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Sample records for nucleoside analog kinase

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

    PubMed

    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

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

    PubMed Central

    Kay, Brian K.; Lavie, Arnon

    2016-01-01

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

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

    PubMed

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

    2016-06-20

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

  4. Polymeric nanogel formulations of nucleoside analogs

    PubMed Central

    Vinogradov, Serguei V

    2008-01-01

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

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

    PubMed

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

    2003-10-31

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

  6. Downregulation of deoxycytidine kinase in cytarabine-resistant mantle cell lymphoma cells confers cross-resistance to nucleoside analogs gemcitabine, fludarabine and cladribine, but not to other classes of anti-lymphoma agents

    PubMed Central

    2014-01-01

    Background Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma associated with poor prognosis. Implementation of high-dose cytarabine (araC) into induction therapy became standard-of-care for all newly diagnosed younger MCL patients. However, many patients relapse even after araC-based regimen. Molecular mechanisms responsible for araC resistance in MCL are unknown and optimal treatment strategy for relapsed/refractory MCL patients remains elusive. Methods Five araC-resistant (R) clones were derived by long-term culture of five MCL cell lines (CTRL) with increasing doses of araC up to 50 microM. Illumina BeadChip and 2-DE proteomic analysis were used to identify gene and protein expression changes associated with araC resistance in MCL. In vitro cytotoxicity assays and experimental therapy of MCL xenografts in immunodeficient mice were used to analyze their relative responsiveness to a set of clinically used anti-MCL drugs. Primary MCL samples were obtained from patients at diagnosis and after failure of araC-based therapies. Results Marked downregulation of deoxycytidine-kinase (DCK) mRNA and protein expression was identified as the single most important molecular event associated with araC-resistance in all tested MCL cell lines and in 50% primary MCL samples. All R clones were highly (20-1000x) cross-resistant to all tested nucleoside analogs including gemcitabine, fludarabine and cladribine. In vitro sensitivity of R clones to other classes of clinically used anti-MCL agents including genotoxic drugs (cisplatin, doxorubicin, bendamustine) and targeted agents (bortezomib, temsirolimus, rituximab) remained unaffected, or was even increased (ibrutinib). Experimental therapy of immunodeficient mice confirmed the anticipated loss of anti-tumor activity (as determined by overall survival) of the nucleoside analogs gemcitabine and fludarabine in mice transplanted with R clone compared to mice transplanted with CTRL cells, while the anti

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

    PubMed Central

    Lu, Q; Inouye, M

    1996-01-01

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

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

    PubMed

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

    2016-09-01

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

  9. Human UMP-CMP kinase 2, a novel nucleoside monophosphate kinase localized in mitochondria.

    PubMed

    Xu, Yunjian; Johansson, Magnus; Karlsson, Anna

    2008-01-18

    Enzyme deficiency in the salvage pathway of deoxyribonucleotide synthesis in mitochondria can cause mtDNA depletion syndromes. We have identified a human mitochondrial UMP-CMP kinase (UMP-CMPK, cytidylate kinase; EC 2.7.4.14), designated as UMP-CMP kinase 2 (UMP-CMPK2). The C-terminal domain of this 449-amino acid protein contains all consensus motifs of a nucleoside monophosphate kinase. Phylogenetic analysis showed that UMP-CMPK2 belonged to a novel nucleoside monophosphate kinase family, which was closer to thymidylate kinase than to cytosolic UMP-CMP kinase. Subcellular localization with green fluorescent protein fusion proteins illustrated that UMP-CMPK2 was localized in the mitochondria of HeLa cells and that the mitochondrial targeting signal was included in the N-terminal 22 amino acids. The enzyme was able to phosphorylate dUMP, dCMP, CMP, and UMP with ATP as phosphate donor, but the kinetic properties were different compared with the cytosolic UMP-CMPK. Its efficacy to convert dUMP was highest, followed by dCMP, whereas CMP and UMP were the poorest substrates. It also phosphorylated the monophosphate forms of the nucleoside analogs ddC, dFdC, araC, BVDU, and FdUrd, which suggests that UMP-CMPK2 may be involved in mtDNA depletion caused by long term treatment with ddC or other pyrimidine analogs. UMP-CMPK2 mRNA expression was exclusively detected in chronic myelogenous leukemia K-562 and lymphoblastic leukemia MOLT-4 among eight studied cancer cell lines. Particular high expression in leukemia cells, dominant expression in bone marrow, and tight correlation with macrophage activation and inflammatory response suggest that UMP-CMPK2 may have other functions in addition to the supply of substrates for mtDNA synthesis. PMID:17999954

  10. Synergistic cytotoxicity of sorafenib with busulfan and nucleoside analogs in human FMS-like tyrosine kinase 3 internal tandem duplications-positive acute myeloid leukemia cells.

    PubMed

    Song, Guiyun; Valdez, Benigno C; Li, Yang; Liu, Yan; Champlin, Richard E; Andersson, Borje S

    2014-11-01

    Clofarabine (Clo), fludarabine (Flu), and busulfan (Bu) are used in pretransplantation conditioning therapy for patients with myeloid leukemia. To further improve their efficacy in FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD)-positive acute myeloid leukemia (AML), we investigated their synergism with sorafenib (Sor). Exposure of FLT3-ITD-positive MV-4-11 and MOLM 13 cells to Bu+Clo+Flu+Sor resulted in synergistic cytotoxicity; no such synergism was observed in the FLT3-wild type THP-1 and KBM3/Bu250(6) cell lines. The drug synergism in MV-4-11 cells could be attributed to activation of DNA damage response, histone 3 modifications, inhibition of prosurvival kinases, and activation of apoptosis. Further, the phosphorylation of kinases, including FLT3, MAPK kinase (MEK), and AKT, was inhibited. The FLT3-ITD substrate STAT5 and its target gene PIM 2 product decreased when cells were exposed to Sor alone, Bu+Clo+Flu, and Bu+Clo+Flu+Sor. The level of the proapoptotic protein p53 upregulated modulator of apoptosis (PUMA) increased, whereas the level of prosurvival protein MCL-1 decreased when cells were exposed to Bu+Clo+Flu+Sor. The interactions of PUMA with MCL-1 and/or BCL-2 were enhanced when cells were exposed to Bu+Clo+Flu or Bu+Clo+Flu+Sor. The changes in the level of these proteins, which are involved in mitochondrial control of apoptosis, correlate with changes in mitochondrial membrane potential. Bu+Clo+Flu+Sor decreased mitochondrial membrane potential by 60% and caused leakage of cytochrome c, second mitochondria-derived activator of caspases (SMAC)/direct IAP Binding protein with low pI (DIABLO), and AIF from the mitochondria to the cytoplasm, caspase activation, and cell death, suggesting the activation of apoptosis. Analogous, synergistic cytotoxicity in response to Bu, Clo, Flu, and Sor was observed in mononuclear cells isolated from FLT3-ITD-positive AML patients. Although our previous studies were aimed at standardizing the

  11. Flow cytomeric measurement of DNA and incorporated nucleoside analogs

    DOEpatents

    Dolbeare, Frank A.; Gray, Joe W.

    1989-01-01

    A method is provided for simultaneously measuring total cellular DNA and incorporated nucleoside analog. The method entails altering the cellular DNA of cells grown in the presence of a nucleoside analog so that single stranded and double stranded portions are present. Separate stains are used against the two portions. An immunochemical stain is used against the single stranded portion to provide a measure of incorporated nucleoside analog, and a double strand DNA-specific stain is used against the double stranded portion to simultaneously provide a measure of total cellular DNA. The method permits rapid flow cytometric analysis of cell populations, rapid identification of cycling and noncycling subpopulations, and determination of the efficacy of S phase cytotoxic anticancer agents.

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

    PubMed Central

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

    2013-01-01

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

  13. Lethal Mutagenesis of HIV with Mutagenic Nucleoside Analogs

    NASA Astrophysics Data System (ADS)

    Loeb, Lawrence A.; Essigmann, John M.; Kazazi, Farhad; Zhang, Jue; Rose, Karl D.; Mullins, James I.

    1999-02-01

    The human immunodeficiency virus (HIV) replicates its genome and mutates at exceptionally high rates. As a result, the virus is able to evade immunological and chemical antiviral agents. We tested the hypothesis that a further increase in the mutation rate by promutagenic nucleoside analogs would abolish viral replication. We evaluated deoxynucleoside analogs for lack of toxicity to human cells, incorporation by HIV reverse transcriptase, resistance to repair when incorporated into the DNA strand of an RNA\\cdot DNA hybrid, and mispairing at high frequency. Among the candidates tested, 5-hydroxydeoxycytidine (5-OH-dC) fulfilled these criteria. In seven of nine experiments, the presence of this analog resulted in the loss of viral replicative potential after 9-24 sequential passages of HIV in human CEM cells. In contrast, loss of viral replication was not observed in 28 control cultures passaged in the absence of the nucleoside analog, nor with other analogs tested. Sequence analysis of a portion of the HIV reverse transcriptase gene demonstrated a disproportionate increase in G -> A substitutions, mutations predicted to result from misincorporation of 5-OH-dC into the cDNA during reverse transcription. Thus, "lethal mutagenesis" driven by the class of deoxynucleoside analogs represented by 5-OH-dC could provide a new approach to treating HIV infections and, potentially, other viral infections.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  16. Inhibition of nucleoside diphosphate kinase in rat liver mitochondria by added 3'-azido-3'-deoxythymidine.

    PubMed

    Valenti, D; Barile, M; Quagliariello, E; Passarella, S

    1999-02-12

    The effect of 3'-azido-3'-deoxythymidine on nucleoside diphosphate kinase of isolated rat liver mitochondria has been studied. This is done by monitoring the increase in the rate of oxygen uptake by nucleoside diphosphate (TDP, UDP, CDP or GDP) addition to mitochondria in state 4. It is shown that 3'-azido-3'-deoxythymidine inhibits the mitochondrial nucleoside diphosphate kinase in a competitive manner, with a Ki value of about 10 microM as measured for each tested nucleoside diphosphate. It is also shown that high concentrations of GDP prevent 3'-azido-3'-deoxythymidine inhibition of the nucleoside diphosphate kinase. PMID:10050777

  17. Theoretical understanding of two-photon-induced fluorescence of isomorphic nucleoside analogs.

    PubMed

    Samanta, Pralok K; Pati, Swapan K

    2015-04-21

    We use ab initio Density Functional Theory (DFT) and Time-dependent DFT (TDDFT) calculations for a detailed understanding of one-photon absorption (1PA) and two-photon absorption (2PA) cross sections of eight different nucleoside analogs. The results are compared and contrasted with the available experimental data. Our calculated results show that the low energy peaks in the absorption spectra mainly arise because of the π-π* electronic transition of the nucleoside analogs. The emission spectra of the nucleoside analogs are also calculated using TDDFT methods. The calculated absorption and emission spectra in the presence of a solvent follow the same trend as those found experimentally. Our results demonstrate that the nucleoside analogs show significantly different electronic and optical properties, although their bonding aspects towards Watson-Crick base pairing remain the same. We also derive the microscopic details of the origin of nonlinear optical properties of the nucleoside analogs. PMID:25785569

  18. Radiolabeled nucleoside analogs in cancer diagnosis and therapy.

    PubMed

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

    1996-09-01

    ]IUdR in cultured cells, animal tumor-model systems, and patients. In vitro, DNA incorporation of 123I- and 125I-labeled IUdR leads to an exponential decrease in cell survival (no shoulder on the survival curve). However, the total number of decays needed to produce a given lethal effect with [123I]IUdR is approximately twice that required with [125I]IUdR. In vivo, the scintigraphic and antineoplastic capabilities of radioiodinated IUdR have been demonstrated in an intraperitoneal murine ovarian tumor model following intraperitoneal injection; in an intracerebral rat gliosarcoma model after intracranial administration; in an intrathecal rat gliosarcoma model after intrathecal infusion; and in a rat transitional cell bladder cancer model following intravesicular infusion. [123I]IUdR, [125I]IUdR, and/or [131I]IUdR have been administered to patients with brain, breast, colorectal, or gastrointestinal cancers (intratumorally); ovarian cancer (intraperitoneally); bladder cancer (intravesically); liver metastases from colorectal cancer (through the hepatic artery, permanent intra-arterial catheter). These studies have confirmed the observations made in animal models. The data indicate that 5-iodo-2'-deoxyuridine radiolabeled with an Auger electron emitter (123I or 125I) may be a useful agent for the scintigraphic diagnosis and/or therapy of neoplastic diseases that are accessible to direct radiopharmaceutical administration. This radiopharmaceutical should serve as a prototype for, and facilitate the development of, other radiolabeled nucleoside analogs. Further investigations are certainly warranted. PMID:8961807

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

    PubMed

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

    2001-04-17

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

  20. Probing the TRAP-RNA interaction with nucleoside analogs.

    PubMed

    Elliott, M B; Gottlieb, P A; Gollnick, P

    1999-10-01

    The trp RNA-binding Attenuation Protein (TRAP) from Bacillus subtilis binds a series of GAG and UAG repeats separated by 2-3 nonconserved spacer nucleotides in trp leader mRNA. To identify chemical groups on the RNA required for stability of the TRAP-RNA complex, we introduced several different nucleoside analogs into each pentamer of the RNA sequence 5'-(UAGCC)-3' repeated 11 times and measured their effect on the TRAP-RNA interaction. Deoxyribonucleoside substitutions revealed that a 2'-hydroxyl group (2'-OH) is required only on the guanosine occupying the third residue of the RNA triplets for high-affinity binding to TRAP. The remaining hydroxyl groups are dispensable. Base analog substitutions identified all of the exocyclic functional groups and N1 nitrogens of adenine and guanine in the second and third nucleotides, respectively, of the triplets as being involved in binding TRAP. In contrast, none of the substitutions made in the first residue caused any detectable changes in affinity, indicating that elements of these bases are not necessary for complex formation and stability. Studies using abasic nucleotides in the first residue of the triplets and in the two spacer residues confirmed that the majority of the specificity and stability of the TRAP-RNA complex is provided by the AG dinucleotide of the triplet repeats. In addition to direct effects on binding, we demonstrate that the N7-nitrogen of adenosine and guanosine in UAG triplet and the 2'-OHs of (UAGCC)11 RNA are involved in the formation of an as yet undetermined structure that interferes with TRAP binding. PMID:10573119

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

    PubMed

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

    2015-10-01

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

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

    PubMed

    Choi, Jung-Suk; Berdis, Anthony J

    2016-01-01

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

  3. Inhibition of Mycoplasma pneumoniae growth by FDA-approved anticancer and antiviral nucleoside and nucleobase analogs

    PubMed Central

    2013-01-01

    Background Mycoplasma pneumoniae (Mpn) is a human pathogen that causes acute and chronic respiratory diseases and has been linked to many extrapulmonary diseases. Due to the lack of cell wall, Mpn is resistant to antibiotics targeting cell wall synthesis such as penicillin. During the last 10 years macrolide-resistant Mpn strains have been frequently reported in Asian countries and have been spreading to Europe and the United States. Therefore, new antibiotics are needed. In this study, 30 FDA-approved anticancer or antiviral drugs were screened for inhibitory effects on Mpn growth and selected analogs were further characterized by inhibition of target enzymes and metabolism of radiolabeled substrates. Results Sixteen drugs showed varying inhibitory effects and seven showed strong inhibition of Mpn growth. The anticancer drug 6-thioguanine had a MIC (minimum inhibitory concentration required to cause 90% of growth inhibition) value of 0.20 μg ml-1, whereas trifluorothymidine, gemcitabine and dipyridamole had MIC values of approximately 2 μg ml-1. In wild type Mpn culture the presence of 6-thioguanine and dipyridamole strongly inhibited the uptake and metabolism of hypoxanthine and guanine while gemcitabine inhibited the uptake and metabolism of all nucleobases and thymidine. Trifluorothymidine and 5-fluorodeoxyuridine, however, stimulated the uptake and incorporation of radiolabeled thymidine and this stimulation was due to induction of thymidine kinase activity. Furthermore, Mpn hypoxanthine guanine phosphoribosyl transferase (HPRT) was cloned, expressed, and characterized. The 6-thioguanine, but not other purine analogs, strongly inhibited HPRT, which may in part explain the observed growth inhibition. Trifluorothymidine and 5-fluorodeoxyuridine were shown to be good substrates and inhibitors for thymidine kinase from human and Mycoplasma sources. Conclusion We have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent

  4. Supramolecular gels made from nucleobase, nucleoside and nucleotide analogs.

    PubMed

    Peters, Gretchen Marie; Davis, Jeffery T

    2016-06-01

    Supramolecular or molecular gels are attractive for various applications, including diagnostics, tissue scaffolding and targeted drug release. Gelators derived from natural products are of particular interest for biomedical purposes, as they are generally biocompatible and stimuli-responsive. The building blocks of nucleic acids (i.e. nucleobases, nucleosides, and nucleotides) are desirable candidates for supramolecular gelation as they readily engage in reversible, noncovalent interactions. In this review, we describe a number of organo- and hydrogels formed through the assembly of nucleosides, nucleotides, and their derivatives. While natural nucleosides and nucleotides generally require derivatization to induce gelation, guanosine and its corresponding nucleotides are well known gelators. This unique gelating ability is due to propensity of the guanine nucleobase to self-associate into stable higher-order assemblies, such as G-ribbons, G4-quartets, and G-quadruplexes. PMID:27146863

  5. Structure of nucleoside diphosphate kinase from pacific shrimp (Litopenaeus vannamei) in binary complexes with purine and pyrimidine nucleoside diphosphates.

    PubMed

    López-Zavala, Alonso A; Quintero-Reyes, Idania E; Carrasco-Miranda, Jesús S; Stojanoff, Vivian; Weichsel, Andrzej; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R

    2014-09-01

    Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012), J. Bioenerg. Biomembr. 44, 325-331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2'-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism. PMID:25195883

  6. Structure of nucleoside diphosphate kinase from pacific shrimp (Litopenaeus vannamei) in binary complexes with purine and pyrimidine nucleoside diphosphates

    PubMed Central

    López-Zavala, Alonso A.; Quintero-Reyes, Idania E.; Carrasco-Miranda, Jesús S.; Stojanoff, Vivian; Weichsel, Andrzej; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R.

    2014-01-01

    Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012 ▶), J. Bioenerg. Biomembr. 44, 325–331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2′-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism. PMID:25195883

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2016-06-01

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

  9. Pseudobond parameters for QM/MM studies involving nucleosides, nucleotides, and their analogs

    NASA Astrophysics Data System (ADS)

    Chaudret, Robin; Parks, Jerry M.; Yang, Weitao

    2013-01-01

    In biological systems involving nucleosides, nucleotides, or their respective analogs, the ribose sugar moiety is the most common reaction site, for example, during DNA replication and repair. However, nucleic bases, which comprise a sizable portion of nucleotide molecules, are usually unreactive during such processes. In quantum mechanical/molecular simulations of nucleic acid reactivity, it may therefore be advantageous to describe specific ribosyl or ribosyl phosphate groups quantum mechanically and their respective nucleic bases with a molecular mechanics potential function. Here, we have extended the pseudobond approach to enable quantum mechanical/molecular mechanical simulations involving nucleotides, nucleosides, and their analogs in which the interface between the two subsystems is located between the sugar and the base, namely, the C(sp3)-N(sp2) bond. The pseudobond parameters were optimized on a training set of 10 molecules representing several nucleotide and nucleoside bases and analogs, and they were then tested on a larger test set of 20 diverse molecules. Particular emphasis was placed on providing accurate geometries and electrostatic properties, including electrostatic potential, natural bond orbital (NBO) and atoms in molecules (AIM) charges and AIM first moments. We also tested the optimized parameters on five nucleotide and nucleoside analogues of pharmaceutical relevance and a small polypeptide (triglycine). Accuracy was maintained for these systems, which highlights the generality and transferability of the pseudobond approach.

  10. Pseudobond parameters for QM/MM studies involving nucleosides, nucleotides, and their analogs

    SciTech Connect

    Chaudret, Robin; Parks, Jerry M; Yang, Weitao

    2013-01-01

    In biological systems involving nucleosides, nucleotides, or their respective analogs, the ribose sugar moiety is the most common reaction site, for example, during DNA replication and repair. How- ever, nucleic bases, which comprise a sizable portion of nucleotide molecules, are usually unreactive during such processes. In quantum mechanical/molecular simulations of nucleic acid reactivity, it may therefore be advantageous to describe specific ribosyl or ribosyl phosphate groups quantum me- chanically and their respective nucleic bases with a molecular mechanics potential function. Here, we have extended the pseudobond approach to enable quantum mechanical/molecular mechanical simulations involving nucleotides, nucleosides, and their analogs in which the interface between the two subsystems is located between the sugar and the base, namely, the C(sp3) N(sp2) bond. The pseudobond parameters were optimized on a training set of 10 molecules representing several nu- cleotide and nucleoside bases and analogs, and they were then tested on a larger test set of 20 diverse molecules. Particular emphasis was placed on providing accurate geometries and electrostatic prop- erties, including electrostatic potential, natural bond orbital (NBO) and atoms in molecules (AIM) charges and AIM first moments. We also tested the optimized parameters on five nucleotide and nu- cleoside analogues of pharmaceutical relevance and a small polypeptide (triglycine). Accuracy was maintained for these systems, which highlights the generality and transferability of the pseudobond approach. 2013 American Institute of Physics. [http://dx.doi.org/10.1063/1.4772182

  11. Pan-Pathway Based Interaction Profiling of FDA-Approved Nucleoside and Nucleobase Analogs with Enzymes of the Human Nucleotide Metabolism

    PubMed Central

    Egeblad, Louise; Welin, Martin; Flodin, Susanne; Gräslund, Susanne; Wang, Liya; Balzarini, Jan; Eriksson, Staffan; Nordlund, Pär

    2012-01-01

    To identify interactions a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs was screened against 23 enzymes of the human nucleotide metabolism using a thermal shift assay. The method was validated with deoxycytidine kinase; eight interactions known from the literature were detected and five additional interactions were revealed after the addition of ATP, the second substrate. The NAL screening gave relatively few significant hits, supporting a low rate of “off target effects.” However, unexpected ligands were identified for two catabolic enzymes guanine deaminase (GDA) and uridine phosphorylase 1 (UPP1). An acyclic guanosine prodrug analog, valaciclovir, was shown to stabilize GDA to the same degree as the natural substrate, guanine, with a ΔTagg around 7°C. Aciclovir, penciclovir, ganciclovir, thioguanine and mercaptopurine were also identified as ligands for GDA. The crystal structure of GDA with valaciclovir bound in the active site was determined, revealing the binding of the long unbranched chain of valaciclovir in the active site of the enzyme. Several ligands were identified for UPP1: vidarabine, an antiviral nucleoside analog, as well as trifluridine, idoxuridine, floxuridine, zidovudine, telbivudine, fluorouracil and thioguanine caused concentration-dependent stabilization of UPP1. A kinetic study of UPP1 with vidarabine revealed that vidarabine was a mixed-type competitive inhibitor with the natural substrate uridine. The unexpected ligands identified for UPP1 and GDA imply further metabolic consequences for these nucleoside analogs, which could also serve as a starting point for future drug design. PMID:22662200

  12. Transgene Expression of Drosophila melanogaster Nucleoside Kinase Reverses Mitochondrial Thymidine Kinase 2 Deficiency*♦

    PubMed Central

    Krishnan, Shuba; Zhou, Xiaoshan; Paredes, João A.; Kuiper, Raoul V.; Curbo, Sophie; Karlsson, Anna

    2013-01-01

    A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK+/− transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK+/−TK2−/− mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK+/−TK2−/− mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency. PMID:23288848

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

    PubMed Central

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

    2001-01-01

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

  14. SELECTIVE INHIBITION OF THE WEST NILE VIRUS METHYLTRANSFERASE BY NUCLEOSIDE ANALOGS

    PubMed Central

    Chen, Hui; Liu, Lihui; Jones, Susan A.; Banavali, Nilesh; Kass, Jorden; Li, Zhong; Zhang, Jing; Kramer, Laura D.; Ghosh, Arun K.; Li, Hongmin

    2012-01-01

    The flavivirus methyltransferase (MTase) sequentially methylates the N7 and 2’-O positions of the viral RNA cap (GpppA-RNA→m7GpppA-RNA→m7GpppAm-RNA), using S-adenosyl-L-methionine (SAM) as a methyl donor. We report here the synthesis and biological evaluation of a series of novel nucleoside analogs. Two of these compounds can effectively and competitively inhibit the WNV MTase with IC50 values in micromolar range and, more importantly, do not inhibit human MTase. The compounds can also suppress the WNV replication in cell culture. PMID:23267828

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

    PubMed

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

    2013-11-15

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

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

    PubMed Central

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

    2015-01-01

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

  17. Relaxed enantioselectivity of human mitochondrial thymidine kinase and chemotherapeutic uses of L-nucleoside analogues.

    PubMed Central

    Verri, A; Priori, G; Spadari, S; Tondelli, L; Focher, F

    1997-01-01

    Our discovery that Herpes virus thymidine kinase (TK) and cellular deoxycytidine kinase lack enantioselectivity, being able to phosphorylate both D- and L-enantiomers of the substrate, suggested the use of unnatural L-nucleoside analogues as antiviral drugs (Herpes, hepatitis and immunodeficiency viruses). Several L-nucleoside analogues have displayed a short-term cytotoxicity much lower than their corresponding D-counterpart. Since the delayed cytotoxicity of a drug often depends on its effects on mitochondrial metabolism, we have investigated the degree of enantioselectivity of human mitochondrial thymidine kinase (mt-TK). We demonstrate that mt-TK does not show an absolute enantioselectivity, being able to recognize, although with lower efficiency, the L-enantiomers of thymidine, deoxycytidine and modified deoxyuridines, such as (E)-5-(2-bromovinyl)-2'-deoxyuridine and 5-iodo-2'-deoxyuridine. Interestingly, the reported negative co-operativity of mt-TK phosphorylating beta-D-2'-deoxythymidine (D-Thd), disappears when the deoxyribose moiety has the inverted configuration, resulting in the preferential phosphorylation of d-Thd even in the presence of high concentrations of the L-enantiomer. This, coupled with the higher Km for beta-L-2'-deoxythymidine (L-Thd), makes mt-TK resistant to high concentrations of L-Thd and L-Thd analogues, minimizing the mitochondria-dependent delayed cytotoxicity that might be caused by the administration of L-nucleoside analogues as antivirals. PMID:9359870

  18. Autophosphorylation of nucleoside diphosphate kinase on non-histidine residues.

    PubMed

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

    1994-10-10

    Recently, several reports appeared which described auto-phosphorylation of NDP kinase on residues different from the active-site histidine. Based on these findings conclusions were drawn with respect to a regulation of enzyme activity and to a possible role as a metastasis suppressor. In this paper we show that although non-histidine autophosphorylation occurs on NDP kinases from mammals, lower eukaryotes and bacteria, less than 0.2% of the subunits are phosphorylated. Using site-directed mutagenesis, we show that the active site histidine is essential for non-histidine autophosphorylation. The low stoichiometry of phosphate incorporation excludes a role of autophosphorylation in regulating overall enzyme activity. PMID:7926021

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

    PubMed Central

    Amutha, Boominathan; Pain, Debkumar

    2003-01-01

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

  20. The Nucleoside Analog D-carba T Blocks HIV-1 Reverse Transcription

    PubMed Central

    Boyer, Paul L.; Vu, B. Christie; Ambrose, Zandrea; Julias, John G.; Warnecke, Svenja; Liao, Chenzhong; Meier, Chris; Marquez, Victor E.; Hughes, Stephen H.

    2009-01-01

    A major pathway for HIV-1 resistance to nucleoside reverse transcriptase inhibitors (NRTIs) involves reverse transcriptase (RT) mutations that enhance ATP-dependent pyrophosphorolysis, which excises NRTIs from the end of viral DNA. We analyzed novel NRTIs for their ability to inhibit DNA synthesis of excision-proficient HIV-1 RT mutants. D-carba T is a carbocyclic nucleoside that has a 3′ hydroxyl on the pseudosugar. The 3′ hydroxyl group allows RT to incorporate additional dNTPs, which should protect D-carba TMP from excision. D-carba T can be converted to the triphosphate form by host cell kinases with moderate efficiency. D-carba T-TP is efficiently incorporated by HIV-1 RT; however, the next dNTP is added slowly to a D-carba TMP at the primer terminus. D-carba T effectively inhibits viral vectors that replicate using NRTI-resistant HIV-1 RTs, and there is no obvious toxicity in cultured cells. NRTIs based on the carbocyclic pseudosugar may offer an effective approach for the treatment of HIV-1 infections. PMID:19678643

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

    PubMed

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

    2010-08-16

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

  2. BCX4430 - A broad-spectrum antiviral adenosine nucleoside analog under development for the treatment of Ebola virus disease.

    PubMed

    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

  3. Stimulation of phospholipase D in rabbit platelet membranes by nucleoside triphosphates and by phosphocreatine: roles of membrane-bound GDP, nucleoside diphosphate kinase and creatine kinase.

    PubMed Central

    Fan, X T; Sherwood, J L; Haslam, R J

    1994-01-01

    Previous work has shown that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and GTP stimulate phospholipase D (PLD) in rabbit platelet membranes and that these effects are greatly enhanced by pretreatment of platelets with phorbol esters that activate protein kinase C [Van der Meulen and Haslam (1990), Biochem. J. 271, 693-700]. In the present study, the effects of Mg2+, various nucleoside triphosphates and phosphocreatine (PCr) were investigated. Platelet membranes containing phospholipids labelled with [3H]glycerol were assayed for PLD in the presence of an optimal Mg2+ concentration (10 mM) by measuring [3H]phosphatidylethanol formation in incubations that included 300 mM ethanol. In membranes from phorbolester-treated platelets, the same maximal increases in PLD activity (5-fold) were seen with 1 microM GTP[S]), and 100 microM GTP. Addition of adenosine 5'-[gamma-thio]triphosphate (ATP[S]), ITP, XTP, UTP and CTP had similar stimulatory effects, but only at > or = 1 mM. In contrast, ATP had a biphasic action, causing a maximal (2-fold) stimulation at 10 microM and smaller effects at higher concentrations; the inhibitory component of the action of ATP was blocked by 2 microM staurosporine. Guanosine 5'-[beta-thio]diphosphate decreased the stimulatory effects of ATP and ATP[S]. UDP, which can inhibit nucleoside diphosphate kinase (NDPK), decreased the activation of PLD by ATP[S], ATP, XTP, CTP and to a lesser extent ITP, but had no effect on the actions of GTP[S] and GTP. Rabbit platelet membranes contained NDPK and addition of [gamma-32P]ATP led to the formation of [32P]GTP in amounts sufficient to explain most or all of the activation of PLD; UDP prevented GTP formation. PCr (0.04-1 mM) also stimulated membrane PLD activity, an effect that was dependent on endogenous membrane-bound creatine kinase (CK). UDP and guanosine 5'-[beta-thio]diphosphate each inhibited this effect of PCr. The results show that in rabbit platelet membranes, CK, NDPK and the GTP

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

    PubMed

    Boissier, Fanny; Georgescauld, Florian; Moynié, Lucile; Dupuy, Jean-William; Sarger, Claude; Podar, Mircea; Lascu, Ioan; Giraud, Marie-France; Dautant, Alain

    2012-06-01

    The nucleoside diphosphate kinase (Ndk) catalyzes the reversible transfer of the γ-phosphate from nucleoside triphosphate to nucleoside diphosphate. Ndks form hexamers or two types of tetramers made of the same building block, namely, the common dimer. The secondary interfaces of the Type I tetramer found in Myxococcus xanthus Ndk and of the Type II found in Escherichia coli Ndk involve the opposite sides of subunits. Up to now, the few available structures of Ndk from thermophiles were hexameric. Here, we determined the X-ray structures of four crystal forms of the Ndk from the hyperthermophilic bacterium Aquifex aeolicus (Aa-Ndk). Aa-Ndk displays numerous features of thermostable proteins and is made of the common dimer but it is a tetramer of Type I. Indeed, the insertion of three residues in a surface-exposed spiral loop, named the Kpn-loop, leads to the formation of a two-turn α-helix that prevents both hexamer and Type II tetramer assembly. Moreover, the side chain of the cysteine at position 133, which is not present in other Ndk sequences, adopts two alternate conformations. Through the secondary interface, each one forms a disulfide bridge with the equivalent Cys133 from the neighboring subunit. This disulfide bridge was progressively broken during X-ray data collection by radiation damage. Such crosslinks counterbalance the weakness of the common-dimer interface. A 40% decrease of the kinase activity at 60°C after reduction and alkylation of the protein corroborates the structural relevance of the disulfide bridge on the tetramer assembly and enzymatic function. PMID:22467275

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

    PubMed

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

    1994-12-23

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

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

    PubMed Central

    Tsume, Yasuhiro; Bermejo, Blanca Borras; Amidon, Gordon L.

    2014-01-01

    Dipeptide monoester prodrugs of floxuridine and gemcitabine were synthesized. Their chemical stability in buffers, enzymatic stability in cell homogenates, permeability in mouse intestinal membrane along with drug concentration in mouse plasma, and anti-proliferative activity in cancer cells were determined and compared to their parent drugs. Floxuridine prodrug was more enzymatically stable than floxuridine and the degradation from prodrug to parent drug works as the rate-limiting step. On the other hand, gemcitabine prodrug was less enzymatically stable than gemcitabine. Those dipeptide monoester prodrugs exhibited 2.4- to 48.7-fold higher uptake than their parent drugs in Caco-2, Panc-1, and AsPC-1 cells. Floxuridine and gemcitabine prodrugs showed superior permeability in mouse jejunum to their parent drugs and exhibited the higher drug concentration in plasma after in situ mouse perfusion. Cell proliferation assays in ductal pancreatic cancer cells, AsPC-1 and Panc-1, indicated that dipeptide prodrugs of floxuridine and gemcitabine were more potent than their parent drugs. The enhanced potency of nucleoside analogs was attributed to their improved membrane permeability. The prodrug forms of 5′-l-phenylalanyl-l-tyrosyl-floxuridine and 5′-l-phenylalanyl-l-tyrosyl-gemcitabine appeared in mouse plasma after the permeation of intestinal membrane and the first-pass effect, suggesting their potential for the development of oral dosage form for anti-cancer agents. PMID:24473270

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

    PubMed

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

    2016-03-18

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

  8. Acanthamoeba polyphaga mimivirus NDK: preliminary crystallographic analysis of the first viral nucleoside diphosphate kinase

    PubMed Central

    Jeudy, Sandra; Coutard, Bruno; Lebrun, Régine; Abergel, Chantal

    2005-01-01

    The complete sequence of the largest known double-stranded DNA virus, Acanthamoeba polyphaga mimivirus, has recently been determined [Raoult et al. (2004 ▶), Science, 306, 1344–1350] and revealed numerous genes not expected to be found in a virus. A comprehensive structural and functional study of these gene products was initiated [Abergel et al. (2005 ▶), Acta Cryst. F61, 212–215] both to better understand their role in the virus physiology and to obtain some clues to the origin of DNA viruses. Here, the preliminary crystallographic analysis of the viral nucleoside diphosphate kinase protein is reported. The crystal belongs to the cubic space group P213, with unit-cell parameter 99.425 Å. The self-rotation function confirms that there are two monomers per asymmetric unit related by a twofold non-crystallographic axis and that the unit cell thus contains four biological entities. PMID:16511098

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

    PubMed

    Georgescauld, Florian; Moynié, Lucile; Habersetzer, Johann; Cervoni, Laura; Mocan, Iulia; Borza, Tudor; Harris, Pernile; Dautant, Alain; Lascu, Ioan

    2013-01-01

    Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg(80)-Asp(93) as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved. PMID:23526954

  10. Intersubunit Ionic Interactions Stabilize the Nucleoside Diphosphate Kinase of Mycobacterium tuberculosis

    PubMed Central

    Georgescauld, Florian; Moynié, Lucile; Habersetzer, Johann; Cervoni, Laura; Mocan, Iulia; Borza, Tudor; Harris, Pernile; Dautant, Alain; Lascu, Ioan

    2013-01-01

    Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg80-Asp93 as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved. PMID:23526954

  11. Evaluation of serum nucleoside diphosphate kinase A for the detection of colorectal cancer

    PubMed Central

    Otero-Estévez, Olalla; De Chiara, Loretta; Barcia-Castro, Leticia; Páez de la Cadena, María; Rodríguez-Berrocal, Francisco Javier; Cubiella, Joaquín; Hernández, Vicent; Martínez-Zorzano, Vicenta Soledad

    2016-01-01

    We previously described the over-expression of nucleoside diphosphate kinase A (NDKA) in tumours and serum from colorectal cancer (CRC) patients, suggesting its use as biomarker. In this study we evaluated the diagnostic accuracy of serum NDKA to detect advanced neoplasia (CRC or advanced adenomas). Furthermore, the performance of NDKA was compared with the faecal immunochemical test (FIT). The study population included a case-control cohort and a screening cohort (511 asymptomatic first-degree relatives of CRC patients that underwent a colonoscopy and a FIT). Serum NDKA was elevated in CRC patients in the case-control cohort (p = 0.002). In the screening cohort, NDKA levels were higher for advanced adenomas (p = 0.010) and advanced neoplasia (p = 0.006) compared to no neoplasia. Moreover, elevated NDKA was associated with severe characteristics of adenomas (≥3 lesions, size ≥ 1 cm or villous component). Setting specificity to 85%, NDKA showed a sensitivity of 30.19% and 29.82% for advanced adenomas and advanced neoplasia, respectively. NDKA combined with FIT (100 ng/mL cut-off) detected advanced adenomas and advanced neoplasia with 45.28% and 49.12% sensitivity, with specificity close to 90%. The combination of serum NDKA and FIT can improve the detection of advanced neoplasia, mainly for lesions located on the proximal colon, in asymptomatic individuals with CRC family-risk. PMID:27222072

  12. Increase of salt dependence of halophilic nucleoside diphosphate kinase caused by a single amino acid substitution.

    PubMed

    Ishibashi, Matsujiro; Hayashi, Tomoe; Yoshida, Chiho; Tokunaga, Masao

    2013-07-01

    Nucleoside diphosphate kinase (HsNDK) from an extremely halophilic archaea, Halobacterium salinarum, is composed of a homo hexamer, assembled as a trimer of basic dimeric units. It requires >2 M NaCl for refolding, although it does not require NaCl for stability or enzymatic activity below 30 °C. A HisN111L mutant with an N-terminal extension sequence containing hexa-His tag, in which Asn111 was replaced with Leu, was designed to be less stable between basic dimeric units. This mutant can lose between 6 and 12 hydrogen bonds between basic dimeric units in the hexamer structure. The HisN111L mutant had enhanced salt requirements for enzymatic activity and refolding even though the secondary structure of the HisN111L mutant was confirmed to be similar to the control, HisNDK, in low and high salt solutions using circular dichroism. We reported previously that G114R and D148C mutants, which had enhanced interactions between basic dimeric units, showed facilitated refolding and stabilization in low salt solution. The results of this study help to elucidate the process for engineering industrial enzymes by controlling subunit-subunit interactions through mutations. PMID:23609188

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

    PubMed Central

    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

    2015-01-01

    A library of sixteen 2nd generation amino- and amido-substituted carboranyl pyrimidine nucleoside analogues, 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 analogues (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 analogue. 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 analogues and will profoundly impact future design strategies for these agents. PMID:26087030

  14. Multicomponent reactions in nucleoside chemistry

    PubMed Central

    Buchowicz, Włodzimierz

    2014-01-01

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

  15. A structural mechanism for dimeric to tetrameric oligomer conversion in Halomonas sp. nucleoside diphosphate kinase

    PubMed Central

    Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo; Matsumoto, Fumiko; Tamada, Taro; Tokunaga, Hiroko; Ishibashi, Matsujiro; Blaber, Michael; Tokunaga, Masao; Kuroki, Ryota

    2012-01-01

    Nucleoside diphosphate kinase (NDK) is known to form homotetramers or homohexamers. To clarify the oligomer state of NDK from moderately halophilic Halomonas sp. 593 (HaNDK), the oligomeric state of HaNDK was characterized by light scattering followed by X-ray crystallography. The molecular weight of HaNDK is 33,660, and the X-ray crystal structure determination to 2.3 and 2.7 Å resolution showed a dimer form which was confirmed in the different space groups of R3 and C2 with an independent packing arrangement. This is the first structural evidence that HaNDK forms a dimeric assembly. Moreover, the inferred molecular mass of a mutant HaNDK (E134A) indicated 62.1–65.3 kDa, and the oligomerization state was investigated by X-ray crystallography to 2.3 and 2.5 Å resolution with space groups of P21 and C2. The assembly form of the E134A mutant HaNDK was identified as a Type I tetramer as found in Myxococcus NDK. The structural comparison between the wild-type and E134A mutant HaNDKs suggests that the change from dimer to tetramer is due to the removal of negative charge repulsion caused by the E134 in the wild-type HaNDK. The higher ordered association of proteins usually contributes to an increase in thermal stability and substrate affinity. The change in the assembly form by a minimum mutation may be an effective way for NDK to acquire molecular characteristics suited to various circumstances. PMID:22275000

  16. SwoHp, a Nucleoside Diphosphate Kinase, Is Essential in Aspergillus nidulans

    PubMed Central

    Lin, Xiaorong; Momany, Cory; Momany, Michelle

    2003-01-01

    The temperature-sensitive swoH1 mutant of Aspergillus nidulans was previously identified in a screen for mutants with defects in polar growth. In the present work, we found that the swoH1 mutant swelled, lysed, and did not produce conidia during extended incubation at the restrictive temperature. When shifted from the permissive to the restrictive temperature, swoH1 showed the temperature-sensitive swelling phenotype only after 8 h at the higher temperature. The swoH gene was mapped to chromosome II and cloned by complementation of the temperature-sensitive phenotype. The sequence showed that swoH encodes a homologue of nucleoside diphosphate kinases (NDKs) from other organisms. Deletion experiments showed that the swoH gene is essential. A hemagglutinin-SwoHp fusion complemented the mutant phenotype, and the purified fusion protein possessed phosphate transferase activity in thin-layer chromatography assays. Sequencing of the mutant allele showed a predicted V83F change. Structural modeling suggested that the swoH1 mutation would lead to perturbation of the NDK active site. Crude cell extracts from the swoH1 mutant grown at the permissive temperature had ∼20% of the NDK activity seen in the wild type and did not show any decrease in activity when assayed at higher temperatures. Though the data are not conclusive, the lack of temperature-sensitive NDK activity in the swoH1 mutant raises the intriguing possibility that the SwoH NDK is required for growth at elevated temperatures rather than for polarity maintenance. PMID:14665452

  17. Efficacy and safety of nucleoside analogs on blocking father-to-infant vertical transmission of hepatitis B virus

    PubMed Central

    CAO, LI-HAU; ZHAO, PEI-LI; LIU, ZHI-MIN; SUN, SHAO-CHUN; XU, DONG-BO; ZHANG, JI-DONG; SHAO, MEI-HUA

    2015-01-01

    The aim of the present study was to observe the efficacy and safety of nucleoside analogs in inhibiting father-to-infant vertical transmission of hepatitis B virus (HBV). Nucleoside analogs compete with HBV DNA polymerase substrate to inhibit DNA polymerase, thus preventing the replication of HBV DNA. A case group and control group were recruited for the study. Between March 2006 and March 2012 at the Liver Disease Center of Qinhuangdao Third Hospital, a total of 201 couples were recruited for the case group. In each case, the father tested positive the following HBV markers: Hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), antibodies against the hepatitis B core antigen (anti-HBc) and HBV DNA. In total, 189 male patients presented with abnormal liver function (94.0%; 189/201). Prior to pregnancy, all the males in the case group were required to test negative for HBV DNA and exhibit normal liver function, while the females were required to test positive for antibodies against HBsAg (anti-HBs). In total, 188 couples comprised the control group. The couples were recruited between March 2006 and March 2012 in the Prenatal Clinic of Qinhuangdao Women's and Children's Hospital. The fathers tested positive for HBsAg, HBeAg, anti-HBc and HBV DNA. With regard to the females, HBsAg tests were all negative and anti-HBs tests were positive. In the case group, there were no HBsAg-positive or HBV DNA-positive newborns, while anti-HBs tests were all positive; thus, the father-to-infant HBV vertical transmission was successfully inhibited. In the control group, 147/188 newborns tested positive for anti-HBs at birth, accounting for 78.2%. In addition, 28 newborns were positive for HBV DNA (14.9%), and 19 newborns tested positive for HBsAg (10.1%). Statistically significant differences were observed between the two groups with regard to these parameters. However, no statistically significant differences in gestational age, birth weight, birth height, 1- and 8-min

  18. Effects of nucleoside analog incorporation on DNA binding to the DNA binding domain of the GATA-1 erythroid transcription factor.

    PubMed

    Foti, M; Omichinski, J G; Stahl, S; Maloney, D; West, J; Schweitzer, B I

    1999-02-01

    We investigate here the effects of the incorporation of the nucleoside analogs araC (1-beta-D-arabinofuranosylcytosine) and ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl] guanine) into the DNA binding recognition sequence for the GATA-1 erythroid transcription factor. A 10-fold decrease in binding affinity was observed for the ganciclovir-substituted DNA complex in comparison to an unmodified DNA of the same sequence composition. AraC substitution did not result in any changes in binding affinity. 1H-15N HSQC and NOESY NMR experiments revealed a number of chemical shift changes in both DNA and protein in the ganciclovir-modified DNA-protein complex when compared to the unmodified DNA-protein complex. These changes in chemical shift and binding affinity suggest a change in the binding mode of the complex when ganciclovir is incorporated into the GATA DNA binding site. PMID:10037146

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

    PubMed

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

    2015-12-01

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

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

    PubMed Central

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

    2015-01-01

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

  1. Characterization of a low molecular mass autophosphorylating protein in cultured sugarcane cells and its identification as a nucleoside diphosphate kinase.

    PubMed Central

    Moisyadi, S; Dharmasiri, S; Harrington, H M; Lukas, T J

    1994-01-01

    A low molecular mass (18 kD) phosphoprotein (pp18) was characterized and purified from cultured sugarcane (Saccharum officinarum L.) cell line H50-7209. Autophosphorylation assays were used to detect pp18 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Only pp18 was detected by a brief in situ phosphorylation method, whereas additional putative protein kinases were detected by an extended method. pp18 was present in both microsomal membrane and soluble fractions and exhibited anomalous turnover of 32P label during in vitro phosphorylation experiments with highest levels present at shorter incubation times. Two major isoforms of the protein were identified in two-dimensional isoelectric focusing/SDS-PAGE of crude extracts and microsomal fractions. The levels of pp18 were enhanced approximately 4-fold by heat shock at 36 degrees C and the elevated pp18 decayed after heat shock was discontinued. pp18 was purified to apparent homogeneity, could be phosphorylated on serine residues, and also exhibited kinase-like activity toward histone H1. The amino acid sequence obtained from a cyanogen bromide digest was greater than 80% identical to nucleoside diphosphate (NDP) kinases from a variety of organisms. Biochemical analysis of the purified protein confirmed the identity as NDP kinase. Thus, NDP kinase appears to be modulated by heat shock in plants. PMID:8016268

  2. Hierarchical self-assembly of switchable nucleolipid supramolecular gels based on environmentally-sensitive fluorescent nucleoside analogs

    NASA Astrophysics Data System (ADS)

    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

  3. Hierarchical self-assembly of switchable nucleolipid supramolecular gels based on environmentally-sensitive fluorescent nucleoside analogs.

    PubMed

    Nuthanakanti, Ashok; Srivatsan, Seergazhi G

    2016-02-14

    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 Hg(2+) 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. PMID:26804191

  4. Distortional binding of transition state analogs to human purine nucleoside phosphorylase probed by magic angle spinning solid-state NMR.

    PubMed

    Vetticatt, Mathew J; Itin, Boris; Evans, Gary B; Schramm, Vern L

    2013-10-01

    Transition state analogs mimic the geometry and electronics of the transition state of enzymatic reactions. These molecules bind to the active site of the enzyme much tighter than substrate and are powerful noncovalent inhibitors. Immucillin-H (ImmH) and 4'-deaza-1'-aza-2'-deoxy-9-methylene Immucillin-H (DADMe-ImmH) are picomolar inhibitors of human purine nucleoside phosphorylase (hPNP). Although both molecules are electronically similar to the oxocarbenium-like dissociative hPNP transition state, DADMe-ImmH is more potent than ImmH. DADMe-ImmH captures more of the transition state binding energy by virtue of being a closer geometric match to the hPNP transition state than ImmH. A consequence of these similarities is that the active site of hPNP exerts greater distortional forces on ImmH than on DADMe-ImmH to "achieve" the hPNP transition state geometry. By using magic angle spinning solid-state NMR to investigate stable isotope-labeled ImmH and DADMe-ImmH, we have explored the difference in distortional binding of these two inhibitors to hPNP. High-precision determinations of internuclear distances from NMR recoupling techniques, rotational echo double resonance, and rotational resonance, have provided unprecedented atomistic insight into the geometric changes that occur upon binding of transition state analogs. We conclude that hPNP stabilizes conformations of these chemically distinct analogs having distances between the cation and leaving groups resembling those of the known transition state. PMID:24043827

  5. Design and synthesis of constrained analogs of LCRF-0004 as potent RON tyrosine kinase inhibitors.

    PubMed

    Raeppel, Stéphane L; Therrien, Eric; Raeppel, Franck

    2015-09-01

    New fused bicyclic lactam head groups as rigidified analogs of thieno[3,2-b]pyridine-based kinase inhibitor LCRF-0004 were designed and synthesized. Depending on the functionalities and the size of these bicyclic head groups, potent inhibitors of RON tyrosine kinase with various level of selectivity against c-Met tyrosine kinase were obtained. PMID:26112445

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

    SciTech Connect

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

    2008-07-31

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

  7. An adenosine kinase inhibitor, ABT-702, inhibits spinal nociceptive transmission by adenosine release via equilibrative nucleoside transporters in rat.

    PubMed

    Otsuguro, Ken-ichi; Tomonari, Yuki; Otsuka, Saori; Yamaguchi, Soichiro; Kon, Yasuhiro; Ito, Shigeo

    2015-10-01

    Adenosine kinase (AK) inhibitor is a potential candidate for controlling pain, but some AK inhibitors have problems of adverse effects such as motor impairment. ABT-702, a non-nucleoside AK inhibitor, shows analgesic effect in animal models of pain. Here, we investigated the effects of ABT-702 on synaptic transmission via nociceptive and motor reflex pathways in the isolated spinal cord of neonatal rats. The release of adenosine from the spinal cord was measured by HPLC. ABT-702 inhibited slow ventral root potentials (sVRPs) in the nociceptive pathway more potently than monosynaptic reflex potentials (MSRs) in the motor reflex pathway. The inhibitory effects of ABT-702 were mimicked by exogenously applied adenosine, blocked by 8CPT (8-cyclopentyl-1,3-dipropylxanthine), an adenosine A1 receptor antagonist, and augmented by EHNA (erythro-9-(2-hydroxy-3-nonyl) adenine), an adenosine deaminase (ADA) inhibitor. Equilibrative nucleoside transporter (ENT) inhibitors reversed the effects of ABT-702, but not those of adenosine. ABT-702 released adenosine from the spinal cord, an effect that was also reversed by ENT inhibitors. The ABT-702-facilitated release of adenosine by way of ENTs inhibits nociceptive pathways more potently than motor reflex pathways in the spinal cord via activation of A1 receptors. This feature is expected to lead to good analgesic effects, but, caution may be required for the use of AK inhibitors in the case of ADA dysfunction or a combination with ENT inhibitors. PMID:26066576

  8. Long Term Expression of Drosophila melanogaster Nucleoside Kinase in Thymidine Kinase 2-deficient Mice with No Lethal Effects Caused by Nucleotide Pool Imbalances*

    PubMed Central

    Krishnan, Shuba; Paredes, João A.; Zhou, Xiaoshan; Kuiper, Raoul V.; Hultenby, Kjell; Curbo, Sophie; Karlsson, Anna

    2014-01-01

    Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2−/−) mice extended the life span of Tk2−/− mice from 3 weeks to at least 20 months. The Dm-dNK+/−Tk2−/− mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK+/−Tk2−/− mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK+/− mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues. PMID:25296759

  9. Modulation of nicotinamide adenine dinucleotide and poly(adenosine diphosphoribose) metabolism by the synthetic "C" nucleoside analogs, tiazofurin and selenazofurin. A new strategy for cancer chemotherapy.

    PubMed Central

    Berger, N A; Berger, S J; Catino, D M; Petzold, S J; Robins, R K

    1985-01-01

    Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and selenazofurin (2-beta-D-ribofuranosylselenazole-4-carboxamide) are synthetic "C" nucleosides whose antineoplastic activity depends on their conversion to tiazofurin-adenine dinucleotide and selenazofurin-adenine dinucleotide which are analogs of NAD. The present study was conducted to determine whether these nucleoside analogs and their dinucleotide derivatives interfere with NAD metabolism and in particular with the NAD-dependent enzyme, poly(ADP-ribose) polymerase. Incubation of L1210 cells with 10 microM tiazofurin or selenazofurin resulted in inhibition of cell growth, reduction of cellular NAD content, and interference with NAD synthesis. Using [14C]nicotinamide to study the uptake of nicotinamide and its conversion to NAD, we showed that the analogs interfere with NAD synthesis, apparently by blocking formation of nicotinamide mononucleotide. The analogs also serve as weak inhibitors of poly(ADP-ribose) polymerase, which is an NAD-utilizing, chromatin-bound enzyme, whose function is required for normal DNA repair processes. Continuous incubation of L1210 cells in tiazofurin or selenazofurin resulted in progressive and synergistic potentiation of the cytotoxic effects of DNA-damaging agents, such as 1,3-bis(2-chloroethyl)-1-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine. These studies provide a basis for designing chemotherapy combinations in which tiazofurin or selenazofurin are used to modulate NAD and poly(ADP-ribose) metabolism to synergistically potentiate the effects of DNA strand-disrupting agents. PMID:3919063

  10. Vanadium-Binding Ability of Nucleoside Diphosphate Kinase from the Vanadium-Rich Fan Worm, Pseudopotamilla occelata.

    PubMed

    Yamaguchi, Nobuo; Yoshinaga, Masafumi; Kamino, Kei; Ueki, Tatsuya

    2016-06-01

    Polychaete fan worms and ascidians accumulate high levels of vanadium ions. Several vanadiumbinding proteins, known as vanabins, have been found in ascidians. However, no vanadium-binding factors have been isolated from the fan worm. In the present study, we sought to identify vanadiumbinding proteins in the branchial crown of the fan worm using immobilized metal ion affinity chromatography. A nucleoside diphosphate kinase (NDK) homolog was isolated and determined to be a vanadium-binding protein. Kinase activity of the NDK homologue, PoNDK, was suppressed by the addition of V(IV), but was unaffected by V(V). The effect of V(IV) on PoNDK precedes its activation by Mg(II). This is the first report to describe the relationship between NDK and V(IV). PoNDK is located in the epidermis of the branchial crown, and its distribution is very similar to that of vanadium. These results suggest that PoNDK is associated with vanadium accumulation and metabolism in P. occelata. PMID:27268980

  11. 1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

    PubMed

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

    1997-05-01

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

  12. Crystal structures of arginine kinase in complex with ADP, nitrate, and various phosphagen analogs.

    PubMed

    Clark, Shawn A; Davulcu, Omar; Chapman, Michael S

    2012-10-12

    Arginine kinase catalyzes the reversible transfer of a phosphoryl group between ATP and l-arginine and is a monomeric homolog of the human enzyme creatine kinase. Arginine and creatine kinases belongs to the phosphagen kinase family of enzymes, which consists of eight known members, each of which is specific for its own phosphagen. Here, the source of phosphagen specificity in arginine kinase is investigated through the use of phosphagen analogs. Crystal structures have been determined for Limulus polyphemus arginine kinase with one of four arginine analogs bound in a transition state analog complex: l-ornithine, l-citrulline, imino-l-ornithine, and d-arginine. In all complexes, the enzyme achieves a closed conformation very similar to that of the cognate transition state analog complex, but differences are observed in the configurations of bound ligands. Arginine kinase exhibits no detectable activity towards ornithine, citrulline, or imino-l-ornithine, and only trace activity towards d-arginine. The crystal structures presented here demonstrate that phosphagen specificity is derived neither from a lock-and-key mechanism nor a modulation of induced-fit conformational changes, but potentially from subtle distortions in bound substrate configurations. PMID:22995310

  13. Purine Nucleoside Analog - Sulfinosine Modulates Diverse Mechanisms of Cancer Progression in Multi-Drug Resistant Cancer Cell Lines

    PubMed Central

    Dačević, Mirjana; Isaković, Aleksandra; Podolski-Renić, Ana; Isaković, Andelka M.; Stanković, Tijana; Milošević, Zorica; Rakić, Ljubisav; Ruždijić, Sabera; Pešić, Milica

    2013-01-01

    Achieving an effective treatment of cancer is difficult, particularly when resistance to conventional chemotherapy is developed. P-glycoprotein (P-gp) activity governs multi-drug resistance (MDR) development in different cancer cell types. Identification of anti-cancer agents with the potential to kill cancer cells and at the same time inhibit MDR is important to intensify the search for novel therapeutic approaches. We examined the effects of sulfinosine (SF), a quite unexplored purine nucleoside analog, in MDR (P-gp over-expressing) non-small cell lung carcinoma (NSCLC) and glioblastoma cell lines (NCI-H460/R and U87-TxR, respectively). SF showed the same efficacy against MDR cancer cell lines and their sensitive counterparts. However, it was non-toxic for normal human keratinocytes (HaCaT). SF induced caspase-dependent apoptotic cell death and autophagy in MDR cancer cells. After SF application, reactive oxygen species (ROS) were generated and glutathione (GSH) concentration was decreased. The expression of key enzyme for GSH synthesis, gamma Glutamyl-cysteine-synthetase (γGCS) was decreased as well as the expression of gst-π mRNA. Consequently, SF significantly decreased the expression of hif-1α, mdr1 and vegf mRNAs even in hypoxic conditions. SF caused the inhibition of P-gp (coded by mdr1) expression and activity. The accumulation of standard chemotherapeutic agent – doxorubicin (DOX) was induced by SF in concentration- and time-dependent manner. The best effect of SF was obtained after 72 h when it attained the effect of known P-gp inhibitors (Dex-verapamil and tariquidar). Accordingly, SF sensitized the resistant cancer cells to DOX in subsequent treatment. Furthermore, SF decreased the experssion of vascular endothelial growth factor (VEGF) on mRNA and protein level and modulated its secretion. In conclusion, the effects on P-gp (implicated in pharmacokinetics and MDR), GSH (implicated in detoxification) and VEGF (implicated in tumor-angiogenesis and

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-07-01

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

  18. [Efficacy of initial antiretroviral therapy based on lopinavir/ritonavir plus 2 nucleoside/nucleotide analogs in patients with human immunodeficiency virus type 1 infection].

    PubMed

    Zamora, Laura; Gatell, José M

    2014-11-01

    Triple combination regimens consisting of lopinavir/ritonavir (LPV/r) plus 2 nucleoside/nucleotide analogs continue to be a valid option in initial antiretroviral therapy. Other protease inhibitors boosted with ritonavir (and in future with cobicistat) have been introduced, as well as other non-nucleoside analogs (rilpivirin) and 3 integrase inhibitors. None of the new regimens have shown superiority over LPV/r or comparisons are lacking. Therefore, regimens including LPV/r continue to be recommended as initial first-line or alternative strategies in most treatment guidelines. Dual combinations with LPV/r (plus raltegravir or lamivudine) are described in another article and can provide a similar response rate to triple combinations, better tolerance, and an improved cost-efficacy ratio, both for initial therapy and in simplification strategies. In contrast, LPV/r or darunavir/r monotherapy does not seem an acceptable option in treatment-naïve patients and is becoming increasingly less acceptable in simplification strategies. PMID:25542868

  19. Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3'-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease.

    PubMed

    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

  20. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A

    PubMed Central

    Borthwick, Lee A.; Kerbiriou, Mathieu; Taylor, Christopher J.; Cozza, Giorgio; Lascu, Ioan; Postel, Edith H.; Cassidy, Diane; Trouvé, Pascal; Mehta, Anil; Robson, Louise; Muimo, Richmond

    2016-01-01

    Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36–54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351–727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia. PMID:26950439

  1. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

    PubMed

    Borthwick, Lee A; Kerbiriou, Mathieu; Taylor, Christopher J; Cozza, Giorgio; Lascu, Ioan; Postel, Edith H; Cassidy, Diane; Trouvé, Pascal; Mehta, Anil; Robson, Louise; Muimo, Richmond

    2016-01-01

    Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia. PMID:26950439

  2. Recognition of CG interrupting site by W-shaped nucleoside analogs (WNA) having the pyrazole ring in an anti-parallel triplex DNA.

    PubMed

    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

  3. Design and synthesis of novel benzoxazole analogs as Aurora B kinase inhibitors.

    PubMed

    An, Ying; Lee, Eun; Yu, Yeongji; Yun, Jieun; Lee, Myeong Youl; Kang, Jong Soon; Kim, Woo-Young; Jeon, Raok

    2016-07-01

    A novel series of benzoxazole analogs was designed and synthesized, and their inhibitory activities against Aurora kinases were evaluated. Some of the tested compounds exhibited a promising activity with respect to the inhibition of Aurora B kinase. A structure-activity relationship study indicated that linker length, regiochemistry, and halogen substitution play important roles in kinase inhibitory potency. The binding modes between representative compounds and Aurora kinases were interpreted through a molecular docking study to explain the inhibitory activity and selectivity for Aurora A and B kinases. Compounds 13l and 13q also show an antiproliferative effect on the human tumor cell lines in a dose-dependent manner. The most potent 13q demonstrated good efficacy in the prostate cancer PC-3 tumor xenograft model. PMID:27209235

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

    PubMed

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

    2014-12-01

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

  5. Post-Translational Phosphorylation of Serine 74 of Human Deoxycytidine Kinase Favors the Enzyme Adopting the Open Conformation Making It Competent for Nucleoside Binding and Release

    SciTech Connect

    Hazra, Saugata; Szewczak, Andrzej; Ort, Stephan; Konrad, Manfred; Lavie, Arnon

    2012-03-26

    Deoxycytidine kinase (dCK) uses either ATP or UTP as a phosphoryl donor to catalyze the phosphorylation of nucleoside acceptors. The kinetic properties of human dCK are modulated in vivo by phosphorylation of serine 74. This residue is a part of the insert region and is distant from the active site. Replacing the serine with a glutamic acid (S74E variant) can mimic phosphorylation of Ser74. To understand how phosphorylation affects the catalytic properties of dCK, we examined the S74E variant of dCK both structurally and kinetically. We observe that the presence of a glutamic acid at position 74 favors the adoption by the enzyme of the open conformation. Glu74 stabilizes the open conformation by directly interacting with the indole side chain of Trp58, a residue that is in the proximity of the base of the nucleoside substrate. The open dCK conformation is competent for the binding of nucleoside but not for phosphoryl transfer. In contrast, the closed conformation is competent for phosphoryl transfer but not for product release. Thus, dCK must make the transition between the open and closed states during the catalytic cycle. We propose a reaction scheme for dCK that incorporates the transition between the open and closed states, and this serves to rationalize the observed kinetic differences between wild-type dCK and the S74E variant.

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

    PubMed

    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

  7. Design, synthesis, and in vitro biological evaluation of novel 6-methyl-7-substituted-7-deaza purine nucleoside analogs as anti-influenza A agents.

    PubMed

    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

  8. Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis.

    PubMed Central

    Sundin, G W; Shankar, S; Chakrabarty, A M

    1996-01-01

    We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-14-->Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined that each of these six mutant Ndks was biochemically active. However, in four cases, the in vivo levels of Ndk were reduced, which consequently affected the growth of 8830R2::Cm in the presence of Tween 20. Two mutant Ndk proteins which could not complement the alginate synthesis defect in 8830R2::Cm were not affected in any characteristic examined in the present study. All of the mutant Ndks characterized which were still biochemically active formed membrane complexes with Pk, resulting in GTP synthesis. Two of the four Ndk activity mutants (His117Gln and Ala125Arg) identified were capable of being truncated to 12 kDa and formed a membrane complex with Pk; however, the complexes formed were inactive for GTP synthesis. The other two Ndk activity mutants could be truncated to 12 kDa but were not detected in membrane fractions. These results further our understanding of the role of Ndk in alginate synthesis and identify amino acid residues in Ndk which have not previously been studied as

  9. The concentrative nucleoside transporter family, SLC28.

    PubMed

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

    2004-02-01

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

  10. The histone deacetylase inhibitor SAHA sensitizes acute myeloid leukemia cells to a combination of nucleoside analogs and the DNA-alkylating agent busulfan.

    PubMed

    Song, Guiyun; Valdez, Benigno C; Li, Yang; Dominguez, Jose R; Corn, Paul; Champlin, Richard E; Andersson, Borje S

    2014-07-01

    Fludarabine (Flu), clofarabine (Clo) and busulfan (Bu) are used in allogeneic hematopoietic stem cell transplant (allo-HSCT). We reported that combining [Flu + Clo + Bu] had a synergistic cytotoxicity in AML cells. We hypothesized that combining [Flu + Clo + Bu] with the histone deacetylase inhibitor SAHA will further enhance cytotoxicity. We exposed the acute myeloid leukemia (AML) cell lines KBM3/Bu250(6) and OCI-AML3 to Flu, Clo, Bu and SAHA alone and in various combinations. [Flu + Clo + Bu + SAHA] resulted in synergistic cytotoxicity, which can be attributed to (1) activated DNA-damage response and cell cycle checkpoint activation through the ATM-CHK2-P53 (or P73) pathway or ATM-CHK2-cdc25-cdc2 pathway, (2) histone modifications and (3) activated apoptosis pathway. The [Flu + Clo + Bu + SAHA] combination causes mitochondrial outer membrane permeabilization, leakage of cytochrome c and Smac/Diablo into the cytosol with caspase activation, and release of apoptosis-inducing factor (AIF) into the nucleus resulting in nuclear fragmentation and cell death. These results provide a mechanistic basis for using SAHA in future clinical trials with double nucleoside analog-busulfan combinations in pretransplant conditioning therapy. PMID:24144307

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

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

    DOE PAGESBeta

    Chauleau, Mathieu; Jacewicz, Agata; Shuman, Stewart

    2015-05-24

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

  13. Crucial roles of thymidine kinase 1 and deoxyUTPase in incorporating the antineoplastic nucleosides trifluridine and 2′-deoxy-5-fluorouridine into DNA

    PubMed Central

    SAKAMOTO, KAZUKI; YOKOGAWA, TATSUSHI; UENO, HIROYUKI; OGUCHI, KEI; KAZUNO, HIROMI; ISHIDA, KEIJI; TANAKA, NOZOMU; OSADA, AKIKO; YAMADA, YUKARI; OKABE, HIROYUKI; MATSUO, KENICHI

    2015-01-01

    Trifluridine (FTD) and 2′-deoxy-5-fluorouridine (FdUrd), a derivative of 5-fluorouracil (5-FU), are antitumor agents that inhibit thymidylate synthase activity and their nucleotides are incorporated into DNA. However, it is evident that several differences occur in the underlying antitumor mechanisms associated with these nucleoside analogues. Recently, TAS-102 (composed of FTD and tipiracil hydrochloride, TPI) was shown to prolong the survival of patients with colorectal cancer who received a median of 2 prior therapies, including 5-FU. TAS-102 was recently approved for clinical use in Japan. These data suggest that the antitumor activities of TAS-102 and 5-FU proceed via different mechanisms. Thus, we analyzed their properties in terms of thymidine salvage pathway utilization, involving membrane transporters, a nucleoside kinase, a nucleotide-dephosphorylating enzyme, and DNA polymerase α. FTD incorporated into DNA with higher efficiency than FdUrd did. Both FTD and FdUrd were transported into cells by ENT1 and ENT2 and were phosphorylated by thymidine kinase 1, which showed a higher catalytic activity for FTD than for FdUrd. deoxyUTPase (DUT) did not recognize dTTP and FTD-triphosphate (F3dTTP), whereas deoxyuridine-triphosphate (dUTP) and FdUrd-triphosphate (FdUTP) were efficiently degraded by DUT. DNA polymerase α incorporated both F3dTTP and FdUTP into DNA at sites aligned with adenine on the opposite strand. FTD-treated cells showed differing nuclear morphologies compared to FdUrd-treated cells. These findings indicate that FTD and FdUrd are incorporated into DNA with different efficiencies due to differences in the substrate specificities of TK1 and DUT, causing abundant FTD incorporation into DNA. PMID:25901475

  14. Crucial roles of thymidine kinase 1 and deoxyUTPase in incorporating the antineoplastic nucleosides trifluridine and 2'-deoxy-5-fluorouridine into DNA.

    PubMed

    Sakamoto, Kazuki; Yokogawa, Tatsushi; Ueno, Hiroyuki; Oguchi, Kei; Kazuno, Hiromi; Ishida, Keiji; Tanaka, Nozomu; Osada, Akiko; Yamada, Yukari; Okabe, Hiroyuki; Matsuo, Kenichi

    2015-01-01

    Trifluridine (FTD) and 2'-deoxy-5-fluorouridine (FdUrd), a derivative of 5-fluorouracil (5-FU), are antitumor agents that inhibit thymidylate synthase activity and their nucleotides are incorporated into DNA. However, it is evident that several differences occur in the underlying antitumor mechanisms associated with these nucleoside analogues. Recently, TAS-102 (composed of FTD and tipiracil hydrochloride, TPI) was shown to prolong the survival of patients with colorectal cancer who received a median of 2 prior therapies, including 5-FU. TAS-102 was recently approved for clinical use in Japan. These data suggest that the antitumor activities of TAS-102 and 5-FU proceed via different mechanisms. Thus, we analyzed their properties in terms of thymidine salvage pathway utilization, involving membrane transporters, a nucleoside kinase, a nucleotide-dephosphorylating enzyme, and DNA polymerase α. FTD incorporated into DNA with higher efficiency than FdUrd did. Both FTD and FdUrd were transported into cells by ENT1 and ENT2 and were phosphorylated by thymidine kinase 1, which showed a higher catalytic activity for FTD than for FdUrd. deoxyUTPase (DUT) did not recognize dTTP and FTD-triphosphate (F3dTTP), whereas deoxyuridine-triphosphate (dUTP) and FdUrd-triphosphate (FdUTP) were efficiently degraded by DUT. DNA polymerase α incorporated both F3dTTP and FdUTP into DNA at sites aligned with adenine on the opposite strand. FTD-treated cells showed differing nuclear morphologies compared to FdUrd-treated cells. These findings indicate that FTD and FdUrd are incorporated into DNA with different efficiencies due to differences in the substrate specificities of TK1 and DUT, causing abundant FTD incorporation into DNA. PMID:25901475

  15. Base-Modified Nucleosides: Etheno Derivatives

    PubMed Central

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

    2016-01-01

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

  16. Synthesis and Anti-HIV-1 Evaluation of Some Novel MC-1220 Analogs as Non-Nucleoside Reverse Transcriptase Inhibitors.

    PubMed

    Loksha, Yasser M; Pedersen, Erik B; Loddo, Roberta; La Colla, Paolo

    2016-05-01

    Some novel MC-1220 analogs were synthesized by condensation of 4,6-dichloro-N-methylpyrimidin-2-amine derivatives (1a,b and 15) and/or 4-chloro-6-methoxy-N,N,5-trimethylpyrimidin-2-amine (2a) with the sodium salt of 2,6-difluorophenylacetonitrile followed by treatment with aqueous sodium hydroxide in methanol, alkylation, reduction, halogenation, and/or acidic hydrolysis. All synthesized compounds were evaluated for their activity against HIV-1. The most active compound in this study was compound 7, which showed activity against HIV-1 comparable to that of MC-1220. The only difference in structure between compound 7 and MC-1220 is a fluoro atom instead of a CH3 group. PMID:26996241

  17. Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene

    SciTech Connect

    Dube, D.K.; Parker, J.D.; French, D.C.; Cahill, D.S.; Dube, S.; Horwitz, M.S.Z.; Munir, K.M.; Loeb, L.A. )

    1991-12-24

    The authors have obtained 42 active artificial mutants of HSV-1 thymidine kinase by replacing codons 166 and 167 with random nucleotide sequences. Codons 166 and 167 are within the putative nucleoside binding site in the HSV-1 tk gene. The spectrum of active mutations indicates that neither Ile{sup 166} nor Ala{sup 167} is absolutely required for thymidine kinase activity. Each of these amino acids can be replaced by some but not all of the 19 other amino acids. The active mutants can be classified as high activity or low activity on two bases: (1) growth of Escherichia coli KY895 in the presence of thymidine and (2) uptake of thymidine by this strain, when harboring plasmids with the random insertions. E. coli KY895 harboring high-activity plasmids or wild-type plasmids can grow in the presence of low amounts of thymidine but are unable to grow in the presence of high amounts of thymidine. The high-activity plasmids also have an enhanced ({sup 3}H)dT uptake. The amounts of thymidine kinase activity in vitro in unfractionated extracts do not correlate with either growth at low thymidine concentration or the rate of thymidine uptake. Heat inactivation studies indicate that the mutant enzymes are without exception more temperature-sensitive than the wild-type enzyme. This thermolability could account for the less than expected thymidine kinase activity in the extracts and suggests that amino acid substitutions at Ile{sup 166} and Ala{sup 167} have produced major changes in protein stability.

  18. Cytokine genotype suggests a role for inflammation in nucleoside analog-associated sensory neuropathy (NRTI-SN) and predicts an individual's NRTI-SN risk.

    PubMed

    Cherry, Catherine L; Rosenow, Ann; Affandi, Jacquita S; McArthur, Justin C; Wesselingh, Steven L; Price, Patricia

    2008-02-01

    Nucleoside analog-associated sensory neuropathy (NRTI-SN) attributed to stavudine, didanosine, or zalcitabine (the dNRTIs) and distal sensory polyneuropathy (DSP) attributed to HIV are clinically indistinguishable. As inflammatory cytokines are involved in DSP, we addressed a role for inflammation in NRTI-SN by determining the alleles of immune-related genes carried by patients with and without NRTI-SN. Demographic details associated with risk of various neuropathies were included in the analysis. Alleles of 14 polymorphisms in 10 genes were determined in Australian HIV patients with definite NRTI-SN (symptom onset <6 months after first dNRTI exposure, n = 16), NRTI-SN-resistant patients (no neuropathy despite >6 months on dNRTIs, n = 20), patients with late onset NRTI-SN (neuropathy onset after >6 months of dNRTIs, n = 19), and HIV-negative controls. Carriage of TNFA-1031*2 was highest in NRTI-SN patients, suggesting potentiation of NRTI-SN. Carriage of IL12B (3' UTR)*2 was higher in NRTI-SN-resistant patients than controls or NRTI-SN patients, suggesting a protective role. BAT1 (intron 10)*2 was more common in NRTI-SN than resistant patients, but neither group differed from controls. This marks the conserved HLA-A1, B8, DR3 haplotype. Of the demographic details considered, increasing height was associated with NRTI-SN risk. A model including cytokine genotype and height predicted NRTI-SN status (p < 0.0001, R(2) = 0.54). Late onset NRTI-SN patients clustered genetically with NRTI-SN-resistant patients, so these patients may be genetically "protected." In addition to patient height, cytokine genotype influenced NRTI-SN risk following dNRTI exposure, suggesting inflammation contributes to NRTI-SN. PMID:18240960

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

    SciTech Connect

    Chauleau, Mathieu; Jacewicz, Agata; Shuman, Stewart

    2015-05-24

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

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

    SciTech Connect

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

    2010-07-16

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

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

    PubMed Central

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

    2014-01-01

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

  2. Simultaneous quantitative determination of 5-aza-2'-deoxycytidine genomic incorporation and DNA demethylation by liquid chromatography tandem mass spectrometry as exposure-response measures of nucleoside analog DNA methyltransferase inhibitors.

    PubMed

    Anders, Nicole M; Liu, Jianyong; Wanjiku, Teresia; Giovinazzo, Hugh; Zhou, Jianya; Vaghasia, Ajay; Nelson, William G; Yegnasubramanian, Srinivasan; Rudek, Michelle A

    2016-06-01

    The epigenetic and anti-cancer activities of the nucleoside analog DNA methyltransferase (DNMT) inhibitors decitabine (5-aza-2'-deoxycytidine, DAC), azacitidine, and guadecitabine are thought to require cellular uptake, metabolism to 5-aza-2'-deoxycytidine triphosphate, and incorporation into DNA. This genomic incorporation can then lead to trapping and degradation of DNMT enzymes, and ultimately, passive loss of DNA methylation. To facilitate measurement of critical exposure-response relationships of nucleoside analog DNMT inhibitors, a sensitive and reliable method was developed to simultaneously quantitate 5-aza-2'-deoxycytidine genomic incorporation and genomic 5-methylcytosine content using LC-MS/MS. Genomic DNA was extracted and digested into single nucleosides. Chromatographic separation was achieved with a Thermo Hyperpcarb porous graphite column (100mm×2.1mm, 5μm) and isocratic elution with a 10mM ammonium acetate:acetonitrile with 0.1% formic acid (70:30, v/v) mobile phase over a 5min total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5-aza-2'-deoxycytidine, 2'-deoxycytidine, and 5-methyl-2'-deoxycytidine. The assay range was 2-400ng/mL for 5-aza-2'-deoxycytidine, 50-10,000ng/mL for 2'-deoxycytidine, and was 5-1000ng/mL for 5-methyl-2'-deoxycytidine. The assay proved to be accurate (93.0-102.2%) and precise (CV≤6.3%) across all analytes. All analytes exhibited long-term frozen digest matrix stability at -70°C for at least 117 days. The method was applied for the measurement of genomic 5-aza-2'-deoxycytidine and 5-methyl-2'-deoxycytidine content following exposure of in vitro cell culture and in vivo animal models to decitabine. PMID:27082761

  3. The Cytidine Analog Fluorocyclopentenylcytosine (RX-3117) Is Activated by Uridine-Cytidine Kinase 2.

    PubMed

    Sarkisjan, Dzjemma; Julsing, Joris R; Smid, Kees; de Klerk, Daniël; van Kuilenburg, André B P; Meinsma, Rutger; Lee, Young B; Kim, Deog J; Peters, Godefridus J

    2016-01-01

    Fluorocyclopentenylcytosine (RX-3117) is an orally available cytidine analog, currently in Phase I clinical trial. RX-3117 has promising antitumor activity in various human tumor xenografts including gemcitabine resistant tumors. RX-3117 is activated by uridine-cytidine kinase (UCK). Since UCK exists in two forms, UCK1 and UCK2, we investigated which form is responsible for RX-3117 phosphorylation. For that purpose we transfected A549 and SW1573 cell lines with UCK-siRNAs. Transfection of UCK1-siRNA efficiently downregulated UCK1-mRNA, but not UCK2-mRNA expression, and did not affect sensitivity to RX-3117. However, transfection of UCK2-siRNA completely downregulated UCK2-mRNA and protein and protected both A549 and SW1573 against RX-3117. UCK enzyme activity in two panels of tumor cell lines and xenograft cells correlated only with UCK2-mRNA expression (r = 0.803 and 0.915, respectively), but not with UCK1-mRNA. Moreover, accumulation of RX-3117 nucleotides correlated with UCK2 expression. In conclusion, RX-3117 is activated by UCK2 which may be used to select patients potentially sensitive to RX-3117. PMID:27612203

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

    PubMed

    van Kuilenburg, André B P; Meinsma, Rutger

    2016-09-01

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

  5. Imatinib Analogs as Potential Agents for PET Imaging of Bcr-Abl/c-KIT Expression at a Kinase Level

    PubMed Central

    Peng, Zhenghong; Maxwell, David S.; Sun, Duoli; Bhanu Prasad, Basvoju A.; Pal, Ashutosh; Wang, Shimei; Balatoni, Julius; Ghosh, Pradip; Lim, Seok T.; Volgin, Andrei; Shavrin, Aleksander; Alauddin, Mian M.; Gelovani, Juri G.; Bornmann, William G.

    2014-01-01

    We synthesized two series of imatinib mesylate (STI-571) analogs to develop a Bcr-Abl and c-KIT receptor-specific labeling agent for positron emission tomography (PET) imaging to measure Bcr-Abl and c-KIT expression levels in a mouse model. The methods of molecular modeling, synthesis of STI-571 and its analogs, in vitro kinase assays, and radiolabeling are described. Molecular modeling revealed that these analogs bind the same Bcr-Abl and c-KIT binding sites as those bound by STI-571. The analogs potently inhibit the tyrosine kinase activity of Bcr-Abl and c-KIT, similarly to STI-571. [18F]-labeled STI-571 was prepared with high specific activity (75 GBq/μmol) by nucleophilic displacement and an average radiochemical yield of 12%. [131I]-labeled STI-571 was prepared with high purity (>95%) and an average radiochemical yield of 23%. The uptake rates of [18F]-STI-571 in K562 cells expressing Abl and in U87WT cells overexpressing c-KIT were significantly higher than those in the U87 cell and could be inhibited by STI-71 (confirming the specificity of uptake). PET scans of K562 and U87WT tumor-bearing mice with [18F]-STI-571 as a contrast agent showed visible tumor uptake and tumor-to-non-target contrast. PMID:24280068

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

    PubMed

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

    2015-10-20

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

  7. Computer-Guided Design, Synthesis, and Protein Kinase C Affinity of a New Salicylate-Based Class of Bryostatin Analogs

    PubMed Central

    2015-01-01

    Bryostatin 1 is in clinical trials for the treatment of cancer and Alzheimer’s disease and is a candidate for a first-in-class approach to HIV/AIDS eradication. It is neither readily available nor optimally suited for clinical use. Using a function oriented synthesis strategy, a new class of bryostatin-inspired analogs was designed with a simplified salicylate-derived subunit, enabling step-economical synthesis (23 total steps) of agents exhibiting bryostatin-like affinity to protein kinase C (PKC). PMID:25238583

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

    PubMed

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

    2005-01-01

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

  9. Novel epigallocatechin gallate (EGCG) analogs activate AMP-activated protein kinase pathway and target cancer stem cells

    PubMed Central

    Chen, Di; Pamu, Sreedhar; Cui, Qiuzhi; Chan, Tak Hang; Dou, Q. Ping

    2012-01-01

    AMP-activated protein kinase (AMPK) is a critical monitor of cellular energy status and also controls processes related to tumor development, including cell cycle progression, protein synthesis, cell growth and survival. Therefore AMPK as an anti-cancer target has received intensive attention recently. It has been reported that the anti-diabetic drug metformin and some natural compounds, such as quercetin, genistein, capsaicin and green tea polyphenol epigallocatechin gallate (EGCG), can activate AMPK and inhibit cancer cell growth. Indeed, natural products have been the most productive source of leads for the development of anti-cancer drugs but perceived disadvantages, such as low bioavailability and week potency, have limited their development and use in the clinic. In this study we demonstrated that synthetic EGCG analogs 4 and 6 were more potent AMPK activators than metformin and EGCG. Activation of AMPK by these EGCG analogs resulted in inhibition of cell proliferation, up-regulation of the cyclin-dependent kinase inhibitor p21, down-regulation of mTOR pathway, and suppression of stem cell population in human breast cancer cells. Our findings suggest that novel potent and specific AMPK activators can be discovered from natural and synthetic sources that have potential to be used for anti-cancer therapy in the clinic. PMID:22459208

  10. Structural modifications of nucleosides in ionic liquids

    PubMed Central

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

    2011-01-01

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

  11. Synthesis and In Vitro Evaluation of 5-[18F]Fluoroalkyl Pyrimidine Nucleosides for Molecular Imaging of Herpes Simplex Virus Type-1 Thymidine Kinase Reporter Gene Expression

    PubMed Central

    Chacko, Ann-Marie; Qu, Wenchao; Kung, Hank F.

    2014-01-01

    Two novel series of 5-fluoroalkyl-2′-deoxyuridines (FPrDU, FBuDU, FPeDU) and 2′-fluoro-2′-deoxy-5-fluoroalkylarabinouridines (FFPrAU, FFBuAU, FFPeAU), having three, four or five methylene units (propyl, butyl, or pentyl) at C-5, were prepared and tested as reporter probes for imaging HSV1-tk gene expression. The Negishi coupling methodology was employed to efficiently synthesize the radiolabeling precursors. All six 5-[18F]fluoroalkyl pyrimidines were prepared readily from 3-N-benzoyl-3′,5′-di-O-benzoyl-protected 5-O-mesylate precursors in 17–35% radiochemical yield (decay-corrected). In vitro studies highlighted that all six [18F]labeled nucleosides selectively accumulated in cells expressing the HSV1-TK protein, with negligible uptake in control cells. [18F]FPrDU, [18F]FBuDU, [18F]FPeDU, and [18F]FFBuAU had the best uptake profiles. Despite selective accumulation in HSV1-tk expressing cells, all 5-fluoroalkyl pyrimidine nucleosides had low to negligible cytotoxic activity (CC50>1000–209 μM). Ultimately, results demonstrated that 5-[18F]fluoropropyl, [18F]fluorobutyl, and [18F]fluoropentyl pyrimidine nucleosides have potential as in vivo HSV1-TK PET reporter probes over a dynamic range of reporter gene expression levels. PMID:18800764

  12. NZ51, a ring-expanded nucleoside analog, inhibits motility and viability of breast cancer cells by targeting the RNA helicase DDX3

    PubMed Central

    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 properties of NZ51 in MCF-7 and MDA-MB-231 breast cancer cell lines. NZ51 treatment decreased cellular motility and cell viability of MCF-7 and MDA-MB-231 cells with IC50 values in the low micromolar range. Biological knockdown of DDX3 in MCF-7 and MDA-MB-231 cells resulted in decreased proliferation rates and reduced clonogenicity. In addition, NZ51 was effective in killing breast cancer cells under hypoxic conditions with the same potency as observed during normoxia. Mechanistic studies indicated that NZ51 did not cause DDX3 degradation, but greatly diminished its functionality. Moreover, in vivo experiments demonstrated that DDX3 knockdown by shRNA resulted in reduced tumor volume and metastasis without altering tumor vascular volume or permeability-surface area. In initial in vivo experiments, NZ51 treatment did not significantly reduce tumor volume. Further studies are needed to optimize drug formulation, dose and delivery. Continuing work will determine the in vitro-in vivo correlation of NZ51 activity and its utility in a clinical setting. PMID:26337079

  13. Triptycene analogs

    NASA Technical Reports Server (NTRS)

    Hua, Duy (Inventor); Perchellet, Jean-Pierre (Inventor)

    2004-01-01

    This invention provides analogs of triptycene which are useful as anticancer drugs, as well as for other uses. The potency of these compounds is in a similar magnitude as daunomycin, a currently used anticancer drug. Each compound of the invention produces one or more desired effects (blocking nucleoside transport, inhibiting nucleic acid or protein syntheses, decreasing the proliferation and viability of cancer cells, inducing DNA fragmentation or retaining their effectiveness against multidrug-resistant tumor cells).

  14. Enhanced Degradation of Dihydrofolate Reductase through Inhibition of NAD Kinase by Nicotinamide Analogs

    PubMed Central

    Hsieh, Yi-Ching; Tedeschi, Philip; AdeBisi Lawal, Rialnat; Banerjee, Debabrata; Scotto, Kathleen; Kerrigan, John E.; Lee, Kuo-Chieh; Johnson-Farley, Nadine; Bertino, Joseph R.

    2013-01-01

    Dihydrofolate reductase (DHFR), because of its essential role in DNA synthesis, has been targeted for the treatment of a wide variety of human diseases, including cancer, autoimmune diseases, and infectious diseases. Methotrexate (MTX), a tight binding inhibitor of DHFR, is one of the most widely used drugs in cancer treatment and is especially effective in the treatment of acute lymphocytic leukemia, non-Hodgkin’s lymphoma, and osteosarcoma. Limitations to its use in cancer include natural resistance and acquired resistance due to decreased cellular uptake and decreased retention due to impaired polyglutamylate formation and toxicity at higher doses. Here, we describe a novel mechanism to induce DHFR degradation through cofactor depletion in neoplastic cells by inhibition of NAD kinase, the only enzyme responsible for generating NADP, which is rapidly converted to NADPH by dehydrogenases/reductases. We identified an inhibitor of NAD kinase, thionicotinamide adenine dinucleotide phosphate (NADPS), which led to accelerated degradation of DHFR and to inhibition of cancer cell growth. Of importance, combination treatment of NADPS with MTX displayed significant synergy in a metastatic colon cancer cell line and was effective in a MTX-transport resistant leukemic cell line. We suggest that NAD kinase is a valid target for further inhibitor development for cancer treatment. PMID:23197646

  15. Nucleoside phosphorylation by the mineral schreibersite

    PubMed Central

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

    2015-01-01

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

  16. Highly ordered crystals of channel-forming membrane proteins, of nucleoside-monophosphate kinases, of FAD-containing oxidoreductases and of sugar-processing enzymes and their mutants

    NASA Astrophysics Data System (ADS)

    Schulz, G. E.; Dreyer, M.; Klein, C.; Kreusch, A.; Mittl, P.; Mu¨ller, C. W.; Mu¨ller-Dieckmann, J.; Muller, Y. A.; Proba, K.; Schlauderer, G.; Spu¨rgin, P.; Stehle, T.; Weiss, M. S.

    1992-08-01

    Preparation and crystallization procedures as well as crystal properties are reported for 12 proteins plus numerous site-directed mutants. The proteins are: the integral membrane protein porin from Rhodobacter capsulatus which diffracts to at least 1.8A˚resolution, porin from Rhodopseudomonas blastica which diffracts to at least 2.0A˚resolution, adenylate kinase from yeast and mutants, adenylate kinase from Escherichia coli and mutants, bovine liver mitochondrial adenylate kinase, guanylate kinase from yeast, uridylate kinase from yeast, glutathione reductase from E. coli and mutants, NADH peroxidase from Streptococcus faecalis containing a sulfenic acid as redox-center, pyruvate oxidase from Lactobacillus plantarum containing FAD and TPP, cyclodextrin glycosyltransferase from Bacillus circulans and mutants, and a fuculose aldolase from E. coli.

  17. Computer-assisted combinatorial design of bicyclic thymidine analogs as inhibitors of Mycobacterium tuberculosis thymidine monophosphate kinase.

    PubMed

    Frecer, Vladimir; Seneci, Pierfausto; Miertus, Stanislav

    2011-01-01

    Thymidine monophosphate kinase (TMPK(mt)) is an essential enzyme for nucleotide metabolism in Mycobacterium tuberculosis, and thus an attractive target for novel antituberculosis agents. In this work, we have explored the chemical space around the 2',3'-bicyclic thymidine nucleus by designing and in silico screening of a virtual focused library selected via structure based methods to identify more potent analogs endowed with favorable ADME-related properties. In all the library members we have exchanged the ribose ring of the template with a cyclopentane moiety that is less prone to enzymatic degradation. In addition, we have replaced the six-membered 2',3'-ring by a number of five-membered and six-membered heterocyclic rings containing alternative proton donor and acceptor groups, to exploit the interaction with the carboxylate groups of Asp9 and Asp163 as well as with several cationic residues present in the vicinity of the TMPK(mt) binding site. The three-dimensional structure of the TMPK(mt) complexed with 5-hydroxymethyl-dUMP, an analog of dTMP, was employed to develop a QSAR model, to parameterize a scoring function specific for the TMPK(mt) target and to select analogues which display the highest predicted binding to the target. As a result, we identified a small highly focused combinatorial subset of bicyclic thymidine analogues as virtual hits that are predicted to inhibit the mycobacterial TMPK in the submicromolar concentration range and to display favorable ADME-related properties. PMID:21082329

  18. Synthesis of Pelorol and Its Analogs and Their Inhibitory Effects on Phosphatidylinositol 3-Kinase

    PubMed Central

    Luo, Yongjie; Chen, Huixuan; Weng, Jiang; Lu, Gui

    2016-01-01

    There are numerous biologically active substances with novel structures and unique physiological functions in marine organisms. These substances are important sources of new lead compounds. Pelorol is a natural product isolated from marine organisms that possesses a novel structure with high bioactivity. In this paper, the synthesis of pelorol has been completed, and the synthesis of some intermediates has been optimized and scaled up. Five pelorol analogs have also been prepared. Preliminary biological activity testing demonstrated that compounds 5 and 6 might be potential lead compounds for cancer therapy. PMID:27338420

  19. Piperazine-based nucleic acid analogs

    DOEpatents

    Schmidt, Jurgen; Silks, Louis A.; Michalczyk, Ryszard

    2005-01-11

    A novel nucleoside analog is disclosed which comprises a piperazine ring in the place of the ring ribose or deoxyribose sugar. Monomers utilizing a broad variety of nucleobases are disclosed, as well as oligomers comprising the monomers disclosed herein linked by a variety of linkages, including amide, phosphonamide, and sulfonamide linkages. A method of synthesizing the nucleoside analogs is also disclosed.

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

    PubMed

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

    2016-01-01

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

  1. Synthesis and characterization of N-parinaroyl analogs of ganglioside GM3 and de-N-acetyl GM3. Interactions with the EGF receptor kinase

    NASA Technical Reports Server (NTRS)

    Song, W.; Welti, R.; Hafner-Strauss, S.; Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    A specific plasma membrane glycosphingolipid, known as ganglioside GM3, can regulate the intrinsic tyrosyl kinase activity of the epidermal growth factor (EGF) receptor; this modulation is not associated with alterations in hormone binding to the receptor. GM3 inhibits EGF receptor tyrosyl kinase activity in detergent micelles, in plasma membrane vesicles, and in whole cells. In addition, immunoaffinity-purified EGF receptor preparations contain ganglioside GM3 (Hanai et al. (1988) J. Biol. Chem. 263, 10915-10921), implying that the glycosphingolipid is intimately associated with the receptor kinase in cell membranes. Both the nature of this association and the molecular mechanism of kinase inhibition remain to be elucidated. In this report, we describe the synthesis of a fluorescent analog of ganglioside GM3, in which the native fatty acid was replaced with trans-parinaric acid. This glycosphingolipid inhibited the receptor kinase activity in a manner similar to that of the native ganglioside. A modified fluorescent glycosphingolipid, N-trans-parinaroyl de-N-acetyl ganglioside GM3, was also prepared. This analog, like the nonfluorescent de-N-acetyl ganglioside GM3, had no effect on receptor kinase activity. Results from tryptophan fluorescence quenching and steady-state anisotropy measurements in membranes containing these fluorescent probes and the human EGF receptor were consistent with the notion that GM3, but not de-N-acetyl GM3, interacts specifically with the receptor in intact membranes.

  2. Effect of fluoxetine and pergolide on expression of nucleoside transporters and nucleic-related enzymes in mouse brain.

    PubMed

    Nagai, Katsuhito; Konishi, Hiroki

    2014-04-01

    Nucleoside transporter (NT) and nucleic-related enzyme (NRE) play key roles in the physiology of nucleosides and the pharmacology of its analogs in mammals. In this study, we examined the effect of fluoxetine, a selective serotonin reuptake inhibitor, and pergolide, a dopamine D receptor agonist, on the expression of NTs and NREs in mouse brain. It was confirmed by the detection of corresponding mRNAs that three equilibrative nucleoside transporter (ENT1-3) isoforms, concentrative nucleoside transporter 2 (CNT2), CNT3, adenosine kinase (AK), and apyrase, but not CNT1, were expressed in brain tissue. Based on an assessment by mRNA determination, the cerebral expression of CNT2 was found to be increased by administration of fluoxetine and pergolide to mice. Furthermore, pergolide increased the expression of ENT2. However, fluoxetine and pergolide had no significant effect on the expression of mRNA for other NTs, AK, and apyrase. Therefore, we concluded that the expression of several NT isoforms, but not NREs, in mouse brain was affected by treatment with fluoxetine and pergolide. PMID:23130601

  3. Anti-parallel triplexes: Synthesis of 8-aza-7-deazaadenine nucleosides with a 3-aminopropynyl side-chain and its corresponding LNA analog.

    PubMed

    Kosbar, Tamer R; Sofan, Mamdouh A; Waly, Mohamed A; Pedersen, Erik B

    2015-05-15

    The phosphoramidites of DNA monomers of 7-(3-aminopropyn-1-yl)-8-aza-7-deazaadenine (Y) and 7-(3-aminopropyn-1-yl)-8-aza-7-deazaadenine LNA (Z) are synthesized, and the thermal stability at pH 7.2 and 8.2 of anti-parallel triplexes modified with these two monomers is determined. When, the anti-parallel TFO strand was modified with Y with one or two insertions at the end of the TFO strand, the thermal stability was increased 1.2°C and 3°C at pH 7.2, respectively, whereas one insertion in the middle of the TFO strand decreased the thermal stability 1.4°C compared to the wild type oligonucleotide. In order to be sure that the 3-aminopropyn-1-yl chain was contributing to the stability of the triplex, the nucleobase X without the aminopropynyl group was inserted in the same positions. In all cases the thermal stability was lower than the corresponding oligonucleotides carrying the 3-aminopropyn-1-yl chain, especially at the end of the TFO strand. On the other hand, the thermal stability of the anti-parallel triplex was dramatically decreased when the TFO strand was modified with the LNA monomer analog Z in the middle of the TFO strand (ΔTm=-9.1°C). Also the thermal stability decreased about 6.1°C when the TFO strand was modified with Z and the Watson-Crick strand with adenine-LNA (A(L)). The molecular modeling results showed that, in case of nucleobases Y and Z a hydrogen bond (1.69 and 1.72Ǻ, respectively) was formed between the protonated 3-aminopropyn-1-yl chain and one of the phosphate groups in Watson-Crick strand. Also, it was shown that the nucleobase Y made a good stacking and binding with the other nucleobases in the TFO and Watson-Crick duplex, respectively. In contrast, the nucleobase Z with LNA moiety was forced to twist out of plane of Watson-Crick base pair which is weakening the stacking interactions with the TFO nucleobases and the binding with the duplex part. PMID:25868748

  4. Crystal structure of human nicotinamide riboside kinase.

    PubMed

    Khan, Javed A; Xiang, Song; Tong, Liang

    2007-08-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD(+) as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 A resolution and in a ternary complex with ADP and tiazofurin at 2.7 A resolution. The active site is located in a groove between the central parallel beta sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations. PMID:17698003

  5. Crystal Structure of Human Nicotinamide Riboside Kinase

    SciTech Connect

    Khan,J.; Xiang, S.; Tong, L.

    2007-01-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD{sup +} as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 {angstrom} resolution and in a ternary complex with ADP and tiazofurin at 2.7 {angstrom} resolution. The active site is located in a groove between the central parallel {beta} sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

  6. Association of Efavirenz Hypersusceptibility with Virologic Response in ACTG 368, a Randomized Trial of Abacavir (ABC) in Combination with Efavirenz (EFV) and Indinavir (IDV) in HIV-infected Subjects with Prior Nucleoside Analog Experience

    PubMed Central

    Demeter, Lisa M.; DeGruttola, Victor; Lustgarten, Stephanie; Bettendorf, Daniel; Fischl, Margaret; Eshleman, Susan; Spreen, William; Nguyen, Bach-Yen; Koval, Christine E.; Eron, Joseph J.; Hammer, Scott; Squires, Kathleen

    2010-01-01

    Purpose To evaluate the association of efavirenz hypersusceptibility (EFV-HS) with clinical outcome in a double-blind, placebo-controlled, randomized trial of EFV plus indinavir (EFV+IDV) vs. EFV+IDV plus abacavir (ABC) in 283 nucleoside-experienced HIV-infected patients. Methods and Results Rates of virologic failure were similar in the 2 arms at week 16 (p=0.509). Treatment discontinuations were more common in the ABC arm (p=0.001). Using logistic regression, there was no association between virologic failure and either baseline ABC resistance or regimen sensitivity score. Using 3 different genotypic scoring systems, EFV-HS was significantly associated with reduced virologic failure at week 16, independent of treatment assignment. In some patients on the nucleoside-sparing arm, the nucleoside-resistant mutant L74V was selected for in combination with the uncommonly occurring EFV-resistant mutant K103N+L100I; L74V was not detected as a minority variant, using clonal sequence analysis, when the nucleoside-sparing regimen was initiated. Conclusions Premature treatment discontinuations in the ABC arm and the presence of EFV-hypersusceptible HIV variants in this patient population likely made it difficult to detect a benefit of adding ABC to EFV+IDV. In addition, L74V, when combined with K103N+L100I, may confer a selective advantage to the virus that is independent of its effects on nucleoside resistance. PMID:18215978

  7. Structural optimization of non-nucleoside DNA methyltransferase inhibitor as anti-cancer agent.

    PubMed

    Zhong, Bo; Vatolin, Sergei; Idippily, Nethrie D; Lama, Rati; Alhadad, Laila A; Reu, Frederic J; Su, Bin

    2016-02-15

    Inhibition of DNA methyltransferase 1 (DNMT1) can reverse the malignant behavior of cancer cells by restoring expression of aberrantly silenced genes that are required for differentiation, senescence, and apoptosis. Clinically used DNMT1 inhibitors decitabine and azacitidine inhibit their target by covalent trapping after incorporation into DNA as azacytidine analogs. These nucleoside compounds are prone to rapid enzymatic inactivation in blood, posing challenges to the development of purely epigenetic dosing schedules. Non-nucleoside compounds that suppress expression or function of DNMT1 may overcome this problem. Using a high-throughput PCR-based site specific chromatin condensation assay, we identified a compound that reactivated Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) in myeloma cells and suppressed expression of DNMT1 from a library of 5120 chemically diverse small molecules. Lead optimization was performed to generate 26 new analogs with lung cancer proliferation and DNMT1 expression as activity readout. Two of the new derivatives showed 2 fold improvement of growth inhibiting potency and also decreased DNMT1 protein levels in lung cancer cells. PMID:26774653

  8. CCM3/PDCD10 Heterodimerizes with Germinal Center Kinase III (GCKIII) Proteins Using a Mechanism Analogous to CCM3 Homodimerization*♦

    PubMed Central

    Ceccarelli, Derek F.; Laister, Rob C.; Mulligan, Vikram Khipple; Kean, Michelle J.; Goudreault, Marilyn; Scott, Ian C.; Derry, W. Brent; Chakrabartty, Avijit; Gingras, Anne-Claude; Sicheri, Frank

    2011-01-01

    CCM3 mutations give rise to cerebral cavernous malformations (CCMs) of the vasculature through a mechanism that remains unclear. Interaction of CCM3 with the germinal center kinase III (GCKIII) subfamily of Sterile 20 protein kinases, MST4, STK24, and STK25, has been implicated in cardiovascular development in the zebrafish, raising the possibility that dysregulated GCKIII function may contribute to the etiology of CCM disease. Here, we show that the amino-terminal region of CCM3 is necessary and sufficient to bind directly to the C-terminal tail region of GCKIII proteins. This same region of CCM3 was shown previously to mediate homodimerization through the formation of an interdigitated α-helical domain. Sequence conservation and binding studies suggest that CCM3 may preferentially heterodimerize with GCKIII proteins through a manner structurally analogous to that employed for CCM3 homodimerization. PMID:21561863

  9. 5-[18F]Fluoroalkyl Pyrimidine Nucleosides: Probes for PET Imaging of Herpes Simplex Virus Type-1 Thymidine Kinase Gene Expression

    PubMed Central

    Chacko, Ann-Marie; Blankemeyer, Eric; Lieberman, Brian P.; Qu, Wenchao; Kung, Hank F.

    2009-01-01

    Introduction The preliminary in vivo evaluation of novel 5-[18F]fluoroalkyl-2’-deoxyuridines ([18F]FPrDU, [18F]FBuDU, [18F]FPeDU; [18F]1a–c, respectively) and 2’-fluoro-2’-deoxy-5-[18F]fluoroalkyl-1-β-D-arabinofuranosyl uracils ([18F]FFPrAU, [18F]FFBuAU, [18F]FFPeAU; [18F]1d–f, respectively) as probes for imaging herpes simplex virus type-1 thymidine kinase (HSV1-tk) gene expression are described. Methods [18F]1a–f were successfully synthesized by a rapid and efficient two step one-pot nucleophilic fluorination reaction using 5-O-mesylate precursors and [18F]F-. For in vivo studies, tumor xenografts were grown in nude mice by implanting RG2 cells stably expressing HSV1-tk (RG2TK+) and wild-type cells (RG2). Results Biodistribution studies at 2 h p.i. revealed that the uptake of [18F]1a–b and [18F]1d–e in RG2TK+ tumors was not significantly different from control tumors. However, [18F]1c and [18F]1f had an average 1.6 and 1.7–fold higher uptake in RG2TK+ tumors than control RG2 tumors. Blood activity curves for [18F]1c and [18F]1f highlight rapid clearance of radioactivity in the blood. Dynamic small animal PET (A-PET) imaging studies of tumor-bearing mice with [18F]1c and [18F]1f showed higher initial uptake (3.5–fold and 1.4–fold, respectively) in RG2TK+ tumors than control tumors, with continued washout of activity from both tumors over time. Conclusions Biological evaluations suggest that [18F]1c and [18F]1f may have limited potential for imaging HSV1-tk gene expression due fast washout of activity from the blood thus significantly decreasing sensitivity and specificity of tracer accumulation in HSV1-tk expressing tumors. PMID:19181266

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

    PubMed Central

    Zammit, V A; Newsholme, E A

    1976-01-01

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

  11. Lack of enantiospecificity of human 2'-deoxycytidine kinase: relevance for the activation of beta-L-deoxycytidine analogs as antineoplastic and antiviral agents.

    PubMed

    Verri, A; Focher, F; Priori, G; Gosselin, G; Imbach, J L; Capobianco, M; Garbesi, A; Spadari, S

    1997-01-01

    We demonstrate that human 2'-deoxycytidine kinase (dCK) is a nonenantioselective enzyme because it phosphorylates beta-D-2'-deoxycytidine (D-dCyd), the natural substrate, and beta-L-2'-deoxycytidine (L-dCyd), its enantiomer, with the same efficiency. Kinetic studies showed that L-dCyd is a competitive inhibitor of the phosphorylation of D-dCyd with a Kl value of 0.12 microM, which is lower than the K(m) value for D-dCyd (1,2 microM). Chemical modifications of either the base or the pentose ring strongly decrease the inhibitory potency of L-dCyd, L-dCyd is resistant to cytidine deaminase and competes in cell cultures with the natural D-dCyd as substrate for dCK, thus reducing the incorporation of exogenous [3H]dCyd into DNA. L-dCyd had no effect on the pool of dTTP deriving from the salvage or from the de novo synthesis, does not inhibit short term RNA and protein syntheses, and shows little or no cytotoxicity. Our results indicate a catalytic similarity between human dCK and herpetic thymidine kinases, enzymes that also lack stereospecificity. This functional analogy underlines the potential role of dCK as activator of L-deoxycytidine analogs as antiviral and antineoplastic agents and lends support to the hypothesis that herpesvirus thymidine kinase might have evolved from a captured cellular dCK gene, developing the ability to phosphorylate thymidine and retaining that to phosphorylate deoxycytidine. PMID:9016355

  12. Improved Protein Kinase C Affinity through Final Step Diversification of a Simplified Salicylate-Derived Bryostatin Analog Scaffold

    PubMed Central

    2015-01-01

    Bryostatin 1, in clinical trials or preclinical development for cancer, Alzheimer’s disease, and a first-of-its-kind strategy for HIV/AIDS eradication, is neither readily available nor optimally suited for clinical use. In preceding work, we disclosed a new class of simplified bryostatin analogs designed for ease of access and tunable activity. Here we describe a final step diversification strategy that provides, in only 25 synthetic steps, simplified and tunable analogs with bryostatin-like PKC modulatory activities. PMID:25238640

  13. Kinetics and mechanism of the acid-catalyzed hydrolysis of a hypermodified nucleoside wyosine and its 5'-monophosphate.

    PubMed Central

    Golankiewicz, B; Zielonacka-Lis, E; Folkman, W

    1985-01-01

    The rates of acid-catalyzed hydrolysis of a hypermodified nucleoside, wyosine and its 5'-monophosphate were determined at various pH, temperature and buffer concentrations. The results show that despite distinct differences in structure and the glycosyl bond stability, the hydrolysis of wyosine proceeds via cleavage of the C-N bond by A-1 mechanism, analogously to simple nucleosides. Unlike majority of other monophosphates studied so far, wyosine 5'-monophosphate is not more stable than respective nucleoside. PMID:4000960

  14. Structural and Functional Analysis of G Protein–Coupled Receptor Kinase Inhibition by Paroxetine and a Rationally Designed Analog

    PubMed Central

    Homan, Kristoff T.; Wu, Emily; Wilson, Michael W.; Singh, Puja; Larsen, Scott D.

    2014-01-01

    Recently we identified the serotonin reuptake inhibitor paroxetine as an inhibitor of G protein–coupled receptor kinase 2 (GRK2) that improves cardiac performance in live animals. Paroxetine exhibits up to 50-fold selectivity for GRK2 versus other GRKs. A better understanding of the molecular basis of this selectivity is important for the development of even more selective and potent small molecule therapeutics and chemical genetic probes. We first sought to understand the molecular mechanisms underlying paroxetine selectivity among GRKs. We directly measured the KD for paroxetine and assessed its mechanism of inhibition for each of the GRK subfamilies and then determined the atomic structure of its complex with GRK1, the most weakly inhibited GRK tested. Our results suggest that the selectivity of paroxetine for GRK2 largely reflects its lower affinity for adenine nucleotides. Thus, stabilization of off-pathway conformational states unique to GRK2 will likely be key for the development of even more selective inhibitors. Next, we designed a benzolactam derivative of paroxetine that has optimized interactions with the hinge of the GRK2 kinase domain. The crystal structure of this compound in complex with GRK2 confirmed the predicted interactions. Although the benzolactam derivative did not significantly alter potency of inhibition among GRKs, it exhibited 20-fold lower inhibition of serotonin reuptake. However, there was an associated increase in the potency for inhibition of other AGC kinases, suggesting that the unconventional hydrogen bond formed by the benzodioxole ring of paroxetine is better accommodated by GRKs. PMID:24220010

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

    PubMed

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

    2015-04-10

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

  16. In Silico Exploration of 1,7-Diazacarbazole Analogs as Checkpoint Kinase 1 Inhibitors by Using 3D QSAR, Molecular Docking Study, and Molecular Dynamics Simulations.

    PubMed

    Gao, Xiaodong; Han, Liping; Ren, Yujie

    2016-01-01

    Checkpoint kinase 1 (Chk1) is an important serine/threonine kinase with a self-protection function. The combination of Chk1 inhibitors and anti-cancer drugs can enhance the selectivity of tumor therapy. In this work, a set of 1,7-diazacarbazole analogs were identified as potent Chk1 inhibitors through a series of computer-aided drug design processes, including three-dimensional quantitative structure-activity relationship (3D-QSAR) modeling, molecular docking, and molecular dynamics simulations. The optimal QSAR models showed significant cross-validated correlation q² values (0.531, 0.726), fitted correlation r² coefficients (higher than 0.90), and standard error of prediction (less than 0.250). These results suggested that the developed models possess good predictive ability. Moreover, molecular docking and molecular dynamics simulations were applied to highlight the important interactions between the ligand and the Chk1 receptor protein. This study shows that hydrogen bonding and electrostatic forces are key interactions that confer bioactivity. PMID:27164065

  17. Part-1: Design, synthesis and biological evaluation of novel bromo-pyrimidine analogs as tyrosine kinase inhibitors.

    PubMed

    Munikrishnappa, Chandrashekar Suradhenupura; Puranik, Sangamesh B; Kumar, G V Suresh; Prasad, Y Rajendra

    2016-08-25

    A novel series of 5-bromo-pyrimidine derivatives (5a-l, 6a-h, 9a-m and 10a-d) were synthesized through multi step reactions starting from 5-bromo-2,4-dichloro pyrimidine. The newly synthesized compounds were characterized using elemental analysis and spectral data (IR, (1)H NMR, (13)C NMR and LC-MS) analysis. The titled compounds were evaluated for their in vitro cytotoxic activity against tumor cell lines panel consisted of HCT116 (human colon cancer cell line), A549 (human lung cancer cell line), K562 (human chronic myeloid leukemia cell line), U937 (human acute monocytic myeloid leukemia cell line), and L02 (human normal cell line) by using MTT assay Mosmann's method. As most of the compounds are highly potent against K562 cells, all the synthesized compounds were evaluated for Bcr/Abl tyrosine kinase inhibitory activity by using well-established ADP-Glo assay method. Dasatinib was utilized as positive control to validate in both biological evaluations. The biological activity revealed that the compounds 5c, 5e, 6g, 9e, 9f and 10c were potent Bcr/Abl kinase inhibitors among the titled compounds. Thus these compounds may be promising lead compounds to be developed as an alternative for current Dasatinib therapy. PMID:27155464

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2011-09-01

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

  20. Nucleoside Inhibitors of Zika Virus.

    PubMed

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

    2016-09-01

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

  1. Computation of relative binding free energy for an inhibitor and its analogs binding with Erk kinase using thermodynamic integration MD simulation.

    PubMed

    Wu, Kuan-Wei; Chen, Po-Chin; Wang, Jun; Sun, Ying-Chieh

    2012-10-01

    In the present study, we carried out thermodynamic integration molecular dynamics simulation for a pair of analogous inhibitors binding with Erk kinase to investigate how computation performs in reproducing the relative binding free energy. The computation with BCC-AM1 charges for ligands gave -1.1 kcal/mol, deviated from experimental value of -2.3 kcal/mol by 1.2 kcal/mol, in good agreement with experimental result. The error of computed value was estimated to be 0.5 kcal/mol. To obtain convergence, switching vdw interaction on and off required approximately 10 times more CPU time than switching charges. Residue-based contributions and hydrogen bonding were analyzed and discussed. Furthermore, subsequent simulation using RESP charge for ligand gave ΔΔG of -1.6 kcal/mol. The computed results are better than the result of -5.6 kcal/mol estimated using PBSA method in a previous study. Based on these results, we further carried out computations to predict ΔΔG for five new analogs, focusing on placing polar and nonpolar functional groups at the meta site of benzene ring shown in the Fig. 1, to see if these ligands have better binding affinity than the above ligands. The computations resulted that a ligand with polar -OH group has better binding affinity than the previous examined ligand by ~2.0 kcal/mol and two other ligands have better affinity by ~1.0 kcal/mol. The predicted better inhibitors of this kind should be of interest to experimentalist for future experimental enzyme and/or cell assays. PMID:22986633

  2. Structure-catalytic activity relationships of dicyclohexylcarboxamidine analogs in phosphorylation and alkylation of nucleosides by a two-step phosphorylating agent, 2-methylthio-4H-1,3,2-benzodioxaphosphorin 2-oxide (MTBO).

    PubMed

    Eto, M; Kawasaki, S

    1986-01-01

    Adenosine borate complex was phosphorylated and o-hydroxybenzylated by 2-methylthio-4H-1,3,2-benzodioxaphosphorin 2-oxide (MTBO) in the presence of 4-morpholine-N,N'-dicyclohexylcarboxamidine (MDC) at first to give 1-(o-hydroxybenzyl)adenosine derivative followed by the rearrangement of the benzyl group to the N-6 amino group to give N6-(o-hydroxybenzyl)adenosine 5'-S-methyl phosphorothiolate. More than 20 analogs of MDC were examined for their catalytic activity in phosphorylation and o-hydroxybenzylation of ribonucleoside by MTBO. Dicyclohexylformamidine (DCF) and n-alkylamino analogs of MDC had no effect on the o-hydroxybenzylation of ribonucleoside by MTBO, but had great effect on the phosphorylation. Dialkylamino and cyclic imino analogs of MDC had high catalytic activities to the both reaction. The dicyclohexylcarboxamidine structure of MDC gave the catalytic ability for phosphorylation by MTBO, while the morpholine moiety had great effect on the selectivity of o-hydroxybenzylation by MTBO. PMID:3562278

  3. Microbial transformation of nucleosides

    NASA Technical Reports Server (NTRS)

    Lamba, S. S.

    1979-01-01

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

  4. [Purine nucleoside phosphorylase].

    PubMed

    Pogosian, L G; Akopian, Zh I

    2013-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  6. Purine nucleoside modulation of functions of human lymphocytes.

    PubMed

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

    1990-09-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2005-07-01

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

  9. A PHASE I STUDY OF THE NOVEL RIBONUCLEOTIDE REDUCTASE INHIBITOR 3-AMINOPYRIDINE-2-CARBOXALDEHYDE THIOSEMICARBAZONE (3-AP, TRIAPINE®) IN COMBINATION WITH THE NUCLEOSIDE ANALOG FLUDARABINE FOR PATIENTS WITH REFRACTORY ACUTE LEUKEMIAS AND AGGRESSIVE MYELOPROLIFERATIVE DISORDERS

    PubMed Central

    Karp, Judith E.; Giles, Francis J.; Gojo, Ivana; Morris, Lawrence; Greer, Jacqueline; Johnson, Bonny; Thein, Mya; Sznol, Mario; Low, Jennifer

    2009-01-01

    Triapine® is a potent ribonucleotide reductase (RR) inhibitor that depletes intracellular deoxyribonculeotide pools, especially dATP. We designed a Phase I trial of Triapine followed by the adenosine analog fludarabine in adults with refractory acute leukemias and aggressive myeloproliferative disorders (MPD). Two schedules were examined: A. Triapine 105mg/m2/day over 4 hours followed by fludarabine daily × 5 (24 patients, fludarabine 15–30 mg/m2/dose); B. Triapine 200mg/m2 over 24 hours followed by 5 days of fludarabine 30 mg/m2/day (9 patients). Complete and partial responses (CR,PR) occurred in Schedule A (5/24, 21%), with CR occurring at the 2 highest fludarabine doses (2/12, 17%). In contrast, no CR or PR occurred in Schedule B. Four of the 5 responses occurred in patients with underlying MPD (4/14, 29%). Drug-related toxicities included fever and metabolic acidosis. Triapine 105 mg/m2 followed by fludarabine 30mg/m2 daily × 5 is active in refractory myeloid malignancies and warrants continuing study for patients with aggressive MPD. PMID:17640728

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

    PubMed

    Ikonen, Satu; Macícková-Cahová, Hana; Pohl, Radek; Sanda, Miloslav; Hocek, Michal

    2010-03-01

    Aqueous Sonogashira cross-coupling reactions of 5-iodopyrimidine or 7-iodo-7-deazaadenine nucleosides with bile acid-derived terminal acetylenes linked via an ester or amide tether gave the corresponding bile acid-nucleoside conjugates. Analogous reactions of halogenated nucleoside triphosphates gave directly bile acid-modified dNTPs. Enzymatic incorporation of these modified nucleotides to DNA was successfully performed using Phusion polymerase for primer extension. One of the dNTPs (dCTP bearing cholic acid) was also efficient for PCR amplification. PMID:20165813

  11. Functional and genetic diversity in the concentrative nucleoside transporter, CNT1, in human populations.

    PubMed

    Gray, Jennifer H; Mangravite, Lara M; Owen, Ryan P; Urban, Thomas J; Chan, Wendy; Carlson, Elaine J; Huang, Conrad C; Kawamoto, Michiko; Johns, Susan J; Stryke, Douglas; Ferrin, Thomas E; Giacomini, Kathleen M

    2004-03-01

    The concentrative nucleoside transporter, CNT1 (SLC28A1), mediates the cellular uptake of naturally occurring pyrimidine nucleosides and many structurally diverse anticancer and antiviral nucleoside analogs. As a first step toward understanding whether genetic variation in CNT1 contributes to variation in the uptake and disposition of clinically used nucleoside analogs, we determined the haplotype structure and functionally analyzed all coding region variants of CNT1 identified in ethnically diverse populations (100 African Americans, 100 European Americans, 30 Asians, 10 Mexican Americans, and 7 Pacific Islanders) (Leabman et al., 2003). A total of 58 coding region haplotypes were identified using PHASE analysis, 44 of which contained at least one amino acid variant. More than half of the coding region haplotypes were population-specific. Using site-directed mutagenesis, 15 protein-altering CNT1 variants, including one amino acid insertion and one base pair (bp) deletion, were constructed and expressed in Xenopus laevis oocytes. All variant transporters took up [3H]thymidine with the exception of CNT1-Ser546Pro, a rare variant, and CNT1-1153del, a single bp deletion found at a frequency of 3% in the African American population. The bp deletion results in a frame-shift followed by a stop-codon. The anticancer nucleoside analog gemcitabine had a reduced affinity for CNT1-Val189Ile (a common CNT1 variant found at a frequency of 26%) compared with reference CNT1 (IC50=13.8 +/- 0.60 microM for CNT1-reference and 23.3 +/- 1.5 microM for CNT1-Val189Ile, p<0.05). These data suggest that common genetic variants of CNT1 may contribute to variation in systemic and intracellular levels of anti-cancer nucleoside analogs. PMID:14978229

  12. Tomato thymidine kinase is subject to inefficient TTP feedback regulation.

    PubMed

    Larsen, N B; Munch-Petersen, B; Piškur, J

    2014-01-01

    A promising suicide gene therapy system to treat gliomas has been reported: the thymidine kinase 1 from tomato (toTK1) combined with the nucleoside analog pro-drug zidovudine (azidothymidine, AZT), which is known to penetrate the blood-brain barrier. Transduction with toTK1 has been found to efficiently increase the sensitivity of human glioblastoma cells to AZT, and nude rats with intracranial glioblastoma grafts have shown significantly improved survival when treated with the toTK1/AZT system. We show in our paper that the strong suicidal effect of AZT together with toTK1 may be explained by reduced TTP-mediated feedback inhibition of the AZT phosphorylation. PMID:24940681

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

    NASA Astrophysics Data System (ADS)

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

    1991-01-01

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

  14. Binding mode prediction of aplysiatoxin, a potent agonist of protein kinase C, through molecular simulation and structure-activity study on simplified analogs of the receptor-recognition domain.

    PubMed

    Ashida, Yoshiki; Yanagita, Ryo C; Takahashi, Chise; Kawanami, Yasuhiro; Irie, Kazuhiro

    2016-09-15

    Aplysiatoxin (ATX) is a naturally occurring tumor promoter isolated from a sea hare and cyanobacteria. ATX binds to, and activates, protein kinase C (PKC) isozymes and shows anti-proliferative activity against human cancer cell lines. Recently, ATX has attracted attention as a lead compound for the development of novel anticancer drugs. In order to predict the binding mode between ATX and protein kinase Cδ (PKCδ) C1B domain, we carried out molecular docking simulation, atomistic molecular dynamics simulation in phospholipid membrane environment, and structure-activity study on a simple acyclic analog of ATX. These studies provided the binding model where the carbonyl group at position 27, the hydroxyl group at position 30, and the phenolic hydroxyl group at position 20 of ATX were involved in intermolecular hydrogen bonding with the PKCδ C1B domain, which would be useful for the rational design of ATX derivatives as anticancer lead compounds. PMID:27436807

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

    NASA Technical Reports Server (NTRS)

    Tohidi, Mahrokh; Orgel, Leslie E.

    1990-01-01

    When 2-prime-deoxythymidine 3-prime, 5-prime-cyclic diphosphate, or the cyclic pyrophosphates of the acyclic nucleoside analogs II and IV are heated to 65-85 C in the presence of imidazole, oligomers with lengths up to 20-30 are formed in excellent yield. This reaction provides a useful source of oligomers for use as templates in aqueous condensation reactions. In the absence of evidence to the contrary, it is assumed that the oligomers are atactic. The potential significance of this reaction in prebiotic chemistry is discussed.

  16. Genomic structure and chromosomal localization of the human deoxycytidine kinase gene

    SciTech Connect

    Song, J.J.; Walker, S.; Gribbin, T. ); Chen, E. Univ. of North Carolina, Chapel Hill ); Johnson, E.E.; Spychala, J.; Mitchell, B.S. )

    1993-01-15

    Deoxycytidine kinase (NTP:deoxycytidine 5[prime]-phosphotransferase, EC 2.7.1.74) is an enzyme that catalyzes phosphorylation of deoxyribonucleosides and a number of nucleoside analogs that are important in antiviral and cancer chemotherapy. Deficiency of this enzyme activity is associated with resistance to these agents, whereas increased enzyme activity is associated with increased activation of such compounds to cytotoxic nucleoside triphosphate derivatives. To characterize the regulation of expression of this gene, we have isolated genomic clones encompassing its entire coding and 5[prime] flanking regions and delinated all the exon/intron boundaries. The gene extends over more than 34 kilobases on chromosome 4 and the coding region is composed of 7 exons ranging in size from 90 to 1544 base pairs (bp). The 5[prime] flanking region is highly G+C-rich and contains four regions that are potential Sp1 binding sites. A 697-bp fragment encompassing 386 bp of 5[prime] upstream region, the 250-bp first exon, and 61 bp of the first intron was demonstrated to promote chloramphenicol acetyltransferase activity in a T-lymphoblast cell line and to have >6-fold greater activity in a Jurkat T-lymphoblast than in a Raji B-lymphoblast cell line. Our data suggest that these 5[prime] sequences may contain elements that are important for the tissue-specific differences in deoxycytidine kinase expression. 32 refs., 4 figs., 2 tabs.

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

    PubMed

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

    1998-01-01

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

  18. Nucleoside transporter proteins as biomarkers of drug responsiveness and drug targets

    PubMed Central

    Pastor-Anglada, Marçal; Pérez-Torras, Sandra

    2015-01-01

    Nucleoside and nucleobase analogs are currently used in the treatment of solid tumors, lymphoproliferative diseases, viral infections such as hepatitis and AIDS, and some inflammatory diseases such as Crohn. Two gene families are implicated in the uptake of nucleosides and nucleoside analogs into cells, SCL28 and SLC29. The former encodes hCNT1, hCNT2, and hCNT3 proteins. They translocate nucleosides in a Na+ coupled manner with high affinity and some substrate selectivity, being hCNT1 and hCNT2 pyrimidine- and purine-preferring, respectively, and hCNT3 a broad selectivity transporter. SLC29 genes encode four members, being hENT1 and hENT2 the only two which are unequivocally implicated in the translocation of nucleosides and nucleobases (the latter mostly via hENT2) at the cell plasma membrane. Some nucleoside-derived drugs can also interact with and be translocated by members of the SLC22 gene family, particularly hOCT and hOAT proteins. Inter-individual differences in transporter function and perhaps, more importantly, altered expression associated with the disease itself might modulate the transporter profile of target cells, thereby determining drug bioavailability and action. Drug transporter pharmacology has been periodically reviewed. Thus, with this contribution we aim at providing a state-of-the-art overview of the clinical evidence generated so far supporting the concept that these membrane proteins can indeed be biomarkers suitable for diagnosis and/or prognosis. Last but not least, some of these transporter proteins can also be envisaged as drug targets, as long as they can show “transceptor” functions, in some cases related to their role as modulators of extracellular adenosine levels, thereby providing a functional link between P1 receptors and transporters. PMID:25713533

  19. Lunar Analog

    NASA Technical Reports Server (NTRS)

    Cromwell, Ronita L.

    2009-01-01

    In this viewgraph presentation, a ground-based lunar analog is developed for the return of manned space flight to the Moon. The contents include: 1) Digital Astronaut; 2) Bed Design; 3) Lunar Analog Feasibility Study; 4) Preliminary Data; 5) Pre-pilot Study; 6) Selection of Stockings; 7) Lunar Analog Pilot Study; 8) Bed Design for Lunar Analog Pilot.

  20. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers

    NASA Astrophysics Data System (ADS)

    Wang, Jun; Bonnesen, Peter V.; Rangel, E.; Vallejo, E.; Sanchez-Castillo, Ariadna; James Cleaves, H., II; Baddorf, Arthur P.; Sumpter, Bobby G.; Pan, Minghu; Maksymovych, Petro; Fuentes-Cabrera, Miguel

    2016-01-01

    Self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N9-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two or more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. These characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Further, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers.

  1. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers

    PubMed Central

    Wang, Jun; Bonnesen, Peter V.; Rangel, E.; Vallejo, E.; Sanchez-Castillo, Ariadna; James Cleaves II, H.; Baddorf, Arthur P.; Sumpter, Bobby G.; Pan, Minghu; Maksymovych, Petro; Fuentes-Cabrera, Miguel

    2016-01-01

    Self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N9-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two or more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. These characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Further, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers. PMID:26725380

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

    PubMed

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

    1994-07-01

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

  3. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers

    DOE PAGESBeta

    Wang, Jun; Bonnesen, Peter V; Rangel, E.; Vallejo, E.; Sanchez-Castillo, Ariadna; Cleaves, II, H. James; Baddorf, Arthur P; Sumpter, Bobby G; Pan, Minghu; Maksymovych, Petro; et al

    2016-01-04

    The self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N9-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two ormore » more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. The resulting characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Moreover, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers.« less

  4. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers.

    PubMed

    Wang, Jun; Bonnesen, Peter V; Rangel, E; Vallejo, E; Sanchez-Castillo, Ariadna; James Cleaves Ii, H; Baddorf, Arthur P; Sumpter, Bobby G; Pan, Minghu; Maksymovych, Petro; Fuentes-Cabrera, Miguel

    2016-01-01

    Self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N(9)-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two or more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. These characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Further, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers. PMID:26725380

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-05-01

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

  7. Extending Thymidine Kinase Activity to the Catalytic Repertoire of Human Deoxycytidine Kinase

    SciTech Connect

    Hazra, Saugata; Sabini, Eliszbetta; Ort, Stephan; Konrad, Manfred; Lavie, Arnon

    2009-03-04

    Salvage of nucleosides in the cytosol of human cells is carried out by deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1). Whereas TK1 is only responsible for thymidine phosphorylation, dCK is capable of converting dC, dA, and dG into their monophosphate forms. Using structural data on dCK, we predicted that select mutations at the active site would, in addition to making the enzyme faster, expand the catalytic repertoire of dCK to include thymidine. Specifically, we hypothesized that steric repulsion between the methyl group of the thymine base and Arg104 is the main factor preventing the phosphorylation of thymidine by wild-type dCK. Here we present kinetic data on several dCK variants where Arg104 has been replaced by select residues, all performed in combination with the mutation of Asp133 to an alanine. We show that several hydrophobic residues at position 104 endow dCK with thymidine kinase activity. Depending on the exact nature of the mutations, the enzyme's substrate preference is modified. The R104M-D133A double mutant is a pyrimidine-specific enzyme due to large K{sub m} values with purines. The crystal structure of the double mutant R104M-D133A in complex with the L-form of thymidine supplies a structural explanation for the ability of this variant to phosphorylate thymidine and thymidine analogs. The replacement of Arg104 by a smaller residue allows L-dT to bind deeper into the active site, making space for the C5-methyl group of the thymine base. The unique catalytic properties of several of the mutants make them good candidates for suicide-gene/protein-therapy applications.

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

    PubMed

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

    2016-05-15

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

  9. Characterization of intestinal absorption of mizoribine mediated by concentrative nucleoside transporters in rats.

    PubMed

    Mori, Nobuhiro; Yokooji, Tomoharu; Kamio, Yoshihiro; Murakami, Teruo

    2008-05-31

    Mizoribine, an imidazole nucleoside, is an inhibitor of purine synthesis and has been used as an orally available immunosuppressive agent in human renal transplantation. In the present study, the intestinal absorption of mizoribine was characterized by examining the contribution of concentrative nucleoside transporters (CNT1, CNT2) in rats. When mizoribine was administered orally in conscious rats, the bioavailability of mizoribine estimated by urinary excretion percentage of unchanged mizoribine was a dose dependent: 53.1+/-6.0% at 5 mg/kg and 24.0+/-5.1% at 20 mg/kg. In in-situ loop studies, the disappearance rate, or absorption rate, of mizoribine from the intestinal lumen was comparable between 1 and 5 mg/kg, but significantly lower at 25 mg/kg. Coadministration of adenosine (a substrate of both CNT1 and CNT2), thymidine (a CNT1 substrate) and inosine (a CNT2 substrate) significantly suppressed the intestinal mizoribine absorption, depending on the nucleoside concentrations coadministered. Gemcitabine (a pyrimidine nucleoside analogue, a CNT1 substrate) and ribavirin (a purine nucleoside analog, a CNT2 substrate) also significantly suppressed the mizoribine intestinal absorption. Bile salts such as sodium cholate and sodium glycocholate (10 mM) also significantly suppressed the intestinal mizoribine absorption, but not ribavirin absorption. Mizoribine is an amphoteric compound, however, the suppression of intestinal absorption by bile salts was not ascribed to the electrostatic interaction or micellar formation between mizoribine and bile salts. In conclusion, the intestinal absorption of mizoribine is mediated by CNT1 and CNT2, and nucleoside-derived drugs such as gemcitabine and ribavirin can suppress the intestinal absorption of mizoribine. Bile salts such as sodium glycocholate were also found to cause interaction with mizoribine. PMID:18371949

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

    PubMed

    Carter, N S; Ben Mamoun, C; Liu, W; Silva, E O; Landfear, S M; Goldberg, D E; Ullman, B

    2000-04-01

    Plasmodium falciparum, the causative agent of the most lethal form of human malaria, is incapable of de novo purine synthesis, and thus, purine acquisition from the host is an indispensable nutritional requirement. This purine salvage process is initiated by the transport of preformed purines into the parasite. We have identified a gene encoding a nucleoside transporter from P. falciparum, PfNT1, and analyzed its function and expression during intraerythrocytic parasite development. PfNT1 predicts a polypeptide of 422 amino acids with 11 transmembrane domains that is homologous to other members of the equilibrative nucleoside transporter family. Southern analysis and BLAST searching of The Institute for Genomic Research (TIGR) malaria data base indicate that PfNT1 is a single copy gene located on chromosome 14. Northern analysis of RNA from intraerythrocytic stages of the parasite demonstrates that PfNT1 is expressed throughout the asexual life cycle but is significantly elevated during the early trophozoite stage. Functional expression of PfNT1 in Xenopus laevis oocytes significantly increases their ability to take up naturally occurring D-adenosine (K(m) = 13.2 microM) and D-inosine (K(m) = 253 microM). Significantly, PfNT1, unlike the mammalian nucleoside transporters, also has the capacity to transport the stereoisomer L-adenosine (K(m) > 500 microM). Inhibition studies with a battery of purine and pyrimidine nucleosides and bases as well as their analogs indicate that PfNT1 exhibits a broad substrate specificity for purine and pyrimidine nucleosides. These data provide compelling evidence that PfNT1 encodes a functional purine/pyrimidine nucleoside transporter whose expression is strongly developmentally regulated in the asexual stages of the P. falciparum life cycle. Moreover, the unusual ability to transport L-adenosine and the vital contribution of purine transport to parasite survival makes PfNT1 an attractive target for therapeutic evaluation. PMID

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

    PubMed

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

    2012-02-01

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

  12. Nucleoside transporters and liver cell growth.

    PubMed

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

    1998-01-01

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

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

    PubMed

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

    1989-08-15

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

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

    PubMed Central

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

    2014-01-01

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

  15. Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase

    PubMed Central

    Van Horn, Wade D.; Sanders, Charles R.

    2013-01-01

    Prokaryotic diacylglycerol kinase (DAGK) and undecaprenol kinase (UDPK) are the lone members of a family of multispan membrane enzymes that are very small, lack relationships to any other family of proteins—including water soluble kinases, and that exhibit an unusual structure and active site architecture. Escherichia coli DAGK plays an important role in recycling diacylglycerol produced as a byproduct of biosynthesis of molecules located in the periplasmic space. UDPK seems to play an analogous role in Gram-positive bacteria, where its importance is evident by the fact that UDPK is essential for biofilm formation by the oral pathogen Streptococcus mutans. DAGK has also long served as a model system for studies of membrane protein biocatalysis, folding, stability, and structure. This review explores our current understanding of the microbial physiology, enzymology, structural biology, and folding of the prokaryotic diacylglycerol kinase family, which is based on over 40 years of studies. PMID:22224599

  16. Prebiotic phosphorylation of nucleosides in formamide

    NASA Technical Reports Server (NTRS)

    Schoffstall, A. M.

    1976-01-01

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

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

    PubMed

    Mohamady, Samy; Taylor, Scott D

    2016-02-01

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

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

    PubMed Central

    Ibarra, Kristie D.; Pfeiffer, Julie K.

    2009-01-01

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

  19. 5-(1-Substituted) alkyl pyrimidine nucleosides as antiviral (herpes) agents.

    PubMed

    Kumar, Rakesh

    2004-10-01

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

  20. Application of germyldesulfonylation reactions to the synthesis of germanium-containing nucleoside analogues.

    PubMed

    Wnuk, Stanislaw F; Sacasa, Pablo R; Restrepo, Jorge

    2009-05-01

    Treatment of the protected (E)-5'-deoxy-5'-[(p-toluenesulfonyl)methylene]uridine and adenosine derivatives with tributyl- or triphenylgermane hydride (AIBN/toluene/Delta) effected radical-mediated germyldesulfonylations to give 5'-(tributyl- or triphenylgermyl)methylene-5'-deoxyuridine and adenosine derivatives as single (E)-isomers. Analogous treatment of 2'-deoxy-2'-[(phenylsulfonyl)methylene]uridine with Ph(3)GeH afforded the corresponding vinyl triphenylgermane product. Stereoselective halodegermylation of the (E)-5'-(tributylgermyl)methylene-5'-deoxy nucleosides with N-iodosuccinimide or N-bromosuccinimide provided the Wittig-type (E)-5'-deoxy-5'-(halomethylene) nucleosides quantitatively, while no halodegermylations was observed with the 5'-deoxy-5'-(triphenylgermyl)methylene counterparts. Treatment of the vinyl trialkylgermanes with aqueous trifluoroacetic acid effected protiodegermylation, while vinyl triarylgermanes were stable under the acidic conditions. PMID:20183601

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

    PubMed Central

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

    2013-01-01

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

  2. New hypoxanthine nucleosides with RNA antiviral activity.

    PubMed

    Nair, V; Ussery, M A

    1992-08-01

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

  3. Mycoplasmas and cancer: focus on nucleoside metabolism

    PubMed Central

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

    2014-01-01

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

  4. Plant thymidine kinase 1: a novel efficient suicide gene for malignant glioma therapy.

    PubMed

    Khan, Zahidul; Knecht, Wolfgang; Willer, Mette; Rozpedowska, Elzbieta; Kristoffersen, Peter; Clausen, Anders Ranegaard; Munch-Petersen, Birgitte; Almqvist, Per M; Gojkovic, Zoran; Piskur, Jure; Ekström, Tomas J

    2010-06-01

    The prognosis for malignant gliomas remains poor, and new treatments are urgently needed. Targeted suicide gene therapy exploits the enzymatic conversion of a prodrug, such as a nucleoside analog, into a cytotoxic compound. Although this therapeutic strategy has been considered a promising regimen for central nervous system (CNS) tumors, several obstacles have been encountered such as inefficient gene transfer to the tumor cells, limited prodrug penetration into the CNS, and inefficient enzymatic activity of the suicide gene. We report here the cloning and successful application of a novel thymidine kinase 1 (TK1) from the tomato plant, with favorable characteristics in vitro and in vivo. This enzyme (toTK1) is highly specific for the nucleoside analog prodrug zidovudine (azidothymidine, AZT), which is known to penetrate the blood-brain barrier. An important feature of toTK1 is that it efficiently phosphorylates its substrate AZT not only to AZT monophosphate, but also to AZT diphosphate, with excellent kinetics. The efficiency of the toTK1/AZT system was confirmed when toTK1-transduced human glioblastoma (GBM) cells displayed a 500-fold increased sensitivity to AZT compared with wild-type cells. In addition, when neural progenitor cells were used as delivery vectors for toTK1 in intracranial GBM xenografts in nude rats, substantial attenuation of tumor growth was achieved in animals exposed to AZT, and survival of the animals was significantly improved compared with controls. The novel toTK1/AZT suicide gene therapy system in combination with stem cell-mediated gene delivery promises new treatment of malignant gliomas. PMID:20154339

  5. Analog earthquakes

    SciTech Connect

    Hofmann, R.B.

    1995-09-01

    Analogs are used to understand complex or poorly understood phenomena for which little data may be available at the actual repository site. Earthquakes are complex phenomena, and they can have a large number of effects on the natural system, as well as on engineered structures. Instrumental data close to the source of large earthquakes are rarely obtained. The rare events for which measurements are available may be used, with modfications, as analogs for potential large earthquakes at sites where no earthquake data are available. In the following, several examples of nuclear reactor and liquified natural gas facility siting are discussed. A potential use of analog earthquakes is proposed for a high-level nuclear waste (HLW) repository.

  6. Concentrative nucleoside transporter 1 (hCNT1) promotes phenotypic changes relevant to tumor biology in a translocation-independent manner

    PubMed Central

    Pérez-Torras, S; Vidal-Pla, A; Cano-Soldado, P; Huber-Ruano, I; Mazo, A; Pastor-Anglada, M

    2013-01-01

    Nucleoside transporters (NTs) mediate the uptake of nucleosides and nucleobases across the plasma membrane, mostly for salvage purposes. The canonical NTs belong to two gene families, SLC29 and SLC28. The former encode equilibrative nucleoside transporter proteins (ENTs), which mediate the facilitative diffusion of natural nucleosides with broad selectivity, whereas the latter encode concentrative nucleoside transporters (CNTs), which are sodium-coupled and show high affinity for substrates with variable selectivity. These proteins are expressed in most cell types, exhibiting apparent functional redundancy. This might indicate that CNTs have specific roles in the physiology of the cell beyond nucleoside salvage. Here, we addressed this possibility using adenoviral vectors to restore tumor cell expression of hCNT1 or a polymorphic variant (hCNT1S546P) lacking nucleoside translocation ability. We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration. Importantly, the translocation-defective transporter triggered these same effects on cell physiology. Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma. These data predict a novel role for a NT protein, hCNT1, which appears to be independent of its role as mediator of nucleoside uptake by cells. Thereby, hCNT1 fits the profile of a transceptor in a substrate translocation-independent manner and is likely to be relevant to tumor biology. PMID:23722537

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

    PubMed

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

    2001-05-01

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

  8. Mechanistic characterization of the tetraacyldisaccharide-1-phosphate 4'-kinase LpxK involved in lipid A biosynthesis.

    PubMed

    Emptage, Ryan P; Pemble, Charles W; York, John D; Raetz, Christian R H; Zhou, Pei

    2013-04-01

    The sixth step in the lipid A biosynthetic pathway involves phosphorylation of the tetraacyldisaccharide-1-phosphate (DSMP) intermediate by the cytosol-facing inner membrane kinase LpxK, a member of the P-loop-containing nucleoside triphosphate (NTP) hydrolase superfamily. We report the kinetic characterization of LpxK from Aquifex aeolicus and the crystal structures of LpxK in complex with ATP in a precatalytic binding state, the ATP analogue AMP-PCP in the closed catalytically competent conformation, and a chloride anion revealing an inhibitory conformation of the nucleotide-binding P-loop. We demonstrate that LpxK activity in vitro requires the presence of a detergent micelle and formation of a ternary LpxK-ATP/Mg(2+)-DSMP complex. Using steady-state kinetics, we have identified crucial active site residues, leading to the proposal that the interaction of D99 with H261 acts to increase the pKa of the imidazole moiety, which in turn serves as the catalytic base to deprotonate the 4'-hydroxyl of the DSMP substrate. The fact that an analogous mechanism has not yet been observed for other P-loop kinases highlights LpxK as a distinct member of the P-loop kinase family, a notion that is also reflected through its localization at the membrane, lipid substrate, and overall structure. PMID:23464738

  9. Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphoregulation of mitochondrial complex I is inhibited by nucleoside reverse transcriptase inhibitors

    SciTech Connect

    Lund, Kaleb C. Wallace, Kendall B.

    2008-01-01

    Nucleoside analog reverse transcriptase inhibitors (NRTIs) are known to directly inhibit mitochondrial complex I activity as well as various mitochondrial kinases. Recent observations that complex I activity and superoxide production are modulated through cAMP-dependent phosphorylation suggests a mechanism through which NRTIs may affect mitochondrial respiration via kinase-dependent protein phosphorylation. In the current study, we examine the potential for NRTIs to inhibit the cAMP-dependent phosphorylation of complex I and the associated NADH:CoQ oxidoreductase activities and rates of superoxide production using HepG2 cells. Phosphoprotein staining of immunocaptured complex I revealed that 3'-azido-3'-deoxythymidine (AZT; 10 and 50 {mu}M), AZT monophosphate (150 {mu}M), and 2',3'-dideoxycytidine (ddC; 1 {mu}M) prevented the phosphorylation of the NDUFB11 subunit of complex I. This was associated with a decrease in complex I activity with AZT and AZT monophosphate only. In the presence of succinate, superoxide production was increased with 2',3'-dideoxyinosine (ddI; 10 {mu}M) and ddC (1 {mu}M). In the presence of succinate + cAMP, AZT showed an inverse dose-dependent effect on superoxide production. None of the NRTIs examined inhibit PKA activity suggesting that the observed effects are due to a direct interaction with complex I. These data demonstrate a direct effect of NRTIs on cAMP-dependent regulation of mitochondrial bioenergetics independent of DNA polymerase-{gamma} activity; in the case of AZT, these observations may provide a mechanism for the observed long-term toxicity with this drug.

  10. Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals. Quantitative basis for Urh1 and purine nucleoside phosphorylase function in NAD+ metabolism.

    PubMed

    Belenky, Peter; Christensen, Kathryn C; Gazzaniga, Francesca; Pletnev, Alexandre A; Brenner, Charles

    2009-01-01

    NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification. PMID:19001417

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

    PubMed

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

    2016-09-01

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

  12. Summary: the modified nucleosides of RNA.

    PubMed Central

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

    1994-01-01

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

  13. 2'-modified nucleosides for site-specific labeling of oligonucleotides

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  15. Variability of gemcitabine accumulation and its relationship to expression of nucleoside transporters in peripheral blood mononuclear cells.

    PubMed

    Choi, Min-Koo

    2012-05-01

    The concentrative nucleoside transporter CNT1 and equilibrated nucleoside transporter ENT1 mediate the cellular uptake of naturally occurring pyrimidine and purine nucleosides and many structurally diverse anticancer and antiviral nucleoside analogs, thereby regulating drug responses or toxicity at the target site. The objectives of this study were to analyze interindividual variations in the cellular accumulation of gemcitabine and to examine the correlation between the uptake of gemcitabine and expression levels of CNT1 and ENT1 transporters. Gemcitabine was a substrate for both CNT1 and ENT1 with higher affinity to CNT1 than to ENT1. The difference in gemcitabine uptake was 4.8-fold in peripheral blood mononuclear cells (PBMCs) from 10 subjects. Among these, the CNT1- and ENT1-mediated uptake of gemcitabine was 14.3- and 16.5-folds, respectively. CNT1-mediated gemcitabine uptake showed a higher correlation with the CNT1 expression level than did ENT1-mediated uptake with ENT1 expression level. In conclusion, CNT1 seemed to be a major contributing factor to gemcitabine uptake in PBMCs and showed 14.3-fold inter-individual variations. However, ENT1-mediated uptake of gemcitabine might compensate for the total uptake of gemcitabine; therefore, the variation in the apparent accumulation of gemcitabine was smaller than that of the individual transporters. PMID:22644860

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

    PubMed

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

    2012-07-01

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

  17. Pharmacological Reversal of Histone Methylation Presensitizes Pancreatic Cancer Cells to Nucleoside Drugs: In Vitro Optimization and Novel Nanoparticle Delivery Studies

    PubMed Central

    Hung, Sau Wai; Bhutia, Yangzom D.; Davis, Franklin; Cho, Jong Hyun; Zastre, Jason; Dhar, Shanta; Chu, Chung K.; Govindarajan, Rajgopal

    2013-01-01

    We evaluated the potential of an investigational histone methylation reversal agent, 3-deazaneplanocin A (DZNep), in improving the chemosensitivity of pancreatic cancer to nucleoside analogs (i.e., gemcitabine). DZNep brought delayed but selective cytotoxicity to pancreatic cancer cells without affecting normal human pancreatic ductal epithelial (HPDE) cells. Co-exposure of DZNep and gemcitabine induced cytotoxic additivity or synergism in both well- and poorly-differentiated pancreatic cell lines by increased apoptosis. In contrast, DZNep exerted antagonism with gemcitabine against HPDE cells with significant reduction in cytotoxicity compared with the gemcitabine-alone regimen. DZNep marginally depended on purine nucleoside transporters for its cytotoxicity, but the transport dependence was circumvented by acyl derivatization. Drug exposure studies revealed that a short priming with DZNep followed by gemcitabine treatment rather than co-treatment of both agents to produce a maximal chemosensitization response in both gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells. DZNep rapidly and reversibly decreased trimethylation of histone H3 lysine 27 but increased trimethylation of lysine 9 in an EZH2- and JMJD1A/2C-dependent manner, respectively. However, DZNep potentiation of nucleoside analog chemosensitization was found to be temporally coupled to trimethylation changes in lysine 27 and not lysine 9. Polymeric nanoparticles engineered to chronologically release DZNep followed by gemcitabine produced pronounced chemosensitization and dose-lowering effects. Together, our results identify that an optimized DZNep exposure can presensitize pancreatic cancer cells to anticancer nucleoside analogs through the reversal of histone methylation, emphasizing the promising clinical utilities of epigenetic reversal agents in future pancreatic cancer combination therapies. PMID:23940717

  18. Synthesis of Conformationally North-Locked Pyrimidine Nucleosides Built on an Oxa-bicyclo[3.1.0]hexane Scaffold

    PubMed Central

    Ludek, Olaf R.; Marquez, Victor E.

    2011-01-01

    Beginning with a known 3-oxabicyclo[3.1.0]hexane scaffold (I), the relocation of the fused cyclopropane ring bond and the shifting of the oxygen atom to an alternative location engendered a new 2-oxabicyclo[3.1.0]hexane template (II) that mimics more closely the tetrahydrofuran ring of conventional nucleosides. The synthesis of this new class of locked nucleosides involved a novel approach that required the isocyanate II (B = NCO) with a hydroxyl-protected scaffold as a pivotal intermediate that was obtained in eleven steps from a known dihydrofuran precursor. The completion of the nucleobases was successfully achieved by quenching the isocyanate with the lithium salts of the corresponding acrylic amides that led to the uracil and thymidine precursors in a single step. Ring closure of these intermediates led to the target, locked nucleosides. The anti-HIV activity of 29 (uridine analogue), 31 (thymidine analogue), and 34 (cytidine analogue) was explored in human osteosarcoma (HOS) cells or modified HOS cells (HOS-313) expressing the herpes simplex virus 1 thymidine kinase (HSV-1 TK). Only the cytidine analogue showed moderate activity in HOS-313 cells, which means that the compounds are not good substrates for the cellular kinases. PMID:22026578

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

    PubMed

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

    2012-09-01

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

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

    PubMed

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

    2006-12-01

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

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

    PubMed

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

    2016-09-01

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

  2. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  3. (Biological applications of nucleosides and nucleotides)

    SciTech Connect

    Srivastava, P.C.

    1990-08-20

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

  4. Labeling and Identification of Direct Kinase Substrates

    PubMed Central

    Carlson, Scott M.; White, Forest M.

    2013-01-01

    Identifying kinase substrates is an important step in mapping signal transduction pathways, but remains a difficult and time-consuming process. Analog-sensitive kinases (AS-kinases) have been used to selectively tag and identify direct kinase substrates in lysates from whole cells. In this approach a gamma-thiol ATP-analog and AS-kinase are used to selectively thiophosphorylate target proteins. Thiophosphate is used as a chemical handle to purify peptides from a tryptic digest, and target proteins are identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Here, we describe an updated strategy for labeling AS-kinase substrates, solid-phase capture of thiophosphorylated peptides, incorporation of stable-isotopic labeling in cell culture (SILAC) for filtering nonspecific background peptides, enrichment of phosphorylated target peptides to identify low-abundance targets, and analysis by LC-MS/MS. PMID:22669844

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

    PubMed

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

    2004-12-01

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

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

    PubMed

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

    2016-03-01

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

  7. Hepatocyte nuclear factor-4alpha and bile acids regulate human concentrative nucleoside transporter-1 gene expression.

    PubMed

    Klein, Kerstin; Kullak-Ublick, Gerd A; Wagner, Martin; Trauner, Michael; Eloranta, Jyrki J

    2009-04-01

    The concentrative nucleoside transporter-1 (CNT1) is a member of the solute carrier 28 (SLC28) gene family and is expressed in the liver, intestine, and kidneys. CNT1 mediates the uptake of naturally occurring pyrimidine nucleosides, but also nucleoside analogs used in anticancer and antiviral therapy. Thus expression levels of CNT1 may affect the pharmacokinetics of these drugs and the outcome of drug therapy. Because little is known about the transcriptional regulation of human CNT1 gene expression, we have characterized the CNT1 promoter with respect to DNA response elements and their binding factors. The transcriptional start site of the CNT1 gene was determined by 5'-RACE. In silico analysis revealed the existence of three putative binding sites for the nuclear receptor hepatocyte nuclear factor-4alpha (HNF-4alpha) within the CNT1 promoter. A luciferase reporter gene construct containing the CNT1 promoter region was transactivated by HNF-4alpha in human cell lines derived from the liver, intestine, and kidneys. Consistent with this, we showed in electromobility shift assays that HNF-4alpha specifically binds to two conserved direct repeat-1 motifs within the proximal CNT1 promoter. In cotransfection experiments, the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha further increased, whereas the bile acid-inducible corepressor small heterodimer partner reduced, HNF-4alpha-dependent CNT1 promoter activity. Consistent with the latter phenomenon, CNT1 mRNA expression levels were suppressed in primary human hepatocytes upon bile acid treatment. Supporting the physiological relevance and species conservation of this effect, ileal Cnt1 mRNA expression was decreased upon bile acid feeding and increased upon bile duct ligation in mice. PMID:19228884

  8. Tissue distribution of concentrative and equilibrative nucleoside transporters in male and female rats and mice.

    PubMed

    Lu, Hong; Chen, Chuan; Klaassen, Curtis

    2004-12-01

    Concentrative nucleoside transporters (Cnts) and equilibrative nucleoside transporters (Ents) have essential physiological functions and are important in disposition of anticancer and antiviral nucleoside analogs. Information on tissue distribution of Cnts and Ents in rodents is sparse. Thus, the present study aimed to determine the distribution of Cnt1-3 and Ent1-3 transcripts in 19 tissues of Sprague-Dawley rats and C57BL/6 mice of both genders. These six transcripts were quantified using the branched DNA signal amplification assay. Cnt1 transcripts were highest in small intestine, followed by kidney and testes, with similar expression in both species. Cnt2 mRNA was expressed highest in the small intestine of both rats and mice, intermediate in liver of rats but not in mice, and lower in thymus and spleen of both species. Cnt3 mRNA has marked species differences, with the highest expression in lung of rats but uterus of mice. Ent1 mRNA was most highly expressed in testes and lung of both species. Ent1 mRNA was highly expressed in liver and pituitary of mice, but not in rats. Ent2 mRNA was highly expressed in testes and brain of both species. Ent3 mRNA was highest in kidney, followed by testes, in both species. Significant gender differences were observed in kidney (mouse) and heart (rat). These studies demonstrate that in general, tissue distribution of Cnt and Ent is similar in rats and mice. However, a few important species and gender differences do exist, which could be responsible for related differences in efficacy and toxicity of substrates for these transporters. PMID:15371301

  9. Functional non-nucleoside adenylyl cyclase inhibitors.

    PubMed

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

    2015-05-01

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

  10. Hepatotoxicity of nucleoside reverse transcriptase inhibitors.

    PubMed

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

    2003-05-01

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

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

    SciTech Connect

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

    2012-07-11

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

  12. Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: Biochemical and genetic characterization

    SciTech Connect

    Cornish, K.V.; Pearlman, R.E.

    1982-08-01

    Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro. Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo. Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect. Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position. Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism.

  13. Membrane-permeable Triphosphate Prodrugs of Nucleoside Analogues.

    PubMed

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

    2016-04-18

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

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

    PubMed

    Baszczyňski, Ondřej; Janeba, Zlatko

    2013-11-01

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

  15. The MUC4 mucin mediates gemcitabine resistance of human pancreatic cancer cells via the Concentrative Nucleoside Transporter family

    PubMed Central

    Skrypek, Nicolas; Duchêne, Bélinda; Hebbar, Mohamed; Leteurtre, Emmanuelle; Van Seuningen, Isabelle; Jonckheere, Nicolas

    2013-01-01

    The fluorinated analog of deoxycytidine, Gemcitabine (Gemzar®), is the main chemotherapy in pancreatic cancer, but survival remains weak mainly because of the high resistance of tumors to the drug. Recent works have shown that the mucin MUC4 may confer an advantage to pancreatic tumor cells by modifying their susceptibility to drugs. However, the cellular mechanism(s) responsible for this MUC4-mediated resistance is unknown. The aim of this work was to identify the cellular mechanisms responsible for gemcitabine resistance linked to MUC4 expression. CAPAN-2 and CAPAN-1 adenocarcinomatous pancreatic cancer cell lines were used to establish stable MUC4-deficient clones (MUC4-KD) by shRNA interference. Measurement of the IC50 index using tetrazolium salt test indicated that MUC4-deficient cells were more sensitive to gemcitabine. This was correlated with increased Bax/BclXL ratio and apoptotic cell number. Expression of Equilibrative/Concentrative Nucleoside Transporter (hENT1, hCNT1/3), deoxycytidine kinase (dCK), ribonucleotide reductase (RRM1/2) and Multidrug-resistance Protein (MRP3/4/5) was evaluated by quantitative RT-PCR (qRT-PCR) and Western-blotting. Alteration of MRP3, MRP4, hCNT1 and hCNT3 expression was observed in MUC4-KD cells but only hCNT1 alteration was correlated to MUC4 expression and sensitivity to gemcitabine. Decreased activation of MAPK, JNK and NF-κB pathways was observed in MUC4-deficient cells in which NF-κB pathway was found to play an important role both in sensitivity to gemcitabine and in hCNT1 regulation. Finally and accordingly to our in vitro data, we found that MUC4 expression was conversely correlated to that of hCNT1 in tissues from patients with pancreatic adenocarcinoma. This work describes a new mechanism of pancreatic cancer cell resistance to gemcitabine in which the MUC4 mucin negatively regulates the hCNT1 transporter expression via the NF-κB pathway. Altogether, these data point out to MUC4 and hCNT1 as potential targets

  16. Distribution of nucleosides in populations of Cordyceps cicadae.

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2005-10-01

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

  18. Equilibrative Nucleoside Transporters 1 and 4: Which One Is a Better Target for Cardioprotection Against Ischemia-Reperfusion Injury?

    PubMed

    Yang, Cui; Leung, George P H

    2015-06-01

    The cardioprotective effects of adenosine and adenosine receptor agonists have been studied extensively. However, their therapeutic outcomes in ischemic heart disease are limited by systemic side effects such as hypotension, bradycardia, and sedation. Equilibrative nucleoside transporter (ENT) inhibitors may be an alternative. By reducing the uptake of extracellular adenosine, ENT1 inhibitors potentiate the cardioprotective effect of endogenous adenosine. They have fewer systemic side effects because they selectively increase the extracellular adenosine levels in ischemic tissues undergoing accelerated adenosine formation. Nonetheless, long-term inhibition of ENT1 may adversely affect tissues that have low capacity for de novo nucleotide biosynthesis. ENT1 inhibitors may also affect the cellular transport, and hence the efficacy, of anticancer and antiviral nucleoside analogs used in chemotherapy. It has been proposed that ENT4 may also contribute to the regulation of extracellular adenosine in the heart, especially under the acidotic conditions associated with ischemia. Like ENT1 inhibitors, ENT4 inhibitors should work specifically on ischemic tissues. Theoretically, ENT4 inhibitors do not affect tissues that rely on ENT1 for de novo nucleotide synthesis. They also have no interaction with anticancer and antiviral nucleosides. Development of specific ENT4 inhibitors may open a new avenue in research on ischemic heart disease therapy. PMID:26070128

  19. Lack of the nucleoside transporter ENT1 results in the Augustine-null blood type and ectopic mineralization.

    PubMed

    Daniels, Geoff; Ballif, Bryan A; Helias, Virginie; Saison, Carole; Grimsley, Shane; Mannessier, Lucienne; Hustinx, Hein; Lee, Edmond; Cartron, Jean-Pierre; Peyrard, Thierry; Arnaud, Lionel

    2015-06-01

    The Augustine-negative alias At(a-) blood type, which seems to be restricted to people of African ancestry, was identified half a century ago but remains one of the last blood types with no known genetic basis. Here we report that a nonsynonymous single nucleotide polymorphism in SLC29A1 (rs45458701) is responsible for the At(a-) blood type. The resulting p.Glu391Lys variation in the last extracellular loop of the equilibrative nucleoside transporter 1 (ENT1; also called SLC29a1) is known not to alter its ability to transport nucleosides and nucleoside analog drugs. Furthermore, we identified 3 individuals of European ancestry who are homozygous for a null mutation in SLC29A1 (c.589+1G>C) and thus have the Augustine-null blood type. These individuals lacking ENT1 exhibit periarticular and ectopic mineralization, which confirms an important role for ENT1/SLC29A1 in human bone homeostasis as recently suggested by the skeletal phenotype of aging Slc29a1(-/-) mice. Our results establish Augustine as a new blood group system and place SLC29A1 as a new candidate gene for idiopathic disorders characterized with ectopic calcification/mineralization. PMID:25896650

  20. Equilibrative Nucleoside Transporters 1 and 4: Which One Is a Better Target for Cardioprotection Against Ischemia–Reperfusion Injury?

    PubMed Central

    Yang, Cui

    2015-01-01

    Abstract: The cardioprotective effects of adenosine and adenosine receptor agonists have been studied extensively. However, their therapeutic outcomes in ischemic heart disease are limited by systemic side effects such as hypotension, bradycardia, and sedation. Equilibrative nucleoside transporter (ENT) inhibitors may be an alternative. By reducing the uptake of extracellular adenosine, ENT1 inhibitors potentiate the cardioprotective effect of endogenous adenosine. They have fewer systemic side effects because they selectively increase the extracellular adenosine levels in ischemic tissues undergoing accelerated adenosine formation. Nonetheless, long-term inhibition of ENT1 may adversely affect tissues that have low capacity for de novo nucleotide biosynthesis. ENT1 inhibitors may also affect the cellular transport, and hence the efficacy, of anticancer and antiviral nucleoside analogs used in chemotherapy. It has been proposed that ENT4 may also contribute to the regulation of extracellular adenosine in the heart, especially under the acidotic conditions associated with ischemia. Like ENT1 inhibitors, ENT4 inhibitors should work specifically on ischemic tissues. Theoretically, ENT4 inhibitors do not affect tissues that rely on ENT1 for de novo nucleotide synthesis. They also have no interaction with anticancer and antiviral nucleosides. Development of specific ENT4 inhibitors may open a new avenue in research on ischemic heart disease therapy. PMID:26070128

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

    PubMed

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

    2015-01-01

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

  2. Transport characteristics of mouse concentrative nucleoside transporter 1.

    PubMed

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

    2010-03-30

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

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

    PubMed

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

    2016-04-14

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

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

    SciTech Connect

    Rowe, M.L.

    1988-01-01

    Uridine- and thymidine-phosphorylating enzymes were measured in developing and germinating seeds of Triticum aestivum v. Arthur and T. aestivum v. Lemhi. Because crude extracts were to be used in the developmental study, characteristics of unpurified nucleoside phosphotransferase (NPTase) were examined. In the developmental study with two varieties of wheat, NPTase activity was found to be very low in all of the true seed tissues during seed maturation. Uridine-phosphorylating activity was due to primarily to uridine kinase. Thymidine phosphorylation was very low in all tissues throughout seed maturation, with a brief appearance by thymidine kinase in the developing embryo. In germinating seeds, uridine-phosphorylating activity was present from earliest stages of germination but showed a decrease in activity followed by a recovery after 48 hours inbibition. Experiments using ({alpha}-{sup 32}P)ATP indicated that uridine kinase was present during early germination but had disappeared by 96 hours. Uridine phosphorylation at later stages of germination was accomplished by NTPase. Thymidine phosphorylation did not begin until after 36 hours of germination and was the result of NPTase activity.

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

    PubMed

    Shakya, Neeraj; Vedi, Satish; Liang, Chao; Yang, Fang; Agrawal, Babita; Kumar, Rakesh

    2014-03-01

    Hepatitis C virus (HCV) infection is one of the major health problems worldwide. If left untreated, it leads to liver cirrhosis, liver cancer and death. Herein, we report synthesis and anti-HCV activity of a new class of pyrimidine nucleosides possessing a 4'-carboxymethyl (9-16, 21 and 23) or 4'-carboxamide function (17-19 and 24). Among these, 10-12 (EC50=33.1-42.4 μM), 14 and 21 (EC50=43.4-59.5 μM) exhibited potent activity in HCV-1a replicon cells without any toxicity to parent Huh-7 cells (CC50=>829-1055 μM). The anti-HCV activities demonstrated by this unusual class of compounds were superior to that of ribavirin (EC50=81.9 μM). Further, the most active analog, 12, was found to interact synergistically with ribavirin to inhibit HCV RNA replication. PMID:24485784

  6. Chutes and Ladders in Hepatitis C Nucleoside Drug Development§

    PubMed Central

    Coats, Steven J.; Garnier-Amblard, Ethel C.; Amblard, Franck; Ehteshami, Maryam; Amiralaei, Sheida; Zhang, Hongwang; Zhou, Longhu; Boucle, Sebastien R. L.; Lu, Xiao; Bondada, Lavanya; Shelton, Jadd R.; Li, Hao; Liu, Peng; Li, Chengwei; Cho, Jong Hyun; Chavre, Satish N.; Zhou, Shaoman; Mathew, Judy; Schinazi, Raymond F.

    2014-01-01

    Chutes and Ladders is an exciting up-and-down-again game in which players race to be the first to the top of the board. Along the way, they will find ladders to help them advance, and chutes that will cause them to move backwards. The development of nucleoside analogs for clinical treatment of hepatitis C presents a similar scenario in which taking shortcuts may help quickly advance a program, but there is always a tremendous risk of being sent backwards as one competes for the finish line. In recent years the treatment options for chronic hepatitis C virus (HCV) infection have expand due to the development of a replicon based in vitro evaluation system, allowing for the identification of multiple drugable viral targets along with a concerted and substantial drug discovery effort. Three major drug targets have reached clinical study for chronic HCV infection: the NS3/4A serine protease, the large phosphoprotein NS5A, and the NS5B RNA-dependent RNA polymerase. Recently, two oral HCV protease inhibitors were approved by the FDA and were the first direct acting anti-HCV agents to result from the substantial research in this area. There are currently many new chemical entities from several different target classes that are being evaluated worldwide in clinical trials for their effectiveness at achieving a sustained virologic response (SVR) (Pham et al., 2004; Radkowski et al., 2005). Clearly the goal is to develop therapies leading to a cure that are safe, widely accessible and available, and effective against all HCV genotypes (GT), and all stages of the disease. Nucleoside analogs that target the HCV NS5B polymerase that have reached human clinical trials is the focus of this review as they have demonstrated significant advantages in the clinic with broader activity against the various HCV GT and a higher barrier to the development of resistant viruses when compared to all other classes of HCV inhibitors. PMID:24275341

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

    PubMed Central

    2012-01-01

    Background Nucleoside analogs used in the chemotherapy of solid tumors, such as the capecitabine catabolite 5′-deoxy-5-fluorouridine (5′-DFUR) trigger a transcriptomic response that involves the aquaglyceroporin aquaporin 3 along with other p53-dependent genes. Here, we examined whether up-regulation of aquaporin 3 (AQP3) mRNA in cancer cells treated with 5′-DFUR represents a collateral transcriptomic effect of the drug, or conversely, AQP3 participates in the activity of genotoxic agents. Methods The role of AQP3 in cell volume increase, cytotoxicity and cell cycle arrest was analyzed using loss-of-function approaches. Results 5′-DFUR and gemcitabine, but not cisplatin, stimulated AQP3 expression and cell volume, which was partially and significantly blocked by knockdown of AQP3. Moreover, AQP3 siRNA significantly blocked other effects of nucleoside analogs, including G1/S cell cycle arrest, p21 and FAS up-regulation, and cell growth inhibition. Short incubations with 5-fluorouracil (5-FU) also induced AQP3 expression and increased cell volume, and the inhibition of AQP3 expression significantly blocked growth inhibition triggered by this drug. To further establish whether AQP3 induction is related to cell cycle arrest and apoptosis, cells were exposed to long incubations with escalating doses of 5-FU. AQP3 was highly up-regulated at doses associated with cell cycle arrest, whereas at doses promoting apoptosis induction of AQP3 mRNA expression was reduced. Conclusions Based on the results, we propose that the aquaglyceroporin AQP3 is required for cytotoxic activity of 5’-DFUR and gemcitabine in the breast cancer cell line MCF7 and the colon adenocarcinoma cell line HT29, and is implicated in cell volume increase and cell cycle arrest. PMID:23017148

  8. Synergistic enhancement of 5-fluorouracil cytotoxicity by deoxyuridine analogs in cancer cells

    PubMed Central

    Matsumoto, Yoshihiro; Rodriguez, Victoria; Whitford, Tracy A.; Beeharry, Neil; Ide, Hiroshi; Tomkinson, Alan E.

    2015-01-01

    5-Fluorouracil (FU) is a halogenated nucleobase analog that is widely used in chemotherapy. Here we show that 5-hydroxymethyl-2′-deoxyuridine (hmUdR) synergistically enhances the activity of FU in cell lines derived from solid tumors but not normal tissues. While the cytotoxicity of FU and hmUdR was not directly related to the amount of the modified bases incorporated into cellular DNA, incubation with this combination resulted in dramatic increase in the number of single strand breaks in replicating cancer cells, leading to NAD-depletion as consequence of poly(ADP-ribose) synthesis and S phase arrest. Cell death resulting from the base/nucleoside combination did not occur by apoptosis, autophagy or necroptosis. Instead, the cells die via necrosis as a result of NAD depletion. The FU-related nucleoside analog, 5-fluoro-2′-deoxyuridine, also displayed synergy with hmUdR, whereas hmUdR could not be replaced by 5-hydroxymethyluracil. Among other 5-modified deoxyuridine analogs tested, 5-formyl-2′-deoxyuridine and, to a lesser extent, 5-hydroxy-2′-deoxyuridine, also acted synergistically with FU, whereas 5-hydroxyethyl-2′-deoxyuridine did not. Together, our results have revealed an unexpected synergistic interaction between deoxyuridine analogs and FU in a cancer cell-specific manner, and suggest that these novel base/nucleoside combinations could be developed into improved FU-based chemotherapies. PMID:25897430

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

    PubMed

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

    1995-01-01

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

  10. Science Teachers' Analogical Reasoning

    NASA Astrophysics Data System (ADS)

    Mozzer, Nilmara Braga; Justi, Rosária

    2013-08-01

    Analogies can play a relevant role in students' learning. However, for the effective use of analogies, teachers should not only have a well-prepared repertoire of validated analogies, which could serve as bridges between the students' prior knowledge and the scientific knowledge they desire them to understand, but also know how to introduce analogies in their lessons. Both aspects have been discussed in the literature in the last few decades. However, almost nothing is known about how teachers draw their own analogies for instructional purposes or, in other words, about how they reason analogically when planning and conducting teaching. This is the focus of this paper. Six secondary teachers were individually interviewed; the aim was to characterize how they perform each of the analogical reasoning subprocesses, as well as to identify their views on analogies and their use in science teaching. The results were analyzed by considering elements of both theories about analogical reasoning: the structural mapping proposed by Gentner and the analogical mechanism described by Vosniadou. A comprehensive discussion of our results makes it evident that teachers' content knowledge on scientific topics and on analogies as well as their pedagogical content knowledge on the use of analogies influence all their analogical reasoning subprocesses. Our results also point to the need for improving teachers' knowledge about analogies and their ability to perform analogical reasoning.

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

    PubMed

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

    2006-12-01

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

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

    PubMed

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

    2015-05-01

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

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

    PubMed Central

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

    2015-01-01

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

  14. ATP-dependent removal of nucleoside reverse transcriptase inhibitors by human immunodeficiency virus type 1 reverse transcriptase.

    PubMed

    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

  15. [An analysis of the DNA synthesized in adenovirus-infected cells under exposure to nucleoside analogs].

    PubMed

    Nosach, L N; Butenko, S I; Timofeeva, M Ia; Diachenko, N S; Tikhomirova, T P

    1989-01-01

    The method of dot DNA-DNA hybridization was used to reveal the inhibition of the synthesis of the adenoviral DNA by 6-azacytidine, cyclocytidine and ribamidyl in the adenovirus-infected cells Hep-2, a degree of which depended on the preparation concentration. PMID:2482929

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

    PubMed

    Thomas, M G; Escalante-Semerena, J C

    2000-08-01

    Computer analysis of the archaeal genome databases failed to identify orthologues of all of the bacterial cobamide biosynthetic enzymes. Of particular interest was the lack of an orthologue of the bifunctional nucleoside triphosphate (NTP):5'-deoxyadenosylcobinamide kinase/GTP:adenosylcobinamide-phosphate guanylyltransferase enzyme (CobU in Salmonella enterica). This paper reports the identification of an archaeal gene encoding a new nucleotidyltransferase, which is proposed to be the nonorthologous replacement of the S. enterica cobU gene. The gene encoding this nucleotidyltransferase was identified using comparative genome analysis of the sequenced archaeal genomes. Orthologues of the gene encoding this activity are limited at present to members of the domain Archaea. The corresponding ORF open reading frame from Methanobacterium thermoautotrophicum Delta H (MTH1152; referred to as cobY) was amplified and cloned, and the CobY protein was expressed and purified from Escherichia coli as a hexahistidine-tagged fusion protein. This enzyme had GTP:adenosylcobinamide-phosphate guanylyltransferase activity but did not have the NTP:AdoCbi kinase activity associated with the CobU enzyme of S. enterica. NTP:adenosylcobinamide kinase activity was not detected in M. thermoautotrophicum Delta H cell extract, suggesting that this organism may not have this activity. The cobY gene complemented a cobU mutant of S. enterica grown under anaerobic conditions where growth of the cell depended on de novo adenosylcobalamin biosynthesis. cobY, however, failed to restore adenosylcobalamin biosynthesis in cobU mutants grown under aerobic conditions where de novo synthesis of this coenzyme was blocked, and growth of the cell depended on the assimilation of exogenous cobinamide. These data strongly support the proposal that the relevant cobinamide intermediates during de novo adenosylcobalamin biosynthesis are adenosylcobinamide-phosphate and adenosylcobinamide-GDP, not adenosylcobinamide

  17. All-trans-retinoic Acid Promotes Trafficking of Human Concentrative Nucleoside Transporter-3 (hCNT3) to the Plasma Membrane by a TGF-β1-mediated Mechanism*

    PubMed Central

    Fernández-Calotti, Paula; Pastor-Anglada, Marçal

    2010-01-01

    Human concentrative nucleoside transporter-3 (hCNT3) is a sodium-coupled nucleoside transporter that exhibits high affinity and broad substrate selectivity, making it the most suitable candidate for mediating the uptake and cytotoxic action of most nucleoside-derived drugs. The drug of this class most commonly used in the treatment of chronic lymphocytic leukemia (CLL) is the pro-apoptotic nucleoside analog fludarabine (Flu), which enters CLL cells primarily through human equilibrative nucleoside transporters (hENTs). Although CLL cells lack hCNT3 activity, they do express this transporter protein, which is located mostly in the cytosol. The aim of our study was to identify agents and mechanisms capable of promoting hCNT3 trafficking to the plasma membrane. Here, we report that all-trans-retinoic acid (ATRA), currently used in the treatment of acute promyelocytic leukemia (APL), increases hCNT3-related activity through a mechanism that involves trafficking of pre-existing hCNT3 proteins to the plasma membrane. This effect is mediated by the autocrine action of transforming growth factor (TGF)-β1, which is transcriptionally activated by ATRA in a p38-dependent manner. TGF-β1 acts through activation of ERK1/2 and the small GTPase RhoA to promote plasma membrane trafficking of the hCNT3 protein. PMID:20172853

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

    SciTech Connect

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

    1982-01-01

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

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

    PubMed

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

    2007-02-12

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

  20. Chemoselective N-Deacetylation of Protected Nucleosides and Nucleotides Promoted by Schwartz's Reagent

    PubMed Central

    Ferrari, Valentina; Serpi, Michaela; McGuigan, Christopher; Pertusati, Fabrizio

    2015-01-01

    Protection and deprotection strategies involving the N-acetyl group are widely utilized in nucleoside and nucleotide chemistry. Herein, we present a mild and selective N-deacetylation methodology, applicable to purine and pyrimidine nucleosides, by means of Schwartz's reagent, compatible with most of the common protecting groups used in nucleoside chemistry. PMID:26492555

  1. The role of human equilibrative nucleoside transporter 1 on the cellular transport of the DNA methyltransferase inhibitors 5-azacytidine and CP-4200 in human leukemia cells.

    PubMed

    Hummel-Eisenbeiss, Johanna; Hascher, Antje; Hals, Petter-Arnt; Sandvold, Marit Liland; Müller-Tidow, Carsten; Lyko, Frank; Rius, Maria

    2013-09-01

    The nucleoside analog 5-azacytidine is an archetypical drug for epigenetic cancer therapy, and its clinical effectiveness has been demonstrated in the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). However, therapy resistance in patients with MDS/AML remains a challenging issue. Membrane proteins that are involved in drug uptake are potential mediators of drug resistance. The responsible proteins for the transport of 5-azacytidine into MDS/AML cells are unknown. We have now systematically analyzed the expression and activity of various nucleoside transporters. We identified the human equilibrative nucleoside transporter 1 (hENT1) as the most abundant nucleoside transporter in leukemia cell lines and in AML patient samples. Transport assays using [¹⁴C]5-azacytidine demonstrated Na⁺-independent uptake of the drug into the cells, which was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a hENT1 inhibitor. The cellular toxicity of 5-azacytidine and its DNA demethylating activity were strongly reduced after hENT1 inhibition. In contrast, the cellular activity of the 5-azacytidine derivative 5-azacytidine-5'-elaidate (CP-4200), a nucleoside transporter-independent drug, persisted after hENT1 inhibition. A strong dependence of 5-azacytidine-induced DNA demethylation on hENT1 activity was also confirmed by array-based DNA methylation profiling, which uncovered hundreds of loci that became demethylated only when hENT1-mediated transport was active. Our data establish hENT1 as a key transporter for the cellular uptake of 5-azacytidine in leukemia cells and raise the possibility that hENT1 expression might be a useful biomarker to predict the efficiency of 5-azacytidine treatments. Furthermore, our data suggest that CP-4200 may represent a valuable compound for the modulation of transporter-related 5-azacytidine resistances. PMID:23814180

  2. Radioactive Decay - An Analog.

    ERIC Educational Resources Information Center

    McGeachy, Frank

    1988-01-01

    Presents an analog of radioactive decay that allows the student to grasp the concept of half life and the exponential nature of the decay process. The analog is devised to use small, colored, plastic poker chips or counters. Provides the typical data and a graph which supports the analog. (YP)

  3. Synthesis and biological investigations of 5-substituted pyrimidine nucleosides coupled to a dihydropyridine/pyridinium salt redox chemical delivery system.

    PubMed

    Kumar, R; Wang, L; Wiebe, L I; Knaus, E E

    2001-11-01

    The syntheses, antiviral activities, and partition coefficients (P) of 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-coupled nucleosides are described. These novel compounds were designed in an effort to enhance the lipophilicity, and thereby the delivery to the CNS, without compromising the anti-HSV-1 activity of the parental nucleosides. We have previously reported the synthesis of 3'-O-(1-methyl-1,4-dihydropyridyl-3- carbonyl) analogs of 5-iodo-(5), 5-vinyl-(6), and (E)-5-(2-iodovinyl)-2'-deoxyuridines (7). We now report the synthesis of 5-iodo-3'-O-(1-methyl-1,4-dihydropyridyl-3- carbonyl)-5'-O-acetyl-2'-deoxyuridine (15) and 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2'-deoxyuridine (17). Quarternization of the 3'-O-(3-pyridylcarbonyl) compounds (10,12) using iodomethane afforded the corresponding 1-methyl pyridinium salts (13,14) which were reduced with sodium dithionite to yield the corresponding 3'-O-1-methyl-1,4-dihydropyridyl-3-carbonyl compounds (15,16). The deprotection of 3'-O-(1-methyl-1,4-dihydropyridyl- 3-carbonyl)-5'-O-t-butyldimethylsilyl-2'-deoxyuridine (16) with Bu4N+F- afforded 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2'-deoxyuridine (17). Compounds 5-7 and 15 were evaluated for their antiviral activity in vitro against HSV-1, HSV-2, HCMV, and VZV, and were found to retain anti-HSV-1, HSV-2 and VZV activity as compared to their parental nucleosides (1-3). In addition, the cellular toxicity of 3'-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-coupled compounds (5-7 and 15) was found to be lower than the parent nucleosides. The lipophilicity of compounds (5-7,15,17) are enhanced substantially, compared to the parent nucleosides, as indicated by an increase in corresponding P values (1-octanol-water) upon replacement of the C-3' hydroxyl by 1-methyl-1,4-dihydropyridyl-3-carbonyl moiety. PMID:11822172

  4. The cystic fibrosis transmembrane recruiter the alter ego of CFTR as a multi-kinase anchor

    PubMed Central

    2007-01-01

    This review focuses on a newly discovered interaction between protein kinases involved in cellular energetics, a process that may be disturbed in cystic fibrosis for unknown reasons. I propose a new model where kinase-mediated cellular transmission of energy provides mechanistic insight to a latent role of the cystic fibrosis transmembrane conductance regulator (CFTR). I suggest that CFTR acts as a multi-kinase recruiter to the apical epithelial membrane. My group finds that, in the cytosol, two protein kinases involved in cell energy homeostasis, nucleoside diphosphate kinase (NDPK) and AMP-activated kinase (AMPK), bind one another. Preliminary data suggest that both can also bind CFTR (function unclear). The disrupted role of this CFTR-kinase complex as ‘membrane transmitter to the cell’ is proposed as an alternative paradigm to the conventional ion transport mediated and CFTR/chloride-centric view of cystic fibrosis pathogenesis. Chloride remains important, but instead, chloride-induced control of the phosphohistidine content of one kinase component (NDPK, via a multi-kinase complex that also includes a third kinase, CK2; formerly casein kinase 2). I suggest that this complex provides the necessary near-equilibrium conditions needed for efficient transmission of phosphate energy to proteins controlling cellular energetics. Crucially, a new role for CFTR as a kinase controller is proposed with ionic concentration acting as a signal. The model posits a regulatory control relay for energy sensing involving a cascade of protein kinases bound to CFTR. PMID:17805562

  5. Cladribine and Fludarabine Nucleoside Change the Levels of CD Antigens on B-Lymphoproliferative Disorders

    PubMed Central

    Cassano, Carlos; Mactier, Swetlana; Mulligan, Stephen P.; Belov, Larissa; Huang, Pauline; Christopherson, Richard I.

    2010-01-01

    The purine analogs, fludarabine nucleoside (FdA), and cladribine (CdA) (1 μM, 24 hours), significantly changed the levels of some surface antigens on the human B-cell lines MEC2 and Raji. Changes in the surface proteins were identified using a Cluster of Differentiation (CD) antibody microarray that captures live cells and confirmed by flow cytometry. For Raji cells, CdA up-regulated CD10, CD54, CD80, and CD86, with repression of CD22, while FdA up-regulated CD20, CD54, CD80, CD86 and CD95. For MEC2 cells, CdA up-regulated CD11a, CD20, CD43, CD45, CD52, CD54, CD62L, CD80, CD86, and CD95, but FdA had no effect. Up-regulation of particular CD antigens induced on a B-cell lymphoproliferative disorder by a purine analog could provide targets for therapeutic antibodies with synergistic cell killing. PMID:22084681

  6. Lack of stereospecificity of suid pseudorabies virus thymidine kinase.

    PubMed Central

    Maga, G; Verri, A; Bonizzi, L; Ponti, W; Poli, G; Garbesi, A; Niccolai, D; Spadari, S; Focher, F

    1993-01-01

    We have partially purified suid pseudorabies virus (PRV) thymidine kinase from infected thymidine kinase- mouse cells, and cytosolic swine thymidine kinase from lymphatic glands, and we have found that PRV thymidine kinase, unlike the host enzyme, shows no stereospecificity for D- and L-beta-nucleosides. In vitro, unnatural L-enantiomers, except L-deoxycytidine, function as specific inhibitors for the viral enzyme in the order: L-thymidine >> L-deoxyguanosine > L-deoxyuridine > L-deoxyadenosine. Contrary to human and swine thymidine kinases and like herpes simplex virus-1 and -2 thymidine kinases, PRV thymidine kinase phosphorylates both the natural (D-) and the unnatural (L-) thymidine enantiomers to their corresponding monophosphates with comparable efficiency. The kinetic parameters Vmax/Km for D- and L-thymidine are 3.7 and 2.3 respectively. Our results demonstrate that the lack of stereospecificity might be a common feature of the thymidine kinases that are encoded by human and animal herpes viruses. These observations could lead to the development of a novel class of antiviral drugs. PMID:8396911

  7. Lack of stereospecificity of suid pseudorabies virus thymidine kinase.

    PubMed

    Maga, G; Verri, A; Bonizzi, L; Ponti, W; Poli, G; Garbesi, A; Niccolai, D; Spadari, S; Focher, F

    1993-09-01

    We have partially purified suid pseudorabies virus (PRV) thymidine kinase from infected thymidine kinase- mouse cells, and cytosolic swine thymidine kinase from lymphatic glands, and we have found that PRV thymidine kinase, unlike the host enzyme, shows no stereospecificity for D- and L-beta-nucleosides. In vitro, unnatural L-enantiomers, except L-deoxycytidine, function as specific inhibitors for the viral enzyme in the order: L-thymidine > L-deoxyguanosine > L-deoxyuridine > L-deoxyadenosine. Contrary to human and swine thymidine kinases and like herpes simplex virus-1 and -2 thymidine kinases, PRV thymidine kinase phosphorylates both the natural (D-) and the unnatural (L-) thymidine enantiomers to their corresponding monophosphates with comparable efficiency. The kinetic parameters Vmax/Km for D- and L-thymidine are 3.7 and 2.3 respectively. Our results demonstrate that the lack of stereospecificity might be a common feature of the thymidine kinases that are encoded by human and animal herpes viruses. These observations could lead to the development of a novel class of antiviral drugs. PMID:8396911

  8. In vitro antiviral activity of adenosine analog NITD008 against tick-borne flaviviruses.

    PubMed

    Lo, Michael K; Shi, Pei-Yong; Chen, Yen-Liang; Flint, Mike; Spiropoulou, Christina F

    2016-06-01

    There are currently no antiviral therapies available for the tick-borne flaviviruses associated with hemorrhagic fevers: Kyasanur Forest disease virus (KFDV), both classical and the Alkhurma hemorrhagic fever virus (AHFV) subtype, and Omsk hemorrhagic fever virus (OHFV). In this brief study, we describe the in vitro antiviral activity of adenosine analog NITD008 against KFDV, AHFV, OHFV, as well as Tick-borne Encephalitis virus (TBEV). Alongside the well-established activity of NITD008 against mosquito-borne flaviviruses, our results have demonstrated the feasibility of identifying nucleoside analog inhibitors that have pan-flavivirus activity. PMID:27016316

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

    PubMed Central

    Riegler, Heike; Geserick, Claudia; Zrenner, Rita

    2011-01-01

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

  10. The GABA transaminase, ABAT, is essential for mitochondrial nucleoside metabolism

    PubMed Central

    Besse, Arnaud; Wu, Ping; Bruni, Francesco; Donti, Taraka; Graham, Brett H.; Craigen, William J.; McFarland, Robert; Moretti, Paolo; Lalani, Seema; Scott, Kenneth L.; Taylor, Robert W.; Bonnen, Penelope E.

    2015-01-01

    Summary ABAT is a key enzyme responsible for catabolism of principal inhibitory neurotransmitter gamma-aminobutyric acid (GABA). We report an essential role for ABAT in a seemingly unrelated pathway, mitochondrial nucleoside salvage, and demonstrate that mutations in this enzyme cause an autosomal recessive neurometabolic disorder and mtDNA depletion syndrome (MDS). We describe a family with encephalomyopathic MDS caused by a homozygous missense mutation in ABAT that results in elevated GABA in subjects’ brains as well as decreased mtDNA levels in subjects’ fibroblasts. Nucleoside rescue and co-IP experiments pinpoint that ABAT functions in the mitochondrial nucleoside salvage pathway to facilitate conversion of dNDPs to dNTPs. Pharmacological inhibition of ABAT through the irreversible inhibitor Vigabatrin caused depletion of mtDNA in photoreceptor cells that was prevented through addition of dNTPs in cell culture media. This work reveals ABAT as a connection between GABA metabolism and nucleoside metabolism and defines a neurometabolic disorder that includes MDS. PMID:25738457