disease. The Nucleoside diphosphate kinases (NDPKs) are enzymes involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform. The objective of this work is obtaining protein's crystals, diffract and process the data for tridimensional structure resolution. Materials and Methods: TcNDPK1 was expressed in E. coli as a fusion protein with Nterminal His-tag. TcNDPK1 was overexpressed and purified by FPLC. Crystallization was assayed by sitting drop and hanging drop vapor diffusion method. Crystals was frozen and diffracted on synchrotron x-ray radiation in Campinas (Brasil). The data set collected was reduced and merged using MOSFLM and SCALA programs. Results and Discussion: His-TcNDPK was overexpressed, purified and crystallized. The crystals are diffracted and collected the data to 3.5A. The crystals belong to the trigonal space group P3, with unit cell parameters a=127.94, b=127.84, c=275.49. Structure determination is under way. These results will provide relevant information that could be the first step in rational drug design for treating Chagas disease.(authors)

  9. Detection of RNA nucleoside modifications with the uridine-specific ribonuclease MC1 from Momordica charantia.

    Science.gov (United States)

    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

  10. Novel phenyl(2-(phenylamino)pyrimidin-4-yl)methanones as potent non-nucleoside reverse transcriptase inhibitors

    Czech Academy of Sciences Publication Activity Database

    Šimon, Petr; Baszczyňski, Ondřej; Šaman, David; Bahador, G.; Stepan, G.; Hu, E.; Lansdon, E.; Jansa, P.; Janeba, Zlatko

    Rome: International Society for Antiviral Research (ISAR), 2015. s. 96. [International Conference on Antiviral Research /28./. 11.05.2015-15.05.2015, Rome] Institutional support: RVO:61388963 Keywords : non-nucleoside reverse transcriptase inhibitors * pyrimidine * HIV -1 Subject RIV: CC - Organic Chemistry

  11. Acyclic nucleoside bisphosphonates as inhibitors of hypoxanthine-guanine-(xanthine) phosphoribosyltransferases: Potential new anti-malarial compounds

    Czech Academy of Sciences Publication Activity Database

    Špaček, Petr; Hocková, Dana; Janeba, Zlatko; Vrbková, Silvie; Edstein, M.; de Jersey, J.; Keough, D. T.; Guddat, L. W.

    Amsterdam : Elsevier, 2012. P2.24-P2.24. [Tetrahedron Symposium: Challenges in Bioorganic & Organic Medicinal Chemistry /13./. 26.06.2012-29.06.2012, Amsterdam] Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleoside phosphonates * biophosphonates Subject RIV: CC - Organic Chemistry

  12. Self-assembled layers of nucleic acid bases and nucleosides at the interfaces and the origin of life

    Czech Academy of Sciences Publication Activity Database

    Vetterl, Vladimír; Ignác, Jan

    Montpellier, 2005. P-232. [15th IUPAB and 5th EBSA International Biophysics Congress. 27.08.2005-01.09.2005, Montpellier] R&D Projects: GA AV ČR(CZ) IBS5004107 Institutional research plan: CEZ:AV0Z50040507 Keywords : self-assembled layers * two-dimensional condensation * nucleosides Subject RIV: BO - Biophysics

  13. Synthesis of conformationally locked L-deoxythreosyl phosphonate nucleosides built on a bicyclo[3.1.0]hexane template.

    Science.gov (United States)

    Saneyoshi, Hisao; Deschamps, Jeffrey R; Marquez, Victor E

    2010-11-19

    Two conformationally locked versions of l-deoxythreosyl phosphonate nucleosides (2 and 3) were synthesized to investigate the preference of HIV reverse transcriptase for a conformation displaying either a fully diaxial or fully diequatorial disposition of substituents. Synthesis of the enantiomeric 4-(6-amino-9H-purin-9-yl)bicyclo[3.1.0]hexan-2-ol carbocyclic nucleoside precursors (diaxially disposed) proceeded straightforwardly from commercially available (1R,4S)-4-hydroxy-2-cyclopent-2-enyl-1-yl acetate employing a hydroxyl-directed Simmons-Smith cyclopropanation that culminated with a Mitsunobu coupling of the purine base. For the more complicated 1-(6-amino-9H-purin-9-yl)bicyclo[3.1.0]hexan-3-ol carbocyclic nucleoside precursors (diequatorially disposed), the obligatory linear approach required the syntheses of key 1-aminobicyclo[3.1.0.]hexan-3-yl benzoate precursors that were assembled via the amide variant of the Kulinkovich reaction involving the intramolecular cyclopropanation of a substituted δ-vinylamide. Completion of the purine ring was achieved by conventional approaches but with much improved yields through the use of a microwave reactor. The syntheses of the phosphonates and the corresponding diphosphates were achieved by conventional means. None of the diphosphates, which were supposed to act as nucleoside triphosphate mimics, could compete with dATP even when present in a 10-fold excess. PMID:20964394

  14. Synthesis and Evaluation of Novel Acyclic Nucleoside Phosphonates as Inhibitors of Plasmodium falciparum and Human 6-Oxopurine Phosphoribosyltransferases

    Czech Academy of Sciences Publication Activity Database

    Kaiser, Martin Maxmilian; Hocková, Dana; Wang, T. H.; Dračínský, Martin; Poštová Slavětínská, Lenka; Procházková, Eliška; Edstein, M. D.; Chavchich, M.; Keough, D. T.; Guddat, L. W.; Janeba, Zlatko

    2015-01-01

    Roč. 10, č. 10 (2015), s. 1707-1723. ISSN 1860-7179 R&D Projects: GA MV VG20102015046; GA ČR GAP207/11/0108 Institutional support: RVO:61388963 Keywords : 6-oxopurine * acyclic nucleoside phosphonates * phosphoribosyltransferases * malaria * phosphoramidates Subject RIV: CC - Organic Chemistry Impact factor: 2.968, year: 2014

  15. Novel type of acyclic nucleoside phosphonates derived from 2-(phosphonomethoxy)-and 2-(phosphonoethoxy)propanoic acid

    Czech Academy of Sciences Publication Activity Database

    Kaiser, Martin Maxmilian; Jansa, Petr; Hocková, Dana; Dračínský, Martin; Janeba, Zlatko

    Amsterdam : Elsevier, 2012. -. [Tetrahedron Symposium: Challenges in Bioorganic & Organic Medicinal Chemistry /13./. 26.06.2012-29.06.2012, Amsterdam] R&D Projects: GA MV VG20102015046; GA ČR GAP207/11/0108 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * antiparasitic * 2-(phosphonomethoxy)propanoic acid * HG(X)PRTase Subject RIV: CC - Organic Chemistry

  16. Synthesis and evaluation of thymidine kinase 1-targeting carboranyl pyrimidine nucleoside analogs for boron neutron capture therapy of cancer.

    Science.gov (United States)

    Agarwal, Hitesh K; Khalil, Ahmed; Ishita, Keisuke; Yang, Weilian; Nakkula, Robin J; Wu, Lai-Chu; Ali, Tehane; Tiwari, Rohit; Byun, Youngjoo; Barth, Rolf F; Tjarks, Werner

    2015-07-15

    A library of sixteen 2nd generation amino- and amido-substituted carboranyl pyrimidine nucleoside analogs, designed as substrates and inhibitors of thymidine kinase 1 (TK1) for potential use in boron neutron capture therapy (BNCT) of cancer, was synthesized and evaluated in enzyme kinetic-, enzyme inhibition-, metabolomic-, and biodistribution studies. One of these 2nd generation carboranyl pyrimidine nucleoside analogs (YB18A [3]), having an amino group directly attached to a meta-carborane cage tethered via ethylene spacer to the 3-position of thymidine, was approximately 3-4 times superior as a substrate and inhibitor of hTK1 than N5-2OH (2), a 1st generation carboranyl pyrimidine nucleoside analog. Both 2 and 3 appeared to be 5'-monophosphorylated in TK1(+) RG2 cells, both in vitro and in vivo. Biodistribution studies in rats bearing intracerebral RG2 glioma resulted in selective tumor uptake of 3 with an intratumoral concentration that was approximately 4 times higher than that of 2. The obtained results significantly advance the understanding of the binding interactions between TK1 and carboranyl pyrimidine nucleoside analogs and will profoundly impact future design strategies for these agents. PMID:26087030

  17. Click, Substitute and Fluoresce: Synthesis and Applications of 2,6-Di-(1,2,3-triazolyl)-purine Nucleosides

    OpenAIRE

    Novosjolova, I; Kovaļovs, A; Bižāne, I; Bizdēna, Ē; Turks, M

    2012-01-01

    A novel class of ditriazolylpurine nucleosides were ob-tained from 2,6-diazido precursors via copper catalyzed azide-alkyne cycloaddition. These intermediates ap-peared to be very reactive towards N- and S-nucleophiles and thus selectively gave C(6)-substituted analogs with triazolyl moiety at C(2)-position. The latter products exhibit interesting fluorescence properties.

  18. Mitochondrial DNA D-loop AG/TC transition mutation in cortical neurons of mice after long-term exposure to nucleoside analogues.

    Science.gov (United States)

    Zhang, Yulin; Wang, Bishi; Liang, Qi; Qiao, Luxin; Xu, Bin; Zhang, Hongwei; Yang, Sufang; Chen, Jun; Guo, Hongliang; Wu, Jian; Chen, Dexi

    2015-10-01

    With the wide application of combined antiretroviral therapy, the prognosis of human immunodeficiency virus (HIV)-1 infected patient has been significantly improved. However, long-term administration of antiretroviral drugs can result in various drug-associated toxicities. Among them, nucleoside analogues were confirmed to inhibit DNA polymerase gamma, resulting in mitochondrial toxicity. Our previous study indicated that long-term exposure of mice to nucleoside analogue could induce mitochondria DNA (mtDNA) loss in cortical neurons. Herein, we further identify mitochondrial toxicity of four nucleoside analogues (zidovudine (AZT), stavudine (D4T), lamivudine (3TC), and didanosine (DDI)) by cloning and sequencing mtDNA D-loop region in mice neurons captured with laser capture microdissection. The results showed that mutation of neuronal mtDNA D-loop sequences increased in mice treated with each of the four nucleoside analogues for 4 months and D4T and DDI induced more severe D-loop lesion than the other two nucleoside analogues. The major type of D-loop point mutations induced by four nucleoside analogues was transition, in particular of "A→G" and "T→C" transition, but the point transition sites were variable. Our findings suggest that long-term exposure to nucleoside analogue can result in mtDNA D-loop region lesion in mouse cortical neurons. PMID:26015313

  19. Early Cellular Responses of Purine Nucleoside-mediated Protection of Hypoxia-induced Injuries of Neuronal PC12 Cells

    Directory of Open Access Journals (Sweden)

    Bettina Tomaselli

    2005-01-01

    Full Text Available Hypoxia in brain may lead to cell death by apoptosis and necrosis. In parallel adenosine, a powerful endogenous neuroprotectant is formed. We wanted to investigate the effect of adenosine and its purine nucleoside relatives, inosine and guanosine on early cellular responses to hypoxia. O2-sensitive neuronal PC12-cells were subjected to chemical hypoxia induced with rotenone, an inhibitor of mitochondrial complex I. Loss of viability after hypoxic insult was impressively rescued by adenosine, guanosine and inosine. PC12-cells mainly express the A2A adenosine receptor. Its inhibition with a specific antagonist (CSC induced cell death of PC12-cells, which could be salvaged by adenosine but not with guanosine or inosine. We have previously demonstrated the important role of mitogen activated protein kinases 1/2 (p42/44 MAPK in purine-mediated rescue. In this study we were interested in the involvement of protein kinases whose activities mediate these processes, including protein kinase A (PKA, phosphoinositide 3-kinase (PI3-K and protein kinase C-related kinases (PRK 1/2. Pharmacological inhibition of PKA and PI3-K increased hypoxia-induced toxicity and likewise also affected the rescue by purine nucleosides. Nerve growth factor (NGF and purine nucleosides induced an activation of PRK 1/2, which to our knowledge indicates for the first time that these kinases are potentially involved in purine nucleoside-mediated rescue of hypoxic neuronal cells. Results suggest that A2A receptor expressing cells are mainly dependent on the purine nucleoside adenosine for their rescue after hypoxic insult. In addition to PKA, PI3-K is an important effector molecule in A2A-mediated signaling and for the rescue of PC12-cells after hypoxic insult.

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

    Science.gov (United States)

    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

  1. Targeting the Plasmodium vivax equilibrative nucleoside transporter 1 (PvENT1 for antimalarial drug development

    Directory of Open Access Journals (Sweden)

    Roman Deniskin

    2016-04-01

    Full Text Available 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

  2. Expression, purification and crystallization of the ecto-enzymatic domain of rat E-NTPDase1 CD39

    International Nuclear Information System (INIS)

    The ecto-enzymatic domain of rat E-NTPDase1 CD39 was expressed and purified and diffraction-quality crystals of the enzyme were obtained. CD39 is a prototype member of the ecto-nucleoside triphosphate diphosphohydrolase family that hydrolyzes extracellular nucleoside diphosphates and triphosphates in the presence of divalent cations. Here, the expression, purification and crystallization of the ecto-enzymatic domain of rat CD39, sCD39, are described. The 67 kDa secreted soluble glycoprotein was recombinantly overexpressed in a glycosylation mutant CHO line, Lec.3.2.8.1, and purified from conditioned media. Diffraction-quality crystals of sCD39 were produced by the vapor-diffusion method using PEG 3350 and ammonium dihydrogen phosphate as precipitants. The enzyme crystallized in a primitive trigonal form in space group P32, with unit-cell parameters a = b = 118.1, c = 81.6 Å and with two sCD39 copies in the asymmetric unit. Several low- to medium-resolution diffraction data sets were collected using an in-house X-ray source. Analysis of the intensity statistics showed that the crystals were invariably merohedrally twinned with a high twin fraction. For initial phasing, a molecular-replacement search was performed against the complete 3.2 Å data set using a maximum-likelihood molecular-replacement method as implemented in Phaser. The initial model of the two sCD39 monomers was placed into the P32 lattice and rigid-body refined and position-minimized with PHENIX

  3. ATP release, generation and hydrolysis in exocrine pancreatic duct cells.

    Science.gov (United States)

    Kowal, J M; Yegutkin, G G; Novak, I

    2015-12-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca(2+) and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide

  4. Golgi-located NTPDase1 of Leishmania major is required for lipophosphoglycan elongation and normal lesion development whereas secreted NTPDase2 is dispensable for virulence.

    Science.gov (United States)

    Sansom, Fiona M; Ralton, Julie E; Sernee, M Fleur; Cohen, Alice M; Hooker, David J; Hartland, Elizabeth L; Naderer, Thomas; McConville, Malcolm J

    2014-12-01

    Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri- and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively. Expression of both proteins as C-terminal GFP fusion proteins revealed that LmNTPDase1 was exclusively targeted to the Golgi apparatus, while LmNTPDase2 was predominantly secreted. An L. major LmNTPDase1 null mutant displayed increased sensitivity to serum complement lysis and exhibited a lag in lesion development when infections in susceptible BALB/c mice were initiated with promastigotes, but not with the obligate intracellular amastigote stage. This phenotype is characteristic of L. major strains lacking lipophosphoglycan (LPG), the major surface glycoconjugate of promastigote stages. Biochemical studies showed that the L. major NTPDase1 null mutant synthesized normal levels of LPG that was structurally identical to wild type LPG, with the exception of having shorter phosphoglycan chains. These data suggest that the Golgi-localized NTPase1 is involved in regulating the normal sugar-nucleotide dependent elongation of LPG and assembly of protective surface glycocalyx. In contrast, deletion of the gene encoding LmNTPDase2 had no measurable impact on parasite virulence in BALB/c mice. These data suggest that the Leishmania major NTPDase enzymes have potentially important roles in the insect stage, but only play a transient or non-major role in pathogenesis in the mammalian host. PMID:25521752

  5. Golgi-located NTPDase1 of Leishmania major is required for lipophosphoglycan elongation and normal lesion development whereas secreted NTPDase2 is dispensable for virulence.

    Directory of Open Access Journals (Sweden)

    Fiona M Sansom

    2014-12-01

    Full Text Available Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases which hydrolyze a broad range of nucleoside tri- and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively. Expression of both proteins as C-terminal GFP fusion proteins revealed that LmNTPDase1 was exclusively targeted to the Golgi apparatus, while LmNTPDase2 was predominantly secreted. An L. major LmNTPDase1 null mutant displayed increased sensitivity to serum complement lysis and exhibited a lag in lesion development when infections in susceptible BALB/c mice were initiated with promastigotes, but not with the obligate intracellular amastigote stage. This phenotype is characteristic of L. major strains lacking lipophosphoglycan (LPG, the major surface glycoconjugate of promastigote stages. Biochemical studies showed that the L. major NTPDase1 null mutant synthesized normal levels of LPG that was structurally identical to wild type LPG, with the exception of having shorter phosphoglycan chains. These data suggest that the Golgi-localized NTPase1 is involved in regulating the normal sugar-nucleotide dependent elongation of LPG and assembly of protective surface glycocalyx. In contrast, deletion of the gene encoding LmNTPDase2 had no measurable impact on parasite virulence in BALB/c mice. These data suggest that the Leishmania major NTPDase enzymes have potentially important roles in the insect stage, but only play a transient or non-major role in pathogenesis in the mammalian host.

  6. 4'-Substituted pyrimidine nucleosides lacking 5'-hydroxyl function as potential anti-HCV agents.

    Science.gov (United States)

    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

  7. Nucleoside analogues are activated by bacterial deoxyribonucleoside kinases in a species-specific manner

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, Anders; On, Stephen L. W.;

    2007-01-01

    bactericidal activity against several clinical bacterial isolates and type strains. We identified and subcloned the genes coding for putative deoxyribonucleoside kinases in Escherichia coli, Pasteurella multocida, Salmonella enterica, Yersinia enterocolitica, Bacillus cereus, Clostridium perfringens and....... The tested Gram-negative bacteria were susceptible to 3"-azido-3"-deoxythymidine (AZT) in the concentration range 0.032-31.6 µM except for a single E. coli isolate and two Pseudomonas aeruginosa isolates which were resistant to the tested AZT concentrations. Purified recombinant S. enterica thymidine...... deoxyadenosine kinase had a Km for gemcitabine of 33.5 µM and kcat/Km of 5.1 × 10^3 s-1 M-1 and activates gemcitabine in vivo. S. enterica and B. cereus are now amongst the first bacteria with a completely characterized set of dNK enzymes. Bacterial dNKs efficiently activate nucleoside analogues in a species...

  8. Discovery, characterization, and lead optimization of 7-azaindole non-nucleoside HIV-1 reverse transcriptase inhibitors.

    Science.gov (United States)

    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

  9. Synthesis and HIV-1 Reverse Transcriptase Inhibitory Activity of Non-Nucleoside Phthalimide Derivatives

    Institute of Scientific and Technical Information of China (English)

    UNGWITAYATORN Jiraporn; WIWAT Chanpen; MATAYATSUK Chutima; PIMTHON Jutarat; PIYAVIRIYAKUL Suratsawadee

    2008-01-01

    A new type of non-nucleoside HIV-1 reverse transcriptase inhibitors in phthalimide series has been synthesized from either the reaction of N-carboethoxyphthalimide with amines or phthalimide with appropriate alkyl halides.The in vitro inhibitory activity of the synthesized compounds was studied by a radiometric assay at a concentration of 200 μg/mL using poly(rA)-oligo(dT) as a template-primer and methyl-[3H]dTTP as a substrate.The three most potent compounds, N-(m,p-dihydroxybenzyl)phthalimide (11), N-[2-(a-furyl)ethyl]phthalimide (29) and N-(5-methylpyrazin-2-ylmethyl)phthalimide (25) exhibited IC50 values of 60.90, 98.10 and 120.75 μg/mL, respecas a substrate).

  10. Simple and rapid synthesis of some nucleoside derivatives: structural and spectral characterization

    Directory of Open Access Journals (Sweden)

    Shagir A. Chowdhury

    2016-03-01

    Full Text Available In our present investigation a new series of nucleoside derivatives (2-13 were synthesized from uridine (1 via only two step reactions by direct acylation method. Firstly, uridine (1 was treated with 4-t-butylbenzoyl chloride in pyridine at -5ºC and afforded the 5´-O-(4-t-butylbenzoyluridine derivative (2 in an excellent yield. In order to obtain newer products, the 5´-O-uridine derivative was further transformed to a series of 2´,3´-di-O-acyl derivatives (2-13 containing a wide variety of functionalities in a single molecular framework. The yields of the compounds were more than 80%. The synthesized titled compounds were characterized by their physical properties, FTIR (Fourier transform infrared spectroscopy, 1H-NMR (Nuclear magnetic resonance spectroscopy and elemental analysis.

  11. Synthesis of 5'-3H-2-Amino-6-cyclopropylaminopurine Nucleosides

    Institute of Scientific and Technical Information of China (English)

    YANG Zhen-Jun; Chung K. Chu; ZHANG Li-He

    2003-01-01

    @@ We have described a novel use of the D4G prodrug (Cyclo-D4G) approach to stabilize the nucleoside and maintain the anti-HIV activity. β-D-Dioxolanyl-2-amino-6-cyclopropylaminopurine (10, DACP) was synthesized as a prodrug of β-D-dioxolanyl guanine (DXG) and showed potent anti-HIV activity in vitro (EC50=0.18 μmol·L-1 The pharmacokinetics of Cyclo-D4G and DACP following intravenous and oral administration of DACP to rhesusmonkeys and rats did not give D4G and DXG as detectable metabolic products by HPLC. Therefore the radio labeled Cyclo-D4G and DACP are necessary for the pharmacokinetics studies in detail.

  12. Chiral analysis and characterization of acyclic nucleoside phosphonates-based antiviral drugs by capillary electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Kašička, Václav; Šolínová, Veronika; Sázelová, Petra; Mikysková, Hana; Koval, Dušan; Břehová, Petra; Krečmerová, Marcela; Janeba, Zlatko; Holý, Antonín

    Bratislava: Slovenská vákuová spoločnosť, 2013 - (Bodor, R.; Okenicová, L.; Staňová, A.), s. 14-17 ISBN 978-80-971179-1-7. [Analytické metódy a zdravie človeka. Medzinárodná konferencia /19./. Rajecké Teplice (SK), 24.06.2013-27.06.2013] R&D Projects: GA ČR(CZ) GAP206/12/0453; GA ČR GA13-17224S; GA MŠk(CZ) ME10040; GA MV VG20102015046 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * chiral analysis * capillary electrophoresis Subject RIV: CB - Analytical Chemistry, Separation

  13. Structural basis for selective inhibition of purine nucleoside phosphorylase from Schistosoma mansoni: kinetic and structural studies.

    Science.gov (United States)

    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

  14. Low liver stiffness among cirrhotic patients with hepatitis B after prolonged treatment with nucleoside analogs

    DEFF Research Database (Denmark)

    Andersen, Ellen Sloth; Weiland, Ola; Leutscher, Peter;

    2011-01-01

    Abstract Objective. Case reports and short-term clinical trials have suggested that treatment for chronic hepatitis B (CHB) may lead to improvement of cirrhosis. The aim of the present study was to measure liver stiffness in patients diagnosed with advanced fibrosis or cirrhosis prior to prolonged...... treatment with nucleoside or nucleotide analogs (NUCs) for CHB. Materials and methods. Patients with CHB and advanced fibrosis or cirrhosis prior to treatment with NUCs for at least 1 year were offered inclusion in the study. We measured liver stiffness using transient elastography (TE) at follow-up. TE cut...... duration was 50.5 months. Among patients with cirrhosis prior to treatment, 26 (49%) had liver stiffness below 11.0 kPa at follow-up, suggesting regression of cirrhosis. Among patients with advanced fibrosis (F3) prior to treatment, 10 (77%) had liver stiffness below 8.1 kPa after treatment, suggesting...

  15. New nucleoside/nucleotide backbone options: a review of recent studies.

    Science.gov (United States)

    Ruane, Peter J; DeJesus, Edwin

    2004-09-01

    The nucleoside/nucleotide reverse transcriptase inhibitor (NRTI/NtRTI) class continues to serve as an important component of the standard of care for HIV infection. Combinations of dual NRTIs/NtRTIs with protease inhibitors (PIs) or nonnucleoside reverse transcriptase inhibitors (NNRTIs) remain the most commonly used regimens in clinical practice. In recent years, clinical outcomes data on previously novel NRTI/NtRTI backbone combinations have provided clinicians with new options to address potency, tolerability, and convenience of antiretroviral therapy. However, the tolerability, drug-drug interactions, and resistance profiles of specific regimens using new NRTI/NtRTI combinations must be weighed against the needs and preferences of individual patients. This review summarizes recent efficacy and safety data on emerging NRTI/NtRTI combination backbones, including tenofovir DF (TDF) with lamivudine (3TC), abacavir with 3TC, didanosine (ddI) with 3TC, ddI with emtricitabine (FTC), and TDF with FTC. PMID:15319666

  16. Aquaporin 3 (AQP3 participates in the cytotoxic response to nucleoside-derived drugs

    Directory of Open Access Journals (Sweden)

    Trigueros-Motos Laia

    2012-09-01

    Full Text Available Abstract 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.

  17. Ab Initio Inverstagation of the Excited States of Nucleobases and Nucleosides

    Science.gov (United States)

    Szalay, Péter G.; Fogarasi, Géza; Watson, Thomas; Perera, Ajith; Lotrich, Victor; Bartlett, Rod J.

    2011-06-01

    Most living bodies are exposed to sunlight, essential life sustaining processes are using this natural radiation. Sunlight has, however, several components (has a broad "spectrum") and in particular the invisible component (UV, ultraviolet) is harmful for living organisms. Scientists around the word are busy to understand what happens in the cell when it is exposed to light: it seems that the building blocks of cells and in particular those carrying the genetic information (DNA and RNA) are highly protected against this exposition. Our research focuses on the spectral properties of the building blocks of DNA and RNA, the so called nucleobases and nucleosides, in order to understand this mechanism. Due to improvement in computer technology both at hardware and software side we are now able to use the most accurate methods of ab initio quantum chemistry to investigate the spectroscopic properties of these building blocks. These calculations provide direct information on the properties of these molecules but also provide important benchmarks for cheaper methods which can be used for even larger systems. We have calculated the excited state properties for the nucleobases (cytosine, guanine and adenine), their complexes with water and with each other (Watson-Crick base pairs and stacks) as well as corresponding nucleosides at the EOM-CCSD(T)/aug-cc-pVDZ level of theory and try to answer the following questions: (1) how the order of excited states varies in different nucleobases; (2) how hydration influences the excitation energy and order of excited states; (3) is there any effect of the sugar substituent; (4) how do close lying other bases change the spectrum. The calculations involve over hundred correlated electrons and up to thousand basis functions. Such calculations are now routinely available with the recently developed ACESIII code and can make use of hundreds or even several thousand of processors. V. Lotrich, N. Flocke, M. Ponton, A. Yau, A. Perera, E. Deumens

  18. Aquaporin 3 (AQP3) participates in the cytotoxic response to nucleoside-derived drugs

    International Nuclear Information System (INIS)

    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. The role of AQP3 in cell volume increase, cytotoxicity and cell cycle arrest was analyzed using loss-of-function approaches. 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. 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

  19. In vivo phenotypic characterisation of nucleoside label-retaining cells in mouse periosteum

    Directory of Open Access Journals (Sweden)

    HM Cherry

    2014-03-01

    Full Text Available Periosteum is known to contain cells that, after isolation and culture-expansion, display properties of mesenchymal stromal/stem cells (MSCs. However, the equivalent cells have not been identified in situ mainly due to the lack of specific markers. Postnatally, stem cells are slow-cycling, long-term nucleoside-label-retaining cells. This study aimed to identify and characterise label-retaining cells in mouse periosteum in vivo. Mice received iodo-deoxy-uridine (IdU via the drinking water for 30 days, followed by a 40-day washout period. IdU+ cells were identified by immunostaining in conjunction with MSC and lineage markers. IdU-labelled cells were detected throughout the periosteum with no apparent focal concentration, and were negative for the endothelial marker von Willebrand factor and the pan-haematopoietic marker CD45. Subsets of IdU+ cells were positive for the mesenchymal/stromal markers vimentin and cadherin-11. IdU+ cells expressed stem cell antigen-1, CD44, CD73, CD105, platelet-derived growth factor receptor-α and p75, thereby displaying an MSC-like phonotype. Co-localisation was not detectable between IdU and the pericyte markers CD146, alpha smooth muscle actin or NG2, nor did IdU co-localise with β-galactosidase in a transgenic mouse expressing this reporter gene in pericytes and smooth muscle cells. Subsets of IdU+ cells expressed the osteoblast-lineage markers Runx2 and osteocalcin. The IdU+ cells expressing osteocalcin were lining the bone and were negative for the MSC marker p75. In conclusion, mouse periosteum contains nucleoside-label-retaining cells with a phenotype compatible with MSCs that are distinct from pericytes and osteoblasts. Future studies characterising the MSC niche in vivo could reveal novel therapeutic targets for promoting bone regeneration/repair.

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

    Science.gov (United States)

    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

  1. Cross-linked polymeric nanogel formulations of 5'-triphosphates of nucleoside analogues: role of the cellular membrane in drug release.

    Science.gov (United States)

    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

  2. Bone mineral density changes in protease inhibitor-sparing vs. nucleoside reverse transcriptase inhibitor-sparing highly active antiretroviral therapy: data from a randomized trial

    DEFF Research Database (Denmark)

    Hansen, Ann-Brit Eg; Obel, N; Nielsen, H;

    2011-01-01

    The aim of the study was to compare changes in bone mineral density (BMD) over 144 weeks in HIV-infected patients initiating nucleoside reverse transcriptase inhibitor (NRTI)-sparing or protease inhibitor-sparing highly active antiretroviral therapy (HAART).......The aim of the study was to compare changes in bone mineral density (BMD) over 144 weeks in HIV-infected patients initiating nucleoside reverse transcriptase inhibitor (NRTI)-sparing or protease inhibitor-sparing highly active antiretroviral therapy (HAART)....

  3. ADME studies of [5‐3H]‐2′‐O‐methyluridine nucleoside in mice: a building block in siRNA therapeutics

    OpenAIRE

    Lozac'h, Frederic; Christensen, Jesper; Faller, Thomas; van de Kerkhof, Esther; Krauser, Joel; Garnier, Maxime; Litherland, Karine; Catoire, Alexandre; Natt, Francois; Hunziker, Jurg; Swart, Piet

    2016-01-01

    Abstract The chemical modification 2′‐O‐methyl of nucleosides is often used to increase siRNA stability towards nuclease activities. However, the metabolic fate of modified nucleosides remains unclear. Therefore, the aim of this study was to determine the mass balance, pharmacokinetic, and absorption, distribution, metabolism, and excretion (ADME)‐properties of tritium‐labeled 2′‐O‐methyluridine, following a single intravenous dose to male CD‐1 mice. The single intravenous administration of [...

  4. Comparative effects of dietary nucleoside-nucleotide mixture and its components on endotoxin induced bacterial translocation and small intestinal injury in protein deficient mice.

    OpenAIRE

    Adjei, A A; Yamauchi, K.; Chan, Y. C.; Konishi, M; Yamamoto, S.

    1996-01-01

    BACKGROUND--Nucleoside-nucleotide mixture has been shown to improve gut morphology and reduce the incidence of bacterial translocation in protein deficient mice. AIMS--To compare the reparative effect of nucleoside-nucleotide mixture and their individual components on maintenance of gut integrity and bacterial translocation based on their differential metabolism and utilisation. METHODS--ICR (CD-1) mice were randomised into eight groups of 10 animals each and fed 20% casein diet (control), pr...

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

    Science.gov (United States)

    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

  6. Internalization of nucleoside phosphates into live cells by complex formation with different CPPs and JBS-nucleoducin.

    Science.gov (United States)

    Mussbach, Franziska; Pietrucha, Regina; Schaefer, Buerk; Reissmann, Siegmund

    2011-01-01

    Nucleoside phosphates can bind to many functional proteins like G-proteins or other GTP-binding proteins in signal transduction or translation processes. Till now internalization of nucleoside phosphates into live cells remains a challenge. We study the internalization of a fluorescent-labelled deoxyuridine triphosphate into HeLa cells and other adhesion and suspension cells. We use different cell-penetrating peptides and a cocktail suitable for formation of non-covalent complexes with the nucleotide. Internalization is observed by fluorescence microscopy, and the uptake efficiency is quantitatively estimated by fluorescence spectroscopy. The applied concentrations of CPPs and the cocktail were checked on cell viability (MTT test) and membrane integrity (bioluminescence test with peptidyl-luciferin), indicating that the CPPs and the complexes with the nucleotide are cytotoxic above certain concentrations. These concentrations depend on CPP and cell type and are the limiting factors for the cargo uptake. PMID:21053144

  7. ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA

    DEFF Research Database (Denmark)

    van den Born, E.; Vagbo, C. B.; Songe-Moller, L.;

    2011-01-01

    Mammals have nine different homologues (ALKBH1-9) of the Escherichia coli DNA repair demethylase AlkB. ALKBH2 is a genuine DNA repair enzyme, but the in vivo function of the other ALKBH proteins has remained elusive. It was recently shown that ALKBH8 contains an additional transfer RNA ( tRNA......) methyltransferase domain, which generates the wobble nucleoside 5-methoxycarbonylmethyluridine (mcm(5)U) from its precursor 5-carboxymethyluridine (cm(5)U). In this study, we report that (R)- and (S)-5-methoxycarbonylhydroxymethyluridine (mchm(5)U), hydroxylated forms of mcm(5)U, are present in mammalian tRNA......(UCG)(Arg), and tRNA(UCC)(Gly), respectively, representing the first example of a diastereomeric pair of modified RNA nucleosides. Through in vitro and in vivo studies, we show that both diastereomers of mchm(5)U are generated from mcm(5)U, and that the AlkB domain of ALKBH8 specifically hydroxylates mcm(5)U into...

  8. TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs

    OpenAIRE

    Huang, Shar-yin N.; Murai, Junko; Dalla Rosa, Ilaria; Dexheimer, Thomas S.; Naumova, Alena; Gmeiner, William H.; Pommier, Yves

    2013-01-01

    Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl–DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs—acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT...

  9. Insights into phosphate cooperativity and influence of substrate modifications on binding and catalysis of hexameric purine nucleoside phosphorylases.

    Directory of Open Access Journals (Sweden)

    Priscila O de Giuseppe

    Full Text Available The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233 displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2'deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2'deoxyribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5' hydroxyl group of adenosine and Arg(43* side chain contributes for the ribosyl radical to adopt an unusual C3'-endo conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl(6 and Br(8 substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser(90 by a threonine in the B. cereus hexameric adenosine phosphorylase (Thr(91 is responsible for the lack of negative cooperativity of phosphate binding

  10. Synthesis of phosphonomethoxyethyl or 1,3-bis(phosphonomethoxy)propan-2-yl lipophilic esters of acyclic nucleoside phosphonates

    Czech Academy of Sciences Publication Activity Database

    Vrbková, Silvie; Dračínský, Martin; Holý, Antonín

    2007-01-01

    Roč. 63, č. 46 (2007), s. 11391-11398. ISSN 0040-4020 R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Grant ostatní: Descartes Prize(XE) HPAW-2002-10096 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * alkoxyalkyl phosphonates * hexadecyloxypropyl ester groups * bisphosphonates Subject RIV: CC - Organic Chemistry Impact factor: 2.869, year: 2007

  11. Syntheses of pyrimidine acyclic nucleoside phosphonates as potent inhibitors of thymidine phosphorylase (PD-ECGF) from SD-lymphoma

    Czech Academy of Sciences Publication Activity Database

    Pomeisl, Karel; Votruba, Ivan; Holý, Antonín; Pohl, Radek

    2007-01-01

    Roč. 26, 8/9 (2007), s. 1025-1028. ISSN 1525-7770. [International Roundtable /17./. Bern, 03.09.2006-07.09.2006] R&D Projects: GA MŠk 1M0508 Grant ostatní: Descartes Prize(XE) HPAW-CT-2002-9001 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * thymidine phosphorylase * pyrimidines * FPMP derivatives * fluorination Subject RIV: CC - Organic Chemistry Impact factor: 0.723, year: 2007

  12. An efficient construction of functionalized DNA by cross-coupling reactions of nucleoside triphosphates followed by primer extension or PCR

    Czech Academy of Sciences Publication Activity Database

    Hocek, Michal; Čapek, Petr; Cahová, Hana; Vrábel, Milan; Gloeckner, Ch.; Marx, A.; Fojta, Miroslav; Havran, Luděk

    Dublin : University Cillege Dublin, 2007. s. 200. [European Symposium on Organic Chemistry /15./. 08.07.2007-13.07.2007, Dublin] R&D Projects: GA MŠk LC512; GA MŠk 1M0508; GA ČR GA203/05/0043 Institutional research plan: CEZ:AV0Z40550506 Keywords : cross-coupling reactions * nucleoside triphosphates Subject RIV: CC - Organic Chemistry

  13. Influence of non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine) on the pharmacodynamic activity of gliclazide in animal models

    OpenAIRE

    Mastan SK; Kumar K Eswar

    2009-01-01

    Abstract Background Type 2 diabetes may occur as a result of HIV infection and/or its treatment. Gliclazide is a widely used drug for the treatment of type 2 diabetes. Efavirenz and nevirapine are widely used non-nucleoside reverse transcriptase inhibitors for the treatment of HIV infection. The role of Efavirenz and nevirapine on the pharmacodynamic activity of gliclazide is not currently known. The objective of this study was to examine the effect of oral administration of efavirenz and nev...

  14. The synthesis of piperidine nucleoside analogs-a comparison of several methods to access the introduction of nucleobases

    Czech Academy of Sciences Publication Activity Database

    Kovačková, Soňa; Dračínský, Martin; Rejman, Dominik

    2011-01-01

    Roč. 67, č. 7 (2011), s. 1485-1500. ISSN 0040-4020 R&D Projects: GA MŠk 2B06065; GA MZd NR9138; GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z40550506 Keywords : piperidine derivatives * nucleoside analogs * nucleobase * proline Subject RIV: CC - Organic Chemistry Impact factor: 3.025, year: 2011

  15. Estimation of apparent binding constant of complexes of selected acyclic nucleoside phosphonates with -cyclodextrin by affinity capillary electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Šolínová, Veronika; Mikysková, Hana; Kaiser, Martin Maxmilian; Janeba, Zlatko; Holý, Antonín; Kašička, Václav

    2016-01-01

    Roč. 37, č. 2 (2016), s. 239-247. ISSN 0173-0835 R&D Projects: GA ČR(CZ) GA13-17224S; GA ČR(CZ) GA15-01948S Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * affinity capillary electrophoresis * binding constant * nucleotide analogs * beta-cyclodextrin Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.028, year: 2014

  16. The HCV Non-Nucleoside Inhibitor Tegobuvir Utilizes a Novel Mechanism of Action to Inhibit NS5B Polymerase Function

    OpenAIRE

    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

    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...

  17. The Ex Vivo Human Placental Transfer of the Anti-HIV Nucleoside Inhibitor Abacavir and the Protease Inhibitor Amprenavir

    OpenAIRE

    Bawdon, R E

    1998-01-01

    Objective: The transfer of abacavir, a new nucleoside inhibitor, and amprenavir, a new protease inhibitor, used for the treatment of human immunodeficiency virus, has been studied in the ex vivo human placental model.Methods: The ex vivo human placental model used C14 antipyrine to determine the transport fraction and clearance index of these compounds at both the peak and trough serum concentrations. The clearance index accumulation and tissue concentrations were determined for each drug by ...

  18. The ex vivo human placental transfer of the anti-HIV nucleoside inhibitor abacavir and the protease inhibitor amprenavir.

    OpenAIRE

    Bawdon, R E

    1998-01-01

    OBJECTIVE: The transfer of abacavir, a new nucleoside inhibitor, and amprenavir, a new protease inhibitor, used for the treatment of human immunodeficiency virus, has been studied in the ex vivo human placental model. METHODS: The ex vivo human placental model used C14 antipyrine to determine the transport fraction and clearance index of these compounds at both the peak and trough serum concentrations. The clearance index accumulation and tissue concentrations were determined for each drug by...

  19. Acyclic nucleoside phosphonates as inhibitors of hypoxanthine-guanine-(xanthine) phosphoribosyltransferase: new anti-malarial chemotherapy leads

    Czech Academy of Sciences Publication Activity Database

    Hocková, Dana; Holý, Antonín; Česnek, Michal; Baszczyňski, Ondřej; Tichý, Tomáš; Krečmerová, Marcela; Janeba, Zlatko; Skinner-Adams, T.; Naesens, L.; Keough, D. T.; de Jersey, J.; Guddat, L. W.

    Praha : ČSCH, 2011. s. 21-21. [Pokroky v organické, bioorganické a farmaceutické chemii - "Liblice 2011" /46./. 11.11.2011-13.11.2011, Lázně Bělohrad] R&D Projects: GA ČR GAP207/11/0108; GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleotide analogues * antiviral activity * antiplasmodial activity * acyclic nucleoside phosphonates Subject RIV: CC - Organic Chemistry

  20. Distinct modulation of telomere length in two T-lymphoblastic leukemia cell lines by cytotoxic nucleoside phosphonates PMEG and PMEDAP

    Czech Academy of Sciences Publication Activity Database

    Hájek, Miroslav; Cvilink, Viktor; Votruba, Ivan; Holý, Antonín; Mertlíková-Kaiserová, Helena

    2010-01-01

    Roč. 643, č. 1 (2010), s. 6-12. ISSN 0014-2999 R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * PMEG * PMEDAP * telomere length * telomerase inhibition Subject RIV: CC - Organic Chemistry Impact factor: 2.737, year: 2010

  1. Purine metabolic enzymes in lymphocytes. III. Effects of immunosuppressants on adenosine deaminase and purine nucleoside phosphorylase activities

    OpenAIRE

    Kurashige, Satonori; Akuzawa, Yuki; Yoshida, Toshiharu; Kodama, Kazue

    1983-01-01

    Mice were treated with a single injection of 6-mercaptopurine riboside (6MP-R), predonine or cyclophosphamide (CY), and the effects of these immunosuppressants on blastogenic responses to phytohemagglutinin P (PHA-P) or bacterial lipopoly saccharide (LPS) and on adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP) activities were studied with spleen lymphocytes. The retardation of blastogenic responses to both PHA-P and LPS were associated with the retardation of both intracellu...

  2. Contents Changes of Triterpenic Acids, Nucleosides, Nucleobases, and Saccharides in Jujube (Ziziphus jujuba) Fruit During the Drying and Steaming Process

    OpenAIRE

    Sheng Guo; Jin-Ao Duan; Ying Zhang; Dawei Qian; Yuping Tang; Zhenhua Zhu; Hanqing Wang

    2015-01-01

    Chinese jujube (Ziziphus jujuba), a medicinal and edible plant, is widely consumed in Asian countries owing to the remarkable health activities of its fruits. To facilitate selection of the suitable processing method for jujube fruits, in this study their contents of triterpenic acids, nucleosides, nucleobases and saccharides after drying and steaming treatment were determined using ultra-high performance liquid chromatography and high performance liquid chromatography coupled with evaporativ...

  3. Enantiopurity analysis of new types of acyclic nucleoside phosphonates by capillary electrophoresis with cyclodextrins as chiral selectors

    Czech Academy of Sciences Publication Activity Database

    Šolínová, Veronika; Kaiser, Martin Maxmilian; Lukáč, Miloš; Janeba, Zlatko; Kašička, Václav

    2014-01-01

    Roč. 37, č. 3 (2014), s. 295-303. ISSN 1615-9306 R&D Projects: GA ČR(CZ) GAP206/12/0453; GA ČR(CZ) GA13-17224S; GA MV VG20102015046 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * CE * chiral analysis * cyclodextrin s * nucleotide analogs Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.737, year: 2014

  4. Direct One-Pot Synthesis of Nucleosides from Unprotected or 5-O-Monoprotected D-Ribose

    Czech Academy of Sciences Publication Activity Database

    Downey, Alan Michael; Richter, C.; Pohl, Radek; Mahrwald, R.; Hocek, Michal

    2015-01-01

    Roč. 17, č. 18 (2015), s. 4604-4607. ISSN 1523-7060 R&D Projects: GA ČR GAP207/11/0344 Institutional support: RVO:61388963 Keywords : nucleosides * cytostatics * biological activity Subject RIV: CC - Organic Chemistry Impact factor: 6.364, year: 2014 http://pubs.acs.org/doi/pdf/10.1021/acs.orglett.5b02332

  5. Influence of Acyclic Nucleoside Phosphonate Antivirals on Gene Expression of Chemokine Receptors CCR5 and CXCR4

    Czech Academy of Sciences Publication Activity Database

    Potměšil, P.; Holý, Antonín; Zídek, Zdeněk

    2015-01-01

    Roč. 61, č. 1 (2015), s. 1-7. ISSN 0015-5500 R&D Projects: GA ČR GA305/03/1470; GA MŠk 1M0508 Institutional support: RVO:61388963 ; RVO:68378041 Keywords : acyclic nucleoside phosphonate * HIV * CCR5 * CXCR4 * cytokine * RT-PCR Subject RIV: CC - Organic Chemistry; FR - Pharmacology ; Medidal Chemistry (UEM-P) Impact factor: 1.000, year: 2014

  6. Anthraquinone as a Redox Label for DNA: Synthesis, Enzymatic Incorporation, and Electrochemistry of Anthraquinone-Modified Nucleosides, Nucleotides, and DNA

    Czech Academy of Sciences Publication Activity Database

    Balintová, Jana; Pohl, Radek; Horáková Brázdilová, Petra; Vidláková, Pavlína; Havran, Luděk; Fojta, Miroslav; Hocek, Michal

    2011-01-01

    Roč. 17, č. 50 (2011), s. 14063-14073. ISSN 0947-6539 R&D Projects: GA MŠk(CZ) LC06035; GA MŠk LC512; GA AV ČR(CZ) IAA400040901 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : anthraquinone * DNA * electrochemistry * nucleosides * oligonucleotides Subject RIV: CC - Organic Chemistry Impact factor: 5.925, year: 2011

  7. Assessment of chiral purity of acyclic nucleoside phosphonates-based anti-AIDS drugs by capillary electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Sázelová, Petra; Šolínová, Veronika; Kašička, Václav; Holý, Antonín

    Baltimore Inner Harbor, MD, 2010. s. 74-75. [International Symposium on Electro- and Liquid Phase-separation Techniques /17./. 29.08.2010-01.09.2010, Baltimore Inner Harbor, MD] R&D Projects: GA ČR(CZ) GA203/08/1428; GA ČR(CZ) GA203/09/0675 Institutional research plan: CEZ:AV0Z40550506 Keywords : chiral analysis * acyclic nucleoside phosphonates * capillary electrophoresis Subject RIV: CB - Analytical Chemistry, Separation

  8. Identification of an Alternative Nucleoside Triphosphate: 5′-Deoxyadenosylcobinamide Phosphate Nucleotidyltransferase in Methanobacterium thermoautotrophicum ΔH

    OpenAIRE

    Michael G Thomas; Escalante-Semerena, Jorge C.

    2000-01-01

    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 nonorth...

  9. Activities of several classes of acyclic nucleoside phosphonates against camelpox virus replication in different cell culture models

    Czech Academy of Sciences Publication Activity Database

    Duraffour, S.; Snoeck, R.; Krečmerová, Marcela; Van Den Oord, J.; De Vos, D.; Holý, Antonín; Crance, J. M.; Garin, D.; De Clercq, E.; Andrei, G.

    2007-01-01

    Roč. 51, č. 12 (2007), s. 4410-4419. ISSN 0066-4804 R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Grant ostatní: FWO(BE) G.0267.04; NIH(US) AI06540-01 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * antivirals * HPMP-5-azacytosine * camelpox virus Subject RIV: CC - Organic Chemistry Impact factor: 4.390, year: 2007

  10. Standardized comparison of the relative impacts of HIV-1 reverse transcriptase (RT) mutations on nucleoside RT inhibitor susceptibility.

    OpenAIRE

    Melikian, George L.; Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W. Jeffrey; Kaufman, David; Towner, William; Troia-Cancio, Paolo V.; Zolopa, Andrew; Robbins, Gregory K.; Kagan, Ron; Israelski, Dennis; Shafer, Robert W.

    2012-01-01

    Determining the phenotypic impacts of reverse transcriptase (RT) mutations on individual nucleoside RT inhibitors (NRTIs) has remained a statistical challenge because clinical NRTI-resistant HIV-1 isolates usually contain multiple mutations, often in complex patterns, complicating the task of determining the relative contribution of each mutation to HIV drug resistance. Furthermore, the NRTIs have highly variable dynamic susceptibility ranges, making it difficult to determine the relative eff...

  11. Non-adenosine nucleoside inosine, guanosine and uridine as promising antiepileptic drugs: a summary of current literature.

    Science.gov (United States)

    Kovacs, Zsolt; Kekesi, Katalin A; Juhasz, Gabor; Barna, Janos; Heja, Laszlo; Lakatos, Renata; Dobolyi, Arpad

    2015-01-01

    Adenosine (Ado) and some non-adenosine (non-Ado) nucleosides including inosine (Ino), guanosine (Guo) and uridine (Urd) are modulatory molecules in the central nervous system (CNS), regulating different physiological and pathophysiological processes in the brain such as sleep and epilepsy. Indeed, different drugs effective on adenosinergic system (e.g., Ado metabolism inhibitors, agonists and antagonists of Ado receptors) are being used in drug development for the treatment of epileptic disorders. Although (i) endogenous Ino, Guo and Urd showed anticonvulsant/antiepileptic effects (e.g., in quinolinic acid - induced seizures and in different epilepsy models such as hippocampal kindling models), and (ii) there is a need to generate new and more effective antiepileptic drugs for the treatment of drug-resistant epilepsies, our knowledge about antiepileptic influence of non-Ado nucleosides is far from complete. Thus, in this review article, we give a short summary of anticonvulsant/antiepileptic effects and mechanisms evoked by Ino, Guo, and Urd. Finally, we discuss some non-Ado nucleoside derivatives and their structures, which may be candidates as potential antiepileptic agents. PMID:25382017

  12. Testing nucleoside analogues as inhibitors of Bacillus anthracis spore germination in vitro and in macrophage cell culture.

    Science.gov (United States)

    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

  13. Determination of nucleosides in Cordyceps sinensis and Ganoderma lucidum by high performance liquid chromatography method

    Directory of Open Access Journals (Sweden)

    Masood Shah Khan

    2015-01-01

    Full Text Available Background: Nucleosides are supportive in the regulation and modulation of various physiological processes in body, they acts as precursors in nucleic acid synthesis, enhance immune response, help in absorption of iron and influence the metabolism of fatty acids. Cordyceps sinensis and Ganoderma lucidum are well-known for its use in traditional medicine of China, Nepal and India. They are rich in nucleosides such as adenine, adenosine, cordycepin, etc. Hence, a simple, economic and accurate high-performance liquid chromatography (HPLC analytical method was proposed for determination of adenine and adenosine for the quality control of plants. Materials and Methods: Chromatographic experiments were conducted on YL9100 HPLC system (South Korea. Reversed-phase chromatography was performed on a C18 column with methanol and dihydrogen phosphate as the mobile phase in isocratic elution method at a flow rate of 1.0 mL/min. Detection was carried out at 254 nm, which gives a sharp peak of adenine and adenosine at a retention time of 6.53 ± 0.02 min and 12.41 ± 0.02, respectively. Results and Discussion: Linear regression analysis data for the calibration plot showed a good linear relationship between response and concentration in the range of 25–200 µg/mL for adenosine and 100–800 µg/mL for adenine with regression coefficient of 0.999 and 0.996, respectively. The adenine was found 0.16% and 0.71% w/w in G. lucidum and in C. sinensis, respectively, and adenosine was found to be 0.14% w/w in G. lucidum whereas absent in C. sinensis. Conclusion: The developed HPLC method for the quantification of adenosine and adenine can be used for the quality control and standardization of crude drug and for the different herbal formulations, in which adenine and adenosine are present as major constituents. The wide linearity range, sensitivity, accuracy, and simple mobile phase imply the method is suitable for routine quantification of adenosine and adenine with

  14. Rigid Adenine Nucleoside Derivatives as Novel Modulators of the Human Sodium Symporters for Dopamine and Norepinephrine.

    Science.gov (United States)

    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

  15. Leishmania donovani Nucleoside Hydrolase terminal domains in cross-protective immunotherapy against Leishmania amazonensis murine infection

    Directory of Open Access Journals (Sweden)

    Dirlei eNico

    2014-06-01

    Full Text Available Nucleoside hydrolases of the Leishmania genus are vital enzymes for the replication of the DNA and conserved phylogenetic markers of the parasites. Leishmania donovani Nucleoside hydrolase (NH36 induced a main CD4+ T cell driven protective response against Leishmania chagasi infection in mice which is directed against its C-terminal domain. In this study, we used the three recombinant domains of NH36: N-terminal domain (F1, amino acids 1-103, central domain (F2 aminoacids 104-198 and C-terminal domain (F3 amino acids 199-314 in combination with saponin and assayed their immunotherapeutic effect on Balb/c mice previously infected with L. amazonensis. We identified that the F1 and F3 peptides determined strong cross-immunotherapeutic effects, reducing the size of footpad lesions to 48% and 64%, and the parasite load in footpads to 82.6% and 81%, respectively. The F3 peptide induced the strongest anti-NH36 antibody response and intradermal response (IDR against L. amazonenis and a high secretion of IFN-γ and TNF-α with reduced levels of IL-10. The F1 vaccine, induced similar increases of IgG2b antibodies and IFN-γ and TNF-α levels, but no IDR and no reduction of IL-10. The multiparameter flow cytometry analysis was used to assess the immune response after immunotherapy and disclosed that the degree of the immunotherapeutic effect is predicted by the frequencies of the CD4+ and CD8+ T cells producing IL-2 or TNF-α or both. Total frequencies and frequencies of double-cytokine CD4 T cell producers were enhanced by F1 and F3 vaccines. Collectively, our multifunctional analysis disclosed that immunotherapeutic protection improved as the CD4 responses progressed from 1+ to 2+, in the case of the F1 and F3 vaccines, and as the CD8 responses changed qualitatively from 1+ to 3+, mainly in the case of the F1 vaccine, providing new correlates of immunotherapeutic protection against cutaneous leishmaniasis in mice based on T-helper TH1 and CD8+ mediated

  16. Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues.

    Science.gov (United States)

    Rinaldo-Matthis, Agnes; Wing, Corin; Ghanem, Mahmoud; Deng, Hua; Wu, Peng; Gupta, Arti; Tyler, Peter C; Evans, Gary B; Furneaux, Richard H; Almo, Steven C; Wang, Ching C; Schramm, Vern L

    2007-01-23

    Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a Km/Kd ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a Km/Kd ratio of 203,300. The tight binding of DADMe-ImmA supports a late SN1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP x ImmA x PO4 and TvPNP x DADMe-ImmA x PO4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 A ionic interaction between a PO4 oxygen and the N1' cation of the hydroxypyrrolidine and is weaker in the TvPNP x ImmA x PO4 structure at 3.5 A. However, the TvPNP x ImmA x PO4 structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP x DADMe-ImmA x PO4. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope-edited difference infrared spectroscopy with [6-18O]ImmH to establish that O6 is the keto tautomer in TvPNP x ImmH x PO4, causing an unfavorable leaving-group interaction

  17. Rigid Adenine Nucleoside Derivatives as Novel Modulators of the Human Sodium Symporters for Dopamine and Norepinephrine

    Science.gov (United States)

    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

  18. Sinefungin, a Natural Nucleoside Analogue of S-Adenosylmethionine, Inhibits Streptococcus pneumoniae Biofilm Growth

    Directory of Open Access Journals (Sweden)

    Mukesh Kumar Yadav

    2014-01-01

    Full Text Available 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.

  19. Current Status of Older and New Purine Nucleoside Analogues in the Treatment of Lymphoproliferative Diseases

    Directory of Open Access Journals (Sweden)

    Pawel Robak

    2009-03-01

    Full Text Available For the past few years more and more new cytotoxic agents active in the treatment of hematological malignancies have been synthesized and become available for either in vitro studies or clinical trials. Among them the class of antineoplastic drugs belonging to the purine nucleoside analogues group (PNAs plays an important role. Three of them: pentostatin (DCF, cladribine (2-CdA and fludarabine (FA were approved by Food and Drug Administration (FDA for the treatment of hematological malignancies. Recently three novel PNAs: clofarabine (CAFdA, nelarabine (ara-G and forodesine (immucillin H, BCX-1777 have been synthesized and introduced into preclinical studies and clinical trials. These agents seem to be useful mainly for the treatment of human T-cell proliferative disorders and they are currently undergoing clinical trials in lymphoid malignancies. However, there are also several studies suggesting the role of these drugs in B-cell malignancies. This review will summarize current knowledge concerning the mechanism of action, pharmacologic properties, clinical activity and toxicity of PNAs accepted for use in clinical practice, as well as new agents available for clinical trials.

  20. Investigation of iron-containing complexes of deoxyribonucleic acid nucleosides by Moessbauer spectroscopy

    International Nuclear Information System (INIS)

    DNA and nucleoside complexes with ferric and ferrous ions were investigated for the concentration of iron ions, ionic strength, temperature, and the nature and spatial configuration of neighbouring atoms of the iron ions in the complexes. Moessbauer spectroscopy was used. The Moessbauer measurements were conducted on lyophilized samples at room temperature (300 K) and on frozen solutions at liquid nitrogen temperature (77 K). Quadrupole splitting was found in all spectra obtained by a Pd(Co) source, with the exception of thymidine, thus indicating that the formation of complexes had not affected the oxidation state of iron ions. A decrease in isomer shift and an increase in quadrupole splitting were found in all spectra obtained by an iron(III) chloride source as well as in all spectra obtained by an iron chloride tetrahydrate source. UV irradiation of the samples prior to the Moessbauer measurements was found to have no effect on the Moessbauer spectra but to result in changes in the oxidation state of iron ions, mainly their valency and the ferrous/ferric ion ratio. The results are shown in a table and in graphs. (L.O.)

  1. Global Conformational Dynamics of HIV-1 Reverse Transcriptase Bound to Non-Nucleoside Inhibitors

    Directory of Open Access Journals (Sweden)

    Peter V. Coveney

    2012-07-01

    Full Text Available HIV-1 Reverse Transcriptase (RT is a multifunctional enzyme responsible for the transcription of the RNA genome of the HIV virus into DNA suitable for incorporation within the DNA of human host cells. Its crucial role in the viral life cycle has made it one of the major targets for antiretroviral drug therapy. The Non-Nucleoside RT Inhibitor (NNRTI class of drugs binds allosterically to the enzyme, affecting many aspects of its activity. We use both coarse grained network models and atomistic molecular dynamics to explore the changes in protein dynamics induced by NNRTI binding. We identify changes in the flexibility and conformation of residue Glu396 in the RNaseH primer grip which could provide an explanation for the acceleration in RNaseH cleavage rate observed experimentally in NNRTI bound HIV-1 RT. We further suggest a plausible path for conformational and dynamic changes to be communicated from the vicinity of the NNRTI binding pocket to the RNaseH at the other end of the enzyme.

  2. Exploring QSAR of Non-Nucleoside Reverse Transcriptase Inhibitors by Neural Networks: TIBO Derivatives

    Directory of Open Access Journals (Sweden)

    Driss Cherqaoui

    2004-01-01

    Full Text Available Abstract: Human Immunodeficiency Virus type 1 (HIV-1 reverse transcriptase is an important target for chemotherapeutic agents against the AIDS disease. 4,5,6,7-Tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H-ones (TIBO derivatives are potent non-nucleoside reverse transcriptase inhibitors (NNRTIs. In the present work, quantitative structure-activity relationship (QSAR analysis for a set of 82 TIBO derivatives has been investigated by means of a three-layered neural network (NN. It has been shown that NN can be a potential tool in the investigation of QSAR analysis compared with the models given in the literature. NN gave good statistical results both in fitting and prediction processes (0.861 ≤ r² ≤ 0.928, 0.839 ≤q² ≤ 0.845. The relevant factors controlling the anti-HIV-1 activity of TIBO derivatives have been identified. The results are along the same lines as those of our previous studies on HEPT derivatives and indicate the importance of the hydrophobic parameter in modeling the QSAR for TIBO derivatives.

  3. Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of Mycobacterium tuberculosis.

    Science.gov (United States)

    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

  4. Tunnel conductance of Watson-Crick nucleoside-base pairs from telegraph noise

    International Nuclear Information System (INIS)

    The use of tunneling signals to sequence DNA is presently hampered by the small tunnel conductance of a junction spanning an entire DNA molecule. The design of a readout system that uses a shorter tunneling path requires knowledge of the absolute conductance across base pairs. We have exploited the stochastic switching of hydrogen-bonded DNA base-nucleoside pairs trapped in a tunnel junction to determine the conductance of individual molecular pairs. This conductance is found to be sensitive to the geometry of the junction, but a subset of the data appears to come from unstrained molecular pairs. The conductances determined from these pairs are within a factor of two of the predictions of density functional calculations. The experimental data reproduces the counterintuitive theoretical prediction that guanine-deoxycytidine pairs (3 H-bonds) have a smaller conductance than adenine-thymine pairs (2 H-bonds). A bimodal distribution of switching lifetimes shows that both H-bonds and molecule-metal contacts break.

  5. Tunnel conductance of Watson-Crick nucleoside-base pairs from telegraph noise.

    Science.gov (United States)

    Chang, Shuai; He, Jin; Lin, Lisha; Zhang, Peiming; Liang, Feng; Young, Michael; Huang, Shuo; Lindsay, Stuart

    2009-05-01

    The use of tunneling signals to sequence DNA is presently hampered by the small tunnel conductance of a junction spanning an entire DNA molecule. The design of a readout system that uses a shorter tunneling path requires knowledge of the absolute conductance across base pairs. We have exploited the stochastic switching of hydrogen-bonded DNA base-nucleoside pairs trapped in a tunnel junction to determine the conductance of individual molecular pairs. This conductance is found to be sensitive to the geometry of the junction, but a subset of the data appears to come from unstrained molecular pairs. The conductances determined from these pairs are within a factor of two of the predictions of density functional calculations. The experimental data reproduces the counterintuitive theoretical prediction that guanine-deoxycytidine pairs (3 H-bonds) have a smaller conductance than adenine-thymine pairs (2 H-bonds). A bimodal distribution of switching lifetimes shows that both H-bonds and molecule-metal contacts break. PMID:19420603

  6. The Dipeptide Monoester Prodrugs of Floxuridine and Gemcitabine—Feasibility of Orally Administrable Nucleoside Analogs

    Directory of Open Access Journals (Sweden)

    Yasuhiro Tsume

    2014-01-01

    Full Text Available 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.

  7. The Dipeptide Monoester Prodrugs of Floxuridine and Gemcitabine—Feasibility of Orally Administrable Nucleoside Analogs

    Science.gov (United States)

    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

  8. The dipeptide monoester prodrugs of floxuridine and gemcitabine-feasibility of orally administrable nucleoside analogs.

    Science.gov (United States)

    Tsume, Yasuhiro; Borras Bermejo, Blanca; 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

  9. Effect of Purine Nucleoside Analogue-Acyclovir on The Sperm Parameters and Testosterone Production in Rats

    Directory of Open Access Journals (Sweden)

    Vahid Nejati

    2013-01-01

    Full Text Available Background: Acyclovir (ACV, a synthetic purine nucleoside analogue derived fromguanosine, is known to be toxic to gonads and the aim of this study was to evaluate theeffect of ACV on the sperm parameters and testosterone production in rat.Materials and Methods: In this experimental study, forty adult male Wistar rats (220± 20 g were randomly divided into five groups (n=8 for each group. One groupserved as control and one group served as sham control [distilled water was intraperitoneally(i.p. injected]. ACV was administered intraperitoneally in the drugtreatment groups (4, 16 and 48 mg/kg/day for 15 days. Eighteen days after the lastinjection, rats were sacrificed by CO2 inhalation. After that, cauda epididymideswere removed surgically. At the end, sperm concentrations in the cauda epididymis,sperm motility, morphology, viability, chromatin quality and DNA integrity wereanalyzed. Serum testosterone concentrations were determined.Results: The results showed that ACV did not affect sperm count, but decreased spermmotility and sperm viability at 16 and 48 mg/kg dose-levels. Sperm abnormalities increasedat 48 mg/kg dose-level of ACV. Further, ACV significantly increases DNA damageat 16 and 48 mg/kg dose-levels and chromatin abnormality at all doses. Besides, asignificant decrease in serum testosterone concentrations was observed at 16 and 48 mg/kg doses.Conclusion: The present results highly support the idea that ACV induces testicular toxicityby adverse effects on the sperm parameters and serum level of testosterone in malerats.

  10. Potential radiosensitizing agents. 6. 2-Nitroimidazole nucleosides: arabinofuranosyl and hexopyranosyl analogues

    International Nuclear Information System (INIS)

    New 2-nitroimidazole nucleosides have been synthesized as radiosensitizers of hypoxic mammalian cells in an attempt to reduce the neurotoxicity and to increase the therapeutic efficacy of this class of agents. The trimethylsilyl derivative of 2-nitroimidazole was condensed with 1-bromo-2,3,5-tri-O-benzoylarabinofuranose in the presence of mercuric cyanide to yield anomeric isomers of arabinofuranosides, which were separated by preparative thin-layer chromatography. Reaction of 2-deoxy-1,3,4,6-tetra-O-acetyl-D-glucose or 3,4,6-tri-O-acetyl-D-glucal with 2-nitroimidazole in the presence of an acid catalyst produced alpha and beta isomers of 2',3'-dideoxy-D-erythro-hex-2'-enopyranosides and an isomeric 3-substituted 1,2,3-trideoxy-D-erythro-hex-1-enopyranose. Hydrolysis of the esters was accomplished with sodium methoxide in methanol at 0 degrees C. The radiosensitizing efficacy of these agents was determined against Chinese hamster (V-79) cells in vitro. The 1-(2',3'-dideoxy-alpha-D-erythro-hex-2'-enopyranosyl)-2-nitroimidazole was the most active agent of this series and was found to be superior to misonidazole as a radiosensitizer

  11. Radiosensitizing effects of nitroimidazole nucleoside analogues on cultured mammalian cells at low radiation doses

    International Nuclear Information System (INIS)

    Radiosensitizing effects of nitroimidazole nucleoside analogues (RK-27, RK-28 and RK-29) were studied using cultured mouse leukemic L5178Y (L5178Y) cells and Chinese hamster V79 (CH V79) cells. There was no significant difference in sensitizer enhancement ratio among the three RK-compounds. The ratios were 1.7 for L5178Y cells and 1.6 for CH V79 cells at 1 mM concentration and were comparable to that of misonidazole. Sensitizer enhancement ratios of RK-28 for L5178Y cells irradiated with 1 to 4 Gy X-rays were, respectively, 1.5 to 1.6 at 0.5 mM concentration, which were nearly the same as those obtained at high doses. RK-28 was as effective for fractionated X-irradiation as for single X-irradiation, assuming complete repair of sublethal damage during incubation at 37degC between sessions of fractionated X-irradiation. The sensitizing effects of RK-28 were additive in combination with neocarzinostatin, an antitumor drug, which has different pharmacological and physiological properties, and enhances the radioresponse of cells. RK-28 is known to be rapidly metabolized and excreted in vivo and is thus expected to be less toxic than misonidazole. It was effective at low radiation doses routinely used in radiotherapy practice and for fractionated X-irradiation, suggesting its clinical utility. (author)

  12. 75Se-substituted purine nucleoside derivatives as tumor diagnostic agents

    International Nuclear Information System (INIS)

    Eight 75Se-substituted purine nucleoside derivatives were represented for the first time and their synthesis was described. The substances were tested in animal experiments for their suitability for tumor diagnostic agents and the change of organ kinetic distribution in dependence on structural characteristics of the compounds was studied. Whole-body retention was essentially higher for all substances possessing an isopropylidene group. Methylation at position 8 results in a preferable excretation via the liver. The substitution of the amino-group by a hydroxyl group increases the excretion rate which is preferably a renal one. The uptake rates recorded for Ehrlich-ascites cell-suspensions give evidence of a dependence on the number of hydropholic groups. Four of the compounds tested show tendencies to a specific uptake in neoplastic tissue, the tumour-muscle quotients are partly in the same order of magnitude like those of 111In or 57Co bleomycine. The tumour-blood quotients do not reach the values of the commercial comaparative preparations. (author)

  13. Triple-Nucleoside Analog Antiretroviral Therapy: Is There Still a Role in Clinical Practice? A Review

    Directory of Open Access Journals (Sweden)

    Kessler Harold A

    2005-06-01

    Full Text Available Abstract The development and widespread clinical use of coformulated abacavir/lamivudine/zidovudine (ABC/3TC/ZDV as Trizivir represented an important advance in the management of HIV-infected patients, especially those with adherence challenges. With a low pill burden, no food restrictions, limited drug-drug interactions, and a favorable resistance profile, ABC/3TC/ZDV remains an alternative option in the US Department of Health and Human Services Consensus Panel Guidelines as initial treatment in antiretroviral-naive patients. Recent data have shown ABC/3TC/ZDV to be less efficacious in suppressing and/or maintaining suppression of virologic replication compared with efavirenz-containing antiretroviral therapy. Although triple-nucleoside/nucleotide reverse transcriptase inhibitor (t-NRTI combinations that do not contain a thymidine analog (ZDV or stavudine have recently shown high virologic failure rates in clinical trials and clinical practice, t-NRTI regimens containing a thymidine analog have consistently been shown to be efficacious.

  14. Effective strand invasion ODN incorporating a new bicyclic nucleoside analogue (WNA).

    Science.gov (United States)

    Aoki, Eriko; Taniguchi, Yosuke; Sasaki, Shigeki

    2007-01-01

    Efficient and specific targeting of DNA sequences by synthetic ligands is a major goal in chemical biology. Triplex-forming oligonucleotides (TFOs) are sequence-specific DNA-binding agents, but are limited to homopurine/homopyrimidine target sequences. We have previously reported two useful analogues (WNA: W-shaped nucleoside analogues), WNA-bT and WNAbC, which recognize a TA and a CG interrupting site forming triplexes with high stability and selectivity, respectively. However, their ability to form triplexes depended on their neighbouring bases in the TFO. Subsequent studies have shown that the sequence-dependency of the WNA analogues, for the formation of triplexes, has been partially solved by use of a WNA analogue bearing a substituted aromatic ring. Investigations into the effects of the substituted aromatic ring of WNA derivatives on the stability of triplexes led to the discovery of strand invasion by the TFO incorporating the new WNA analogue to form a highly stable duplex. PMID:18029683

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

    Science.gov (United States)

    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

  16. Complexes of cadmium(II) and mercury(II) with polyamines, nucleosides and nucleotides

    International Nuclear Information System (INIS)

    Computer analysis of potentiometric titration data was applied for determination of stability constants of Cd(II) and Hg(II) complexes in binary systems with polyamines (PA), nucleosides (Nuc) and nucleotides (NMP). For the systems of Hg(II) and PA an untypical increase in the complex stability with increasing ring size was observed and interpreted as the mercury preference to formation of linear complexes. Results of potentiometric and 13C NMR studies for complexes of both metals indicate the involvement of all donor nitrogen atoms of di- and triamines in the coordination, leading to formation of N2 and N3 type chromophores, respectively. Monodentate complexes of Hg(II) with Cyd are formed already at very low pH (complexes with Cd from pH about 4). In the systems with AMP apart from nitrogen donor atoms, also the phosphate groups are involved in coordination. In the solid complexes of Cd(II) and Hg(II) with PA all donor atoms from the polyamines were found to be involved in the coordination and the presence of nitrate ions was established both in the inner and in the outer coordination spheres. (author)

  17. Searching for novel scaffold of triazole non-nucleoside inhibitors of HIV-1 reverse transcriptase.

    Science.gov (United States)

    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

  18. Nucleosides from Anthopleura stell%绿海葵中的核苷类成分

    Institute of Scientific and Technical Information of China (English)

    禚如朋; 付宏征; 张礼和; 林文翰

    2001-01-01

    目的 为了寻找抗肿瘤的活性成分。方法 利用溶剂法和色谱法对绿海葵进行了研究。结果 分离得到5 个核苷,经波谱学解析鉴定为2-羟基嘌呤核苷(Ⅰ),脱氧肌苷(Ⅱ),1-甲基黄嘌呤核苷(Ⅲ),脱氧鸟苷(Ⅳ),脱氧核糖胸腺嘧啶(Ⅴ)。结论 Ⅲ为一新天然产物,其余均为海洋生物中首次发现。%To study the chemical constituents of sea anemone, Anthopleura stell Verrill.Methods The chemical constituents were isolated and purified by chromatographic methods and their structures elucidated by chemical evidences and spectra data.Results 5 nucleosides were obtained and identified from the ethanolic extract as 2-hydroxy purine (Ⅰ); deoxyinosine (Ⅱ); 1-methylxanthosine (Ⅲ); deoxyguanosine (Ⅳ), and deoxyribo-thymidine (Ⅴ).Conclusion Compound Ⅲ was a new natural product, while the others were found from sea anemone for the first time.

  19. A structural study of lamellar phases formed by nucleoside-functionalized lipids

    CERN Document Server

    Berti, D; Baglioni, P; Dante, S; Hauss, T

    2002-01-01

    We report a neutron-scattering investigation of lamellar phases formed by novel phospholipids bearing nucleosides at the polar-head-group region. These nucleolipids can interact through stacking and H-bond interactions, following a pattern that resembles base-base coupling in natural nucleic acids (DNA, RNA), i.e. they have similar recognition properties. Bilayer stacks formed of DPP-adenosine, DPP-uridine and their 1:1 mixture were investigated after equilibration in a 98% relative humidity atmosphere. The DPP-adenosine spectrum can be accounted for (in analogy to DPPC) by a lamellar phase with a smectic period of about 60 A. DPP-uridine displays a not so straightforward behavior that we have tentatively ascribed to the coexistence of lamellae with different smectic periods. In the 1:1 mixture the lamellar mesophase of DPP-uridine is retained, suggesting a specific interaction of the uridine polar-head group with the adenosine moiety of DPP-adenosine. It should be stressed that this behavior can be considere...

  20. Tunnel conductance of Watson-Crick nucleoside-base pairs from telegraph noise

    Science.gov (United States)

    Chang, Shuai; He, Jin; Lin, Lisha; Zhang, Peiming; Liang, Feng; Young, Michael; Huang, Shuo; Lindsay, Stuart

    2009-05-01

    The use of tunneling signals to sequence DNA is presently hampered by the small tunnel conductance of a junction spanning an entire DNA molecule. The design of a readout system that uses a shorter tunneling path requires knowledge of the absolute conductance across base pairs. We have exploited the stochastic switching of hydrogen-bonded DNA base-nucleoside pairs trapped in a tunnel junction to determine the conductance of individual molecular pairs. This conductance is found to be sensitive to the geometry of the junction, but a subset of the data appears to come from unstrained molecular pairs. The conductances determined from these pairs are within a factor of two of the predictions of density functional calculations. The experimental data reproduces the counterintuitive theoretical prediction that guanine-deoxycytidine pairs (3 H-bonds) have a smaller conductance than adenine-thymine pairs (2 H-bonds). A bimodal distribution of switching lifetimes shows that both H-bonds and molecule-metal contacts break.

  1. Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

    Energy Technology Data Exchange (ETDEWEB)

    Mastrangelo, Eloise; Bollati, Michela; Milani, Mario [Department of Biomolecular Sciences and Biotechnology, CNR-INFM, University of Milano, Via Celoria 26, 20133 Milano (Italy); Lamballeire, Xavier de; Brisbare, Nadege [Unité des Virus Emergents, Faculté de Médecine, 27 Boulevard Jean Moulin, 13005 Marseille (France); Dalle, Karen; Lantez, Violaine; Egloff, Marie-Pierre; Coutard, Bruno; Canard, Bruno [Laboratoire Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS ESIL, Case 932, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France); Gould, Ernest; Forrester, Naomi [CEH Oxford, Mansfield Road, Oxford OX1 3SR (United Kingdom); Bolognesi, Martino, E-mail: martino.bolognesi@unimi.it [Department of Biomolecular Sciences and Biotechnology, CNR-INFM, University of Milano, Via Celoria 26, 20133 Milano (Italy)

    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.

  2. Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies.

    Science.gov (United States)

    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

  3. The therapeutic efficiency of nucleotides and nucleosides in UV radiation edema of mice

    International Nuclear Information System (INIS)

    The influence of several nucleotides and nucleosides on UV radiation edemas of mice was studied with the aid of a staining test. In the first test series, amounts equimolar to 20 mg thymidine were injected i.p. It was found that thymidine, ATP, ADP and A5'MP had a significant influence which uridine did not have. The NAD dose of 54.8 mg was lethal in all 10 animals and the ATP dose of 42 mg in three out of 10 animals, while ADP and A5'MP had the effect of a reversible retardation of movements. The most effective substances of this series were ATP and ADP. In the second test series, the substances were equimolar to 1.8 mg thymidine. All substances tested, i.e. thymidine, adenosin, adenosin-cyclophosphate, NAD, NADH, ATP, ADP and A5'MP had a significant effect. Except for NAD, to which the animals reacted with a slight retardation, all substances were well tolerated. NAD and ADP were the most effective. In a third test series, dose-efficiency curves were established for thymidine and ATP. ATP was significantly more effective in equimolar doses. This finding is discussed. (orig.)

  4. Capillary bioreactors based on human purine nucleoside phosphorylase: a new approach for ligands identification and characterization.

    Science.gov (United States)

    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

  5. Pyrimidine nucleoside phosphorylation in developing seeds and germinating seedlings of wheat

    International Nuclear Information System (INIS)

    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 [α-32P]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

  6. Antiviral therapy for chronic hepatitis B: Combination of nucleoside analogs and interferon

    Institute of Scientific and Technical Information of China (English)

    Satoru; Hagiwara; Naoshi; Nishida; Masatoshi; Kudo

    2015-01-01

    The ideal goal of chronic hepatitis B(CHB) treatment should be suppression of emergence of hepatocellular carcinoma through the disappearance of hepatitis B s antigen(HBs Ag) rather than the control of serum hepatitis B virus-DNA level. For this purpose, various types of combination therapies using nucleoside analogs(NAs) and interferon(IFN) have been conducted. The therapeutic effects of combination of two different kinds of agents are better than those of the monotherapy using NAs or IFN alone, probably because different pharmaceutical properties might act in a coordinated manner. Recently, combination therapies with NAs and IFN and sequential therapies with NAs administration followed by IFN therapy have been routinely employed. We previously reported that combination therapy using entecavir(ETV) and pegylated(PEG)-IFN showed antiviral effects in 71% of CHB patients; the effect of this combination was better than that using lamivudine(LAM) and PEG-IFN. This is partially explained by the better antiviral effects of ETV than those of LAM. In our analysis, the cohort of CHB consisted of the patients who showed a flare-up of hepatitis before antiviral therapy, and their baseline HBs Ag levels were relatively low. Therefore, in addition to the combination of the agents, the appropriate selection of patients is critical to achieve a good viral response.

  7. Hyperglycemia alters E-NTPDases, ecto-5'-nucleotidase, and ectosolic and cytosolic adenosine deaminase activities and expression from encephala of adult zebrafish (Danio rerio).

    Science.gov (United States)

    Capiotti, Katiucia Marques; Siebel, Anna Maria; Kist, Luiza Wilges; Bogo, Maurício Reis; Bonan, Carla Denise; Da Silva, Rosane Souza

    2016-06-01

    Hyperglycemia is the main feature for the diagnosis of diabetes mellitus (DM). Some studies have demonstrated the relationship between DM and dysfunction on neurotransmission systems, such as the purinergic system. In this study, we evaluated the extracellular nucleotide hydrolysis and adenosine deamination activities from encephalic membranes of hyperglycemic zebrafish. A significant decrease in ATP, ADP, and AMP hydrolyses was observed at 111-mM glucose-treated group, which returned to normal levels after 7 days of glucose withdrawal. A significant increase in ecto-adenosine deaminase activity was observed in 111-mM glucose group, which remain elevated after 7 days of glucose withdrawal. The soluble-adenosine deaminase activity was significantly increased just after 7 days of glucose withdrawal. We also evaluated the gene expressions of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-5'-nucleotidase, ADA, and adenosine receptors from encephala of adult zebrafish. The entpd 2a.1, entpd 2a.2, entpd 3, and entpd 8 mRNA levels from encephala of adult zebrafish were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expressions of adenosine receptors (adora 1 , adora 2aa , adora 2ab , and adora 2b ) were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expression of ADA (ada 2a.1) was decreased in glucose withdrawal group. Maltodextrin, used as a control, did not affect the expression of adenosine receptors, ADA and E-NTPDases 2, 3, and 8, while the expression of ecto-5'-nucleotidase was slightly increased and the E-NTPDases 1 decreased. These findings demonstrated that hyperglycemia might affect the ecto-nucleotidase and adenosine deaminase activities and gene expression in zebrafish, probably through a mechanism involving the osmotic effect, suggesting that the modifications caused on purinergic system may also contribute to the diabetes-induced progressive cognitive impairment. PMID:26769247

  8. Inhibitors for the bacterial ectonucleotidase Lp1NTPDase from Legionella pneumophila.

    Science.gov (United States)

    Fiene, Amelie; Baqi, Younis; Malik, Enas M; Newton, Patrice; Li, Wenjin; Lee, Sang-Yong; Hartland, Elizabeth L; Müller, Christa E

    2016-09-15

    Legionella pneumophila is an aerobic, Gram-negative bacterium of the genus Legionella, which constitutes the major causative agent of Legionnaires' disease. Recently a nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila was identified and termed Lp1NTPDase; it was found to be a structural and functional homolog of mammalian NTPDases catalyzing the hydrolysis of ATP to ADP and ADP to AMP. Its activity is believed to contribute to the virulence of Legionella pneumophila. Therefore Lp1NTPDase inhibitors are considered as novel antibacterial drugs. However, only weakly potent compounds are available so far. In the present study, a capillary electrophoresis (CE)-based enzyme assay for monitoring the Lp1NTPDase activity was established. The enzymatic reaction was performed in a test tube followed by separation of substrate and products by CE and subsequent quantification by UV analysis. After kinetic characterization of the enzyme, a series of 1-amino-4-ar(alk)ylamino-2-sulfoanthraquinone derivatives structurally related to the anthraquinone dye Reactive Blue 2, a non-selective ecto-NTPDase inhibitor, was investigated for inhibitory activity on Lp1NTPDase using the CE-based enzyme assay. Derivatives bearing a large lipophilic substituent (e.g., fused aromatic rings) in the 4-position of the 1-amino-2-sulfoanthraquinone showed the highest inhibitory activity. Compounds with IC50 values in the low micromolar range were identified. The most potent inhibitor was 1-amino-4-[phenanthrene-9-yl-amino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (28, PSB-16131), with an IC50-value of 4.24μM. It represents the most potent Lp1NTPDase inhibitor described to date. These findings may serve as a starting point for further optimization. Lp1NTPDase inhibition provides a novel approach for the (immuno)therapy of Legionella infections. PMID:27522579

  9. CrATP interferes in the promastigote-macrophage interaction in Leishmania amazonensis infection.

    Science.gov (United States)

    Ennes-Vidal, V; Castro, R O S; Britto, C; Barrabin, H; D'Avila-Levy, C M; Moreira, O C

    2011-07-01

    Recent have shown the relationship between Ecto-Nucleoside-Triphosphate-Diphosphohydrolases (Ecto-NTPDases or ecto-nucleotidases) and virulence and infectivity in trypanosomatids. In this work, the inhibition of the ecto-ATPase activities and promastigote growth of Leishmania amazonensis by CrATP was characterized. Furthermore, this compound was used to investigate the role of ecto-nucleotidase in the interaction of L. amazonensis with resident peritoneal macrophages obtained from BALB/c mice. CrATP partially inhibits the ecto-ATPase activity, presenting Ki values of 575·7±199·1 and 383·5±79·0 μm, in the presence or absence of 5 mm MgCl2, respectively. The apparent Kms for ATP (2·9±0·5 mm to Mg2+-dependent ecto-ATPase and 0·4±0·2 mm to Mg2+-independent ecto-ATPase activities) are not significantly altered by CrATP, suggesting a reversible non-competitive inhibition of both enzymes. When CrATP was added to the cultivation medium at 500 μm, it drastically inhibited the cellular growth. The interaction of promastigote forms of L. amazonensis with BALB/c peritoneal macrophages is strongly affected by CrATP. When the parasites were treated with 500 μm CrATP before interacting with macrophages, the adhesion and endocytic indices were strongly reduced to 53·0±14·8% and 39·8±1·1%, respectively. These results indicate that ecto-nucleotidase plays an important role in the infection process caused by Leishmania amazonensis. PMID:21679488

  10. The F0F1 ATP Synthase Complex Localizes to Membrane Rafts in Gonadotrope Cells.

    Science.gov (United States)

    Allen-Worthington, Krystal; Xie, Jianjun; Brown, Jessica L; Edmunson, Alexa M; Dowling, Abigail; Navratil, Amy M; Scavelli, Kurt; Yoon, Hojean; Kim, Do-Geun; Bynoe, Margaret S; Clarke, Iain; Roberson, Mark S

    2016-09-01

    Fertility in mammals requires appropriate communication within the hypothalamic-pituitary-gonadal axis and the GnRH receptor (GnRHR) is a central conduit for this communication. The GnRHR resides in discrete membrane rafts and raft occupancy is required for signaling by GnRH. The present studies use immunoprecipitation and mass spectrometry to define peptides present within the raft associated with the GnRHR and flotillin-1, a key raft marker. These studies revealed peptides from the F0F1 ATP synthase complex. The catalytic subunits of the F1 domain were validated by immunoprecipitation, flow cytometry, and cell surface biotinylation studies demonstrating that this complex was present at the plasma membrane associated with the GnRHR. The F1 catalytic domain faces the extracellular space and catalyzes ATP synthesis when presented with ADP in normal mouse pituitary explants and a gonadotrope cell line. Steady-state extracellular ATP accumulation was blunted by coadministration of inhibitory factor 1, limiting inorganic phosphate in the media, and by chronic stimulation of the GnRHR. Steady-state extracellular ATP accumulation was enhanced by pharmacological inhibition of ecto-nucleoside triphosphate diphosphohydrolases. Kisspeptin administration induced coincident GnRH and ATP release from the median eminence into the hypophyseal-portal vasculature in ovariectomized sheep. Elevated levels of extracellular ATP augmented GnRH-induced secretion of LH from pituitary cells in primary culture, which was blocked in media containing low inorganic phosphate supporting the importance of extracellular ATP levels to gonadotrope cell function. These studies indicate that gonadotropes have intrinsic ability to metabolize ATP in the extracellular space and extracellular ATP may serve as a modulator of GnRH-induced LH secretion. PMID:27482602

  11. The chemoenzymatic synthesis of clofarabine and related 2′-deoxyfluoroarabinosyl nucleosides: the electronic and stereochemical factors determining substrate recognition by E. coli nucleoside phosphorylases

    OpenAIRE

    Fateev, Ilja V; Konstantin V. Antonov; Konstantinova, Irina D; Tatyana I. Muravyova; Frank Seela; Esipov, Roman S; Anatoly I. Miroshnikov; 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, 2FAra-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 nucleosid...

  12. Interaction of monoclonal antibodies directed against bromodeoxyuridine with pyrimidine bases, nucleosides, and DNA

    International Nuclear Information System (INIS)

    Although antibodies directed against bromodeoxyuridine (BrdU) are being used in both clinical and basic research laboratories as tools to study and monitor DNA synthesis, little is known about the epitopes with which they react. Four monoclonal antibodies directed against BrdU were produced and were characterized to learn more about the epitopes on BrdU which are important for antibody recognition, to identify compounds other than BrdU which react with the antibodies and which might interfere with immunologic assays for BrdU, and to characterize the reaction of these antibodies with BrdU-containing DNA. By radioimmunoassays, the antibodies generally reacted well with 5-iododeoxyuridine, 5-fluorodeoxyuridine, and 5-nitrouracil. However, none of the antibodies reacted well with uridine - indicating that a substituent on uridine C5 was essential for antibody reactivity - or with 5-bromo or iodo-cytosine, indicating that the region around pyrimidine C4 is important for antibody recognition. Although the antibodies reacted with 5-halogen-substituted uracil bases, the antibodies reacted much better with the corresponding halogenated nucleosides, indicating that the sugar moiety was important for recognition. The presence of a triphosphate group of C'5 of BrdU (i.e., BrdUTP) did not detectably alter antibody recognition. S1 nuclease treatment of purified DNA suggested that all four monoclonal antibodies reacted exclusively with single-stranded regions of BrdU-containing DNA. Comparison of detecting DNA synthesis by [3H]TdR incorporation followed by autoradiography with that by BrdU incorporation followed by indirect immunofluorescence indicated that the latter technique was both an accurate and a sensitive measure of DNA synthesis

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

    Science.gov (United States)

    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

  14. Triazole pyrimidine nucleosides as inhibitors of Ribonuclease A. Synthesis, biochemical, and structural evaluation.

    Science.gov (United States)

    Parmenopoulou, Vanessa; Chatzileontiadou, Demetra S M; Manta, Stella; Bougiatioti, Stamatina; Maragozidis, Panagiotis; Gkaragkouni, Dimitra-Niki; Kaffesaki, Eleni; Kantsadi, Anastassia L; Skamnaki, Vassiliki T; Zographos, Spyridon E; Zounpoulakis, Panagiotis; Balatsos, Nikolaos A A; Komiotis, Dimitris; Leonidas, Demetres D

    2012-12-15

    Five ribofuranosyl pyrimidine nucleosides and their corresponding 1,2,3-triazole derivatives have been synthesized and characterized. Their inhibitory action to Ribonuclease A has been studied by biochemical analysis and X-ray crystallography. These compounds are potent competitive inhibitors of RNase A with low μM inhibition constant (K(i)) values with the ones having a triazolo linker being more potent than the ones without. The most potent of these is 1-[(β-D-ribofuranosyl)-1,2,3-triazol-4-yl]uracil being with K(i) = 1.6 μM. The high resolution X-ray crystal structures of the RNase A in complex with three most potent inhibitors of these inhibitors have shown that they bind at the enzyme catalytic cleft with the pyrimidine nucleobase at the B(1) subsite while the triazole moiety binds at the main subsite P(1), where P-O5' bond cleavage occurs, and the ribose at the interface between subsites P(1) and P(0) exploiting interactions with residues from both subsites. The effect of a susbsituent group at the 5-pyrimidine position at the inhibitory potency has been also examined and results show that any addition at this position leads to a less efficient inhibitor. Comparative structural analysis of these RNase A complexes with other similar RNase A-ligand complexes reveals that the triazole moiety interactions with the protein form the structural basis of their increased potency. The insertion of a triazole linker between the pyrimidine base and the ribose forms the starting point for further improvement of these inhibitors in the quest for potent ribonucleolytic inhibitors with pharmaceutical potential. PMID:23122937

  15. O2 Protonation Controls Threshold Behavior for N-Glycosidic Bond Cleavage of Protonated Cytosine Nucleosides.

    Science.gov (United States)

    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

  16. Identification of an alternative nucleoside triphosphate: 5'-deoxyadenosylcobinamide phosphate nucleotidyltransferase in Methanobacterium thermoautotrophicum delta H.

    Science.gov (United States)

    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

  17. Metabolites of purine nucleoside phosphorylase (NP in serum have the potential to delineate pancreatic adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Shaiju K Vareed

    Full Text Available Pancreatic Adenocarcinoma (PDAC, the fourth highest cause of cancer related deaths in the United States, has the most aggressive presentation resulting in a very short median survival time for the affected patients. Early detection of PDAC is confounded by lack of specific markers that has motivated the use of high throughput molecular approaches to delineate potential biomarkers. To pursue identification of a distinct marker, this study profiled the secretory proteome in 16 PDAC, 2 carcinoma in situ (CIS and 7 benign patients using label-free mass spectrometry coupled to 1D-SDS-PAGE and Strong Cation-Exchange Chromatography (SCX. A total of 431 proteins were detected of which 56 were found to be significantly elevated in PDAC. Included in this differential set were Parkinson disease autosomal recessive, early onset 7 (PARK 7 and Alpha Synuclein (aSyn, both of which are known to be pathognomonic to Parkinson's disease as well as metabolic enzymes like Purine Nucleoside Phosphorylase (NP which has been exploited as therapeutic target in cancers. Tissue Microarray analysis confirmed higher expression of aSyn and NP in ductal epithelia of pancreatic tumors compared to benign ducts. Furthermore, extent of both aSyn and NP staining positively correlated with tumor stage and perineural invasion while their intensity of staining correlated with the existence of metastatic lesions in the PDAC tissues. From the biomarker perspective, NP protein levels were higher in PDAC sera and furthermore serum levels of its downstream metabolites guanosine and adenosine were able to distinguish PDAC from benign in an unsupervised hierarchical classification model. Overall, this study for the first time describes elevated levels of aSyn in PDAC as well as highlights the potential of evaluating NP protein expression and levels of its downstream metabolites to develop a multiplex panel for non-invasive detection of PDAC.

  18. Radiolysis of aqueous solutions of nucleosides halogenated at the sugar moiety

    International Nuclear Information System (INIS)

    The pulse radiolysis of aqueous solutions of nucleosides halogenated at the sugar moiety (2'-bromo-2'-deoxyuridine 4, 3'-deoxy-3'-iodothymidine 5, 5'-deoxy-5'-iodouridine 6) has been studied. G(Hal) were determined by conductometry varying the experimental conditions (pH, saturation with Ar, N2O or air, addition of t-butanol). The results indicate that solvated electrons both add to the nucleobases and eliminate halogen ions from the halogenated sugar moiety. In the case of 4(and possibly of 5) the radical anion of the base transfers (k approximately 105s-1) an electron to the sugar-bound halogen atom thus cleaving the C-Hal bond. In competition with this reaction there is a protonation of the radical anion of the base by protons and by water. For the latter reaction constant of k = 5 x 103 M-1s-1 was estimated. Compound 4 has also been investigated by product analysis after 60-Co-γ-irradiation. In aerated solutions erythrose is formed with a G-value of 0.12. Its precursor radical is the 2'-radical generated from 4 by dissociative electron capture which reacts with O2 to the corresponding peroxyl radical. Erythrose is formed after a sequence of reactions, one of which involves the scission of the C-1'-C-2'bond. Under this condition G(HBr) as measured by pulse radiolysis is 0.8. Thus erythrose is formed in 15 per cent yield with respect to its precursor radical. This result is of importance in assessing the precursor radical of a similar product observed in irradiated DNA. (author)

  19. Biochemical mechanism of action of certain glucoside and nucleoside dialdehyde inhibitors

    International Nuclear Information System (INIS)

    The periodate-oxidation products of 4-methyl umbelliferyl 7-β-D-glucoside (MUGox), n-dodecyl β-D-glucoside (DGox), n-amyl-β-D-glucoside (AGox), 6,7 dihydroxy 6-β-D-glucoside (esculinox), 4-hydroxy phenyl β-D-glucoside (arbutinox), salicylaldehyde-β-D-glucoside (helicinox), indoxyl-β-D0-D-glucoside (IGox) and β-Naphthyl α-D-glucoside (NαGox) were synthesized and screened for activity against L1210 leukemia in-vitro. MUGox, DGox, Arbutinox, and Helicinox were the most active, having IC50 values of 40, 48, 44, and 47 μM, respectively. MUGox was found to inhibit DNA, RNA and protein synthesis significantly in L1210 cells. Its effects on the in-vitro activity of 2-cycle purified bovine brain tubulin were examined. MUGOx inhibited tubulin assembly only minimally at 100 μM but stabilized assembled microtubules from depolymerization at the same concentration. MUGox was a noncompetitive inhibitor of wheat germ RNA polymerase II and E. coli RNA polymerase with respect to [3H]UMP incorporation into RNA. MUGox bound one essential lysine in the wheat germ enzyme and two essential lysines in the E. coli enzyme. DGox and AGox produced mixed inhibition of wheat germ RNA polymerase II. The purine nucleoside analog 6-S-(acetylaminoethyl-1-naphthylamino-5-sulfonyl) thioinosine dicarboxaldehyde (AMPR-OP) noncompetitively blocked 0X174 DNA binding to E. coli RNA polymerase. It is tentatively concluded that AMPR-OP bound two lysine residues in the template binding subsite in addition to one lysine in the initiation subsite

  20. Virological and immunological outcomes at 3 years after starting antiretroviral therapy with regimens containing non-nucleoside reverse transcriptase inhibitor, protease inhibitor, or both in INITIO: open-label randomised trial

    DEFF Research Database (Denmark)

    Yeni, P; Cooper, DA; Aboulker, J-P;

    2006-01-01

    antiretroviral therapy with two nucleoside analogue reverse transcriptase inhibitors (didanosine+stavudine) plus either a non-nucleoside reverse transcriptase inhibitor (efavirenz, EFV) or a protease inhibitor (nelfinavir, NFV), or both (EFV/NFV), in patients with HIV-1 infection who had not previously received...

  1. Mutation in the phosphoribosylpyrophosphate synthetase gene (prs) that results in simultaneous requirements for purine and pyrimidine nucleosides, nicotinamide nucleotide, histidine, and tryptophan in Escherichia coli

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    1988-01-01

    , histidine, tryptophan, and nicotinamide mononucleotide were all added to the growth medium. Viability of the strain was dependent upon mutations in genes of the nucleoside salvage pathways that improved the utilization of exogenous nucleosides. The properties of the strain are those expected of a PRPP...

  2. Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2',4'-bridged nucleosides.

    Science.gov (United States)

    Kuwahara, Masayasu; Obika, Satoshi; Nagashima, Jun-ichi; Ohta, Yuki; Suto, Yoshiyuki; Ozaki, Hiroaki; Sawai, Hiroaki; Imanishi, Takeshi

    2008-08-01

    In order to systematically analyze the effects of nucleoside modification of sugar moieties in DNA polymerase reactions, we synthesized 16 modified templates containing 2',4'-bridged nucleotides and three types of 2',4'-bridged nucleoside-5'-triphospates with different bridging structures. Among the five types of thermostable DNA polymerases used, Taq, Phusion HF, Vent(exo-), KOD Dash and KOD(exo-), the KOD Dash and KOD(exo-) DNA polymerases could smoothly read through the modified templates containing 2'-O,4'-C-methylene-linked nucleotides at intervals of a few nucleotides, even at standard enzyme concentrations for 5 min. Although the Vent(exo-) DNA polymerase also read through these modified templates, kinetic study indicates that the KOD(exo-) DNA polymerase was found to be far superior to the Vent(exo-) DNA polymerase in accurate incorporation of nucleotides. When either of the DNA polymerase was used, the presence of 2',4'-bridged nucleotides on a template strand substantially decreased the reaction rates of nucleotide incorporations. The modified templates containing sequences of seven successive 2',4'-bridged nucleotides could not be completely transcribed by any of the DNA polymerases used; yields of longer elongated products decreased in the order of steric bulkiness of the modified sugars. Successive incorporation of 2',4'-bridged nucleotides into extending strands using 2',4'-bridged nucleoside-5'-triphospates was much more difficult. These data indicate that the sugar modification would have a greater effect on the polymerase reaction when it is adjacent to the elongation terminus than when it is on the template as well, as in base modification. PMID:18583360

  3. Fast Simultaneous Determination of 13 Nucleosides and Nucleobases in Cordyceps sinensis by UHPLC–ESI–MS/MS

    OpenAIRE

    Shi-Yu Zong; Han Han; Bing Wang; Ning Li; Tina Ting-Xia Dong; Tong Zhang; TSIM, KARL W. K.

    2015-01-01

    A reliable ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UHPLC–ESI–MS/MS) method for the fast simultaneous determination of 13 nucleosides and nucleobases in Cordyceps sinensis (C. sinensis) with 2-chloroadenosine as internal standard was developed and validated. Samples were ultrasonically extracted in an ice bath thrice, and the optimum analyte separation was performed on an ACQUITY UPLCTM HSS C18 column (100 mm × 2.1 mm, 1.8 μm) with gradie...

  4. Inhibitory activities of three classes of acyclic nucleoside phosphonates against murine polyomavirus and primate simian virus 40 strains

    Czech Academy of Sciences Publication Activity Database

    Lebeau, I.; Andrei, G.; Krečmerová, Marcela; De Clercq, E.; Holý, Antonín; Snoeck, R.

    2007-01-01

    Roč. 51, č. 6 (2007), s. 2268-2273. ISSN 0066-4804 R&D Projects: GA AV ČR 1QS400550501; GA MŠk 1M0508 Grant ostatní: FWO(BE) G.0267.04; NIH(US) AI 062540-01; René Descartes Prize 2001(XE) HPAV-2002-100096 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * polyomavirus * 5-azacytosine Subject RIV: CC - Organic Chemistry Impact factor: 4.390, year: 2007

  5. Novel conformationally locked nucleosides and nucleotides based on bicyclo[3.2.1]octane scaffold as a pseudosugar moiety

    Czech Academy of Sciences Publication Activity Database

    Šála, Michal; Dejmek, Milan; Procházková, Eliška; Hřebabecký, Hubert; Rybáček, Jiří; Dračínský, Martin; Novák, Pavel; Rosenbergová, Šárka; Fukal, J.; Sychrovský, Vladimír; Rosenberg, Ivan; Nencka, Radim

    Praha : Institute of Organic Chemistry and Biochemistry AS CR, v. v. i, 2014 - (Hocek, M.), s. 379-380 ISBN 978-80-86241-50-0. - (Collection Symposium Series. 14). [Symposium on Chemistry of Nucleic Acid Components /16./. Český Krumlov (CZ), 08.06.2014-13.06.2014] R&D Projects: GA ČR GPP207/12/P625; GA TA ČR TA03010598 Institutional support: RVO:61388963 Keywords : locked nucleosides * LNA Subject RIV: CC - Organic Chemistry

  6. Human equilibrative nucleoside transporter 1 and carcinoma of the ampulla of Vater: expression differences in tumour histotypes

    OpenAIRE

    Perrone, G.; Morini, S; Santini, D; Rabitti, C; Vincenzi, B; R. Alloni; Antinori, A.; Magistrelli, P; Lai, R.; Cass, C; Mackey, J R; Coppola, R.; Tonini, G; Onetti Muda, A.

    2010-01-01

    The human equilibrative nucleoside transporter 1 (hENT1) is the major means by which gemcitabine enters human cells; recent evidence exists that hENT1 is expressed in carcinoma of the ampulla of Vater and that it should be considered as a molecular prognostic marker for patients with resected ampullary cancer. Aim of the present study is to evaluate the variations of hENT1 expression in ampullary carcinomas and to correlate such variations with histological subtypes and clinicopathological pa...

  7. 4′-Ethynyl Nucleoside Analogs: Potent Inhibitors of Multidrug-Resistant Human Immunodeficiency Virus Variants In Vitro

    OpenAIRE

    Kodama, Ei-Ichi; Kohgo, Satoru; Kitano, Kenji; Machida, Haruhiko; Gatanaga, Hiroyuki; Shigeta, Shiro; Matsuoka, Masao; OHRUI, Hiroshi; Mitsuya, Hiroaki

    2001-01-01

    A series of 4′-ethynyl (4′-E) nucleoside analogs were designed, synthesized, and identified as being active against a wide spectrum of human immunodeficiency viruses (HIV), including a variety of laboratory strains of HIV-1, HIV-2, and primary clinical HIV-1 isolates. Among such analogs examined, 4′-E-2′-deoxycytidine (4′-E-dC), 4′-E-2′-deoxyadenosine (4′-E-dA), 4′-E-2′-deoxyribofuranosyl-2,6-diaminopurine, and 4′-E-2′-deoxyguanosine were the most potent and blocked HIV-1 replication with 50%...

  8. Acyclic Nucleoside Phosphonates as Inhibitors of Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase: New Anti-Malarial Chemotherapy Leads

    Czech Academy of Sciences Publication Activity Database

    Hocková, Dana; Holý, Antonín; Česnek, Michal; Baszczyňski, Ondřej; Tichý, Tomáš; Krečmerová, Marcela; Janeba, Zlatko; Skinner-Adams, T. S.; Naesens, L.; Keough, D. T.; de Jersey, J.; Guddat, L. W.

    Praha : Institute of Organic Chemistry and Biochemistry AS CR, v. v. i., 2011 - (Hocek, M.), s. 171-174 ISBN 978-80-86241-37-1. - (Collection Symposium Series. 12). [Chemistry of Nucleic Acid Components /15./. Český Krumlov (CZ), 05.06.2011-10.06.2011] R&D Projects: GA MŠk 1M0508; GA ČR GAP207/11/0108 Institutional research plan: CEZ:AV0Z40550506 Keywords : ANPs * acyclic nucleoside phosphonates * anti-malarial * Plasmodium * HGXPRTase Subject RIV: CC - Organic Chemistry

  9. Synthesis and characterization of mixed ligand complexes of bio-metals with pyrimidine nucleoside (uridine) and amino acids

    Indian Academy of Sciences (India)

    P Rabindra Reddy; A Mohan Reddy

    2000-12-01

    The mixed ligand complexes of Cu(II), Ni(II) and Co(II) with uridine and amino acids, L-alanine, L-phenylalanine and L-tryptophan were synthesized and characterized by elemental analysis, conductivity data, infrared spectra, electronic spectra and magnetic susceptibility data. In these complexes, the nucleoside (uridine) acts as a monodentate ligand coordinating through O(4) under the conditions of investigation, whereas the amino acids coordinate through the carboxylate oxygen and the amino nitrogen. Distorted octahedral geometry for Cu(II) and octahedral geometries for both Ni(II) and Co(II) are proposed.

  10. Enantiopurity analysis of anti-AIDS drugs and related acyclic nucleoside phosphonates-based antivirotics by capillary electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Kašička, Václav; Šolínová, Veronika; Sázelová, Petra; Mikysková, Hana; Koval, Dušan; Holý, Antonín

    Alphaville: AlphaGraphics, 2013 - (Guzman, N.; Tavares, M.). s. 32-32 [LACE 2013. Latin-American Symposium on Biotechnology, Biomedical, Biopharmaceutical, and Industrial Applications of Capillary Electrophoresis and Microchip Technology /19./. 29.11.2013-03.12.2013, Lima] R&D Projects: GA ČR(CZ) GAP206/12/0453; GA ČR(CZ) GA13-17224S; GA MŠk(CZ) ME10040; GA MV VG20102015046 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * capillary electrophoresis * chiral analysis Subject RIV: CB - Analytical Chemistry, Separation

  11. Comparison of Transition Metal-Mediated Oxidation Reactions of Guanine in Nucleoside and Single-Stranded Oligodeoxynucleotide Contexts

    OpenAIRE

    Ghude, Pranjali; Schallenberger, Mark A.; Fleming, Aaron M.; Muller, James G.; Burrows, Cynthia J.

    2011-01-01

    As the most readily oxidized of DNA’s four natural bases, guanine is a prime target for attack by reactive oxygen species (ROS) and transition metal-mediated oxidants. The oxidation products of a modified guanosine nucleoside and of a single-stranded oligodeoxynucleotide, 5′-d(TTTTTTTGTTTTTTT)-3′ have been studied using oxidants that include CoII, NiII, and IrIV compounds as well as photochemically generated oxidants such as sulphate radical, electron-transfer agents (riboflavin) and singlet ...

  12. Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2′,4′-bridged nucleosides

    OpenAIRE

    Kuwahara, Masayasu; Obika, Satoshi; Nagashima, Jun-ichi; Ohta, Yuki; SUTO, Yoshiyuki; Ozaki, Hiroaki; Sawai, Hiroaki; Imanishi, Takeshi

    2008-01-01

    In order to systematically analyze the effects of nucleoside modification of sugar moieties in DNA polymerase reactions, we synthesized 16 modified templates containing 2′,4′-bridged nucleotides and three types of 2′,4′-bridged nucleoside-5′-triphospates with different bridging structures. Among the five types of thermostable DNA polymerases used, Taq, Phusion HF, Vent(exo-), KOD Dash and KOD(exo-), the KOD Dash and KOD(exo-) DNA polymerases could smoothly read through the modified templates ...

  13. Molecular Recognition of Bases and Nucleosides by Mono-substituted Mononuclear Complexes of 1,4,7,10-Tetraaza-cyclododecane

    Institute of Scientific and Technical Information of China (English)

    向清祥; 余孝其; 吴江; 刘培岩; 夏传琴; 谢如刚

    2003-01-01

    The mononuclear macrocyclic polyamine metal complexes 5a-5e have been shown to form stable 1 : 1 complexes with bases and nucleosides. Their binding constants (K) were determined by UV-visible spectrometric titration. The results show that recognition ability of the complexes 5a--5e for uracil, U (Uridine), dT (Thymidine) is higher than that for the other bases or nucleosides (such as Cytidine, Guanosine, Adenosine). The metal ion also plays an important role for the recognition ability of complexes.

  14. Qualitative and quantitative analysis of glucosinolates and nucleosides in Radix Isatidis by HPLC and liquid chromatography tandem mass spectrometry

    Directory of Open Access Journals (Sweden)

    Xiuming Wang

    2013-09-01

    Full Text Available Multi-component fingerprinting and quantitation of the glucosinolates and nucleosides in samples of Radix Isatidis have been carried out using high-performance liquid chromatography with diode-array detection and electrospray ionization tandem mass spectrometry (HPLC–DAD–ESI/MS. Five nucleosides together with one glucosinolate were identified by comparing retention times, ultraviolet spectra, mass spectra and/or empirical molecular formulae of reference compounds. Quantitation of these six compounds was carried out simultaneously by HPLC on a Phenomenex Luna C18 column using gradient elution with methanol and water and detection at 254 nm. All calibration curves were linear (r>0.9994 within test ranges. Limits of detection and quantitation were 0.33 ng and 2.50 ng on column, respectively. Intra- and inter-day precision (as relative standard deviation for all analytes was <2.19% with recoveries in the range 99.6%–101.8% at three concentration levels. The validated method was successfully applied to fingerprinting and assay of 25 batches of Radix Isatidis sourced from different geographical regions of China. The method is simple and reliable and has potential value in the quality control of Radix Isatidis.

  15. Crystallization and preliminary X-ray structural analysis of nucleoside triphosphate hydrolases from Neospora caninum and Toxoplasma gondii

    International Nuclear Information System (INIS)

    Recombinant nucleoside triphosphate hydrolases from N. caninum and T. gondii have been purified and crystallized for X-ray structure analysis. The nucleoside triphosphate hydrolases that are produced by Neospora caninum (NcNTPase) and Toxoplasma gondii (TgNTPase-I) have a different physiological function from the ubiquitous ecto-ATPases. The recombinant enzymes were crystallized at 293 K using polyethylene glycol 3350 as a precipitant and X-ray diffraction data sets were collected for NcNTPase (to 2.8 Å resolution) and TgNTPase-I (to 3.1 Å resolution) at 100 K using synchrotron radiation. The crystals of NcNTPase and TgNTPase-I belonged to the orthorhombic space group I222 (unit-cell parameters a = 93.6, b = 140.8, c = 301.1 Å) and the monoclinic space group P21 (unit-cell parameters a = 87.1, b = 123.5, c = 120.2 Å, β = 96.6°), respectively, with two NcNTPase (VM = 3.7 Å3 Da−1) and four TgNTPase-I (VM = 2.7 Å3 Da−1) molecules per asymmetric unit. SAD phasing trials using a data set (λ = 0.97904 Å) collected from a crystal of selenomethionylated NcNTPase gave an initial electron-density map of sufficient quality to build a molecular model of NcNTPase

  16. Characterization of Nucleosides and Nucleobases in Natural Cordyceps by HILIC–ESI/TOF/MS and HILIC–ESI/MS

    Directory of Open Access Journals (Sweden)

    Bin Yang

    2013-08-01

    Full Text Available A method combining hydrophilic interaction chromatography (HILIC and electrospray ionization mass spectrometry (ESI-MS was developed for the characterization and determination of natural Cordyceps. Separation was achieved on a Waters Xbridge Amide column with gradient elution. Identification of 15 target nucleosides and nucleobases was based on retention time, UV spectra and mass measurements of the protonated molecules ([M+H]+ and main fragment ions (ESI-TOF/MS. Eight non-target compounds were tentatively identified by ESI-TOF/MS. The 15 target compounds were quantified by HILIC-ESI-MS/MS using time-programmed selective ion monitoring or multiple reaction monitoring in positive-ion mode under optimized mass conditions. This technique showed good linearity, repeatability and recovery. This approach was also successfully implemented in the analysis of nucleosides and nucleobases in 12 batches of natural Cordyceps samples that were collected from different regions in China. The developed HILIC-ESI-MS method exhibited clear advantages in identifying and determining highly polar bioactive components in Cordyceps, as well as their quality control.

  17. Fast Simultaneous Determination of 13 Nucleosides and Nucleobases in Cordyceps sinensis by UHPLC-ESI-MS/MS.

    Science.gov (United States)

    Zong, Shi-Yu; Han, Han; Wang, Bing; Li, Ning; Dong, Tina Ting-Xia; Zhang, Tong; Tsim, Karl W K

    2015-01-01

    A reliable ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the fast simultaneous determination of 13 nucleosides and nucleobases in Cordyceps sinensis (C. sinensis) with 2-chloroadenosine as internal standard was developed and validated. Samples were ultrasonically extracted in an ice bath thrice, and the optimum analyte separation was performed on an ACQUITY UPLC(TM) HSS C18 column (100 mm × 2.1 mm, 1.8 μm) with gradient elution. All targeted analytes were separated in 5.5 min. Furthermore, all calibration curves showed good linear regression (r > 0.9970) within the test ranges, and the limits of quantitation and detection of the 13 analytes were less than 150 and 75 ng/mL, respectively. The relative standard deviations (RSDs) of intra- and inter-day precisions were <6.23%. Recoveries of the quantified analytes ranged within 85.3%-117.3%, with RSD < 6.18%. The developed UHPLC-ESI-MS/MS method was successfully applied to determine nucleosides and nucleobases in 11 batches of C. sinensis samples from different regions in China. The range for the total content in the analyzed samples was 1329-2057 µg/g. PMID:26690105

  18. Fast Simultaneous Determination of 13 Nucleosides and Nucleobases in Cordyceps sinensis by UHPLC–ESI–MS/MS

    Directory of Open Access Journals (Sweden)

    Shi-Yu Zong

    2015-12-01

    Full Text Available A reliable ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UHPLC–ESI–MS/MS method for the fast simultaneous determination of 13 nucleosides and nucleobases in Cordyceps sinensis (C. sinensis with 2-chloroadenosine as internal standard was developed and validated. Samples were ultrasonically extracted in an ice bath thrice, and the optimum analyte separation was performed on an ACQUITY UPLCTM HSS C18 column (100 mm × 2.1 mm, 1.8 μm with gradient elution. All targeted analytes were separated in 5.5 min. Furthermore, all calibration curves showed good linear regression (r > 0.9970 within the test ranges, and the limits of quantitation and detection of the 13 analytes were less than 150 and 75 ng/mL, respectively. The relative standard deviations (RSDs of intra- and inter-day precisions were <6.23%. Recoveries of the quantified analytes ranged within 85.3%–117.3%, with RSD < 6.18%. The developed UHPLC–ESI–MS/MS method was successfully applied to determine nucleosides and nucleobases in 11 batches of C. sinensis samples from different regions in China. The range for the total content in the analyzed samples was 1329–2057 µg/g.

  19. Enzymatic regioselective acylation of nucleosides in biomass-derived 2-methyltetrahydrofuran: kinetic study and enzyme substrate recognition.

    Science.gov (United States)

    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

  20. Oxidation half-reaction of aqueous nucleosides and nucleotides via photoelectron spectroscopy augmented by ab initio calculations.

    Science.gov (United States)

    Schroeder, Christi A; Pluhařová, Eva; Seidel, Robert; Schroeder, William P; Faubel, Manfred; Slavíček, Petr; Winter, Bernd; Jungwirth, Pavel; Bradforth, Stephen E

    2015-01-14

    Oxidative damage to DNA and hole transport between nucleobases in oxidized DNA are important processes in lesion formation for which surprisingly poor thermodynamic data exist, the relative ease of oxidizing the four nucleobases being one such example. Theoretical simulations of radiation damage and charge transport in DNA depend on accurate values for vertical ionization energies (VIEs), reorganization energies, and standard reduction potentials. Liquid-jet photoelectron spectroscopy can be used to directly study the oxidation half-reaction. The VIEs of nucleic acid building blocks are measured in their native buffered aqueous environment. The experimental investigation of purine and pyrimidine nucleotides, nucleosides, pentose sugars, and inorganic phosphate demonstrates that photoelectron spectra of nucleotides arise as a spectral sum over their individual chemical components; that is, the electronic interactions between each component are effectively screened from one another by water. Electronic structure theory affords the assignment of the lowest energy photoelectron band in all investigated nucleosides and nucleotides to a single ionizing transition centered solely on the nucleobase. Thus, combining the measured VIEs with theoretically determined reorganization energies allows for the spectroscopic determination of the one-electron redox potentials that have been difficult to establish via electrochemistry. PMID:25551179

  1. Mitochondrial toxicities of nucleoside analogue reverse transcriptase inhibitors in AIDS cases

    Directory of Open Access Journals (Sweden)

    Yogesh S Marfatia

    2014-01-01

    Full Text Available The development of antiretroviral therapy (ART has been one of the most dramatic progressions in the history of medicine. Concomitant with this momentous therapeutic advance, the mitochondrial toxicities of ART were recognized as an important clinical entity. Aim: The aim was to study the mitochondrial toxicities in terms of peripheral neuropathy (PN, lipodystrophy (LD, hepatic steatosis, lactic academia (LA, and pancreatitis developing in AIDS cases on nucleoside analog reverse transcriptase inhibitors (NRTIs based ART regimens. Materials and Methods: An observational study, which included 90 AIDS cases, receiving first line ART regimens containing two NRTIs (zidovudine [AZT]/stavudine [d4T] with lamivudine [3TC] and one nonNRTIs (nevirapine/efavirenz was conducted at Skin-VD outpatient department of a tertiary care hospital attached to a Medical College. Thorough history was taken, and clinical examination was done. Cases were subjected to measurements of abdominal girth and mid-arm circumference, liver function tests, blood sugar, lipid profile, serum lactate, and amylase levels. Results: Of 90 cases on ART, 66% were males and 34% were females. Mitochondrial toxicities developed in 26 (30% cases out of 90, which included 3 (7% out of 42 cases on AZT + 3TC and 23 (48% out of 48 cases on d4T + 3TC. Most common toxicity was PN seen in 20 (22% cases; male cases developed PN at a lower CD4 count than female cases. LD was observed in total of 13 (14.5% cases; deposition of fat in the abdomen in seven cases and at the nape of the neck (buffalo hump in one case while loss of fat from extremities was seen in seven cases and loss of buccal fat in seven cases. Women presented more with fat accumulation (breast and abdomen, while men with loss of fat (limbs and buttocks. Both PN and LD were more common in d4T based regimen. LA was reported in one case on d4T. Hepatic steatosis was seen in three cases and pancreatitis in one case receiving AZT. Conclusion

  2. Purine Nucleoside Phosphorylase mediated molecular chemotherapy and conventional chemotherapy: A tangible union against chemoresistant cancer

    International Nuclear Information System (INIS)

    Late stage Ovarian Cancer is essentially incurable primarily due to late diagnosis and its inherent heterogeneity. Single agent treatments are inadequate and generally lead to severe side effects at therapeutic doses. It is crucial to develop clinically relevant novel combination regimens involving synergistic modalities that target a wider repertoire of cells and lead to lowered individual doses. Stemming from this premise, this is the first report of two- and three-way synergies between Adenovirus-mediated Purine Nucleoside Phosphorylase based gene directed enzyme prodrug therapy (PNP-GDEPT), docetaxel and/or carboplatin in multidrug-resistant ovarian cancer cells. The effects of PNP-GDEPT on different cellular processes were determined using Shotgun Proteomics analyses. The in vitro cell growth inhibition in differentially treated drug resistant human ovarian cancer cell lines was established using a cell-viability assay. The extent of synergy, additivity, or antagonism between treatments was evaluated using CalcuSyn statistical analyses. The involvement of apoptosis and implicated proteins in effects of different treatments was established using flow cytometry based detection of M30 (an early marker of apoptosis), cell cycle analyses and finally western blot based analyses. Efficacy of the trimodal treatment was significantly greater than that achieved with bimodal- or individual treatments with potential for 10-50 fold dose reduction compared to that required for individual treatments. Of note was the marked enhancement in apoptosis that specifically accompanied the combinations that included PNP-GDEPT and accordingly correlated with a shift in the expression of anti- and pro-apoptotic proteins. PNP-GDEPT mediated enhancement of apoptosis was reinforced by cell cycle analyses. Proteomic analyses of PNP-GDEPT treated cells indicated a dowregulation of proteins involved in oncogenesis or cancer drug resistance in treated cells with accompanying upregulation of

  3. A randomized trial comparing initial HAART regimens of nelfinavir/nevirapine and ritonavir/saquinavir in combination with two nucleoside reverse transcriptase inhibitors

    DEFF Research Database (Denmark)

    Kirk, Ole; Lundgren, Jens D; Pedersen, Court;

    2003-01-01

    BACKGROUND: A triple-class HAART regimen may be associated with a better virological effect than conventional regimens, but may also lead to toxicity and more profound resistance. METHODS: Randomized, controlled, open-label trial of 233 protease inhibitor- and non-nucleoside reverse transcriptase...

  4. Viral resuppression and detection of drug resistance following interruption of a suppressive non-nucleoside reverse transcriptase inhibitor-based regimen

    DEFF Research Database (Denmark)

    Fox, Zoe; Phillips, Andrew; Cohen, Cal;

    2008-01-01

    BACKGROUND: Interruption of a non-nucleoside reverse transcriptase inhibitor (NNRTI)-regimen is often necessary, but must be performed with caution because NNRTIs have a low genetic barrier to resistance. Limited data exist to guide clinical practice on the best interruption strategy to use...

  5. An efficient method for the construction of functionalized DNA bearing amino acid groups through cross-coupling reactions of nucleoside triphosphates followed by primer extension or PCR

    Czech Academy of Sciences Publication Activity Database

    Čapek, Petr; Cahová, Hana; Pohl, Radek; Hocek, Michal; Gloeckner, Ch.; Marx, A.

    2007-01-01

    Roč. 13, č. 21 (2007), s. 6196-6203. ISSN 0947-6539 R&D Projects: GA MŠk LC512; GA ČR GA203/05/0043 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleoside triphosphates * cross-coupling * DNA Subject RIV: CC - Organic Chemistry Impact factor: 5.330, year: 2007

  6. Synthesis and Evaluation of the Biological Profile of Novel Analogues of Nucleosides and of Potential Mimetics of Sugar Phosphates and Nucleotides

    Czech Academy of Sciences Publication Activity Database

    Xavier, N.M.; Lucas, S.D.; Jorda, Radek; Schwarz, S.; Loesche, A.; Csuk, R.; Oliveira, M.C.

    2015-01-01

    Roč. 26, č. 19 (2015), s. 2663-2672. ISSN 0936-5214 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : nucleosides * nucleotides * carbohydrates Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.419, year: 2014

  7. Conformationally locked aryl C-nucleosides: synthesis of phosphoramidite monomers and incorporation into single-stranded DNA and LNA (locked nucleic acid)

    DEFF Research Database (Denmark)

    Babu, B. Ravindra; Prasad, Ashok K.; Trikha, Smriti;

    2002-01-01

    Synthesis of a series of LNA-type beta-configured C-aryl nucleosides, i.e., 2'- O,4'-C-methylene-beta-D-ribofuranosyl derivatives containing phenyl, 4-fluoro-3-methylphenyl, 1-naphthyl, 1-pyrenyl and 2,4,5-trimethylphenyl groups as aglycons, has been accomplished. The key synthetic step consisted...

  8. Synthesis of acyclic nucleoside phosphonates bearing (N-methyl)anthraniloyl substituent as potential inhibitors of adenylate cyclase toxin from Bordetella Pertussis

    Czech Academy of Sciences Publication Activity Database

    Břehová, Petra; Šmídková, Markéta; Mertlíková-Kaiserová, Helena; Dračínský, Martin; Janeba, Zlatko

    Praha: Czech Chemical Society, 2015. s. 61. [Liblice 2015. Advances in Organic , Bioorganic and Pharmaceutical Chemistry /50./. 06.11.2015-08.11.2015, Olomouc] R&D Projects: GA MV VG20102015046 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * adenylate cyclase toxin * prodrugs Subject RIV: CC - Organic Chemistry

  9. Method of preparing highly active and thermostable preparations of liver uridin-kinase usable for enzymic synthesis of radioactive nucleoside-5'-phosphates

    International Nuclear Information System (INIS)

    A method is described of preparing a high-activity uridine kinase for the enzymic synthesis of radioactive nucleoside-5m-phosphates of the pyrimidine series. The preparation is separated from male rat liver after intraperitoneal application of 5'-azacytidine. Examples are given showing detailed procedures for the conversion of uridine and 6-azauridine to the corresponding 5'-phosphates. (L.K.)

  10. Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture

    DEFF Research Database (Denmark)

    Biondi, R M; Engel, M; Sauane, M;

    1996-01-01

    Although a number of nucleoside diphosphate kinases (NDPKs) have been reported to act as inhibitors of metastasis or as a transcription factor in mammals, it is not known whether these functions are linked to their enzymatic activity or how this protein is regulated. In this report, we show that ...... on histidine residues, however, only the B isoform appeared to be serine phosphorylated....

  11. The synthesis of double-headed nucleosides by the CuAAC reaction and their effect in secondary nucleic acid structures

    DEFF Research Database (Denmark)

    Jørgensen, Anna Søndergaard; Shaikh, Khalil Isak; Enderlin, Gerald; Ivarsen, Elise; Kumar, Surender; Nielsen, Poul

    2011-01-01

    Four double-headed nucleosides were prepared by the CuAAC reaction. Hereby, a triazole-containing linker connects an additional thymine or adenine to the 2´-position of 2´-deoxyuridine, a thymine to the 5´-position of thymidine and a thymine to the 6¢-position of an LNA-thymidine monomer. Whereas...

  12. Sugar-modified derivatives of cytostatic 6-(het)aryl-7-deazapurine nucleosides: 2'-C-methylribonucleosides, arabinonucleosides and 2'-deoxy-2'-fluoroarabinonucleosides

    Czech Academy of Sciences Publication Activity Database

    Nauš, Petr; Perlíková, Pavla; Pohl, Radek; Hocek, Michal

    2011-01-01

    Roč. 76, č. 8 (2011), s. 957-988. ISSN 0010-0765 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleosides * cross-coupling * antitumor agents * purines * 7-deazapurines * 7H-Pyrrolo[2,3d]pyrimidine * cytostatic agents Subject RIV: CC - Organic Chemistry Impact factor: 1.283, year: 2011

  13. The effect of the glycosylation position on the base pairing and supramolecular structure of the 5‧-deoxyribosyl Janus-type AT nucleosides

    Science.gov (United States)

    Zhou, Xinglong; Liu, Jiang; Guo, Xiurong; Meng, Liying; Chu, Liangyin; Chen, Qianming; Zhao, Hang; He, Yang

    2015-11-01

    The intrinsic structural diversity of the nucleosides is one of the essential properties for their wide participation in myriads of chemical or biochemical processes. Two unique regio-isomeric Janus-type AT nucleosides with the 5‧-deoxyribose attached on N1 or N3 position has been synthesized through Vorbrueggen or transglycosylation reactions. These two isomers display different supramolecular morphologies in solution state from their N8 glycosylated counterpart. The underneath structural details of the N3 glycosylated isomer were revealed by the single-crystal X-ray analysis. In addition to a novel base pair pattern was identified which is entirely different from the reverse Watson-Crick base pair adopted by its N8 isomer, an interesting water-filled columnar nanotuble-like structure was also found in its solid state. This study not only enriches the structural varieties of AT Janus-type nucleosides, but also provides specific information concerning the effect of the sugar glycosylation position on the properties of these new type of pyrimido[4,5-d]pyrimidine nucleosides.

  14. Cooperation and competition between adenylate kinase, nucleoside diphosphokinase, electron transport, and ATP synthase in plant mitochondria studied by 31P-nuclear magnetic resonance

    International Nuclear Information System (INIS)

    Nucleotide metabolism in potato (Solanum tuberosum) mitochondria was studied using 31P-nuclear magnetic resonance spectroscopy and the O2 electrode. Immediately following the addition of ADP, ATP synthesis exceeded the rate of oxidative phosphorylation, fueled by succinate oxidation, due to mitochondrial adenylate kinase (AK) activity two to four times the maximum activity of ATP synthase. Only when the AK reaction approached equilibrium was oxidative phosphorylation the primary mechanism for net ATP synthesis. A pool of sequestered ATP in mitochondria enabled AK and ATP synthase to convert AMP to ATP in the presence of exogenous inorganic phosphate. During this conversion, AK activity can indirectly influence rates of oxidation of both succinate and NADH via changes in mitochondrial ATP. Mitochondrial nucleoside diphosphokinase, in cooperation with ATP synthase, was found to facilitate phosphorylation of nucleoside diphosphates other than ADP at rates similar to the maximum rate of oxidative phosphorylation. These results demonstrate that plant mitochondria contain all of the machinery necessary to rapidly regenerate nucleoside triphosphates from AMP and nucleoside diphosphates made during cellular biosynthesis and that AK activity can affect both the amount of ADP available to ATP synthase and the level of ATP regulating electron transport

  15. The HopQ1 effector's nucleoside hydrolase-like domain is required for bacterial virulence in arabidopsis and tomato, but not host recognition in tobacco.

    Directory of Open Access Journals (Sweden)

    Wei Li

    Full Text Available Bacterial pathogens deliver multiple effector proteins into host cells to facilitate bacterial growth. HopQ1 is an effector from Pseudomonas syringae pv. tomato DC3000 that is conserved across multiple bacterial pathogens which infect plants. HopQ1's central region possesses some homology to nucleoside hydrolases, but possesses an alternative aspartate motif not found in characterized enzymes. A structural model was generated for HopQ1 based on the E. coli RihB nucleoside hydrolase and the role of HopQ1's potential catalytic residues for promoting bacterial virulence and recognition in Nicotiana tabacum was investigated. Transgenic Arabidopsis plants expressing HopQ1 exhibit enhanced disease susceptibility to DC3000. HopQ1 can also promote bacterial virulence on tomato when naturally delivered from DC3000. HopQ1's nucleoside hydrolase-like domain alone is sufficient to promote bacterial virulence, and putative catalytic residues are required for virulence promotion during bacterial infection of tomato and in transgenic Arabidopsis lines. HopQ1 is recognized and elicits cell death when transiently expressed in N. tabacum. Residues required to promote bacterial virulence were dispensable for HopQ1's cell death promoting activities in N. tabacum. Although HopQ1 has some homology to nucleoside hydrolases, we were unable to detect HopQ1 enzymatic activity or nucleoside binding capability using standard substrates. Thus, it is likely that HopQ1 promotes pathogen virulence by hydrolyzing alternative ribose-containing substrates in planta.

  16. 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.

    Science.gov (United States)

    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

  17. Liver injury in HIV-1-infected patients receiving non-nucleosides reverse transcriptase inhibitors-based antiretroviral therapy

    Institute of Scientific and Technical Information of China (English)

    LI Zai-cun; LI Hong-jun; DAI Li-li; GAO Yan-qing; CAI Wei-ping; LI Hai-ying; HUANG Xiao-jie; ZHANG Tong; WU Hao

    2010-01-01

    Background Liver injury is one of the most important adverse effects of antiretroviral therapy, leading to therapy changing or discontinuation. Data on liver injury in human immunodeficiency virus-1-infected patients receiving antiretroviral therapy are limited in China. The purpose of this study was to investigate the features of liver injury in human immunodeficiency virus type 1-infected patients receiving non-nucleosides reverse transcriptase inhibitors-based antiretroviral therapy in China.Methods Seventy-five patients on antiretroviral therapy containing non-nucleosides reverse transcriptase inhibitors were retrospectively studied. The patients were divided into 2 groups: group 1 (with liver injury, n=45) and group 2(without liver injury, n=30). The features of liver injury were analyzed. The sex, age, baseline CD4 counts, hepatitis B virus (HBV) and/or hepatitis C virus (HCV) co-infection, hepatotoxic drug use and nevirapine or efavirenz use were compared between two groups.Results Forty-five patients (60.0%), 31 (68.9%) males and 14 (31.1%) females, aged 12 to 52 years (averaged (3g±9)years), experienced at least one episode of liver injury. Forty (53.3%) patients were co-infected with HBV and/or HCV, 42 (56%) patients had concomitant use of antituberculosis drugs or cotrimoxazole, 46 (61.3%) and 29 (38.7%) patients received regimen containing nevirapine and efavirenz, respectively. Grade 1 liver injuries were observed in 26 (57.8%)patients, grade 2 in 16 (35.6%), grade 3 in 2 (4.0%) and grade 4 in 1 (2.2%). Three (6.7%) patients discontinued highly active antiretroviral therapy (HAART) due to liver injury. In group 1, there were 29 (64.4%) patients co-infected with HBV and/or HCV, 32 (71.1%) patients received regimen containing nevirapine, and 30 (66.7%) patients had concomitant use of anti-tuberculosis drugs or cotrimoxazole, respectively, significantly higher than those in group 2 (11 (36.7%), 14 (46.7%)and 12 (40%), respectively; P=0.018, 0.033, 0

  18. High level of oxidized nucleosides in thyroid mitochondrial DNA; damaging effects of Fenton reaction substrates

    Directory of Open Access Journals (Sweden)

    Karbownik-Lewińska Małgorzata

    2012-12-01

    Full Text Available Abstract Background The mitochondrial DNA (mtDNA lies in close proximity to the free radical-producing electron transport chain, thus, it is highly prone to oxidative damage. Oxyphilic type of follicular thyroid carcinoma consists of cells filled – almost exclusively – with aberrant mitochondria. In turn, bivalent iron (Fe2+ and hydrogen peroxide (H2O2 are indispensable for thyroid hormone synthesis, therefore being available in physiological conditions presumably at high concentrations. They participate in Fenton reaction (Fe2++H2O2→Fe3++·OH + OH-, resulting in the formation of the most harmful free radical – hydroxyl radical (·OH. The same substrates may be used to experimentally induce oxidative damage to macromolecules. The aim of the study was to evaluate the background level of oxidative damage to mtDNA and the damaging effects of Fenton reaction substrates. Methods Thyroid mtDNA was incubated in the presence of either H2O2 [100, 10, 1.0, 0.5, 0.1, 0.001, 0.00001 mM] or FeSO4 (Fe2+ [300, 150, 30, 15, 3.0, 1.5 μM], or in the presence of those two factors used together, namely, in the presence of Fe2+ [30 μM] plus H2O2 [100, 10, 1.0, 0.5, 0.1, 0.001, 0.00001 mM], or in the presence of H2O2 [0.5 mM] plus Fe2+ [300, 150, 30, 15, 3.0, 1.5 μM]. 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG concentration, as the index of DNA damage, was measured by HPLC. Results Both Fenton reaction substrates, used separately, increased 8-oxodG level for the highest H2O2 concentration of 100 mM and in Fe2+ concentration-dependent manner [300, 150, and 30 μM]. When Fe2+ and H2O2 were applied together, Fe2+ enhanced H2O2 damaging effect to a higher degree than did H2O2 on Fe2+ effect. Conclusions The level of oxidized nucleosides in thyroid mtDNA is relatively high, when compared to nuclear DNA. Both substrates of Fenton reaction, i.e. ferrous ion and hydrogen peroxide, increase oxidative damage to mtDNA, with stronger damaging effect exerted by

  19. Imaging expression of adenoviral HSV1-tk suicide gene transfer using the nucleoside analogue FIRU

    International Nuclear Information System (INIS)

    Substrates for monitoring HSV1-tk gene expression include uracil and acycloguanosine derivatives.The most commonly used uracil derivative to monitor HSV1-tk gene transfer is 1-(2-fluoro-2-deoxy-β-D-arabinofuranosyl)-5-[*I]iodouracil (fialuridine; I*-FIAU), where the asterisk denotes any of the radioactive iodine isotopes that can be used. We have previously studied other nucleosides with imaging properties as good as or better than FIAU, including 1-(2-fluoro-2-deoxy-β-D-ribofuranosyl)-5-[*I]iodouracil (FIRU). The first aim of this study was to extend the biodistribution data of 123I-labelled FIRU. Secondly, we assessed the feasibility of detecting differences in HSV1-tk gene expression levels following adenoviral gene transfer in vivo with 123I-FIRU. 9L rat gliosarcoma cells were stably transfected with the HSV1-tk gene (9L-tk+). 123I-FIRU was prepared by radioiodination of 1-(2-fluoro-2-deoxy-β-D-ribofuranosyl)-5-tributylstannyl uracil (FTMRSU; precursor compound) and purified using an activated Sep-Pak column. Incubation of 9L-tk+ cells and the parental 9L cells with 123I-FIRU resulted in a 100-fold higher accumulation of radioactivity in the 9L-tk+ cells after an optimum incubation time of 4 h. NIH-bg-nu-xid mice were then inoculated subcutaneously with HSV1-tk (-) 9L cells or HSV1-tk (+) 9L-tk+ cells into both flanks. Biodistribution studies and gamma camera imaging were performed at 15 min and 1, 2, 4 and 24 h p.i. At 15 min, the tumour/muscle, tumour/blood and tumour/brain ratios were 5.2, 1.0 and 30.3 respectively. Rapid renal clearance of the tracer from the body resulted in increasing tumour/muscle, tumour/blood and tumour/brain ratios, reaching values of 32.2, 12.5 and 171.6 at 4 h p.i. A maximum specific activity of 22%ID/g tissue was reached in the 9L-tk+ tumours 4 h after 123I-FIRU injection. Two Ad5-based adenoviral vectors containing the HSV1-tk gene were constructed: a replication-incompetent vector with the transgene in the former E1 region

  20. Optical isomers of a new 2-nitroimidazole nucleoside analog (PR-350 series): radiosensitization efficiency and toxicity

    International Nuclear Information System (INIS)

    Purpose: A new 2-nitroimidazole nucleoside radiosensitizer, PR-350 (1-[1',3',4'-trihydroxy-2'-butoxy]-methyl-2-nitroimidazole), has been reported to be as efficient as and less toxic than etanidazole. This compound is racemic, and it was recently optically resolved into two isomers, PR-68 (2'R,3'S type) and PR-69 (2'S,3'R type). The other two isomers, PR-28 (2'S,3'S type) and PR-44 (2'R,3'R type), were asymmetrically synthesized. In the present study, we investigated the properties, sensitizing activity, and toxicity of PR-350 and the four optical isomers in comparison with those of other 2-nitroimidazole hypoxic cell radiosensitizers, etanidazole, KU-2285, KIN-804, and RP-170. Because PR-350 and PR-28 can be industrially synthesized, we evaluated whether either of these two drugs are suitable for further investigation. Methods and Materials: In an in vitro study, EMT-6 cells were irradiated at a dose of 1-3 Gy under hypoxic conditions in the presence of the drugs at a concentration of 1 mM. A combined cytokinesis-block micronucleus and chromosomal aberration assay was performed. To assess the in vivo effects, colony assay and growth delay assay were performed using SCCVII tumor-bearing C3H mice. The mice received 16-24 Gy 10-40 min after administration of 50-200 mg/kg of the drugs. Toxicity and pharmacokinetics in mice were also investigated. Results: The sensitizer enhancement ratio (SER) in the in vitro cytokinesis-block micronucleus assay increased in the following order: PR-69 (1.27) ≅ PR-28 (1.31) ≅ PR-44 (1.38) ≅ PR-350 (1.41) ≅ PR-68 (1.47) 50 in mice were 5.8 g/kg for PR-350, approximately 7 g/kg for PR-28, 4 g/kg for PR-68, and 6 g/kg for PR-44 and PR-69. The concentration of PR-28 in the murine sciatic nerve was lower than that of PR-350. Conclusion: In vivo radiosensitizing activity differed among the four optical isomers, which appeared to be due, at least in part, to differences in lipophilicity. Although PR-28 was the least toxic, its low

  1. Evaluation of technetium-99m/rhenium labelled nucleoside analogues as potential radiotracers in oncology

    International Nuclear Information System (INIS)

    Over the last decade, suicide gene therapy has emerged as a very promising method to treat cancer. This therapy consists of introducing new genetic material into the nucleus of cancer cells so that they express a therapeutic protein. The protein leads to a therapeutic effect upon interaction with a prodrug. The most widely used system is the combination of the enzyme Herpes Simplex Virus Thymidine Kinase of type 1 (HSV1-TK) with the nontoxic antiviral prodrug gancyclovir. The efficiency of protein expression is crucial for a successful therapy. Several Positron Emission Tomography (PET) tracers such as 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine [18F]FHBG) or [124I]iodo-2'-fluoro-2'-deoxy-l-β-D-arabino-furanosyluracil (124I]FIAU) have been shown to be suitable probes for reporting HSV1-TK expression. One aspect of this work was to develop Single Photon Emission Tomography (SPET) reporter probes based on the inexpensive technetium-99m whose physical properties are well-suited for diagnosis (t1/2 = 6.02 h; Eγ = 140 keV). A series of complexes was prepared by derivatizing the precursor 5'-amino-5'-deoxythymidine at position N5' in order to introduce spacers of various lengths (∼ 0-30 aangstroem) carrying tridentate metal chelating entities such as iminodiacetic acid and picolylamine-N-monoacetic acid. The nucleoside derivatives were reacted with the precursors [ReBr3(CO)3]2- and [99mTc(OH2)3(CO)3]+ to form water-stable organometallic thymidine complexes. Unexpectedly, most of the compounds showed no inhibition of HSV1-TK but a mixed inhibition of the human cytosolic thymidine kinase with Kiu values ranging from 4.4-334 μM. Competitive inhibition of HSV1-TK was only observed for the thymidine analogue in which the base and the metal core were separated by a spacer of approximately 30 aangstroem length (Ki = 16.3 ± 4.6 μM). This compound also exhibited a mixed inhibition of the hTK1 with Kic = 73 ± 20 μM. When tested in vitro for cell uptake in transfected

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

    Science.gov (United States)

    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.

  3. Incorporation of 3H-labeled nucleosides and 3H-labeled deoxynucleosides into detergent soluble DNA

    International Nuclear Information System (INIS)

    Detergent soluble DNA from splenocytes of immunologically stimulated mice has been shown to incorporate [3H]dThd more rapidly than detergent insoluble DNA. In this report the author compares the incorporation of other 3H-labeled nucleosides and 3H-labeled deoxynucleosides and the distribution of 3H in the different size classes of detergent soluble DNA. The order of incorporation into DS DNA is [3H]dThd > [3H]dCyd > [3H]Ado > [3H]dGuo approx. = [3H]Cyd > [3H]dAdo > [3H]Guo. The author also shows that the previously reported slight enrichment in Gua + Cyt content is not due to preferential incorporation of dGuo or of dCyd into any one size class

  4. Enantioseparation of newly synthesized acyclic nucleoside phosphonates-based antivirals by capillary electrophoresis using cyclodextrins as chiral selectors

    Czech Academy of Sciences Publication Activity Database

    Šolínová, Veronika; Kaiser, Martin Maxmilian; Lukáč, Miloš; Janeba, Zlatko; Kašička, Václav

    Bratislava : Slovenská vákuová spoločnosť, 2013 - (Bodor, R.; Okenicová, L.; Staňová, A.), s. 245-247 ISBN 978-80-971179-1-7. [Analytické metódy a zdravie človeka. Medzinárodná konferencia /19./. Rajecké Teplice (SK), 24.06.2013-27.06.2013] R&D Projects: GA ČR(CZ) GAP206/12/0453; GA ČR GA13-17224S; GA MV VG20102015046 Grant ostatní: AV ČR CSIC(CZ) 2008CZ0019 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * chiral analysis * capillary electrophoresis Subject RIV: CB - Analytical Chemistry, Separation

  5. Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase

    DEFF Research Database (Denmark)

    Biondi, R M; Walz, K; Issinger, O G;

    1996-01-01

    We have critically analyzed current methodologies for distinguishing histidine and serine phosphorylated residues in proteins and report a simple technique that assures a reliable discrimination. Electro-transfer of a phosphorylated enzyme to Immobilon membranes and its treatment at pH 1 and 14 in...... buffers containing 5% methanol allows unambiguous distinction between serine/threonine and histidine phosphorylation (O-phosphomonoesters and phosphoramide, respectively) since under these conditions only one type of residue is dephosphorylated. The addition of 5% methanol to all buffers was indispensable...... to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase (NDP kinase) phosphorylation. In this enzyme, autophosphorylation of active site histidine is an accepted intermediate step in the...

  6. Hierarchical self-assembly of switchable nucleolipid supramolecular gels based on environmentally-sensitive fluorescent nucleoside analogs

    Science.gov (United States)

    Nuthanakanti, Ashok; Srivatsan, Seergazhi G.

    2016-02-01

    Exquisite recognition and folding properties have rendered nucleic acids as useful supramolecular synthons for the construction of programmable architectures. Despite their proven applications in nanotechnology, scalability and fabrication of nucleic acid nanostructures still remain a challenge. Here, we describe a novel design strategy to construct new supramolecular nucleolipid synthons by using environmentally-sensitive fluorescent nucleoside analogs, based on 5-(benzofuran-2-yl)uracil and 5-(benzo[b]thiophen-2-yl)uracil cores, as the head group and fatty acids, attached to the ribose sugar, as the lipophilic group. These modified nucleoside-lipid hybrids formed organogels driven by hierarchical structures such as fibers, twisted ribbons, helical ribbons and nanotubes, which depended on the nature of fatty acid chain and nucleobase modification. NMR, single crystal X-ray and powder X-ray diffraction studies revealed the coordinated interplay of various non-covalent interactions invoked by modified nucleobase, sugar and fatty acid chains in setting up the pathway for the gelation process. Importantly, these nucleolipid gels retained or displayed aggregation-induced enhanced emission and their gelation behavior and photophysical properties could be reversibly switched by external stimuli such as temperature, ultrasound and chemicals. Furthermore, the switchable nature of nucleolipid gels to chemical stimuli enabled the selective two channel recognition of fluoride and Hg2+ ions through visual phase transition and fluorescence change. Fluorescent organogels exhibiting such a combination of useful features is rare, and hence, we expect that this innovative design of fluorescent nucleolipid supramolecular synthons could lead to the emergence of a new family of smart optical materials and probes.Exquisite recognition and folding properties have rendered nucleic acids as useful supramolecular synthons for the construction of programmable architectures. Despite their

  7. Synthesis and Biological Evaluation of 2-Oxo/Thioxoquinoxaline and 2-Oxo/Thioxoquinoxaline-Based Nucleoside Analogues.

    Science.gov (United States)

    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

  8. The HCV non-nucleoside inhibitor Tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase function.

    Directory of Open Access Journals (Sweden)

    Christy M Hebner

    Full Text Available 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.

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

    Science.gov (United States)

    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

  10. Quantifying the length and variance of the eukaryotic cell cycle phases by a stochastic model and dual nucleoside pulse labelling.

    Directory of Open Access Journals (Sweden)

    Tom Serge Weber

    2014-07-01

    Full Text Available A fundamental property of cell populations is their growth rate as well as the time needed for cell division and its variance. The eukaryotic cell cycle progresses in an ordered sequence through the phases G1, S, G2, and M, and is regulated by environmental cues and by intracellular checkpoints. Reflecting this regulatory complexity, the length of each phase varies considerably in different kinds of cells but also among genetically and morphologically indistinguishable cells. This article addresses the question of how to describe and quantify the mean and variance of the cell cycle phase lengths. A phase-resolved cell cycle model is introduced assuming that phase completion times are distributed as delayed exponential functions, capturing the observations that each realization of a cycle phase is variable in length and requires a minimal time. In this model, the total cell cycle length is distributed as a delayed hypoexponential function that closely reproduces empirical distributions. Analytic solutions are derived for the proportions of cells in each cycle phase in a population growing under balanced growth and under specific non-stationary conditions. These solutions are then adapted to describe conventional cell cycle kinetic assays based on pulse labelling with nucleoside analogs. The model fits well to data obtained with two distinct proliferating cell lines labelled with a single bromodeoxiuridine pulse. However, whereas mean lengths are precisely estimated for all phases, the respective variances remain uncertain. To overcome this limitation, a redesigned experimental protocol is derived and validated in silico. The novelty is the timing of two consecutive pulses with distinct nucleosides that enables accurate and precise estimation of both the mean and the variance of the length of all phases. The proposed methodology to quantify the phase length distributions gives results potentially equivalent to those obtained with modern phase

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

    Science.gov (United States)

    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

  12. The HCV non-nucleoside inhibitor Tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase function.

    Science.gov (United States)

    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

  13. ATP-dependent removal of nucleoside reverse transcriptase inhibitors by human immunodeficiency virus type 1 reverse transcriptase.

    Science.gov (United States)

    Naeger, Lisa K; Margot, Nicolas A; Miller, Michael D

    2002-07-01

    Removal of nucleoside chain terminator inhibitors mediated by human immunodeficiency virus (HIV) reverse transcriptase (RT) using ATP as an acceptor molecule has been proposed as a novel mechanism of HIV resistance. Recombinant wild-type and mutant HIV type 1 (HIV-1) RT enzymes with thymidine analog resistance mutations D67N, K70R, and T215Y were analyzed for their ability to remove eight nucleoside reverse transcriptase inhibitors in the presence of physiological concentrations of ATP. The order for the rate of removal of the eight inhibitors by the mutant RT enzyme was zidovudine (AZT) > stavudine (d4T) > zalcitabine (ddC) > abacavir > amdoxovir (DAPD) > lamivudine (3TC) > didanosine (ddI) > tenofovir. Thymidine analogs AZT and d4T were the most significantly removed by the mutant enzyme, suggesting that removal of these inhibitors by the ATP-dependent removal mechanism contributes to the AZT and d4T resistance observed in patients with HIV expressing thymidine analog resistance mutations. ATP-dependent removal of tenofovir was 22- to 35-fold less efficient than removal of d4T and AZT, respectively. The addition of ATP and the next complementary deoxynucleoside triphosphate caused a reduction of ATP-mediated removal of d4T, ddC, and DAPD, while AZT and abacavir removal was unaffected. The reduction of d4T, ddC, and DAPD removal in the presence of the deoxynucleoside triphosphate could explain the minor changes in susceptibility to these drugs observed in conventional in vitro phenotypic assays using cells that have higher deoxynucleoside triphosphate pools. The minimal removal of abacavir, ddC, DAPD, 3TC, ddI, and tenofovir is consistent with the minor changes in susceptibility to these drugs observed for HIV mutants with thymidine analog resistance mutations. PMID:12069972

  14. The influence of X-ray radiation and physical training upon the molecular heterogeneity of nucleoside-phosphate kinases in subcellular hepatic fractions

    International Nuclear Information System (INIS)

    The different molecular forms of nucleoside-monophosphate kinases (FK 2.7.4) and nucleoside-diphosphate (KF 1.7.4.6) which are responsible for the final steps of pyrimidine nucleotide synthesis were determined in mitochondrial rat hepatic supernatant under condition of combined influence of X-ray and maximum physical exercises (running up to complete exhaustion). The maximum activity of investigated nucleosidephosphate kinases were observed in intact animals in that fractions which were eluted with trys-HCl buffer solution (0.075 and 1.0 M, pH 7.4). X-ray radiation and physical exercises caused the deviation of chromatographic data of maximal enzymatic activities. The drastic lowering of investigated enzymes was observed after X-ray irradiation and maximum physical exhaustion. This fact is in favour for the suppression of the final steps of pirimidine nucleotides synthesis under explored conditions of experimental investigations. 15 refs.; 2 tabs

  15. 2-Aryl-8-aza-3-deazaadenosine analogues of 5'-O-[N-(salicyl)sulfamoyl]adenosine: Nucleoside antibiotics that block siderophore biosynthesis in Mycobacterium tuberculosis.

    Science.gov (United States)

    Krajczyk, Anna; Zeidler, Joanna; Januszczyk, Piotr; Dawadi, Surendra; Boshoff, Helena I; Barry, Clifton E; Ostrowski, Tomasz; Aldrich, Courtney C

    2016-07-15

    A series of 5'-O-[N-(salicyl)sulfamoyl]-2-aryl-8-aza-3-deazaadenosines were designed to block mycobactin biosynthesis in Mycobacterium tuberculosis (Mtb) through inhibition of the essential adenylating enzyme MbtA. The synthesis of the 2-aryl-8-aza-3-deazaadenosine nucleosides featured sequential copper-free palladium-catalyzed Sonogashira coupling of a precursor 4-cyano-5-iodo-1,2,3-triazolonucleoside with terminal alkynes and a Minakawa-Matsuda annulation reaction. These modified nucleosides were shown to inhibit MbtA with apparent Ki values ranging from 6.1 to 25nM and to inhibit Mtb growth under iron-deficient conditions with minimum inhibitory concentrations ranging from 12.5 to >50μM. PMID:27265685

  16. Determination of nucleosides and nucleotides in baby foods by hydrophilic interaction chromatography coupled to tandem mass spectrometry in the presence of hydrophilic ion-pairing reagents.

    Science.gov (United States)

    Mateos-Vivas, María; Rodríguez-Gonzalo, Encarnación; Domínguez-Álvarez, Javier; García-Gómez, Diego; Carabias-Martínez, Rita

    2016-11-15

    In this work we propose a rapid and efficient method for the joint determination of nucleosides and nucleotides in dairy and non-dairy baby foods based on hydrophilic interaction chromatography coupled to tandem mass spectrometry in the presence of diethylammonium (DEA) as a hydrophilic ion-pairing reagent (IP-HILIC-MS/MS). Sample treatment of the baby food included dilution with water and centrifugal ultrafiltration (CUF) with an additional washing step that notably improved the global performance of the process. Later dilution of the extract with acetonitrile allowed adequate separation in the HILIC system. With the proposed treatment, we obtained extraction recoveries higher than 80% and, additionally, no matrix effects were observed. The CUF-IP-HILIC-MS/MS method was validated according to the 2002/657/EC decision and was used for the quantification of nucleotides and nucleosides in sixteen samples of commercial baby foods. PMID:27283702

  17. Synthesis of branched 9-[2-(2-phosphonoethoxy)ethyl]purines as a new class of acyclic nucleoside phosphonates which inhibit Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase

    Czech Academy of Sciences Publication Activity Database

    Hocková, Dana; Holý, Antonín; Masojídková, Milena; Keough, D. T.; de Jersey, J.; Guddat, L. W.

    2009-01-01

    Roč. 17, č. 17 (2009), s. 6218-6232. ISSN 0968-0896 R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * drug design * phosphoribosyltransferase * enzyme inhibitors * purine salvage pathway Subject RIV: CC - Organic Chemistry Impact factor: 2.822, year: 2009

  18. Pd-catalyzed Suzuki-Miyaura coupling reaction in the synthesis of 5-aryl substituted pyrimidine acyclic nucleoside phosphonates as potential multisubstrate inhibitors of thymidine phosphorylase

    Czech Academy of Sciences Publication Activity Database

    Pomeisl, Karel; Holý, Antonín; Pohl, Radek

    Dublin : University College Dublin, 2007. s. 329. [European Symposium on Organic Chemistry /15./. 08.07.2007-13.07.2007, Dublin] R&D Projects: GA MŠk 1M0508 Grant ostatní: Descartes Prize(XE) HPAW-CT-2002-9001 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * thymidine phosphorylase * Suzuki coupling Subject RIV: CC - Organic Chemistry

  19. Sonogashira cross-coupling in the synthesis of acyclic nucleoside phosphonates: preparation of 6-[(phosphonomethoxy)alkynyl]- and 6-[(phosphonomethoxy)alkyl]pyrimidines

    Czech Academy of Sciences Publication Activity Database

    Hocková, Dana; Masojídková, Milena; Holý, Antonín

    2005-01-01

    Roč. 70, č. 2 (2005), 247-257. ISSN 0010-0765 R&D Projects: GA AV ČR(CZ) IBS4055109 Grant ostatní: René Descartes Prize 2001(XE) HPAW-2002-90001 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleotide analogues * acyclic nucleoside phosphonates * pyrimidines Subject RIV: CC - Organic Chemistry Impact factor: 0.949, year: 2005

  20. Preparation of nucleoside-pyridine hybrids and pyridine attached acylureas from an unexpected uracil ring-opening and pyridine ring-forming sequence

    Institute of Scientific and Technical Information of China (English)

    Xue Sen Fan; Xia Wang; Xin Ying Zhang; Dong Feng; Ying Ying Qu

    2009-01-01

    Novel pyrimidine nucleoside-3,5-dicyanopyridine hybrids (4) or pyridine attached acylureas (5) were selectively and efficiently prepared from the reaction of 2'-deoxyuridin-5-yl-methylene malonortitrile (1), malononitrile (2) and thiophenol (3) or from an unexpected uracil ring-opening and pyridine ring-forming sequence via the reaction of 1 and 3. It is the first time such a sequence has ever been reported.

  1. Mechanistic studies on the synergistic cytotoxicity of the nucleoside analogs gemcitabine and clofarabine in multiple myeloma: Relevance of p53 and its clinical implications

    OpenAIRE

    Valdez, Benigno C.; Wang, Guiyun; Murray, David; Nieto, Yago; Li, Yang; Shah, Jatin; Turturro, Francesco; Wang, Michael; Weber, Donna M.; Champlin, Richard E.; Qazilbash, Muzaffar H; Andersson, Borje S.

    2013-01-01

    Hematopoietic stem cell transplantation (HSCT) is an established treatment for multiple myeloma (MM), a plasma cell malignancy. To identify an improved pretransplant conditioning regimen, we investigated the cytotoxicity of gemcitabine (Gem) and clofarabine (Clo) combinations toward MM cell lines and patient cell samples. A strong synergism of the two nucleoside analogs, when combined at their approximate IC10 concentrations, was observed. This synergism could be partly due to the observed Ge...

  2. Direct analysis in real time (DART) mass spectrometry of nucleotides and nucleosides: elucidation of a novel fragment [C5H5O]+ and its in-source adducts.

    Science.gov (United States)

    Curtis, Matthew; Minier, Mikael A; Chitranshi, Priyanka; Sparkman, O David; Jones, Patrick R; Xue, Liang

    2010-08-01

    Direct analysis in real time (DART) mass spectrometry is a recently developed innovative technology, which has shown broad applications for fast and convenient analysis of complex samples. Due to the ease of sample preparation, we have recently initiated an investigation of the feasibility of detecting nucleotides and nucleosides using the DART-AccuTOF instrument, which we will refer to as the DART mass spectrometer. Our experimental results reveal that the ions representing the intact molecules of nucleotides are not detectable in either positive-ion or negative-ion mode. Instead, all four natural nucleotides fragment in the DART ion source, and a common fragment ion, [C(5)H(5)O](+) (1), is observed, which is probably formed via multiple-elimination reactions. Interestingly, 1 can form adducts with nucleobases in different molar ratios in the DART ion source. In contrast to nucleotides, the ions representing the intact molecules of nucleosides are detected in both positive-ion and negative-ion mode using DART mass spectrometry. Surprisingly, the fragmentation pattern of nucleosides is different from that of nucleotides in the DART ion source. In the cases of nucleosides (under positive-ion conditions), the production of 1 is not observed, indicating that the phosphate group plays an important role for the multiple eliminations observed in the spectra of nucleotides. The in-source reactions described in the present work show the complexity of the conditions in the DART ion source, and we hope that our results illustrate a better understanding about DART mass spectrometry. PMID:20451406

  3. Characterization of hepatitis B virus inhibition by novel 2'-fluoro-2',3'-unsaturated beta-D- and L-nucleosides.

    Science.gov (United States)

    Pai, S Balakrishna; Pai, Rekha B; Xie, Meng-yu; Beker, Tolunay; Shi, Junxing; Tharnish, Philip M; Chu, Chung K; Schinazi, Raymond F

    2005-01-01

    The clinical emergence of lamivudine and adefovir resistance mutations on prolonged therapy further necessitates the development of additional drugs for the treatment of hepatitis B virus (HBV) infections. We have evaluated a number of novel 2'-fluoro-2',3'-unsaturated D- and L-nucleosides for their anti-HBV activity in the HepG2-2.2.15 cell system. The most potent nucleosides were beta-L-2'-fluoro-2',3'-dideoxy-2',3'-didehydrocy-tidine (L-2'-Fd4C) and beta-L-2'-fluoro-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine (L-2'-Fd4FC) with median effective concentrations (EC50) of 0.002 microM and 0.004 microM, respectively. The D-enantiomers of the 2'-fluoro-substituted cytidine analogues in this series showed activity, with the 5-fluorocytidine (D-2'-Fd4FC) being the most potent (EC50 = 0.05 microM). The active compounds were not cytotoxic to a number of cell lines or to bone marrow progenitor cells. Furthermore, mitochondrial DNA synthesis and function were not affected by these nucleosides. L-2'-Fd4C did not affect viral transcription, implying that it does not inhibit cellular RNA polymerase II. Studies with the HBV polymerase in core particles revealed that the 5'-triphosphates of L-2'-Fd4C and D-2'-Fd4FC produced a dose-dependent inhibition of the incorporation of 32P-dCTP into the HBV DNA, indicating that the mechanism of action of these compounds is through specific inhibition of viral DNA synthesis. This class of nucleosides, which exhibit potent antiviral activity and a favourable safety profile, have potential for the treatment of HBV infections and warrant further development. PMID:16004081

  4. The effect of non-alcoholic fatty liver disease on virologic response in patients with hepatitis B e antigen-positive chronic hepatitis B treated with nucleoside analogues

    Institute of Scientific and Technical Information of China (English)

    陈梅琴

    2014-01-01

    Objective To investigate the effect of non-alcoholic fatty liver disease(NAFLD)on virologic response in chronic hepatitis B patients treated with nucleoside analogues.Methods Three hundred and thirty-two treatment-naive patients with hepatitis B e antigen(HBeAg)-positive chronic hepatitis B(CHB)who visited clinic or hospitalized in the First Affiliated Hospital of Wenzhou Medical College from January 2007 to December 2009

  5. Stacked base-pair structures of adenine nucleosides stabilized by the formation of hydrogen-bonding network involving the two sugar groups

    International Nuclear Information System (INIS)

    Highlights: ► A combination of laser desorption and supersonic jet-cooling is used to produce base pairs of adenine nucleosides. ► Stacked base-pair structure of N6,N6-dimethyladnosine is identified by IR vibrational spectroscopy. ► Anharmonic vibrational calculation is employed to analyze the vibrational mode coupling in the stacked base pair. - Abstract: We have employed a laser desorption technique combined with supersonic-jet cooling for producing base pairs of adenine nucleosides, adenosine (Ado) and N6,N6-dimethyladenosine (DMAdo) under low-temperature conditions. The resulting base pairs are then ionized through resonant two-photon ionization (R2PI) and analyzed by time-of-flight mass spectrometry. It is found that dimers of these adenine nucleosides are stable, especially in the case of DMAdo, with respect to those of the corresponding bases, i.e., adenine and N6,N6-dimethyladenine. Structural analysis of the DMAdo dimer is performed based on the IR–UV double resonance measurements and theoretical calculations. The result demonstrates that the dimer possesses a stacked structure being stabilized by the formation of hydrogen-bonding network involving the two sugar groups. The occurrence of the frequency shift and broadening is explained satisfactorily based on the anharmonic coupling of the OH stretching modes with specific bending modes and low-frequency modes of base and sugar moieties

  6. Volumetric properties of the nucleosides adenosine, cytidine, and uridine in aqueous solution at T = 298.15 K and p = (10 to 120) MPa

    International Nuclear Information System (INIS)

    Highlights: ► Sound speeds were measured for aqueous solutions of nucleosides at high pressures. ► Volumetric properties were derived for T = 298.15 K and p = (10 to 120) MPa. ► The isothermal compression of adenosine is positive and increases as p increases. ► The isothermal compressions for cytidine and uridine change sign as p increases. -- Abstract: Sound speeds have been measured for aqueous solutions of the nucleosides adenosine, cytidine, and uridine at T = 298.15 K and at the pressures p = (10, 20, 40, 60, 80, 100, and 120) MPa. Using methods described in previous work, these sound speeds were used to derive the partial molar volumes at infinite dilution, V2o, the partial molar isentropic compressions at infinite dilution, KS,2o, and the partial molar isothermal compressions at infinite dilution, KT,2o{KT,2o=-(∂V2o/∂p)T}, for the nucleosides in aqueous solution at elevated pressures. The V2o and KT,2o results were rationalized in terms of the likely solute–water interactions. The V2o results were also critically compared with those calculated using the revised Helgeson–Kirkham–Flowers (HKF) equation of state

  7. Design and synthesis of novel 5-substituted acyclic pyrimidine nucleosides as potent and selective inhibitors of hepatitis B virus.

    Science.gov (United States)

    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

  8. Pharmacokinetics of the Experimental Non-Nucleosidic DNA Methyl Transferase Inhibitor N-Phthalyl-l-Tryptophan (RG 108) in Rats.

    Science.gov (United States)

    Schneeberger, Yvonne; Stenzig, Justus; Hübner, Florian; Schaefer, Andreas; Reichenspurner, Hermann; Eschenhagen, Thomas

    2016-05-01

    DNA methyl transferase (DNMT) inhibitors can re-establish the expression of tumour suppressor genes in malignant diseases, but might also be useful in other diseases. Inhibitors in clinical use are nucleosidic cytotoxic agents that need to be integrated into the DNA of dividing cells. Here, we assessed the in vivo kinetics of a non-nucleosidic inhibitor that is potentially free of cytotoxic effects and does not require cell division. The non-specific DNMT inhibitor N-phthalyl-l-tryptophan (RG 108) was injected subcutaneously in rats. Blood was drawn 0, 0.5, 1, 2, 4, 6, 8 and 24 hr after injection and RG 108 in plasma was measured by high-performance liquid chromatography coupled to mass spectrometry. Trough levels and area under the curve (AUC) were significantly higher with multiple-dose administration and cytochrome inhibition. In this group, time to maximal plasma concentration (tmax , mean ± S.D.) was 37.5 ± 15 min., terminal plasma half-life was approximately 3.7 h (60% CI: 2.1-15.6 h), maximal plasma concentration (Cmax ) was 61.3 ± 7.6 μM, and AUC was 200 ± 54 μmol·h/l. RG 108 peak levels were not influenced by cytochrome inhibition or multiple-dose administration regimens. Maximal tissue levels (Cmax in μmol/kg) were 6.9 ± 6.7, 1.6 ± 0.4 and 3.4 ± 1.1 in liver, skeletal and heart muscle, respectively. We conclude that despite its high lipophilicity, RG 108 can be used for in vivo experiments, appears safe and yields plasma and tissue levels in the range of the described 50% inhibitory concentration of around 1 to 5 μM. RG 108 can therefore be a useful tool for in vivo DNMT inhibition. PMID:26525153

  9. Arabidopsis SOI33/AtENT8 Gene Encodes a Putative Equilibrative Nucleoside Transporter That Is Involved in Cytokinin Transport In Planta

    Institute of Scientific and Technical Information of China (English)

    Jiaqiang SUN; Naoya HIROSE; Xingchun WANG; Pei WEN; Li XUE; Hitoshi SAKAKIBARA; Jianru ZUO

    2005-01-01

    The plant phytohormone cytokinin plays an important role in many facets of plant growth and development by regulating cell division and differentiation. Recent studies have shed significant light into the mechanisms of cytokinin metabolism and signaling. However, little is known about how the hormone is transported in planta, although it has been proposed that the hormone is presumably transported in nucleoside-conjugated forms. Here, we report the identification and characterization of cytokinin transport ers in Arabidopsis. We previously reported that a gain-of-function mutation in the PGA22/AtIPT8 gene caused overproduction of cytokinins in planta. In an effort to screen for suppressor of pga22/atipt8 (soi) mutants, we identified a mutant soi33-1. Molecular and genetic analyses indicated that SOI33 encodes a putative equilibrative nucleoside transporter (ENT), previously designated as AtENT8. Members of this small gene family are presumed to be involved in the transport of nucleosides in eukaryotic cells. Under conditions of nitrogen starvation, loss-of-function mutations in SOI33/AtENT8 or in a related gene AtENT3 cause a reduced sensitivity to the nucleoside-type cytokinins isopentenyladenine riboside (iPR) and trans zeatin riboside (tZR), but display a normal response to the free base-type cytokinins isopentenyladenine (iP) and trans-zeatin (tZ). Conversely, overexpression of SOI33/AtENT8 renders transgenic plants hyper sensitive to iPR but not to iP. An in planta measurement experiment indicated that uptake efficiency of 3H labeled iPR was reduced more than 40% in soi33 and atent3 mutants. However, a mutation inAtENT1 had no substantial effect on the cytokinin response and iPR uptake efficiency. Our results suggest that SOI33/ AtENT8 and AtENT3 are involved in the transport of nucleoside-type cytokinins in Arabidopsis.

  10. Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors

    Directory of Open Access Journals (Sweden)

    Nadya Urakova

    2016-04-01

    Full Text Available Rabbit haemorrhagic disease virus (RHDV is a calicivirus that causes acute infections in both domestic and wild European rabbits (Oryctolagus cuniculus. The virus causes significant economic losses in rabbit farming and reduces wild rabbit populations. The recent emergence of RHDV variants capable of overcoming immunity to other strains emphasises the need to develop universally effective antivirals to enable quick responses during outbreaks until new vaccines become available. The RNA-dependent RNA polymerase (RdRp is a primary target for the development of such antiviral drugs. In this study, we used cell-free in vitro assays to examine the biochemical characteristics of two rabbit calicivirus RdRps and the effects of several antivirals that were previously identified as human norovirus RdRp inhibitors. The non-nucleoside inhibitor NIC02 was identified as a potential scaffold for further drug development against rabbit caliciviruses. Our experiments revealed an unusually high temperature optimum (between 40 and 45 °C for RdRps derived from both a pathogenic and a non-pathogenic rabbit calicivirus, possibly demonstrating an adaptation to a host with a physiological body temperature of more than 38 °C. Interestingly, the in vitro polymerase activity of the non-pathogenic calicivirus RdRp was at least two times higher than that of the RdRp of the highly virulent RHDV.

  11. Standardized Comparison of the Relative Impacts of HIV-1 Reverse Transcriptase (RT) Mutations on Nucleoside RT Inhibitor Susceptibility

    Science.gov (United States)

    Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W. Jeffrey; Kaufman, David; Towner, William; Troia-Cancio, Paolo V.; Zolopa, Andrew; Robbins, Gregory K.; Kagan, Ron; Israelski, Dennis; Shafer, Robert W.

    2012-01-01

    Determining the phenotypic impacts of reverse transcriptase (RT) mutations on individual nucleoside RT inhibitors (NRTIs) has remained a statistical challenge because clinical NRTI-resistant HIV-1 isolates usually contain multiple mutations, often in complex patterns, complicating the task of determining the relative contribution of each mutation to HIV drug resistance. Furthermore, the NRTIs have highly variable dynamic susceptibility ranges, making it difficult to determine the relative effect of an RT mutation on susceptibility to different NRTIs. In this study, we analyzed 1,273 genotyped HIV-1 isolates for which phenotypic results were obtained using the PhenoSense assay (Monogram, South San Francisco, CA). We used a parsimonious feature selection algorithm, LASSO, to assess the possible contributions of 177 mutations that occurred in 10 or more isolates in our data set. We then used least-squares regression to quantify the impact of each LASSO-selected mutation on each NRTI. Our study provides a comprehensive view of the most common NRTI resistance mutations. Because our results were standardized, the study provides the first analysis that quantifies the relative phenotypic effects of NRTI resistance mutations on each of the NRTIs. In addition, the study contains new findings on the relative impacts of thymidine analog mutations (TAMs) on susceptibility to abacavir and tenofovir; the impacts of several known but incompletely characterized mutations, including E40F, V75T, Y115F, and K219R; and a tentative role in reduced NRTI susceptibility for K64H, a novel NRTI resistance mutation. PMID:22330916

  12. Morpholino, piperidino, and pyrrolidino derivatives of pyrimidine nucleosides as inhibitors of ribonuclease A: synthesis, biochemical, and crystallographic evaluation.

    Science.gov (United States)

    Samanta, Anirban; Leonidas, Demetres D; Dasgupta, Swagata; Pathak, Tanmaya; Zographos, Spyros E; Oikonomakos, Nikos G

    2009-02-26

    Six 5'-deoxy-5'-morpholine, piperidine, and pyrrolidine of pyrimidine nucleosides have been synthesized and characterized. Their inhibitory action to ribonuclease A has been studied by biochemical analysis and X-ray crystallography. These compounds are moderate inhibitors of RNase A with mid-to-upper micromolar inhibition constants (K(i)). The high resolution X-ray crystal structures of the RNase A-inhibitor complexes have shown that all inhibitors bind at the enzyme catalytic cleft with the pyrimidine nucleobase at the B(1)R(2) subsites while the 5' group binds away from the main subsite P(1), where P-O(5') bond cleavage occurs, toward the solvent close to subsite P(0). Structure-activity relationship analysis has demonstrated that the compounds with the larger group in the 5' position are more potent. Comparative structural analysis of these RNase A complexes with other similar RNase A-ligand complexes provides a structural explanation of their potency and suggests ways to improve their efficiency and selectivity. These inhibitors can be the starting point for the development of compounds that can be used as pharmaceuticals against pathologies associated with RNase A homologues such as human angiogenin, which is implicated in tumor induced neovascularization. PMID:19173562

  13. Antiviral therapy with nucleotide/nucleoside analogues in chronic hepatitis B: A meta-analysis of prospective randomized trials.

    Science.gov (United States)

    Bedre, Renesh H; Raj, Utkarsh; Misra, Sri Prakash; Varadwaj, Pritish Kumar

    2016-03-01

    Nucleotide/nucleoside analogues (antiviral therapy) are used in the therapy of HBeAg positive and HBeAg negative chronic hepatitis B. We analyzed ten selected randomized controlled with 2557 patients to estimate the effect of antiviral drugs in chronic hepatitis B with compared to placebo. Virological response, biochemical response, histological response, seroconversion of HBeAg, and loss of HBeAg were estimated as primary efficacy measures. The included studies were subjected for heterogeneity and publication bias. The heterogeneity was assessed with χ2 and I(2) statistics. Publication bias was assessed by funnel plot. Greater rates of improvement obtained in antiviral group for virological response [43.96 % vs. 3.15 %, RR = 0.57, 95 % CI = 0.54-0.61, p-value pharyngitis [22.22 % vs. 18.23 %, OR = 1.12, 95 % CI = 0.86-1.45, p-value = 0.40]. Excluding adverse events, all primary efficacy measures shown statistical significant result for chronic hepatitis treatment (p-value <0.05). Antiviral therapy provided significant benefit for the treatment of chronic hepatitis B with no measurable adverse effects. PMID:27083430

  14. Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening.

    Science.gov (United States)

    Qi, Yanfei; Li, Youxin; Bao, James J

    2016-08-01

    A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237-0.833 U/ml and 9.013-10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity. PMID:27173606

  15. Contents Changes of Triterpenic Acids, Nucleosides, Nucleobases, and Saccharides in Jujube (Ziziphus jujuba Fruit During the Drying and Steaming Process

    Directory of Open Access Journals (Sweden)

    Sheng Guo

    2015-12-01

    Full Text Available Chinese jujube (Ziziphus jujuba, a medicinal and edible plant, is widely consumed in Asian countries owing to the remarkable health activities of its fruits. To facilitate selection of the suitable processing method for jujube fruits, in this study their contents of triterpenic acids, nucleosides, nucleobases and saccharides after drying and steaming treatment were determined using ultra-high performance liquid chromatography and high performance liquid chromatography coupled with evaporative light scattering detector methods. The results showed that except for sucrose, the content levels of most analytes were increasing in the jujube fruits during drying treatment at 45 °C. The levels of cyclic nucleotides such as adenosine 3′,5′-cyclic monophosphate and guanosine 3′,5′-cyclic monophosphate, were significantly decreased after the fruits were steamed. Therefore, owing to the bioactivities of these components for human health, the dried fruits would be the better choice as medicinal material or functional food, and dried jujube fruit should not be further steamed.

  16. BCX4430 - A broad-spectrum antiviral adenosine nucleoside analog under development for the treatment of Ebola virus disease.

    Science.gov (United States)

    Taylor, Raymond; Kotian, Pravin; Warren, Travis; Panchal, Rekha; Bavari, Sina; Julander, Justin; Dobo, Sylvia; Rose, Angela; El-Kattan, Yahya; Taubenheim, Brian; Babu, Yarlagadda; Sheridan, William P

    2016-01-01

    The adenosine nucleoside analog BCX4430 is a direct-acting antiviral drug under investigation for the treatment of serious and life-threatening infections from highly pathogenic viruses, such as the Ebola virus. Cellular kinases phosphorylate BCX4430 to a triphosphate that mimics ATP; viral RNA polymerases incorporate the drug's monophosphate nucleotide into the growing RNA chain, causing premature chain termination. BCX4430 is active in vitro against many RNA viral pathogens, including the filoviruses and emerging infectious agents such as MERS-CoV and SARS-CoV. In vivo, BCX4430 is active after intramuscular, intraperitoneal, and oral administration in a variety of experimental infections. In nonclinical studies involving lethal infections with Ebola virus, Marburg virus, Rift Valley fever virus, and Yellow Fever virus, BCX4430 has demonstrated pronounced efficacy. In experiments conducted in several models, both a reduction in the viral load and an improvement in survival were found to be related to the dose of BCX4430. A Phase 1 clinical trial of intramuscular administration of BCX4430 in healthy subjects is currently ongoing. PMID:27095300

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

    Science.gov (United States)

    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

  18. Contents Changes of Triterpenic Acids, Nucleosides, Nucleobases, and Saccharides in Jujube (Ziziphus jujuba) Fruit During the Drying and Steaming Process.

    Science.gov (United States)

    Guo, Sheng; Duan, Jin-Ao; Zhang, Ying; Qian, Dawei; Tang, Yuping; Zhu, Zhenhua; Wang, Hanqing

    2015-01-01

    Chinese jujube (Ziziphus jujuba), a medicinal and edible plant, is widely consumed in Asian countries owing to the remarkable health activities of its fruits. To facilitate selection of the suitable processing method for jujube fruits, in this study their contents of triterpenic acids, nucleosides, nucleobases and saccharides after drying and steaming treatment were determined using ultra-high performance liquid chromatography and high performance liquid chromatography coupled with evaporative light scattering detector methods. The results showed that except for sucrose, the content levels of most analytes were increasing in the jujube fruits during drying treatment at 45 °C. The levels of cyclic nucleotides such as adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, were significantly decreased after the fruits were steamed. Therefore, owing to the bioactivities of these components for human health, the dried fruits would be the better choice as medicinal material or functional food, and dried jujube fruit should not be further steamed. PMID:26703531

  19. In vivo protective effect of Uridine, a pyrimidine nucleoside, on genotoxicity induced by Levodopa/Carbidopa in mice.

    Science.gov (United States)

    Orenlili Yaylagul, Esra; Cansev, Mehmet; Celikler Kasimogullari, Serap

    2015-08-01

    Parkinson's disease (PD) is a common neurodegenerative disorder that affects millions of people all over the world. Motor symptoms of PD are most commonly controlled by L-3,4-dihydroxyphenylalanine (Levodopa, L-DOPA), a precursor of dopamine, plus a peripherally-acting aromatic-L-amino-acid decarboxylase (dopa decarboxylase) inhibitor, such as carbidopa. However, chronic treatment with a combination of Levodopa plus carbidopa has been demonstrated to cause a major complication, namely abnormal involuntary movements. On the other hand, the effect of this treatment on bone marrow cells is unknown. Therefore, in this study, we aimed to investigate possible genotoxic effects of Levodopa and Carbidopa using male Balb/C mice. Our results showed that Levodopa alone or in combination with carbidopa caused genotoxicity in in vivo micronucleus test (mouse bone marrow) and Comet assay (blood cells). Furthermore, we showed that simultaneous administration of uridine, a pyrimidine nucleoside, reversed the genotoxic effect of Levodopa and Carbidopa in both assays. Our data show for the first time that Levodopa plus carbidopa combination causes genotoxicity which is reversed by uridine treatment. These findings might enhance our understanding for the complications of a common Parkinson's treatment and confer benefit in terms of reducing a possible genotoxic effect of this treatment. PMID:25976300

  20. Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors

    Science.gov (United States)

    Urakova, Nadya; Netzler, Natalie; Kelly, Andrew G.; Frese, Michael; White, Peter A.; Strive, Tanja

    2016-01-01

    Rabbit haemorrhagic disease virus (RHDV) is a calicivirus that causes acute infections in both domestic and wild European rabbits (Oryctolagus cuniculus). The virus causes significant economic losses in rabbit farming and reduces wild rabbit populations. The recent emergence of RHDV variants capable of overcoming immunity to other strains emphasises the need to develop universally effective antivirals to enable quick responses during outbreaks until new vaccines become available. The RNA-dependent RNA polymerase (RdRp) is a primary target for the development of such antiviral drugs. In this study, we used cell-free in vitro assays to examine the biochemical characteristics of two rabbit calicivirus RdRps and the effects of several antivirals that were previously identified as human norovirus RdRp inhibitors. The non-nucleoside inhibitor NIC02 was identified as a potential scaffold for further drug development against rabbit caliciviruses. Our experiments revealed an unusually high temperature optimum (between 40 and 45 °C) for RdRps derived from both a pathogenic and a non-pathogenic rabbit calicivirus, possibly demonstrating an adaptation to a host with a physiological body temperature of more than 38 °C. Interestingly, the in vitro polymerase activity of the non-pathogenic calicivirus RdRp was at least two times higher than that of the RdRp of the highly virulent RHDV. PMID:27089358

  1. Ligand similarity guided receptor selection enhances docking accuracy and recall for non-nucleoside HIV reverse transcriptase inhibitors.

    Science.gov (United States)

    Stanton, Richard A; Nettles, James H; Schinazi, Raymond F

    2015-11-01

    Non-nucleoside reverse transcriptase inhibitors (NNRTI) are allosteric inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), a viral polymerase essential to infection. Despite the availability of >150 NNRTI-bound RT crystal structures, rational design of new NNRTI remains challenging because of the variability of their induced fit, hydrophobic binding patterns. Docking NNRTI yields inconsistent results that vary markedly depending on the receptor structure used, as only 27% of the >20k cross-docking calculations we performed using known NNRTI were accurate. In order to determine if a hospitable receptor for docking could be selected a priori, we evaluated more than 40 chemical descriptors for their ability to pre-select a best receptor for NNRTI cross-docking. The receptor selection was based on similarity scores between the bound- and target-ligands generated by each descriptor. The top descriptors were able to double the probability of cross-docking accuracy over random receptor selection. Additionally, recall of known NNRTI from a large library of similar decoys was increased using the same approach. The results demonstrate the utility of pre-selecting receptors when docking into difficult targets. Graphical Abstract Cross-docking accuracy increases when using chemical descriptors to determine the NNRTI with maximum similarity to the new compound and then docking into its respective receptor. PMID:26450349

  2. Cloning, expression and preliminary crystallographic studies of the potential drug target purine nucleoside phosphorylase from Schistosoma mansoni.

    Science.gov (United States)

    Pereira, Humberto M; Cleasby, Anne; Pena S, Sérgio D J; Franco G, Glória R; Garratt, Richard C

    2003-06-01

    The parasite Schistosoma mansoni, unlike its mammalian hosts, lacks the de novo pathway for purine biosynthesis and depends on salvage pathways for its purine requirements. The gene encoding one enzyme of this pathway, purine nucleoside phosphorylase from S. mansoni (SmPNP) was identified, fully sequenced and cloned into the bacterial expression vector pMAL c2G to produce a protein in fusion with maltose-binding protein. The recombinant fusion protein was expressed at high levels and was purified in a single step by amylose resin affinity chromatography. After factor Xa cleavage, SmPNP was purified using a cation-exchange column and crystallized by hanging-drop vapour diffusion using polyethylene glycol 1500 as precipitant in the presence of 20% glycerol in acetate buffer. The use of the non-detergent sulfobetaine 195 (NDSB 195) as an additive had a marked effect on the size of the resulting crystals. Two data sets were obtained, one from a crystal grown in the absence of NDSB 195 and one from a crystal grown in its presence. The crystals are isomorphous and belong to the space group P2(1)2(1)2(1). It is intended to use the structures in the discovery and development of specific inhibitors of SmPNP. PMID:12777786

  3. Standardized comparison of the relative impacts of HIV-1 reverse transcriptase (RT) mutations on nucleoside RT inhibitor susceptibility.

    Science.gov (United States)

    Melikian, George L; Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W Jeffrey; Kaufman, David; Towner, William; Troia-Cancio, Paolo V; Zolopa, Andrew; Robbins, Gregory K; Kagan, Ron; Israelski, Dennis; Shafer, Robert W

    2012-05-01

    Determining the phenotypic impacts of reverse transcriptase (RT) mutations on individual nucleoside RT inhibitors (NRTIs) has remained a statistical challenge because clinical NRTI-resistant HIV-1 isolates usually contain multiple mutations, often in complex patterns, complicating the task of determining the relative contribution of each mutation to HIV drug resistance. Furthermore, the NRTIs have highly variable dynamic susceptibility ranges, making it difficult to determine the relative effect of an RT mutation on susceptibility to different NRTIs. In this study, we analyzed 1,273 genotyped HIV-1 isolates for which phenotypic results were obtained using the PhenoSense assay (Monogram, South San Francisco, CA). We used a parsimonious feature selection algorithm, LASSO, to assess the possible contributions of 177 mutations that occurred in 10 or more isolates in our data set. We then used least-squares regression to quantify the impact of each LASSO-selected mutation on each NRTI. Our study provides a comprehensive view of the most common NRTI resistance mutations. Because our results were standardized, the study provides the first analysis that quantifies the relative phenotypic effects of NRTI resistance mutations on each of the NRTIs. In addition, the study contains new findings on the relative impacts of thymidine analog mutations (TAMs) on susceptibility to abacavir and tenofovir; the impacts of several known but incompletely characterized mutations, including E40F, V75T, Y115F, and K219R; and a tentative role in reduced NRTI susceptibility for K64H, a novel NRTI resistance mutation. PMID:22330916

  4. Cross-validated stepwise regression for identification of novel non-nucleoside reverse transcriptase inhibitor resistance associated mutations

    Directory of Open Access Journals (Sweden)

    Van Houtte Margriet

    2011-10-01

    Full Text Available Abstract Background Linear regression models are used to quantitatively predict drug resistance, the phenotype, from the HIV-1 viral genotype. As new antiretroviral drugs become available, new resistance pathways emerge and the number of resistance associated mutations continues to increase. To accurately identify which drug options are left, the main goal of the modeling has been to maximize predictivity and not interpretability. However, we originally selected linear regression as the preferred method for its transparency as opposed to other techniques such as neural networks. Here, we apply a method to lower the complexity of these phenotype prediction models using a 3-fold cross-validated selection of mutations. Results Compared to standard stepwise regression we were able to reduce the number of mutations in the reverse transcriptase (RT inhibitor models as well as the number of interaction terms accounting for synergistic and antagonistic effects. This reduction in complexity was most significant for the non-nucleoside reverse transcriptase inhibitor (NNRTI models, while maintaining prediction accuracy and retaining virtually all known resistance associated mutations as first order terms in the models. Furthermore, for etravirine (ETR a better performance was seen on two years of unseen data. By analyzing the phenotype prediction models we identified a list of forty novel NNRTI mutations, putatively associated with resistance. The resistance association of novel variants at known NNRTI resistance positions: 100, 101, 181, 190, 221 and of mutations at positions not previously linked with NNRTI resistance: 102, 139, 219, 241, 376 and 382 was confirmed by phenotyping site-directed mutants. Conclusions We successfully identified and validated novel NNRTI resistance associated mutations by developing parsimonious resistance prediction models in which repeated cross-validation within the stepwise regression was applied. Our model selection

  5. The Lipid-Lowering Efficacy of Switching Within Non-Nucleoside Reverse Transcriptase Inhibitors in HIV-Infected Patients

    Directory of Open Access Journals (Sweden)

    A. M. Bain

    2008-01-01

    Full Text Available The objective of present research is to evaluate the lipid lowering efficacy and safety of switching within non-nucleoside reverse transcriptase inhibitors (NNRTI in HIV-infected patients. This is a multicenter, retrospective study utilizing a comprehensive electronic patient registry to identify all adult HIV-infected patients seen from October 1, 1998 through October 1, 2006, who substituted efavirenz for nevirapine (EFV→NVP or vice-versa (NVP→EFV, without change in other antiretrovirals. Lipid profiles before and after the switch were analyzed. A total of 124 patients were identified with 14 male (EFV→NVP, n = 9; NVP→EFV, n = 5 patients meeting the strict criteria for inclusion. An EFV→NVP switch resulted in significant reductions in TC -16% and non-HDL -25% (p≤0.02 and a trend towards a reduction in LDL-C -12%, TG -27%, TC/HDL -23%, TG/HDL -48% and an increase in HDL-C +15% without any changes to BMI, viral or immunological control. However, a NVP→EFV switch appeared to result in a non-significant worsening of LDL-C +29%, HDL-C -8%, TG +36%, non-HDL +28%, TC/HDL +57% and TG/HDL +46%. Lastly, more patients achieved their lipid goals when switched from EFV to NVP. These data suggest that switching from EFV to NVP-based HAART is associated with lipid improvement, however, switching from NVP to EFV-based HAART is associated with worsening of serum lipids.

  6. Human equilibrative nucleoside transporter 1 and carcinoma of the ampulla of Vater: expression differences in tumour histotypes

    Directory of Open Access Journals (Sweden)

    G. Perrone

    2010-09-01

    Full Text Available The human equilibrative nucleoside transporter 1 (hENT1 is the major means by which gemcitabine enters human cells; recent evidence exists that hENT1 is expressed in carcinoma of the ampulla of Vater and that it should be considered as a molecular prognostic marker for patients with resected ampullary cancer. Aim of the present study is to evaluate the variations of hENT1 expression in ampullary carcinomas and to correlate such variations with histological subtypes and clinicopathological parameters. Forty-one ampullary carcinomas were histologically classified into intestinal, pancreaticobiliary and unusual types. hENT1 and Ki67 expression were evaluated by immunohistochemistry, and apoptotic cells were identified by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labelling (TUNEL method. hENT1 overexpression was detected in 63.4% ampullary carcinomas. A significant difference in terms of hENT1 and Ki67 expression was found between intestinal vs. pancreaticobiliary types (P=0.03 and P=0.009 respectively. Moreover, a significant statistical positive correlation was found between apoptotic and proliferative Index (P=0.036, while no significant correlation was found between hENT1 and apoptosis. Our results on hENT1 expression suggest that classification of ampullary carcinoma by morphological subtypes may represent an additional tool in prospective clinical trials aimed at examining treatment efficacy; in addition, data obtained from Ki67 and TUNEL suggest a key role of hENT1 in tumour growth of ampullary carcinoma.

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

    Science.gov (United States)

    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

  8. Nucleoside-derived antagonists to A3 adenosine receptors lower mouse intraocular pressure and act across species.

    Science.gov (United States)

    Wang, Zhao; Do, Chi Wai; Avila, Marcel Y; Peterson-Yantorno, Kim; Stone, Richard A; Gao, Zhan-Guo; Joshi, Bhalchandra; Besada, Pedro; Jeong, Lak Shin; Jacobson, Kenneth A; Civan, Mortimer M

    2010-01-01

    The purpose of the study was to determine whether novel, selective antagonists of human A3 adenosine receptors (ARs) derived from the A3-selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the Servo-Null Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells. Five agonist-based A3AR antagonists lowered mouse IOP measured with SNMS tonometry by 3-5 mm Hg within minutes of topical application. Of the five agonist derivatives, LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other four compounds, consonant with slower, smaller responses previously measured non-invasively following topical application of A3AR agonists and the dihydropyridine A3AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 min. Like MRS 1191, agonist-based A3AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A3ARs derived from the potent, selective A3 agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, five derivatives of the A3AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivatives of the highly-selective A3AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivatives are promising A3 antagonists for study in multiple animal models. PMID:19878673

  9. Oligonucleotides with cyclohexene-nucleoside building blocks: crystallization and preliminary X-ray studies of a left-handed sequence GTGTACAC

    International Nuclear Information System (INIS)

    A novel oligonucleotide containing cyclohexene-nucleoside building blocks has been crystallized. Crystals belong to space group R3 and diffract to 1.7 Å. Cyclohexene nucleic acids contain a cyclohexene ring instead of the normal β-d-2′-deoxyribose. The cyclohexene oligonucleotide GTGTACAC was synthesized using phosphoramidite chemistry and standard protecting groups. Crystals of GTGTACAC were obtained at 289 K by the hanging-drop vapour-diffusion technique. The crystals diffract to 1.7 Å resolution and belong to the trigonal space group R3, with unit-cell parameters a = 41.434, c = 66.735 Å

  10. Cytotoxicity and cellular uptake of pyrimidine nucleosides for imaging herpes simplex type-1 thymidine kinase (HSV-1 TK) expression in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Morin, Kevin W.; Duan Weili; Xu Lihua; Zhou Aihua; Moharram, Sameh; Knaus, Edward E.; McEwan, Alexander J.B.; Wiebe, Leonard I. E-mail: leonard.wiebe@ualberta.ca

    2004-07-01

    In vivo transfer of the herpes simplex virus type-1 thymidine kinase (HSV-1 TK) gene, with subsequent administration of antiviral drugs such as ganciclovir, has emerged as a promising gene therapy protocol for treating proliferative disorders. The in vitro cytotoxicities (IC{sub 50}) for two series of 5-iodo- and (E)-5-(2-iodovinyl)-substituted 2'-deoxy- and 2'-deoxy-2'-fluoro-pyrimidine nucleosides ranged from millimolar to low nanomolar concentrations in mammalian tumor cell lines (KBALB; R-970-5; 143B; EMT-6) and their counterparts engineered to express HSV-1 TK (KBALB-STK; 143B-LTK). Their HSV-1 TK selectivity indices ranged from one (nonselective) to one million (highly selective) based on cytotoxicity, with FIRU being the least toxic to all cell lines, and FIAU being most toxic. HSV-1 TK selectivity, based on uptake, ranged from 10 to 140, with IVDU being most selective for HSV-1 TK expressing cells, followed by IVFRU, FIRU, FIAU, IVFAU and finally IUDR. Phosphorylation of [{sup 125}I]FIAU led to incorporation of the radiolabel into nucleic acids, whereas IVFRU and FIRU radioactivity was trapped primarily in the nucleotide pool. These data indicate that cytotoxicity does not depend on initial metabolic trapping (e.g., phosphorylation), but on elaboration of the mononucleotides to more cytotoxic anabolites. Lipophilicities and nucleoside transport rates of the six nucleosides tested were within narrow ranges. This supports the premise that cellular biochemistry, and not cellular bioavailability, is responsible for the observed broad range of cytotoxicity and trapping. In vivo biodistribution studies with 5-[{sup 125}I]iodo-2'-fluoro-2'-deoxyribouridine (FIRU), 5-[{sup 125}I]iodo-2'-fluoro-2'-deoxyarabinouridine (FIAU) and (E)-5-(2-[{sup 125}I]iodovinyl)-2'-fluoro-2'-deoxyuridine (IVFRU) demonstrate selective accumulation of all three radiotracers in HSV-1 TK-expressing KBABK-STK tumors, compared to their very low

  11. Engineering of halophilic enzymes: Two acidic amino acid residues at the carboxy-terminal region confer halophilic characteristics to Halomonas and Pseudomonas nucleoside diphosphate kinases

    OpenAIRE

    Tokunaga, Hiroko; Arakawa, Tsutomu; Tokunaga, Masao

    2008-01-01

    Nucleoside diphosphate kinase from Halomonas sp. 593 (HaNDK) exhibits halophilic characteristics. Residues 134 and 135 in the carboxy-terminal region of HaNDK are Glu–Glu, while those of its homologous counterpart of non-halophilic Pseudomonas NDK (PaNDK) are Ala–Ala. The double mutation, E134A-E135A, in HaNDK results in the loss of the halophilic characteristics, and, conversely, the double mutation of A134E-A135E in PaNDK confers halophilic characters to this enzyme, indicating that the cha...

  12. NZ51, a ring-expanded nucleoside analog, inhibits motility and viability of breast cancer cells by targeting the RNA helicase DDX3

    OpenAIRE

    Xie, Min; Vesuna, Farhad; Botlagunta, Mahendran; Bol, Guus Martinus; Irving, Ashley; Bergman, Yehudit; Hosmane, Ramachandra S.; Kato, Yoshinori; Winnard, Paul T.; Raman, Venu

    2015-01-01

    DDX3X (DDX3), a human RNA helicase, is over expressed in multiple breast cancer cell lines and its expression levels are directly correlated to cellular aggressiveness. NZ51, a ring-expanded nucleoside analogue (REN) has been reported to inhibit the ATP dependent helicase activity of DDX3. Molecular modeling of NZ51 binding to DDX3 indicated that the 5:7-fused imidazodiazepine ring of NZ51 was incorporated into the ATP binding pocket of DDX3. In this study, we investigated the anticancer prop...

  13. Positive interactive radiosensitization observed in vitro with the combination of two nucleoside analogs (E)-2'-deoxy-2'-(fluoromethylene) cytidine (FMdC) and iododeoxyuridine (IdUrd).

    OpenAIRE

    Coucke, Philippe; Cottin, E.; Azria, D; Martineau, P.; Adamer, F; Decosterd, L-A; Buchegger, F; Do, H-P

    2004-01-01

    (E)-20-Deoxy-20-(fluoromethylene) cytidine (FMdC), an inhibitor of ribonucleotide diphosphate reductase (RR), is a potent radiation-sensitiser acting through alterations in the deoxyribonucleoside triphosphate (dNTP) pool in the de novo pathway to DNA synthesis. The activity of thymidine kinase (TK), a key enzyme in the ‘salvage pathway’, is known to increase in response to a lowering of dATP induced by FMdC. Nucleoside analogues such as iododeoxyuridine (IdUrd) are incorporated into DNA a...

  14. C-C Bond Formation: Synthesis of C5 Substituted Pyrimidine and C8 Substituted Purine Nucleosides Using Water Soluble Pd-imidate Complex.

    Science.gov (United States)

    Gayakhe, Vijay; Ardhapure, Ajaykumar V; Kapdi, Anant R; Sanghvi, Yogesh S; Serrano, Jose Luis; Schulzke, Carola

    2016-01-01

    The synthesis of a highly efficient, water soluble [Pd(Sacc)2 (TPA)2 ] complex for C-C bond formation is described. Additionally, application of the [Pd(Sacc)2 (TPA)2 ] complex for Suzuki-Miyaura arylation of all four nucleosides (5-iodo-2'-deoxyuridine [5-IdU], 5-iodo-2'-deoxycytidine [5-IdC], 8-bromo-2'-deoxyadenosine, and 8-bromo-2'-deoxyguanosine) with various aryl/heteroaryl boronic acids in plain water under milder conditions is demonstrated. © 2016 by John Wiley & Sons, Inc. PMID:27248782

  15. Synthesis of 5′-Functionalized Nucleosides: S-Adenosylhomocysteine Analogues with the Carbon-5′ and Sulfur Atoms Replaced by a Vinyl or Halovinyl Unit

    OpenAIRE

    Wnuk, Stanislaw F.; Sacasa, Pablo R.; Lewandowska, Elzbieta; Andrei, Daniela; Cai, Sumin; Borchardt, Ronald T.

    2008-01-01

    Adenosine and uridine analogues functionalized with alkenyl or fluoroalkenyl chain at C5′ were prepared employing cross-metathesis, Negishi couplings and Wittig reactions. Metathesis of the protected 5′-deoxy-5′-methyleneadenosine or uridine analogues with six-carbon amino acids (homoallylglycines) in the presence of Grubbs catalysts gave nucleoside analogues with the C5′-C6′ double bond. Alternatively, the Pd-catalyzed cross-coupling between the protected 5′-deoxy-5′-(iodomethylene) nucleosi...

  16. Selectivity and affinity of DNA triplex forming oligonucleotides containing the nucleoside analogues 2'-O-methyl-5-(3-amino-1-propynyl)uridine and 2'-O-methyl-5-propynyluridine.

    Science.gov (United States)

    Li, Hong; Miller, Paul S; Seidman, Michael M

    2008-11-21

    Triplex forming oligonucleotides (TFOs) containing the nucleoside analogues 2'-O-methyl-5-propynyluridine (1) and 2'-O-methyl-5-(3-amino-1-propynyl)uridine (2) were synthesized. The affinity and selectivity of triplex formation by these TFOs were studied by gel shift analysis, T(m) value measurement, and association rate assays. The results show that the introduction of 1 and 2 into TFOs can improve the stability of the triplexes under physiological conditions. Optimized distribution of 1 or 2 in the TFOs combined with a cluster of contiguous nucleosides with 2'-aminoethoxy sugars resulted in formation of triplexes with further enhanced stability and improved selectivity. PMID:18972052

  17. A new sensitive 32P-postlabeling assay based on the specific enzymatic conversion of bulky DNA lesions to radiolabeled dinucleotides and nucleoside 5'-monophosphates

    International Nuclear Information System (INIS)

    A new sensitive 32P-postlabelling assay for DNA adducts has been developed. When DNA containing bulky adducts, X1, X2, .....Xn, is digested with nuclease P1 at pH 5, normal nucleotides are released as 5'-monophosphates, pN, while adducts are excised as 5'-phosphorylated dinucleotides, pXipN, because inter-nucleotide linkages on the 3' side of X resist attack by nuclease P1. Addition of prostatic acid phosphatase to such a digest results in 5'-dephosphorylation of the nucleotides to normal nucleosides, N, and adducted dinucleotides, XipN, carrying a 5'-terminal free hydroxyl group. The dinucleotides but not nucleosides are converted to 5'-32P-labeled dinucleotides,[32P]pXipN, by T4 polynucleotide kinase-catalyzed [32P]posphate transfer from [γ-32P]ATP. Upon mapping on polyethyleneimine-cellulose anion-exchange TLC, the labeled dinucleotide adducts produce characteristic autoradiographic fingerprints. Alternatively, they are further digested with snake venom phosphodiesterase to yield 5'-monophosphates, [32P]pXi and pN. TLC profiles of the monophosphate adducts are distinct from those of the dinucleotides. These reactions provide the basis of the new 32P-postlabeling scheme, which is compared in this paper with a previously reported protocol yielding adducts in the form of 5'-32P-labeled 3',5'-bisphosphates, [32P]pXip. (author)

  18. Systematic evaluation of methyl ester bioisosteres in the context of developing alkenyldiarylmethanes (ADAMs) as non-nucleoside reverse transcriptase inhibitors (NNRTIs) for anti-HIV-1 chemotherapy.

    Science.gov (United States)

    Hoshi, Ayako; Sakamoto, Takeshi; Takayama, Jun; Xuan, Meiyan; Okazaki, Mari; Hartman, Tracy L; Buckheit, Robert W; Pannecouque, Christophe; Cushman, Mark

    2016-07-01

    The alkenyldiarylmethanes (ADAMs) are a class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) targeting HIV-1. Four chemically and metabolically stabilized ADAMs incorporating N-methoxyimidoyl halide replacements of the methyl esters of the lead compound were previously reported. In this study, twenty-five new ADAMs were synthesized in order to investigate the biological consequences of installing nine different methyl ester bioisosteres at three different locations. Attempts to define a universal rank order of methyl ester bioisosteres and discover the 'best' one in terms of inhibitory activity versus HIV-1 reverse transcriptase (RT) led to the realization that the potencies are critically dependent on the surrounding structure at each location, and therefore the definition of universal rank order is impossible. This investigation produced several new non-nucleoside reverse transcriptase inhibitors in which all three of the three methyl esters of the lead compound were replaced by methyl ester bioisosteres, resulting in compounds that are more potent as HIV-1 RT inhibitors and antiviral agents than the lead compound itself and are expected to also be more metabolically stable than the lead compound. PMID:27234889

  19. Simultaneous Quantitation of Free Amino Acids, Nucleosides and Nucleobases in Sipunculus nudus by Ultra-High Performance Liquid Chromatography with Triple Quadrupole Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Yahui Ge

    2016-03-01

    Full Text Available To evaluate the nutritional and functional value of Sipunculus nudus, a rapid, simple and sensitive analytical method was developed using ultra-high performance liquid chromatography coupled with a triple quadrupole mass detection in multiple-reaction monitoring mode for the simultaneous quantitative determination of 25 free amino acids and 16 nucleosides and nucleobases in S. nudus within 20 min, which was confirmed to be reproducible and accurate. The limits of detection (LODs and quantification (LOQs were between 0.003–0.229 μg/mL and 0.008–0.763 μg/mL for the 41 analytes, respectively. The established method was applied to analyze 19 batches of S. nudus samples from four habitats with two different processing methods. The results showed that S. nudus contained a variety of free amino acids, nucleosides and nucleobases in sufficient quantity and reasonable proportion. They also demonstrated that the contents of these compounds in different parts of S. nudus were significantly discriminating, which were in the order: (highest coelomic fluid > body wall > intestine (lowest. The method is simple and accurate, and could serve as a technical support for establishing quality control of S. nudus and other functional seafoods. Moreover, the research results also laid foundation for further exploitation and development of S. nudus.

  20. Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3'-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease.

    Science.gov (United States)

    Yamamoto, Junpei; Takahata, Chiaki; Kuraoka, Isao; Hirota, Kouji; Iwai, Shigenori

    2016-01-01

    Nucleoside/nucleotide analogs that lack the 3'-hydroxy group are widely utilized for HIV therapy. These chain-terminating nucleoside analogs (CTNAs) block DNA synthesis after their incorporation into growing DNA, leading to the antiviral effects. However, they are also considered to be DNA damaging agents, and tyrosyl-DNA phosphodiesterase 1, a DNA repair enzyme, is reportedly able to remove such CTNA-modifications of DNA. Here, we have synthesized phosphoramidite building blocks of representative CTNAs, such as acyclovir, abacavir, carbovir, and lamivudine, and oligonucleotides with the 3'-CTNAs were successfully synthesized on solid supports. Using the chemically synthesized oligonucleotides, we investigated the excision of the 3'-CTNAs in DNA by the human excision repair cross complementing protein 1-xeroderma pigmentosum group F (ERCC1-XPF) endonuclease, which is one of the main components of the nucleotide excision repair pathway. A biochemical analysis demonstrated that the ERCC1-XPF endonuclease cleaved 2-7 nt upstream from the 3'-blocking CTNAs, and that DNA synthesis by the Klenow fragment was resumed after the removal of the CTNAs, suggesting that ERCC1-XPF participates in the repair of the CTNA-induced DNA damage. PMID:27294910

  1. Levels of human equilibrative nucleoside transporter-1 are higher in proliferating regions of A549 tumor cells grown as tumor xenografts in vivo

    International Nuclear Information System (INIS)

    3’-Fluoro-3’-deoxythymidine (FLT) has been proposed for positron emission tomography (PET)-based identification of tumor chemosensitivity that is mediated by the human equilibrative nucleoside transporter-1 (ENT1). ENT1 facilitates transport of FLT into cells and elevated levels of FLT are associated with both larger FLT-PET signals and increased response to nucleoside-based chemotherapies. FLT-PET is also used as a measure of tumor proliferation. The present study examined the extent to which ENT1 levels vary in a proliferation-dependent manner in tumor cells in vivo. Methods: The human adenocarcinoma cell line A549 was used to establish tumor xenografts in nude mice. FLT uptake was measured in vivo using PET, and further examined ex vivo using autoradiography. FLT uptake patterns were compared to immunohistochemical (IHC) analysis of ENT1 and the proliferation markers Ki67 and BrdU. Results: Regional differences in FLT uptake matched differences in IHC proliferation markers. All cells stained for ENT1, but the staining intensity was twice as high for Ki67+ cells than for Ki67− cells. Conclusions: Under in vivo conditions, proliferating regions of tumors show increased FLT uptake and higher ENT1 levels than nonproliferating tumor regions.

  2. Efavirenz Has the Highest Anti-Proliferative Effect of Non-Nucleoside Reverse Transcriptase Inhibitors against Pancreatic Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Markus Hecht

    Full Text Available Cancer prevention and therapy in HIV-1-infected patients will play an important role in future. The non-nucleoside reverse transcriptase inhibitors (NNRTI Efavirenz and Nevirapine are cytotoxic against cancer cells in vitro. As other NNRTIs have not been studied so far, all clinically used NNRTIs were tested and the in vitro toxic concentrations were compared to drug levels in patients to predict possible anti-cancer effects in vivo.Cytotoxicity was studied by Annexin-V-APC/7AAD staining and flow cytometry in the pancreatic cancer cell lines BxPC-3 and Panc-1 and confirmed by colony formation assays. The 50% effective cytotoxic concentrations (EC50 were calculated and compared to the blood levels in our patients and published data.The in vitro EC50 of the different drugs in the BxPC-3 pancreatic cancer cells were: Efavirenz 31.5 μmol/l (= 9944 ng/ml, Nevirapine 239 μmol/l (= 63,786 ng/ml, Etravirine 89.0 μmol/l (= 38,740 ng/ml, Lersivirine 543 μmol/l (= 168,523 ng/ml, Delavirdine 171 μmol/l (= 78,072 ng/ml, Rilpivirine 24.4 μmol/l (= 8941 ng/ml. As Efavirenz and Rilpivirine had the highest cytotoxic potential and Nevirapine is frequently used in HIV-1 positive patients, the results of these three drugs were further studied in Panc-1 pancreatic cancer cells and confirmed with colony formation assays. 205 patient blood levels of Efavirenz, 127 of Rilpivirine and 31 of Nevirapine were analyzed. The mean blood level of Efavirenz was 3587 ng/ml (range 162-15,363 ng/ml, of Rilpivirine 144 ng/ml (range 0-572 ng/ml and of Nevirapine 4955 ng/ml (range 1856-8697 ng/ml. Blood levels from our patients and from published data had comparable Efavirenz levels to the in vitro toxic EC50 in about 1 to 5% of all patients.All studied NNRTIs were toxic against cancer cells. A low percentage of patients taking Efavirenz reached in vitro cytotoxic blood levels. It can be speculated that in HIV-1 positive patients having high Efavirenz blood levels pancreatic

  3. Influence of non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine on the pharmacodynamic activity of gliclazide in animal models

    Directory of Open Access Journals (Sweden)

    Mastan SK

    2009-10-01

    Full Text Available Abstract Background Type 2 diabetes may occur as a result of HIV infection and/or its treatment. Gliclazide is a widely used drug for the treatment of type 2 diabetes. Efavirenz and nevirapine are widely used non-nucleoside reverse transcriptase inhibitors for the treatment of HIV infection. The role of Efavirenz and nevirapine on the pharmacodynamic activity of gliclazide is not currently known. The objective of this study was to examine the effect of oral administration of efavirenz and nevirapine on blood glucose and investigate their effect on the activity of gliclazide in rats (normal and diabetic and rabbits to evaluate the safety and effectiveness of the combination. Methods Studies in normal and alloxan induced diabetic rats were conducted with oral doses of 2 mg/kg bd. wt. of gliclazide, 54 mg/kg bd. wt. of efavirenz or 18 mg/kg bd. wt. of nevirapine and their combination with adequate washout periods in between treatments. Studies in normal rabbits were conducted with 5.6 mg/1.5 kg bd. wt. of gliclazide, 42 mg/1.5 kg bd. wt. of efavirenz or 14 mg/1.5 kg bd. wt. of nevirapine and their combination given orally. Blood samples were collected at regular time intervals in rats from retro orbital puncture and by marginal ear vein puncture in rabbits. All the blood samples were analysed for blood glucose by GOD/POD method. Results Efavirenz and nevirapine alone have no significant effect on the blood glucose level in rats and rabbits. Gliclazide produced hypoglycaemic/antidiabetic activity in normal and diabetic rats with peak activity at 2 h and 8 h and hypoglycaemic activity in normal rabbits at 3 h. In combination, efavirenz reduced the effect of gliclazide in rats and rabbits, and the reduction was more significant with the single dose administration of efavirenz than multiple dose administration. In combination, nevirapine has no effect on the activity of gliclazide in rats and rabbits. Conclusion Thus, it can be concluded that the

  4. Sensitive assessment of the virologic outcomes of stopping and restarting non-nucleoside reverse transcriptase inhibitor-based antiretroviral therapy.

    Directory of Open Access Journals (Sweden)

    Anna Maria Geretti

    Full Text Available BACKGROUND: Non-nucleoside reverse transcriptase inhibitor (NNRTI-resistant mutants have been shown to emerge after interruption of suppressive NNRTI-based antiretroviral therapy (ART using routine testing. The aim of this study was to quantify the risk of resistance by sensitive testing and correlate the detection of resistance with NNRTI concentrations after treatment interruption and virologic responses after treatment resumption. METHODS: Resistance-associated mutations (RAMs and NNRTI concentrations were studied in plasma from 132 patients who interrupted suppressive ART within SMART. RAMs were detected by Sanger sequencing, allele-specific PCR, and ultra-deep sequencing. NNRTI concentrations were measured by sensitive high-performance liquid chromatography. RESULTS: Four weeks after NNRTI interruption, 19/31 (61.3% and 34/39 (87.2% patients showed measurable nevirapine (>0.25 ng/ml or efavirenz (>5 ng/ml concentrations, respectively. Median eight weeks after interruption, 22/131 (16.8% patients showed ≥1 NNRTI-RAM, including eight patients with NNRTI-RAMs detected only by sensitive testing. The adjusted odds ratio (OR of NNRTI-RAM detection was 7.62 (95% confidence interval [CI] 1.52, 38.30; p = 0.01 with nevirapine or efavirenz concentrations above vs. below the median measured in the study population. Staggered interruption, whereby nucleos(tide reverse transcriptase inhibitors (NRTIs were continued for median nine days after NNRTI interruption, did not prevent NNRTI-RAMs, but increased detection of NRTI-RAMs (OR 4.25; 95% CI 1.02, 17.77; p = 0.03. After restarting NNRTI-based ART (n = 90, virologic suppression rates <400 copies/ml were 8/13 (61.5% with NNRTI-RAMs, 7/11 (63.6% with NRTI-RAMs only, and 51/59 (86.4% without RAMs. The ORs of re-suppression were 0.18 (95% CI 0.03, 0.89 and 0.17 (95% CI 0.03, 1.15 for patients with NNRTI-RAMs or NRTI-RAMs only respectively vs. those without RAMs (p = 0.04. CONCLUSIONS

  5. hTERT-targeted E. coli purine nucleoside phosphorylase gene/6-methylpurine deoxyribose therapy for pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    ZHOU Jia-hua; TANG Bo; LIU Xun-liang; HE Dao-wei; YANG De-tong

    2007-01-01

    Background Pancreatic cancer is one of the most common tumors and has a 5-year survival for all stages of less than 5%. Most patients with pancreatic cancer are diagnosed at an advanced stage and therefore are not candidates for surgical resection. In recent years, investigation into alternative treatment strategies for this aggressive disease has led to advances in the field of gene therapy for pancreatic cancer. E. coli purine nucleoside phosphorylase/6-methylpurine deoxyribose (ePNP/MePdR) is a suicide gene/prodrug system where PNP enzyme cleaves nontoxic MePdR into cytotoxic membrane-permeable compounds 6-methylpurine (MeP) with high bystander activity, hTERT is expressed in cell lines and tissues for telomerase activity. In this study we examined the efficacy of ePNP under the control of hTERT promoter sequences and assessed the selective killing effects of the ePNP/prodrug MePdR system on pancreatic tumors.Methods Recombinant pET-PNP was established. The protein of E. coli PNPase was expressed and an antibody to E.coli PNPase was prepared. Transcriptional activities of hTERT promoter sequences were analyzed using a luciferase reporter gene. A recombinant phTERT-ePNP vector was constructed. The ePNP/MePdR system affects SW1990 human pancreatic cancer cell lines in vitro.Results The hTERT promoter had high transcriptional activity and conferred specificity on cancer cell lines. The antibody to E. coli PNPase was demonstrated to be specific for the ePNP protein. The MePdR treatment induced a high in vitro cytotoxicity on the sole hTERT-ePNP-producing cell lines and affected SW1990 cells in a dose-dependent manner.Conclusions The hTERT promoter control of the ePNP/MePdR system can provide a beneficial anti-tumor treatment in pancreatic cancer cell lines including a good bystander killing effect.

  6. Protective effect of taurine on hypochlorous acid toxicity to nuclear nucleoside triphosphatase in isolated nuclei from rat liver

    Institute of Scientific and Technical Information of China (English)

    Ju-Xiang Li; Yong-Zheng Pang; Chao-Shu Tang; Zai-Quan Li

    2004-01-01

    AIM: Taurine has been shown to be an effective scavenger of hypochlorous acid (HOCI). The role of HOCI is well established in tissue damage associated with inflammation and injury. In the present study, the effect of HOCI on nuclear nucleoside triphosphatase of hepatocytes and the ability of taurine to prevent this effect were investigated.METHODS: Isolated hepatic nuclei from rat liver were exposed to HOCI with or without taurine. The NTPase activity on nuclear envelope was assayed using ATP and GTP as substrates, respectively.RESULTS: The first series of experiments evaluated the toxicity of HOCl and the efficacy of taurine to protect NTPase.HOCI at 10-9-5×10-6 mol/L reduced nuclear NTPase activities in a concentration dependent manner (ATP and GTP as substrates) (P<0.01). HOCI at 10-6 mol/L reduced the NTPase activity by 65% (ATP as substrate) and 76% (GTP as substrate). Taurine (10-7 to 10-4 mol/L) was tested for protection against HOCI at 10-6 mol/L and the nuclei treated with 5x10-4 mol/L taurine exhibited only 20% and 12% reduction in NTPase activities compared to untreated controls. A second study was performed comparing taurine to glutathione (GSH). GSH and HOCI at 10-6 mol/L exhibited 46% and 67.4% reduction in NTPase activities compared with control. GSH (10-4 mol/L) which was incubated with the nuclei and HOCi still exhibited 44.2% and 44.8% reduction in NTPase activities of untreated control. Taurine with HOCI only exhibited 15.2% and 17.1% reduction in NTPase activities, which provided more powerful protection against HOCI than GSH. The third experiment was undertaken to evaluate the specificity of taurine against HOCI. Incubation of rat hepatic nuclei with Fe3+/H2O2 (1 m mol/L vS 5μ mol/L) resulted in a decrease in nuclear NTPase activities (P<0.01).When hepatic nuclei were incubated with Tau (10-4 mol/L) and Fe3+/H2O2 (1m mol/L vS 5μ mol/L), nuclear NTPase activities were only slightly increased as compared with that of incubation with Fe3+/H

  7. The nucleoside diphosphate kinase gene Nme3 acts as quantitative trait locus promoting non-Mendelian inheritance.

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    Hermann Bauer

    Full Text Available The t-haplotype, a variant form of the t-complex region on mouse chromosome 17, acts as selfish genetic element and is transmitted at high frequencies (> 95% from heterozygous (t/+ males to their offspring. This phenotype is termed transmission ratio distortion (TRD and is caused by the interaction of the t-complex responder (Tcr with several quantitative trait loci (QTL, the t-complex distorters (Tcd1 to Tcd4, all located within the t-haplotype region. Current data suggest that the distorters collectively impair motility of all sperm derived from t/+ males; t-sperm is rescued by the responder, whereas (+-sperm remains partially dysfunctional. Recently we have identified two distorters as regulators of RHO small G proteins. Here we show that the nucleoside diphosphate kinase gene Nme3 acts as a QTL on TRD. Reduction of the Nme3 dosage by gene targeting of the wild-type allele enhanced the transmission rate of the t-haplotype and phenocopied distorter function. Genetic and biochemical analysis showed that the t-allele of Nme3 harbors a mutation (P89S that compromises enzymatic activity of the protein and genetically acts as a hypomorph. Transgenic overexpression of the Nme3 t-allele reduced t-haplotype transmission, proving it to be a distorter. We propose that the NME3 protein interacts with RHO signaling cascades to impair sperm motility through hyperactivation of SMOK, the wild-type form of the responder. This deleterious effect of the distorters is counter-balanced by the responder, SMOK(Tcr, a dominant-negative protein kinase exclusively expressed in t-sperm, thus permitting selfish behaviour and preferential transmission of the t-haplotype. In addition, the previously reported association of NME family members with RHO signaling in somatic cell motility and metastasis, in conjunction with our data involving RHO signaling in sperm motility, suggests a functional conservation between mechanisms for motility control in somatic cells and

  8. Evaluation of Novel Acyclic Nucleoside Phosphonates against Human and Animal Gammaherpesviruses Revealed an Altered Metabolism of Cyclic Prodrugs upon Epstein-Barr Virus Reactivation in P3HR-1 Cells

    Czech Academy of Sciences Publication Activity Database

    Coen, N.; Duraffour, S.; Naesens, L.; Krečmerová, Marcela; Van Den Oord, J.; Snoeck, R.; Andrei, G.

    2013-01-01

    Roč. 87, č. 22 (2013), s. 12422-12432. ISSN 0022-538X R&D Projects: GA MPO FR-TI4/625 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonate * gammaherpesvirus * Epstein-Barr virus * Kaposi's sarcoma * HPMP-5-azaC * cidofovir Subject RIV: EE - Microbiology, Virology Impact factor: 4.648, year: 2013

  9. Acyclic nucleoside phosphonates as a new class of anti-malarial compounds: Inhibition of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase by 9-[2-(2-phosphonoethoxy)ethyl]-purines and their branched derivatives

    Czech Academy of Sciences Publication Activity Database

    Hocková, Dana; Holý, Antonín; Keough, D. T.; Naesens, L.; Skinner-Adams, T. S.; de Jersey, J.; Guddat, L. W.

    Praha : -, 2009. -. ISBN 978-80-02-02160-5. [ESOC 2009. European Symposium on Organic Chemistry /16./. 12.07.2009-16.07.2009, Praha] Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * anti-malarial compounds Subject RIV: CC - Organic Chemistry

  10. Use of nucleoside reverse transcriptase inhibitors and risk of myocardial infarction in HIV-infected patients enrolled in the D:A:D study : a multi-cohort collaboration

    NARCIS (Netherlands)

    Sabin, Caroline A; Worm, Signe W; Weber, Rainer; Reiss, Peter; El-Sadr, Wafaa; Dabis, Francois; De Wit, Stephane; Law, Matthew; D'Arminio Monforte, Antonella; Friis-Møller, Nina; Kirk, Ole; Pradier, Christian; Weller, Ian; Phillips, Andrew N; Lundgren, Jens D; Schölvinck, Elisabeth H.

    2008-01-01

    BACKGROUND: Whether nucleoside reverse transcriptase inhibitors increase the risk of myocardial infarction in HIV-infected individuals is unclear. Our aim was to explore whether exposure to such drugs was associated with an excess risk of myocardial infarction in a large, prospective observational c

  11. Assessment of expression of selected Bcl-2 family proteins in lymphoid infiltration in patients with B-cell chronic lymphocytic leukaemia treated with nucleoside analogues.

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    Janusz Kłoczko

    2008-12-01

    Full Text Available B-cell chronic lymphocytic leukaemia (B-CLL is characterized by clonal growth and accumulation of mature lymphoid cells due to disturbance in genetically regulated form of cell death called apoptosis. The intrinsic mechanism of apoptosis is controlled by Bcl-2 family proteins. Purine nucleoside analogues induce the apoptosis in cells in a state of quiescence. The aim of the study was to assess expression of selected Bcl-2 family proteins in neoplastic infiltration in bone marrow in patients with B-CLL treated with nucleoside analogues. The study comprised examination of bone marrow obtained routinely by trephine biopsy from 18 patients with B-CLL diagnosed before administration of purine nucleoside analogues treatment and after its completion. Expression of Bcl-2, Bcl-x and Bax proteins was examined. Lymphoid cells in bone marrow were present in all patients before administration of treatment. After treatment in two patients bone marrow was infiltrated in diffuse pattern, whereas other patients presented nodular pattern of infiltration. The difference between stage of infiltration before and after treatment was statistically significant (p<0.002. High percentage of infiltration cells with positive anti Bcl-2 reaction from 42.0% in one patient to 85.33+/-3.06% in four patients before treatment was observed. After treatment percentage of infiltration cells with positive anti Bcl-2 antibody reaction was from 33.0+/-18.38% in two patients to 99.0% in one patient. Positive correlation between stage of infiltration and expression of Bcl-2 protein was confirmed before and after treatment. Such correlations were not observed in case of Bax and Bcl-x. Strong staining of immunohistochemical reaction of cells in lymphoid infiltration with Bcl-2 antibody was confirmed. There was a difference between Bcl-/Bax ratio before and after treatment. Immunohistochemical assessment of expression of Bcl-2 family proteins in cells of lymphoid infiltration in bone

  12. Applications of nucleoside-based molecular probes for the in vivo assessment of tumour biochemistry using positron emission tomography (PET

    Directory of Open Access Journals (Sweden)

    Leonard I. Wiebe

    2007-05-01

    Full Text Available Positron emission tomography (PET is a non-invasive nuclear imaging technique. In PET, radiolabelled molecules decay by positron emission. The gamma rays resulting from positron annihilation are detected in coincidence and mapped to produce three dimensional images of radiotracer distribution in the body. Molecular imaging with PET refers to the use of positron-emitting biomolecules that are highly specific substrates for target enzymes, transport proteins or receptor proteins. Molecular imaging with PET produces spatial and temporal maps of the target-related processes. Molecular imaging is an important analytical tool in diagnostic medical imaging, therapy monitoring and the development of new drugs. Molecular imaging has its roots in molecular biology. Originally, molecular biology meant the biology of gene expression, but now molecular biology broadly encompasses the macromolecular biology and biochemistry of proteins, complex carbohydrates and nucleic acids. To date, molecular imaging has focused primarily on proteins, with emphasis on monoclonal antibodies and their derivative forms, small-molecule enzyme substrates and components of cell membranes, including transporters and transmembrane signalling elements. This overview provides an introduction to nucleosides, nucleotides and nucleic acids in the context of molecular imaging.A tomografia por emissão de pósitrons (TEP é uma técnica de imagem não invasiva da medicina nuclear. A TEP utiliza moléculas marcadas com emissores de radiação beta positiva (pósitrons. As radiações gama medidas que resultam do aniquilamento dos pósitrons são detectadas por um sistema de coincidência e mapeadas para produzir uma imagem tridimensional da distribuição do radiotraçador no corpo. A imagem molecular com TEP refere-se ao uso de biomoléculas marcadas com emissor de pósitron que são substratos altamente específicos para alvos como enzimas, proteínas transportadoras ou receptores prot

  13. Lamivudine plus a boosted-protease inhibitor as simplification strategy in HIV-infected patients with toxicity to nucleoside analogues

    Directory of Open Access Journals (Sweden)

    J Casado

    2012-11-01

    Full Text Available Purpose of the study: Dual therapy with lamivudine plus a PI boosted with ritonavir (PI/r could be an alternative to standard triple therapy or PI/r monotherapy as a simplification strategy in patients with toxicity to nucleoside analogues (NA. Methods: Retrospective cohort study of 44 HIV-infected patients on suppressive HAART, with no chronic HBV, who simplified to this dual therapy since 2008. Virological and immunological outcome, lipids and renal changes were evaluated. Summary of results: Mean age was 50 years (38-70, 66% were male, and the median time of HIV infection was 18.6 years. The median nadir CD4+ count was 150 cells/ml (2–407. At inclusion, patients were receiving therapy with lamivudine plus atazanavir/r in 5 cases, lopinavir/r in 12, and darunavir/r in 27, and they had an HIV RNA level<50 copies/ml for a median time of 794 days (129–2344, 90% >6 months. The NA discontinued was tenofovir (27, didanosine (12, AZT (3, and d4T (2. The reasons for changing were toxicity in 76% of cases, especially renal impairment. They had received a mean of 8 regimens before (2–20, and 55% were in CDC-stage C. In 11 cases, history of resistance was available (to NA in 7 cases, including the 210W mutation in four. The mutations 184V was not observed, but four patients (9% had a previous failure to therapy including 3tC. Mutations in the protease gene were observed in 8 patients (2 to 7 mutations, the most frequent 77I and 93L, without resistance to the current PI/r. During 62.8 patient-years of follow-up (median, 802 days, only 2 patients failed (4.5%, due to incomplete adherence, at 27 and 141 days. Of note, these two patients had no previous failed with 3tC or PI. Overall, CD4+ count increased for 55 cells/ml. No new adverse events were observed, but total cholesterol (from 180 to 246 mg/dl, p=0.007 and triglycerides (from 166 to 195, p=0.01 increased during the first 24 weeks with improvement at 48 weeks. On the other hand, estimated

  14. QSAR Studies on 6-(1-Naphthylmethyl) Substituted S-DABO Derivatives as Novel Non-nucleoside HIV-1 Reverse Transcriptase Inhibitors

    Institute of Scientific and Technical Information of China (English)

    YIN Li-Qin; YU Shi-Wen; YAO Ling-Feng; HE Yan-Ping; XIE Xiao-Guang

    2008-01-01

    The AM1 and B3LYP methods were employed to calculate the structural pro- perties of 20 6-(1-naphthylmethyl) substituted S-DABO derivatives with β-carbonyl group on the C(2) side chain as novel potent non-nucleoside HIV-1 reverse transcriptase inhibitors. The correlation analysis (CA) and stepwise multiple regression analysis (SMR) were performed. The QSAR models indicate that the physicochemical parameters of QC9, MRR1, ELUMO, ∏R2 and μ have significant influence on the activities of these derivatives. The substitution of hydrophobic R2 and bulky aromatic R1 to form a conjugated system with the frame of those S-DABO series compounds should be considered to design new potent compounds for anti-HIV-1.

  15. Discovery of the Aryl-phospho-indole IDX899, a Highly Potent Anti-HIV Non-nucleoside Reverse Transcriptase Inhibitor.

    Science.gov (United States)

    Dousson, Cyril; Alexandre, François-René; Amador, Agnès; Bonaric, Séverine; Bot, Stéphanie; Caillet, Catherine; Convard, Thierry; da Costa, Daniel; Lioure, Marie-Pierre; Roland, Arlène; Rosinovsky, Elodie; Maldonado, Sébastien; Parsy, Christophe; Trochet, Christophe; Storer, Richard; Stewart, Alistair; Wang, Jingyang; Mayes, Benjamin A; Musiu, Chiara; Poddesu, Barbara; Vargiu, Luana; Liuzzi, Michel; Moussa, Adel; Jakubik, Jocelyn; Hubbard, Luke; Seifer, Maria; Standring, David

    2016-03-10

    Here, we describe the design, synthesis, biological evaluation, and identification of a clinical candidate non-nucleoside reverse transcriptase inhibitors (NNRTIs) with a novel aryl-phospho-indole (APhI) scaffold. NNRTIs are recommended components of highly active antiretroviral therapy (HAART) for the treatment of HIV-1. Since a major problem associated with NNRTI treatment is the emergence of drug resistant virus, this work focused on optimization of the APhI against clinically relevant HIV-1 Y181C and K103N mutants and the Y181C/K103N double mutant. Optimization of the phosphinate aryl substituent led to the discovery of the 3-Me,5-acrylonitrile-phenyl analogue RP-13s (IDX899) having an EC50 of 11 nM against the Y181C/K103N double mutant. PMID:26804933

  16. Discovery of Potent Non-Nucleoside Inhibitors of Dengue Viral RNA-Dependent RNA Polymerase from a Fragment Hit Using Structure-Based Drug Design.

    Science.gov (United States)

    Yokokawa, Fumiaki; Nilar, Shahul; Noble, Christian G; Lim, Siew Pheng; Rao, Ranga; Tania, Stefani; Wang, Gang; Lee, Gladys; Hunziker, Jürg; Karuna, Ratna; Manjunatha, Ujjini; Shi, Pei-Yong; Smith, Paul W

    2016-04-28

    The discovery and optimization of non-nucleoside dengue viral RNA-dependent-RNA polymerase (RdRp) inhibitors are described. An X-ray-based fragment screen of Novartis' fragment collection resulted in the identification of a biphenyl acetic acid fragment 3, which bound in the palm subdomain of RdRp. Subsequent optimization of the fragment hit 3, relying on structure-based design, resulted in a >1000-fold improvement in potency in vitro and acquired antidengue activity against all four serotypes with low micromolar EC50 in cell-based assays. The lead candidate 27 interacts with a novel binding pocket in the palm subdomain of the RdRp and exerts a promising activity against all clinically relevant dengue serotypes. PMID:26984786

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

    Science.gov (United States)

    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

  18. Evaluation of intensity and energy interaction parameters for the complexation of Pr(III) with selected nucleoside and nucleotide through absorption spectral studies.

    Science.gov (United States)

    Bendangsenla, N; Moaienla, T; David Singh, Th; Sumitra, Ch; Rajmuhon Singh, N; Indira Devi, M

    2013-02-15

    The interactions of Pr(III) with nucleosides and nucleotides have been studied in different organic solvents employing absorption difference and comparative absorption spectrophotometry. The magnitudes of the variations in both energy and intensity interaction parameters were used to explore the degree of outer and inner sphere co-ordination, incidence of covalency and the extent of metal 4f-orbital involvement in chemical bonding. Various electronic spectral parameters like Slater-Condon (F(k)), Racah (E(k)), Lande parameter (ξ(4f)), Nephelauxatic ratio (β), bonding (b(1/2)), percentage covalency (δ) and intensity parameters like oscillator strength (P) and Judd Ofelt electronic dipole intensity parameter (T(λ), λ=2,4,6) have been evaluated. The variation of these evaluated parameters were employed to interpret the nature of binding of Pr(III) with different ligands i.e. Adenosine/ATP in presence and absence of Ca(2+). PMID:23257345

  19. 氟代烟酰胺核苷磷酸酯的一釜合成%One-pot synthesis of fluoro-substituted nicotinamide nucleoside phosphates

    Institute of Scientific and Technical Information of China (English)

    陈哲; 杨振军; 张亮仁; 张礼和

    2012-01-01

    目的 改进氟代烟酰胺核苷单磷酸酯( la.ara-F NMN)的合成方法并合成一系列新的氟代烟酰胺核苷磷酸酯.方法 以氟代糖3为原料,经溴代、去保护和单磷酸化反应得到ara-F NMN.以烟酰胺核苷6为关键中间体,经磷酸化和酯化反应的一釜合成,得到系列氟代烟酰胺核苷磷酸二酯、磷酸三酯和硫代磷酸二酯.结果与结论 优化了合成方法并对新化合物的结构进行了确证.%Fluoro-substituted nucleotides(la,lb) are potent CD38 inhibitors. However, they display poor permeability and less resistant to hydrolysis. It is of great interest to develop specific and generally applicable inhibitors of CD38 with membrane permeability, as well as improved anti-hydrolysis ability. In this study, a new class of fluoro-substituted nicotinamide nucleoside phosphates(1c,2) .including phosphodiesters,thio-phosphodiesters and phosphotriesters, has been synthesized from nicotinamide nucleoside 6 by a one-pot reaction of phosphorylation and esterification. All target compounds were characterized by NMR and HRMS. The synthesis of ara-F NMN(1a) has also been improved,in which the synthesis was performed by starting from the corresponding fluoro-substituted sugar 3,then followed by bromination with HBr,coupling with nicotinamide, deprotection with K2CO3/MeOH and monophosphorylation with POCl3 .successively. The detailed biological investigation and structure-activity relationship of these novel NAD analogues are underway.

  20. MD simulation studies to investigate iso-energetic conformational behaviour of modified nucleosides m2G and m22G present in tRNA

    Directory of Open Access Journals (Sweden)

    Rohit S Bavi

    2013-02-01

    Full Text Available Modified nucleic acid bases are most commonly found in tRNA. These may contain modifications from simple methylation to addition of bulky groups. Methylation of the four canonical nucleotide bases at a wide variety of positions is particularly prominent among the known modification. Methylation of N2 group of guanine is a relatively common modification in tRNA and rRNA. N2-methylguanosine (m2G is the second most often encountered nucleoside in E. coli tRNAs. N2, N2-dimethylguanosine (m22G is found in the majority of eukaryotic tRNAs and involved in forming base pair interactions with adjacent bases. Hence, in order to understand the structural significance of these methylated nucleic acid bases we have carried out molecular dynamics simulation to see the salvation effect. The results obtained shows iso-energetic conformational behaviors for m2G and m22G. The simulation trajectory of m2G shows regular periodical fluctuations suggesting that m2G is equally stable as either s-cis or s-trans rotamers. The two rotamers of m2G may interact canonically or non-canonically with opposite base as s-trans m2G26:C/A/U44 and s-cis m2G26:A/U44. The free rotations around the C-N bond could be the possible reason for these iso-energetic conformations. Dimethylation of G has almost no influence on base pairing with either A or U. Thus, these results reveal that modified nucleosides m2G and m22G may play an important role to prevent tRNA from adopting the unusual mitochondrial like conformation.

  1. In vitro and ex vivo inhibition of human telomerase by anti-HIV nucleoside reverse transcriptase inhibitors (NRTIs but not by non-NRTIs.

    Directory of Open Access Journals (Sweden)

    Kyle R Hukezalie

    Full Text Available Telomerase is a specialized reverse transcriptase responsible for the de novo synthesis of telomeric DNA repeats. In addition to its established reverse transcriptase and terminal transferase activities, recent reports have revealed unexpected cellular activities of telomerase, including RNA-dependent RNA polymerization. This telomerase characteristic, distinct from other reverse transcriptases, indicates that clinically relevant reverse transcriptase inhibitors might have unexpected telomerase inhibition profiles. This is particularly important for the newer generation of RT inhibitors designed for anti-HIV therapy, which have reported higher safety margins than older agents. Using an in vitro primer extension assay, we tested the effects of clinically relevant HIV reverse transcriptase inhibitors on cellular telomerase activity. We observed that all commonly used nucleoside reverse transcriptase inhibitors (NRTIs, including zidovudine, stavudine, tenofovir, didanosine and abacavir, inhibit telomerase effectively in vitro. Truncated telomere synthesis was consistent with the expected mode of inhibition by all tested NRTIs. Through dose-response experiments, we established relative inhibitory potencies of NRTIs on in vitro telomerase activity as compared to the inhibitory potencies of the corresponding dideoxynucleotide triphosphates. In contrast to NRTIs, the non-nucleoside reverse transcriptase inhibitors (NNRTIs nevirapine and efavirenz did not inhibit the primer extension activity of telomerase, even at millimolar concentrations. Long-term, continuous treatment of human HT29 cells with select NRTIs resulted in an accelerated loss of telomere repeats. All tested NRTIs exhibited the same rank order of inhibitory potencies on telomerase and HIV RT, which, according to published data, were orders-of-magnitude more sensitive than other DNA polymerases, including the susceptible mitochondria-specific DNA polymerase gamma. We concluded that

  2. A multi-step process of viral adaptation to a mutagenic nucleoside analogue by modulation of transition types leads to extinction-escape.

    Directory of Open Access Journals (Sweden)

    Rubén Agudo

    Full Text Available Resistance of viruses to mutagenic agents is an important problem for the development of lethal mutagenesis as an antiviral strategy. Previous studies with RNA viruses have documented that resistance to the mutagenic nucleoside analogue ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide is mediated by amino acid substitutions in the viral polymerase that either increase the general template copying fidelity of the enzyme or decrease the incorporation of ribavirin into RNA. Here we describe experiments that show that replication of the important picornavirus pathogen foot-and-mouth disease virus (FMDV in the presence of increasing concentrations of ribavirin results in the sequential incorporation of three amino acid substitutions (M296I, P44S and P169S in the viral polymerase (3D. The main biological effect of these substitutions is to attenuate the consequences of the mutagenic activity of ribavirin -by avoiding the biased repertoire of transition mutations produced by this purine analogue-and to maintain the replicative fitness of the virus which is able to escape extinction by ribavirin. This is achieved through alteration of the pairing behavior of ribavirin-triphosphate (RTP, as evidenced by in vitro polymerization assays with purified mutant 3Ds. Comparison of the three-dimensional structure of wild type and mutant polymerases suggests that the amino acid substitutions alter the position of the template RNA in the entry channel of the enzyme, thereby affecting nucleotide recognition. The results provide evidence of a new mechanism of resistance to a mutagenic nucleoside analogue which allows the virus to maintain a balance among mutation types introduced into progeny genomes during replication under strong mutagenic pressure.

  3. ADME studies of [5-(3)H]-2'-O-methyluridine nucleoside in mice: a building block in siRNA therapeutics.

    Science.gov (United States)

    Lozac'h, Frederic; Christensen, Jesper; Faller, Thomas; van de Kerkhof, Esther; Krauser, Joel; Garnier, Maxime; Litherland, Karine; Catoire, Alexandre; Natt, Francois; Hunziker, Jurg; Swart, Piet

    2016-02-01

    The chemical modification 2'-O-methyl of nucleosides is often used to increase siRNA stability towards nuclease activities. However, the metabolic fate of modified nucleosides remains unclear. Therefore, the aim of this study was to determine the mass balance, pharmacokinetic, and absorption, distribution, metabolism, and excretion (ADME)-properties of tritium-labeled 2'-O-methyluridine, following a single intravenous dose to male CD-1 mice. The single intravenous administration of [5-(3)H]-2'-O-methyluridine was well tolerated in mice. Radioactivity was rapidly and widely distributed throughout the body and remained detectable in all tissues investigated throughout the observation period of 48 h. After an initial rapid decline, blood concentrations of total radiolabeled components declined at a much slower rate. [(3)H]-2'-O-Methyluridine represented a minor component of the radioactivity in plasma (5.89% of [(3)H]-AUC 0-48 h). Three [(3)H]-2'-O-methyluridine metabolites namely uridine (M1), cytidine (M2), and uracil (M3) were the major circulating components representing 32.8%, 8.11%, and 23.6% of radioactivity area under the curve, respectively. The highest concentrations of total radiolabeled components and exposures were observed in kidney, spleen, pineal body, and lymph nodes. The mass balance, which is the sum of external recovery of radioactivity in excreta and remaining radioactivity in carcass and cage wash, was complete. Renal excretion accounted for about 52.7% of the dose with direct renal excretion of the parent in combination with metabolism to the endogenous compounds cytidine, uracil, cytosine, and cytidine. PMID:26977299

  4. Design, synthesis, and in vitro biological evaluation of novel 6-methyl-7-substituted-7-deaza purine nucleoside analogs as anti-influenza A agents.

    Science.gov (United States)

    Lin, Cai; Sun, Chenghai; Liu, Xiao; Zhou, Yiqian; Hussain, Muzammal; Wan, Junting; Li, Minke; Li, Xue; Jin, Ruiliang; Tu, Zhengchao; Zhang, Jiancun

    2016-05-01

    Among many subtypes of influenza A viruses, influenza A(H1N1) and A(H3N2) subtypes are currently circulating among humans (WHO report 2014-15). Therapeutically, the emergence of viral resistance to currently available drugs (adamantanes and neuraminidase inhibitors) has heightened alarms for developing novel drugs that could address diverse targets in the viral replication cycle in order to improve treatment outcomes. To this regard, the design and synthesis of nucleoside analog inhibitors as potential anti-influenza A agents is a very active field of research nowadays. In this study, we designed and synthesized a series of hitherto unknown 6-methyl-7-substituted-7-deaza purine nucleoside analogs, and evaluated for their biological activities against influenza A virus strains, H1N1 and H3N2. From the viral inhibition assay, we identified some effective compounds, among which, compounds 5x (IC50 = 5.88 μM and 6.95 μM for H1N1 and H3N2, respectively) and 5z (IC50 = 3.95 μM and 3.61 μM for H1N1 and H3N2, respectively) demonstrated potent anti-influenza A activity. On the basis of selectivity index, we conceive that compound 5x may serve as a chemical probe of interest for further lead optimization studies with a general aim of developing novel and effective anti-influenza A virus agents. PMID:26802557

  5. Differential maintenance of the M184V substitution in the reverse transcriptase of human immunodeficiency virus type 1 by various nucleoside antiretroviral agents in tissue culture.

    Science.gov (United States)

    Petrella, Marco; Oliveira, Maureen; Moisi, Daniela; Detorio, Mervi; Brenner, Bluma G; Wainberg, Mark A

    2004-11-01

    The M184V substitution in human immunodeficiency virus type 1 reverse transcriptase (RT) is rapidly selected in tissue culture following serial passage of wild-type virus in the presence of increasing concentrations of lamivudine (3TC). M184V is also associated with several alterations of RT enzymatic function in vitro that may adversely affect viral fitness or replication capacity, which creates a potential rationale for its maintenance once it has been selected by antiviral chemotherapy. However, the relative effectiveness of nucleoside RT inhibitors that are structurally unrelated to 3TC in selecting and/or maintaining M184V has not been investigated. In the present study, we have studied the abilities of a variety of drugs, i.e., zalcitabine (ddC), didanosine (ddI), abacavir (ABC), and the novel nucleoside SPD754, in addition to 3TC, to maintain the presence of M184V in tissue culture and have shown that SPD754, ABC, and 3TC are able to preserve M184V in mixed dual infections consisting of wild-type viruses and clinical isolates which contained the M184V mutation. Moreover, M184V could also be maintained in these cultures when a subtherapeutic concentration of 3TC (i.e., 0.05 microM) was used. In contrast, neither ddI nor ddC was able to maintain M184V to the same extent as the other drugs after 10 weeks of tissue culture in mixtures of wild-type viruses and isolates containing M184V in different proportions. PMID:15504840

  6. Sustained First Remission in an Adolescent With Hepatosplenic T-Cell Lymphoma Treated With T-Cell Leukemia Induction, Nucleoside Analog-Based Consolidation, and Early Hematopoietic Stem Cell Transplant

    OpenAIRE

    Schafer, Eric; Chen, Allen; Arceci, Robert J.

    2009-01-01

    Hepatosplenic T-cell lymphoma (HTCL) is a rare malignancy. Prognosis is poor with only a few case reports of long-term survivors. While HTCL universally involves the bone marrow, the condition has been most often treated with multimodal lymphoma specific chemotherapy. We report a durable, sustained first remission in an adolescent treated for HTCL who received induction therapy according to a high risk T-cell leukemia regimen, a nucleoside analog-based consolidation, and allogeneic transplant...

  7. Simultaneous Qualitative Assessment and Quantitative Analysis of Metabolites (Phenolics, Nucleosides and Amino Acids) from the Roots of Fresh Gastrodia elata Using UPLC-ESI-Triple Quadrupole Ion MS and ESI- Linear Ion Trap High-Resolution MS

    OpenAIRE

    Sha Chen; Jun Qiu Liu; Hui Xiao; Jun Zhang; An Liu

    2016-01-01

    A sensitive, effective and optimized method, based on ultra performance liquid chromatography (UPLC) coupled with ESI-triple quadrupole ion MS and ESI-linear ion trap high-resolution MS, has been developed for the simultaneous quantitative and qualitative determination of phenolics, nucleosides and amino acids in the roots of fresh Gastrodia elata. Optimization of the analytical method provided higher separation efficiency and better peak resolution for the targeted compounds. The simultaneou...

  8. 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).

    Science.gov (United States)

    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

  9. 食品中核苷类成分的药理作用研究进展%Recent Advances in Research on Pharmaceutical Functions of Nucleosides and Nucleotides in Dietary Foods

    Institute of Scientific and Technical Information of China (English)

    张雪梅; 杨丰庆; 夏之宁

    2012-01-01

    Dietary intake of nucleosides and nucleotides plays an important role in maintaining the functions of bone marrow hematopoietic cells,intestinal mucosa and brain.Food is one of the major supply sources of nucleosides and nucleotides for human body.In this article,the physiological functions of nucleosides and nucleotides in immune,metabolism,and hepatic,cardiovascular and nerve systems and anti-virus and anti-bacteria activity are reviewed.%日常摄入的核苷及核苷酸类成分在维持骨髓造血细胞、肠黏膜以及脑组织的正常功能方面具有重要的作用。而膳食是为人体提供该类成分的主要来源之一。本文综述了膳食中核苷及核苷酸对人体免疫、代谢、肝脏、心血管及神经系统等所发挥的生理作用,以及其抗菌和抗病毒等其他生理活性。

  10. The case for addressing primary resistance mutations to non-nucleoside reverse transcriptase inhibitors to treat children born from mothers living with HIV in sub-Saharan Africa

    Directory of Open Access Journals (Sweden)

    Khady Kébé

    2014-01-01

    Full Text Available The prevalence of human immunodeficiency virus (HIV drug resistance mutations (DRMs was estimated in 25 untreated infants who were living with HIV-1, younger than 13 months and living in Senegal. Antiretroviral DRMs were detected in 8 of 25 (32% children. Non-nucleoside reverse transcriptase inhibitor (NNRTI DRMs were present in all (100% children whose viruses harboured DRMs: K103N in 43%; Y181C, K101E and V106M each in 29%; and Y188L in 14%. The D67N thymidine-analogue mutation was observed in only two children whose mothers had received chemoprophylaxis of mother-to-child transmission (MTCT. The proportion of children whose viruses harboured DRMs was then 6.5-fold higher in children whose mother–child couples had received nevirapine (NVP-based chemoprophylaxis than in other couples without prophylaxis [7 of 13 (53.8% vs. 1 of 12 (8.3%]. These findings point to the absolute need to address primary resistance mutations in case of virological failure in young children treated by antiretroviral drugs, and to make more effective treatment regimens available to NVP-exposed infants living with HIV-1 in Senegal.

  11. Structure-based optimization and derivatization of 2-substituted quinolone-based non-nucleoside HCV NS5B inhibitors with submicromolar cellular replicon potency.

    Science.gov (United States)

    Cheng, Yu; Shen, Jian; Peng, Run-Ze; Wang, Gui-Feng; Zuo, Jian-Ping; Long, Ya-Qiu

    2016-06-15

    HCV NS5B polymerase is an attractive and validated target for anti-HCV therapy. Starting from our previously identified 2-aryl quinolones as novel non-nucleoside NS5B polymerase inhibitors, structure-based optimization furnished 2-alkyl-N-benzyl quinolones with improved antiviral potency by employing privileged fragment hybridization strategy. The N-(4-chlorobenzyl)-2-(methoxymethyl)quinolone derivative 5f proved to be the best compound of this series, exhibiting a selective sub-micromolar antiviral effect (EC50=0.4μM, SI=10.8) in Huh7.5.1 cells carrying a HCV genotype 2a. Considering the undesirable pharmacokinetic property of the highly substituted quinolones, a novel chemotype of 1,6-naphthyridine-4,5-diones were evolved via scaffold hopping, affording brand new structure HCV inhibitors with compound 6h (EC50 (gt2a)=2.5μM, SI=7.2) as a promising hit. Molecular modeling studies suggest that both of 2-alkyl quinolones and 1,6-naphthyridine-4,5-diones function as HCV NS5B thumb pocket II inhibitors. PMID:27133482

  12. Design, Synthesis, and Evaluation of Thiophene[3,2-d]pyrimidine Derivatives as HIV-1 Non-nucleoside Reverse Transcriptase Inhibitors with Significantly Improved Drug Resistance Profiles.

    Science.gov (United States)

    Kang, Dongwei; Fang, Zengjun; Li, Zhenyu; Huang, Boshi; Zhang, Heng; Lu, Xueyi; Xu, Haoran; Zhou, Zhongxia; Ding, Xiao; Daelemans, Dirk; De Clercq, Erik; Pannecouque, Christophe; Zhan, Peng; Liu, Xinyong

    2016-09-01

    We designed and synthesized a series of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase inhibitors (NNRTIs) with a piperidine-substituted thiophene[3,2-d]pyrimidine scaffold, employing a strategy of structure-based molecular hybridization and substituent decorating. Most of the synthesized compounds exhibited broad-spectrum activity with low (single-digit) nanomolar EC50 values toward a panel of wild-type (WT), single-mutant, and double-mutant HIV-1 strains. Compound 27 was the most potent; compared with ETV, its antiviral efficacy was 3-fold greater against WT, 5-7-fold greater against Y181C, Y188L, E138K, and F227L+V106A, and nearly equipotent against L100I and K103N, though somewhat weaker against K103N+Y181C. Importantly, 27 has lower cytotoxicity (CC50 > 227 μM) and a huge selectivity index (SI) value (ratio of CC50/EC50) of >159101. 27 also showed favorable, drug-like pharmacokinetic and safety properties in rats in vivo. Molecular docking studies and the structure-activity relationships provide important clues for further molecular elaboration. PMID:27541578

  13. Bone mineral density changes in protease inhibitor-sparing vs. nucleoside reverse transcriptase inhibitor-sparing highly active antiretroviral therapy: data from a randomized trial

    DEFF Research Database (Denmark)

    Hansen, Ab; Obel, N; Nielsen, H;

    2011-01-01

    Objective The aim of the study was to compare changes in bone mineral density (BMD) over 144 weeks in HIV-infected patients initiating nucleoside reverse transcriptase inhibitor (NRTI)-sparing or protease inhibitor-sparing highly active antiretroviral therapy (HAART). Methods Sixty-three HAART......, compared with -3.2% (95% CI -4.4 to -2.1) and -1.9% (95% CI -3.5 to -0.3) in the protease inhibitor-sparing arm. Hip BMD declined until week 48 before stabilizing. In the NRTI-sparing arm, BMD had decreased by -5.1% (95% CI -7.1 to -3.1) at week 48 and -4.5% (95% CI -6.9 to -2.1) at week 144, compared with...... -6.1% (95% CI -8.2 to -4.0) and -5.0% (95% CI -6.8 to -3.1) in the protease inhibitor-sparing arm. There were no significant differences between arms. Low baseline CD4 cell count was independently associated with spine (P=0.007) and hip (P=0.04) BMD loss and low body mass index with hip BMD loss (P=0...

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

    Science.gov (United States)

    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.

  15. The σ-hole phenomenon of halogen atoms forms the structural basis of the strong inhibitory potency of C5 halogen substituted glucopyranosyl nucleosides towards glycogen phosphorylase b.

    Science.gov (United States)

    Kantsadi, Anastasia L; Hayes, Joseph M; Manta, Stella; Skamnaki, Vicky T; Kiritsis, Christos; Psarra, Anna-Maria G; Koutsogiannis, Zissis; Dimopoulou, Athina; Theofanous, Stavroula; Nikoleousakos, Nikolaos; Zoumpoulakis, Panagiotis; Kontou, Maria; Papadopoulos, George; Zographos, Spyros E; Komiotis, Dimitris; Leonidas, Demetres D

    2012-04-01

    C5 halogen substituted glucopyranosyl nucleosides (1-(β-D-glucopyranosyl)-5-X-uracil; X=Cl, Br, I) have been discovered as some of the most potent active site inhibitors of glycogen phosphorylase (GP), with respective K(i) values of 1.02, 3.27, and 1.94 μM. The ability of the halogen atom to form intermolecular electrostatic interactions through the σ-hole phenomenon rather than through steric effects alone forms the structural basis of their improved inhibitory potential relative to the unsubstituted 1-(β-D-glucopyranosyl)uracil (K(i) =12.39 μM), as revealed by X-ray crystallography and modeling calculations exploiting quantum mechanics methods. Good agreement was obtained between kinetics results and relative binding affinities calculated by QM/MM-PBSA methodology for various substitutions at C5. Ex vivo experiments demonstrated that the most potent derivative (X=Cl) toward purified GP has no cytotoxicity and moderate inhibitory potency at the cellular level. In accordance, ADMET property predictions were performed, and suggest decreased polar surface areas as a potential means of improving activity in the cell. PMID:22267166

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

    Science.gov (United States)

    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.

  17. Recognition of CG interrupting site by W-shaped nucleoside analogs (WNA) having the pyrazole ring in an anti-parallel triplex DNA.

    Science.gov (United States)

    Taniguchi, Yosuke; Uchida, Yuko; Takaki, Tomoko; Aoki, Eriko; Sasaki, Shigeki

    2009-10-01

    We have previously developed W-shaped nucleoside analogs (WNA) for recognition of TA and CG interrupting sites, which are the intrinsic limitation for the formation of a stable triplex DNA by the natural triplex-forming oligonucleotide (TFO). However, the stabilization effect of WNA is dependent on the neighboring nucleobases at both sides of the WNA analogs within the TFO. Considering that the base is located at the hindered site constructed of three bases of the target duplex and the TFO, it was expected that replacement of the pyrimidine base of the WNA analog with a smaller pyrazole ring might avoid steric repulsion to produce a greater stability for the triplex. In this study, the new WNA analogs bearing the pyrazole ring, 3-aminopyrazole (AP), and 4-methyl-3-pyrazole-5-on (MP) were synthesized, incorporated into the TFOs, then their stabilizing effects on the triplexes were evaluated. A remarkable success was illustrated by the fact that the TFO containing WNA-betaAP in the 3'G-WNA-G-5' sequence formed a stable triplex with selectivity to the CG interrupting site where the previous WNA-betaC did not induce the triplex formation. PMID:19736014

  18. Effects of the protonation state in the interaction of an HIV-1 reverse transcriptase (RT) amino acid, Lys101, and a non nucleoside RT inhibitor, GW420867X.

    Science.gov (United States)

    Galembeck, Sérgio E; Bickelhaupt, F Matthias; Fonseca Guerra, Célia; Galembeck, Eduardo

    2014-07-01

    Interactions between an inhibitor and amino acids from a binding pocket could help not only to understand the nature of these interactions, but also to support the design of new inhibitors. In this paper, we explore the key interaction between a second generation non-nucleoside reverse transcriptase inhibitor (NNRTI), GW420867X, and HIV-1 RT amino acid Lys101 (K101), by quantum mechanical methods. The neutral, protonated, and zwitterionic complexes of GW420867X-K101 were studied. The interaction energies were determined by SCS-MP2/def2-cc-pVQZ, and the electron density was analyzed by natural bond orbital (NBO), atoms in molecules (AIM) and reduced gradient analysis. A large increase in the interaction was observed with the tautomerization of neutral or neutral protonated species. The monomers interact by two medium-strength hydrogen bonds, one partially covalent and another noncovalent. There are some van der Waals intramolecular interactions that are topologically unstable. The nature of the intermolecular interactions was also analyzed using quantitative molecular orbital (MO) theory in combination with an energy decomposition analysis (EDA) based on dispersion-corrected density functional theory (DFT) at BLYP-D/TZ2P. PMID:24965933

  19. Study on Suitability of KOD DNA Polymerase for Enzymatic Production of Artificial Nucleic Acids Using Base/Sugar Modified Nucleoside Triphosphates

    Directory of Open Access Journals (Sweden)

    Satoshi Obika

    2010-11-01

    Full Text Available Recently, KOD and its related DNA polymerases have been used for preparing various modified nucleic acids, including not only base-modified nucleic acids, but also sugar-modified ones, such as bridged/locked nucleic acid (BNA/LNA which would be promising candidates for nucleic acid drugs. However, thus far, reasons for the effectiveness of KOD DNA polymerase for such purposes have not been clearly elucidated. Therefore, using mutated KOD DNA polymerases, we studied here their catalytic properties upon enzymatic incorporation of nucleotide analogues with base/sugar modifications. Experimental data indicate that their characteristic kinetic properties enabled incorporation of various modified nucleotides. Among those KOD mutants, one achieved efficient successive incorporation of bridged nucleotides with a 2′-ONHCH2CH2-4′ linkage. In this study, the characteristic kinetic properties of KOD DNA polymerase for modified nucleoside triphosphates were shown, and the effectiveness of genetic engineering in improvement of the enzyme for modified nucleotide polymerization has been demonstrated.

  20. Quantitative assessment of the use of modified nucleoside triphosphates in expression profiling: differential effects on signal intensities and impacts on expression ratios

    Directory of Open Access Journals (Sweden)

    Dorris David

    2002-07-01

    Full Text Available Abstract Background The power of DNA microarrays derives from their ability to monitor the expression levels of many genes in parallel. One of the limitations of such powerful analytical tools is the inability to detect certain transcripts in the target sample because of artifacts caused by background noise or poor hybridization kinetics. The use of base-modified analogs of nucleoside triphosphates has been shown to increase complementary duplex stability in other applications, and here we attempted to enhance microarray hybridization signal across a wide range of sequences and expression levels by incorporating these nucleotides into labeled cRNA targets. Results RNA samples containing 2-aminoadenosine showed increases in signal intensity for a majority of the sequences. These results were similar, and additive, to those seen with an increase in the hybridization time. In contrast, 5-methyluridine and 5-methylcytidine decreased signal intensities. Hybridization specificity, as assessed by mismatch controls, was dependent on both target sequence and extent of substitution with the modified nucleotide. Concurrent incorporation of modified and unmodified ATP in a 1:1 ratio resulted in significantly greater numbers of above-threshold ratio calls across tissues, while preserving ratio integrity and reproducibility. Conclusions Incorporation of 2-aminoadenosine triphosphate into cRNA targets is a promising method for increasing signal detection in microarrays. Furthermore, this approach can be optimized to minimize impact on yield of amplified material and to increase the number of expression changes that can be detected.