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. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML.

PubMed

Liyanage, Sanduni U; Hurren, Rose; Voisin, Veronique; Bridon, Gaëlle; Wang, Xiaoming; Xu, ChangJiang; MacLean, Neil; Siriwardena, Thirushi P; Gronda, Marcela; Yehudai, Dana; Sriskanthadevan, Shrivani; Avizonis, Daina; Shamas-Din, Aisha; Minden, Mark D; Bader, Gary D; Laposa, Rebecca; Schimmer, Aaron D

2017-05-11

Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2'3'-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML. © 2017 by The American Society of Hematology.

Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML

PubMed Central

Liyanage, Sanduni U.; Hurren, Rose; Voisin, Veronique; Bridon, Gaëlle; Wang, Xiaoming; Xu, ChangJiang; MacLean, Neil; Siriwardena, Thirushi P.; Gronda, Marcela; Yehudai, Dana; Sriskanthadevan, Shrivani; Avizonis, Daina; Shamas-Din, Aisha; Minden, Mark D.; Bader, Gary D.; Laposa, Rebecca

2017-01-01

Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2′3′-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2′3′-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML. PMID:28283480

Crystal Structure and Biochemical Characterization of a Mycobacterium smegmatis AAA-Type Nucleoside Triphosphatase Phosphohydrolase (Msm0858).

PubMed

Unciuleac, Mihaela-Carmen; Smith, Paul C; Shuman, Stewart

2016-05-15

AAA proteins (ATPases associated with various cellular activities) use the energy of ATP hydrolysis to drive conformational changes in diverse macromolecular targets. Here, we report the biochemical characterization and 2.5-Å crystal structure of a Mycobacterium smegmatis AAA protein Msm0858, the ortholog of Mycobacterium tuberculosis Rv0435c. Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates. The Msm0858 structure comprises (i) an N-terminal domain (amino acids [aa] 17 to 201) composed of two β-barrel modules and (ii) two AAA domains, D1 (aa 212 to 473) and D2 (aa 476 to 744), each of which has ADP in the active site. Msm0858-ADP is a monomer in solution and in crystallized form. Msm0858 domains are structurally homologous to the corresponding modules of mammalian p97. However, the position of the N-domain modules relative to the AAA domains in the Msm0858-ADP tertiary structure is different and would impede the formation of a p97-like hexameric quaternary structure. Mutational analysis of the A-box and B-box motifs indicated that the D1 and D2 AAA domains are both capable of ATP hydrolysis. Simultaneous mutations of the D1 and D2 active-site motifs were required to abolish ATPase activity. ATPase activity was effaced by mutation of the putative D2 arginine finger, suggesting that Msm0858 might oligomerize during the ATPase reaction cycle. A truncated variant Msm0858 (aa 212 to 745) that lacks the N domain was characterized as a catalytically active homodimer. Recent studies have underscored the importance of AAA proteins (ATPases associated with various cellular activities) in the physiology of mycobacteria. This study reports the ATPase activity and crystal structure of a previously uncharacterized mycobacterial AAA protein, Msm0858. Msm0858 consists of an N-terminal β-barrel domain and two AAA domains, each with ADP bound in the active site. Msm0858 is a

Synthesis and antiviral activity of 3'-deoxy-3'-C-hydroxymethyl nucleosides.

PubMed

Bamford, M J; Coe, P L; Walker, R T

1990-09-01

A series of 3'-branched-chain sugar nucleosides, in particular 3'-deoxy-3'-C-hydroxmethyl nucleosides, have been synthesized and evaluated as antiviral agents. Reaction of 1-(2,3-epoxy-5-O-trityl-beta-D-lyxo-pentofuranosyl) derivatives 12 and 13, of uracil and thymine, respectively, with 5,6-dihydro-2-lithio-5-methyl-1,3,5-dithiazine 14 afforded the corresponding 3'-functionalized nucleosides 15 and 16, respectively. Replacement of the trityl group with tertbutyldiphenylsilyl allowed high yielding hydrolysis of the 3'-function to give the 3'-deoxy-3'-C-formyl-beta-D-arabino-pentofuranosyl nucleosides 21 and 22. Desilylation afforded the 1-(3-deoxy-3-C-formyl-beta- D-lyxo-pentofuranosyl) 3',5'-O-hemiacetal nucleosides 33 and 34, respectively. Reduction of the formyl group of 21 and 22, followed by desilylation, yielded the 3'-deoxy-3'-C-(hydroxymethyl)-beta-D-arabino- pentofuranosyl) analogues 7 and 8, respectively. The uracil base moiety of 7 was converted to 5-iodouracil and then to (E)-5-(2-bromovinyl)uracil to furnish an analogue 10 of BVaraU. The 1-(3-deoxy-3-C-(hydroxymethyl)-beta-D-lyxo-pentofuranosyl) and 1-(2,3-dideoxy-3-C-(hydroxymethyl)-beta-D-erythro-pentofuranosyl) derivatives of uracil (31 and 6, respectively) and 5-iodouracil (32 and 9, respectively) were also obtained. All novel, fully deprotected nucleoside analogues were evaluated for antiviral activity against human immunodeficiency virus type-1, herpes simplex virus types-1 and -2, varicella zoster virus, human cytomegalovirus and influenza A. Of the compounds tested only (E)-5-(2-bromovinyl)-1-[3-deoxy- 3-C-(hydroxymethyl)-beta-D-arabino-pentofuranosyl]uracil (10) inhibited VZV (alone), but did so at concentrations well below the cytotoxicity threshold.

Modulatory action of α-tocopherol on erythrocyte membrane adenosine triphosphatase against radiation damage in oral cancer.

PubMed

Chitra, Subramaniam; Shyamaladevi, Chennam Srinivasulu

2011-03-01

To investigate the possible effects of α-tocopherol on erythrocyte membrane adenosine triphosphatases against radiation damage in oral cancer patients. Adenosine triphosphatase activities were analysed in oral cancer patients before and after radiotherapy (at a dosage of 6000 cGY in five fractions per week for a period of six weeks) and after supplemented with α-tocopherol (400 IU per day for entire period of radiotherapy). The membrane bound enzymes such as Na(+)/K(+)-ATPase, Ca(2+)-ATPase, Mg(2+)-ATPase and some trace elements were altered in oral cancer patients before and after radiotherapy. Supplemented with α-tocopherol modulates the erythrocyte membrane which is damaged by radiotherapy which suggests that α-tocopherol protects the erythrocyte membrane from radiation damage in oral cancer patients.

Carnitine modulates crucial myocardial adenosine triphosphatases and acetylcholinesterase enzyme activities in choline-deprived rats.

PubMed

Strilakou, Athina A; Tsakiris, Stylianos T; Kalafatakis, Konstantinos G; Stylianaki, Aikaterini T; Karkalousos, Petros L; Koulouris, Andreas V; Mourouzis, Iordanis S; Liapi, Charis A

2014-01-01

Choline is an essential nutrient, and choline deficiency has been associated with cardiovascular morbidity. Choline is also the precursor of acetylcholine (cholinergic component of the heart's autonomic nervous system), whose levels are regulated by acetylcholinesterase (AChE). Cardiac contraction-relaxation cycles depend on ion gradients established by pumps like the adenosine triphosphatases (ATPases) Na(+)/K(+)-ATPase and Mg(2+)-ATPase. This study aimed to investigate the impact of dietary choline deprivation on the activity of rat myocardial AChE (cholinergic marker), Na(+)/K(+)-ATPase, and Mg(2+)-ATPase, and the possible effects of carnitine supplementation (carnitine, structurally relevant to choline, is used as an adjunct in treating cardiac diseases). Adult male albino Wistar rats were distributed among 4 groups, and were fed a standard or choline-deficient diet for one month with or without carnitine in their drinking water (0.15% w/v). The enzyme activities were determined spectrophotometrically in the myocardium homogenate. Choline deficiency seems to affect the activity of the aforementioned parameters, but only the combination of choline deprivation and carnitine supplementation increased myocardial Na(+)/K(+)-ATPase activity along with a concomitant decrease in the activities of Mg(2+)-ATPase and AChE. The results suggest that carnitine, in the setting of choline deficiency, modulates cholinergic myocardial neurotransmission and the ATPase activity in favour of cardiac work efficiency.

A cross-linking study of the Ca2+, Mg2+-activated adenosine triphosphatase of Escherichia coli.

PubMed

Bragg, P D; Hou, C

1980-05-01

The solubilized Ca2+,Mg2+-activated adenosine triphosphatase of Escherichia coli is composed of five subunits designated alpha, beta, gamma, delta and epsilon in order of decreasing molecular weight. The subunit structure of the enzyme has been investigated by the use of the cleavable cross-linking agents dithiobis(succinimidyl propionate), methyl-4-mercaptobutyrimidate, dimethyl-3,3'-dithiobispropionimidate, disuccinimidyl tartarate, and cupric 1,10-phenanthrolinate. The products of cross-linking were analyzed by two different two-dimensional gel electrophoresis systems. The following cross-linked subunit dimers were observed: alpha 2, beta 2, alpha beta, alpha delta, beta gamma, beta delta, beta epsilon and gamma epsilon. These results, together with other published data, are discussed in relation to a model of the arrangement of the subunits in the ATPase molecule.

Pentose phosphates in nucleoside interconversion and catabolism.

PubMed

Tozzi, Maria G; Camici, Marcella; Mascia, Laura; Sgarrella, Francesco; Ipata, Piero L

2006-03-01

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway, or are supplied by nucleoside phosphorylases. The two main pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, are readily interconverted by the action of phosphopentomutase. Ribose-5-phosphate is the direct precursor of 5-phosphoribosyl-1-pyrophosphate, for both de novo and 'salvage' synthesis of nucleotides. Phosphorolysis of deoxyribonucleosides is the main source of deoxyribose phosphates, which are interconvertible, through the action of phosphopentomutase. The pentose moiety of all nucleosides can serve as a carbon and energy source. During the past decade, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. We review herein the experimental knowledge on the molecular mechanisms by which (a) ribose-1-phosphate, produced by purine nucleoside phosphorylase acting catabolically, is either anabolized for pyrimidine salvage and 5-fluorouracil activation, with uridine phosphorylase acting anabolically, or recycled for nucleoside and base interconversion; (b) the nucleosides can be regarded, both in bacteria and in eukaryotic cells, as carriers of sugars, that are made available though the action of nucleoside phosphorylases. In bacteria, catabolism of nucleosides, when suitable carbon and energy sources are not available, is accomplished by a battery of nucleoside transporters and of inducible catabolic enzymes for purine and pyrimidine nucleosides and for pentose phosphates. In eukaryotic cells, the modulation of pentose phosphate production by nucleoside catabolism seems to be affected by developmental and physiological factors on enzyme levels.

[The use of complex interval models for predicting activity of non-nucleoside reverse transcriptase activity].

PubMed

Burliaeva, E V; Tarkhov, A E; Burliaev, V V; Iurkevich, A M; Shvets, V I

2002-01-01

Searching of new anti-HIV agents is still crucial now. In general, researches are looking for inhibitors of certain HIV's vital enzymes, especially for reverse transcriptase (RT) inhibitors. Modern generation of anti-HIV agents represents non-nucleoside reverse transcriptase inhibitors (NNRTIs). They are much less toxic than nucleoside analogues and more chemically stable, thus being slower metabolized and emitted from the human body. Thus, search of new NNRTIs is actual today. Synthesis and study of new anti-HIV drugs is very expensive. So employment of the activity prediction techniques for such a search is very beneficial. This technique allows predicting the activities for newly proposed structures. It is based on the property model built by investigation of a series of known compounds with measured activity. This paper presents an approach of activity prediction based on "structure-activity" models designed to form a hypothesis about probably activity interval estimate. This hypothesis formed is based on structure descriptor domains, calculated for all energetically allowed conformers for each compound in the studied sef. Tetrahydroimidazobenzodiazipenone (TIBO) derivatives and phenylethyltiazolyltiourea (PETT) derivatives illustrated the predictive power of this method. The results are consistent with experimental data and allow to predict inhibitory activity of compounds, which were not included into the training set.

Synthesis, anti-HIV activity studies, and in silico rationalization of cyclobutane-fused nucleosides.

PubMed

Figueras, Antoni; Miralles-Llumà, Rosa; Flores, Ramon; Rustullet, Albert; Busqué, Félix; Figueredo, Marta; Font, Josep; Alibés, Ramon; Maréchal, Jean-Didier

2012-06-01

The present work describes some recent approaches to novel 3-oxabicyclo[3.2.0]heptane-type nucleosides structurally similar to the potent anti-HIV agent stavudine (d4T). To gain knowledge at the molecular level relevant for further synthetic designs, the lack of activity of these compounds was investigated by computational approaches accounting for three main physiological requirements of anti-HIV nucleosides: their drug-likeness, their activation process, and their subsequent interaction with HIV reverse transcriptase (HIV-RT). Our results show that the inclusion of the fused cyclobutane at the 2'- and 3'-positions of the sugar portion provides drug-like compounds. Nonetheless, the presence of this cyclobutane moiety prevents binding orientations consistent with the catalytic activation for at least one of the enzymes known to activate d4T. To the best of our knowledge, this is the first study to explicitly consider the simulation of the entire activation process to rationalize anti-HIV activities. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Steroid hormones are novel nucleoside transport inhibitors by competition with nucleosides for their transporters.

PubMed

Kaneko, Masahiro; Hakuno, Fumihiko; Kamei, Hiroyasu; Yamanaka, Daisuke; Chida, Kazuhiro; Minami, Shiro; Coe, Imogen R; Takahashi, Shin-Ichiro

2014-01-10

Nucleoside transport is important for nucleic acid synthesis in cells that cannot synthesize nucleosides de novo, and for entry of many cytotoxic nucleoside analog drugs used in chemotherapy. This study demonstrates that various steroid hormones induce inhibition of nucleoside transport in mammalian cells. We analyzed the inhibitory effects of estradiol (E2) on nucleoside transport using SH-SY5Y human neuroblastoma cells. We observed inhibitory effects after acute treatment with E2, which lasted in the presence of E2. However, when E2 was removed, the effect immediately disappeared, suggesting that E2 effects are not mediated through the canonical regulatory pathway of steroid hormones, such as transcriptional regulation. We also discovered that E2 could competitively inhibit thymidine uptake and binding of the labeled nucleoside transporter inhibitor, S-[4-nitrobenzyl]-6-thioinosine (NBTI), indicating that E2 binds to endogenous nucleoside transporters, leading to inhibition of nucleoside transport. We then tested the effects of various steroids on nucleoside uptake in NBTI-sensitive cells, SH-SY5Y and NBTI-insensitive cells H9c2 rat cardiomyoblasts. We found E2 and progesterone clearly inhibited both NBTI-sensitive and insensitive uptake at micromolar concentrations. Taken together, we concluded that steroid hormones function as novel nucleoside transport inhibitors by competition with nucleosides for their transporters. Copyright © 2013 Elsevier Inc. All rights reserved.

Synthesis and antiviral activity of certain second generation methylenecyclopropane nucleosides

PubMed Central

Williams, John D.; Khan, Atiyya R.; Harden, Emma A.; Hartline, Caroll B.; Jefferson, Geraldine M.; Keith, Kathy A.; Prichard, Mark N.; Zemlicka, Jiri; Peet, Norton P.; Bowlin, Terry L.

2012-01-01

A second-generation series of substituted methylenecyclopropane nucleosides (MCPNs) has been synthesized and evaluated for antiviral activity against a panel of human herpesviruses, and for cytotoxicity. Although alkylated 2,6-diaminopurine analogs showed little antiviral activity, the compounds containing ether and thioether substituents at the 6-position of the purine did demonstrate potent and selective antiviral activity against several different human herpesviruses. In the 6-alkoxy series, antiviral activity depended on the length of the ether carbon chain, with the optimum chain length being about four carbon units long. For the corresponding thioethers, compounds containing secondary thioethers were more potent than those with primary thioethers. PMID:22607883

THE LOCALIZATION OF MYOFIBRILLAR ATPASE ACTIVITY IN THE FLIGHT MUSCLES OF THE BLOWFLY, CALLIPHORA ERYTHROCEPHALA

PubMed Central

Tice, Lois W.; Smith, David S.

1965-01-01

The distribution of ATPase activity in the asynchronous flight muscles of Calliphora erythrocephala (Diptera) was studied at a fine structural level, using preparations of teased fibers, both unfixed and after brief fixation in hydroxyadipaldehyde, incubated in a medium for the histochemical demonstration of myosin or actomyosin ATPase. In relaxed fibrils, activity was found confined to the A bands and was absent from the H zones as well as from the Z and I band regions. At high magnification, deposits of final product, lead phosphate, appeared primarily related to the thick filaments, or to short lateral extensions from them. Evidence was gathered which indicated that this enzyme activity was that of a triphosphatase which did not act on dinucleoside or non-nucleoside substrates. PMID:4221034

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

PubMed Central

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

2003-01-01

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

[Purine and pyrimidine nucleoside phosphorylases - remarkable enzymes still not fully understood].

PubMed

Bzowska, Agnieszka

2015-01-01

Purine and pyrimidine nucleoside phosphorylases catalyze the reversible phosphorolytic cleavage of the glycosidic bond of purine and pyrimidine nucleosides, and are key enzymes of the nucleoside salvage pathway. This metabolic route is the less costly alternative to the de novo synthesis of nucleosides and nucleotides, supplying cells with these important building blocks. Interest in nucleoside phosphorylases is not only due to their important role in metabolism of nucleosides and nucleotides, but also due to the potential medical use of the enzymes (all phosphorylases in activating prodrugs - nucleoside and nucleic base analogs, high-molecular mass purine nucleoside phosphorylases in gene therapy of some solid tumors) and their inhibitors (as selective immunosuppressive, anticancer and antiparasitic agents, and preventing inactivation of other nucleoside drugs). Phosphorylases are also convenient tools for efficient enzymatic synthesis of otherwise inaccessible nucleoside analogues. In this paper the contribution of Professor David Shugar and some of his colleagues and coworkers in studies of these remarkable enzymes carried out over nearly 40 years is discussed on the background of global research in this field.

Structure-Activity Relationships of Acyclic Selenopurine Nucleosides as Antiviral Agents.

PubMed

Sahu, Pramod K; Umme, Tamima; Yu, Jinha; Kim, Gyudong; Qu, Shuhao; Naik, Siddhi D; Jeong, Lak Shin

2017-07-12

A series of acyclic selenopurine nucleosides 3a - f and 4a - g were synthesized based on the bioisosteric rationale between oxygen and selenium, and then evaluated for antiviral activity. Among the compounds tested, seleno-acyclovir ( 4a ) exhibited the most potent anti-herpes simplex virus (HSV)-1 (EC 50 = 1.47 µM) and HSV-2 (EC 50 = 6.34 µM) activities without cytotoxicity up to 100 µM, while 2,6-diaminopurine derivatives 4e - g exhibited significant anti-human cytomegalovirus (HCMV) activity, which is slightly more potent than the guanine derivative 4d , indicating that they might act as prodrugs of seleno-ganciclovir ( 4d ).

Nucleoside pyrophosphatase activity associated with pig kidney alkaline phosphatase

PubMed Central

Wass, Milica; Butterworth, P. J.

1971-01-01

1. A study was made of the hydrolysis, at pH9.0, of ATP and ADP catalysed by pig kidney alkaline phosphatase. Both of these nucleoside pyrophosphates are substrates for the enzyme; Km values are 4×10−5m for ATP and 6.3×10−5m for ADP. Vmax. for ADP is approximately double that of ATP. 2. Above 0.1mm approximately, both ATP and ADP are inhibitory, but the inhibition is reversible by the addition of Mg2+ ions to form MgATP2− or MgADP− complexes. The complexes, besides being non-inhibitory, are also substrates for the enzyme with Km values identical with those of the respective free nucleotides. 3. Mg2+ ions are inhibitory when present in excess of ATP or ADP. The degree of inhibition is greater with ATP as substrate, but with both ATP and ADP a mixed competitive–non-competitive type of inhibition is observed. 4. It is suggested that under normal conditions the enzyme is inhibited by cellular concentrations of ATP plus ADP but that an increase in the concentration of Mg2+ ions stimulates activity by relieving nucleoside pyrophosphate inhibition. The properties may be of importance in the regulation of the transport of bivalent cations. PMID:4331861

Simultaneous quantification and splenocyte-proliferating activities of nucleosides and bases in Cervi cornu Pantotrichum

PubMed Central

Zong, Ying; Wang, Yu; Li, Hang; Li, Na; Zhang, Hui; Sun, Jiaming; Niu, Xiaohui; Gao, Xiaochen

2014-01-01

Background: Cervi Cornu Pantotrichum has been a well known traditional Chinese medicine, which is young horn of Cervus Nippon Temminck (Hualurong: HLR). At present, the methods used for the quality control of Cervi Cornu Pantotrichum show low specificity. Objective: To describe a holistic method based on chemical characteristics and splenocyte-proliferating activities to evaluate the quality of HLR. Materials and Methods: The nucleosides and bases from HLR were identified by high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS), and six of them were chosen to be used for simultaneous HPLC quantification according to the results of proliferation of mouse splenocytes in vitro. Results: In this study, eight nucleosides and bases have been identified. In addition, uracil, hypoxanthine, uridine, inosine, guanosine, and adenosine were chosen to be used for simultaneous HPLC quantification. Simultaneous quantification of these six substances was performed on ten groups of HLR under the condition of a TIANHE Kromasil C18 column (5 μm, 4.6 mm × 250 mm i.d.) and a gradient elution of water and acetonitrile. Of the ten groups, HLR displayed the highest total nucleoside contents (TNC, sum of adenosine and uracil, 0.412 mg/g) with the strongest splenocyte-proliferating activities. Conclusion: These results suggest that TNC (such as particularly highly contained adenosine and uracil) in HLR has a certain correlation with the activity of splenocyte-proliferating, and it may be used as a quality control for HLR. This comprehensive method could be applied to other traditional Chinese medicines to ameliorate their quality control. PMID:25422536

Alkaline phosphatase, 5'-nucleotidase and magnesium-dependent adenosine triphosphatase activities in the transitional epithelium of the rat urinary bladder.

PubMed

Zhang, S X; Kobayashi, T; Okada, T; García del Saz, E; Seguchi, H

1991-07-01

The cerium-based method was used to demonstrate cytochemically the ultrastructural localization of alkaline phosphatase (ALPase), 5'-nucleotidase (5'-Nase) and magnesium-dependent adenosine triphosphatase (Mg-ATPase) on the transitional epithelium of the rat urinary bladder. The reaction product for ALPase was found on the plasma membrane of all epithelial cells, except the luminal surface of superficial cells. The activity of 5'-Nase appeared on the plasma membrane of all bladder transitional epithelial cells, including the free surface of superficial cells. The Mg-ATPase reaction product was seen on the plasma membrane of superficial, intermediate and basal cells, but never on the luminal surface of superficial cells and it was only occasionally seen on the basal surface. The possible functions of these phosphatases have been discussed, and it was emphasized that the 5'-Nase activity present on the luminal surface of superficial cells may play a special role in the membrane movement of these cells in the transitional epithelium.

Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity

PubMed Central

Fowler, Benjamin J.; Gelfand, Bradley D.; Kim, Younghee; Kerur, Nagaraj; Tarallo, Valeria; Hirano, Yoshio; Amarnath, Shoba; Fowler, Daniel H.; Radwan, Marta; Young, Mark T.; Pittman, Keir; Kubes, Paul; Agarwal, Hitesh K.; Parang, Keykavous A.; Hinton, David R.; Bastos-Carvalho, Ana; Li, Shengjian; Yasuma, Tetsuhiro; Mizutani, Takeshi; Yasuma, Reo; Wright, Charles; Ambati, Jayakrishna

2014-01-01

Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)-derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium (RPE) in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease (GVHD), and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases. PMID:25414314

Structural relatedness of three ion-transport adenosine triphosphatases around their active sites of phosphorylation.

PubMed

Walderhaug, M O; Post, R L; Saccomani, G; Leonard, R T; Briskin, D P

1985-03-25

Three membrane-bound adenosine triphosphatases were investigated for homology in the sequence of four amino acids about the active site of phosphorylation. The ATPases were as follows: sodium-potassium-dependent ATPase from dog kidney, Na,K-ATPase; hydrogen-potassium-dependent ATPase from hog gastric mucosa, H,K-ATPase, an ATPase similar to Na,K-ATPase; and an ATPase activity in the plasma membrane of corn, Zea mays, roots (CR-ATPase), a higher plant ATPase. A membrane preparation containing an ATPase of Acholeplasma laidlawii, a prokaryote, (AL) was also investigated. For most of the experiments, the preparations were phosphorylated from [gamma-32P]ATP, denatured in acid, and subjected to proteolytic digestion. Radioactive phosphopeptides were separated by high voltage paper electrophoresis and characterized by sensitivity to chemical reagents. In gastric H,K-ATPase, the aspartate residue at the active site was determined directly by labeling with [3H]borohydride. A common sequence around the active site was found for Na,K-ATPase, H,K-ATPase, and CR-ATPase. This sequence, -Cys-(Ser/Thr)-Asp(P)-Lys-, is similar to that in the calcium ion-transport ATPase of sarcoplasmic reticulum. The AL membrane preparation showed an acylphosphate that turned over rapidly after a chase of labeled membranes with unlabeled ATP. The corresponding sequence was different from that of the three ATPases. An acylphosphate was on two polypeptides with molecular weights of about 80,000 and 60,000; these appear not to correspond to subunits of a Na+-stimulated ATPase in this organism (Lewis, R. N. A. H., and McElhaney, R. N. (1983) Biochim. Biophys. Acta 735, 113-122).

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

Dependence of renal (Na+ + k+)-adenosine triphosphatase activity on thyroid status.

PubMed

Lo, S C; August, T R; Liberman, U A; Edelman, I S

1976-12-25

In thyroidectomized rats, a single injection of L-2,,5,2'-triiodothyronine (T3) (50mug/100 g body weight) elicited at 45% increase in (Na+ + k+)-dependent adenosine triphosphatase (NaK-ATPase) activity of the membrane-rich fraction of renal cortex at the optimal time of response, 48 h after injection. Three successive doses of T3 (50 mug/100 g body weight), given on alternate days, increased NaK-ATPase by 67% in the renal cortex but had no significant effect on the outer medulla or the papilla. Moreover, T3 had no effect on Mg2+-dependent adenosine trisphatase (MgATPase) in cortex, cedulla, or papilla. Three doses of T3 (50 mug/100 g body weight) given on alternate days to thyroidectomized rats elecited a 134, 79, and 46% increase in Vmax for ATP, Na4, and K+, respectively. There were no changes in the Km for ATP or the K1/2 values for Na+ and K+. Two methods were used to estimate the effect of T3 on the number of NaK-ATPase units (assumed to represent the number of Na+ pump sites); rat renal plasma membrane fractions were incubated with [gamma-32P]ATP, Mg2+, and Na+; the 32P-labeled membrane protein yeild was quantitatively dependent on Na+ and was hydrolyzed on addition of K+. There was a linear correlation between the specific activity of NaK-ATPase (Vmax) and the amount of phosphorylated intermediate formed, in renal cortical membrane fractions from thyroidectomized rats given T3 or the diluent. There was also a linear correlation between the specific activity of NaK-ATPase (Vmax) and the amount of [3H]ouabain specifically bound (Na+-, Mg2+-, APT-dependent) to the NaK-ATPase preparation. Injection of T3 resulted in a 70% increase in NaK-ATPase activity, a 79% increase in formation of the phosphorylated intermediate, and a 65% increase in the [H]ouabain specifically bound to the NaK-ATPase system. The T3-dependent increases in Vmax for ATP, Na+, and K+ and the proportionate increases in the phosphorylated intermediate and in the amount of [3H]ouabain bound

Area, age and gender dependence of the nucleoside system in the brain: a review of current literature.

PubMed

Kovács, Zsolt; Juhász, Gábor; Palkovits, Miklós; Dobolyi, Arpád; Kékesi, Katalin A

2011-01-01

Nucleosides, such as uridine, inosine, guanosine and adenosine, may participate in the regulation of sleep, cognition, memory and nociception, the suppression of seizures, and have also been suggested to play a role in the pathophysiology of some neurodegenerative and neuropsychiatric diseases. Under pathological conditions, levels of nucleosides change extremely in the brain, indicating their participation in the pathophysiology of disorders like Alzheimer's disease, Parkinson's disease and schizophrenia. These findings have resulted in an increasing attention to the roles of nucleosides in the central nervous system. The specific effects of nucleosides depend on the expression of their receptors and transporters in neuronal and glial cells, as well as their extracellular concentrations in the brain. A complex interlinked metabolic network and transporters of nucleosides may balance nucleoside levels in the brain tissue under normal conditions and enable the fine modulation of neuronal and glial processes via nucleoside receptor signaling mechanisms. Brain levels of nucleosides were found to vary when measured in a variety of different brain regions. In addition, nucleoside levels also depend on age and gender. Furthermore, distributions of nucleoside transporters and receptors as well as nucleoside metabolic enzyme activities demonstrate the area, age and gender dependence of the nucleoside system, suggesting different roles of nucleosides in functionally different brain areas. The aim of this review article is to summarize our present knowledge of the area-, age- and gender-dependent distribution of nucleoside levels, nucleoside metabolic enzyme activity, nucleoside receptors and nucleoside transporters in the brain.

Nucleoside phosphorylation in amide solutions

NASA Technical Reports Server (NTRS)

Schoffstall, A. M.; Kokko, B.

1978-01-01

The paper deals with phosphorylation in possible prebiotic nonaqueous solvents. To this end, phosphorylation of nucleosides using inorganic phosphates in amide solutions is studied at room and elevated temperatures. Reaction proceeds most readily in formamide and N-methylformamide. Products obtained at elevated temperature are nucleotides, nucleoside 2',3'-cyclic phosphates, and when the phosphate concentration is high, nucleoside diphosphates. At room temperature, adenosine afforded a mixture of nucleotides, but none of the cyclic nucleotide. Conditions leading to the highest relative percentage of cyclic nucleotide involve the use of low concentrations of phosphate and an excess of nucleoside.

[Effects of ifenprodil on the adenosine triphosphatase of guinea pig liver mitochondria].

PubMed

Yamashita, Y; Miyake, Y; Mitsuhiro, S; Furukawa, T

1992-07-01

The effects of ifenprodil on adenosine triphosphatase (ATPase) activity were examined using guinea pig liver mitochondria. 1) Intact mitochondrial ATPase activity was stimulated by ifenprodil in a concentration-dependent manner, this effect being further potentiated with dinitrophenol. The stimulation by ifenprodil appeared with only ATP among four nucleotides as substrate. Mg2+ and Ca2+ attenuated the effect of ifenprodil. Ifenprodil abolished the KCN-induced inhibition. 2) Heat-treated mitochondrial ATPase activity, kept for 60 min at 50 degrees C, was decreased in a concentration-dependent manner by ifenprodil. The inhibitory effect of ifenprodil was abolished by Mg2+ and Ca2+. These results indicate that ifenprodil has two behaviors, acceleration of a latent ATPase and inhibition of an activated ATPase. These findings, together with our previous data, suggest that ifenprodil seems to affect the actions of Mg2+ and Ca2+ on mitochondrial ATPase by directly affecting the membrane, and these mechanisms may be involved in its anti-cyanide effect.

Quantum chemical study of leaving group activation in T. vivax nucleoside hydrolase

NASA Astrophysics Data System (ADS)

Loverix, Stefan; Versees, Wim; Steyaert, Jan; Geerlings, Paul

General acid catalysis is a powerful and widely used strategy in enzymatic nucleophilic displacement reactions. However, in the nucleoside hydrolase of the parasite Trypanosoma vivax, crystallographic and mutagenesis studies failed to identify a general acid. The only groups in the vicinity of the leaving group that contribute to catalysis are (i) the indole side chain of Trp260, and (ii) the 5'-group of the substrate's ribose moiety. The x-ray structure of the slow Asp10Ala mutant of nucleoside hydrolase with the substrate inosine bound in the active site displays a face-to-face aromatic stacking interaction between Trp260 and the purine base of the substrate, as well as a peculiar C4'-endo ribose pucker that allows the 5'-OH group to accept an intramolecular hydrogen bond from the C8 of the purine. The first interaction (aromatic stacking) has been shown to raise the pKa of the leaving purine. Here, we present a DFT study showing that the 5'-OH group of ribose fulfills a similar role, rather than stabilizing the oxocarbenium-like transition state.

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

PubMed

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

2015-01-01

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

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

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

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 drugsmore » (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.« less

North- and South-Bicyclo[3.1.0]Hexene Nucleosides: The Effect of Ring Planarity on Anti-HIV Activity | Center for Cancer Research

Cancer.gov

The picture shows the locked north (blue) and south (red) bicyclo[3.1.0]hexane nucleosides in the normal pseudorotational cycle, and the corresponding shift to a smaller cycle (nmax=7°) caused by the insertion of a double bond. The former nucleosides are inactive, while the flattening of the embedded cyclopentene ring provides active compounds against HOS cells infected with

Antiviral properties of deazaadenine nucleoside derivatives.

PubMed

Vittori, S; Dal Ben, D; Lambertucci, C; Marucci, G; Volpini, R; Cristalli, G

2006-01-01

Viral infections have menaced human beings since time immemorial, and even today new viral strains that cause lethal diseases are being discovered with alarming frequency. One major example is HIV, the etiological agent of AIDS, which spread up in the last two decades. Very recently, other virus based diseases such as avian flu have spread fear around the world, and hemorrhagic fevers from central Africa serious threaten human health because of their very deadly effects. New antiviral agents are still greatly needed to counter these menaces. Many scientists are involved in this field of research, and many of the recently discovered effective antiviral compounds are nucleoside analogues. Among those derivatives, deazapurine nucleoside analogues have demonstrated potent inhibitory effect of viral replication. This review reports on recently generated data from preparing and testing deazapurine nucleoside derivatives as inhibitors in virus replication systems. Although most of the reported data have been produced in antiHIV, antiHCMV, and antiHSV biological testing, very recently other new important fields of application have been discovered, all in topical subjects of strong interest. In fact, deazapurine nucleosides have been found to be active as chemotherapeutics for some veterinary systemic viral infections, for which no antiviral drugs are licensed yet. Furthermore, they demonstrated efficacy in the inhibition of Hepatitis C virus replication. Finally, these compounds showed high potency as virucides against Ebola Virus, curing Ebola infected mice with a single dose administration.

Improving Nucleoside Analogs via Lipid Conjugation; Is fatter any better?

PubMed Central

Alexander, Peter; Kucera, Gregory; Pardee, Timothy S.

2016-01-01

In the past few decades, nucleoside analog drugs have been used to treat a large variety of cancers. These antimetabolite drugs mimic nucleosides and interfere with chain lengthening upon incorporation into the DNA or RNA of actively replicating cells. However, efficient delivery of these drugs is limited due to their pharmacokinetic properties, and tumors often develop drug resistance. In addition, nucleoside analogs are generally hydrophilic, resulting in poor bioavailability and impaired blood-brain barrier penetration. Conjugating these drugs to lipids modifies their pharmacokinetic properties and may improve in vivo efficacy. This review will cover recent advances in the field of conjugation of phospholipids to nucleoside analogs. This includes conjugation of myristic acid, 12-thioethyldodecanoic acid, 5-elaidic acid esters, phosphoramidate, and self-emulsifying formulations. Relevant in vitro and in vivo data will be discussed for each drug, as well as any available data from clinical trials. PMID:26829896

Calcium-regulated nuclear enzymes: potential mediators of phytochrome-induced changes in nuclear metabolism?

NASA Technical Reports Server (NTRS)

Roux, S. J.

1992-01-01

Calcium ions have been proposed to serve as important regulatory elements in stimulus-response coupling for phytochrome responses. An important test of this hypothesis will be to identify specific targets of calcium action that are required for some growth or development process induced by the photoactivated form of phytochrome (Pfr). Initial studies have revealed that there are at least two enzymes in pea nuclei that are stimulated by Pfr in a Ca(2+)-dependent fashion, a calmodulin-regulated nucleoside triphosphatase and a calmodulin-independent but Ca(2+)-dependent protein kinase. The nucleoside triphosphatase appears to be associated with the nuclear envelope, while the protein kinase co-purifies with a nuclear fraction highly enriched for chromatin. This short review summarizes the latest findings on these enzymes and relates them to what is known about Pfr-regulated nuclear metabolism.

Anti-herpesvirus activity profile of 4'-thioarabinofuranosyl purine and uracil nucleosides and activity of 1-beta-D-2'-fluoro-4'-thioarabinofuranosyl guanine and 2,6-diaminopurine against clinical isolates of human cytomegalovirus.

PubMed

Machida, H; Ashida, N; Miura, S; Endo, M; Yamada, K; Kitano, K; Yoshimura, Y; Sakata, S; Ijichi, O; Eizuru, Y

1998-08-01

Newly synthesized 4'-thio- and 2'-fluoro-4'-thioarabinofuranosyl purine and pyrimidine nucleosides were compared with the corresponding 4'-oxo type arabinosyl nucleosides for anti-herpesvirus and anti-cell proliferative potencies. 4'-Thioarabinosyl- and 2'-fluoro-4'-thioarabinofuranosyl 5-substituted uracils had selective antiviral activities, but were not superior to 4'-oxo nucleosides, except for the activity of 5-ethyl-uracil 4'-thio nucleosides against herpes simplex virus. Furthermore, 4'-thio substituted derivatives of sorivudine (BV-araU) and related compounds, and 2'-fluoro-5-methyl-arabinosyluracil exhibited reduced activity against varicella-zoster virus compared with the parent compounds. The 4'-thioarabinosyluracils, except for 5-methyluracil derivatives, were inactive against human cytomegalovirus (HCMV). 4'-Thioarabinofuranosyl guanine and diaminopurine had the most potent anti-HCMV and anti-proliferative activities, whereas arabinosyl guanine and diaminopurine had only marginal antiviral activity. 2'-Fluoro-4'-thioarabinofuranosyl derivatives of guanine (4'-thio-FaraG) and 2,6-diaminopurine (4'-thio-FaraDAP), however, had particularly high activity against all herpesviruses tested with anti-proliferative activity equipotent to that of arabinosyl guanine and diaminopurine. 4'-Thio- and 2'-fluoro-4'-thioarabinofuranosyladenines exhibited biological activities similar to that of arabinosyladenine. Both 4'-thio-FaraG and 4'-thio-FaraDAP had a 6-fold lower ED50 than ganciclovir against clinical isolates of HCMV. A ganciclovir-resistant isolate, obtained from a patient who had received long-term ganciclovir-treatment, was susceptible to 4'-thio-FaraG and 4'-thio-FaraDAP.

The Role of Transporters in the Toxicity of Nucleoside and Nucleotide Analogs

PubMed Central

Koczor, Christopher A; Torres, Rebecca A

2013-01-01

Introduction Two families of nucleoside analogs have been developed to treat viral infections and cancer, but these compounds can cause tissue and cell-specific toxicity related to their uptake and subcellular activity which are dictated by host enzymes and transporters. Cellular uptake of these compounds requires nucleoside transporters that share functional similarities but differ in substrate specificity. Tissue-specific cellular expression of these transporters enables nucleoside analogs to produce their tissue specific toxic effects, a limiting factor in the treatment of retroviruses and cancer. Areas Covered This review discusses the families of nucleoside transporters and how they mediate cellular uptake of nucleoside analogs. Specific focus is placed on examples of known cases of transporter-mediated cellular toxicity and classification of the toxicities resulting. Efflux transporters are also explored as a contributor to analog toxicity and cell-specific effects. Expert Opinion Efforts to modulate transporter uptake/clearance remain long-term goals of oncologists and virologists. Accordingly, subcellular approaches that either increase or decrease intracellular nucleoside analog concentrations are eagerly sought and include transporter inhibitors and targeting transporter expression. However, additional understanding of nucleoside transporter kinetics, tissue expression, and genetic polymorphisms are required to design better molecules and better therapies. PMID:22509856

Identification of Non-nucleoside Human Ribonucleotide Reductase Modulators

DOE PAGES

Ahmad, Md. Faiz; Huff, Sarah E.; Pink, John; ...

2015-10-21

Ribonucleotide reductase (RR) catalyzes the rate-limiting step of dNTP synthesis and is an established cancer target. Drugs targeting RR are mainly nucleoside in nature. In this study, we sought to identify non-nucleoside small-molecule inhibitors of RR. Using virtual screening, binding affinity, inhibition, and cell toxicity, we have discovered a class of small molecules that alter the equilibrium of inactive hexamers of RR, leading to its inhibition. Several unique chemical categories, including a phthalimide derivative, show micromolar IC 50s and K Ds while demonstrating cytotoxicity. A crystal structure of an active phthalimide binding at the targeted interface supports the noncompetitive modemore » of inhibition determined by kinetic studies. Furthermore, the phthalimide shifts the equilibrium from dimer to hexamer. Finally, together, these data identify several novel non-nucleoside inhibitors of human RR which act by stabilizing the inactive form of the enzyme.« less

New carbocyclic nucleoside analogues with a bicyclo[2.2.1]heptane fragment as sugar moiety; synthesis, X-ray crystallography and anticancer activity.

PubMed

Tănase, Constantin I; Drăghici, Constantin; Căproiu, Miron Teodor; Shova, Sergiu; Mathe, Christophe; Cocu, Florea G; Enache, Cristian; Maganu, Maria

2014-01-01

An amine group was synthesized starting from an optically active bicyclo[2.2.1]heptane compound, which was then used to build the 5 atoms ring of a key 6-chloropurine intermediate. This was then reacted with ammonia and selected amines obtaining new adenine- and 6-substituted adenine conformationally constrained carbocyclic nucleoside analogues with a bicyclo[2.2.1]heptane skeleton in the sugar moiety. X-ray crystallography confirmed an exo-coupling of base to the ring and a L configuration of the nucleoside analogues. The compounds were tested for anticancer activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Nucleoside-Lipid-Based Nanocarriers for Sorafenib Delivery

NASA Astrophysics Data System (ADS)

Benizri, Sebastien; Ferey, Ludivine; Alies, Bruno; Mebarek, Naila; Vacher, Gaelle; Appavoo, Ananda; Staedel, Cathy; Gaudin, Karen; Barthélémy, Philippe

2018-01-01

Although the application of sorafenib, a small inhibitor of tyrosine protein kinases, to cancer treatments remains a worldwide option in chemotherapy, novel strategies are needed to address the low water solubility (< 5 μM), toxicity, and side effects issues of this drug. In this context, the use of nanocarriers is currently investigated in order to overcome these drawbacks. In this contribution, we report a new type of sorafenib-based nanoparticles stabilized by hybrid nucleoside-lipids. The solid lipid nanoparticles (SLNs) showed negative or positive zeta potential values depending on the nucleoside-lipid charge. Transmission electron microscopy of sorafenib-loaded SLNs revealed parallelepiped nanoparticles of about 200 nm. Biological studies achieved on four different cell lines, including liver and breast cancers, revealed enhanced anticancer activities of Sorafenib-based SLNs compared to the free drug. Importantly, contrast phase microscopy images recorded after incubation of cancer cells in the presence of SLNs at high concentration in sorafenib (> 80 μM) revealed a total cancer cell death in all cases. These results highlight the potential of nucleoside-lipid-based SLNs as drug delivery systems.

Desulfurization of 2-thiouracil nucleosides: conformational studies of 4-pyrimidinone nucleosides.

PubMed

Kraszewska, Karina; Kaczyńska, Iwona; Jankowski, Stefan; Karolak-Wojciechowska, Janina; Sochacka, Elzbieta

2011-04-01

4-Pyrimidinone ribofuranoside (H(2)o(4)U) and 4-pyrimidinone 2'-deoxyribofuranoside (dH(2)o(4)U) were synthesized by the oxidative desulfurization of parent 2-thiouracil nucleosides with m-chloroperbenzoic acid. The crystal structures of H(2)o(4)U and dH(2)o(4)U and their conformations in solution were determined and compared with corresponding 2-thiouracil and uracil nucleosides. The absence of a large 2-thiocarbonyl/2-carbonyl group in the nucleobase moiety results in C2'-endo puckering of the ribofuranose ring (S conformer) in the crystal structure of H(2)o(4)U, which is not typical of RNA nucleosides. Interestingly, the hydrogen bonding network in the crystals of dH(2)o(4)U stabilizes the sugar moiety conformation in the C3'-endo form (N conformer), rarely found in DNA nucleosides. In aqueous solution, dH(2)o(4)U reveals a similar population of the C2'-endo conformation (65%) to that of 2'-deoxy-2-thiouridine (62%), while the 62% population of the S conformer for H(2)o(4)U is significantly different from that of the parent 2-thiouridine, for which the N conformer is dominant (71%). Such a difference may be of biological importance, as the desulfurization process of natural tRNA 2-thiouridines may occur under conditions of oxidative stress in the cell and may influence the decoding process. Copyright © 2011 Elsevier Ltd. All rights reserved.

Retained sensitivity to cytotoxic pyrimidine nucleoside analogs in thymidine kinase 2 deficient human fibroblasts.

PubMed

Bjerke, Mia; Solaroli, Nicola; Lesko, Nicole; Balzarini, Jan; Johansson, Magnus; Karlsson, Anna

2010-01-01

Thymidine kinase 2 (TK2) is a mitochondrial deoxyribonucleoside kinase that phosphorylates several nucleoside analogs used in anti-viral and anti-cancer therapy. A fibroblast cell line with decreased TK2 activity was investigated in order to obtain insights in the effects of TK2 deficiency on nucleotide metabolism. The role of TK2 for the sensitivity against cytotoxic nucleoside analogs was also investigated. The TK2 deficient cells retained their sensitivity against all pyrimidine nucleoside analogs tested. This study suggests that nucleoside analog phosphorylation mediated by TK2 may be less important, compared to other deoxyribonucleoside kinases, for the cytotoxic effects of these compounds.

Modulation of the human equilibrative nucleoside transporter1 (hENT1) activity by IL-4 and PMA in B cells from chronic lymphocytic leukemia.

PubMed

Fernández Calotti, Paula; Galmarini, Carlos María; Cañones, Cristian; Gamberale, Romina; Saénz, Daniel; Avalos, Julio Sánchez; Chianelli, Mónica; Rosenstein, Ruth; Giordano, Mirta

2008-02-15

Nucleoside transporters (NTs) are essential for the uptake of therapeutic nucleoside analogs, broadly used in cancer treatment. The mechanisms responsible for NT regulation are largely unknown. IL-4 is a pro-survival signal for chronic lymphocytic leukemia (CLL) cells and has been shown to confer resistance to nucleoside analogs. The aim of this study was to investigate whether IL-4 is able to modulate the expression and function of the human equilibrative NT1 (hENT1) in primary cultures of CLL cells and, consequently, to affect cytotoxicity induced by therapeutic nucleosides analogs. We found that treatment with IL-4 (20 ng/ml for 24 h) increased mRNA hENT1 expression in CLL cells without affecting that of normal B cells. Given that the enhanced mRNA levels of hENT1 in CLL cells did not result in increased transport activity, we examined the possibility that hENT1 induced by IL-4 may require post-translational modifications to become active. We found that the acute stimulation of PKC in IL-4-treated CLL cells by short-term incubation with PMA significantly increased hENT1 transport activity and favoured fludarabine-induced apoptosis. By contrast, and in line with previous reports, IL-4 plus PMA protected CLL cells from a variety of cytotoxic agents. Our findings indicate that the combined treatment with IL-4 and PMA enhances hENT1 activity and specifically sensitizes CLL cells to undergo apoptosis induced by fludarabine.

Metabolism of Exogenous Purine Bases and Nucleosides by Salmonella typhimurium

PubMed Central

Hoffmeyer, J.; Neuhard, J.

1971-01-01

Purine-requiring mutants of Salmonella typhimurium LT2 containing additional mutations in either adenosine deaminase or purine nucleoside phosphorylase have been constructed. From studies of the ability of these mutants to utilize different purine compounds as the sole source of purines, the following conclusions may be drawn. (i) S. typhimurium does not contain physiologically significant amounts of adenine deaminase and adenosine kinase activities. (ii) The presence of inosine and guanosine kinase activities in vivo was established, although the former activity appears to be of minor significance for inosine metabolism. (iii) The utilization of exogenous purine deoxyribonucleosides is entirely dependent on a functional purine nucleoside phosphorylase. (iv) The pathway by which exogenous adenine is converted to guanine nucleotides in the presence of histidine requires a functional purine nucleoside phosphorylase. Evidence is presented that this pathway involves the conversion of adenine to adenosine, followed by deamination to inosine and subsequent phosphorolysis to hypoxanthine. Hypoxanthine is then converted to inosine monophosphate by inosine monophosphate pyrophosphorylase. The rate-limiting step in this pathway is the synthesis of adenosine from adenine due to lack of endogenous ribose-l-phosphate. PMID:4928005

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

PubMed

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

2014-05-01

Purine nucleoside phosphorylase (PNP) and xanthine oxidase (XO) are key enzymes involved in the purine salvage pathway. PNP metabolizes purine bases to synthetize purine nucleotides whereas XO catalyzes the oxidation of purines to uric acid. In humans, PNP activity is reported to be high in erythrocytes and XO activity to be low in plasma; however, XO activity increases after ischemic events. XO activity in plasma of northern elephant seals has been reported during prolonged fasting and rest and voluntary associated apneas. The objective of this study was to analyze circulating PNP and XO activities in marine mammals adapted to tolerate repeated cycles of ischemia/reperfusion associated with diving (bottlenose dolphin, northern elephant seal) in comparison with semiaquatic (river otter) and terrestrial mammals (human, pig). PNP activities in plasma and erythrocytes, as well as XO activity in plasma, from all species were quantified by spectrophotometry. No clear relationship in circulating PNP or XO activity could be established between marine, semiaquatic and terrestrial mammals. Erythrocytes from bottlenose dolphins and humans are highly permeable to nucleosides and glucose, intraerythrocyte PNP activity may be related to a release of purine nucleotides from the liver. High-energy costs will probably mean a higher ATP degradation rate in river otters, as compared to northern elephant seals or dolphins. Lower erythrocyte PNP activity and elevated plasma XO activity in northern elephant seal could be associated with fasting and/or sleep- and dive-associated apneas. Copyright © 2014 Elsevier Inc. All rights reserved.

Ergopeptines bromocriptine and ergovaline and the dopamine type-2 receptor inhibitor domperidone inhibit bovine equilibrative nucleoside transporter 1-like activity.

PubMed

Miles, Edwena D; Xue, Yan; Strickland, James R; Boling, James A; Matthews, James C

2011-09-14

Neotyphodium coenophialum-infected tall fescue contains ergopeptines. Except for interactions with biogenic amine receptors (e.g., dopamine type-2 receptor, D2R), little is known about how ergopeptines affect animal metabolism. The effect of ergopeptines on bovine nucleoside transporters (NT) was evaluated using Madin-Darby bovine kidney (MDBK) cells. Equilibrative NT1 (ENT1)-like activity accounted for 94% of total NT activity. Inhibitory competition (IC(50)) experiments found that this activity was inhibited by both bromocriptine (a synthetic model ergopeptine and D2R agonist) and ergovaline (a predominant ergopeptine of tall fescue). Kinetic inhibition analysis indicated that bromocriptine inhibited ENT1-like activity through a competitive and noncompetitive mechanism. Domperidone (a D2R antagonist) inhibited ENT1 activity more in the presence than in the absence of bromocriptine and displayed an IC(50) value lower than that of bromocriptine or ergovaline, suggesting that inhibition was not through D2R-mediated events. These novel mechanistic findings imply that cattle consuming endophyte-infected tall fescue have reduced ENT1 activity and, thus, impaired nucleoside metabolism.

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

NASA Astrophysics Data System (ADS)

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

2013-09-01

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

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.

Effects of omeprazole treatment on nucleoside transporter expression and adenosine uptake in rat gastric mucosa.

PubMed

Redzic, Zoran B; Hasan, Fuad A; Al-Sarraf, Hameed

2009-05-01

Increased adenosine concentration inhibits gastric acid secretion in rat via adenosine A1 and A2A receptors, whereas achlorhydria suppresses A1 and A2A receptor gene expression. This study aimed to examine the effects of omeprazole-induced achlorhydria on the expression and functional activity of nucleoside transporters in rat gastric mucosa. Wistar rats were treated for either 1 or 3 days with 0.4 mmol/kg omeprazole via gavage; controls were treated with vehicle. The expression of nucleoside transporters at the transcript level was explored by quantitative real-time polymerase chain reaction assays; the functional activity of nucleoside transporters in gastric mucosa was explored by observing [3H]adenosine uptake in vitro. Gastric mucosa expressed rat equilibrative nucleoside transporter (rENT) 1 and 2, and rat concentrative nucleoside transporter (rCNT) 1, 2, and 3 at the transcript level, and the estimated values for the threshold cycles for target amplification (Ct) were 31.5 +/- 2, 28.5 +/- 2.1, 32.9 +/- 2.2, 29.1 +/- 2, and 28.9 +/- 2.5, respectively (n = 3 or 4). The Ct value for rat beta-actin was 21.9 +/- 1.8 (n = 4). In vitro uptake of [3H]adenosine by gastric mucosa samples consisted of Na+-dependent and Na+-independent components. One-day omeprazole treatment caused no change in nucleoside transporter mRNA levels or in [3H]adenosine uptake. Three-day omeprazole treatments, however, led to a 12-fold and 17-fold increase in rENT2 and rCNT1 mRNA levels, respectively. Samples taken after 3 days of treatment also took up significantly more [3H]adenosine than did samples from the corresponding control. In conclusion, the possible modification of nucleoside transport activities by changes in intraluminal acidity may have significance as part of a purinergic regulatory feedback mechanism in the control of gastric acid secretion.

Dual door entry to exciplex emission in a chimeric DNA duplex containing non-nucleoside-nucleoside pair.

PubMed

Bag, Subhendu Sekhar; Talukdar, Sangita; Kundu, Rajen; Saito, Isao; Jana, Subhashis

2014-01-25

Dual door entry to exciplex formation was established in a chimeric DNA duplex wherein a fluorescent non-nucleosidic base surrogate () is paired against a fluorescent nucleosidic base surrogate (). Packing of the nucleobases via intercalative stacking interactions led to an exciplex emission either via FRET from the donor or direct excitation of the FRET acceptor .

Modulators of Nucleoside Metabolism in the Therapy of Brain Diseases

PubMed Central

Boison, Detlev

2010-01-01

Nucleoside receptors are known to be important targets for a variety of brain diseases. However, the therapeutic modulation of their endogenous agonists by inhibitors of nucleoside metabolism represents an alternative therapeutic strategy that has gained increasing attention in recent years. Deficiency in endogenous nucleosides, in particular of adenosine, may causally be linked to a variety of neurological diseases and neuropsychiatric conditions ranging from epilepsy and chronic pain to schizophrenia. Consequently, augmentation of nucleoside function by inhibiting their metabolism appears to be a rational therapeutic strategy with distinct advantages: (i) in contrast to specific receptor modulation, the increase (or decrease) of the amount of a nucleoside will affect several signal transduction pathways simultaneously and therefore have the unique potential to modify complex neurochemical networks; (ii) by acting on the network level, inhibitors of nucleoside metabolism are highly suited to fine-tune, restore, or amplify physiological functions of nucleosides; (iii) therefore inhibitors of nucleoside metabolism have promise for the “soft and smart” therapy of neurological diseases with the added advantage of reduced systemic side effects. This review will first highlight the role of nucleoside function and dysfunction in physiological and pathophysiological situations with a particular emphasis on the anticonvulsant, neuroprotective, and antinociceptive roles of adenosine. The second part of this review will cover pharmacological approaches to use inhibitors of nucleoside metabolism, with a special emphasis on adenosine kinase, the key regulator of endogenous adenosine. Finally, novel gene-based therapeutic strategies to inhibit nucleoside metabolism and focal treatment approaches will be discussed. PMID:21401494

Kinetics and regulation of the myofibrillar adenosine triphosphatase.

PubMed Central

Goodno, C C; Wall, C M; Perry, S V

1978-01-01

1. The steady-state kinetic behaviour of the ATPase (adenosine triphosphatase) of intact myofibrils was studied in the presence of both high and low concentrations of Ca2+ (0.25 mM and less than 10 nM respectively). 2. Kinetic data were collected over the initial linear phase of the assay, which lasts for 20--60s. To obtain consistent data we found it necessary to use either fresh myofibril preparations or preparations that had been stored in the presence of thiol compounds. 3. When assayed in the presence of 0.25 mM-Ca2+, the myofibrillar ATPase obeyed Michaelis-Menten kinetics over the range 0.03--5.0 mM-MgATP (Km 16 +/- 6 micrometer, V 0.4 +/- 0.1 mumol/min per mg). 4. At low Ca2+ concentrations (less than 10 nM) the myofibrillar ATPase displayed pronounced substrate inhibition, which was not observed at high Ca2+ concentrations. Thus increasing the MgATP concentration had the net effect of decreasing the ATPase activity at low Ca2+ relative to that at high Ca2+. This preferential effect of MgATP on the low-Ca2+ ATPase may be important in Ca2+ control. 5. The substrate inhibition that was observed at low Ca2+ was lost on storage or thiol modification of the myofibrils. 6. Under physiological conditions (2 mM-MgATP, I 0.15, pH 7.0), the ATPase of fresh and thiol-protected myofibrils displayed approx. 100-fold activation by Ca2+. PMID:154323

Nucleoside Derived Antibiotics to Fight Microbial Drug Resistance: New Utilities for an Established Class of Drugs?

PubMed

Serpi, Michaela; Ferrari, Valentina; Pertusati, Fabrizio

2016-12-08

Novel antibiotics are urgently needed to combat the rise of infections due to drug-resistant microorganisms. Numerous natural nucleosides and their synthetically modified analogues have been reported to have moderate to good antibiotic activity against different bacterial and fungal strains. Nucleoside-based compounds target several crucial processes of bacterial and fungal cells such as nucleoside metabolism and cell wall, nucleic acid, and protein biosynthesis. Nucleoside analogues have also been shown to target many other bacterial and fungal cellular processes although these are not well characterized and may therefore represent opportunities to discover new drugs with unique mechanisms of action. In this Perspective, we demonstrate that nucleoside analogues, cornerstones of anticancer and antiviral treatments, also have great potential to be repurposed as antibiotics so that an old drug can learn new tricks.

Nucleoside antibiotics: biosynthesis, regulation, and biotechnology.

PubMed

Niu, Guoqing; Tan, Huarong

2015-02-01

The alarming rise in antibiotic-resistant pathogens has coincided with a decline in the supply of new antibiotics. It is therefore of great importance to find and create new antibiotics. Nucleoside antibiotics are a large family of natural products with diverse biological functions. Their biosynthesis is a complex process through multistep enzymatic reactions and is subject to hierarchical regulation. Genetic and biochemical studies of the biosynthetic machinery have provided the basis for pathway engineering and combinatorial biosynthesis to create new or hybrid nucleoside antibiotics. Dissection of regulatory mechanisms is leading to strategies to increase the titer of bioactive nucleoside antibiotics. Copyright © 2014. Published by Elsevier Ltd.

Aspartic acid based nucleoside phosphoramidate prodrugs as potent inhibitors of hepatitis C virus replication.

PubMed

Maiti, Munmun; Maiti, Mohitosh; Rozenski, Jef; De Jonghe, Steven; Herdewijn, Piet

2015-05-14

In view of a persistent threat to mankind, the development of nucleotide-based prodrugs against hepatitis C virus (HCV) is considered as a constant effort in many medicinal chemistry groups. In an attempt to identify novel nucleoside phosphoramidate analogues for improving the anti-HCV activity, we have explored, for the first time, aspartic acid (Asp) and iminodiacetic acid (IDA) esters as amidate counterparts by considering three 2'-C-methyl containing nucleosides, 2'-C-Me-cytidine, 2'-C-Me-uridine and 2'-C-Me-2'-fluoro-uridine. Synthesis of these analogues required protection for the vicinal diol functionality of the sugar moiety and the amino group of the cytidine nucleoside to regioselectively perform phosphorylation reaction at the 5'-hydroxyl group. Anti-HCV data demonstrate that the Asp-based phosphoramidates are ∼550 fold more potent than the parent nucleosides. The inhibitory activity of the Asp-ProTides was higher than the Ala-ProTides, suggesting that Asp would be a potential amino acid candidate to be considered for developing novel antiviral prodrugs.

Isolation of Escherichia coli mutants with an adenosine triphosphatase insensitive to aurovertin.

PubMed Central

Satre, M; Klein, G; Vignais, P V

1978-01-01

Energy-transducing adenosine triphosphatase (ATPase) from Escherichia coli is inhibited by aurovertin. Aurovertin-resistant mutants were generated by nitrosoguanidine mutagenesis of E. coli AN180, whose growth on a nonfermentable carbon source was blocked by aurovertin. The ATPase activity of cell extracts from 15 different mutants (designated MA1, MA2, MA3, etc.) was found to be at least 20 times less sensitive to aurovertin than that from the parent strain. The aurovertin-resistant mutants did not show cross-resistance towards a number of ATPase inhibitors including azide, dicyclohexylcarbodiimide, quercetin, 7-chloro-4-nitrobenzofurazan, and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. Aurovertin inhibited the energization brought about by addition of ATP to E. coli AN180 membrane vesicles; it was without effect on MA1 and MA2 membrane vesicles energized by ATP. The mutation in MA1, like other mutations of the ATPase complex, maps in the unc region of the bacterial chromosome. PMID:148459

Identification of a nucleoside analog active against adenosine kinase–expressing plasma cell malignancies

PubMed Central

Sadek, Jouliana; Hernandez-Hopkins, Denise; Akar, Gunkut; Barelli, Peter J.; Sahai, Michelle A.; Zhou, Hufeng; Totonchy, Jennifer; Jayabalan, David; Niesvizky, Ruben; Guasparri, Ilaria; Liu, Yifang; Sei, Shizuko; Shoemaker, Robert H.; Elemento, Olivier; Kaye, Kenneth M.

2017-01-01

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase–inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI–sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers. PMID:28504647

Identification of a nucleoside analog active against adenosine kinase-expressing plasma cell malignancies.

PubMed

Nayar, Utthara; Sadek, Jouliana; Reichel, Jonathan; Hernandez-Hopkins, Denise; Akar, Gunkut; Barelli, Peter J; Sahai, Michelle A; Zhou, Hufeng; Totonchy, Jennifer; Jayabalan, David; Niesvizky, Ruben; Guasparri, Ilaria; Hassane, Duane; Liu, Yifang; Sei, Shizuko; Shoemaker, Robert H; Warren, J David; Elemento, Olivier; Kaye, Kenneth M; Cesarman, Ethel

2017-06-01

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase-inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI-sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers.

A Transient Kinetic Approach to Investigate Nucleoside Inhibitors of Mitochondrial DNA polymerase γ

PubMed Central

Anderson, Karen S.

2010-01-01

Nucleoside analogs play an essential role in treating human immunodeficiency virus (HIV) infection since the beginning of the AIDS epidemic and work by inhibition of HIV-1 reverse transcriptase (RT), a viral polymerase essential for DNA replication. Today, over 90% of all regimens for HIV treatment contain at least one nucleoside. Long-term use of nucleoside analogs has been associated with adverse effects including mitochondrial toxicity due to inhibition of the mitochondrial polymerase, DNA polymerase gamma (mtDNA pol ©). In this review, we describe our efforts to delineate the molecular mechanism of nucleoside inhibition of HIV-1 RT and mtDNA pol © based upon a transient kinetic approach using rapid chemical quench methodology. Using transient kinetic methods, the maximum rate of polymerization (kpol), the dissociation constant for the ground state binding (Kd), and the incorporation efficiency (kpol/Kd) can be determined for the nucleoside analogs and their natural substrates. This analysis allowed us to develop an understanding of the structure activity relationships that allow correlation between the structural and stereochemical features of the nucleoside analog drugs with their mechanistic behavior toward the viral polymerase, RT, and the host cell polymerase, mtDNA pol γ. An in-depth understanding of the mechanisms of inhibition of these enzymes is imperative in overcoming problems associated with toxicity. PMID:20573564

Substrate specificity of pyrimidine nucleoside phosphorylases of NP-II family probed by X-ray crystallography and molecular modeling

NASA Astrophysics Data System (ADS)

Balaev, V. V.; Lashkov, A. A.; Prokofev, I. I.; Gabdulkhakov, A. G.; Seregina, T. A.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

2016-09-01

Pyrimidine nucleoside phosphorylases, which are widely used in the biotechnological production of nucleosides, have different substrate specificity for pyrimidine nucleosides. An interesting feature of these enzymes is that the three-dimensional structure of thymidine-specific nucleoside phosphorylase is similar to the structure of nonspecific pyrimidine nucleoside phosphorylase. The three-dimensional structures of thymidine phosphorylase from Salmonella typhimurium and nonspecific pyrimidine nucleoside phosphorylase from Bacillus subtilis in complexes with a sulfate anion were determined for the first time by X-ray crystallography. An analysis of the structural differences between these enzymes demonstrated that Lys108, which is involved in the phosphate binding in pyrimidine nucleoside phosphorylase, corresponds to Met111 in thymidine phosphorylases. This difference results in a decrease in the charge on one of the hydroxyl oxygens of the phosphate anion in thymidine phosphorylase and facilitates the catalysis through SN2 nucleophilic substitution. Based on the results of X-ray crystallography, the virtual screening was performed for identifying a potent inhibitor (anticancer agent) of nonspecific pyrimidine nucleoside phosphorylase, which does not bind to thymidine phosphorylase. The molecular dynamics simulation revealed the stable binding of the discovered compound—2-pyrimidin-2-yl-1H-imidazole-4-carboxylic acid—to the active site of pyrimidine nucleoside phosphorylase.

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

PubMed Central

Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

1993-01-01

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

Structure-activity relationship of uridine-based nucleoside phosphoramidate prodrugs for inhibition of dengue virus RNA-dependent RNA polymerase.

PubMed

Wang, Gang; Lim, Siew Pheng; Chen, Yen-Liang; Hunziker, Jürg; Rao, Ranga; Gu, Feng; Seh, Cheah Chen; Ghafar, Nahdiyah Abdul; Xu, Haoying; Chan, Katherine; Lin, Xiaodong; Saunders, Oliver L; Fenaux, Martijn; Zhong, Weidong; Shi, Pei-Yong; Yokokawa, Fumiaki

2018-05-03

To identify a potent and selective nucleoside inhibitor of dengue virus RNA-dependent RNA polymerase, a series of 2'- and/or 4'-ribose sugar modified uridine nucleoside phosphoramidate prodrugs and their corresponding triphosphates were synthesized and evaluated. Replacement of 2'-OH with 2'-F led to be a poor substrate for both dengue virus and human mitochondrial RNA polymerases. Instead of 2'-fluorination, the introduction of fluorine at the ribose 4'-position was found not to affect the inhibition of the dengue virus polymerase with a reduction in uptake by mitochondrial RNA polymerase. 2'-C-ethynyl-4'-F-uridine phosphoramidate prodrug displayed potent anti-dengue virus activity in the primary human peripheral blood mononuclear cell-based assay with no significant cytotoxicity in human hepatocellular liver carcinoma cell lines and no mitochondrial toxicity in the cell-based assay using human prostate cancer cell lines. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

PubMed

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

2015-10-09

Cladribine, 2-chloro-2'-deoxyadenosine, is a highly efficacious, clinically used nucleoside for the treatment of hairy cell leukemia. It is also being evaluated against other lymphoid malignancies and has been a molecule of interest for well over half a century. In continuation of our interest in the amide bond-activation in purine nucleosides via the use of (benzotriazol-1yl-oxy)tris(dimethylamino)phosphonium hexafluorophosphate, we have evaluated the use of O⁶-(benzotriazol-1-yl)-2'-deoxyguanosine as a potential precursor to cladribine and its analogues. These compounds, after appropriate deprotection, were assessed for their biological activities, and the data are presented herein. Against hairy cell leukemia (HCL), T-cell lymphoma (TCL) and chronic lymphocytic leukemia (CLL), cladribine was the most active against all. The bromo analogue of cladribine showed comparable activity to the ribose analogue of cladribine against HCL, but was more active against TCL and CLL. The bromo ribose analogue of cladribine showed activity, but was the least active among the C6-NH₂-containing compounds. Substitution with alkyl groups at the exocyclic amino group appears detrimental to activity, and only the C6 piperidinyl cladribine analogue demonstrated any activity. Against adenocarcinoma MDA-MB-231 cells, cladribine and its ribose analogue were most active.

Flexibility of the sugar moiety of nucleosides at high pressures

NASA Astrophysics Data System (ADS)

Lee, Scott

2007-03-01

In this poster we review our recent high pressure experiments on deoxyadenosine, adenosine, deoxycytidine and cytidine via mid-infrared absorption. These experiments reveal the presence of phase transitions near 2 GPa in these four different nucleosides. The spectroscopic evidence indicates that the sugar pucker changes at the phase transition in all four nucleosides. Differences between the deoxyribose nucleosides and the ribose nucleosides are compared to the known differences in the conformational flexibility of DNA and RNA.

Evaluation of Anti-HIV-1 Mutagenic Nucleoside Analogues*

PubMed Central

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

2015-01-01

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

Evaluation of anti-HIV-1 mutagenic nucleoside analogues.

PubMed

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

2015-01-02

Because of their high mutation rates, RNA viruses and retroviruses replicate close to the threshold of viability. Their existence as quasi-species has pioneered the concept of "lethal mutagenesis" that prompted us to synthesize pyrimidine nucleoside analogues with antiviral activity in cell culture consistent with an accumulation of deleterious mutations in the HIV-1 genome. However, testing all potentially mutagenic compounds in cell-based assays is tedious and costly. Here, we describe two simple in vitro biophysical/biochemical assays that allow prediction of the mutagenic potential of deoxyribonucleoside analogues. The first assay compares the thermal stabilities of matched and mismatched base pairs in DNA duplexes containing or not the nucleoside analogues as follows. A promising candidate should display a small destabilization of the matched base pair compared with the natural nucleoside and the smallest gap possible between the stabilities of the matched and mismatched base pairs. From this assay, we predicted that two of our compounds, 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine, should be mutagenic. The second in vitro reverse transcription assay assesses DNA synthesis opposite nucleoside analogues inserted into a template strand and subsequent extension of the newly synthesized base pairs. Once again, only 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine are predicted to be efficient mutagens. The predictive potential of our fast and easy first line screens was confirmed by detailed analysis of the mutation spectrum induced by the compounds in cell culture because only compounds 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine were found to increase the mutation frequency by 3.1- and 3.4-fold, respectively. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Lipid requirement of the membrane sodium-plus-potassium ion-dependent adenosine triphosphatase system.

PubMed Central

Wheeler, K P; Walker, J A; Barker, D M

1975-01-01

The dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) on lipid has been examined in a number of different ways, with the use of various preparations from kidney tissue. The main findings were as follows. (1) The ATPase activities of the preparations examined were closely correlated with their total phospholipid content. (2) Extraction of the ATPase with deoxycholate or Lubrol W, combined with suitable salt-fractionation and washing procedures, removed phospholipid, cholesterol and enzymic activity in parallel; but activity was completely lost before all lipid had been removed. (3) The loss of activity could not be attributed to inhibition by residual detergent. (4) No selective removal of any particular phospholipid class by detergent could be detected. (5) Consistent reactivation of the Lubrol-extracted enzymes was obtained by adding dispersions of exogenous phospholipid, but only some, bearing a net negative charge, such as phosphatidylserine and phosphatidylglycerol, were effective. (6) The degree of reactivation was correlated with the amount of residual activity remaining after lipid depletion. (7) Partial purification of the ATPase, giving a 50-fold increase in specific activity, was not accompanied by selective enhancement of any particular class of phospholipid. We conclude that although the ATPase is dependent on phospholipid, only the reactivation results provide evidence for specificity. PMID:125082

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

PubMed Central

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

2014-01-01

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

Intracellular studies of the nucleoside reverse transcriptase inhibitor active metabolites: a review.

PubMed

Rodriguez Orengo, J F; Santana, J; Febo, I; Diaz, C; Rodriguez, J L; Garcia, R; Font, E; Rosario, O

2000-03-01

Nucleoside reverse transcriptase inhibitors (NRTIs) plasma concentrations do not correlate with clinical efficacy or toxicity. These agents need to be phosphorylated to become active against HIV-infection. Thus, the characterization of the NRTIs intracellular metabolite pharmacological parameters will provide a better understanding that could lead to the development of more rational dose regimens in the HIV-infected population. Furthermore, intracellular measurements of NRTIs may provide a better marker with respect to clinical efficacy and toxicity than plasma concentrations. Thus, in this article we review the latest information regarding the intracellular pharmacological parameters of zidovudine (ZDV) and lamivudine (3TC) active metabolites in HIV-infected patients including the results from our recent clinical studies. We will start the discussion with ZDV and 3TC clinical efficacy, followed by systemic pharmacokinetics studies. We will then discuss the in vitro and in vivo intracellular studies with particular emphasis in the method development to measure these metabolites and we will conclude with the most current data from our clinical trials.

Anti-herpesvirus activity of the acyclic nucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine.

PubMed Central

Smee, D F; Martin, J C; Verheyden, J P; Matthews, T R

1983-01-01

The antiherpetic effects of a novel purine acyclic nucleoside, 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), were compared with those of acyclovir in cell cultures and in mice. The modes of action of DHPG and acyclovir were similar in that herpes thymidine kinase phosphorylated each compound, and both agents selectively inhibited viral over host cell DNA synthesis. In 50% plaque reduction assays in Vero cells, the drugs inhibited herpes simplex virus types 1 and 2 thymidine kinase-positive strains at 0.2 to 2.4 microM. DHPG was markedly more active than acyclovir against human cytomegalovirus (50% inhibitory doses were 7 and 95 microM, respectively). Each nucleoside inhibited uninfected cell macromolecule synthesis and cell proliferation at concentrations far above those required to inhibit herpes simplex virus replication. Although the two compounds had many similarities in their behavior in vitro, the important difference was the superior performance of DHPG against herpesvirus-induced encephalitis and vaginitis in vivo. Thus, mortality in mice infected with herpesvirus type 2 was reduced 50% by daily doses of 7 to 10 mg of DHPG/kg, whereas an equally effective daily dose of acyclovir was approximately 500 mg/kg. DHPG at a daily dose of 50 mg/kg was also superior to acyclovir at 100 mg/kg per day in its inhibition of herpetic vaginal lesions in mice. PMID:6307132

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

PubMed

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

1990-10-01

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

Nucleoside Inhibitors of Tick-Borne Encephalitis Virus

PubMed Central

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

2015-01-01

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

Sodium-potassium-activated adenosine triphosphatase of electrophorus electric organ. X. Immunochemical properties of the Lubrol-solubilized enzume and its constituent polypeptides.

PubMed

Jean, D H; Albers, R W; Koval, G J

1975-02-10

Detergent (Lubrol WX)-solubilized sodium-potassium-activated adenosine triphosphatase ((Na+ + K+)-ATPase) of electrophorus electric organ contains two major constituent polypeptides with molecular weights of 96,000 and 58,000 which can be readily demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These two polypeptides can be clearly separated and can be obtained in milligram quantities by preparative sodium dodecyl sulfate gel electrophoresis. The separated polypeptides, after removal of sodium dodecyl sulfate, and Lubrol-solubilized (Na+ + K+)-ATPase activity to some degree. Moreover, the degree of inhibition is directly proportional to the increasing amounts of antisera. The inhibition is maximal 4 weeks after the first injection. Immunodiffusion in 1% agar gel indicated that only Lubrol-solubilized enzyme antiserum, but not 58,000-dalton or 96,00-dalton polypeptide antiserum, gives one major precipitin band. However, specific complex formation between each polypeptide antiserum and Lubrol-solubilized enzyme occurs. This was demonstrated indirectly. After incubating Lubrol-solubilized enzyme with increasing amounts of polypeptide antisera at 37 degrees for 15 min, they were placed in the side wells of an immunodiffusion plate with antiserum against Lubrol-solubilized enzyme in the central well. The intensity of the precipitin band decreased with increasing amounts of polypeptide antisera. Thus, the results indicate that both 96,000-dalton and 58,000-dalton polypeptides are integral subunits of (Na+ + K+)-ATPase.

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

PubMed Central

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

2016-01-01

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

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.

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

PubMed

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

2016-05-01

A series of novel aryloxyphosphoramidate nucleoside prodrugs based on l-aspartic acid and l-glutamic acid as amino acid motif has been synthesized and evaluated for antitumoral activity. Depending on the cancer cell line studied and on the nature of the parent nucleoside compound (gemcitabine, 5-iodo-2'-deoxy-uridine, floxuridine or brivudin), the corresponding ProTides are endowed with an improved or decreased cytotoxic activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fluoro carbocyclic nucleosides: synthesis and antiviral activity of 2'- and 6'-fluoro carbocyclic pyrimidine nucleosides including carbocyclic 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil and carbocyclic 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil.

PubMed

Borthwick, A D; Evans, D N; Kirk, B E; Biggadike, K; Exall, A M; Youds, P; Roberts, S M; Knight, D J; Coates, J A

1990-01-01

The racemic carbocyclic 2'-fluoroarabinosyl pyrimidine nucleosides 8, 9 (C-FIAU), 12, and 13 (C-FMAU) and the 2'-fluororibosyl pyrimidine nucleosides 17, 20, and 21 were prepared from their respective protected 2'-fluoro amino diols 5 and 14. The carbocyclic 2'-2'-difluorothymidine analogue 27 was obtained from the protected difluoro amino diol 24 which was prepared from the ketone 23 and (diethylamino)sulfur trifluoride (DAST). The chiral carbocyclic 2'-deoxy-6'-fluorouridines 33, 34, 38, and 39 were synthesized from the protected 6'-fluoro amino diols 30 and 36, which were prepared by reduction of the azides 28 and 35. C-FMAU (13) and C-FIAU (9) were active in vitro against HSV-1 with ID50 values of 4.4 and 11 micrograms/mL, respectively, but they were inactive against HSV-2. The cytidine analogues 12 and 20 displayed modest activity in vitro against HSV-1 and HSV-2 but were inactive against human influenza A virus.

Formation of nucleoside 5'-polyphosphates under potentially prebiological conditions

NASA Technical Reports Server (NTRS)

Lohrmann, R.

1976-01-01

The characteristics and efficiencies of biochemical reactions involving nucleoside 5'-diphosphates and -triphosphates (important substrates of RNA and DNA synthesis) under conditions corresponding to the primitive prebiotic earth are investigated. Urea catalysis of the formation of linear inorganic polyphosphates and metal ions promoting the reactions are discussed. Linear polyphosphate was incubated with Mg(++) in the presence of a nucleoside 5'-phosphate, to yield nucleoside 5'-polyphosphates when products are dried, while Mg(++) prompts depolymerization to trimetaphosphate in aqueous solutions. Plausible biogenetic pathways are examined.

OCTN1 is a high-affinity carrier of nucleoside analogs

PubMed Central

Drenberg, Christina D.; Gibson, Alice A.; Pounds, Stanley B.; Shi, Lei; Rhinehart, Dena P.; Li, Lie; Hu, Shuiying; Du, Guoqing; Nies, Anne T.; Schwab, Matthias; Pabla, Navjotsingh; Blum, William; Gruber, Tanja A.; Baker, Sharyn D.; Sparreboom, Alex

2017-01-01

Resistance to xenobiotic nucleosides used to treat acute myeloid leukemia (AML) and other cancers remains a major obstacle to clinical management. One process suggested to participate in resistance is reduced uptake into tumor cells via nucleoside transporters, although precise mechanisms are not understood. Through transcriptomic profiling, we determined that low expression of the ergothioneine transporter OCTN1 (SLC22A4; ETT) strongly predicts poor event-free survival and overall survival in multiple cohorts of AML patients receiving treatment with the cytidine nucleoside analog cytarabine. Cell biological studies confirmed OCTN1-mediated transport of cytarabine and various structurally-related cytidine analogs, such as 2′deoxycytidine and gemcitabine, occurs through a saturable process that is highly sensitive to inhibition by the classic nucleoside transporter inhibitors dipyridamole and nitrobenzylmercaptopurine ribonucleoside (NBMPR). Our findings have immediate clinical implications given the potential of the identified transport system to help refine strategies that could improve patient survival across multiple cancer types where nucleoside analogs are used in cancer treatment. PMID:28209616

Recognition of Artificial Nucleobases by E. coli Purine Nucleoside Phosphorylase versus its Ser90Ala Mutant in the Synthesis of Base-Modified Nucleosides.

PubMed

Fateev, Ilja V; Kharitonova, Maria I; Antonov, Konstantin V; Konstantinova, Irina D; Stepanenko, Vasily N; Esipov, Roman S; Seela, Frank; Temburnikar, Kartik W; Seley-Radtke, Katherine L; Stepchenko, Vladimir A; Sokolov, Yuri A; Miroshnikov, Anatoly I; Mikhailopulo, Igor A

2015-09-14

A wide range of natural purine analogues was used as probe to assess the mechanism of recognition by the wild-type (WT) E. coli purine nucleoside phosphorylase (PNP) versus its Ser90Ala mutant. The results were analyzed from viewpoint of the role of the Ser90 residue and the structural features of the bases. It was found that the Ser90 residue of the PNP 1) plays an important role in the binding and activation of 8-aza-7-deazapurines in the synthesis of their nucleosides, 2) participates in the binding of α-D-pentofuranose-1-phosphates at the catalytic site of the PNP, and 3) catalyzes the dephosphorylation of intermediary formed 2-deoxy-α-D-ribofuranose-1-phosphate in the trans-2-deoxyribosylation reaction. 5-Aza-7-deazaguanine manifested excellent substrate activity for both enzymes, 8-amino-7-thiaguanine and 2-aminobenzothiazole showed no substrate activity for both enzymes. On the contrary, the 2-amino derivatives of benzimidazole and benzoxazole are substrates and are converted into the N1- and unusual N2-glycosides, respectively. 9-Deaza-5-iodoxanthine showed moderate inhibitory activity of the WT E. coli PNP, whereas 9-deazaxanthine and its 2'-deoxyriboside are weak inhibitors. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features.

PubMed

Narczyk, Marta; Bertoša, Branimir; Papa, Lucija; Vuković, Vedran; Leščić Ašler, Ivana; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Luić, Marija; Štefanić, Zoran

2018-04-01

Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probably, in part, due to their complex oligomeric nature and a whole spectrum of active site conformations related to interactions with different ligands. Here we report an extensive structural characterization of the apo forms of hexameric PNP from Helicobacter pylori (HpPNP), as well as its complexes with phosphate (P i ) and an inhibitor, formycin A (FA), together with kinetic, binding, docking and molecular dynamics studies. X-ray structures show previously unseen distributions of open and closed active sites. Microscale thermophoresis results indicate that a two-site model describes P i binding, while a three-site model is needed to characterize FA binding, irrespective of P i presence. The latter may be related to the newly found nonstandard mode of FA binding. The ternary complex of the enzyme with P i and FA shows, however, that P i binding stabilizes the standard mode of FA binding. Surprisingly, HpPNP has low affinity towards the natural substrate adenosine. Molecular dynamics simulations show that P i moves out of most active sites, in accordance with its weak binding. Conformational changes between nonstandard and standard binding modes of nucleoside are observed during the simulations. Altogether, these findings show some unique features of HpPNP and provide new insights into the functioning of the active sites, with implications for understanding the complex mechanism of catalysis of this enzyme. The atomic coordinates and structure factors have been deposited in the Protein Data Bank: with accession codes 6F52 (HpPNPapo_1), 6F5A (HpPNPapo_2), 6F5I (HpPNPapo_3), 5LU0 (HpPNP_PO4), 6F4W (HpPNP_FA) and 6F4X (HpPNP_PO4_FA). Purine nucleoside orthophosphate ribosyl transferase, EC2.4.2.1, UniProtID: P56463. © 2018 Federation of European Biochemical Societies.

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.

4'-alpha-C-Branched N,O-nucleosides: synthesis and biological properties.

PubMed

Chiacchio, Ugo; Genovese, Filippo; Iannazzo, Daniela; Piperno, Anna; Quadrelli, Paolo; Antonino, Corsaro; Romeo, Roberto; Valveri, Vincenza; Mastino, Antonio

2004-07-15

The synthesis of 4'-alpha-C-branched N,O-nucleosides has been described, based on the 1,3-dipolar cycloaddition of nitrones with vinyl acetate followed by coupling with silylated nucleobases, The obtained compounds have been evaluated for their activity against HSV-1, HSV-2, HTLV-1. Cytotoxicity and apoptotic activity have been also investigated: compound 10c shows moderate apoptotic activity in Molt-3 cells.

Differential effects of lipid depletion on membrane sodium-plus-potassium ion-dependent adenosine triphosphatase and potassium ion-dependent phosphatase.

PubMed Central

Wheeler, K P; Walker, J A

1975-01-01

The phospholipid-dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) and associated K-+-dependent phosphatase activity (EC 3.6.1.7) have been compared. Unlike the (Na-++K-+)-dependent ATPase activities, the K-+-dependent phosphatase activities of a number of different preparations were not closely correlated with their total phospholipid contents. After partial lipid depletion with a single extraction in Lubrol W the residual ATPase and phosphatase activities were correlated, but their magnitudes were quite different: on average only about 5% of the former remained compared with 50% of the latter. A similar differential effect on these activities was found after extraction with deoxycholate. In contrast with the ATPase, consistent restoration of the phosphatase activity of Lubrol-extracted enzymes by added exogenous phospholipids was not observed. We conclude that, although the K-+-dependent phosphatase may be lipid-dependent, the lipid requirement must be different from that of the complete ATPase system, and this difference should help investigations of their relationship. PMID:167727

Decreased erythrocyte nucleoside transport and hENT1 transporter expression in glucose 6-phosphate dehydrogenase deficiency.

PubMed

Al-Ansari, Mohammad; Craik, James D

2015-01-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is associated with erythrocyte sensitivity to oxidative damage and hemolytic crises. In β-thalassemia major, where hemoglobin instability imposes oxidative stress, erythrocytes show reduced hENT1 nucleoside transporter expression and decreased nucleoside uptake. This study investigated hENT1 expression and nucleoside transport in G6PD-deficient erythrocytes to determine if decreased hENT1 activity might be a contributory feature in the variable pathology of this enzymopathy. Uptake of (3)H-uridine was measured at room temperature using an inhibitor-oil stop protocol and 5-s incubations. Erythrocyte membranes were analyzed by SDS-PAGE and nucleoside (hENT1), glucose (GLUT-1), and anion exchange (Band 3) transporter polypeptides quantitated on immunoblots. In G6PD-deficient cells, uridine uptake (mean 8.18, 95 % CI 5.6-10.7 vs controls mean 12.35, 95 % CI 9.2-15.5, pmol uridine/gHb/min; P = 0.031) and expression of hENT1 (mean 50.4 %, 95 % CI 38.1-62.7 %, arbitrary units n = 11 vs controls mean 95.23 %, 95 % CI 88.38-102.1 % arbitrary units, n = 8; P < 0.001) were significantly lower; expression of GLUT-1 (mean 106.9 %, vs control mean 99.75 %; P = 0.308) and Band 3 polypeptides (mean 100.1 %, vs control mean 102.84 %; P = 0.329) were unchanged. Nucleoside transporter activity in human erythrocytes sustains intracellular purine nucleotide levels and assists in control of plasma adenosine levels; decreased hENT1 expression and activity in G6PD-deficiency could affect red metabolism and influence a wide spectrum of responses mediated by adenosine receptors.

Determination of redox potentials for the Watson-Crick base pairs, DNA nucleosides, and relevant nucleoside analogues.

PubMed

Crespo-Hernandez, Carlos E; Close, David M; Gorb, Leonid; Leszczynski, Jerzy

2007-05-17

Redox potentials for the DNA nucleobases and nucleosides, various relevant nucleoside analogues, Watson-Crick base pairs, and seven organic dyes are presented based on DFT/B3LYP/6-31++G(d,p) and B3YLP/6-311+G(2df,p)//B3LYP/6-31+G* levels of calculations. The values are determined from an experimentally calibrated set of equations that correlate the vertical ionization (electron affinity) energy of 20 organic molecules with their experimental reversible oxidation (reduction) potential. Our results are in good agreement with those estimated experimentally for the DNA nucleosides in acetonitrile solutions (Seidel et al. J. Phys. Chem. 1996, 100, 5541). We have found that nucleosides with anti conformation exhibit lower oxidation potentials than the corresponding syn conformers. The lowering in the oxidation potential is due to the formation of an intramolecular hydrogen bonding interaction between the 5'-OH group of the sugar and the N3 of the purine bases or C2=O of the pyrimidine bases in the syn conformation. Pairing of adenine or guanine with its complementary pyrimidine base decreases its oxidation potential by 0.15 or 0.28 V, respectively. The calculated energy difference between the oxidation potential for the G.C base pair and that of the guanine base is in good agreement with the experimental value estimated recently (0.34 V: Caruso, T.; et al. J. Am. Chem. Soc. 2005, 127, 15040). The complete and consistent set of reversible redox values determined in this work for the DNA constituents is expected to be of considerable value to those studying charge and electronic energy transfer in DNA.

Nature's combinatorial biosynthesis and recently engineered production of nucleoside antibiotics in Streptomyces.

PubMed

Chen, Shawn; Kinney, William A; Van Lanen, Steven

2017-04-01

Modified nucleosides produced by Streptomyces and related actinomycetes are widely used in agriculture and medicine as antibacterial, antifungal, anticancer and antiviral agents. These specialized small-molecule metabolites are biosynthesized by complex enzymatic machineries encoded within gene clusters in the genome. The past decade has witnessed a burst of reports defining the key metabolic processes involved in the biosynthesis of several distinct families of nucleoside antibiotics. Furthermore, genome sequencing of various Streptomyces species has dramatically increased over recent years. Potential biosynthetic gene clusters for novel nucleoside antibiotics are now apparent by analysis of these genomes. Here we revisit strategies for production improvement of nucleoside antibiotics that have defined mechanisms of action, and are in clinical or agricultural use. We summarize the progress for genetically manipulating biosynthetic pathways for structural diversification of nucleoside antibiotics. Microorganism-based biosynthetic examples are provided and organized under genetic principles and metabolic engineering guidelines. We show perspectives on the future of combinatorial biosynthesis, and present a working model for discovery of novel nucleoside natural products in Streptomyces.

New carbocyclic N(6)-substituted adenine and pyrimidine nucleoside analogues with a bicyclo[2.2.1]heptane fragment as sugar moiety; synthesis, antiviral, anticancer activity and X-ray crystallography.

PubMed

Tănase, Constantin I; Drăghici, Constantin; Cojocaru, Ana; Galochkina, Anastasia V; Orshanskaya, Jana R; Zarubaev, Vladimir V; Shova, Sergiu; Enache, Cristian; Maganu, Maria

2015-10-01

New nucleoside analogues with an optically active bicyclo[2.2.1]heptane skeleton as sugar moiety and 6-substituted adenine were synthesized by alkylation of 6-chloropurine intermediate. Thymine and uracil analogs were synthesized by building the pyrimidine ring on amine 1. X-ray crystallography confirmed an exo-coupling of the thymine to the ring and an L configuration of the nucleoside analogue. The library of compounds was tested for their inhibitory activity against influenza virus A∖California/07/09 (H1N1)pdm09 and coxsackievirus B4 in cell culture. Compounds 13a and 13d are the most promising for their antiviral activity against influenza, and compound 3c against coxsackievirus B4. Compounds 3b and 3g were tested for anticancer activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Susceptibility of recombinant porcine endogenous retrovirus reverse transcriptase to nucleoside and non-nucleoside inhibitors.

PubMed

Wilhelm, M; Fishman, J A; Pontikis, R; Aubertin, A M; Wilhelm, F X

2002-12-01

Transplantation of organs, tissues or cells from pigs to humans could be a potential solution to the shortage of human organs for transplantation. Porcine endogenous retroviruses (PERVs) remain a major safety concern for porcine xenotransplantation. Thus, finding drugs that could be used as virological prophylaxis (or therapy) against PERV replication would be desirable. One of the most effective ways to block retroviral multiplication is to inhibit the enzyme reverse transcriptase (RT) which catalyzes the reverse transcription of viral RNA to proviral double-stranded DNA. We report here the cloning and expression of PERV RT and its susceptibility to several inhibitors. Our data demonstrate PERV susceptibility in vitro to the triphosphorylated nucleoside analog of zidovudine (AZT) and to ddGTP and to a lesser extent to ddTTP but almost no susceptibility to the non-nucleoside RT inhibitors tested.

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.

Efficacy of non-nucleoside reverse transcriptase inhibitor-based highly active antiretroviral therapy in Thai HIV-infected children aged two years or less.

PubMed

Puthanakit, Thanyawee; Aurpibul, Linda; Sirisanthana, Thira; Sirisanthana, Virat

2009-03-01

Twenty-six Thai HIV-infected children, aged 2 years or less were prospectively enrolled to receive non-nucleoside reverse transcription inhibitor-based highly active antiretroviral therapy (HAART). Twenty-two children (85%) had World Health Organization clinical stage 3 or 4. The median baseline CD4 cell percentage and plasma HIV RNA were 17% and 5.9 log 10 copies/mL, respectively. The median age at HAART initiation was 9.8 months (range, 1.5-24.0). One child died. The mean CD4 cell percentages at 24, 48, and 96 weeks of treatment were 26%, 31%, and 37%, respectively. The proportions of children with virologic suppression (<400 copies/mL) at week 24 and 48 were 14/26 (54%) and 19/26 (73%), respectively. Non-nucleoside reverse transcription inhibitor-based HAART is safe and effective in HIV-infected young children in a resource-limited setting.

Intrinsic electrophilic properties of nucleosides: Photoelectron spectroscopy of their parent anions

NASA Astrophysics Data System (ADS)

Stokes, Sarah T.; Li, Xiang; Grubisic, Andrej; Ko, Yeon Jae; Bowen, Kit H.

2007-08-01

The nucleoside parent anions 2'-deoxythymidine-, 2'-deoxycytidine-, 2'-deoxyadenosine-, uridine-, cytidine-, adenosine-, and guanosine- were generated in a novel source, employing a combination of infrared desorption, electron photoemission, and a gas jet expansion. Once mass selected, the anion photoelectron spectrum of each of these was recorded. In the three cases in which comparisons were possible, the vertical detachment energies and likely adiabatic electron affinities extracted from these spectra agreed well with the values calculated both by Richardson et al. [J. Am. Chem. Soc. 126, 4404 (2004)] and by Li et al. [Radiat. Res. 165, 721 (2006)]. Through the combination of our experimental results and their theoretical calculations, several implications emerge. (1) With the possible exception of dG-, the parent anions of nucleosides exist, and they are stable. (2) These nucleoside anions are valence anions, and in most cases the negative charge is closely associated with the nucleobase moiety. (3) The nucleoside parent anions we have generated and studied are the negative ions of canonical, neutral nucleosides, similar to those found in DNA.

Intrinsic electrophilic properties of nucleosides: photoelectron spectroscopy of their parent anions.

PubMed

Stokes, Sarah T; Li, Xiang; Grubisic, Andrej; Ko, Yeon Jae; Bowen, Kit H

2007-08-28

The nucleoside parent anions 2(')-deoxythymidine(-), 2(')-deoxycytidine(-), 2(')-deoxyadenosine(-), uridine(-), cytidine(-), adenosine(-), and guanosine(-) were generated in a novel source, employing a combination of infrared desorption, electron photoemission, and a gas jet expansion. Once mass selected, the anion photoelectron spectrum of each of these was recorded. In the three cases in which comparisons were possible, the vertical detachment energies and likely adiabatic electron affinities extracted from these spectra agreed well with the values calculated both by Richardson et al. [J. Am. Chem. Soc. 126, 4404 (2004)] and by Li et al. [Radiat. Res. 165, 721 (2006)]. Through the combination of our experimental results and their theoretical calculations, several implications emerge. (1) With the possible exception of dG(-), the parent anions of nucleosides exist, and they are stable. (2) These nucleoside anions are valence anions, and in most cases the negative charge is closely associated with the nucleobase moiety. (3) The nucleoside parent anions we have generated and studied are the negative ions of canonical, neutral nucleosides, similar to those found in DNA.

Use of 2'-spirocyclic ethers in HCV nucleoside design.

PubMed

Du, Jinfa; Chun, Byoung-Kwon; Mosley, Ralph T; Bansal, Shalini; Bao, Haiying; Espiritu, Christine; Lam, Angela M; Murakami, Eisuke; Niu, Congrong; Micolochick Steuer, Holly M; Furman, Phillip A; Sofia, Michael J

2014-03-13

Conformationally restricted 2'-spironucleosides and their prodrugs were synthesized as potential anti-HCV agents. Although the replicon activity of the new agents containing pyrimidine bases was modest, the triphosphate of a 2'-oxetane cytidine analogue demonstrated potent intrinsic biochemical activity against the NS5B polymerase, with IC50 = 8.48 μM. Activity against NS5B bearing the S282T mutation was reduced. Phosphoramidate prodrugs of a 2'-oxetane 2-amino-6-O-methyl-purine nucleoside demonstrated potent anti-HCV activity in vitro, and the corresponding triphosphate retained similar potent activity against both wild-type and S282T HCV NS5B polymerase.

The chemistry of nicotinamide adenine dinucleotide (NAD) analogues containing C-nucleosides related to nicotinamide riboside.

PubMed

Pankiewicz, Krzysztof W; Watanabe, Kyoichi A; Lesiak-Watanabe, Krystyna; Goldstein, Barry M; Jayaram, Hiremagalur N

2002-04-01

Oncolytic C-nucleosides, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and benzamide riboside (3-beta-D-ribofuranosylbenzamide) are converted in cell into active metabolites thiazole-4-carboxamide- and benzamide adenine dinucleotide, TAD and BAD, respectively. TAD and BAD as NAD analogues were found to bind at the nicotinamide adenine dinucleotide (cofactor NAD) site of inosine monophosphate dehydrogenase (IMPDH), an important target in cancer treatment. The synthesis and evaluation of anticancer activity of a number of C-nucleosides related to tiazofurin and nicotinamide riboside then followed and are reviewed herein. Interestingly, pyridine C-nucleosides (such as C-nicotinamide riboside) are not metabolized into the corresponding NAD analogues in cell. Their conversion by chemical methods is described. As dinucleotides these compounds show inhibition of IMPDH in low micromolar level. Also, the synthesis of BAD in metabolically stable bis(phosphonate) form is discussed indicating the usefulness of such preformed inhibitors in drug development. Among tiazofurin analogues, Franchetti and Grifantini found, that the replacement of the sulfur by oxygen (as in oxazafurin) but not the removal of nitrogen (tiophenfurin) of the thiazole ring resulted in inactive compounds. The anti cancer activity of their synthetic dinucleotide analogues indicate that inactive compounds are not only poorly metabolized in cell but also are weak inhibitors of IMPDH as dinucleotides.

Quantitative high-performance liquid chromatography of nucleosides in biological materials.

PubMed

Gehrke, C W; Kuo, K C; Davis, G E; Suits, R D; Waalkes, T P; Borek, E

1978-03-21

A rigorous, comprehensive, and reliable reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the analysis of ribonucleosides in urine (psi, m1A, m1I, m2G, A, m2(2)G). An initial isolation of ribonucleosides with an affinity gel containing an immobilized phenylboronic acid was used to improve selectivity and sensitivity. Response for all nucleosides was linear from 0.1 to 50 nmoles injected and good quantitation was obtained for 25 microliter or less of sample placed on the HPLC column. Excellent precision of analysis for urinary nucleosides was achieved on matrix dependent and independent samples, and the high resolution of the reversed-phase column allowed the complete separation of 9 nucleosides from other unidentified UV absorbing components at the 1-ng level. Supporting experimental data are presented on precision, recovery, chromatographic methods, minimum detection limit, retention time, relative molar response, sample clean-up, stability of nucleosides, boronate gel capacity, and application to analysis of urine from patients with leukemia and breast cancer. This method is now being used routinely for the determination of the concentration and ratios of nucleosides in urine from patients with different types of cancer and in chemotherapy response studies.

Metabolomic profile and nucleoside composition of Cordyceps nidus sp. nov. (Cordycipitaceae): A new source of active compounds

PubMed Central

Danies, Giovanna; Sierra, Rocio; Schauer, Nicolas; Trenkamp, Sandra

2017-01-01

Cordyceps sensu lato is a genus of arthropod-pathogenic fungi, which have been used traditionally as medicinal in Asia. Within the genus, Ophiocordyceps sinensis is the most coveted and expensive species in China. Nevertheless, harvesting wild specimens has become a challenge given that natural populations of the fungus are decreasing and because large-scale culture of it has not yet been achieved. The worldwide demand for products derived from cultivable fungal species with medicinal properties has increased recently. In this study, we propose a new species, Cordyceps nidus, which parasitizes underground nests of trapdoor spiders. This species is phylogenetically related to Cordyceps militaris, Cordyceps pruinosa, and a sibling species of Cordyceps caloceroides. It is found in tropical rainforests from Bolivia, Brazil, Colombia and Ecuador. We also investigated the medicinal potential of this fungus based on its biochemical properties when grown on four different culture media. The metabolic profile particularly that of nucleosides, in polar and non-polar extracts was determined by UPLC, and then correlated to their antimicrobial activity and total phenolic content. The metabolome showed a high and significant dependency on the substrate used for fungal growth. The mass intensities of nucleosides and derivative compounds were higher in natural culture media in comparison to artificial culture media. Among these compounds, cordycepin was the predominant, showing the potential use of this species as an alternative to O. sinensis. Furthermore, methanol fractions showed antimicrobial activity against gram-positive bacteria, and less than 3.00 mg of gallic acid equivalents per g of dried extract were obtained when assessing its total phenolic content by modified Folin-Ciocalteu method. The presence of polyphenols opens the possibility of further exploring the antioxidant capacity and the conditions that may enhance this characteristic. The metabolic composition and

Metabolomic profile and nucleoside composition of Cordyceps nidus sp. nov. (Cordycipitaceae): A new source of active compounds.

PubMed

Chiriví, Juan; Danies, Giovanna; Sierra, Rocio; Schauer, Nicolas; Trenkamp, Sandra; Restrepo, Silvia; Sanjuan, Tatiana

2017-01-01

Cordyceps sensu lato is a genus of arthropod-pathogenic fungi, which have been used traditionally as medicinal in Asia. Within the genus, Ophiocordyceps sinensis is the most coveted and expensive species in China. Nevertheless, harvesting wild specimens has become a challenge given that natural populations of the fungus are decreasing and because large-scale culture of it has not yet been achieved. The worldwide demand for products derived from cultivable fungal species with medicinal properties has increased recently. In this study, we propose a new species, Cordyceps nidus, which parasitizes underground nests of trapdoor spiders. This species is phylogenetically related to Cordyceps militaris, Cordyceps pruinosa, and a sibling species of Cordyceps caloceroides. It is found in tropical rainforests from Bolivia, Brazil, Colombia and Ecuador. We also investigated the medicinal potential of this fungus based on its biochemical properties when grown on four different culture media. The metabolic profile particularly that of nucleosides, in polar and non-polar extracts was determined by UPLC, and then correlated to their antimicrobial activity and total phenolic content. The metabolome showed a high and significant dependency on the substrate used for fungal growth. The mass intensities of nucleosides and derivative compounds were higher in natural culture media in comparison to artificial culture media. Among these compounds, cordycepin was the predominant, showing the potential use of this species as an alternative to O. sinensis. Furthermore, methanol fractions showed antimicrobial activity against gram-positive bacteria, and less than 3.00 mg of gallic acid equivalents per g of dried extract were obtained when assessing its total phenolic content by modified Folin-Ciocalteu method. The presence of polyphenols opens the possibility of further exploring the antioxidant capacity and the conditions that may enhance this characteristic. The metabolic composition and

Selenium-Mediated Dehalogenation of Halogenated Nucleosides and its Relevance to the DNA Repair Pathway.

PubMed

Mondal, Santanu; Manna, Debasish; Mugesh, Govindasamy

2015-08-03

Halogenated nucleosides can be incorporated into the newly synthesized DNA of replicating cells and therefore are commonly used in the detection of proliferating cells in living tissues. Dehalogenation of these modified nucleosides is one of the key pathways involved in DNA repair mediated by the uracil-DNA glycosylase. Herein, we report the first example of a selenium-mediated dehalogenation of halogenated nucleosides. We also show that the mechanism for the debromination is remarkably different from that of deiodination and that the presence of a ribose or deoxyribose moiety in the nucleosides facilitates the deiodination. The results described herein should help in understanding the metabolism of halogenated nucleosides in DNA and RNA. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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

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

2008-01-01

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

Synthesis of conformationally North-locked pyrimidine nucleosides built on an oxabicyclo[3.1.0]hexane scaffold.

PubMed

Ludek, Olaf R; Marquez, Victor E

2012-01-20

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 11 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.

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

PubMed

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

2015-01-01

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

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

PubMed Central

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

2012-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2012-08-01

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

Selective loss of nucleoside carrier expression in rat hepatocarcinomas.

PubMed

Dragan, Y; Valdés, R; Gomez-Angelats, M; Felipe, A; Javier Casado, F; Pitot, H; Pastor-Anglada, M

2000-08-01

Evidence that hepatoma cell lines show differential expression of concentrative nucleoside transporters (CNT1 and CNT2) prompted us to study the transporter proteins in 2 models of hepatocarcinogenesis, the chemically induced Solt and Farber model and the albumin-SV40 large T antigen (Alb-SV40) transgenic rat. CNT1 expression was lower in tumor biopsy specimens from Alb-SV40 rat livers than in normal tissue. Immunocytochemistry revealed that the CNT1 protein was indeed absent in the tumor lesions. CNT1 was also absent in a cell line, L25, derived from the Alb-SV40 transgenic rat liver tumors, whereas another cell line, L37, derived from the normal-appearing parenchyma, retained the expression of both carrier isoforms. The protein expression correlated with the nucleoside transport properties of these cell lines. Moreover, although CNT2 expression was highly dependent on the growth characteristics of the 2 cell lines, as was CNT1 (albeit to a lower extent) in L37 cells, it was not expressed in L25 cells at any stage of cell growth. In contrast to the transgenic model of hepatocarcinogenesis, in the chemically induced tumors the expression of CNT2 was lower, although still detectable. In summary, these data indicate that hepatocarcinogenesis leads to a selective loss or diminished expression of nucleoside carrier isoforms, a feature that may be relevant to our understanding of the molecular basis of the bioavailability of those drugs that are nucleoside derivatives and may be substrates of these carriers. The transport properties and isoform-expression profile of the L25 and L37 cell lines make them suitable hepatocyte culture models with which to study nucleoside transport processes and drug sensitivity.

Structural Determinants of the 5′-Methylthioinosine Specificity of Plasmodium Purine Nucleoside Phosphorylase

PubMed Central

Donaldson, Teraya M.; Ting, Li-Min; Zhan, Chenyang; Shi, Wuxian; Zheng, Renjian; Almo, Steven C.; Kim, Kami

2014-01-01

Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5′-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5′-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5′-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5′-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket. PMID:24416224

Synthesis of Purine Nucleoside and Nucleotide Analogs as Antiparasitic Agents.

DTIC Science & Technology

1979-09-01

was to conduct studies on the synthesis of purine nucleoside and nucleotide analogs as anti- parasitic agents. The primary target compounds were 5...antiparasitic agents. - Jaffe has proposed that the susceptibility of pathogenic helminths and protozoa to fraudulent purine, in contrast to pyrimidine...8217-substituted derivatives are thus designed to inhibit nucleoside and nucleotide kinases as well as other parasitic enzymes. Mammalian cells, onthe

Testing Nucleoside Analogues as Inhibitors of Bacillus anthracis Spore Germination In Vitro and in Macrophage Cell Culture ▿

PubMed Central

Alvarez, Zadkiel; Lee, Kyungae; Abel-Santos, Ernesto

2010-01-01

Bacillus anthracis, the etiological agent of anthrax, has a dormant stage in its life cycle known as the endospore. When conditions become favorable, spores germinate and transform into vegetative bacteria. In inhalational anthrax, the most fatal manifestation of the disease, spores enter the organism through the respiratory tract and germinate in phagosomes of alveolar macrophages. Germinated cells can then produce toxins and establish infection. Thus, germination is a crucial step for the initiation of pathogenesis. B. anthracis spore germination is activated by a wide variety of amino acids and purine nucleosides. Inosine and l-alanine are the two most potent nutrient germinants in vitro. Recent studies have shown that germination can be hindered by isomers or structural analogues of germinants. 6-Thioguanosine (6-TG), a guanosine analogue, is able to inhibit germination and prevent B. anthracis toxin-mediated necrosis in murine macrophages. In this study, we screened 46 different nucleoside analogues as activators or inhibitors of B. anthracis spore germination in vitro. These compounds were also tested for their ability to protect the macrophage cell line J774a.1 from B. anthracis cytotoxicity. Structure-activity relationship analysis of activators and inhibitors clarified the binding mechanisms of nucleosides to B. anthracis spores. In contrast, no structure-activity relationships were apparent for compounds that protected macrophages from B. anthracis-mediated killing. However, multiple inhibitors additively protected macrophages from B. anthracis. PMID:20921305

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

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

Tran, Timothy H.; Christoffersen, S.; Allan, Paula W.

2011-09-20

Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 {angstrom} resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamermore » with an ?/? monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is {approx}7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.« less

Classification of lung cancer patients and controls by chromatography of modified nucleosides in serum

USGS Publications Warehouse

McEntire, John E.; Kuo, Kenneth C.; Smith, Mark E.; Stalling, David L.; Richens, Jack W.; Zumwalt, Robert W.; Gehrke, Charles W.; Papermaster, Ben W.

1989-01-01

A wide spectrum of modified nucleosides has been quantified by high-performance liquid chromatography in serum of 49 male lung cancer patients, 35 patients with other cancers, and 48 patients hospitalized for nonneoplastic diseases. Data for 29 modified nucleoside peaks were normalized to an internal standard and analyzed by discriminant analysis and stepwise discriminant analysis. A model based on peaks selected by a stepwise discriminant procedure correctly classified 79% of the cancer and 75% of the noncancer subjects. It also demonstrated 84% sensitivity and 79% specificity when comparing lung cancer to noncancer subjects, and 80% sensitivity and 55% specificity in comparing lung cancer to other cancers. The nucleoside peaks having the greatest influence on the models varied dependent on the subgroups compared, confirming the importance of quantifying a wide array of nucleosides. These data support and expand previous studies which reported the utility of measuring modified nucleoside levels in serum and show that precise measurement of an array of 29 modified nucleosides in serum by high-performance liquid chromatography with UV scanning with subsequent data modeling may provide a clinically useful approach to patient classification in diagnosis and subsequent therapeutic monitoring.

Origin, Utilization, and Recycling of Nucleosides in the Central Nervous System

ERIC Educational Resources Information Center

Ipata, Piero Luigi

2011-01-01

The brain relies on the salvage of preformed purine and pyrimidine rings, mainly in the form of nucleosides, to maintain its nucleotide pool in the proper qualitative and quantitative balance. The transport of nucleosides from blood into neurons and glia is considered to be an essential prerequisite to enter their metabolic utilization in the…

Nucleobase and nucleoside transport and integration into plant metabolism

PubMed Central

Girke, Christopher; Daumann, Manuel; Niopek-Witz, Sandra; Möhlmann, Torsten

2014-01-01

Nucleotide metabolism is an essential process in all living organisms. Besides newly synthesized nucleotides, the recycling (salvage) of partially degraded nucleotides, i.e., nucleosides and nucleobases serves to keep the homeostasis of the nucleotide pool. Both types of metabolites are substrates of at least six families of transport proteins in Arabidopsis thaliana (Arabidopsis) with a total of 49 members. In the last years several members of such transport proteins have been analyzed allowing to present a more detailed picture of nucleoside and nucleobase transport and the physiological function of these processes. Besides functioning in nucleotide metabolism it turned out that individual members of the before named transporters exhibit the capacity to transport a wide range of different substrates including vitamins and phytohormones. The aim of this review is to summarize the current knowledge on nucleobase and nucleoside transport processes in plants and integrate this into nucleotide metabolism in general. Thereby, we will focus on those proteins which have been characterized at the biochemical level. PMID:25250038

[Quantitative analysis of nucleosides in four Cordyceps genus by HPLC].

PubMed

Qian, Zheng-Ming; Li, Wen-Qing; Wang, Chuan-Xi; Zhou, Miao-Xia; Sun, Min-Tian; Gao, Hao; Li, Wen-Jia

2016-07-01

To compare the main nucleosides in Cordyceps genus herbs (C. sinensis, C. millitaris, Hirsutella sinensis and C. sobolifera), an HPLC method for simultaneous determination of uridine, inosine, guanosine, adenosine and cordycepine in Cordyceps genus herbs was developed. The sample was extracted with 0.5% phosphoric acid solution to prepare test solution. The separation was performed on a Zorbax SB-Aq (4.6 mm×150 mm, 5 μm) column with gradient elution by 0.04 mol•L⁻¹ potassium dihydrogen phosphate solution and acetonitrile, column temperature 30 ℃,flow rate 0.8 mL•min⁻¹,and detection wavelength 260 nm. The content of nucleosides in four Cordyceps genus herbs was evaluated by fingerprint analysis and hierarchical cluster analysis (HCA). The calibration curves of five nucleosides showed good linear regression (r>0.99) and the average recoveries were between 95.0% and 105.0%. The contents of the five nucleosides in the four Cordyceps genus herbs were different and could be obviously distinguished by HCA. The fingerprint analysis result showed that the similarity between C. sinensis and the others was less than 0.9. The method was accurate and reliable, which can be used for quality control of Cordyceps genus herbs. Copyright© by the Chinese Pharmaceutical Association.

Compositions containing nucleosides and manganese and their uses

DOEpatents

Daly, Michael J.; Gaidamakova, Elena K.; Matrosova, Vera Y.; Levine, Rodney L.; Wehr, Nancy B.

2015-11-17

This invention encompasses methods of preserving protein function by contacting a protein with a composition comprising one or more purine or pyrimidine nucleosides (such as e.g., adenosine or uridine) and an antioxidant (such as e.g., manganese). In addition, the invention encompasses methods of treating and/or preventing a side effect of radiation exposure and methods of preventing a side effect of radiotherapy comprising administration of a pharmaceutically effective amount of a composition comprising one or more purine or pyrimidine nucleosides (such as e.g., adenosine or uridine) and an antioxidant (such as e.g., manganese) to a subject in need thereof. The compositions may comprise D. radiodurans extracts.

Manganese(II) Chloride Alters Nucleotide and Nucleoside Catabolism in Zebrafish (Danio rerio) Adult Brain.

PubMed

Altenhofen, Stefani; Nabinger, Débora Dreher; Pereira, Talita Carneiro Brandão; Leite, Carlos Eduardo; Bogo, Maurício Reis; Bonan, Carla Denise

2018-05-01

ATP and adenosine, the main signaling molecules of purinergic system, are involved in toxicological effects induced by metals. The manganese (Mn) exposure induces several cellular changes, which could interfere with signaling pathways, such as the purinergic system. In this study, we evaluated the effects of exposure to manganese(II) chloride (MnCl 2 ) during 96 h on nucleoside triphosphate diphosphohydrolase (NTPDase), ecto-5'-nucleotidase, and adenosine deaminase (ADA) activities, followed by analyzing the gene expression patterns of NTPDases (entpd1, entpd2a.1, entpd2a.2, entpd2-like, entpd3) and ADA (ADA 1 , ADA 2.1 , ADA 2.2 , ADAasi, ADAL) families in zebrafish brain. In addition, the brain metabolism of nucleotides and nucleosides was evaluated after MnCl 2 exposure. The results showed that MnCl 2 exposure during 96 h inhibited the NTPDase (1.0 and 1.5 mM) and ecto-ADA (0.5, 1.0, and 1.5 mM) activities, further decreasing ADA2.1 expression at all MnCl 2 concentrations analyzed. Purine metabolism was also altered by the action of MnCl 2 . An increased amount of ADP appeared at all MnCl 2 concentrations analyzed; however, AMP and adenosine levels are decreased at the concentrations of 1.0 and 1.5 mM MnCl 2 , whereas decreased inosine (INO) levels were observed at all concentrations tested. The findings of this study demonstrated that MnCl 2 may inhibit NTPDase and ecto-ADA activities, consequently modulating nucleotide and nucleoside levels, which may contribute for the toxicological effects induced by this metal.

An ATP-dependent ligase with substrate flexibility involved in assembly of the peptidyl nucleoside antibiotic polyoxin.

PubMed

Gong, Rong; Qi, Jianzhao; Wu, Pan; Cai, You-Sheng; Ma, Hongmin; Liu, Yang; Duan, He; Wang, Meng; Deng, Zixin; Price, Neil P J; Chen, Wenqing

2018-04-27

Polyoxin (POL) is an unusual peptidyl nucleoside antibiotic, in which peptidyl moiety and nucleoside skeleton are linked by an amide bond. However, their biosynthesis remains poorly understood. Here, we report the deciphering of PolG as an ATP-dependent ligase responsible for the assembly of POL. A polG mutant is capable of accumulating multiple intermediates, including the peptidyl moiety (carbamoylpolyoxamic acid, CPOAA) and the nucleosides skeletons (POL-C and the previously overlooked thymine POL-C). We further demonstrated that PolG employs an ATP-dependent mechanism for amide bond formation, and that the generation of the hybrid nucleoside antibiotic, POL-N, is also governed by PolG. Finally, we determined that the deduced ATP-binding sites are functionally essential for PolG, and that they are highly conserved in a number of related ATP-dependent ligases. These insights have allowed us proposed a catalytic mechanism for the assembly of peptidyl nucleoside antibiotic via an acyl-phosphate intermediate, and have opened the way for the combinatorial biosynthesis/pathway engineering of this group of nucleoside antibiotics. Importance POL is well known for its remarkable antifungal bioactivities and unusual structural features. Actually, elucidation of the POL assembly logic not only provides the enzymatic basis for further biosynthetic understanding of related peptidyl nucleoside antibiotics, but also contributes to the rational generation of more hybrid nucleoside antibiotics via synthetic biology strategy. Copyright © 2018 American Society for Microbiology.

Human Cytomegalovirus Resistance to Deoxyribosylindole Nucleosides Maps to a Transversion Mutation in the Terminase Subunit-Encoding Gene UL89

PubMed Central

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

2014-01-01

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

Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC–UV–MS

PubMed Central

Russell, Susan P.; Limbach, Patrick A.

2013-01-01

Post-transcriptional chemical covalent modification of adenosine, guanosine, uridine and cytidine occurs frequently in all types of ribonucleic acids (RNAs). In ribosomal RNA (rRNA) and transfer RNA (tRNA) these modifications make important contributions to RNA structure and stability and to the accuracy and efficiency of protein translation. The functional dynamics, synergistic nature and regulatory roles of these posttranscriptional nucleoside modifications within the cell are not well characterized. These modifications are present at very low levels and isolation of individual nucleosides for analysis requires a complex multi-step approach. The focus of this study is to characterize the reproducibility of a liquid chromatography method used to isolate and quantitatively characterize modified nucleosides in tRNA and rRNA when nucleoside detection is performed using ultraviolet and mass spectrometric detection (UV and MS, respectively). Despite the analytical challenges of sample isolation and dynamic range, quantitative profiling of modified nucleosides obtained from bacterial tRNAs and rRNAs is feasible at relative standard deviations of 5% RSD or less. PMID:23500350

A general approach to the synthesis of 5-S-functionalized pyrimidine nucleosides and their analogues.

PubMed

Kananovich, Dzmitry G; Reino, Alli; Ilmarinen, Kaja; Rõõmusoks, Marko; Karelson, Mati; Lopp, Margus

2014-08-14

A general and efficient approach was developed for the introduction of S-functionality at the C-5 position of cytosine and uracil nucleosides and their analogues. The key step is a palladium-catalyzed C-S coupling of the corresponding 5-bromo nucleoside derivative and alkyl thiol. The butyl 3-mercaptopropionate coupling products were further converted to the corresponding disulphides, the stable precursors of 5-mercaptopyrimidine nucleosides.

Relationship between Na+-dependent respiration and Na+ + K+-adenosine triphosphatase activity in the action of thyroid hormone on rat jejunal mucosa.

PubMed Central

Liberman, U A; Asano, Y; Lo, C S; Edelman, I S

1979-01-01

Administration of three successive doses of triiodothyronine (T3) (50 micrograms/100 g body wt), given on alternate days to thyroidectomized and euthyroid rats, stimulated oxygen consumption (QO2) and Na+ transport-dependent respiration (QO2 [5]) in the stripped jejunal mucosa, a preparation that consisted mostly of epithelial cells. The increase in QO2(t) accounted for 57% of the increment in QO2 in the transition from the hypothyroid to the euthyroid state and for 29% of the increment in the transition from the euthyroid to the hyperthyroid state. Administration of T3 to hypothyroid rats also increased the yield of epithelial cells. Injection of T3 into thyroidectomized and euthyroid rats increased the specific activity (at Vmax) of the (Na+ + K+)-dependent adenosine triphosphatase (NaK-ATPase) in jejunal crude membrane preparations. No significant change was recorded in the activity of Mg-ATPase in the same preparation. The ratio of QO2/NaK-ATPase and QO2(t)/NaK-ATPase in the various thyroid states remained constant, indicating proportionate increased in the respiratory and enzymatic indices. The effect of administration of T3 to thyroidectomized rats on the number of NaK-ATPase units (recovered in the crude membrane preparation) was estimated by: (a) Na+ + Mg++ + ATP-dependent binding of [3H]-ouabain to crude membrane fractions, and (b) the amount of the phosphorylated intermediate formed in the NaK-ATPase reaction from AT32P(gamma). Estimates were obtained of the maximal number of [3H]ouabain binding sites (Nm) and dissociation constants (Kd). Nm for [3H]ouabain and Nak-ATPase specific activity increased to about the same extent after T3 administration to thyroidectomized rats, with no change in the apparent Kd values. The amount of phosphorylated intermediate formed in jejunal crude membrane preparations also increased significantly. Thus, thyroid hormone administration may increase the number of active Na+pump sites in the plasma membrane. The apparent

Relationship between Na+-dependent respiration and Na+ + K+-adenosine triphosphatase activity in the action of thyroid hormone on rat jejunal mucosa.

PubMed

Liberman, U A; Asano, Y; Lo, C S; Edelman, I S

1979-07-01

Administration of three successive doses of triiodothyronine (T3) (50 micrograms/100 g body wt), given on alternate days to thyroidectomized and euthyroid rats, stimulated oxygen consumption (QO2) and Na+ transport-dependent respiration (QO2 [5]) in the stripped jejunal mucosa, a preparation that consisted mostly of epithelial cells. The increase in QO2(t) accounted for 57% of the increment in QO2 in the transition from the hypothyroid to the euthyroid state and for 29% of the increment in the transition from the euthyroid to the hyperthyroid state. Administration of T3 to hypothyroid rats also increased the yield of epithelial cells. Injection of T3 into thyroidectomized and euthyroid rats increased the specific activity (at Vmax) of the (Na+ + K+)-dependent adenosine triphosphatase (NaK-ATPase) in jejunal crude membrane preparations. No significant change was recorded in the activity of Mg-ATPase in the same preparation. The ratio of QO2/NaK-ATPase and QO2(t)/NaK-ATPase in the various thyroid states remained constant, indicating proportionate increased in the respiratory and enzymatic indices. The effect of administration of T3 to thyroidectomized rats on the number of NaK-ATPase units (recovered in the crude membrane preparation) was estimated by: (a) Na+ + Mg++ + ATP-dependent binding of [3H]-ouabain to crude membrane fractions, and (b) the amount of the phosphorylated intermediate formed in the NaK-ATPase reaction from AT32P(gamma). Estimates were obtained of the maximal number of [3H]ouabain binding sites (Nm) and dissociation constants (Kd). Nm for [3H]ouabain and Nak-ATPase specific activity increased to about the same extent after T3 administration to thyroidectomized rats, with no change in the apparent Kd values. The amount of phosphorylated intermediate formed in jejunal crude membrane preparations also increased significantly. Thus, thyroid hormone administration may increase the number of active Na+pump sites in the plasma membrane. The apparent

Microwave-assisted synthesis of novel purine nucleosides as selective cholinesterase inhibitors.

PubMed

Schwarz, S; Csuk, R; Rauter, A P

2014-04-21

Alzheimer's disease (AD), the most common form of senile dementia, is characterized by high butyrylcholinesterase (BChE) levels in the brain in later AD stages, for which no treatment is available. Pursuing our studies on selective BChE inhibitors, that may contribute to understand the role of this enzyme in disease progression, we present now microwave-assisted synthesis and anticholinesterase activity of a new nucleoside series embodying 6-chloropurine or 2-acetamido-6-chloropurine linked to D-glucosyl, D-galactosyl and D-mannosyl residues. It was designed to assess the contribution of sugar stereochemistry, purine structure and linkage to the sugar for cholinesterase inhibition efficiency and selectivity. Compounds were subjected to Ellman's assay and their inhibition constants determined. The α-anomers were the most active compounds, while selectivity for BChE or acetylcholinesterase (AChE) inhibition could be tuned by the purine base, by the glycosyl moiety and by N(7)-ligation. Some of the nucleosides were far more potent than the drug galantamine, and the most promising competitive and selective BChE inhibitor, the N(7)-linked 2-acetamido-α-D-mannosylpurine, showed a Ki of 50 nM and a selectivity factor of 340 fold for BChE over AChE.

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

Protective Mechanisms of S. lycopersicum Aqueous Fraction (Nucleosides and Flavonoids) on Platelet Activation and Thrombus Formation: In Vitro, Ex Vivo and In Vivo Studies.

PubMed

Fuentes, Eduardo; Pereira, Jaime; Alarcón, Marcelo; Valenzuela, Claudio; Pérez, Pablo; Astudillo, Luis; Palomo, Iván

2013-01-01

The purpose of this research was to investigate mechanisms of antiplatelet action of bioactive principle from S. lycopersicum. Aqueous fraction had a high content of nucleosides (adenosine, guanosine, and adenosine 5'-monophosphate) by HPLC analysis. Also aqueous fraction presented flavonoids content. Aqueous fraction inhibited platelet activation by 15 ± 6% (P < 0.05). Fully spread of human platelets on collagen in the presence of aqueous fraction was inhibited from 15 ± 1 to 9 ± 1  μ m(2) (P < 0.001). After incubation of whole blood with aqueous fraction, the platelet coverage was inhibited by 55 ± 12% (P < 0.001). Platelet ATP secretion and aggregation were significantly inhibited by the aqueous fraction. At the same concentrations that aqueous fraction inhibits platelet aggregation, levels of sCD40L significantly decreased and the intraplatelet cAMP levels increased. In addition, SQ22536, an adenylate cyclase inhibitor, attenuated the effect of aqueous fraction toward ADP-induced platelet aggregation and intraplatelet level of cAMP. Platelet aggregation ex vivo (human study) and thrombosis formation in vivo (murine model) were inhibited by aqueous fraction. Finally, aqueous fraction may be used as a functional ingredient adding antiplatelet activities (nucleosides and flavonoids) to processed foods.

Protective Mechanisms of S. lycopersicum Aqueous Fraction (Nucleosides and Flavonoids) on Platelet Activation and Thrombus Formation: In Vitro, Ex Vivo and In Vivo Studies

PubMed Central

Fuentes, Eduardo; Pereira, Jaime; Alarcón, Marcelo; Valenzuela, Claudio; Pérez, Pablo; Astudillo, Luis; Palomo, Iván

2013-01-01

The purpose of this research was to investigate mechanisms of antiplatelet action of bioactive principle from S. lycopersicum. Aqueous fraction had a high content of nucleosides (adenosine, guanosine, and adenosine 5′-monophosphate) by HPLC analysis. Also aqueous fraction presented flavonoids content. Aqueous fraction inhibited platelet activation by 15 ± 6% (P < 0.05). Fully spread of human platelets on collagen in the presence of aqueous fraction was inhibited from 15 ± 1 to 9 ± 1 μm2 (P < 0.001). After incubation of whole blood with aqueous fraction, the platelet coverage was inhibited by 55 ± 12% (P < 0.001). Platelet ATP secretion and aggregation were significantly inhibited by the aqueous fraction. At the same concentrations that aqueous fraction inhibits platelet aggregation, levels of sCD40L significantly decreased and the intraplatelet cAMP levels increased. In addition, SQ22536, an adenylate cyclase inhibitor, attenuated the effect of aqueous fraction toward ADP-induced platelet aggregation and intraplatelet level of cAMP. Platelet aggregation ex vivo (human study) and thrombosis formation in vivo (murine model) were inhibited by aqueous fraction. Finally, aqueous fraction may be used as a functional ingredient adding antiplatelet activities (nucleosides and flavonoids) to processed foods. PMID:24159349

Fe(II)-dependent, uridine-5'-monophosphate α-ketoglutarate dioxygenases in the synthesis of 5'-modified nucleosides.

PubMed

Yang, Zhaoyong; Unrine, Jason; Nonaka, Koichi; Van Lanen, Steven G

2012-01-01

Several nucleoside antibiotics from various actinomycetes contain a high-carbon sugar nucleoside that is putatively derived via C-5'-modification of the canonical nucleoside. Two prominent examples are the 5'-C-carbamoyluridine- and 5'-C-glycyluridine-containing nucleosides, both families of which were discovered using screens aimed at finding inhibitors of bacterial translocase I involved in the assembly of the bacterial peptidoglycan cell wall. A shared open reading frame was identified whose gene product is similar to enzymes of the nonheme, Fe(II)-, and α-ketoglutarate-dependent dioxygenases. The enzyme LipL from the biosynthetic pathway for A-90289, a 5'-C-glycyluridine-containing nucleoside, was functionally characterized as an UMP:α-ketoglutarate dioxygenase, providing the enzymatic imperative for the generation of a nucleoside-5'-aldehdye that serves as a downstream substrate for an aldol or aldol-type reaction leading to the high-carbon sugar scaffold. The functional assignment of LipL and the homologous enzymes-including bioinformatic analysis, iron detection and quantification, and assay development for biochemical characterization-is presented herein. Copyright © 2012 Elsevier Inc. All rights reserved.

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

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

Abramchik, Yu. A., E-mail: inna@ns.crys.ras.ru; Timofeev, V. I., E-mail: espiov@ibch.ru; Zhukhlistova, N. E., E-mail: tostars@mail.ru

2015-07-15

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

Expression and purification of human and Saccharomyces cerevisiae equilibrative nucleoside transporters.

PubMed

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

2018-02-01

Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides also act as ligands for purinergic receptors. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that aid in regulating plasmalemmal flux of purine and pyrimidine nucleosides and nucleobases. ENTs exhibit broad substrate selectivity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. To determine how ENT-mediated transport occurs at the molecular level, greater chemical insight and assays employing purified protein are essential. This article focuses on the expression and purification of human ENT1, human ENT2, and Saccharomyces cerevisiae ScENT1 using novel expression and purification strategies to isolate recombinant ENTs. ScENT1, hENT1, and hENT2 were expressed in W303 Saccharomyces cerevisiae cells and detergent solubilized from the membrane. After detergent extraction, these ENTs were further purified using immobilized metal affinity chromatography and size exclusion chromatography. This effort resulted in obtaining quantities of purified protein sufficient for future biophysical analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Furan Decorated Nucleoside Analogues as Fluorescent Probes: synthesis, photophysical evaluation and site-specific incorporation

PubMed Central

Greco, Nicholas J.; Tor, Yitzhak

2007-01-01

The synthesis and photophysical evaluation of modified nucleoside analogues in which a five-membered heterocycle (furan, thiophene, oxazole and thiazole) is attached to the 5 position of 2′-deoxyuridine are reported. The furan containing derivative is identified as the most promising responsive nucleoside of this family due to its emission quantum efficiency and degree of sensitivity to its microenvironment. The furan moiety was then attached to the 5 position of 2′-deoxycytidine as well as the 8 position of adenosine and guanosine. Photophysical evaluation of these four furan containing nucleoside analogues reveal distinct differences in the absorption, emission and quantum efficiency depending upon the class of nucleoside (pyrimidine or purine). Comparing the photophysical properties of all furan containing nucleosides, identifies the furan thymidine analogue, 5-(fur-2-yl)-2′-deoxyuridine, as the best candidate for use as a responsive fluorescent probe in nucleic acids. 5-(fur-2-yl)-2′-deoxyuridine was then converted to the corresponding phosphoramidite and site specifically incorporated into DNA oligonucleotides with greater than 88% coupling efficiency. Such furan-modified oligonucleotides form stable duplexes upon hybridization to their complementary DNA strands and display favorable fluorescent features. PMID:18431439

A convenient synthesis of a novel nucleoside analogue: 4-(alpha-diformyl-methyl)-1-(beta-D-ribofuranosyl)-2-pyrimidinone.

PubMed

Gao, K; Orgel, L E

2000-01-01

The nucleoside analogue 4-(alpha-diformyl-methyl)-1-(beta-D-ribofuranosyl)-2-pyrimidinone (5) was prepared from the corresponding 4-methyl pyrimidinone nucleoside by means of the Vilsmeier reaction. The unprotected nucleoside can be phosphorylated directly with phosphorus oxychloride in triethyl phosphate.

A convenient synthesis of a novel nucleoside analogue: 4-(alpha-diformyl-methyl)-1-(beta-D-ribofuranosyl)-2-pyrimidinone

NASA Technical Reports Server (NTRS)

Gao, K.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

2000-01-01

The nucleoside analogue 4-(alpha-diformyl-methyl)-1-(beta-D-ribofuranosyl)-2-pyrimidinone (5) was prepared from the corresponding 4-methyl pyrimidinone nucleoside by means of the Vilsmeier reaction. The unprotected nucleoside can be phosphorylated directly with phosphorus oxychloride in triethyl phosphate.

The Synthesis and Study of Azole Carboxamide Nucleosides as Agents Active Against RNA Viruses.

DTIC Science & Technology

1986-09-15

solvent such as nitromethane gave a nucleoside product , identified as 1-(2,3,5-tri-O-benzoyl-o--D-ribofuranosyl)-l,2,4- triazol-3(2H)-one (20, BL-00307...at 2220 cm . Treatment of 26 with NH4OH/H 202 solution, and purification of the reaction product by chromatography on silica gel furnished 1-(2-deoxy...30) in 74% yield. Treatment of 30 with NH4OH/H202 solution, and purification of the reaction product by chroma- tography on silica gel furnished 1

Molecular moment similarity between several nucleoside analogs of thymidine and thymidine. sil@watson.ibm.com.

PubMed

Silverman, B D; Pitman, M C; Platt, D E

1999-06-01

Molecular moment descriptors of the shape and charge distributions of twenty five nucleoside structures have been examined. The structures include thymidine as well as the difluorotoluene nucleoside analog which has been found to pair efficiently with adenine by polymerase catalysis. The remaining twenty three structures have been chosen to be as structurally similar to thymidine and to the difluorotoluene nucleoside analog as possible. The moment descriptors which include a description of the relationship of molecular charge to shape show the difluorotoluene nucleoside to be one of the most proximate molecules to thymidine in the space of the molecular moments. The calculations, therefore, suggest that polymerase specificity might be not only a consequence of molecular steric features alone but also of the molecular electrostatic environment and its registration with molecular shape.

Regioselective Synthesis of Pyrazolo[3,4-D]Pyrimidine Based Carbocyclic Nucleosides as Possible Antiviral Agent.

PubMed

Kasula, Mohan; Samunuri, Ramakrishnamraju; Chakravarty, Harapriya; Bal, Chandralata; Baba, Masanori; Jha, Ashok Kumar; Sharon, Ashoke

2016-01-01

Carbocyclic nucleosides are considered as nucleoside mimetic having high therapeutic potentials, however diverse exploration is still limited due to their synthetic difficulties. The major challenges are associated with the preparation of new base and carbocyclic sugar key intermediates. The modified base may provide conformational advantage to achieve better nucleoside mimetics and may also help in increasing the drug-like properties. In this manuscript, we report the use of acetamidine hydrochloride to synthesize 6-methyl-4-amino-pyrazolo[3,4-d]pyrimidine base and regioselective synthesis of six new carbocyclic nucleosides (6a-f) for antiviral evaluation. Theoretical investigations were carried out on the basis of thermodynamic and kinetic stability using MM based energy optimizations and QM based transition state search for the significant regioselectivity, which was further experimentally analyzed by NOE and UV spectroscopy.

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

2009-01-02

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.

Scaffold Repurposing of Nucleosides (Adenosine Receptor Agonists): Enhanced Activity at the Human Dopamine and Norepinephrine Sodium Symporters.

PubMed

Tosh, Dilip K; Janowsky, Aaron; Eshleman, Amy J; Warnick, Eugene; Gao, Zhan-Guo; Chen, Zhoumou; Gizewski, Elizabeth; Auchampach, John A; Salvemini, Daniela; Jacobson, Kenneth A

2017-04-13

We have repurposed (N)-methanocarba adenosine derivatives (A 3 adenosine receptor (AR) agonists) to enhance radioligand binding allosterically at the human dopamine (DA) transporter (DAT) and inhibit DA uptake. We extended the structure-activity relationship of this series with small N 6 -alkyl substitution, 5'-esters, deaza modifications of adenine, and ribose restored in place of methanocarba. C2-(5-Halothien-2-yl)-ethynyl 5'-methyl 9 (MRS7292) and 5'-ethyl 10 (MRS7232) esters enhanced binding at DAT (EC 50 ∼ 35 nM) and at the norepinephrine transporter (NET). 9 and 10 were selective for DAT compared to A 3 AR in the mouse but not in humans. At DAT, the binding of two structurally dissimilar radioligands was enhanced; NET binding of only one radioligand was enhanced; SERT radioligand binding was minimally affected. 10 was more potent than cocaine at inhibiting DA uptake (IC 50 = 107 nM). Ribose analogues were weaker in DAT interaction than the corresponding bicyclics. Thus, we enhanced the neurotransmitter transporter activity of rigid nucleosides while reducing A 3 AR affinity.

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

2016-01-04

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.

Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells.

PubMed

Rehan, Shahid; Jaakola, Veli-Pekka

2015-10-01

Human equilibrative nucleoside transporter-1 (hENT1) is the major plasma membrane transporter involved in transportation of natural nucleosides as well as nucleoside analog drugs, used in anti-cancer and anti-viral therapies. Despite extensive biochemical and pharmacological studies, little is known about the structure-function relationship of this protein. The major obstacles to purification include a low endogenous expression level, the lack of an efficient expression and purification protocol, and the hydrophobic nature of the protein. Here, we report protein expression, purification and functional characterization of hENT1 from Sf9 insect cells. hENT1 expressed by Sf9 cells is functionally active as demonstrated by saturation binding with a Kd of 1.2±0.2nM and Bmax of 110±5pmol/mg for [(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR). We also demonstrate purification of hENT1 using FLAG antibody affinity resin in lauryl maltose neopentyl glycol detergent with a Kd of 4.3±0.7nM. The yield of hENT1 from Sf9 cells was ∼0.5mg active transporter per liter of culture. The purified protein is functionally active, stable, homogenous and appropriate for further biophysical and structural studies. Copyright © 2015 Elsevier Inc. All rights reserved.

Cloning and functional expression of a gene encoding a P1 type nucleoside transporter from Trypanosoma brucei.

PubMed

Sanchez, M A; Ullman, B; Landfear, S M; Carter, N S

1999-10-15

Nucleoside transporters are likely to play a central role in the biochemistry of the parasite Trypanosoma brucei, since these protozoa are unable to synthesize purines de novo and must salvage them from their hosts. Furthermore, nucleoside transporters have been implicated in the uptake of antiparasitic and experimental drugs in these and other parasites. We have cloned the gene for a T. brucei nucleoside transporter, TbNT2, and shown that this permease is related in sequence to mammalian equilibrative nucleoside transporters. Expression of the TbNT2 gene in Xenopus oocytes reveals that the permease transports adenosine, inosine, and guanosine and hence has the substrate specificity of the P1 type nucleoside transporters that have been previously characterized by uptake assays in intact parasites. TbNT2 mRNA is expressed in bloodstream form (mammalian host stage) parasites but not in procyclic form (insect stage) parasites, indicating that the gene is developmentally regulated during the parasite life cycle. Genomic Southern blots suggest that there are multiple genes related in sequence to TbNT2, implying the existence of a family of nucleoside transporter genes in these parasites.

An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors

PubMed Central

Wendelsdorf, Katherine V.; Song, Zhuo; Cao, Yang; Samuels, David C.

2009-01-01

Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-γ hypothesis states that this toxicity stems from the analogs' inhibition of the mitochondrial DNA polymerase (polymerase-γ) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-γ with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication. PMID:19132079

Chemical and Conformational Diversity of Modified Nucleosides Affects tRNA Structure and Function.

PubMed

Väre, Ville Y P; Eruysal, Emily R; Narendran, Amithi; Sarachan, Kathryn L; Agris, Paul F

2017-03-16

RNAs are central to all gene expression through the control of protein synthesis. Four major nucleosides, adenosine, guanosine, cytidine and uridine, compose RNAs and provide sequence variation, but are limited in contributions to structural variation as well as distinct chemical properties. The ability of RNAs to play multiple roles in cellular metabolism is made possible by extensive variation in length, conformational dynamics, and the over 100 post-transcriptional modifications. There are several reviews of the biochemical pathways leading to RNA modification, but the physicochemical nature of modified nucleosides and how they facilitate RNA function is of keen interest, particularly with regard to the contributions of modified nucleosides. Transfer RNAs (tRNAs) are the most extensively modified RNAs. The diversity of modifications provide versatility to the chemical and structural environments. The added chemistry, conformation and dynamics of modified nucleosides occurring at the termini of stems in tRNA's cloverleaf secondary structure affect the global three-dimensional conformation, produce unique recognition determinants for macromolecules to recognize tRNAs, and affect the accurate and efficient decoding ability of tRNAs. This review will discuss the impact of specific chemical moieties on the structure, stability, electrochemical properties, and function of tRNAs.

Metabolic engineering of an industrial polyoxin producer for the targeted overproduction of designer nucleoside antibiotics.

PubMed

Qi, Jianzhao; Liu, Jin; Wan, Dan; Cai, You-Sheng; Wang, Yinghu; Li, Shunying; Wu, Pan; Feng, Xuan; Qiu, Guofu; Yang, Sheng-Ping; Chen, Wenqing; Deng, Zixin

2015-09-01

Polyoxin and nikkomycin are naturally occurring peptidyl nucleoside antibiotics with potent antifungal bioactivity. Both exhibit similar structural features, having a nucleoside skeleton and one or two peptidyl moieties. Combining the refactoring of the polyoxin producer Streptomyces aureochromogenes with import of the hydroxypyridylhomothreonine pathway of nikkomycin allows the targeted production of three designer nucleoside antibiotics designated as nikkoxin E, F, and G. These structures were determined by NMR and/or high resolution mass spectrometry. Remarkably, the introduction of an extra copy of the nikS gene encoding an ATP-dependent ligase significantly enhanced the production of the designer antibiotics. Moreover, all three nikkoxins displayed improved bioactivity against several pathogenic fungi as compared with the naturally-occurring antibiotics. These data provide a feasible model for high efficiency generation of nucleoside antibiotics related to polyoxins and nikkomycins in a polyoxin cell factory via synthetic biology strategy. © 2015 Wiley Periodicals, Inc.

Profiling of modified nucleosides from ribonucleic acid digestion by supercritical fluid chromatography coupled to high resolution mass spectrometry.

PubMed

Laboureur, Laurent; Guérineau, Vincent; Auxilien, Sylvie; Yoshizawa, Satoko; Touboul, David

2018-02-16

A method based on supercritical fluid chromatography coupled to high resolution mass spectrometry for the profiling of canonical and modified nucleosides was optimized, and compared to classical reverse-phase liquid chromatography in terms of separation, number of detected modified nucleosides and sensitivity. Limits of detection and quantification were measured using statistical method and quantifications of twelve nucleosides of a tRNA digest from E. coli are in good agreement with previously reported data. Results highlight the complementarity of both separation techniques to cover the largest view of nucleoside modifications for forthcoming epigenetic studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Fission yeast RNA triphosphatase reads an Spt5 CTD code

DOE PAGES

Doamekpor, Selom K.; Schwer, Beate; Sanchez, Ana M.; ...

2014-11-20

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

Fission yeast RNA triphosphatase reads an Spt5 CTD code

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

Doamekpor, Selom K.; Schwer, Beate; Sanchez, Ana M.

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

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

DOE PAGES

Wang, Jun; Bonnesen, Peter V; Rangel, E.; ...

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

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

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

Wang, Jun; Bonnesen, Peter V; Rangel, E.

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

Quantitative Detection of Nucleoside Analogues by Multi-enzyme Biosensors using Time-Resolved Kinetic Measurements.

PubMed

Muthu, Pravin; Lutz, Stefan

2016-04-05

Fast, simple and cost-effective methods for detecting and quantifying pharmaceutical agents in patients are highly sought after to replace equipment and labor-intensive analytical procedures. The development of new diagnostic technology including portable detection devices also enables point-of-care by non-specialists in resource-limited environments. We have focused on the detection and dose monitoring of nucleoside analogues used in viral and cancer therapies. Using deoxyribonucleoside kinases (dNKs) as biosensors, our chemometric model compares observed time-resolved kinetics of unknown analytes to known substrate interactions across multiple enzymes. The resulting dataset can simultaneously identify and quantify multiple nucleosides and nucleoside analogues in complex sample mixtures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural and thermodynamic analysis of modified nucleosides in self-assembled DNA cross-tiles.

PubMed

Hakker, Lauren; Marchi, Alexandria N; Harris, Kimberly A; LaBean, Thomas H; Agris, Paul F

2014-01-01

DNA Holliday junctions are important natural strand-exchange structures that form during homologous recombination. Immobile four-arm junctions, analogs to Holliday junctions, have been designed to self-assemble into cross-tile structures by maximizing Watson-Crick base pairing and fixed crossover points. The cross-tiles, self-assembled from base pair recognition between designed single-stranded DNAs, form higher order lattice structures through cohesion of self-associating sticky ends. These cross-tiles have 16 unpaired nucleosides in the central loop at the junction of the four duplex stems. The importance of the centralized unpaired nucleosides to the structure's thermodynamic stability and self-assembly is unknown. Cross-tile DNA nanostructures were designed and constructed from nine single-stranded DNAs with four shell strands, four arms, and a central loop containing 16 unpaired bases. The 16 unpaired bases were either 2'-deoxyribothymidines, 2'-O-methylribouridines, or abasic 1',2'-dideoxyribonucleosides. Thermodynamic profiles and structural base-stacking contributions were assessed using UV absorption spectroscopy during thermal denaturation and circular dichroism spectroscopy, respectively, and the resulting structures were observed by atomic force microscopy. There were surprisingly significant changes in the thermodynamic and structural properties of lattice formation as a result of altering only the 16 unpaired, centralized nucleosides. The 16 unpaired 2'-O-methyluridines were stabilizing and produced uniform tubular structures. In contrast, the abasic nucleosides were destabilizing producing a mixture of structures. These results strongly indicate the importance of a small number of centrally located unpaired nucleosides within the structures. Since minor modifications lead to palpable changes in lattice formation, DNA cross-tiles present an easily manipulated structure convenient for applications in biomedical and biosensing devices.

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

PubMed

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

2016-06-01

A series of new silicon(IV) phthalocyanines (SiPcs) di-substituted axially with different nucleoside moieties have been synthesized and evaluated for their singlet oxygen quantum yields (ΦΔ) and in vitro photodynamic activities. The adenosine-substituted SiPc shows a lower photosensitizing efficiency (ΦΔ=0.35) than the uridine- and cytidine-substituted analogs (ΦΔ=0.42-0.44), while the guanosine-substituted SiPc exhibits a weakest singlet oxygen generation efficiency with a ΦΔ value down to 0.03. On the other hand, replacing axial adenosines with chloro-modified adenosines and purines can result in the increase of photogenerating singlet oxygen efficiencies of SiPcs. The formed SiPcs 1 and 2, which contain monochloro-modified adenosines and dichloro-modified purines respectively, appear as efficient photosensitizers with ΦΔ of 0.42-0.44. Both compounds 1 and 2 present high photocytotoxicities against HepG2 and BGC823 cancer cells with IC50 values ranging from 9nM to 33nM. The photocytotoxicities of these two compounds are remarkably higher than the well-known anticancer photosensitizer, chlorin e6 (IC50=752nM against HepG2 cells) in the same condition. As revealed by confocal microscopy, for both cell lines, compound 1 can essentially bind to mitochondria, while compound 2 is just partially localized in mitochondria. In addition, the two compounds induce cell death of HepG2 cells likely through apoptosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Formation of nucleoside 5'-polyphosphates from nucleotides and trimetaphosphate

NASA Technical Reports Server (NTRS)

Lohrmann, R.

1975-01-01

Nucleoside 5'-polyphosphates (N5PP) formed when solutions of nucleoside 5'-phosphates (N5P) and trimetaphosphate (TMP) are dessicated at room temperature are studied by paper chromatography, electrophoresis, and metal catalytic reactions. Divalent Mg ion exhibited superior catalytic function to other divalent metal ions in the reaction. Major reaction products are indicated. The importance of the N5PP series, TMP, and N5-triphosphate as substrates of RNA and DNA synthesis, and under postulated prebiotic conditions likely to obtain during prebiological ages of the earth, is emphasized and discussed. Alternate drying and wetting, evaporation from a prebiotic puddle, concentration of solubles in the remaining liquid phase, metal catalysis, and the role of these substances in the formation of amino acids and long-chain polyphosphates are considered.

Engineering the expression level of cytosolic nucleoside diphosphate kinase in transgenic Solanum tuberosum roots alters growth, respiration and carbon metabolism.

PubMed

Dorion, Sonia; Clendenning, Audrey; Rivoal, Jean

2017-03-01

Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme that catalyzes the transfer of the γ-phosphate from a donor nucleoside triphosphate to an acceptor nucleoside diphosphate. In this study we used a targeted metabolomic approach and measurement of physiological parameters to report the effects of the genetic manipulation of cytosolic NDPK (NDPK1) expression on physiology and carbon metabolism in potato (Solanum tuberosum) roots. Sense and antisense NDPK1 constructs were introduced in potato using Agrobacterium rhizogenes to generate a population of root clones displaying a 40-fold difference in NDPK activity. Root growth, O 2 uptake, flux of carbon between sucrose and CO 2 , levels of reactive oxygen species and some tricarboxylic acid cycle intermediates were positively correlated with levels of NDPK1 expression. In addition, NDPK1 levels positively affected UDP-glucose and cellulose contents. The activation state of ADP-glucose pyrophosphorylase, a key enzyme in starch synthesis, was higher in antisense roots than in roots overexpressing NDPK1. Further analyses demonstrated that ADP-glucose pyrophosphorylase was more oxidized, and therefore less active, in sense clones than antisense clones. Consequently, antisense NDPK1 roots accumulated more starch and the starch to cellulose ratio was negatively affected by the level of NDPK1. These data support the idea that modulation of NDPK1 affects the distribution of carbon between starch and cellulose biosynthetic pathways. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

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

PubMed Central

Addepalli, Balasubrahmanym; Lesner, Nicholas P.; Limbach, Patrick A.

2015-01-01

A codon-optimized recombinant ribonuclease, MC1 is characterized for its uridine-specific cleavage ability to map nucleoside modifications in RNA. The published MC1 amino acid sequence, as noted in a previous study, was used as a template to construct a synthetic gene with a natural codon bias favoring expression in Escherichia coli. Following optimization of various expression conditions, the active recombinant ribonuclease was successfully purified as a C-terminal His-tag fusion protein from E. coli [Rosetta 2(DE3)] cells. The isolated protein was tested for its ribonuclease activity against oligoribonucleotides and commercially available E. coli tRNATyr I. Analysis of MC1 digestion products by ion-pairing reverse phase liquid-chromatography coupled with mass spectrometry (IP-RP-LC-MS) revealed enzymatic cleavage of RNA at the 5′-termini of uridine and pseudouridine, but cleavage was absent if the uridine was chemically modified or preceded by a nucleoside with a bulky modification. Furthermore, the utility of this enzyme to generate complementary digestion products to other common endonucleases, such as RNase T1, which enables the unambiguous mapping of modified residues in RNA is demonstrated. PMID:26221047

Supplementation of Nucleosides During Selection can Reduce Sequence Variant Levels in CHO Cells Using GS/MSX Selection System.

PubMed

Tang, Danming; Lam, Cynthia; Louie, Salina; Hoi, Kam Hon; Shaw, David; Yim, Mandy; Snedecor, Brad; Misaghi, Shahram

2018-01-01

In the process of generating stable monoclonal antibody (mAb) producing cell lines, reagents such as methotrexate (MTX) or methionine sulfoximine (MSX) are often used. However, using such selection reagent(s) increases the possibility of having higher occurrence of sequence variants in the expressed antibody molecules due to the effects of MTX or MSX on de novo nucleotide synthesis. Since MSX inhibits glutamine synthase (GS) and results in both amino acid and nucleoside starvation, it is questioned whether supplementing nucleosides into the media could lower sequence variant levels without affecting titer. The results show that the supplementation of nucleosides to the media during MSX selection decreased genomic DNA mutagenesis rates in the selected cells, probably by reducing nucleotide mis-incorporation into the DNA. Furthermore, addition of nucleosides enhance clone recovery post selection and does not affect antibody expression. It is further observed that nucleoside supplements lowered DNA mutagenesis rates only at the initial stage of the clone selection and do not have any effect on DNA mutagenesis rates after stable cell lines are established. Therefore, the data suggests that addition of nucleosides during early stages of MSX selection can lower sequence variant levels without affecting titer or clone stability in antibody expression. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interspecies conservation of retinal guanosine 5'-triphosphatase. Characterization by photoaffinity labelling and tryptic-peptide mapping.

PubMed Central

McMurray, M M; Hansen, J S; Haley, B E; Takemoto, D J; Takemoto, L J

1985-01-01

Light-activated hydrolysis of cyclic GMP is achieved through the photoexcitation of rhodopsin, a process which then triggers the replacement of GDP for GTP by a retinal guanosine 5'-triphosphatase referred to as 'transducin'. The transducin-GTP complex then switches on the phosphodiesterase [Fung, Hurley & Stryer (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 152-156]. The bovine transducin consists of an alpha-subunit (39000 Mr), which is a GTP-binding component, together with a beta-(37000 Mr) and a gamma-subunit (10000 Mr). We have purified retinal transducin from cow, pig, chick and frog. The enzyme specific activities and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic profiles indicate that this enzyme is similar in all species except the frog. Whereas the bovine, pig and chick transducins consist of major 37000- and 39000-Mr components, that of the frog consists of a single 75000-Mr component. Labelling of the GTP-binding components with the photoaffinity label 8-azidoguanosine [gamma-32P]triphosphate demonstrated that the 37000-Mr components of the cow, pig and chick and the 75000-Mr component of the frog were major GTP-binding components. In addition, peptide maps of radioiodinated tryptic peptides indicate that the frog 75000-Mr protein is highly related to the pig transducin. These results demonstrate evolutionary conservation of retinal transducin and the presence of a higher-Mr, but nonetheless highly conserved form, of transducin in the frog. The relationship of this component to the recently reported rod-outer-segment inhibitor protein [Yamazaki, Stein, Chernoff & Bitensky (1983) J. Biol. Chem. 258, 8188-8194] is discussed. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2983663

An ATP-dependent ligase with substrate flexibility involved in assembly of the peptidyl nucleoside antibiotic polyoxin

USDA-ARS?s Scientific Manuscript database

Polyoxin (POL) is an unusual nucleoside antibiotic, in which peptidyl moiety and nucleoside skeleton are linked by an amide bond. However, their biosynthesis remains poorly understood. Here, we report the deciphering of PolG as an ATP-dependent ligase responsible for the assembly of POL. A polG muta...

In search of Flavivirus inhibitors part 2: tritylated, diphenylmethylated and other alkylated nucleoside analogues.

PubMed

Saudi, Milind; Zmurko, Joanna; Kaptein, Suzanne; Rozenski, Jef; Neyts, Johan; Van Aerschot, Arthur

2014-04-09

Several flaviviruses, such as the yellow fever virus and the dengue virus cause severe and potentially lethal infection in man. Following up on our initial hit 3',5'-bistritylated uridine 1, a series of alkylated nucleoside analogues were synthesized and evaluated for their in vitro antiviral activities against dengue fever virus and yellow fever virus. Hereto, alkyl and aryl groups were attached at various positions of the sugar ring combined with subtle variation of the heterocyclic base. Among the new series of derivatives, 3',5'-di-O-trityl-5-fluoro-2'-deoxyuridine (39) was the most efficient in this series and inhibited both yellow fever virus and dengue virus replication with a 50% effective concentration (EC₅₀) of ∼1 μg/mL without considerable cytotoxicity. The other fluorinated derivatives proved more toxic. Almost all diphenylmethylated pyrimidine nucleosides with 3',5'-di-O-benzhydryl-2'-deoxyuridine (50) as the example were endowed with strong cytotoxic effects down to 1 μg/mL. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Engineering of an industrial polyoxin producer for the rational production of hybrid peptidyl nucleoside antibiotics.

PubMed

Zhai, Lipeng; Lin, Shuangjun; Qu, Dongjing; Hong, Xuechuan; Bai, Linquan; Chen, Wenqing; Deng, Zixin

2012-07-01

Polyoxins and nikkomycins are potent antifungal peptidyl nucleoside antibiotics, which inhibit fungal cell wall biosynthesis. They consist of a nucleoside core and one or two independent peptidyl moieties attached to the core at different sites. Making mutations and introducing heterologous genes into an industrial Streptomyces aureochromogenes polyoxin producer, resulted in the production of four polyoxin-nikkomycin hybrid antibiotics designated as polyoxin N and nikkoxin B-D, whose structures were confirmed using high resolution MS and NMR. Two of the hybrid antibiotics, polyoxin N and nikkoxin D, were significantly more potent against some human or plant fungal pathogens than their parents. The data provides an example for rational generation of novel peptidyl nucleoside antibiotics in an industrial producer. Copyright © 2012 Elsevier Inc. All rights reserved.

Recent advances in the development of next generation non-nucleoside reverse transcriptase inhibitors.

PubMed

Tarby, Christine M

2004-01-01

Since their discovery, non-nucleoside reverse transcriptase inhibitors (NNRTIs) have become one of the cornerstones of highly active anti-retroviral therapy (HAART). Currently, three NNRTI agents, efavirenz, nevirapine and delavirdine are commercially available. Efavirenz and nevirapine, used in combination with nucleoside reverse transcriptase inhibitors (NRTIs), provide durable regimens with efficacy comparable to protease inhibitor (PI) containing therapies. When virological failure occurs following treatment with an NNRTI, the resistance mutations can confer reduced sensitivity to the entire agent class. Therefore, the strategy for the development of next generation NNRTIs has been to focus on compounds which have improved potencies against the clinically relevant viral mutants. Agents with improved virological profiles and which maintain the ease of administration and favorable safety profiles of the current agents should find use in anti-retroviral naïve patients as well as in components of salvage regimens in the anti-retroviral experienced patient. This review summarizes the recent developments with compounds in clinical trials as of January 2002 as well as to summarize information on new agents appearing in the primary and patent literature between January 2001 and December 2002.

Synthesis of a new family of acyclic nucleoside phosphonates, analogues of TPases transition states.

PubMed

Dayde, Bénédicte; Benzaria, Samira; Pierra, Claire; Gosselin, Gilles; Surleraux, Dominique; Volle, Jean-Noël; Pirat, Jean-Luc; Virieux, David

2012-05-07

A 6-step procedure was developed for the synthesis of a new family of acyclic nucleoside phosphonates (ANPs), "PHEEPA" [(2-pyrimidinyl-2-(2-hydroxyethoxy)ethyl)phosphonic acids] in overall yields ranging from 4.5% to 32%. These compounds, which possess on one side a hydroxy function and on the other side a phosphonate group, can be considered either as potential antiviral agents or as transition state analogues of nucleoside phosphorylases such as thymidine phosphorylase.

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.

pH-Cleavable Nucleoside Lipids: A New Paradigm for Controlling the Stability of Lipid-Based Delivery Systems.

PubMed

Oumzil, Khalid; Benizri, Sébastien; Tonelli, Giovanni; Staedel, Cathy; Appavoo, Ananda; Chaffanet, Max; Navailles, Laurence; Barthélémy, Philippe

2015-11-01

Lipid-based delivery systems are an established technology with considerable clinical acceptance and several applications in human. Herein, we report the design, synthesis and evaluation of novel orthoester nucleoside lipids (ONLs) for the modulation of liposome stability. The ONLs contain head groups with 3'-orthoester nucleoside derivatives featuring positive or negative charges. The insertion of the orthoester function in the NL structures allows the formation of pH-sensitive liposomes. ONL-based liposomes can be hydrolyzed to provide nontoxic products, including nucleoside derivatives and hexadecanol. To allow the release to be tunable at different hydrolysis rates, the charge of the polar head structure is modulated, and the head group can be released at a biologically relevant pH. Crucially, when ONLs are mixed with natural phosphocholine lipids (PC), the resultant liposome evolves toward the formation of a hexadecanol/PC lamellar system. Biological evaluation shows that stable nucleic acid lipid particles (SNALPs) formulated with ONLs and siRNAs can effectively enter into tumor cells and release their nucleic acid payload in response to an intracellular acidic environment. This results in a much higher antitumor activity than conventional SNALPs. The ability to use pH-cleavable nucleolipids to control the stability of lipid-based delivery systems represents a promising approach for the intracellular delivery of drug cargos. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses.

PubMed

Pardi, Norbert; Hogan, Michael J; Naradikian, Martin S; Parkhouse, Kaela; Cain, Derek W; Jones, Letitia; Moody, M Anthony; Verkerke, Hans P; Myles, Arpita; Willis, Elinor; LaBranche, Celia C; Montefiori, David C; Lobby, Jenna L; Saunders, Kevin O; Liao, Hua-Xin; Korber, Bette T; Sutherland, Laura L; Scearce, Richard M; Hraber, Peter T; Tombácz, István; Muramatsu, Hiromi; Ni, Houping; Balikov, Daniel A; Li, Charles; Mui, Barbara L; Tam, Ying K; Krammer, Florian; Karikó, Katalin; Polacino, Patricia; Eisenlohr, Laurence C; Madden, Thomas D; Hope, Michael J; Lewis, Mark G; Lee, Kelly K; Hu, Shiu-Lok; Hensley, Scott E; Cancro, Michael P; Haynes, Barton F; Weissman, Drew

2018-06-04

T follicular helper (Tfh) cells are required to develop germinal center (GC) responses and drive immunoglobulin class switch, affinity maturation, and long-term B cell memory. In this study, we characterize a recently developed vaccine platform, nucleoside-modified, purified mRNA encapsulated in lipid nanoparticles (mRNA-LNPs), that induces high levels of Tfh and GC B cells. Intradermal vaccination with nucleoside-modified mRNA-LNPs encoding various viral surface antigens elicited polyfunctional, antigen-specific, CD4 + T cell responses and potent neutralizing antibody responses in mice and nonhuman primates. Importantly, the strong antigen-specific Tfh cell response and high numbers of GC B cells and plasma cells were associated with long-lived and high-affinity neutralizing antibodies and durable protection. Comparative studies demonstrated that nucleoside-modified mRNA-LNP vaccines outperformed adjuvanted protein and inactivated virus vaccines and pathogen infection. The incorporation of noninflammatory, modified nucleosides in the mRNA is required for the production of large amounts of antigen and for robust immune responses. © 2018 Pardi et al.

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

2016-08-07

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.

Assessment of nucleosides as putative tumor biomarkers in prostate cancer screening by CE-UV.

PubMed

Buzatto, Adriana Zardini; de Oliveira Silva, Mariana; Poppi, Ronei Jesus; Simionato, Ana Valéria Colnaghi

2017-05-01

Cancer is responsible for millions of deaths worldwide, but most base diseases may be cured if detected early. Screening tests may be used to identify early-stage malignant neoplasms. However, the major screening tool for prostate cancer, the prostate-specific antigen test, has unsuitable sensitivity. Since cancer cells may affect the pattern of consumption and excretion of nucleosides, such biomolecules are putative biomarkers that can be used for diagnosis and treatment evaluation. Using a previously validated method for the analysis of nucleosides in blood serum by capillary electrophoresis with UV-vis spectroscopy detection, we investigated 60 samples from healthy individuals and 42 samples from prostate cancer patients. The concentrations of nucleosides in both groups were compared and a multivariate partial least squares-discriminant analysis classification model was optimized for prediction of prostate cancer. The validation of the model with an independent sample set resulted in the correct classification of 82.4% of the samples, with sensitivity of 90.5% and specificity of 76.7%. A significant downregulation of 5-methyluridine and inosine was observed, which can be indicative of the carcinogenic process. Therefore, such analytes are potential candidates for prostate cancer screening. Graphical Abstract Separation of the studied nucleosides and the internal standard 8-Bromoguanosine by CE-UV (a); classification of the external validation samples (30 from healthy volunteers and 21 from prostate cancer patients) by the developed Partial Least Square - Discriminant Analysis (PLS-DA) model with accuracy of 82.4% (b); Receiver Operating Characteristics (ROC) curve (c); and Variable Importance in the Projection (VIP) values for the studied nucleosides (d). A significant down-regulation of 5- methyluridine (5mU) and inosine (I) was observed, which can be indicative of the presence of prostate tumors.

Development of a purification procedure for the isolation of nucleosides from urine prior to mass spectrometric analysis.

PubMed

Dudley, E; El-Shakawi, S; Games, D E; Newton, R P

2000-03-01

A chromatographic separation of nucleosides from urine has been developed in order to facilitate their mass spectrometric analysis for clinical diagnosis. A number of chromatographic resins were studied in order to develop an effective and efficient purification procedure. The optimized sequential protocol comprises a centrifugation, acidification and neutralization step, followed by application of an affinity chromatographic column and finally further separation on an acidic cation exchange column and a basic anion exchanger. This scheme shows effective clean-up of a standard radiolabelled nucleoside with a recovery of 92.5%, and recovery of nucleosides added to urine samples before extraction showed recoveries of 72-82%.

Telbivudine, a nucleoside analog inhibitor of HBV polymerase, has a different in vitro cross-resistance profile than the nucleotide analog inhibitors adefovir and tenofovir.

PubMed

Seifer, Maria; Patty, April; Serra, Ilaria; Li, Bin; Standring, David N

2009-02-01

Telbivudine, a nucleoside analog inhibitor of the viral polymerase of hepatitis B virus (HBV), has been approved for the treatment of chronic HBV infection, along with the nucleoside inhibitors lamivudine and entecavir, and the nucleotide inhibitors adefovir and tenofovir. The resistance profiles of these agents were investigated via drug treatment of HepG2 cells stably transfected with wild-type or mutant HBV genomes bearing known resistance mutations. Telbivudine was not active against HBV strains bearing lamivudine mutations L180M/M204V/I but remained active against the M204V single mutant in vitro, potentially explaining the difference in resistance profiles between telbivudine and lamivudine. Against HBV genomes with known telbivudine-resistance mutations, M204I and L80I/M204I, telbivudine, lamivudine and entecavir lost 353- to >1000-fold activity whereas adefovir and tenofovir exhibited no more than 3-5-fold change. Conversely, against HBV cell lines expressing adefovir resistance mutations N236T and A181V, or the A194T mutant associated with resistance to tenofovir, telbivudine remained active as shown by respective fold-changes of 0.5 (N236T) and 1.0 (A181V and A194T). These in vitro results indicate that nucleoside and nucleotide drugs have different cross-resistance profiles. The addition of telbivudine to ongoing adefovir therapy could provide effective antiviral therapy to patients who develop adefovir resistance.

Improved treatment of nucleosides and nucleotides in the OPLS-AA force field

NASA Astrophysics Data System (ADS)

Robertson, Michael J.; Tirado-Rives, Julian; Jorgensen, William L.

2017-09-01

DFT calculations have been used to develop improved descriptions of the torsional energetics for nucleosides and nucleotides in the OPLS-AA force field. Scans of nucleotide dihedral angles (γ, χ, and β) and methyl phosphates provided the bases for the new torsional parameters. In addition, the angle-bending parameters of phosphodiesters and ribose were updated, and adjustments were made to existing carbohydrate torsions to better capture the sugar puckering landscape of ribose. MD simulations of nucleosides with the new parameters demonstrate a significant improvement in the ribose sugar puckering and χ angle distributions. Additionally, energy-minimization of protein-nucleotide crystal structures with the new parameters produced accurate poses.

Chromophoric Nucleoside Analogues: Synthesis and Characterization of 6-Aminouracil-Based Nucleodyes.

PubMed

Freeman, Noam S; Moore, Curtis E; Wilhelmsson, L Marcus; Tor, Yitzhak

2016-06-03

Nucleodyes, visibly colored chromophoric nucleoside analogues, are reported. Design criteria are outlined and the syntheses of cytidine and uridine azo dye analogues derived from 6-aminouracil are described. Structural analysis shows that the nucleodyes are sound structural analogues of their native nucleoside counterparts, and photophysical studies demonstrate that the nucleodyes are sensitive to microenvironmental changes. Quantum chemical calculations are presented as a valuable complementary tool for the design of strongly absorbing nucleodyes, which overlap with the emission of known fluorophores. Förster critical distance (R0) calculations determine that the nucleodyes make good FRET pairs with both 2-aminopurine (2AP) and pyrrolocytosine (PyC). Additionally, unique tautomerization features exhibited by 5-(4-nitrophenylazo)-6-oxocytidine (8) are visualized by an extraordinary crystal structure.

Parallel Prebiotic Origin of Canonical and Non-Canonical Purine Nucleosides

NASA Astrophysics Data System (ADS)

Becker, S.; Carell, T.

2017-07-01

RNA of all living organisms is highly modified. It is unclear if these non-canonical bases are ancestors of an early Earth or biological inventions. We investigated a prebiotic pathway that leads to canonical and non-canonical purine nucleosides.

Characterization of nucleosides and nucleobases in fruits of Ziziphus jujuba by UPLC-DAD-MS.

PubMed

Guo, Sheng; Duan, Jin-Ao; Tang, Yu-Ping; Zhu, Zhen-Hua; Qian, Ye-Fei; Yang, Nian-Yun; Shang, Er-Xin; Qian, Da-Wei

2010-10-13

The fruit of Ziziphus jujuba , named dazao in Chinese, has been utilized as food as well as crude drugs in China for thousands of years. To explore the profiles of the nucleosides and nucleobases in this fruit, an ultraperformance liquid chromatograph coupled with a photodiode array detector and electrospray ionization-mass spectrometer method (UPLC-DAD-MS) has been established and validated in this paper. The validated method was successfully applied for the simultaneous characterization and quantitation of 9 nucleosides and nucleobases in 49 dazao samples, which comprised 43 cultivars from 26 cultivation regions. Furthermore, principal component analysis (PCA) was performed to classify the samples on the basis of the contents of the nine analyzed compounds. The results showed that almost all of these dazao samples were rich in nucleosides and nucleobases, although their contents were obviously various, and the proposed method could serve as a prerequisite for quality control of jujube products.

Pd-catalyzed versus uncatalyzed, PhI(OAc)2-mediated cyclization reactions of N6-([1,1'-biaryl]-2-yl)adenine nucleosides.

PubMed

Satishkumar, Sakilam; Poudapally, Suresh; Vuram, Prasanna K; Gurram, Venkateshwarlu; Pottabathini, Narender; Sebastian, Dellamol; Yang, Lijia; Pradhan, Padmanava; Lakshman, Mahesh K

2017-11-09

In this work we have assessed reactions of N 6 -([1,1'-biaryl]-2-yl)adenine nucleosides with Pd(OAc) 2 and PhI(OAc) 2 , via a Pd II /Pd IV redox cycle. The substrates are readily obtained by Pd/Xantphos-catalyzed reaction of adenine nucleosides with 2-bromo-1,1'-biaryls. In PhMe, the N 6 -biarylyl nucleosides gave C6-carbazolyl nucleoside analogues by C-N bond formation with the exocyclic N 6 nitrogen atom. In the solvent screening for the Pd-catalyzed reactions, an uncatalyzed process was found to be operational. It was observed that the carbazolyl products could also be obtained in the absence of a metal catalyst by reaction with PhI(OAc) 2 in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). Thus, under Pd catalysis and in HFIP, reactions proceed to provide carbazolyl nucleoside analogues, with some differences. If reactions of N 6 -biarylyl nucleoside substrates were conducted in MeCN, formation of aryl benzimidazopurinyl nucleoside derivatives was observed in many cases by C-N bond formation with the N 1 ring nitrogen atom of the purine (carbazole and benzimidazole isomers are readily separated by chromatography). Whereas Pd II /Pd IV redox is responsible for carbazole formation under the metal-catalyzed conditions, in HFIP and MeCN radical cations and/or nitrenium ions can be intermediates. An extensive set of radical inhibition experiments was conducted and the data are presented.

Characterization of nucleobases and nucleosides in the fruit of Alpinia oxyphylla collected from different cultivation regions.

PubMed

Song, Wenjing; Li, Yonghui; Wang, Jianguo; Li, Zeyou; Zhang, Junqing

2014-03-01

The fruit of Alpinia oxyphylla, known as Yizhi, Yakuchi and Ikji in Chinese, Japanese, and Korean, respectively, has been utilized as an important drug for the treatment of diarrhea, dyspepsia, spermatorrhea, kidney asthenia and abdominal pain in East Asian traditional medicine for thousands of years. Since the therapeutic effects of A. oxyphylla are attributed to multiple components and nucleobases and nucleosides exhibit various bioactivities, it is necessary to explore the chemical characterization of nucleobases and nucleosides in this herb. Herein, 10 common nucleobases and nucleosides, including cytidine, adenosine, thymidine, inosine, guanosine, 2'-deoxyinosine, guanine, adenine, cytosine, and hypoxanthine, were quantified simultaneously in the fruit of A. oxyphylla collected from different geographical regions. Changes in their contents were discussed, and hierarchical cluster analysis (HCA) was performed to classify all samples on the basis of the contents of the investigated analytes. The results indicated that there was a large variation in the contents of nucleobases and nucleosides among the herbs from different regions, and the samples collected from the same cultivation region were mostly classified in one cluster. The method can be used for comprehensive quality evaluation of A. oxyphylla. Copyright © 2013 John Wiley & Sons, Ltd.

Porphyromonas gingivalis attenuates ATP-mediated inflammasome activation and HMGB1 release through expression of a nucleoside-diphosphate kinase

PubMed Central

Johnson, Larry; Atanasova, Kalina R.; Bui, Phuong Q.; Lee, Jungnam; Hung, Shu-Chen; Yilmaz, Özlem; Ojcius, David M.

2015-01-01

Many intracellular pathogens evade the innate immune response in order to survive and proliferate within infected cells. We show that Porphyromonas gingivalis, an intracellular opportunistic pathogen, uses a nucleoside-diphosphate kinase (NDK) homolog to inhibit innate immune responses due to stimulation by extracellular ATP, which acts as a danger signal that binds to P2X7 receptors and induces activation of an inflammasome and caspase-1. Thus, infection of gingival epithelial cells (GECs) with wild-type P. gingivalis results in inhibition of ATP-induced caspase-1 activation. However, ndk-deficient P. gingivalis is less effective than wild-type P. gingivalis in reducing ATP-mediated caspase-1 activation and secretion of the proinflammatory cytokine, IL-1β, from infected GECs. Furthermore, P. gingivalis NDK modulates release of high-mobility group protein B1 (HMGB1), a pro-inflammatory danger signal, which remains associated with chromatin in healthy cells. Unexpectedly, infection with either wild-type or ndk-deficient P. gingivalis causes release of HMGB1 from the nucleus to the cytosol. But HMGB1 is released to the extracellular space when uninfected GECs are further stimulated with ATP, and there is more HMGB1 released from the cells when ATP-treated cells are infected with ndk-deficient mutant than wild-type P. gingivalis. Our results reveal that NDK plays a significant role in inhibiting P2X7-dependent inflammasome activation and HMGB1 release from infected GECs. PMID:25828169

Biocatalytic separation of N-7/N-9 guanine nucleosides.

PubMed

Singh, Sunil K; Sharma, Vivek K; Olsen, Carl E; Wengel, Jesper; Parmar, Virinder S; Prasad, Ashok K

2010-11-19

Vorbrüggen coupling of trimethylsilylated 2-N-isobutanoylguanine with peracetylated pentofuranose derivatives generally gives inseparable N-7/N-9 glycosyl mixtures. We have shown that the two isomers can be separated biocatalytically by Novozyme-435-mediated selective deacetylation of the 5'-O-acetyl group of peracetylated N-9 guanine nucleosides.

Interleukin28B and inosine triphosphatase help to personalize hepatitis C treatment.

PubMed

Tanaka, Yasuhito

2011-01-01

In the therapy using a combination of pegylated interferon-α and ribavirin (PEG-IFN/RBV) for chronic hepatitis C (CHC), approximately 50% of CHC patients infected with high viremia of HCV genotype 1 reached sustained viral response. The recent discovery revealed by a genome-wide association study technology provides the unexpected role of IL28B and inosine triphosphatase (ITPA) in HCV infection. The former single nucleotide polymorphisms (SNPs) around the IL28B gene could improve the diagnostics on the prediction of spontaneous clearance and the response to anti-HCV treatment, suggesting that these findings could be strong evidence to enhance the development of a novel therapeutic strategy and the basic study of IFN-λs. Interestingly, the discovered IL28B SNPs revealed the enigma that the viral clearance rate was dependent on ethnicity. The latter functional SNP in ITPA locus was the most significant SNP associated with RBV-induced anemia as well as IFN-induced thrombocytopenia. Note that severe Hb decline, which is mainly found in ITPA-CC patients, was inversely correlated with platelet reduction, contributing to an association between severe anemia and relative reactive increase in platelet count. These data may provide a valuable pharmacogenetic diagnostic tool for tailoring PEG-IFN/RBV dosing to minimize drug-induced adverse events and for further optimization of clinical anti-HCV chemotherapeutics. Copyright © 2011 S. Karger AG, Basel.

Synthesis and antiviral evaluation of novel 2,3-dihydroxypropyl nucleosides from 2- and 4-thiouracils.

PubMed

Abdel-Rahman, Adel A-H; El-Etrawy, Abd-Allah Sh; Abdel-Megied, Ahmed E-S; Zeid, Ibrahim F; El Ashry, El Sayed H

2008-12-01

Regioselective alkylation of 2-thiouracils 1a-c and 4-thiouracils 7a,b with 2,3-O-isopropylidene-2,3-dihydroxypropyl chloride (2) afforded 2-[[(2,2-Dimethyl-1,3-dioxolan-4-yl) methyl]thio]pyrimidin-4(1H)-ones 3a-c and 4-[[(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl]thio] pyrimidin-2(1H)-ones 8a,b, respectively. Further alkylation with 2 and/or 2,3-O-isopropylidine-1-O-(4-toluenesulfonyl)-glycerol (4) gave the acyclo N-nucleosides 5a-c and 9a,b whose deprotection afforded 6a-c and 10a,b. 2-(Methylthio)pyrimidin-4(1H)-ones 11a-c and 4-(methylthio)pyrimidin-2(1H)-ones 14a,b were treated with 2 and/or 4 to give 12a-c and 15a,b which were deprotected to give 13a-c and 16a,b. Pyrimidine-2,4(1H,3H)-dithiones 17a-c were treated with two equivalents of 2 to give 2,4-bis[[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]thio] pyrimidines 18a-c. Deprotection of compounds 18a-c gave 2,4-bis[(2,3-dihydroxypropyl)thio]pyrimidines 19a-c. The activity of the deprotected nucleosides against Hepatitis B virus was evaluated and showed moderate inhibition activity against HBV with mild cytotoxicity.

Hypoxanthine enters human vascular endothelial cells (ECV 304) via the nitrobenzylthioinosine-insensitive equilibrative nucleoside transporter.

PubMed Central

Osses, N; Pearson, J D; Yudilevich, D L; Jarvis, S M

1996-01-01

The transport properties of the nucleobase hypoxanthine were examined in the human umbilical vein endothelial cell line ECV 304. Initial rates of hypoxanthine influx were independent of extracellular cations: replacement of Na+ with Li+, Rb+, N-methyl-D-glucamine or choline had no significant effect on hypoxanthine uptake by ECV 304 cells. Kinetic analysis demonstrated the presence of a single saturable system for the transport of hypoxanthine in ECV 304 cells with an apparent K(m) of 320 +/- 10 microM and a Vmax of 5.6 +/- 0.9 pmol/10(6) cells per s. Hypoxanthine uptake was inhibited by the nucleosides adenosine, uridine and thymidine (apparent Ki 41 +/- 6, 240 +/- 27 and 59 +/- 8 microM respectively) and the nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole (apparent Ki 2.5 +/- 0.3, 11 +/- 3 and 0.16 +/- 0.006 microM respectively), whereas the nucleobases adenine, guanine and thymine had little effect (50% inhibition at > 1 mM). ECV 304 cells were also shown to transport adenosine via both the NBMPR-sensitive and -insensitive nucleoside carriers. Hypoxanthine specifically inhibited adenosine transport via the NBMPR-insensitive system in a competitive manner (apparent Ki 290 +/- 14 microM). These results indicate that hypoxanthine entry into ECV 304 endothelial cells is mediated by the NBMPR-insensitive nucleoside carrier present in these cells. PMID:8760371

Optical activity and electronic absorption spectra of some simple nucleosides related to cytidine and uridine: all-valence-shell molecular orbital calculations.

PubMed Central

Miles, D W; Redington, P K; Miles, D L; Eyring, H

1981-01-01

The circular dichroism and electronic absorption of three simple model systems for cytidine and uridine have been measured to 190 nm. The molecular spectral properties (excitation wavelengths, oscillator strengths, rotational strengths, and polarization directions) and electronic transitional patterns were investigated by using wave functions of the entire nucleoside with the goal of establishing the reliability of the theoretical method. The computed electronic absorption quantities were shown to be in satisfactory agreement with experimental data. It was found that the computed optical rotatory strengths of the B2u and E1u electronic transitions and lowest observed n-pi transition are in good agreement with experimental values. Electronic transitions were characterized by their electronic transitional patterns derived from population analysis of the transition density matrix. The theoretical rotational strengths associated with the B2u and E1u transitions stabilize after the use of just a few singly excited configurations in the configuration interaction basis and, hypothetically, are more reliable as indicators of conformation in pyrimidine nucleosides related to cytidine. PMID:6950393

Comparative activities of several nucleoside analogs against duck hepatitis B virus in vitro.

PubMed Central

Yokota, T; Konno, K; Chonan, E; Mochizuki, S; Kojima, K; Shigeta, S; de Clercq, E

1990-01-01

Duck hepatitis B virus (DHBV) replication in primary duck hepatocytes was monitored by examining the synthesis of both DHBV DNA and DHBV core antigen. Several nucleoside analogs which were previously shown to inhibit the replication of DNA viruses (i.e., herpesviruses) and retroviruses were examined for their inhibitory effects on the synthesis of DHBV core antigen in primary duck hepatocytes. (S)-9-(3-Hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine, 2',3'-dideoxyadenosine, and 2',3'-dideoxycytidine inhibited DHBV core antigen synthesis at concentrations that were significantly lower than those found to be toxic to the primary hepatocytes. Of all the compounds tested, (S)-HPMPA showed the lowest 50% effective concentration (0.5 micrograms/ml). The selectivity index or ratio of the 50% cytotoxic concentration to the 50% effective concentration of (S)-HPMPA was greater than 300. (S)-HPMPA not only inhibited DHBV core antigen but also DHBV DNA synthesis in DHBV-infected hepatocytes. PMID:2201250

Phosphorylation of deoxycytidine kinase on Ser-74: impact on kinetic properties and nucleoside analog activation in cancer cells.

PubMed

Amsailale, Rachid; Van Den Neste, Eric; Arts, Angélique; Starczewska, Eliza; Bontemps, Françoise; Smal, Caroline

2012-07-01

Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme in the activation of several therapeutic nucleoside analogs (NA). Its activity can be increased in vivo by Ser-74 phosphorylation, a property that could be used for enhancing NA activation and clinical efficacy. In line with this, studies with recombinant dCK showed that mimicking Ser-74 phosphorylation by a S74E mutation increases its activity toward pyrimidine analogs. However, purine analogs had not been investigated. Here, we show that the S74E mutation increased the k(cat) for cladribine (CdA) by 8- or 3-fold, depending on whether the phosphoryl donor was ATP or UTP, for clofarabine (CAFdA) by about 2-fold with both ATP and UTP, and for fludarabine (F-Ara-A) by 2-fold, but only with UTP. However, the catalytic efficiencies (k(cat)/Km) were not, or slightly, increased. The S74E mutation also sensitized dCK to feed-back inhibition by dCTP, regardless of the phosphoryl donor. Importantly, we did not observe an increase of endogenous dCK activity toward purine analogs after in vivo-induced increase of Ser-74 phosphorylation. Accordingly, treatment of CLL cells with aphidicolin, which enhances dCK activity through Ser-74 phosphorylation, did not modify the conversion of CdA or F-Ara-A into their active triphosphate form. Nevertheless, the same treatment enhanced activation of gemcitabine (dFdC) into dFdCTP in CLL as well as in HCT-116 cells and produced synergistic cytotoxicity. We conclude that increasing phosphorylation of dCK on Ser-74 might constitute a valuable strategy to enhance the clinical efficacy of some NA, like dFdC, but not of CdA or F-Ara-A. Copyright © 2012 Elsevier Inc. All rights reserved.

Nucleobases, nucleosides, and nucleotides: versatile biomolecules for generating functional nanomaterials.

PubMed

Pu, Fang; Ren, Jinsong; Qu, Xiaogang

2018-02-21

The incorporation of biomolecules into nanomaterials generates functional nanosystems with novel and advanced properties, presenting great potential for applications in various fields. Nucleobases, nucleosides and nucleotides, as building blocks of nucleic acids and biological coenzymes, constitute necessary components of the foundation of life. In recent years, as versatile biomolecules for the construction or regulation of functional nanomaterials, they have stimulated interest in researchers, due to their unique properties such as structural diversity, multiplex binding sites, self-assembly ability, stability, biocompatibility, and chirality. In this review, strategies for the synthesis of nanomaterials and the regulation of their morphologies and functions using nucleobases, nucleosides, and nucleotides as building blocks, templates or modulators are summarized alongside selected applications. The diverse applications range from sensing, bioimaging, and drug delivery to mimicking light-harvesting antenna, the construction of logic gates, and beyond. Furthermore, some perspectives and challenges in this emerging field are proposed. This review is directed toward the broader scientific community interested in biomolecule-based functional nanomaterials.

Simultaneous analysis of nucleobases, nucleosides and ginsenosides in ginseng extracts using supercritical fluid chromatography coupled with single quadrupole mass spectrometry.

PubMed

Huang, Yang; Zhang, Tingting; Zhao, Yumei; Zhou, Haibo; Tang, Guangyun; Fillet, Marianne; Crommen, Jacques; Jiang, Zhengjin

2017-09-10

Nucleobases, nucleosides and ginsenosides, which have a significant impact on the physiological activity of organisms, are reported to be the active components of ginseng, while they are less present in ginseng extracts. Few analytical methods have been developed so far to simultaneously analyze these three classes of compounds with different polarities present in ginseng extracts. In the present study, a simple and efficient analytical method was successfully developed for the simultaneous separation of 17 nucleobases, nucleosides and ginsenosides in ginseng extracts using supercritical fluid chromatography coupled with single quadrupole mass spectrometry (SFC-MS). The effect of various experimental factors on the separation performance, such as the column type, temperature and backpressure, the type of modifier and additive, and the concentration of make-up solvent were systematically investigated. Under the selected conditions, the developed method was successfully applied to the quality evaluation of 14 batches of ginseng extracts from different origins. The results obtained for the different batches indicate that this method could be employed for the quality assessment of ginseng extracts. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

QSAR-Driven Design and Discovery of Novel Compounds With Antiplasmodial and Transmission Blocking Activities.

PubMed

Lima, Marilia N N; Melo-Filho, Cleber C; Cassiano, Gustavo C; Neves, Bruno J; Alves, Vinicius M; Braga, Rodolpho C; Cravo, Pedro V L; Muratov, Eugene N; Calit, Juliana; Bargieri, Daniel Y; Costa, Fabio T M; Andrade, Carolina H

2018-01-01

Malaria is a life-threatening infectious disease caused by parasites of the genus Plasmodium , affecting more than 200 million people worldwide every year and leading to about a half million deaths. Malaria parasites of humans have evolved resistance to all current antimalarial drugs, urging for the discovery of new effective compounds. Given that the inhibition of deoxyuridine triphosphatase of Plasmodium falciparum ( Pf dUTPase) induces wrong insertions in plasmodial DNA and consequently leading the parasite to death, this enzyme is considered an attractive antimalarial drug target. Using a combi-QSAR (quantitative structure-activity relationship) approach followed by virtual screening and in vitro experimental evaluation, we report herein the discovery of novel chemical scaffolds with in vitro potency against asexual blood stages of both P. falciparum multidrug-resistant and sensitive strains and against sporogonic development of P. berghei . We developed 2D- and 3D-QSAR models using a series of nucleosides reported in the literature as Pf dUTPase inhibitors. The best models were combined in a consensus approach and used for virtual screening of the ChemBridge database, leading to the identification of five new virtual Pf dUTPase inhibitors. Further in vitro testing on P. falciparum multidrug-resistant (W2) and sensitive (3D7) parasites showed that compounds LabMol-144 and LabMol-146 demonstrated fair activity against both strains and presented good selectivity versus mammalian cells. In addition, LabMol-144 showed good in vitro inhibition of P. berghei ookinete formation, demonstrating that hit-to-lead optimization based on this compound may also lead to new antimalarials with transmission blocking activity.

Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates.

PubMed Central

Grossmann, K; Friedrich, H; Seitz, U

1980-01-01

The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I. PMID:7470092

Analysis of nucleoside-binding proteins by ligand-specific elution from dye resin: application to Mycobacterium tuberculosis aldehyde dehydrogenases.

PubMed

Kim, Chang-Yub; Webster, Cecelia; Roberts, Justin K M; Moon, Jin Ho; Alipio Lyon, Emily Z; Kim, Heungbok; Yu, Minmin; Hung, Li-Wei; Terwilliger, Thomas C

2009-12-01

We show that Cibacron Blue F3GA dye resin chromatography can be used to identify ligands that specifically interact with proteins from Mycobacterium tuberculosis, and that the identification of these ligands can facilitate structure determination by enhancing the quality of crystals. Four native Mtb proteins of the aldehyde dehydrogenase (ALDH) family were previously shown to be specifically eluted from a Cibacron Blue F3GA dye resin with nucleosides. In this study we characterized the nucleoside-binding specificity of one of these ALDH isozymes (recombinant Mtb Rv0223c) and compared these biochemical results with co-crystallization experiments with different Rv0223c-nucleoside pairings. We found that the strongly interacting ligands (NAD and NADH) aided formation of high-quality crystals, permitting solution of the first Mtb ALDH (Rv0223c) structure. Other nucleoside ligands (AMP, FAD, adenosine, GTP and NADP) exhibited weaker binding to Rv0223c, and produced co-crystals diffracting to lower resolution. Difference electron density maps based on crystals of Rv0223c with various nucleoside ligands show most share the binding site where the natural ligand NAD binds. From the high degree of similarity of sequence and structure compared to human mitochondrial ALDH-2 (BLAST Z-score = 53.5 and RMSD = 1.5 A), Rv0223c appears to belong to the ALDH-2 class. An altered oligomerization domain in the Rv0223c structure seems to keep this protein as monomer whereas native human ALDH-2 is a multimer.

High yield expression and purification of equilibrative nucleoside transporter 7 (ENT7) from Arabidopsis thaliana.

PubMed

Girke, Christopher; Arutyunova, Elena; Syed, Maria; Traub, Michaela; Möhlmann, Torsten; Lemieux, M Joanne

2015-09-01

Equilibrative nucleoside transporters (ENTs) facilitate the import of nucleosides and their analogs into cells in a bidirectional, non-concentrative manner. However, in contrast to their name, most characterized plant ENTs act in a concentrative manner. A direct characterization of any ENT protein has been hindered due to difficulties in overexpression and obtaining pure recombinant protein. The equilibrative nucleoside transporter 7 from Arabidopsis thaliana (AtENT7) was expressed in Xenopus laevis oocytes to assess mechanism of substrate uptake. Recombinant protein fused to enhanced green fluorescent protein (eGFP) was expressed in Pichia pastoris to characterize its oligomeric state by gel filtration and substrate binding by microscale thermophoresis (MST). AtENT7 expressed in X. laevis oocytes works as a classic equilibrative transporter. The expression of AtENT7-eGFP in the P. pastoris system yielded milligram amounts of pure protein that exists as stable homodimers. The concentration dependent binding of purine and pyrimidine nucleosides to the purified recombinant protein, assessed by MST, confirmed that AtENT7-eGFP is properly folded. For the first time the binding of nucleobases was observed for AtENT7. The availability of pure recombinant AtENT7 will permit detailed kinetic and structural studies of this unique member of the ENT family and, given the functional similarity to mammalian ENTs, will serve as a good model for understanding the structural basis of translocation mechanism for the family. Copyright © 2015 Elsevier B.V. All rights reserved.

Application of a coupled enzyme assay to characterize nicotinamide riboside kinases.

PubMed

Dölle, Christian; Ziegler, Mathias

2009-02-15

The recently identified nicotinamide riboside kinases (Nrks) constitute a distinct pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis. Here we present the combination of an established optical adenosine triphosphatase (ATPase) test, the pyruvate kinase/lactate dehydrogenase system, with the Nrk-catalyzed reaction to determine kinetic properties of these enzymes, in particular affinities for ATP. The assay allows variation of both nucleoside and phosphate donor substrates, thereby providing major advantages for the characterization of these enzymes. We confirm previously established kinetic parameters and identify differences in substrate selectivity between the two human Nrk isoforms. The proposed assay is inexpensive and may be applied for high-throughput screening.

Cytosine arabinoside influx and nucleoside transport sites in acute leukemia.

PubMed

Wiley, J S; Jones, S P; Sawyer, W H; Paterson, A R

1982-02-01

Although cytosine arabinoside (araC) can induce a remission in a majority of patients presenting with acute myeloblastic leukemia (AML), a minority fail to respond and moreover the drug has less effect in acute lymphoblastic leukemia (ALL). The carrier-mediated influx of araC into purified blasts from patients with AML, ALL, and acute undifferentiated leukemia (AUL) has been compared to that of normal lymphocytes and polymorphs. Blasts showed a larger mediated influx of araC than mature cells, since mean influxes for myeloblasts and lymphoblasts were 6- and 2.3-fold greater than polymorphs and lymphocytes, respectively. Also, the mean influx for myeloblasts was fourfold greater than the mean for lymphoblasts. The number of nucleoside transport sites was estimated for each cell type by measuring the equilibrium binding of [(3)H]nitrobenzylthioinosine (NBMPR), which inhibits nucleoside fluxes by binding with high affinity to specific sites on the transport mechanism. The mean binding site numbers for myeloblasts and lymphoblasts were 5- and 2.8-fold greater, respectively, than for the mature cells of the same maturation series. The mean number of NBMPR binding sites for myeloblasts was fourfold greater than for lymphoblasts. Patients with AUL were heterogeneous since blasts from some gave values within the myeloblastic range and others within the lymphoblastic range. The araC influx correlated closely with the number of NBMPR binding sites measured in the same cells on the same day. Transport parameters were measured on blasts from 15 patients with AML or AUL who were then treated with standard induction therapy containing araC. Eight patients entered complete remission, while seven failed therapy, among whom were the three patients with the lowest araC influx (<0.4 pmol/10(7) cells per min) and NBMPR binding (<3,000 sites/cell) for the treated group. In summary, myeloblasts have both higher araC transport rates and more nucleoside transport sites than lymphoblasts

Molecular dynamics and crystallization phenomenon of supercooled and glassy DNA and RNA nucleosides: β-adenosine, β-thymidine, and β-uridine

NASA Astrophysics Data System (ADS)

Adrjanowicz, K.; Wojnarowska, Z.; Grzybowska, K.; Hawelek, L.; Kaminski, K.; Paluch, M.; Kasprzycka, A.; Walczak, K.

2011-11-01

Nucleosides are chemical compounds that have an extremely important biological role; they can be found in all types of living organisms. They are crucial components from which DNA and RNA acids are built. In addition, nucleosides are key regulators of many physiological processes. In this paper, the molecular dynamics in the liquid and glassy state of three selected nucleosides, β-adenosine, β-thymidine, and β-uridine, was investigated by means of dielectric spectroscopy. Our results revealed multiple relaxation processes associated with different types of molecular motions. Besides the primary α relaxation, two secondary modes in the glassy states of examined compounds were identified. Crystallization progress monitored by dielectric spectroscopy and x-ray diffraction technique at isostructural relaxation conditions revealed that the examined nucleosides possess completely different tendencies to recrystallize from the liquid as well as the glassy state. We have also made an attempt to predict the time scale of molecular motion below the glass transition temperatures of the respective nucleosides to discuss their potential stability at room temperature over prolonged storage time. Finally, combination of molecular mobility studies with evaluation of thermodynamic parameters from calorimetric measurements allowed us to discuss the fundamental roles of both kinetic and thermodynamic factors in governing the physical stability of the glassy state.

A kinetic study on the chemical cleavage of nucleoside diphosphate sugars.

PubMed

Huhta, Eija; Parjanen, Atte; Mikkola, Satu

2010-03-30

Nucleoside diphosphate sugars serve in essential roles in metabolic processes. They have, therefore, been used in mechanistic studies on glycosylation reactions, and their analogues have been synthesised as enzyme and receptor inhibitors. Despite extensive biochemical research, little is known about their chemical reactions. In the present work the chemical cleavage of two different types of nucleoside diphosphate sugars has been studied. UDP-Glc is phosphorylated at the anomeric carbon, whereas in ADP-Rib C-1 is unsubstituted, allowing hence the equilibrium between cyclic hemiacetal and acyclic carbonyl forms. Due to the structural difference, these substrates react via different pathways under slightly alkaline conditions: while UDP-Glc reacts exclusively by a nucleophilic attack of a glucose hydroxyl group on the diphosphate moiety, ADP-Rib undergoes a complex reaction sequence that involves isomerisation processes of the acyclic ribose sugar and results in a release of ADP. Copyright 2009 Elsevier Ltd. All rights reserved.

Docking and 3-D QSAR studies on indolyl aryl sulfones. Binding mode exploration at the HIV-1 reverse transcriptase non-nucleoside binding site and design of highly active N-(2-hydroxyethyl)carboxamide and N-(2-hydroxyethyl)carbohydrazide derivatives.

PubMed

Ragno, Rino; Artico, Marino; De Martino, Gabriella; La Regina, Giuseppe; Coluccia, Antonio; Di Pasquali, Alessandra; Silvestri, Romano

2005-01-13

Three-dimensional quantitative structure-activity relationship (3-D QSAR) studies and docking simulations were developed on indolyl aryl sulfones (IASs), a class of novel HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (Silvestri, et al. J. Med. Chem. 2003, 46, 2482-2493) highly active against wild type and some clinically relevant resistant strains (Y181C, the double mutant K103N-Y181C, and the K103R-V179D-P225H strain, highly resistant to efavirenz). Predictive 3-D QSAR models using the combination of GRID and GOLPE programs were obtained using a receptor-based alignment by means of docking IASs into the non-nucleoside binding site (NNBS) of RT. The derived 3-D QSAR models showed conventional correlation (r(2)) and cross-validated (q(2)) coefficients values ranging from 0.79 to 0.93 and from 0.59 to 0.84, respectively. All described models were validated by an external test set compiled from previously reported pyrryl aryl sulfones (Artico, et al. J. Med. Chem. 1996, 39, 522-530). The most predictive 3-D QSAR model was then used to predict the activity of novel untested IASs. The synthesis of six designed derivatives (prediction set) allowed disclosure of new IASs endowed with high anti-HIV-1 activities.

Do non-nucleoside reverse transcriptase inhibitors contribute to lipodystrophy?

PubMed

Nolan, David

2005-01-01

Lipodystrophy complications, including lipoatrophy (pathological fat loss) and metabolic complications, have emerged as important long-term toxicities associated with antiretroviral therapy in the current era. The wealth of data that has accumulated over the past 6 years has now clarified the contribution of specific antiretroviral drugs to the risk of these clinical endpoints, with evidence that lipoatrophy is strongly associated with the choice of nucleoside reverse transcriptase inhibitor therapy (specifically, stavudine and to a lesser extent zidovudine). The aetiological basis of metabolic complications of antiretroviral therapy has proven to be complex, in that the risk appears to be modulated by a number of lifestyle factors that have made the metabolic syndrome highly prevalent in the general population, with additional contributions from HIV disease status itself, as well as from individual drugs within the HIV protease inhibitor class. The currently licensed non-nucleoside reverse transcriptase inhibitor (NNRTI) drugs, efavirenz and nevirapine, have been proven to have a favourable safety profile in terms of lipodystrophy complications. However, it must be noted that NNRTI drugs also have individual toxicity profiles that must be accounted for when considering and/or monitoring their use in the treatment of HIV infection.

[Comparative study on specific chromatograms and main nucleosides of cultivated and wild Cordyceps sinensis].

PubMed

Zan, Ke; Huang, Li-Li; Guo, Li-Nong; Liu, Jie; Zheng, Jian; Ma, Shuang-Cheng; Qian, Zheng-Ming; Li, Wen-Jia

2017-10-01

This study is to establish the HPLC specific chromatogram and determine four main nucleosides of wild and cultivated Cordyceps sinensis. Uridine, inosine, guanosine and adenosine were selected as reference substance. HPLC analysis was performed on a Waters XSelect HSS T3 C₁₈ (4.6 mm×250 mm, 5 μm), with a mobile phase consisting of water(A)-acetonitrile (B) at a flow rate of 0.6 mL•min⁻¹ (0-5 min,0% B;5-15 min,0%-10% B, 15-30 min,10%-20% B, 30-33 min, 20%-50% B, 33-35 min, 50%-0% B, 35-40 min, 0% B). The detection wavelength was 260 nm and the column temperature was controlled at 30 ℃, and the injection volume was 5 μL. HPLC specific chromatogram of wild and cultivated C. sinensis was established and four main nucleosides were simultaneously determined by the above method. Specific chromatograms and contents of four main nucleosides showed no significant differences between cultivated and wild C. sinensis. These results can provide scientific evidences for further development and utilization of cultivated C. sinensis. Copyright© by the Chinese Pharmaceutical Association.

Function-Oriented Synthesis: How to Design Simplified Analogues of Antibacterial Nucleoside Natural Products?

PubMed

Ichikawa, Satoshi

2016-06-01

It is important to pursue function-oriented synthesis (FOS), a strategy for the design of less structurally complex targets with comparable or superior activity that can be made in a practical manner, because compared to synthetic drugs, many biologically relevant natural products possess large and complex chemical structures that may restrict chemical modifications in a structure-activity relationship study. In this account, we describe recent efforts to simplify complex nucleoside natural products including caprazamycins. Considering the structure-activity relationship study with several truncated analogues, three types of simplified derivatives, namely, oxazolidine, isoxazolidine, and lactam-fused isoxazolidine-containing uridine derivatives, were designed and efficiently synthesized. These simplified derivatives have exhibited promising antibacterial activities. A significant feature of our studies is the rational and drastic simplification of the molecular architecture of caprazamycins. This study provides a novel strategy for the development of a new type of antibacterial agent effective against drug-resistant bacteria. © 2016 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A method for isolation of rat lymphocyte-rich mononuclear cells from lung tissue useful for determination of nucleoside triphosphate diphosphohydrolase activity.

PubMed

Jaques, Jeandre Augusto Dos S; Peres Rezer, João Felipe; Ruchel, Jader Betsch; Gutierres, Jessié; Bairros, André Valle; Gomes Farias, Iria Luiza; Almeida da Luz, Sonia Cristina; Mello Bertoncheli, Claudia de; Chitolina Schetinger, Maria Rosa; Morsch, Vera Maria; Leal, Daniela Bitencourt Rosa

2011-03-01

Methods for the isolation of peripheral blood mononuclear cells (PBMCs) and human lung mononuclear cells (LMCs) have been proposed previously. This study describes a method that allows the separation of lymphocyte-rich LMCs from rats. Trypan blue was applied to determine cell viability. White blood cell and differential cell counts were also performed. Relationships between nucleoside triphosphate diphosphohydrolase (NTPDase, EC 3.6.1.5) activities expressed in milligrams of protein, millions of cells, and millions of viable cells were examined as linear correlations. The lung tissue yielded 82.46% lymphocytes, 8.6% macrophages, 2.20% monocytes, and 1.27% polymorphonuclear cells (PMNs). In LMCs, a very strong correlation was observed as follows: between NTPDase activity, as determined using ATP or ADP as a substrate, expressed in milligrams of protein and that expressed in millions of cells (r ≥ 0.91), between that expressed in milligrams of protein and that expressed in millions of viable cells (r ≥ 0.91), and between that expressed in millions of cells and that expressed in millions of viable cells (r ≥ 0.98). Based on our results, we affirm that NTPDase activity could be expressed in millions of viable cells, millions of cells, or milligrams of protein. 2010 Elsevier Inc. All rights reserved.

Synthesis of novel 3'-azido-3'-deoxy-α-L-ribo configured nucleosides: A comparative study between chemical and chemo-enzymatic methodologies.

PubMed

Rana, Neha; Kumar, Manish; Singh, Ankita; Maity, Jyotirmoy; Shukla, Poonam; Prasad, Ashok K

2018-05-03

Syntheses of novel 3'-azido-3'-deoxy-2'-O,4'-C-methylene-α-L-ribofuranosyl nucleosides have been carried out from 3'-azido-3'-deoxy-4'-C-hydroxymethyl-β-D-xylofuranosyl nucleosides following both chemical and chemo-enzymatic methodologies. The precursor nucleoside in turn was synthesized from a common glycosyl donor 4-C-acetoxymethyl-1,2,5-tri-O-acetyl-3-azido-3-deoxy-α,β-D-xylofuranose, which was obtained by the acetolysis of 4-C-acetoxymethyl-5-O-acetyl-3-azido-3-deoxy-1,2-O-isopropylidene-α-D-xylofuranose in 96% yield. It has been observed that a chemo-enzymatic pathway for the synthesis of targeted nucleosides is much more efficient than a chemical pathway, leading to the improvement in yield for the synthesis of 3'-azido-3'-deoxy-α-L-ribofuranosyl thymine and uracil from 49 to 89% and 55 to 93%, respectively.

Six related nucleoside/nucleobase transporters from Trypanosoma brucei exhibit distinct biochemical functions.

PubMed

Sanchez, Marco A; Tryon, Rob; Green, Joy; Boor, Ilja; Landfear, Scott M

2002-06-14

Purine nucleoside and nucleobase transporters are of fundamental importance for Trypanosoma brucei and related kinetoplastid parasites because these protozoa are not able to synthesize purines de novo and must salvage the compounds from their hosts. In the studies reported here, we have identified a family of six clustered genes in T. brucei that encode nucleoside/nucleobase transporters. These genes, TbNT2/927, TbNT3, TbNT4, TbNT5, TbNT6, and TbNT7, have predicted amino acid sequences that show high identity to each other and to TbNT2, a P1 type nucleoside transporter recently identified in our laboratory. Expression in Xenopus laevis oocytes revealed that TbNT2/927, TbNT5, TbNT6, and TbNT7 are high affinity adenosine/inosine transporters with K(m) values of <5 microm. In addition, TbNT5, and to a limited degree TbNT6 and TbNT7, also mediate the uptake of the nucleobase hypoxanthine. Ribonuclease protection assays showed that mRNA from all of the six members of this gene family are expressed in the bloodstream stage of the T. brucei life cycle but that TbNT2/927 and TbNT5 mRNAs are also expressed in the insect stage of the life cycle. These results demonstrate that T. brucei expresses multiple purine transporters with distinct substrate specificities and different patterns of expression during the parasite life cycle.

Non-nucleoside inhibitors of the measles virus RNA-dependent RNA polymerase: synthesis, structure-activity relationships, and pharmacokinetics.

PubMed

Ndungu, J Maina; Krumm, Stefanie A; Yan, Dan; Arrendale, Richard F; Reddy, G Prabhakar; Evers, Taylor; Howard, Randy; Natchus, Michael G; Saindane, Manohar T; Liotta, Dennis C; Plemper, Richard K; Snyder, James P; Sun, Aiming

2012-05-10

The measles virus (MeV), a member of the paramyxovirus family, is an important cause of pediatric morbidity and mortality worldwide. In an effort to provide therapeutic treatments for improved measles management, we previously identified a small, non-nucleoside organic inhibitor of the viral RNA-dependent RNA polymerase by means of high-throughput screening. Subsequent structure-activity relationship (SAR) studies around the corresponding pyrazole carboxamide scaffold led to the discovery of 2 (AS-136a), a first generation lead with low nanomolar potency against life MeV and attractive physical properties suitable for development. However, its poor water solubility and low oral bioavailability (F) in rat suggested that the lead could benefit from further SAR studies to improve the biophysical characteristics of the compound. Optimization of in vitro potency and aqueous solubility led to the discovery of 2o (ERDRP-00519), a potent inhibitor of MeV (EC(50) = 60 nM) with an aqueous solubility of approximately 60 μg/mL. The agent shows a 10-fold exposure (AUC/C(max)) increase in the rat model relative to 2, displays near dose proportionality in the range of 10-50 mg/kg, and exhibits good oral bioavailability (F = 39%). The significant solubility increase appears linked to the improved oral bioavailability.

CoMFA and CoMSIA 3D-QSAR studies on S(6)-(4-nitrobenzyl)mercaptopurine riboside (NBMPR) analogs as inhibitors of human equilibrative nucleoside transporter 1 (hENT1).

PubMed

Gupte, Amol; Buolamwini, John K

2009-01-15

3D-QSAR (CoMFA and CoMSIA) studies were performed on human equlibrative nucleoside transporter (hENT1) inhibitors displaying K(i) values ranging from 10,000 to 0.7nM. Both CoMFA and CoMSIA analysis gave reliable models with q2 values >0.50 and r2 values >0.92. The models have been validated for their stability and robustness using group validation and bootstrapping techniques and for their predictive abilities using an external test set of nine compounds. The high predictive r2 values of the test set (0.72 for CoMFA model and 0.74 for CoMSIA model) reveals that the models can prove to be a useful tool for activity prediction of newly designed nucleoside transporter inhibitors. The CoMFA and CoMSIA contour maps identify features important for exhibiting good binding affinities at the transporter, and can thus serve as a useful guide for the design of potential equilibrative nucleoside transporter inhibitors.

Human immunodeficiency virus types 1 and 2 exhibit comparable sensitivities to Zidovudine and other nucleoside analog inhibitors in vitro.

PubMed

Smith, Robert A; Gottlieb, Geoffrey S; Anderson, Donovan J; Pyrak, Crystal L; Preston, Bradley D

2008-01-01

Using an indicator cell assay that directly quantifies viral replication, we show that human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2, respectively) exhibit similar sensitivities to 3'-azido-3'-deoxythymidine (zidovudine) as well as other nucleoside analog inhibitors of reverse transcriptase. These data support the use of nucleoside analogs for antiviral therapy of HIV-2 infection.

Label-free assay based on immobilized capillary enzyme reactor of Leishmania infantum nucleoside triphosphate diphosphohydrolase (LicNTPDase-2-ICER-LC/UV).

PubMed

Magalhães, Luana; de Oliveira, Arthur Henrique Cavalcante; de Souza Vasconcellos, Raphael; Mariotini-Moura, Christiane; de Cássia Firmino, Rafaela; Fietto, Juliana Lopes Rangel; Cardoso, Carmen Lúcia

2016-01-01

Nucleoside triphosphate diphosphohydrolase (NTPDase) is an enzyme belonging to the apyrase family that participates in the hydrolysis of the nucleosides di- and triphosphate to the corresponding nucleoside monophosphate. This enzyme underlies the virulence of parasites such as Leishmania. Recently, an NTPDase from Leishmania infantum (LicNTPDase-2) was cloned and expressed and has been considered as a new drug target for the treatment of leishmaniasis. With the intent of developing label-free online screening methodologies, LicNTPDase-2 was covalently immobilized onto a fused silica capillary tube in the present study to create an immobilized capillary enzyme reactor (ICER) based on LicNTPDase-2 (LicNTPDase-2-ICER). To perform the activity assays, a multidimensional chromatographic method was developed employing the LicNTPDase-2-ICER in the first dimension, and an analytical Ascentis C8 column was used in the second dimension to provide analytical separation of the substrates and products. The validated LicNTPDase-2-ICER method provided the following kinetic parameters of the immobilized enzyme: KM of 2.2 and 1.8mmolL(-1) for the ADP and ATP substrates, respectively. Suramin (1mmolL(-1)) was also shown to inhibit 32.9% of the enzymatic activity. The developed method is applicable to kinetic studies and enables the recognition of the ligands. Furthermore, a comparison of the values of LicNTPDase-2-ICER with those obtained with an LC method using free enzyme in solution showed that LicNTPDase-2-ICER-LC/UV was an accurate and reproducible method that enabled automated measurements for the rapid screening of ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

Modified nucleoside dependent Watson-Crick and wobble codon binding by tRNALysUUU species.

PubMed

Yarian, C; Marszalek, M; Sochacka, E; Malkiewicz, A; Guenther, R; Miskiewicz, A; Agris, P F

2000-11-07

Nucleoside modifications are important to the structure of all tRNAs and are critical to the function of some tRNA species. The transcript of human tRNA(Lys3)(UUU) with a UUU anticodon, and the corresponding anticodon stem and loop domain (ASL(Lys3)(UUU)), are unable to bind to poly-A programmed ribosomes. To determine if specific anticodon domain modified nucleosides of tRNA(Lys) species would restore ribosomal binding and also affect thermal stability, we chemically synthesized ASL(Lys) heptadecamers and site-specifically incorporated the anticodon domain modified nucleosides pseudouridine (Psi(39)), 5-methylaminomethyluridine (mnm(5)U(34)) and N6-threonylcarbamoyl-adenosine (t(6)A(37)). Incorporation of t(6)A(37) and mnm(5)U(34) contributed structure to the anticodon loop, apparent by increases in DeltaS, and significantly enhanced the ability of ASL(Lys3)(UUU) to bind poly-A programmed ribosomes. Neither ASL(Lys3)(UUU)-t(6)A(37) nor ASL(Lys3)(UUU)-mnm(5)U(34) bound AAG programmed ribosomes. Only the presence of both t(6)A(37) and mnm(5)U(34) enabled ASL(Lys3)(UUU) to bind AAG programmed ribosomes, as well as increased its affinity for poly-A programmed ribosomes to the level of native Escherichia coli tRNA(Lys). The completely unmodified anticodon stem and loop of human tRNA(Lys1,2)(CUU) with a wobble position-34 C bound AAG, but did not wobble to AAA, even when the ASL was modified with t(6)A(37). The data suggest that tRNA(Lys)(UUU) species require anticodon domain modifications in the loop to impart an ordered structure to the anticodon for ribosomal binding to AAA and require a combination of modified nucleosides to bind AAG.

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

PubMed

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

2010-11-19

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

Absence of a universal mechanism of mitochondrial toxicity by nucleoside analogs.

PubMed

Lund, Kaleb C; Peterson, LaRae L; Wallace, Kendall B

2007-07-01

Nucleoside analogs are associated with various mitochondrial toxicities, and it is becoming increasingly difficult to accommodate these differences solely in the context of DNA polymerase gamma inhibition. Therefore, we examined the toxicities of zidovudine (AZT) (10 and 50 microM; 2.7 and 13.4 microg/ml), didanosine (ddI) (10 and 50 microM; 2.4 and 11.8 microg/ml), and zalcitabine (ddC) (1 and 5 microM; 0.21 and 1.1 microg/ml) in HepG2 and H9c2 cells without the presumption of mitochondrial DNA (mtDNA) depletion. Ethidium bromide (EtBr) (0.5 microg/ml; 1.3 microM) was used as a positive control. AZT treatment resulted in metabolic disruption (increased lactate and superoxide) and increased cell mortality with decreased proliferation, while mtDNA remained unchanged or increased (HepG2 cells; 50 microM AZT). ddC caused pronounced mtDNA depletion in HepG2 cells but not in H9c2 cells and increased mortality in HepG2 cells, but no significant metabolic disruption in either cell type. ddI caused a moderate depletion of mtDNA in both cell types but showed no other effects. EtBr exposure resulted in metabolic disruption, increased cell mortality with decreased cell proliferation, and mtDNA depletion in both cell types. We conclude that nucleoside analogs display unique toxicities within and between culture models, and therefore, care should be taken when generalizing about the mechanisms of nucleoside reverse transcriptase inhibitor toxicity. Additionally, mtDNA abundance does not necessarily correlate with metabolic disruption, especially in cell culture; careful discernment is recommended in this regard.

Investigation of the binding between pepsin and nucleoside analogs by spectroscopy and molecular simulation.

PubMed

Li, Zhen; Li, Zhigang; Yang, Lingling; Xie, Yuanzhe; Shi, Jie; Wang, Ruiyong; Chang, Junbiao

2015-03-01

In this paper, the interactions of pepsin with CYD (cytidine) or nucleoside analogs, including FNC (2'-deoxy-2'-β-fluoro-4'-azidocytidine) and CMP (cytidine monophosphate), were investigated by fluorescence, UV-visible absorption and synchronous fluorescence spectroscopy under mimic physiological conditions. The results indicated that FNC (CYD/CMP) caused the fluorescence quenching by the formation of complex. The binding constants and thermo-dynamic parameters at three different temperatures were obtained. The hydrophobic and electrostatic interactions were the predominant intermolecular forces to stabilize the complex. The F atom in FNC might weaken the binding of nucleoside analog to pepsin. Results showed that CYD was the strongest quencher and bound to pepsin with higher affinity.

Synthesis of some purine and pyrimidine nucleosides of 3-amino-2,3,6-trideoxy-L-lyxo-hexopyranose (daunosamine).

PubMed

Lazzari, E; Vigevani, A; Arcamone, F

1977-06-01

The daunosaminyl analogue of the antibiotic puromycin and the nucleoside derivatives of daunosamine with adenine, thymine, and cytosine have been synthesised. The nucleoside derivatives of 6-dimethylaminopurine, thymine, and cytosine were prepared by melting the protected daunosamine with the protected base in vacuo. Daunosaminyladenine was obtained by condensing N-trifluoroacetyl-O-trifluoroacetyl-alpha-daunosaminyl chloride either with N6-benzoyl-9-chloromercuryadenine in boiling xylene or with N6-benzoyladenine in dichloromethane at room temperature in the presence of a molecular sieve. In each reaction, the beta-anomeric nucleoside was obtained, as shown by p.m.r. data. The protecting groups were removed with barium hydroxide or methanolic ammonia to give the free aminonucleosides in good yield. 9-beta-Daunosaminyl-6-dimethylaminopurine was coupled to N-benzylocyxcarbonyl-O-methyltyrosine, giving, after hydrogenolysis, the daunosaminyl analogue of puromycin.

Inosine triphosphatase genetic variants are protective against anemia during antiviral therapy for HCV2/3 but do not decrease dose reductions of RBV or increase SVR.

PubMed

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

2011-02-01

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

Low frequency Raman study of the nucleosides

NASA Astrophysics Data System (ADS)

Koontz, Craig; Lee, Scott

2011-04-01

In both transcription and replication, the two helices of the DNA molecule move apart. Consequently, vibrations involving the relative motions of large portions of the molecule with respect to one another are of intrinsic interest. Such vibrations have relatively low frequencies because they involve weak bonds and large masses. Low frequency modes are difficult to observe in Raman spectroscopy because they are very close to the signal from the Rayleigh scattered light (which is very intense). In this poster, we will describe our results for the eight nucleosides: adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxycytidine, uracil and deoxythymidine.

Low frequency Raman study of the nucleosides

NASA Astrophysics Data System (ADS)

Koontz, Craig; Lee, Scott

2011-03-01

In both transcription and replication, the two helices of the DNA molecule move apart. Consequently, vibrations involving the relative motions of large portions of the molecule with respect to one another are of intrinsic interest. Such vibrations have relatively low frequencies because they involve weak bonds and large masses. Low frequency modes are difficult to observe in Raman spectroscopy because they are very close to the signal from the Rayleigh scattered light (which is very intense). In this poster, we will describe our results for the eight nucleosides: adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxycytidine, uracil and deoxythymidine.

Low frequency Raman study of the nucleosides

NASA Astrophysics Data System (ADS)

Koontz, Craig; Lee, Scott

2010-10-01

In both transcription and replication, the two helices of the DNA molecule move apart. Consequently, vibrations involving the relative motions of large portions of the molecule with respect to one another are of intrinsic interest. Such vibrations have relatively low frequencies because they involve weak bonds and large masses. Low frequency modes are difficult to observe in Raman spectroscopy because they are very close to the signal from the Rayleigh scattered light (which is very intense). In this poster, we will describe our results for the eight nucleosides: adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxycytidine, uracil and deoxythymidine.

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

PubMed Central

Gaston, Kirk W; Limbach, Patrick A

2014-01-01

The analysis of ribonucleic acids (RNA) by mass spectrometry has been a valuable analytical approach for more than 25 years. In fact, mass spectrometry has become a method of choice for the analysis of modified nucleosides from RNA isolated out of biological samples. This review summarizes recent progress that has been made in both nucleoside and oligonucleotide mass spectral analysis. Applications of mass spectrometry in the identification, characterization and quantification of modified nucleosides are discussed. At the oligonucleotide level, advances in modern mass spectrometry approaches combined with the standard RNA modification mapping protocol enable the characterization of RNAs of varying lengths ranging from low molecular weight short interfering RNAs (siRNAs) to the extremely large 23 S rRNAs. New variations and improvements to this protocol are reviewed, including top-down strategies, as these developments now enable qualitative and quantitative measurements of RNA modification patterns in a variety of biological systems. PMID:25616408

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry.

PubMed

Gaston, Kirk W; Limbach, Patrick A

2014-01-01

The analysis of ribonucleic acids (RNA) by mass spectrometry has been a valuable analytical approach for more than 25 years. In fact, mass spectrometry has become a method of choice for the analysis of modified nucleosides from RNA isolated out of biological samples. This review summarizes recent progress that has been made in both nucleoside and oligonucleotide mass spectral analysis. Applications of mass spectrometry in the identification, characterization and quantification of modified nucleosides are discussed. At the oligonucleotide level, advances in modern mass spectrometry approaches combined with the standard RNA modification mapping protocol enable the characterization of RNAs of varying lengths ranging from low molecular weight short interfering RNAs (siRNAs) to the extremely large 23 S rRNAs. New variations and improvements to this protocol are reviewed, including top-down strategies, as these developments now enable qualitative and quantitative measurements of RNA modification patterns in a variety of biological systems.

Methylated nucleosides in tRNA and tRNA methyltransferases

PubMed Central

Hori, Hiroyuki

2014-01-01

To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon-anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed. PMID:24904644

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

PubMed Central

Hebner, Christy M.; Han, Bin; Brendza, Katherine M.; Nash, Michelle; Sulfab, Maisoun; Tian, Yang; Hung, Magdeleine; Fung, Wanchi; Vivian, Randall W.; Trenkle, James; Taylor, James; Bjornson, Kyla; Bondy, Steven; Liu, Xiaohong; Link, John; Neyts, Johan; Sakowicz, Roman; Zhong, Weidong; Tang, Hengli; Schmitz, Uli

2012-01-01

Tegobuvir (TGV) is a novel non-nucleoside inhibitor (NNI) of HCV RNA replication with demonstrated antiviral activity in patients with genotype 1 chronic HCV infection. The mechanism of action of TGV has not been clearly defined despite the identification of resistance mutations mapping to the NS5B polymerase region. TGV does not inhibit NS5B enzymatic activity in biochemical assays in vitro, suggesting a more complex antiviral mechanism with cellular components. Here, we demonstrate that TGV exerts anti-HCV activity utilizing a unique chemical activation and subsequent direct interaction with the NS5B protein. Treatment of HCV subgenomic replicon cells with TGV results in a modified form of NS5B with a distinctly altered mobility on a SDS-PAGE gel. Further analysis reveals that the aberrantly migrating NS5B species contains the inhibitor molecule. Formation of this complex does not require the presence of any other HCV proteins. The intensity of the aberrantly migrating NS5B species is strongly dependent on cellular glutathione levels as well as CYP 1A activity. Furthermore analysis of NS5B protein purified from a heterologous expression system treated with TGV by mass spectrometry suggests that TGV undergoes a CYP- mediated intracellular activation step and the resulting metabolite, after forming a glutathione conjugate, directly and specifically interacts with NS5B. Taken together, these data demonstrate that upon metabolic activation TGV is a specific, covalent inhibitor of the HCV NS5B polymerase and is mechanistically distinct from other classes of the non-nucleoside inhibitors (NNI) of the viral polymerase. PMID:22720059

Determination of 6-thioguanosine diphosphate and triphosphate and nucleoside diphosphate kinase activity in erythrocytes: novel targets for thiopurine therapy?

PubMed

Karner, Susanne; Shi, Shaojun; Fischer, Christine; Schaeffeler, Elke; Neurath, Markus F; Herrlinger, Klaus R; Hofmann, Ute; Schwab, Matthias

2010-04-01

6-Thioguanine nucleotides are the sum of 6-thioguanosine 5'-monophosphate (TGMP), -diphosphate (TGDP), and -triphosphate (TGTP) representing essential metabolites involved in drug action of thiopurines. Elevated levels of TGDP have been associated with poor response to azathioprine therapy in patients with inflammatory bowel disease. The conversion of TGDP to TGTP is supposed to be catalyzed by nucleoside diphosphate kinase (NDPK). The aim of this work was to investigate simultaneously individual 6-thioguanosine phosphate levels and NDPK activity in red blood cells (RBCs) of patients on azathioprine therapy. Ion-pair high-performance liquid chromatography methods with fluorescence and ultraviolet detection were applied to quantify individual levels of 6-thioguanosine 5'-phosphates and NDPK activity, respectively, in RBCs. Recombinantly expressed NDPK isoforms A and B were unequivocally identified to catalyze the formation of TGTP (30.6 +/- 3.88 nmol x min x mg for NDPK A versus 41.2 +/- 1.05 nmol x min x mg for NDPK B). Comprehensive analyses on the stability of TGMP, TGDP, and TGTP and the reproducibility of NDPK activity in RBCs were performed to provide a reliable sampling protocol for clinical practice. Of note, isolation of RBCs within 6 hours followed by immediate storage at -80 degrees C is crucial for prevention of degradation of 5'-phosphates. In a clinical study of 37 patients on azathioprine, TGTP was the predominant 6-thioguanosine phosphate in RBCs. In contrast, three patients showed TGTP/(TGDP + TGTP) ratios of 57.2%, 64.3%, and 66% corresponding to elevated TGDP levels. NDPK activity ranged from 4.1 to 11.3 nmol x min x mg hemoglobin. No correlation between NDPK activity and the 6-thioguanosine phosphate levels was found. The question whether interindividual variability of NDPK activity may explain differences in 6-thioguanosine 5'-phosphates levels has to be investigated in a prospective large-scale study.

Enzymatic separation of epimeric 4-C-hydroxymethylated furanosugars: Synthesis of bicyclic nucleosides.

PubMed

Rana, Neha; Kumar, Manish; Khatri, Vinod; Maity, Jyotirmoy; Prasad, Ashok K

2017-01-01

Conversion of D-glucose to 4- C- hydroxymethyl-1,2- O -isopropylidene-α-D-ribofuranose, which is a key precursor for the synthesis of different types of bicyclic/spiro nucleosides, led to the formation of an inseparable 1:1 mixture of the desired product and 4- C- hydroxymethyl-1,2- O -isopropylidene-α-D-xylofuranose. A convenient environment friendly Novozyme ® -435 catalyzed selective acetylation methodology has been developed for the separation of an epimeric mixture of ribo - and xylotrihydroxyfuranosides in quantitative yields. The structure of both the monoacetylated epimers, i.e., 5- O -acetyl-4- C -hydroxymethyl-1,2- O -isopropylidene-α-D-ribo- and xylofuranose obtained by enzymatic acetylation, has been confirmed by an X-ray study on their corresponding 4- C - p -toluenesulfonyloxymethyl derivatives. Furthermore, the two separated epimers were used for the convergent synthesis of two different types of bicyclic nucleosides, which confirms their synthetic utility.

Design and synthesis of N₁-aryl-benzimidazoles 2-substituted as novel HIV-1 non-nucleoside reverse transcriptase inhibitors.

PubMed

Monforte, Anna-Maria; Ferro, Stefania; De Luca, Laura; Lo Surdo, Giuseppa; Morreale, Francesca; Pannecouque, Christophe; Balzarini, Jan; Chimirri, Alba

2014-02-15

A series of novel N1-aryl-2-arylthioacetamido-benzimidazoles were synthesized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Some of them proved to be effective in inhibiting HIV-1 replication at submicromolar and nanomolar concentration acting as HIV-1 non-nucleoside RT inhibitors (NNRTIs), with low cytotoxicity. The preliminary structure-activity relationship (SAR) of these new derivatives was discussed and rationalized by docking studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

A novel Raman spectrophotometric method for quantitative measurement of nucleoside triphosphate hydrolysis.

PubMed

Jenkins, R H; Tuma, R; Juuti, J T; Bamford, D H; Thomas, G J

1999-01-01

A novel spectrophotometric method, based upon Raman spectroscopy, has been developed for accurate quantitative determination of nucleoside triphosphate phosphohydrolase (NTPase) activity. The method relies upon simultaneous measurement in real time of the intensities of Raman marker bands diagnostic of the triphosphate (1115 cm(-1)) and diphosphate (1085 cm(-1)) moieties of the NTPase substrate and product, respectively. The reliability of the method is demonstrated for the NTPase-active RNA-packaging enzyme (protein P4) of bacteriophage phi6, for which comparative NTPase activities have been estimated independently by radiolabeling assays. The Raman-determined rate for adenosine triphosphate substrate (8.6 +/- 1.3 micromol x mg(-1) x min(-1) at 40 degrees C) is in good agreement with previous estimates. The versatility of the Raman method is demonstrated by its applicability to a variety of nucleotide substrates of P4, including the natural ribonucleoside triphosphates (ATP, GTP) and dideoxynucleoside triphosphates (ddATP, ddGTP). Advantages of the present protocol include conservative sample requirements (approximately 10(-6) g enzyme/protocol) and relative ease of data collection and analysis. The latter conveniences are particularly advantageous for the measurement of activation energies of phosphohydrolase activity.

Synthesis, characterization and cytotoxic properties of platinum(II) complexes containing the nucleosides adenosine and cytidine.

PubMed

Montagner, Diego; Gandin, Valentina; Marzano, Cristina; Longato, Bruno

2011-06-01

Cytidine (cyt) and adenosine (ado) react with cis-[L(2)Pt(μ-OH)](2)(NO(3))(2) (L=PMe(3), PPh(3)) in various solvents to give the nucleoside complexes cis-[L(2)Pt{cyt(-H),N(3)N(4)}](3)(NO(3))(3) (L=PMe(3), 1),cis-[L(2)Pt{cyt(-H),N(4)}(cyt,N(3))]NO(3) (L=PPh(3), 2), cis-[L(2)Pt{ado(-H),N(1)N(6)}](2)(NO(3))(2) (L=PMe(3), 3) and cis-[L(2)Pt{ado(-H),N(6)N(7)}]NO(3) (L=PPh(3), 4). When the condensation reaction is carried out in solution of nitriles (RCN, R=Me, Ph) the amidine derivatives cis-[(PPh(3))(2)PtNH=C(R){cyt(-2H)}]NO(3) (R=Me, 5a; R=Ph, 5b) and cis-[(PPh(3))(2)PtNH=C(R){ado(-2H)}]NO(3) (R=Me, 6a: R=Ph, 6b) are quantitatively formed. The coordination mode of these nucleosides, characterized in solution by multinuclear NMR spectroscopy and mass spectrometry, is similar to that previously observed for the nucleobases 1-methylcytosine (1-MeCy) and 9-methyladenine (9-MeAd). The cytotoxic properties of the new complexes, and those of the nucleobase analogs, cis-[(PPh(3))(2)PtNH=C(R){1-MeCy(-2H)}]NO(3) (R=Me, 7a: R=Ph, 7b), cis-[(PPh(3))(2)PtNH=C(R){9-MeAd(-2H)}]NO(3) (R=Me, 8a: R=Ph, 8b) have been investigated in a wide panel of human cancer cells. Interestingly, whereas the Pt(II) nucleoside complexes (1-4) did not show appreciable cytotoxicity, the corresponding amidine derivatives (7a, 7b, 8a, 8b, 5b, and 6b) exhibited a significant in vitro antitumor activity. Copyright © 2011 Elsevier Inc. All rights reserved.

Synthesis of Conformationally North-Locked Pyrimidine Nucleosides Built on an Oxabicyclo[3.1.0]hexane Scaffold | Center for Cancer Research

Cancer.gov

Beginning with a known 3-oxabicyclo[3.1.0]-hexane scaffold, 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 that mimics more closely the tetrahydrofuran ring of conventional nucleosides. The synthesis of this new class of locked nucleosides involved a novel

Active potassium transport coupled to active sodium transport in vesicles reconstituted from purified sodium and potassium ion-activated adenosine triphosphatase from the rectal gland of Squalus acanthias.

PubMed

Hilden, S; Hokin, L E

1975-08-25

Vesicles containing a purified shark rectal gland (sodium + potassium)-activated adenosine triphosphatase-(NaK ATPase) were prepared by dialyzing for 2 days egg lecithin, cholate, and the NaK ATPase purified from the rectal gland of Squalus acanthias. These vesicles were capable of both Na+ and K+ transport. Studies of K+ transport were made by measuring the ATP-stimulated transport outward of 42K+ or 86Rb+. Vesicles were preloaded with isotope by equilibration at 4 degrees for 1 to 3 days. Transport of 42K+ or 86Rb+ was initiated by addition of MgATP to the vesicles. The ATP-dependent exit of either isotope was the same. Experiments are presented which show that this loss of isotope was not due to changes in ion binding but rather due to a loss in the amount of ion trapped in the vesicular volume. The transport of K+ was dependent on external Mg2+. CTP was almost as effective as ATP in stimulating K+ transport, while UTP was relatively ineffective. These effects of nucleotides parallel their effects on Na+ accumulation and their effectiveness as substrates for the enzyme. Potassium transport was inhibited by ouabain and required the presence of Na+. The following asymmetries were seen: (a) addition of external Mg2+ supported K+ transport; (b) ouabain inhibited K+ transport only if it was present inside the vesicles; (c) addition of external Na+ to the vesicles stimulated K+ transport. External Li+ was ineffective as a Na+ substitute. The specific requirement of external Na+ for K+ transport indicates that K+ exit is coupled to Na+ entry. Changes in the internal vesicular ion concentrations were studied with vesicles prepared in 20 mM NaCl and 50 mM KCl. After 1 hour of transport at 25 degrees, a typical Na+ concentration in the vesicles in the presence of ATP was 72 mM. A typical K+ concentration in the vesicles was 10 mM as measured with 42K+ or 6 mM as measured with 86Rb+. The following relationships have been calculated for Na+ transport, K+ transport and ATP

Absence of a Universal Mechanism of Mitochondrial Toxicity by Nucleoside Analogs▿

PubMed Central

Lund, Kaleb C.; Peterson, LaRae L.; Wallace, Kendall B.

2007-01-01

Nucleoside analogs are associated with various mitochondrial toxicities, and it is becoming increasingly difficult to accommodate these differences solely in the context of DNA polymerase gamma inhibition. Therefore, we examined the toxicities of zidovudine (AZT) (10 and 50 μM; 2.7 and 13.4 μg/ml), didanosine (ddI) (10 and 50 μM; 2.4 and 11.8 μg/ml), and zalcitabine (ddC) (1 and 5 μM; 0.21 and 1.1 μg/ml) in HepG2 and H9c2 cells without the presumption of mitochondrial DNA (mtDNA) depletion. Ethidium bromide (EtBr) (0.5 μg/ml; 1.3 μM) was used as a positive control. AZT treatment resulted in metabolic disruption (increased lactate and superoxide) and increased cell mortality with decreased proliferation, while mtDNA remained unchanged or increased (HepG2 cells; 50 μM AZT). ddC caused pronounced mtDNA depletion in HepG2 cells but not in H9c2 cells and increased mortality in HepG2 cells, but no significant metabolic disruption in either cell type. ddI caused a moderate depletion of mtDNA in both cell types but showed no other effects. EtBr exposure resulted in metabolic disruption, increased cell mortality with decreased cell proliferation, and mtDNA depletion in both cell types. We conclude that nucleoside analogs display unique toxicities within and between culture models, and therefore, care should be taken when generalizing about the mechanisms of nucleoside reverse transcriptase inhibitor toxicity. Additionally, mtDNA abundance does not necessarily correlate with metabolic disruption, especially in cell culture; careful discernment is recommended in this regard. PMID:17470651

Elucidation of Different Binding Modes of Purine Nucleosides to Human Deoxycytidine Kinase

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

Sabini, Elisabetta; Hazra, Saugata; Konrad, Manfred

2008-07-30

Purine nucleoside analogues of medicinal importance, such as cladribine, require phosphorylation by deoxycytidine kinase (dCK) for pharmacological activity. Structural studies of ternary complexes of human dCK show that the enzyme conformation adjusts to the different hydrogen-bonding properties between dA and dG and to the presence of substituent at the 2-position present in dG and cladribine. Specifically, the carbonyl group in dG elicits a previously unseen conformational adjustment of the active site residues Arg104 and Asp133. In addition, dG and cladribine adopt the anti conformation, in contrast to the syn conformation observed with dA. Kinetic analysis reveals that cladribine is phosphorylatedmore » at the highest efficiency with UTP as donor. We attribute this to the ability of cladribine to combine advantageous properties from dA (favorable hydrogen-bonding pattern) and dG (propensity to bind to the enzyme in its anti conformation), suggesting that dA analogues with a substituent at the 2-position are likely to be better activated by human dCK.« less

Computer-generated Model of Purine Nucleoside Phosphorylase (PNP)

NASA Technical Reports Server (NTRS)

1987-01-01

Purine Nucleoside Phosphorylase (PNP) is an important target enzyme for the design of anti-cancer and immunosuppressive drugs. Bacterial PNP, which is slightly different from the human enzyme, is used to synthesize chemotherapuautic agents. Knowledge of the three-dimensional structure of the bacterial PNP molecule is useful in efforts to engineer different types of PNP enzymes, that can be used to produce new chemotherapeutic agents. This picture shows a computer model of bacterial PNP, which looks a lot like a display of colorful ribbons. Principal Investigator was Charles Bugg.

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. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Fluorescence polarization immunoassays for monitoring nucleoside triphosphate diphosphohydrolase (NTPDase) activity.

PubMed

Fiene, Amelie; Baqi, Younis; Lecka, Joanna; Sévigny, Jean; Müller, Christa E

2015-01-07

The following members of the ecto-nucleoside triphosphate diphosphohydrolase family, NTPDase1 (CD39), NTPDase-2, -3, and -8, play an important role in purinergic signal transduction by regulating extracellular nucleotide levels. Potent and selective NTPDase inhibitors are required as pharmacological tools and have potential as novel drugs, e.g. for anti-cancer and anti-bacterial therapy. We have developed fast and sensitive NTPDase fluorescence polarization (FP) immunoassays using the natural substrates (ATP or ADP). During the NTPDase1-catalyzed reaction, the substrate is dephosphorylated to ADP which is further dephosphorylated yielding AMP as the final product (by NTPDase1). NTPDase3 and -8 yield AMP and ADP, while NTPDase2 results mainly in the formation of ADP. Direct quantification of the respective product, AMP or ADP, is achieved by displacement of an appropriate fluorescent tracer nucleotide from a specific antibody leading to a change in fluorescence polarization. The assays are highly sensitive and can be performed with low substrate concentrations (20 μM ATP or 10 μM ADP) below the KM values of NTPDases, which simplifies the identification of novel competitive inhibitors. Optimized antibody and enzyme concentrations allow the reproducible detection of 2 μM ADP and 1 μM AMP (at 10% substrate conversion). Validation of the assays yielded excellent Z'-factors greater than 0.70 for all investigated NTPDase subtypes indicating high robustness of the analytical method. Furthermore, we tested a standard inhibitor and performed a first exemplary screening campaign with a library consisting of >400 compounds (Z'-factor: 0.87, hit rate 0.5%). Thereby we demonstrated the suitability of the FP assay for IC50 value determination and high-throughput screening in a 384-well format. The new FP assays were shown to be superior to current standard assays.

Enzymatic separation of epimeric 4-C-hydroxymethylated furanosugars: Synthesis of bicyclic nucleosides

PubMed Central

Rana, Neha; Kumar, Manish; Khatri, Vinod; Maity, Jyotirmoy

2017-01-01

Conversion of D-glucose to 4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose, which is a key precursor for the synthesis of different types of bicyclic/spiro nucleosides, led to the formation of an inseparable 1:1 mixture of the desired product and 4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-xylofuranose. A convenient environment friendly Novozyme®-435 catalyzed selective acetylation methodology has been developed for the separation of an epimeric mixture of ribo- and xylotrihydroxyfuranosides in quantitative yields. The structure of both the monoacetylated epimers, i.e., 5-O-acetyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribo- and xylofuranose obtained by enzymatic acetylation, has been confirmed by an X-ray study on their corresponding 4-C-p-toluenesulfonyloxymethyl derivatives. Furthermore, the two separated epimers were used for the convergent synthesis of two different types of bicyclic nucleosides, which confirms their synthetic utility. PMID:29062429

Binding of nickel /II/ to 5-prime-nucleoside monophosphates and related compounds. [role in origin of life

NASA Technical Reports Server (NTRS)

Orenberg, J. B.; Kjos, K. M.; Winkler, R.; Link, J.; Lawless, J. G.

1982-01-01

The interactions of Ni(II) cation with a representative suite of purine bases and the respective nucleosides and nucleotides have been studied by ultraviolet difference spectroscopy. Apparent association constants were determined for each system at pH 7.0, using computer linear regression coupled with an iteration technique. The specificity of binding of Ni(2+) for the purine nucleotides studied at pH 7.0 was 5-prime-GMP greater than 5-prime-AMP; a similar ordering was also found for the respective nucleosides and bases. In this study binding was not observed for the suite of pyramidines used, although an Ni(2+) -cytidine complex has been observed (Fiskin and Beer, 1965). It was also found that Ni(2+) bound more strongly to the purine 5-prime-nucleotides than to the respective nucleosides and bases. These trends are explained in terms of metal-ligand bonds and available bonding positions on the ligands. A role for metal-ion-nucleotide types of complexes is suggested in the processes that might have given rise to the origin of life.

Metabolic signature of breast cancer cell line MCF-7: profiling of modified nucleosides via LC-IT MS coupling.

PubMed

Bullinger, Dino; Neubauer, Hans; Fehm, Tanja; Laufer, Stefan; Gleiter, Christoph H; Kammerer, Bernd

2007-11-29

Cancer, like other diseases accompanied by strong metabolic disorders, shows characteristic effects on cell turnover rate, activity of modifying enzymes and DNA/RNA modifications, resulting also in elevated amounts of excreted modified nucleosides. For a better understanding of the impaired RNA metabolism in breast cancer cells, we screened these metabolites in the cell culture supernatants of the breast cancer cell line MCF-7 and compared it to the human mammary epithelial cells MCF-10A. The nucleosides were isolated and analyzed via 2D-chromatographic techniques: In the first dimension by cis-diol specific boronate affinity extraction and subsequently by reversed phase chromatography coupled to an ion trap mass spectrometer. Besides the determination of ribonucleosides, additional compounds with cis-diol structure, deriving from cross-linked biochemical pathways, like purine-, histidine- and polyamine metabolism were detected. In total, 36 metabolites were identified by comparison of fragmentation patterns and retention time. Relation to the internal standard isoguanosine yielded normalized area ratios for each identified compound and enabled a semi-quantitative metabolic signature of both analyzed cell lines.13 of the identified 26 modified ribonucleosides were elevated in the cell culture supernatants of MCF-7 cells, with 5-methyluridine, N2,N2,7-trimethylguanosine, N6-methyl-N6-threonylcarbamoyladenosine and 3-(3-aminocarboxypropyl)-uridine showing the most significant differences. 1-ribosylimidazole-4-acetic acid, a histamine metabolite, was solely found in the supernatants of MCF-10A cells, whereas 1-ribosyl-4-carboxamido-5-aminoimidazole and S-adenosylmethionine occurred only in supernatants of MCF-7 cells. The obtained results are discussed against the background of pathological changes in cell metabolism, resulting in new perspectives for modified nucleosides and related metabolites as possible biomedical markers for breast carcinoma in vivo.

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

Uridine Nucleoside Thiation: Gas-Phase Structures and Energetics

NASA Astrophysics Data System (ADS)

Hamlow, Lucas; Lee, Justin; Rodgers, M. T.; Berden, Giel; Oomens, Jos

2016-06-01

The naturally occurring thiated uridine nucleosides, 4-thiouridine (s4Urd) and 2-thiouridine (s2Urd), play important roles in the function and analysis of a variety of RNAs. 2-Thiouridine and its C5 modified analogues are commonly found in tRNAs and are believed to play an important role in codon recognition possibly due to their different structure, which has been shown by NMR to be predominantly C3'-endo. 2-Thiouridine may also play an important role in facilitating nonenzymatic RNA replication and transcription. 4-Thiouridine is a commonly used photoactivatable crosslinker that is often used to study RNA-RNA and RNA-protein cross-linking behavior. Differences in the base pairing between uracil and 4-thiouracil with adenine and guanine are an important factor in their role as a cross linker. The photoactivity of s4Urd may also aid in preventing near-UV lethality in cells. An understanding of their intrinsic structure in the gas-phase may help further elucidate the roles these modified nucleosides play in the regulation of RNAs. In this work, infrared multiple photon dissociation (IRMPD) action spectra of the protonated forms of s2Urd and s4Urd were collected in the IR fingerprint region. Structural information is determined by comparison with theoretical linear IR spectra generated from density functional theory calculations using molecular modeling to generate low-energy candidate structures. Present results are compared with analogous results for the protonated forms of uridine and 2'-deoxyuridine as well as solution phase NMR data and crystal structures.

Identification and Characterization of a Secondary Sodium-Binding Site and the Main Selectivity Determinants in the Human Concentrative Nucleoside Transporter 3.

PubMed

Arimany-Nardi, C; Claudio-Montero, A; Viel-Oliva, A; Schmidtke, P; Estarellas, C; Barril, X; Bidon-Chanal, A; Pastor-Anglada, M

2017-06-05

The family of concentrative Na + /nucleoside cotransporters in humans is constituted by three subtypes, namely, hCNT1, hCNT2, and hCNT3. Besides their different nucleoside selectivity, hCNT1 and hCNT2 have a Na + /nucleoside stoichiometry of 1:1, while for hCNT3 it is 2:1. This distinct stoichiometry of subtype 3 might hint the existence of a secondary sodium-binding site that is not present in the other two subtypes, but to date their three-dimensional structures remain unknown and the residues implicated in Na + binding are unclear. In this work, we have identified and characterized the Na + binding sites of hCNT3 by combining molecular modeling and mutagenesis studies. A model of the transporter was obtained by homology modeling, and key residues of two sodium-binding sites were identified and verified with a mutagenesis strategy. The structural model explains the altered sodium-binding properties of the hCNT3C602R polymorphic variant and supports previously generated data identifying the determinant residues of nucleoside selectivity, paving the way to understand how drugs can target this plasma membrane transporter.

Differential modulation of P-glycoprotein expression and activity by non-nucleoside HIV-1 reverse transcriptase inhibitors in cell culture.

PubMed

Störmer, Elke; von Moltke, Lisa L; Perloff, Michael D; Greenblatt, David J

2002-07-01

This study investigated the effects of the non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTI) nevirapine (NVR), efavirenz (EFV), and delavirdine (DLV) on P-glycoprotein (P-gp) activity and expression to anticipate P-gp related drug-drug interactions associated with combination therapy. NNRTIs were evaluated as P-gp substrates by measuring differential transport across Caco-2 cell monolayers. Inhibition of P-gp mediated rhodaminel23 (Rh123) transport in Caco-2 cells was used to assess P-gp inhibition by NNRTIs. Induction of P-gp expression and activity in LS180V cells following 3-day exposure to NNRTIs was measured by western blot analysis and cellular Rh123 uptake, respectively. The NNRTIs showed no differential transport between the basolateral to apical and apical to basolateral direction. NNRTI transport in either direction was not affected by the P-gp inhibitor verapamil. DLV inhibited Rh123 transport, causing a reduction to 15% of control at 100 microM (IC50 = 30 microM). NVR caused a concentration-dependent induction of P-gp expression in LS180V cells resulting in a 3.5-fold increase in immunoreactive P-gp at 100 microM NVR. Induction attributable to EFV and DLV was quantitatively smaller. NVR significantly reduced cellular uptake of Rh123 into LS180V cells, indicating increased drug efflux due to induced P-gp activity; effects of EFV and DLV were smaller. Acute DLV treatment of LS180V cells previously induced with NVR or ritonavir did not reverse the decreased Rh123 cell accumulation. NNRTIs show differential effects on P-gp activity and expression in vitro. Clinical studies are required to elucidate the clinical importance of potential drug interactions.

Addressing the selectivity and toxicity of antiviral nucleosides.

PubMed

Feng, Joy Y

2018-01-01

Nucleoside and nucleotide analogs have played significant roles in antiviral therapies and are valued for their impressive potency and high barrier to resistance. They have been approved for treatment of herpes simplex virus-1, HIV, HBV, HCV, and influenza, and new drugs are being developed for the treatment of RSV, Ebola, coronavirus MERS, and other emerging viruses. However, this class of compounds has also experienced a high attrition rate in clinical trials due to toxicity. In this review, we discuss the utility of different biochemical and cell-based assays and provide recommendations for assessing toxicity liability before entering animal toxicity studies.

FT-IR spectra of the anti-HIV nucleoside analogue d4T (Stavudine). Solid state simulation by DFT methods and scaling by different procedures

NASA Astrophysics Data System (ADS)

Alcolea Palafox, M.; Kattan, D.; Afseth, N. K.

2018-04-01

A theoretical and experimental vibrational study of the anti-HIV d4T (stavudine or Zerit) nucleoside analogue was carried out. The predicted spectra in the three most stable conformers in the biological active anti-form of the isolated state were compared. Comparison of the conformers with those of the natural nucleoside thymidine was carried out. The calculated spectra were scaled by using different scaling procedures and three DFT methods. The TLSE procedure leads to the lowest error and is thus recommended for scaling. With the population of these conformers the IR gas-phase spectra were predicted. The crystal unit cell of the different polymorphism forms of d4T were simulated through dimer forms by using DFT methods. The scaled spectra of these dimer forms were compared. The FT-IR spectrum was recorded in the solid state in the 400-4000 cm-1 range. The respective vibrational bands were analyzed and assigned to different normal modes of vibration by comparison with the scaled vibrational values of the different dimer forms. Through this comparison, the polymorphous form of the solid state sample was identified. The study indicates that d4T exist only in the ketonic form in the solid state. The results obtained were in agreement with those determined in related anti-HIV nucleoside analogues.

Synthesis and solution conformation studies of the modified nucleoside N(4),2'-O-dimethylcytidine (m(4)Cm) and its analogues.

PubMed

Mahto, Santosh K; Chow, Christine S

2008-10-01

The dimethylated ribosomal nucleoside m(4)Cm and its monomethylated analogues Cm and m(4)C were synthesized. The conformations (syn vs anti) of the three modified nucleosides and cytidine were determined by CD and 1D NOE difference spectroscopy. The ribose sugar puckers were determined by the use of proton coupling constants. The position of modification (e.g., O vs N methylation) was found to have an effect on the sugar pucker of cytidine.

Discovery of novel inhibitors for Leishmania nucleoside diphosphatase kinase (NDK) based on its structural and functional characterization

NASA Astrophysics Data System (ADS)

Mishra, Arjun K.; Singh, Nidhi; Agnihotri, Pragati; Mishra, Shikha; Singh, Saurabh P.; Kolli, Bala K.; Chang, Kwang Poo; Sahasrabuddhe, Amogh A.; Siddiqi, M. I.; Pratap, J. Venkatesh

2017-06-01

Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100 mM Bis-Tris propane pH 6.0 and 50 mM MgCl2. It belongs to the hexagonal space group P6322 with unit cell parameters a = b = 115.18, c = 62.18 Å and α = β = 90°, γ = 120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and Rfree values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation -based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed 45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.

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.

SAMHD1 enhances nucleoside-analogue efficacy against HIV-1 in myeloid cells

PubMed Central

Ordonez, Paula; Kunzelmann, Simone; Groom, Harriet C. T.; Yap, Melvyn W.; Weising, Simon; Meier, Chris; Bishop, Kate N.; Taylor, Ian A.; Stoye, Jonathan P.

2017-01-01

SAMHD1 is an intracellular enzyme that specifically degrades deoxynucleoside triphosphates into component nucleoside and inorganic triphosphate. In myeloid-derived dendritic cells and macrophages as well as resting T-cells, SAMHD1 blocks HIV-1 infection through this dNTP triphosphohydrolase activity by reducing the cellular dNTP pool to a level that cannot support productive reverse transcription. We now show that, in addition to this direct effect on virus replication, manipulating cellular SAMHD1 activity can significantly enhance or decrease the anti-HIV-1 efficacy of nucleotide analogue reverse transcription inhibitors presumably as a result of modulating dNTP pools that compete for recruitment by viral polymerases. Further, a variety of other nucleotide-based analogues, not normally considered antiretrovirals, such as the anti-herpes drugs Aciclovir and Ganciclovir and the anti-cancer drug Clofarabine are now revealed as potent anti-HIV-1 agents, under conditions of low dNTPs. This in turn suggests novel uses for nucleotide analogues to inhibit HIV-1 in differentiated cells low in dNTPs. PMID:28220857

Complete inactivation of HIV-1 using photo-labeled non-nucleoside reverse transcriptase inhibitors.

PubMed

Rios, Adan; Quesada, Jorge; Anderson, Dallas; Goldstein, Allan; Fossum, Theresa; Colby-Germinario, Susan; Wainberg, Mark A

2011-01-01

We demonstrate that a photo-labeled derivative of the non-nucleoside reverse transcriptase inhibitor (NNRTI) dapivirine termed DAPY, when used together with exposure to ultraviolet light, was able to completely and irreversibly inactivate both HIV-1 RT activity as well as infectiousness in each of a T cell line and peripheral blood mononuclear cells. Control experiments using various concentrations of DAPY revealed that a combination of exposure to ultraviolet light together with use of the specific, high affinity photo-labeled compound was necessary for complete inactivation to occur. This method of HIV RT inactivation may have applicability toward preservation of an intact viral structure and warrants further investigation in regard to the potential of this approach to elicit a durable, broad protective immune response. Copyright © 2010 Elsevier B.V. All rights reserved.

Inhibitory effect of extracellular purine nucleotide and nucleoside concentrations on T cell proliferation

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

Weiler, Monica; Schmetzer, Helga; German Research Center for Environmental Health, Munich

The release of nucleic acids and derivatives after tissue-injury may affect cellular immune-response. We studied the impact of extracellular ribo-, desoxyribonucleotides and nucleosides on T-cell immunity. Peripheral-blood-mononuclear-cells (PBMCs) or isolated CD3{sup +}T-cells obtained from 6 healthy donors were stimulated via CD3/CD28 Dynabeads or dendritic cells (DCs) in the presence or absence of pyrimidine-, purine-nucleotides and -nucleosides (range 2–200 µM). Addition of deoxy-, guanosine-triphosphate (dGTP, GTP) and guanosine resulted concentration dependent in a complete, adenosine-triphosphate (ATP) in a partial inhibition of the induced T-cell-proliferation. Deoxyadenosine-triphosphate (dATP), adenosine and the pyrimidine-ribo- and -deoxyribonucleotides displayed no inhibitory capacity. Inhibitory effects of dGTP andmore » GTP, but not of guanosine and ATP were culture-media-dependent and could be almost abrogated by use of the serum-free lymphocyte-culture-media X-Vivo15 instead of RPMI1640 with standard-supplementation. In contrast to RPMI1640, X-Vivo15 resulted in a significant down-regulation of the cell-surface-located ectonucleotidases CD39 (Ecto-Apyrase) and CD73 (Ecto-5′-Nucleotidase), critical for the extracellular nucleotides-hydrolysis to nucleosides, explaining the loss of inhibition mediated by dGTP and GTP, but not Guanosine. In line with previous findings ATP was found to exert immunosuppressive effects on T-cell-proliferation. Purine-nucleotides, dGTP and GTP displayed a higher inhibitory capacity, but seem to be strictly dependent on the microenvironmental conditions modulating the responsiveness of the respective T-lymphocytes. Further evaluation of experimental and respective clinical settings should anticipate these findings.« less

New Cerebroside and Nucleoside Derivatives from a Red Sea Strain of the Marine Cyanobacterium Moorea producens.

PubMed

Youssef, Diaa T A; Ibrahim, Sabrin R M; Shaala, Lamiaa A; Mohamed, Gamal A; Banjar, Zainy M

2016-03-09

In the course of our ongoing efforts to identify marine-derived bioactive compounds, the marine cyanobacterium Moorea producens was investigated. The organic extract of the Red Sea cyanobacterium afforded one new cerebroside, mooreaside A (1), two new nucleoside derivatives, 3-acetyl-2'-deoxyuridine (2) and 3-phenylethyl-2'-deoxyuridine (3), along with the previously reported compounds thymidine (4) and 2,3-dihydroxypropyl heptacosanoate (5). The structures of the compounds were determined by different spectroscopic studies (UV, IR, 1D, 2D NMR, and HRESIMS), as well as comparison with the literature data. Compounds 1-5 showed variable cytotoxic activity against three cancer cell lines.

Plant nucleoside 5'-phosphoramidate hydrolase; simple purification from yellow lupin (Lupinus luteus) seeds and properties of homogeneous enzyme.

PubMed

Guranowski, Andrzej; Wojdyła, Anna M; Rydzik, Anna M; Stepiński, Janusz; Jemielity, Jacek

2011-01-01

Adenosine 5'-phosphoramidate (NH₂-pA) is an uncommon natural nucleotide of poorly understood biochemistry and function. We studied a plant enzyme potentially involved in the catabolism of NH₂-pA. A fast and simple method comprising extraction of yellow lupin (Lupinus luteus) seed-meal with a low ionic strength buffer, ammonium sulfate and acetone fractionations, removal of contaminating proteins by heat denaturation, and affinity chromatography on AMP-agarose, yielded homogenous nucleoside 5'-phosphoramidase. Mass spectrometric analysis showed that the lupin hydrolase exhibits closest similarity to Arabidopsis thaliana Hint1 protein. The substrate specificity of the lupin enzyme, in particular its ability to split the P-S bond in adenosine 5'-phosphorothioate, is typical of known Hint1 proteins. Adenosine 5'-phosphofluoride and various derivatives of guanosine 5'-phosphoramidate were also substrates. Neither common divalent metal cations nor 10 mM EDTA or EGTA affected the hydrolysis of NH₂-pA. The enzyme functions as a homodimer (2 x 15,800 Da). At the optimum pH of 7.0, the K(m) for NH₂-pA was 0.5 µM and k(cat) 0.8 s⁻¹ (per monomer active site). The properties of the lupin nucleoside 5'-phosphoramidase are compared with those of its counterparts from other organisms.

Structural characterization of purine nucleoside phosphorylase from human pathogen Helicobacter pylori.

PubMed

Štefanić, Zoran; Mikleušević, Goran; Luić, Marija; Bzowska, Agnieszka; Leščić Ašler, Ivana

2017-08-01

Microaerophilic bacterium Helicobacer pylori is a well known human pathogen involved in the development of many diseases. Due to the evergrowing infection rate and increase of H. pylori antibiotic resistence, it is of utmost importance to find a new way to attack and eradicate H. pylori. The purine metabolism in H. pylori is solely dependant on the salvage pathway and one of the key enzymes in this pathway is purine nucleoside phosphorylase (PNP). In this timely context, we report here the basic biochemical and structural characterization of recombinant PNP from the H. pylori clinical isolate expressed in Escherichia coli. Structure of H. pylori PNP is typical for high molecular mass PNPs. However, its activity towards adenosine is very low, thus resembling more that of low molecular mass PNPs. Understanding the molecular mechanism of this key enzyme may lead to the development of new drug strategies and help in the eradication of H. pylori. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure-based design, synthesis, and biological evaluation of novel pyrrolyl aryl sulfones: HIV-1 non-nucleoside reverse transcriptase inhibitors active at nanomolar concentrations.

PubMed

Artico, M; Silvestri, R; Pagnozzi, E; Bruno, B; Novellino, E; Greco, G; Massa, S; Ettorre, A; Loi, A G; Scintu, F; La Colla, P

2000-05-04

Pyrrolyl aryl sulfones (PASs) have been recently reported as a new class of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitors acting at the non-nucleoside binding site of this enzyme (Artico, M.; et al. J. Med. Chem. 1996, 39, 522-530). Compound 3, the most potent inhibitor within the series (EC(50) = 0.14 microM, IC(50) = 0.4 microM, and SI > 1429), was then selected as a lead compound for a synthetic project based on molecular modeling studies. Using the three-dimensional structure of RT cocrystallized with the alpha-APA derivative R95845, we derived a model of the RT/3 complex by taking into account previously developed structure-activity relationships. Inspection of this model and docking calculations on virtual compounds prompted the design of novel PAS derivatives and related analogues. Our computational approach proved to be effective in making qualitative predictions, that is in discriminating active versus inactive compounds. Among the compounds synthesized and tested, 20 was the most active one, with EC(50) = 0.045 microM, IC(50) = 0.05 microM, and SI = 5333. Compared with the lead 3, these values represent a 3- and 8-fold improvement in the cell-based and enzyme assays, respectively, together with the highest selectivity achieved so far in the PAS series.

The Synthesis and Study of New Ribavirin Derivatives and Related Nucleoside Azole Carboxamides as Agents Active against RNA Viruses.

DTIC Science & Technology

1981-09-01

No other nucleoside product was detected. 26 When 4 was subjected to hydrogenation 3 7 with Pd/C (10%), a mixture of products was obtained even after...prolonged reduction. O2N N 02JC> ______H2 N A 3 PtO 2 0 Bz • + SzO 08 45 BOBz 3 NaOEt NH 3 O2N N N2H4H 2N N N NNN No O NO ON 6 7 The major product was...1,2,4-triazole. The minor product was found to be 3-amino-l-(2,3,3-tri-O-benzovl- 8-D-ribofuranosyl)-l,2,4-triazole (5, BJ-91120). In an effort to improve

Gemcitabine and Nucleos(t)ide Synthesis Inhibitors Are Broad-Spectrum Antiviral Drugs that Activate Innate Immunity.

PubMed

Shin, Hye Jin; Kim, Chonsaeng; Cho, Sungchan

2018-04-20

Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos(t)ide synthesis pathways, resulting in the depletion or imbalance of (d)NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos(t)ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos(t)ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs.

Synthesis and solution conformation studies of the modified nucleoside N4, 2′-O-dimethylcytidine (m4Cm) and its analogues

PubMed Central

Mahto, Santosh K.; Chow, Christine S.

2008-01-01

The dimethylated ribosomal nucleoside m4Cm and its monomethylated analogues Cm and m4C were synthesized. The conformations (syn versus anti) of the three modified nucleosides and cytidine were determined by CD and 1D NOE difference spectroscopy. The ribose sugar puckers were determined by the use of proton coupling constants. The position of modification (e.g., O versus N methylation) was found to have an effect on the sugar pucker of cytidine. PMID:18805697

Characterisation of nucleosides and nucleobases in Mactra veneriformis by high performance liquid chromatography coupled with diode array detector-mass spectrometry (HPLC-DAD-MS).

PubMed

Liu, Rui; Ji, Jing; Wang, Lingchong; Chen, Shiyong; Guo, Sheng; Wu, Hao

2012-11-15

Mactra veneriformis has been used as sea food and traditional Chinese medicine (TCM) for thousands of years in China. In the present study, a high performance liquid chromatograph coupled with photodiode array detector and electrospray ionisation-mass spectrometer (HPLC-DAD-ESI-MS) method was established for detection of the nucleosides and nucleobases in M. veneriformis from four aquaticultural area of Jiangsu during different harvest time of one year. The validated method was successfully applied to identifying 10 nucleosides and nucleobases in 48 M. veneriformis samples. Quantitative analysis showed that nucleosides and nucleobases are rich in all M. veneriformis samples. However, their contents vary in different areas and harvest times. Principal component analysis (PCA) was used to classify the 48 samples based on the contents of the nucleosides and nucleobases. As a result, the samples could be mainly clustered into four groups, which was similar as aquaticultural areas classification. Based on the results, present method might be applicable for the quality control of M. veneriformis, or even other marine shellfish aquiculture and their products, and the quality of M. veneriformis might be more related with aquaticultural areas. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

Active methamphetamine use is associated with transmitted drug resistance to non-nucleoside reverse transcriptase inhibitors in individuals with HIV infection of unknown duration.

PubMed

Cachay, Edward R; Moini, Niousha; Kosakovsky Pond, Sergei L; Pesano, Rick; Lie, Yolanda S; Aiem, Heidi; Butler, David M; Letendre, Scott; Mathews, Wm Christopher; Smith, Davey M

2007-01-01

Frequent methamphetamine use among recently HIV infected individuals is associated with transmitted drug resistance (TDR) to non-nucleoside reverse transcriptase inhibitors (NNRTI); however, the reversion time of TDR to drug susceptible HIV may exceed 3 years. We assessed whether recreational substance use is associated with detectable TDR among individuals newly diagnosed with HIV infection of unknown duration. Cross-sectional analysis. Subjects were enrolled at the University California, San Diego Early Intervention Program. Demographic, clinical and substance use data were collected using structured interviews. Genotypic resistance testing was performed using GeneSeq, Monogram Biosciences. We analyzed the association between substance use and TDR using bivariate analyses and the corresponding transmission networks using phylogenetic models. Between April 2004 and July 2006, 115 individuals with genotype data were enrolled. The prevalence of alcohol, marijuana and methamphetamine use were 98%, 71% and 64% respectively. Only active methamphetamine use in the 30 days prior to HIV diagnosis was independently associated with TDR to NNRTI (OR: 6.6; p=0.002). Despite not knowing the duration of their HIV infection, individuals reporting active methamphetamine use in the 30 days prior to HIV diagnosis are at an increased risk of having HIV strains that are resistant to NNRTI.

Destruction of Peptides and Nucleosides in Reactions with Low-Energy Electrons

NASA Astrophysics Data System (ADS)

Muftakhov, M. V.; Shchukin, P. V.

2018-05-01

Mass-spectrometry of negative ions is used to study dissociative electron capture by molecules of several nucleosides, simplest di- and tripeptides, and modified dipeptides. Energy domains and efficiencies of dissociative capture are determined for the objects under study, and threshold energies of several fragmentation processes are estimated. It is shown that cytidine and peptides are stable against fragmentation due to simple bond breaking at electron energies ranging from 0 to 1 eV.

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

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

Timofeev, V. I., E-mail: tostars@mail.ru; Abramchik, Yu. A., E-mail: ugama@yandex.ru; Zhukhlistova, N. E., E-mail: inna@ns.crys.ras.ru

2016-03-15

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

Labeled Nucleoside Triphosphates with Reversibly Terminating Aminoalkoxyl Groups

PubMed Central

Hutter, Daniel; Kim, Myong-Jung; Karalkar, Nilesh; Leal, Nicole A.; Chen, Fei; Guggenheim, Evan; Visalakshi, Visa; Olejnik, Jerzy; Gordon, Steven; Benner, Steven A.

2013-01-01

Nucleoside triphosphates having a 3′-ONH2 blocking group have been prepared with and without fluorescent tags on their nucleobases. DNA polymerases were identified that accepted these, adding a single nucleotide to the 3′-end of a primer in a template-directed extension reaction that then stops. Nitrite chemistry was developed to cleave the 3′-ONH2 group under mild conditions to allow continued primer extension. Extension-cleavage-extension cycles in solution were demonstrated with untagged nucleotides and mixtures of tagged and untagged nucleotides. Multiple extension-cleavage-extension cycles were demonstrated on an Intelligent Bio-Systems Sequencer, showing the potential of the 3′-ONH2 blocking group in “next generation sequencing”. PMID:21128174

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

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

Hazra, Saugata; Szewczak, Andrzej; Ort, Stephan

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 adoptionmore » 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.« less

Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease.

PubMed

Jochmans, Dirk; Anders, Maria; Keuleers, Inge; Smeulders, Liesbeth; Kräusslich, Hans-Georg; Kraus, Günter; Müller, Barbara

2010-10-15

Current antiretroviral therapy against human immunodeficiency virus (HIV-1) reduces viral load and thereby prevents viral spread, but it cannot eradicate proviral genomes from infected cells. Cells in immunological sanctuaries as well as cells producing low levels of virus apparently contribute to a reservoir that maintains HIV persistence in the presence of highly active antiretroviral therapy. Thus, accelerated elimination of virus producing cells may represent a complementary strategy to control HIV infection. Here we sought to exploit HIV protease (PR) related cytotoxicity in order to develop a strategy for drug induced killing of HIV producing cells. PR processes the viral Gag and Gag-Pol polyproteins during virus maturation, but is also implicated in killing of virus producing cells through off-target cleavage of host proteins. It has been observed previously that micromolar concentrations of certain non-nucleoside reverse transcriptase inhibitors (NNRTIs) can stimulate intracellular PR activity, presumably by enhancing Gag-Pol dimerization. Using a newly developed cell-based assay we compared the degree of PR activation displayed by various NNRTIs. We identified inhibitors showing higher potency with respect to PR activation than previously described for NNRTIs, with the most potent compounds resulting in ~2-fold increase of the Gag processing signal at 250 nM. The degree of enhancement of intracellular Gag processing correlated with the compound's ability to enhance RT dimerization in a mammalian two-hybrid assay. Compounds were analyzed for their potential to mediate specific killing of chronically infected MT-4 cells. Levels of cytotoxicity on HIV infected cells determined for the different NNRTIs corresponded to the relative degree of drug induced intracellular PR activation, with CC50 values ranging from ~0.3 μM to above the tested concentration range (10 μM). Specific cytotoxicity was reverted by addition of PR inhibitors. Two of the most active

Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease

PubMed Central

2010-01-01

Background Current antiretroviral therapy against human immunodeficiency virus (HIV-1) reduces viral load and thereby prevents viral spread, but it cannot eradicate proviral genomes from infected cells. Cells in immunological sanctuaries as well as cells producing low levels of virus apparently contribute to a reservoir that maintains HIV persistence in the presence of highly active antiretroviral therapy. Thus, accelerated elimination of virus producing cells may represent a complementary strategy to control HIV infection. Here we sought to exploit HIV protease (PR) related cytotoxicity in order to develop a strategy for drug induced killing of HIV producing cells. PR processes the viral Gag and Gag-Pol polyproteins during virus maturation, but is also implicated in killing of virus producing cells through off-target cleavage of host proteins. It has been observed previously that micromolar concentrations of certain non-nucleoside reverse transcriptase inhibitors (NNRTIs) can stimulate intracellular PR activity, presumably by enhancing Gag-Pol dimerization. Results Using a newly developed cell-based assay we compared the degree of PR activation displayed by various NNRTIs. We identified inhibitors showing higher potency with respect to PR activation than previously described for NNRTIs, with the most potent compounds resulting in ~2-fold increase of the Gag processing signal at 250 nM. The degree of enhancement of intracellular Gag processing correlated with the compound's ability to enhance RT dimerization in a mammalian two-hybrid assay. Compounds were analyzed for their potential to mediate specific killing of chronically infected MT-4 cells. Levels of cytotoxicity on HIV infected cells determined for the different NNRTIs corresponded to the relative degree of drug induced intracellular PR activation, with CC50 values ranging from ~0.3 μM to above the tested concentration range (10 μM). Specific cytotoxicity was reverted by addition of PR inhibitors. Two of

The importance of nucleoside hydrolase enzyme (NH) in studies to treatment of Leishmania: A review.

PubMed

Figueroa-Villar, José D; Sales, Edijane M

2017-02-01

Leishmania is a genus of trypanosomes, which are responsible for leishmaniasis disease, a major trypanosome infection in humans. In recent years, published studies have shown that the search for new drugs for Leishmania treatments has intensified. Through technique modeling it has been possible to develop new compounds, which act as nucleoside hydrolase (NH) inhibitors. The effect of these enzymes is the hydrolysis of certain RNA nucleotides, which include uridine and inosine, necessary for the protozoa to transform certain nucleosides obtained from infected individuals into nucleobases for the preparation of their DNA. The obtention of NH inhibitors is very important to eliminate leishmaniasis disease in infected individuals. The aim of this study is to discuss the research and development of new agents for the treatment of Leishmania, and to stimulate the formulation of new NH inhibitors. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

2'-Deoxyribosyltransferase from Leishmania mexicana, an efficient biocatalyst for one-pot, one-step synthesis of nucleosides from poorly soluble purine bases.

PubMed

Crespo, N; Sánchez-Murcia, P A; Gago, F; Cejudo-Sanches, J; Galmes, M A; Fernández-Lucas, Jesús; Mancheño, José Miguel

2017-10-01

Processes catalyzed by enzymes offer numerous advantages over chemical methods although in many occasions the stability of the biocatalysts becomes a serious concern. Traditionally, synthesis of nucleosides using poorly water-soluble purine bases, such as guanine, xanthine, or hypoxanthine, requires alkaline pH and/or high temperatures in order to solubilize the substrate. In this work, we demonstrate that the 2'-deoxyribosyltransferase from Leishmania mexicana (LmPDT) exhibits an unusually high activity and stability under alkaline conditions (pH 8-10) across a broad range of temperatures (30-70 °C) and ionic strengths (0-500 mM NaCl). Conversely, analysis of the crystal structure of LmPDT together with comparisons with hexameric, bacterial homologues revealed the importance of the relationships between the oligomeric state and the active site architecture within this family of enzymes. Moreover, molecular dynamics and docking approaches provided structural insights into the substrate-binding mode. Biochemical characterization of LmPDT identifies the enzyme as a type I NDT (PDT), exhibiting excellent activity, with specific activity values 100- and 4000-fold higher than the ones reported for other PDTs. Interestingly, LmPDT remained stable during 36 h at different pH values at 40 °C. In order to explore the potential of LmPDT as an industrial biocatalyst, enzymatic production of several natural and non-natural therapeutic nucleosides, such as vidarabine (ara A), didanosine (ddI), ddG, or 2'-fluoro-2'-deoxyguanosine, was carried out using poorly water-soluble purines. Noteworthy, this is the first time that the enzymatic synthesis of 2'-fluoro-2'-deoxyguanosine, ara G, and ara H by a 2'-deoxyribosyltransferase is reported.

UHPLC-TQ-MS Coupled with Multivariate Statistical Analysis to Characterize Nucleosides, Nucleobases and Amino Acids in Angelicae Sinensis Radix Obtained by Different Drying Methods.

PubMed

Zhu, Shaoqing; Guo, Sheng; Duan, Jin-Ao; Qian, Dawei; Yan, Hui; Sha, Xiuxiu; Zhu, Zhenhua

2017-06-01

To explore the nutrients in roots of Angelica sinensis (Angelicae Sinensis Radix, ASR), a medicinal and edible plant, and evaluate its nutritional value, a rapid and reliable UHPLC-TQ-MS method was established and used to determine the potential nutritional compounds, including nucleosides, nucleobases and amino acids, in 50 batches of ASR samples obtained using two drying methods. The results showed that ASR is a healthy food rich in nucleosides, nucleobases and amino acids, especially arginine. The total average content of nucleosides and nucleobases in all ASR samples was 3.94 mg/g, while that of amino acids reached as high as 61.79 mg/g. Principle component analysis showed that chemical profile differences exist between the two groups of ASR samples prepared using different drying methods, and the contents of nutritional compounds in samples dried with the tempering-intermittent drying processing method (TIDM) were generally higher than those dried using the traditional solar processing method. The above results suggest that ASR should be considered an ideal healthy food and TIDM could be a suitable drying method for ASR when taking nucleosides, nucleobases and amino acids as the major consideration for their known human health benefits.

Nucleoside Analog-treated Chronic Hepatitis B Patients showed Reduced Expression of PECAM-1 Gene in Peripheral Blood Mononuclear Cells in Bangladesh

PubMed Central

Tabassum, Shahina; Ullah Munshi, Saif; Hossain, Marufa; Imam, Akhter

2014-01-01

ABSTRACT Background and aim Assessment of therapeutic response is important for monitoring the prognosis and to take decision for cessation of nucleoside analogues therapy in chronic hepatitis B patients. In addition to serum alanine aminotransferase (ALT), hepatitis B virus (HBV) deoxyribonucleic acid (DNA) load and HBeAg status, identification of molecular markers associated with host immune response would be essential to assess therapeutic response. In this regard the current study was performed with the aim to detect expression of platelet endothelial cell adhesion molecule (PECAM)-I gene in peripheral blood monocytes (PBMCs) of treated chronic hepatitis B patients and also to correlate expression of this gene with serum HBV DNA load and serum ALT levels. Materials and methods The study analyzed 60 chronic hepatitis B (CHB) patients, including 30 untreated and 30 nucleoside analogs treated and 10 healthy controls. PECAM-1 gene expression/ transcripts were detected by conventional RT-PCR. Results The expression PECAM-1 mRNA in the PBMCs of CHB patients was significantly higher in untreated (3.17 ± 0.75) than the treated patients (1.64 ± 0.29) (p < 0.01). Expression of PECAM-1 was positively correlated with serum ALT levels of both untreated (r = 0.580) and treated (r = 0.566) CHB patients. Moreover, in both untreated and treated groups, these gene expressions were positively correlated to serum HBV DNA load with the correlation coefficient r = 0.545 and r = 0.591 respectively. Conclusion PECAM-1 may be used as a biomarker for assessment of inflammatory activity as well as therapeutic response in CHB patients. How to cite this article: Sultana N, Tabassum S, Munshi SU, Hossain M, Imam A. Nucleoside Analog-treated Chronic Hepatitis B Patients showed Reduced Expression of PECAM-1 Gene in Peripheral Blood Mononuclear Cells in Bangladesh. Euroasian J Hepato-Gastroenterol 2014;4(2):87-91. PMID:29699354

Synthesis and Evaluation of a Novel Adenosine-Ribose Probe for Global-Scale Profiling of Nucleoside and Nucleotide-Binding Proteins

PubMed Central

Mahajan, Shikha; Manetsch, Roman; Merkler, David J.; Stevens Jr., Stanley M.

2015-01-01

Proteomics is a powerful approach used for investigating the complex molecular mechanisms of disease pathogenesis and progression. An important challenge in modern protein profiling approaches involves targeting of specific protein activities in order to identify altered molecular processes associated with disease pathophysiology. Adenosine-binding proteins represent an important subset of the proteome where aberrant expression or activity changes of these proteins have been implicated in numerous human diseases. Herein, we describe an affinity-based approach for the enrichment of adenosine-binding proteins from a complex cell proteome. A novel N 6-biotinylated-8-azido-adenosine probe (AdoR probe) was synthesized, which contains a reactive group that forms a covalent bond with the target proteins, as well as a biotin tag for affinity enrichment using avidin chromatography. Probe specificity was confirmed with protein standards prior to further evaluation in a complex protein mixture consisting of a lysate derived from mouse neuroblastoma N18TG2 cells. Protein identification and relative quantitation using mass spectrometry allowed for the identification of small variations in abundance of nucleoside- and nucleotide-binding proteins in these samples where a significant enrichment of AdoR-binding proteins in the labeled proteome from the neuroblastoma cells was observed. The results from this study demonstrate the utility of this method to enrich for nucleoside- and nucleotide-binding proteins in a complex protein mixture, pointing towards a unique set of proteins that can be examined in the context of further understanding mechanisms of disease, or fundamental biological processes in general. PMID:25671571

The search for and identification of amino acids, nucleobases and nucleosides in samples returned from Mars

NASA Technical Reports Server (NTRS)

Gehrke, Charles W.; Ponnamperuma, Cyril; Kuo, Kenneth C.; Stalling, David L.; Zumwalt, Robert W.

1989-01-01

An investigation of the returned Mars samples for biologically important organic compounds, with emphasis on amino acid, the puring and pyrimidine bases, and nucleosides is proposed. These studies would be conducted on subsurface samples obtained by drilling past the surface oxidizing layer with emphasis on samples containing the larges quantities of organic carbon as determined by the rover gas chromatographic mass spectrometer (GCMS). Extraction of these molecules from the returned samples will be performed using the hydrothermal extraction technique described by Cheng and Ponnamperuma. More rigorous extraction methods will be developed and evaluated. For analysis of the extract for free amino acids or amino acids present in a bound or peptidic form, aliquots will be analyzed by capillary GCMS both before and after hydrolysis with 6N hydrochloric acid. Establishment of the presence of amino acids would then lead to the next logical step which would be the use of chiral stationary gas chromatography phases to determine the enatiomeic composition of the amino acids present, and thus potentially establish their biotic or abiotic origin. Confirmational analyses for amino acids would include ion-exchange and reversed-phase liquid chromatographic analysis. For analyses of the returned Mars samples for nucleobases and nucleosides, affinity and reversed-phase liquid chromatography would be utilized. This technology coupled with scanning UV detection for identification, presents a powerful tool for nucleobase and nucleoside analysis. Mass spectrometric analysis of these compounds would confirm their presence in samples returned form Mars.

Combination nucleoside/nucleotide reverse transcriptase inhibitors for treatment of HIV infection.

PubMed

Akanbi, Maxwell O; Scarsi, Kimberly K; Scarci, Kimberly; Taiwo, Babafemi; Murphy, Robert L

2012-01-01

The combination of two nucleoside/nucleotide reverse transcriptase inhibitors (N(t)RTIs) and a third agent from another antiretroviral class is currently recommended for initial antiretroviral therapy. In general, N(t)RTIs remain relevant in subsequent regimens. There are currently six nucleoside reverse transcriptase inhibitors and one nucleotide reverse transcriptase inhibitor drug entities available, and several formulations that include two or more N(t)RTIs in a fixed-dose combination. These entities have heterogeneous pharmacological and clinical properties. Accordingly, toxicity, pill burden, dosing frequency, potential drug-drug interaction, preexisting antiretroviral drug resistance and comorbid conditions should be considered when constructing a regimen. This approach is critical in order to optimize virologic efficacy and clinical outcomes. This article reviews N(t)RTI combinations used in the treatment of HIV-infected adults. The pharmacological properties of each N(t)RTI, and the clinical trials that have influenced treatment guidelines are discussed. It is likely that N(t)RTIs will continue to dominate the global landscape of HIV treatment and prevention, despite emerging interest in N(t)RTI-free combination therapy. Clinical domains where only few alternatives to N(t)RTIs exist include treatment of HIV/HBV coinfection and HIV-2. There is a need for novel N(t)RTIs with enhanced safety and resistance profiles compared with current N(t)RTIs.

Active Methamphetamine Use is Associated with Transmitted Drug Resis-tance to Non-Nucleoside Reverse Transcriptase Inhibitors in Individuals with HIV Infection of Unknown Duration

PubMed Central

Cachay, Edward R; Moini, Niousha; Kosakovsky Pond, Sergei L; Pesano, Rick; Lie, Yolanda S; Aiem, Heidi; Butler, David M; Letendre, Scott; Mathews, Wm. Christopher; Smith, Davey M

2007-01-01

Background: Frequent methamphetamine use among recently HIV infected individuals is associated with transmitted drug resistance (TDR) to non-nucleoside reverse transcriptase inhibitors (NNRTI); however, the reversion time of TDR to drug susceptible HIV may exceed 3 years. We assessed whether recreational substance use is associated with detectable TDR among individuals newly diagnosed with HIV infection of unknown duration. Design: Cross-sectional analysis. Methods: Subjects were enrolled at the University California, San Diego Early Intervention Program. Demographic, clinical and substance use data were collected using structured interviews. Genotypic resistance testing was performed using GeneSeq™, Monogram Biosciences. We analyzed the association between substance use and TDR using bivariate analyses and the corresponding transmission networks using phylogenetic models. Results: Between April 2004 and July 2006, 115 individuals with genotype data were enrolled. The prevalence of alcohol, marijuana and methamphetamine use were 98%, 71% and 64% respectively. Only active methamphetamine use in the 30 days prior to HIV diagnosis was independently associated with TDR to NNRTI (OR: 6.6; p=0.002). Conclusion: Despite not knowing the duration of their HIV infection, individuals reporting active methamphetamine use in the 30 days prior to HIV diagnosis are at an increased risk of having HIV strains that are resistant to NNRTI. PMID:18923691

Helicase-primase inhibitors for herpes simplex virus: looking to the future of non-nucleoside inhibitors for treating herpes virus infections.

PubMed

Biswas, Subhajit; Sukla, Soumi; Field, Hugh J

2014-01-01

Helicase-primase inhibitors (HPIs) are the first new family of potent herpes virus (herpes simplex and varicella-zoster virus) inhibitors to go beyond the preliminary stages of investigation since the emergence of the original nucleoside analog inhibitors. To consider the clinical future of HPIs, this review puts the exciting new findings with two HPIs, amenamevir and pritelivir, into the historical context of antiviral development for the prevention and treatment of herpes simplex virus over the last century and, on this basis, the authors speculate on the potential evolution of these and other non-nucleoside inhibitors in the future.

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.

A new nucleoside analog, 9-[[2-hydroxy-1-(hydroxymethyl)ethoxyl]methyl]guanine, highly active in vitro against herpes simplex virus types 1 and 2.

PubMed Central

Smith, K O; Galloway, K S; Kennell, W L; Ogilvie, K K; Radatus, B K

1982-01-01

A novel nucleoside analog, 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]-guanine (BIOLF-62), was found to have potent antiviral activity against herpes simplex virus types 1 and 2 at concentrations well below cytotoxic levels. For example, the Patton strain of herpes simplex virus type 1 was susceptible at concentrations 140- to 2,900-fold below that which inhibited cell division by 50%, depending upon the cell line used for assay. Different herpesvirus strains varied considerably in their susceptibility to the drug, as did results obtained with the same virus strain in different cell lines. BIOLF-62 compared favorably with 5-iodo-2'-deoxyuridine and acyclovir with respect to ratios of viral to cell inhibitory drug concentrations. Patterns of drug resistance to herpesvirus mutants suggested that the primary mode of action of BIOLF-62 is different from that of known antiviral compounds. Human adenovirus type 2, varicella-zoster virus, and Epstein-Barr virus were inhibited by this drug but at concentrations within the cell inhibitory range. Vaccinia virus and human cytomegalovirus were not inhibited at high drug concentrations. PMID:6289741

Comparative characterization of nucleotides, nucleosides and nucleobases in Abelmoschus manihot roots, stems, leaves and flowers during different growth periods by UPLC-TQ-MS/MS.

PubMed

Du, Le-Yue; Qian, Da-Wei; Jiang, Shu; Shang, Er-Xin; Guo, Jian-Ming; Liu, Pei; Su, Shu-Lan; Duan, Jin-Ao; Zhao, Min

2015-12-01

Nucleotides, nucleosides and nucleobases have been proven as important bioactive compounds related to many physiological processes. Abelmoschus manihot (L.) Medicus from the family of Malvaceae is an annual herbal plant of folk medicine widely distributed in Oceania and Asia. However, up to now, no detailed information could be available for the types and contents of nucleotides, nucleosides and nucleobases contained in A. manihot roots, stems, leaves as well as the flowers. In the present study, an UPLC-TQ-MS/MS method was established for detection of the twelve nucleotides, nucleosides and nucleobases. The validated method was successfully applied to identify the 12 analytes in different parts of A. manihot harvested at ten growth periods. 2'-deoxyinosine was not detected in all of the A. manihot samples. The data demonstrated that the distribution and concentration of the 12 compounds in A. manihot four parts were arranged in a decreasing order as leaf>flower>stem>root. Based on the results, the leaves and flowers of A. manihot could be developed as health products possessed nutraceutical and bioactive properties in the future. This method might also be utilized for the quality control of the A. manihot leaves and other herbal medicines being rich in nucleotides, nucleosides and nulecobases. Copyright © 2015 Elsevier B.V. All rights reserved.

Zebrafish-based identification of the antiseizure nucleoside inosine from the marine diatom Skeletonema marinoi

PubMed Central

Khamma, Supitcha; Marcourt, Laurence; Righi, Davide; Romano, Giovanna; Esposito, Francesco; Ianora, Adrianna; Queiroz, Emerson F.; Wolfender, Jean-Luc; Crawford, Alexander D.

2018-01-01

With the goal of identifying neuroactive secondary metabolites from microalgae, a microscale in vivo zebrafish bioassay for antiseizure activity was used to evaluate bioactivities of the diatom Skeletonema marinoi, which was recently revealed as being a promising source of drug-like small molecules. A freeze-dried culture of S. marinoi was extracted by solvents with increasing polarities (hexane, dichloromethane, methanol and water) and these extracts were screened for anticonvulsant activity using a larval zebrafish epilepsy model with seizures induced by the GABAA antagonist pentylenetetrazole. The methanolic extract of S. marinoi exhibited significant anticonvulsant activity and was chosen for bioassay-guided fractionation, which associated the bioactivity with minor constituents. The key anticonvulsant constituent was identified as the nucleoside inosine, a well-known adenosine receptor agonist with previously reported antiseizure activities in mice and rat epilepsy models, but not reported to date as a bioactive constituent of microalgae. In addition, a UHPLC-HRMS metabolite profiling was used for dereplication of the other constituents of S. marinoi. Structures of the isolated compounds were elucidated by nuclear magnetic resonance and high-resolution spectrometry. These results highlight the potential of zebrafish-based screening and bioassay-guided fractionation to identify neuroactive marine natural products. PMID:29689077

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

The spread of Ras activity triggered by activation of a single dendritic spine.

PubMed

Harvey, Christopher D; Yasuda, Ryohei; Zhong, Haining; Svoboda, Karel

2008-07-04

In neurons, individual dendritic spines isolate N-methyl-d-aspartate (NMDA) receptor-mediated calcium ion (Ca2+) accumulations from the dendrite and other spines. However, the extent to which spines compartmentalize signaling events downstream of Ca2+ influx is not known. We combined two-photon fluorescence lifetime imaging with two-photon glutamate uncaging to image the activity of the small guanosine triphosphatase Ras after NMDA receptor activation at individual spines. Induction of long-term potentiation (LTP) triggered robust Ca2+-dependent Ras activation in single spines that decayed in approximately 5 minutes. Ras activity spread over approximately 10 micrometers of dendrite and invaded neighboring spines by diffusion. The spread of Ras-dependent signaling was necessary for the local regulation of the threshold for LTP induction. Thus, Ca2+-dependent synaptic signals can spread to couple multiple synapses on short stretches of dendrite.

l-2',3'-Didehydro-2',3'-dideoxy-3'-fluoronucleosides: synthesis, anti-HIV activity, chemical and enzymatic stability, and mechanism of resistance.

PubMed

Chong, Youhoon; Gumina, Giuseppe; Mathew, Judy S; Schinazi, Raymond F; Chu, Chung K

2003-07-17

As antiviral nucleosides containing a 2',3'-unsaturated sugar moiety with 2'-fluoro substitution are endowed with increased stabilization of the glycosyl bond, it was of interest to investigate the influence of the fluorine atom at the 3'-position. Various pyrimidine and purine L-3'-fluoro-2',3'-unsaturated nucleosides were synthesized from their precursors, L-3',3'-difluoro-2',3'-dideoxy nucleosides, by elimination of hydrogen fluoride. In the L-3',3'-difluoro-2',3'-dideoxy nucleoside series, cytidine 16 and 5-fluorocytidine 18 analogues showed modest antiviral activity (EC(50) 11.5 and 8.8 microM, respectively) when evaluated against HIV-1 in human peripheral blood mononuclear (PBM) cells. In the 2',3'-unsaturated series, L-3'-fluoro-2',3'-didehydro-2',3'-dideoxycytidine 24 and 5-fluorocytidine 26 showed highly potent antiviral activity (EC(50) 0.089 and 0.018 microM, respectively) without significant cytotoxicity. The guanosine analogue 48 showed only marginal anti-HIV activity with some cytotoxicity (EC(50) 38.5 microM, and IC(50) 17.4, 58.4, 36.5 microM in PBM, CEM, and Vero cells, respectively). The cytidine 24 and 5-fluorocytidine 26 analogues, however, showed significantly decreased antiviral activity against the clinically important lamivudine-resistant variants (HIV-1(M184V)). Molecular modeling studies demonstrated that the 3'-fluoro atom of the L-3'-fluoro-2',3'-unsaturated nucleoside is within the hydrogen bonding distance with the amide backbone of Asp185, which favors the binding of the nucleoside triphosphate to the wild-type RT. This favorable binding mode, however, cannot be maintained when the triphosphate of 3'-fluoro 2',3'-unsaturated nucleoside binds to the active site of M184V RT because the bulky side chain of Val184 occupies the space needed for the nucleotide. The biological results suggest that, in addition to the sugar conformation, the base moiety may also play a role in their interaction with the M184V RT.

[Determination of 5 nucleosides components in culture of Paecilomyces hepialid by HPLC].

PubMed

Yang, Dan; Ma, Yun-shu; Huang, Ting-ting; Chen, Cheng

2015-08-01

The concentration of 5 nucleosides, uracil, uridine, guanidine, adenine and adenosine in culture of Paecilomyces hepialid was determined by the developed method of HPLC. The HPLC method was performed on a Waters SunFire C18 (4.6 mm x 250 mm, 5 μm) column with methanol-water gradient elution as the mobile phase. The detection wavelength was 260 nm and the colunmn temperature was controlled at 30 °C. The linear range was 10.00-200.00 mg · L(-1) (r = 0.9994) for uracil, 10.10-202.00 mg · L(-1) (r = 0.9992) for uridine, 10.00-200.00 mg · L(-1) (r = 0.9991) for guanidine, 10.30-206.00 mg · L(-1) (r = 0.9992) for adenine and 10.45-209.00 mg · L(-1) (r = 0.9991) for adenosine, respectively. The RSD of precision was 0.032%, 0.035%, 0.039%, 0.049%, 0.00080%, respectively. The average recoveries of uracil, guanidine, adenine, and adenosine were 97.34%, 99.10%, 101.6%, 98.61% and 100.2% with RSD of 1.3%, 2.1%, 0.96%, 0.95%, and 1.3% respectively. The method showed high sensitivity, good selectivity, linearity and repeatability, which was suitable for the content analysis of 5 nucleosides components in P. hepialid and its extracts.

RubisCO of a nucleoside pathway known from Archaea is found in diverse uncultivated phyla in bacteria

PubMed Central

Wrighton, Kelly C; Castelle, Cindy J; Varaljay, Vanessa A; Satagopan, Sriram; Brown, Christopher T; Wilkins, Michael J; Thomas, Brian C; Sharon, Itai; Williams, Kenneth H; Tabita, F Robert; Banfield, Jillian F

2016-01-01

Metagenomic studies recently uncovered form II/III RubisCO genes, originally thought to only occur in archaea, from uncultivated bacteria of the candidate phyla radiation (CPR). There are no isolated CPR bacteria and these organisms are predicted to have limited metabolic capacities. Here we expand the known diversity of RubisCO from CPR lineages. We report a form of RubisCO, distantly similar to the archaeal form III RubisCO, in some CPR bacteria from the Parcubacteria (OD1), WS6 and Microgenomates (OP11) phyla. In addition, we significantly expand the Peregrinibacteria (PER) II/III RubisCO diversity and report the first II/III RubisCO sequences from the Microgenomates and WS6 phyla. To provide a metabolic context for these RubisCOs, we reconstructed near-complete (>93%) PER genomes and the first closed genome for a WS6 bacterium, for which we propose the phylum name Dojkabacteria. Genomic and bioinformatic analyses suggest that the CPR RubisCOs function in a nucleoside pathway similar to that proposed in Archaea. Detection of form II/III RubisCO and nucleoside metabolism gene transcripts from a PER supports the operation of this pathway in situ. We demonstrate that the PER form II/III RubisCO is catalytically active, fixing CO2 to physiologically complement phototrophic growth in a bacterial photoautotrophic RubisCO deletion strain. We propose that the identification of these RubisCOs across a radiation of obligately fermentative, small-celled organisms hints at a widespread, simple metabolic platform in which ribose may be a prominent currency. PMID:27137126

RubisCO of a nucleoside pathway known from Archaea is found in diverse uncultivated phyla in bacteria

DOE PAGES

Wrighton, Kelly C.; Castelle, Cindy J.; Varaljay, Vanessa A.; ...

2016-05-03

Metagenomic studies recently uncovered form II/III RubisCO genes, originally thought to only occur in archaea, from uncultivated bacteria of the candidate phyla radiation (CPR). There are no isolated CPR bacteria and these organisms are predicted to have limited metabolic capacities. Here we expand the known diversity of RubisCO from CPR lineages. We report a form of RubisCO, distantly similar to the archaeal form III RubisCO, in some CPR bacteria from the Parcubacteria (OD1), WS6 and Microgenomates (OP11) phyla. In addition, we significantly expand the Peregrinibacteria (PER) II/III RubisCO diversity and report the first II/III RubisCO sequences from the Microgenomates andmore » WS6 phyla. To provide a metabolic context for these RubisCOs, we reconstructed near-complete ( > 93%) PER genomes and the first closed genome for a WS6 bacterium, for which we propose the phylum name Dojkabacteria. Genomic and bioinformatic analyses suggest that the CPR RubisCOs function in a nucleoside pathway similar to that proposed in Archaea. Detection of form II/III RubisCO and nucleoside metabolism gene transcripts from a PER supports the operation of this pathway in situ. We demonstrate that the PER form II/III RubisCO is catalytically active, fixing CO 2 to physiologically complement phototrophic growth in a bacterial photoautotrophic RubisCO deletion strain. We propose that the identification of these RubisCOs across a radiation of obligately fermentative, small-celled organisms hints at a widespread, simple metabolic platform in which ribose may be a prominent currency.« less

Prolonged lifespan with enhanced exploratory behavior in mice overexpressing the oxidized nucleoside triphosphatase hMTH1.

PubMed

De Luca, Gabriele; Ventura, Ilenia; Sanghez, Valentina; Russo, Maria Teresa; Ajmone-Cat, Maria Antonietta; Cacci, Emanuele; Martire, Alberto; Popoli, Patrizia; Falcone, Germana; Michelini, Flavia; Crescenzi, Marco; Degan, Paolo; Minghetti, Luisa; Bignami, Margherita; Calamandrei, Gemma

2013-08-01

The contribution that oxidative damage to DNA and/or RNA makes to the aging process remains undefined. In this study, we used the hMTH1-Tg mouse model to investigate how oxidative damage to nucleic acids affects aging. hMTH1-Tg mice express high levels of the hMTH1 hydrolase that degrades 8-oxodGTP and 8-oxoGTP and excludes 8-oxoguanine from both DNA and RNA. Compared to wild-type animals, hMTH1-overexpressing mice have significantly lower steady-state levels of 8-oxoguanine in both nuclear and mitochondrial DNA of several organs, including the brain. hMTH1 overexpression prevents the age-dependent accumulation of DNA 8-oxoguanine that occurs in wild-type mice. These lower levels of oxidized guanines are associated with increased longevity and hMTH1-Tg animals live significantly longer than their wild-type littermates. Neither lipid oxidation nor overall antioxidant status is significantly affected by hMTH1 overexpression. At the cellular level, neurospheres derived from adult hMTH1-Tg neural progenitor cells display increased proliferative capacity and primary fibroblasts from hMTH1-Tg embryos do not undergo overt senescence in vitro. The significantly lower levels of oxidized DNA/RNA in transgenic animals are associated with behavioral changes. These mice show reduced anxiety and enhanced investigation of environmental and social cues. Longevity conferred by overexpression of a single nucleotide hydrolase in hMTH1-Tg animals is an example of lifespan extension associated with healthy aging. It provides a link between aging and oxidative damage to nucleic acids. © 2013 John Wiley & Sons Ltd and the Anatomical Society.

Broad-spectrum non-nucleoside inhibitors for caliciviruses.

PubMed

Netzler, Natalie E; Enosi Tuipulotu, Daniel; Eltahla, Auda A; Lun, Jennifer H; Ferla, Salvatore; Brancale, Andrea; Urakova, Nadya; Frese, Michael; Strive, Tanja; Mackenzie, Jason M; White, Peter A

2017-10-01

Viruses of the Caliciviridae cause significant and sometimes lethal diseases, however despite substantial research efforts, specific antivirals are lacking. Broad-spectrum antivirals could combat multiple viral pathogens, offering a rapid solution when no therapies exist. The RNA-dependent RNA polymerase (RdRp) is an attractive antiviral target as it is essential for viral replication and lacks mammalian homologs. To focus the search for pan-Caliciviridae antivirals, the RdRp was probed with non-nucleoside inhibitors (NNIs) developed against hepatitis C virus (HCV) to reveal both allosteric ligands for structure-activity relationship enhancement, and highly-conserved RdRp pockets for antiviral targeting. The ability of HCV NNIs to inhibit calicivirus RdRp activities was assessed using in vitro enzyme and murine norovirus cell culture assays. Results revealed that three NNIs which bound the HCV RdRp Thumb I (TI) site also inhibited transcriptional activities of six RdRps spanning the Norovirus, Sapovirus and Lagovirus genera of the Caliciviridae. These NNIs included JTK-109 (RdRp inhibition range: IC 50 4.3-16.6 μM), TMC-647055 (IC 50 range: 18.8-45.4 μM) and Beclabuvir (IC 50 range: 23.8->100 μM). In silico studies and site-directed mutagenesis indicated the JTK-109 binding site was within the calicivirus RdRp thumb domain, in a pocket termed Site-B, which is highly-conserved within all calicivirus RdRps. Additionally, RdRp inhibition assays revealed that JTK-109 was antagonistic with the previously reported RdRp inhibitor pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) tetrasodium salt (PPNDS), that also binds to Site-B. Moreover, like JTK-109, PPNDS was also a potent inhibitor of polymerases from six viruses spanning the three Caliciviridae genera tested (IC 50 range: 0.1-2.3 μM). Together, this study demonstrates the potential for de novo development of broad-spectrum antivirals that target the highly-conserved RdRp thumb pocket

Nucleoside-(5'→P) methylenebisphosphonodithioate analogues: synthesis and chemical properties.

PubMed

Meltzer, Diana; Nadel, Yael; Lecka, Joanna; Amir, Aviran; Sévigny, Jean; Fischer, Bilha

2013-09-06

Nucleoside-(5'→P) methylenebisphosphonodithioate analogues are bioisosteres of natural nucleotides. The potential therapeutic applications of these analogues are limited by their relative instability. With a view toward improving their chemical and metabolic stability as well as their affinity toward zinc ions, we developed a novel nucleotide scaffold, nucleoside-5'-tetrathiobisphosphonate. We synthesized P1-(uridine/adenosine-5')-methylenebisphosphonodithioate, 2 and 3, and P1,P2-di(uridine/adenosine-5')-methylenebisphosphonodithioate, 4 and 5. Using (1)H and (31)P NMR-monitored Zn(2+)/Mg(2+) titrations, we found that 5 coordinated Zn(2+) by both N7 nitrogen atoms and both dithiophosphonate moieties, whereas 3 coordinated Zn(2+) by an N7 nitrogen atom and Pβ. Both 3 and 5 did not coordinate Mg(2+) ions. (31)P NMR-monitored kinetic studies showed that 3 was more stable at pD 1.5 than 5, with t(1/2) of 44 versus 9 h, respectively, and at pD 11 both showed no degradation for at least 2 weeks. However, 5 was more stable than 3 under an air-oxidizing atmosphere, with t1/2 of at least 3 days versus 14 h, respectively. Analogues 3 and 5 were highly stable to NPP1,3 and NTPDase1,2,3,8 hydrolysis (0-7%). However, they were found to be poor ectonucleotidase inhibitors. Although 3 and 5 did not prove to be effective inhibitors of zinc-containing NPP1/3, which is involved in the pathology of osteoarthritis and diabetes, they may be promising zinc chelators for the treatment of other health disorders involving an excess of zinc ions.

Photoelectron and computational studies of the copper-nucleoside anionic complexes, Cu(-)(cytidine) and Cu(-)(uridine).

PubMed

Li, Xiang; Ko, Yeon-Jae; Wang, Haopeng; Bowen, Kit H; Guevara-García, Alfredo; Martínez, Ana

2011-02-07

The copper-nucleoside anions, Cu(-)(cytidine) and Cu(-)(uridine), have been generated in the gas phase and studied by both experimental (anion photoelectron spectroscopy) and theoretical (density functional calculations) methods. The photoelectron spectra of both systems are dominated by single, intense, and relatively narrow peaks. These peaks are centered at 2.63 and 2.71 eV for Cu(-)(cytidine) and Cu(-)(uridine), respectively. According to our calculations, Cu(-)(cytidine) and Cu(-)(uridine) species with these peak center [vertical detachment energy (VDE)] values correspond to structures in which copper atomic anions are bound to the sugar portions of their corresponding nucleosides largely through electrostatic interactions; the observed species are anion-molecule complexes. The combination of experiment and theory also reveal the presence of a slightly higher energy, anion-molecule complex isomer in the case of the Cu(-)(cytidine). Furthermore, our calculations found that chemically bond isomers of these species are much more stable than their anion-molecule complex counterparts, but since their calculated VDE values are larger than the photon energy used in these experiments, they were not observed.

Photoelectron and computational studies of the copper-nucleoside anionic complexes, Cu-(cytidine) and Cu-(uridine)

NASA Astrophysics Data System (ADS)

Li, Xiang; Ko, Yeon-Jae; Wang, Haopeng; Bowen, Kit H.; Guevara-García, Alfredo; Martínez, Ana

2011-02-01

The copper-nucleoside anions, Cu-(cytidine) and Cu-(uridine), have been generated in the gas phase and studied by both experimental (anion photoelectron spectroscopy) and theoretical (density functional calculations) methods. The photoelectron spectra of both systems are dominated by single, intense, and relatively narrow peaks. These peaks are centered at 2.63 and 2.71 eV for Cu-(cytidine) and Cu-(uridine), respectively. According to our calculations, Cu-(cytidine) and Cu-(uridine) species with these peak center [vertical detachment energy (VDE)] values correspond to structures in which copper atomic anions are bound to the sugar portions of their corresponding nucleosides largely through electrostatic interactions; the observed species are anion-molecule complexes. The combination of experiment and theory also reveal the presence of a slightly higher energy, anion-molecule complex isomer in the case of the Cu-(cytidine). Furthermore, our calculations found that chemically bond isomers of these species are much more stable than their anion-molecule complex counterparts, but since their calculated VDE values are larger than the photon energy used in these experiments, they were not observed.

Nelfinavir and non-nucleoside reverse transcriptase inhibitor-based salvage regimens in heavily HIV pretreated patients

PubMed Central

Baril, Jean-Guy; Lefebvre, Eric A; Lalonde, Richard G; Shafran, Stephen D; Conway, Brian

2003-01-01

OBJECTIVE: To assess the efficacy of nelfinavir mesylate (NFV) in combination with delavirdine mesylate (DLV) or efavirenz (EFV) and other antiretroviral agents following virological failure on other protease inhibitor (PI)-based regimens. DESIGN: Multicentre, retrospective chart review. METHODS: One hundred-one patients who were naive to both NFV and non-nucleoside reverse transcriptase inhibitors (NNRTIs) and who initiated NFV plus DLV or EFV-based salvage regimens were reviewed. Response to treatment was defined as a reduction in HIV ribonucleic acid (RNA) levels to unquantifiable levels (less than 50 copies/mL, less than 400 copies/mL, less than 500 copies/mL) on at least one occasion after the initiation of salvage therapy. Baseline correlates of response, including prior duration of HIV infection, prior number of regimens, viral load and CD4 cell counts were also evaluated. RESULTS: Patients had a mean duration of HIV infection of 10 years, a mean duration of prior therapy of four years, a median of four prior nucleoside reverse transcriptase inhibitors and a median of two prior PIs. At the time of review the mean duration of salvage therapy was 63.4 weeks. Virological suppression was achieved in 59 (58.4%) patients within a mean of eight weeks and maintained for a mean of 44.9 weeks (the mean follow-up was 78 weeks). Of the non-responders, 16 (38%) achieved a less than 1 log10 decrease in HIV RNA levels. Although there was no association between baseline correlates, response rate (75.7%) was significantly higher in patients with HIV RNA levels of 50,000 copies/mL or lower and CD4 counts greater than 200 cells/mm3. CONCLUSION: NFV/NNRTI-based highly active antiretroviral therapy regimens are an effective therapy in many patients who have experienced virological breakthroughs on at least one prior PI-based regimen. PMID:18159457

Structural optimization of N1-aryl-benzimidazoles for the discovery of new non-nucleoside reverse transcriptase inhibitors active against wild-type and mutant HIV-1 strains.

PubMed

Monforte, Anna Maria; De Luca, Laura; Buemi, Maria Rosa; Agharbaoui, Fatima E; Pannecouque, Christophe; Ferro, Stefania

2018-02-01

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are recommended components of preferred combination antiretroviral therapies used for the treatment of human immunodeficiency virus (HIV) infection. These regimens are extremely effective in suppressing virus replication. Recently, our research group identified some N 1 -aryl-2-arylthioacetamido-benzimidazoles as a novel class of NNRTIs. In this research work we report the design, the synthesis and the structure-activity relationship studies of new compounds (20-34) in which some structural modifications have been introduced in order to investigate their effects on reverse transcriptase (RT) inhibition and to better define the features needed to increase the antiviral activity. Most of the new compounds proved to be highly effective in inhibiting both RT enzyme at nanomolar concentrations and HIV-1 replication in MT4 cells with minimal cytotoxicity. Among them, the most promising N 1 -aryl-2-arylthioacetamido-benzimidazoles and N 1 -aryl-2-aryloxyacetamido-benzimidazoles were also tested toward a panel of single- and double-mutants strain responsible for resistance to NNRTIs, showing in vitro antiviral activity toward single mutants L100I, K103N, Y181C, Y188L and E138K. The best results were observed for derivatives 29 and 33 active also against the double mutants F227L and V106A. Computational approaches were applied in order to rationalize the potency of the new synthesized inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Discovery of Potent Non-Nucleoside Inhibitors of Dengue Viral RNA-Dependent RNA Polymerase from a Fragment Hit Using Structure-Based Drug Design.

PubMed

Yokokawa, Fumiaki; Nilar, Shahul; Noble, Christian G; Lim, Siew Pheng; Rao, Ranga; Tania, Stefani; Wang, Gang; Lee, Gladys; Hunziker, Jürg; Karuna, Ratna; Manjunatha, Ujjini; Shi, Pei-Yong; Smith, Paul W

2016-04-28

The discovery and optimization of non-nucleoside dengue viral RNA-dependent-RNA polymerase (RdRp) inhibitors are described. An X-ray-based fragment screen of Novartis' fragment collection resulted in the identification of a biphenyl acetic acid fragment 3, which bound in the palm subdomain of RdRp. Subsequent optimization of the fragment hit 3, relying on structure-based design, resulted in a >1000-fold improvement in potency in vitro and acquired antidengue activity against all four serotypes with low micromolar EC50 in cell-based assays. The lead candidate 27 interacts with a novel binding pocket in the palm subdomain of the RdRp and exerts a promising activity against all clinically relevant dengue serotypes.

The structural modification of DNA nucleosides by nonenzymatic glycation: an in vitro study based on the reactions of glyoxal and methylglyoxal with 2'-deoxyguanosine.

PubMed

Li, Yuyuan; Cohenford, Menashi A; Dutta, Udayan; Dain, Joel A

2008-01-01

Methylglyoxal and glyoxal are generated from the oxidation of carbohydrates and lipids, and like D-glucose have been shown to nonenzymatically react with proteins to form advanced glycation end products (AGEs). AGEs can occur both in vitro and in vivo, and these compounds have been shown to exacerbate many of the long-term complications of diabetes. Earlier studies in our laboratory reported D-glucose, D-galactose, and D/L-glyceraldehyde formed AGEs with nucleosides. The objective of this study was to focus on purines and pyrimidines and to analyze these DNA nucleoside derived AGE adducts with glyoxal or methylglyoxal using a combination of analytical techniques. Studies using UV and fluorescence spectroscopy along with mass spectrometry provided for a thorough analysis of the nucleoside AGEs and demonstrated that methylglyoxal and glyoxal reacted with 2'-deoxyguanosine via the classic Amadori pathway, and did not react appreciably with 2'-deoxyadenosine, 2'-deoxythymidine, and 2'-deoxycytidine. Additional findings revealed that methylglyoxal was more reactive than glyoxal.

Novel Carboranyl Derivatives of Nucleoside Mono- and Diphosphites and Phosphonates: A Synthetic Investigation

PubMed Central

Vyakaranam, Kamesh; Hosmane, Narayan S.

2004-01-01

A number of nucleoside mono- and diphosphites and phosphonates containing 1,2-dicarbadodecaborane (12) (la-6b) at 5'-position of the sugar moiety have been synthesized in good yields. Experimental details along with the spectroscopic and analytical data, supporting the formation of the title compounds, are presented. These constitute a new generation of boron compounds that are envisioned to be useful in cancer treatment via Boron Neutron Capture Therapy (BNCT). PMID:18365067

Mechanochemistry of nucleosides, nucleotides and related materials

PubMed Central

Eguaogie, Olga; Conlon, Patrick F; Gîlea, Manuela A; Liang, Yipei

2018-01-01

The application of mechanical force to induce the formation and cleavage of covalent bonds is a rapidly developing field within organic chemistry which has particular value in reducing or eliminating solvent usage, enhancing reaction rates and also in enabling the preparation of products which are otherwise inaccessible under solution-phase conditions. Mechanochemistry has also found recent attention in materials chemistry and API formulation during which rearrangement of non-covalent interactions give rise to functional products. However, this has been known to nucleic acids science almost since its inception in the late nineteenth century when Miescher exploited grinding to facilitate disaggregation of DNA from tightly bound proteins through selective denaturation of the latter. Despite the wide application of ball milling to amino acid chemistry, there have been limited reports of mechanochemical transformations involving nucleoside or nucleotide substrates on preparative scales. A survey of these reactions is provided, the majority of which have used a mixer ball mill and display an almost universal requirement for liquid to be present within the grinding vessel. Mechanochemistry of charged nucleotide substrates, in particular, provides considerable benefits both in terms of efficiency (reducing total processing times from weeks to hours) and by minimising exposure to aqueous conditions, access to previously elusive materials. In the absence of large quantities of solvent and heating, side-reactions can be reduced or eliminated. The central contribution of mechanochemistry (and specifically, ball milling) to the isolation of biologically active materials derived from nuclei by grinding will also be outlined. Finally non-covalent associative processes involving nucleic acids and related materials using mechanochemistry will be described: specifically, solid solutions, cocrystals, polymorph transitions, carbon nanotube dissolution and inclusion complex formation

The public health approach to identify antiretroviral therapy failure: high-level nucleoside reverse transcriptase inhibitor resistance among Malawians failing first-line antiretroviral therapy

PubMed Central

Hosseinipour, Mina C.; van Oosterhout, Joep J.G.; Weigel, Ralf; Phiri, Sam; Kamwendo, Debbie; Parkin, Neil; Fiscus, Susan A.; Nelson, Julie A.E.; Eron, Joseph J.; Kumwenda, Johnstone

2010-01-01

Background Over 150 000 Malawians have started antiretroviral therapy (ART), in which first-line therapy is stavudine/lamivudine/nevirapine. We evaluated drug resistance patterns among patients failing first-line ART on the basis of clinical or immunological criteria in Lilongwe and Blantyre, Malawi. Methods Patients meeting the definition of ART failure (new or progressive stage 4 condition, CD4 cell count decline more than 30%, CD4 cell count less than that before treatment) from January 2006 to July 2007 were evaluated. Among those with HIV RNA of more than 1000 copies/ml, genotyping was performed. For complex genotype patterns, phenotyping was performed. Results Ninety-six confirmed ART failure patients were identified. Median (interquartile range) CD4 cell count, log10 HIV-1 RNA, and duration on ART were 68 cells/μl (23–174), 4.72 copies/ml (4.26–5.16), and 36.5 months (26.6–49.8), respectively. Ninety-three percent of samples had nonnucleoside reverse transcriptase inhibitor mutations, and 81% had the M184V mutation. The most frequent pattern included M184V and nonnucleoside reverse transcriptase inhibitor mutations along with at least one thymidine analog mutation (56%). Twenty-three percent of patients acquired the K70E or K65R mutations associated with tenofovir resistance; 17% of the patients had pan-nucleoside resistance that corresponded to K65R or K70E and additional resistance mutations, most commonly the 151 complex. Emergence of the K65R and K70E mutations was associated with CD4 cell count of less than 100 cells/μl (odds ratio 6.1) and inversely with the use of zidovudine (odds ratio 0.18). Phenotypic susceptibility data indicated that the nucleoside reverse transcriptase inhibitor backbone with the highest activity for subsequent therapy was zidovudine/lamivudine/tenofovir, followed by lamivudine/tenofovir, and then abacavir/didanosine. Conclusion When clinical and CD4 cell count criteria are used to monitor first-line ART failure

Nucleotides and nucleosides in ovine and caprine milk during lactation.

PubMed

Plakantara, S; Michaelidou, A-M; Polychroniadou, A; Menexes, G; Alichanidis, E

2010-06-01

The aim of this study was to determine the nucleoside and nucleotide content in ovine and caprine milks at the colostral, transitional, and mature stages of lactation. Samples from 18 dairy sheep and 18 dairy goats were collected at 1, 2, 3, 4, 5, and 15 d postpartum. Separation and quantitation of the 5'-nucleotides (NT) and the nucleosides (NS) was performed by reverse phase HPLC. For each compound measured, considerable interindividual variation was recorded in both species of milk. The total NS content ranged from 57 to 132 micromol/L and from 54 to 119 micromol/L in ovine and caprine milk, respectively. The major NS identified in both species of milk was uridine, representing more than 60% of the total NS pool. The mean levels of inosine and guanosine were comparable between ewe and goat milk. Instead, the mean level of cytidine across the sampling period was much higher in ewe milk (11.9 micromol/L compared with 4.5 micromol/L in goat milk) and exhibited a peak value on the fourth day of lactation. The adenosine content was at least 3-fold higher in caprine milk compared with its ovine counterpart. The total NS and orotic acid contents did not differ significantly between the 2 species. However, in the case of total NT content, interspecies differences were significant, with NT levels ranging from 294 to 441 micromol/L in ovine milk and from 166 to 366 micromol/L in caprine milk. The NT content in colostrum (1-3 d) of both species was higher than in mature milk (15 d), and uridine monophosphate was the dominant NT in all samples. 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Hydrolytic Glycosidic Bond Cleavage in RNA Nucleosides: Effects of the 2'-Hydroxy Group and Acid-Base Catalysis.

PubMed

Lenz, Stefan A P; Kohout, Johnathan D; Wetmore, Stacey D

2016-12-22

Despite the inherent stability of glycosidic linkages in nucleic acids that connect the nucleobases to sugar-phosphate backbones, cleavage of these bonds is often essential for organism survival. The current study uses DFT (B3LYP) to provide a fundamental understanding of the hydrolytic deglycosylation of the natural RNA nucleosides (A, C, G, and U), offers a comparison to DNA hydrolysis, and examines the effects of acid, base, or simultaneous acid-base catalysis on RNA deglycosylation. By initially examining HCOO - ···H 2 O mediated deglycosylation, the barriers for RNA hydrolysis were determined to be 30-38 kJ mol -1 higher than the corresponding DNA barriers, indicating that the 2'-OH group stabilizes the glycosidic bond. Although the presence of HCOO - as the base (i.e., to activate the water nucleophile) reduces the barrier for uncatalyzed RNA hydrolysis (i.e., unactivated H 2 O nucleophile) by ∼15-20 kJ mol -1 , the extreme of base catalysis as modeled using a fully deprotonated water molecule (i.e., OH - nucleophile) decreases the uncatalyzed barriers by up to 65 kJ mol -1 . Acid catalysis was subsequently examined by selectively protonating the hydrogen-bond acceptor sites of the RNA nucleobases, which results in an up to ∼80 kJ mol -1 barrier reduction relative to the corresponding uncatalyzed pathway. Interestingly, the nucleobase proton acceptor sites that result in the greatest barrier reductions match sites typically targeted in enzyme-catalyzed reactions. Nevertheless, simultaneous acid and base catalysis is the most beneficial way to enhance the reactivity of the glycosidic bonds in RNA, with the individual effects of each catalytic approach being weakened, additive, or synergistic depending on the strength of the base (i.e., degree of water nucleophile activation), the nucleobase, and the hydrogen-bonding acceptor site on the nucleobase. Together, the current contribution provides a greater understanding of the reactivity of the glycosidic

Development and evaluation of a hydrophilic interaction liquid chromatography-MS/MS method to quantify 19 nucleobases and nucleosides in rat plasma.

PubMed

Du, Yan; Li, Yin-Jie; Hu, Xun-Xiu; Deng, Xu; Qian, Zeng-Ting; Li, Zheng; Guo, Meng-Zhe; Tang, Dao-Quan

2017-04-01

As essential endogenous compounds, nucleobases and nucleosides fulfill various functions in living organisms. This study presents the development and validation of a new hydrophilic interaction liquid chromatography tandem mass spectrometry method for simultaneous quantification of 19 nucleobases and nucleosides in rat plasma. For the sample preparation, 15 kinds of protein precipitants were evaluated according to the chromatographic profile and ion response of analytes. The optimization of chromatographic separation was respectively performed using reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography mode; each separation mode included two test columns with different stationary phases. The chromatographic profile and parameters such as half-width (W 1/2 ), capacity factor (K') and tailing factor (f t ) were used to evaluate the separation efficiencies. Furthermore, the adopted composition of two mobile phase systems and the concentrations of the additives in the optimum buffer system were also investigated. The developed method was fully validated and successfully applied quantitatively to determine 19 nucleobases and nucleosides in plasma from normal and diabetic nephropathy (DN) rats. Significant differences between normal and DN rats were found in plasma levels of cytosine, xanthine, thymidine, adenosine, guanosine, inosine and 8-hydroxy-2'-deoxyguanosine. This information may provide a useful reference for the discovery of potential biomarkers of DN. Copyright © 2016 John Wiley & Sons, Ltd.

The flavivirus NS2B-NS3 protease-helicase as a target for antiviral drug development.

PubMed

Luo, Dahai; Vasudevan, Subhash G; Lescar, Julien

2015-06-01

The flavivirus NS3 protein is associated with the endoplasmic reticulum membrane via its close interaction with the central hydrophilic region of the NS2B integral membrane protein. The multiple roles played by the NS2B-NS3 protein in the virus life cycle makes it an attractive target for antiviral drug discovery. The N-terminal region of NS3 and its cofactor NS2B constitute the protease that cleaves the viral polyprotein. The NS3 C-terminal domain possesses RNA helicase, nucleoside and RNA triphosphatase activities and is involved both in viral RNA replication and virus particle formation. In addition, NS2B-NS3 serves as a hub for the assembly of the flavivirus replication complex and also modulates viral pathogenesis and the host immune response. Here, we review biochemical and structural advances on the NS2B-NS3 protein, including the network of interactions it forms with NS5 and NS4B and highlight recent drug development efforts targeting this protein. This article forms part of a symposium in Antiviral Research on flavivirus drug discovery. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of the plant ribokinase and discovery of a role for Arabidopsis ribokinase in nucleoside metabolism

DOE PAGES

Riggs, John W.; Rockwell, Nathan C.; Cavales, Philip C.; ...

2016-09-06

Ribose can be used for energy or as a component of several important biomolecules, but for it to be used in either capacity it must first be phosphorylated by ribokinase (RBSK). RBSK proteins are part of the phosphofructokinase-B (pfkB) family of carbohydrate kinases. Sequence comparisons of pfkB proteins from the model plant Arabidopsis thaliana with the human and Escherichia coli RBSK identified a single candidate RBSK, At1g17160 (AtRBSK). AtRBSK is more similar to predicted RBSKs from other plant species and known mammalian and prokaryotic RBSK than to all other PfkB proteins in Arabidopsis. AtRBSK contains a predicted chloroplast transit peptide,more » and we confirmed plastid localization using AtRBSK fused to YFP. Structure prediction software verified that the AtRBSK sequence mapped onto a known RBSK structure. Kinetic parameters of purified recombinant AtRBSK were determined to be K mribose = 150 μm ± 17 μm, K mATP = 45 μm ± 5.6 μm, and k cat = 2.0 s ₋1. Substrate inhibition was observed for AtRBSK (K iATP = 2.44 mm ± 0.36 mm), as has been demonstrated for other RBSK proteins. Ribose accumulated in Arabidopsis plants lacking AtRBSK. Such plants grew normally unless media was supplemented with ribose, which led to chlorosis and growth inhibition. Both chlorosis and ribose accumulation were abolished upon the introduction of a transgene expressing AtRBSK-MYC, demonstrating that the loss of protein is responsible for ribose hypersensitivity. Lastly, ribose accumulation in plants lacking AtRBSK was reduced in plants also deficient in the nucleoside ribohydrolase NSH1, linking AtRBSK activity to nucleoside metabolism.« less

Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food

PubMed Central

Mahor, Durga; Priyanka, Anu; Prasad, Gandham S; Thakur, Krishan Gopal

2016-01-01

Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a

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.

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

DOE PAGES

Chauleau, Mathieu; Jacewicz, Agata; Shuman, Stewart

2015-05-24

DNA 3' pp 5'G caps synthesized by the 3'-PO 4/5'-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'-PO 4 ligases, is also a DNA 3' de-capping enzyme, converting DNAppG to DNA 3'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,more » 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.« less

Problem-Solving Test: Catalytic Activities of a Human Nuclear Enzyme

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2011-01-01

Terms to be familiar with before you start to solve the test: ion exchange chromatography, polynucleotides, oligonucleotides, radioactive labeling, template, primer, DNA polymerase, reverse transcriptase, helicase, nucleoside triphosphates, nucleoside diphosphates, nucleoside monophosphates, nucleosides, 5'-end and 3'-end, bacteriophage,…

Products of the direct reaction of the diazonium ion of a metabolite of the carcinogen N-nitrosomorpholine with purines of nucleosides and DNA.

PubMed

Zink, Charles N; Soissons, Nicolas; Fishbein, James C

2010-07-19

A number of putative purine nucleoside and nucleobase adducts of the diazonium ion derived from 3-hydroxy-N-nitrosomorpholine have been synthesized as dimethylacetals. These are converted, in most cases nearly quantitatively, to the aldehydes, or in two cases to their derivatives, on treatment with mild acid to yield standards for a quantitative investigation of alkylation of purine nucleosides and DNA by the above metabolite of the powerful carcinogen N-nitrosomorpholine. The stability of the resulting nucleobase ethoxyacetaldehyde (EA) adducts has been characterized under a number of conditions with respect to their propensity to decompose. The stabilities, compared to that of the previously characterized adduct of the model benzimidazole, are generally unexceptional. Deposition of adducts on purine nucleosides and DNA were quantified in reactions in which 3-hydroperoxy-N-nitrosomorpholine was reduced to the hydroxy metabolite by a water-soluble phosphine at 21 +/- 2 degrees C. The adduct profile is highly similar to that observed from simpler alpha-hydroxy metabolites of acyclic dialkylnitrosamines, with the three most abundant ethoxyacetaldehyde (EA) adducts in reactions of duplex DNA being N7-EA-Gua approximately O(6)-EA-Gua > N3-EA-Ade. The initial rate kinetics of formation of hydroxyethyl (HE) lesions from the initially formed EA lesions have been determined in the case of the major products in the cases of both the nucleoside and DNA adducts. The rates of formation of HE adducts are accelerated in DNA, relative to the nucleosides in the cases of the N7-EA-Ade, N7-EA-Gua, and O(6)-EA-Gua adducts by factors of 7, 14, and 54, respectively. The initial rates of depurination of the N3-EA-Ade, N7-EA-Gua, and N7-EA-Gua adducts have also been quantified, and they are unexceptional in comparison with what has been previously reported for simple alkyl adducts. The adduct profiles reported here stand in significant contrast to what has been reported previously for

Establishment of new transmissible and drug-sensitive human immunodeficiency virus type 1 wild types due to transmission of nucleoside analogue-resistant virus.

PubMed

de Ronde, A; van Dooren, M; van Der Hoek, L; Bouwhuis, D; de Rooij, E; van Gemen, B; de Boer, R; Goudsmit, J

2001-01-01

Sequence analysis of human immunodeficiency virus type 1 (HIV-1) from 74 persons with acute infections identified eight strains with mutations in the reverse transcriptase (RT) gene at positions 41, 67, 68, 70, 215, and 219 associated with resistance to the nucleoside analogue zidovudine (AZT). Follow-up of the fate of these resistant HIV-1 strains in four newly infected individuals revealed that they were readily replaced by sensitive strains. The RT of the resistant viruses changed at amino acid 215 from tyrosine (Y) to aspartic acid (D) or serine (S), with asparagine (N) as a transient intermediate, indicating the establishment of new wild types. When we introduced these mutations and the original threonine (T)-containing wild type into infectious molecular clones and assessed their competitive advantage in vitro, the order of fitness was in accord with the in vivo observations: 215Y < 215D = 215S = 215T. As detected by real-time nucleic acid sequence-based amplification with two molecular beacons, the addition of AZT or stavudine (d4T) to the viral cultures favored the 215Y mutant in a dose-dependent manner. Our results illustrate that infection with nucleoside analogue-resistant HIV leads in newly infected individuals to mutants that are sensitive to nucleoside analogues, but only a single mutation removed from drug-resistant HIV. Such mutants were shown to be transmissible, stable, and prone to rapid selection for resistance to AZT or d4T as soon as antiretroviral therapy was administered. Monitoring of patients for the presence of new HIV-1 wild types with D, S, or N residues at position 215 may be warranted in order to estimate the threat to long-term efficacy of regimens including nucleoside analogues.

The pretranslocation ribosome is targeted by GTP-bound EF-G in partially activated form

PubMed Central

Hauryliuk, Vasili; Mitkevich, Vladimir A.; Eliseeva, Natalia A.; Petrushanko, Irina Yu.; Ehrenberg, Måns; Makarov, Alexander A.

2008-01-01

Translocation of the tRNA·mRNA complex through the bacterial ribosome is driven by the multidomain guanosine triphosphatase elongation factor G (EF-G). We have used isothermal titration calorimetry to characterize the binding of GDP and GTP to free EF-G at 4°C, 20°C, and 37°C. The binding affinity of EF-G is higher to GDP than to GTP at 4°C, but lower at 37°C. The binding enthalpy and entropy change little with temperature in the case of GDP binding but change greatly in the case of GTP binding. These observations are compatible with a large decrease in the solvent-accessible hydrophobic surface area of EF-G on GTP, but not GDP, binding. The explanation we propose is the locking of the switch 1 and switch 2 peptide loops in the G domain of EF-G to the γ-phosphate of GTP. From these data, in conjunction with previously reported structural data on guanine nucleotide-bound EF-G, we suggest that EF-G enters the pretranslocation ribosome as an “activity chimera,” with the G domain activated by the presence of GTP but the overall factor conformation in the inactive form typical of a GDP-bound multidomain guanosine triphosphatase. We propose that the active overall conformation of EF-G is attained only in complex with the ribosome in its “ratcheted state,” with hybrid tRNA binding sites. PMID:18836081

Short Communication: Transplacental Nucleoside Analogue Exposure and Mitochondrial Parameters in HIV-Uninfected Children

PubMed Central

Brogly, Susan B.; DiMauro, Salvatore; Van Dyke, Russell B.; Williams, Paige L.; Naini, Ali; Libutti, Daniel E.; Choi, Julia; Chung, Michelle

2011-01-01

Abstract Transplacental nucleoside analogue exposure can affect infant mitochondrial DNA (mtDNA). We evaluated mitochondria in peripheral blood mononuclear cells of children with and without clinical signs of mitochondrial dysfunction (MD) and antiretroviral (ARV) exposure. We previously identified 20 children with signs of MD (cases) among 1037 HIV-uninfected children born to HIV-infected women. We measured mtDNA copies/cell and oxidative phosphorylation (OXPHOS) NADH dehydrogenase (complex I) and cytochrome c oxidase (complex IV) protein levels and enzyme activities, determined mtDNA haplogroups and deletions in 18 of 20 cases with stored samples and in sex- and age-matched HIV-uninfected children, both ARV exposed and unexposed, (1) within 18 months of birth and (2) at the time of presentation of signs of MD. In specimens drawn within 18 months of birth, mtDNA levels were higher and OXPHOS protein levels and enzyme activities lower in cases than controls. In contrast, at the time of MD presentation, cases and ARV-exposed controls had lower mtDNA levels, 214 and 215 copies/cell, respectively, than ARV-unexposed controls, 254 copies/cell. OXPHOS protein levels and enzyme activities were lower in cases than exposed controls, and higher in cases than unexposed controls, except for complex IV activity, which was higher in cases. Haplotype H was less frequent among cases (6%) than controls (31%). No deletions were found. The long-term significance of these small but potentially important alterations should continue to be studied as these children enter adolescence and adulthood. PMID:21142587

The binding of the primary water of hydration to nucleosides, CsDNA and potassium hyaluronate

NASA Astrophysics Data System (ADS)

Lukan, A. M.; Cavanaugh, D.; Whitson, K. B.; Marlowe, R. L.; Lee, S. A.; Anthony, L.; Rupprecht, A.; Mohan, V.

1998-03-01

Differential scanning calorimetry (DSC) has been used to study the eight nucleosides, CsDNA and KHA hydrated at 59% relative humidity. Thermograms were measured between 25 and 180 ^oC for scan rates of 1, 2, 5, 10 and 20 K/min. A broad endothermic transition (due to the desorption of the water) near 80 ^oC was observed for all runs. The average enthalpy of desorption per water molecule was evaluated from the area under the peak. A Kissinger analysis of these data yielded the net activation energy for desorption. Both parameters were very similar for the two biopolymers. Rayleigh scattering of Mossbauer radiation (RSMR) data(G. Albanese et al. ) Hyperfine Int. 95, 97 (1995) were analyzed via a simple harmonic oscillator model to evaluate the effective force constant of the water bound to the biopolymer. This analysis suggests that the effective force constant of water bound to HA is much larger (about 5 times) than for water bound to DNA.

Characterization of nucleosides and nucleobases in natural Cordyceps by HILIC-ESI/TOF/MS and HILIC-ESI/MS.

PubMed

Zhao, Heng-Qiang; Wang, Xiao; Li, Hong-Mei; Yang, Bin; Yang, Hong-Jun; Huang, Luqi

2013-08-15

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

Probing Human Telomeric DNA and RNA Topology and Ligand Binding in a Cellular Model by Using Responsive Fluorescent Nucleoside Probes.

PubMed

Manna, Sudeshna; Panse, Cornelia H; Sontakke, Vyankat A; Sangamesh, Sarangamath; Srivatsan, Seergazhi G

2017-08-17

The development of biophysical systems that enable an understanding of the structure and ligand-binding properties of G-quadruplex (GQ)-forming nucleic acid sequences in cells or models that mimic the cellular environment would be highly beneficial in advancing GQ-directed therapeutic strategies. Herein, the establishment of a biophysical platform to investigate the structure and recognition properties of human telomeric (H-Telo) DNA and RNA repeats in a cell-like confined environment by using conformation-sensitive fluorescent nucleoside probes and a widely used cellular model, bis(2-ethylhexyl) sodium sulfosuccinate reverse micelles (RMs), is described. The 2'-deoxy and ribonucleoside probes, composed of a 5-benzofuran uracil base analogue, faithfully report the aqueous micellar core through changes in their fluorescence properties. The nucleoside probes incorporated into different loops of H-Telo DNA and RNA oligonucleotide repeats are minimally perturbing and photophysically signal the formation of respective GQ structures in both aqueous buffer and RMs. Furthermore, these sensors enable a direct comparison of the binding affinity of a ligand to H-Telo DNA and RNA GQ structures in the bulk and confined environment of RMs. These results demonstrate that this combination of a GQ nucleoside probe and easy-to-handle RMs could provide new opportunities to study and devise screening-compatible assays in a cell-like environment to discover GQ binders of clinical potential. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of a procedure for the isolation and enrichment of modified nucleosides and nucleobases from urine prior to their determination by capillary electrophoresis-mass spectrometry.

PubMed

Rodríguez-Gonzalo, Encarnación; Hernández-Prieto, Raquel; García-Gómez, Diego; Carabias-Martínez, Rita

2014-01-01

A sample treatment step based on solid-phase extraction (SPE) with polymeric sorbents has been developed for the simultaneous isolation and preconcentration of nucleosides and nucleobases from urine prior to analyses by CE-ESI-MS. In most reported methods nucleosides are isolated from urine by SPE in affinity mode, using an immobilized phenylboronic acid group, which specifically binds cis-diols. However, this is not applicable to non-cis-diol compounds. Here, different types of polymeric sorbents were evaluated for the simultaneous extraction of nucleosides and nucleobases from urine. The best results were obtained with Isolute ENV+, a hydroxylated styrene-divylbenzene polymer, whose retention capacity can be attributed mainly to hydrophobic interactions, and thus it can be applied to a broad range of compounds, regardless of whether they present or not to the cis-diol group in their structure. Other parameters such as the elution solvent and sample volume were optimized. We also studied the influence of the addition of isotopically labeled internal standards (ILISs) before or after the extraction step. The detection limits achieved were in the 0.04-0.17μg/mL range for a sample size of 2.0mL and relative standard deviations were 4-22%. The whole method developed, SPE prior to CE-ESI-MS, was applied to human urine samples from healthy volunteers. We conclude that SPE with polymeric sorbents prior to the electrophoretic CE-ESI-MS methodology constitutes a fast, valid and reliable approach for the simultaneously extraction of urinary nucleosides and nucleobases. Copyright © 2013 Elsevier B.V. All rights reserved.

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

PubMed

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

2015-12-04

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

Ionization energies of aqueous nucleic acids: photoelectron spectroscopy of pyrimidine nucleosides and ab initio calculations.

PubMed

Slavícek, Petr; Winter, Bernd; Faubel, Manfred; Bradforth, Stephen E; Jungwirth, Pavel

2009-05-13

Vertical ionization energies of the nucleosides cytidine and deoxythymidine in water, the lowest ones amounting in both cases to 8.3 eV, are obtained from photoelectron spectroscopy measurements in aqueous microjets. Ab initio calculations employing a nonequilibrium polarizable continuum model quantitatively reproduce the experimental spectra and provide molecular interpretation of the individual peaks of the photoelectron spectrum, showing also that lowest ionization originates from the base. Comparison of calculated vertical ionization potentials of pyrimidine bases, nucleosides, and nucleotides in water and in the gas phase underlines the dramatic effect of bulk hydration on the electronic structure. In the gas phase, the presence of sugar and, in particular, of phosphate has a strong effect on the energetics of ionization of the base. Upon bulk hydration, the ionization potential of the base in contrast becomes rather insensitive to the presence of the sugar and phosphate, which indicates a remarkable screening ability of the aqueous solvent. Accurate aqueous-phase vertical ionization potentials provide a significant improvement to the corrected gas-phase values used in the literature and represent important information in assessing the threshold energies for photooxidation and oxidation free energies of solvent-exposed DNA components. Likewise, such energetic data should allow improved assessment of delocalization and charge-hopping mechanisms in DNA ionized by radiation.

Pyrrole-indolinone SU11652 targets the nucleoside diphosphate kinase from Leishmania parasites.

PubMed

Vieira, Plínio Salmazo; Souza, Tatiana de Arruda Campos Brasil; Honorato, Rodrigo Vargas; Zanphorlin, Letícia Maria; Severiano, Kelven Ulisses; Rocco, Silvana Aparecida; de Oliveira, Arthur Henrique Cavalcante; Cordeiro, Artur Torres; Oliveira, Paulo Sérgio Lopes; de Giuseppe, Priscila Oliveira; Murakami, Mário Tyago

2017-07-01

Nucleoside diphosphate kinases (NDKs) are key enzymes in the purine-salvage pathway of trypanosomatids and have been associated with the maintenance of host-cell integrity for the benefit of the parasite, being potential targets for rational drug discovery and design. The NDK from Leishmania major (LmNDK) and mutants were expressed and purified to homogeneity. Thermal shift assays were employed to identify potential inhibitors for LmNDK. Calorimetric experiments, site-directed mutagenesis and molecular docking analysis were performed to validate the interaction and to evaluate the structural basis of ligand recognition. Furthermore, the anti-leishmanial activity of the newly identified and validated compound was tested in vitro against different Leishmania species. The molecule SU11652, a Sunitinib analog, was identified as a potential inhibitor for LmNDK and structural studies indicated that this molecule binds to the active site of LmNDK in a similar conformation to nucleotides, mimicking natural substrates. Isothermal titration calorimetry experiments combined with site-directed mutagenesis revealed that the residues H50 and H117, considered essential for catalysis, play an important role in ligand binding. In vitro cell studies showed that SU11652 had similar efficacy to Amphotericin b against some Leishmania species. Together, our results indicate the pyrrole-indolinone SU11652 as a promising scaffold for the rational design of new drugs targeting the enzyme NDK from Leishmania parasites. Copyright © 2017 Elsevier Inc. All rights reserved.

Efficient assessment of modified nucleoside stability under conditions of automated oligonucleotide synthesis: characterization of the oxidation and oxidative desulfurization of 2-thiouridine.

PubMed

Sochacka, E

2001-01-01

In order to efficiently assess the chemical stability of modified nucleosides to the reagents and conditions of automated oligonucleotide synthesis, we designed, developed and tested a scheme in which the modified nucleoside, directly attached to a solid support, is exposed to the cyclic chemistry of the instrument. Stability of 2-thiouridine against different oxidizers was investigated. Tertbutyl hydroperoxide (1 M) in anhydrous acetonitrile was a more effective oxidizer for the incorporation of 2-thiouridine into oligonucleotide chains than the same oxidizer in methylene chloride. Carbon tetrachloride/water in the presence of a basic catalyst was superior in maintaining the thiocarbonyl function, but its utility for RNA synthesis has yet to be fully tested, whereas 2-phenylsulfonyloxaziridine was a very efficient reagent for oxidative desulfurization of 2-thiouridine.

Regioselective and stereoselective route to N2-β-tetrazolyl unnatural nucleosides via SN2 reaction at the anomeric center of Hoffer's chlorosugar.

PubMed

Bag, Subhendu Sekhar; Talukdar, Sangita; Anjali, S J

2016-04-15

We are reporting a regioselective and stereoselective route to N2-β-tetrazolyl aromatic donor/acceptor unnatural nucleosides as new class of possible DNA base analogs. The SN2 substitution reaction at the anomeric center of Hoffer's chlorosugar with various 5-substituted aromatic tetrazoles in THF in presence of K2CO3 proceeds with regioselectivity at N2-tetrazoles and stereoselectivity at α-chlorosugar with very good yield. The stereoelectronic and steric effects play a crucial role for the observed outcome which is also supported from a theoretical (DFT) study. The methodology is simple, eco-compatible and the tetrazolyl unnatural nucleosides might find applications in decorating DNA for various biotechnological and DNA based material science applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of temperature modulations on TEMPO-mediated regioselective oxidation of unprotected carbohydrates and nucleosides.

PubMed

Yadav, Mahipal; Liotta, Charles L; Krishnamurthy, Ramanarayanan

2018-02-02

Regioselective oxidation of unprotected and partially protected oligosaccharides is a much sought-after goal. Herein, we report a notable improvement in the efficiency of TEMPO-catalyzed oxidation by modulating the temperature of the reaction. Mono-, di-, and tri-saccharides are oxidized regioselectively in yields of 75 to 92%. The present method is simple to implement and is also applicable for selective oxidations of other mono- and poly-hydroxy compounds including unprotected and partially protected nucleosides. Copyright © 2018 Elsevier Ltd. All rights reserved.

Problem-solving test: catalytic activities of a human nuclear enzyme.

PubMed

Szeberényi, József

2011-01-01

Terms to be familiar with before you start to solve the test: ion exchange chromatography, polynucleotides, oligonucleotides, radioactive labeling, template, primer, DNA polymerase, reverse transcriptase, helicase, nucleoside triphosphates, nucleoside diphosphates, nucleoside monophosphates, nucleosides, 5′-end and 3′-end, bacteriophage, polyacrylamide gel electrophoresis, urea, autoradiography, proofreading, telomerase, endonucleases, exonucleases, primase, topoisomerases, and excinuclease.

Positive regulation of deoxycytidine kinase activity by phosphorylation of Ser-74 in B-cell chronic lymphocytic leukaemia lymphocytes.

PubMed

Smal, Caroline; Van Den Neste, Eric; Maerevoet, Marie; Poiré, Xavier; Théate, Ivan; Bontemps, Françoise

2007-08-08

Deoxycytidine kinase (dCK) activates several antileukaemic nucleoside analogues. We have recently reported that the activity of dCK, overexpressed in HEK 293T cells, correlates with its phosphorylation level on Ser-74. Here, we show that dCK from B-cell chronic lymphocytic leukaemia (B-CLL) lymphocytes can be detected by an anti-phospho-Ser-74 antibody and that interindividual variability in dCK activity is related to its phosphorylation level on Ser-74. Moreover, pharmacological intervention modified Ser-74 phosphorylation, in close parallel with changes in dCK activity. These results suggest that activation of dCK via phosphorylation of Ser-74 might constitute a new therapeutic strategy to enhance activation and efficacy of nucleoside analogues.

Effect of Ginkgo biloba extract 50 on immunity and antioxidant enzyme activities in ischemia reperfusion rats.

PubMed

Lu, Shaoping; Guo, Xia; Zhao, Pinting

2011-11-02

The aim of the study was to investigate the effect of Ginkgo biloba extract 50 (GBE50), a well-known natural antioxidant, against immunity and antioxidant enzyme activities in ischemia reperfusion (IR) rats. Rats were then divided into six groups fed for 15 days with the same diet: three groups (IV, V, VI) were treated by different doses of GBE50 suspension [20, 40, or 60 mg/kg body weight by oral gavage every day at a fixed time (10.00 a.m.)] (equal to 5, 10 and 20 times, respectively, the maximum recommended human dose), and three groups (I, II, III) were untreated. At the end of the experiment, rats' hearts were subjected to 30 min of ischemia followed by 90 min of reperfusion. Results showed that IR significantly enhanced heart rate, S-T height, myocardium (myeloperoxidase) MPO activity and blood interleukin-8 (IL-8), tumor necrosis factor Alpha (TNF-a), interleukin-1β (IL-1β) levels, blood aspartate transaminase (AST), lactate dehydrogenase (LDH), and creatinine kinase (CK) activities, reduced myocardium sodium-potassium adenosine triphosphatase (Na(+)-K(+)-ATPase), calcium-magnesium adenosine triphosphatase (Ca(2+)-Mg(2+)-ATPase) activities and antioxidant enzyme activities in IR group (III) compared to sham control group (II). Pretreatment of GBE50 markedly significantly reduced heart rate, S-T height, myocardium MPO activity and blood IL-8, TNF-a, IL-1β levels, blood AST, LDH, and CK activities, enhanced myocardium Na(+)-K(+)-ATPase, Ca(2+)-Mg(2+)-ATPase activities and antioxidant enzyme activities in IR group (II) compared to IR group (III). The results suggested that the GBE50 may reduce the oxidative stress in the reperfused myocardium, and increased immunity and antioxidant activities in IR rats.

SYNTHESIS AND ANTIVIRAL EVALUATION OF 9-(S)-[3-ALKOXY-2-(PHOSPHONOMETHOXY)PROPYL]NUCLEOSIDE ALKOXYALKYL ESTERS: INHIBITORS OF HEPATITIS C VIRUS AND HIV-1 REPLICATION

PubMed Central

Valiaeva, Nadejda; Wyles, David L.; Schooley, Robert T.; Hwu, Julia B.; Beadle, James R.; Prichard, Mark N.

2011-01-01

We reported previously that octadecyloxyethyl 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]adenine (ODE-(S)-HPMPA) was active against genotype 1b and 2a hepatitis C virus (HCV) replicons. This is surprising because acyclic nucleoside phosphonates have been regarded as having antiviral activity only against double stranded DNA viruses, HIV and HBV. We synthesized octadecyloxyethyl 9-(S)-[3-methoxy-2-(phosphonomethoxy)propyl]-adenine and found it to be active in genotype 1b and 2a HCV replicons with EC50 values of 1-2 μM and a CC50 of>150 μM. Analogs with substitutions at the 3′-hydroxyl larger than methyl or ethyl, or with other purine bases were less active but most compounds had significant antiviral activity against HIV-1 in vitro. The most active anti-HIV compound was octadecyloxyethyl 9-(R)-[3-methoxy-2-(phosphonomethoxy)propyl]guanine with an EC50 <0.01 nanomolar and a selectivity index of>4.4 million. PMID:21719300

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

Transgene expression of Drosophila melanogaster nucleoside kinase reverses mitochondrial thymidine kinase 2 deficiency.

PubMed

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

2013-02-15

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.

Alteration of paraoxonase, arylesterase and lactonase activities in people around fluoride endemic area of Tamil Nadu, India.

PubMed

Arulkumar, Mani; Vijayan, Raji; Penislusshiyan, Sakayanathan; Sathishkumar, Palanivel; Angayarkanni, Jayaraman; Palvannan, Thayumanavan

2017-08-01

Toxicity due to excess fluoride concentration in drinking water is of great concern in people who rely only on the ground water as their water source in many region of the world. We collected samples and examined the toxicity of fluoride in a population residing at Salem, Dharmapuri and Krishnagiri districts of Tamil Nadu, India and measured HDL bound enzyme (PON1), erythrocyte membrane bound enzymes (acetylcholinesterase, AChE) and adenosine 5' triphosphatase (ATPases), plasma enzyme (butyrylcholinesterase, BChE) and rate limiting enzyme in heme biosynthesis (delta aminolevulinic acid dehydratase, δ-ALAD) activities. In fluorosis patients, formation of lipid peroxidation product was more in erythrocytes than in plasma. The observation further revealed that there was 50% reduction in the activity of HDL bound anti atherogenic enzyme-paraoxonase (PON1). The activities of membrane bound and signaling enzymes (acetylcholinesterase - AChE and adenosine 5' triphosphatase - ATPase) of erythrocyte were also diminished. These results suggested that there was defectiveness in the signaling and energy metabolism in fluorosis patients. Altered isoenzyme pattern of lactate dehydrogenase (LDH) in fluorosis samples was observed. Furthermore, the result suggested that both the heart (LDH 1) and liver (LDH 5) were most affected by fluoride toxicity. The study also provided reference values for tests which are used to predict the severity of fluoride toxicity. The toxic effect of fluoride was due to the collective effects on vital protective system rather than single factor. Copyright © 2017 Elsevier B.V. All rights reserved.

Five putative nucleoside triphosphate diphosphohydrolase genes are expressed in Trichomonas vaginalis.

PubMed

Frasson, Amanda Piccoli; Dos Santos, Odelta; Meirelles, Lúcia Collares; Macedo, Alexandre José; Tasca, Tiana

2016-01-01

Trichomonas vaginalis is a protozoan that parasitizes the human urogenital tract causing trichomoniasis, the most common non-viral sexually transmitted disease. The parasite has unique genomic characteristics such as a large genome size and expanded gene families. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) is an enzyme responsible for hydrolyzing nucleoside tri- and diphosphates and has already been biochemically characterized in T. vaginalis. Considering the important role of this enzyme in the production of extracellular adenosine for parasite uptake, we evaluated the gene expression of five putative NTPDases in T. vaginalis. We showed that all five putative TvNTPDase genes (TvNTPDase1-5) were expressed by both fresh clinical and long-term grown isolates. The amino acid alignment predicted the presence of the five crucial apyrase conserved regions, transmembrane domains, signal peptides, phosphorylation and catalytic sites. Moreover, a phylogenetic analysis showed that TvNTPDase sequences make up a clade with NTPDases intracellularly located. Biochemical NTPDase activity (ATP and ADP hydrolysis) is responsive to the serum-restrictive conditions and the gene expression of TvNTPDases was mostly increased, mainly TvNTPDase2 and TvNTPDase4, although there was not a clear pattern of expression among them. In summary, the present report demonstrates the gene expression patterns of predicted NTPDases in T. vaginalis. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

2-Aryl-8-aza-3-deazaadenosine Analogues of 5’-O-[N-(Salicyl)sulfamoyl]adenosine: Nucleoside Antibiotics that Block Siderophore Biosynthesis in Mycobacterium tuberculosis

PubMed Central

Krajczyk, Anna; Zeidler, Joanna; Januszczyk, Piotr; Dawadi, Surendra; Boshoff, Helena I.; Barry, Clifton E.; Ostrowski, Tomasz; Aldrich, Courtney C.

2016-01-01

A series of 5’-O-[N-(salicyl)sulfamoyl]-2-aryl-8-aza-3-deazaadenosines were designed to block mycobactin biosynthesis in Mycobacterium tuberculosis (Mtb) through inhibition of the essential adenylating enzyme MbtA. The synthesis of the 2-aryl-8-aza-3-deazaadenosine nucleosides featured sequential copper-free palladium-catalyzed Sonogashira coupling of a precursor 4-cyano-5-iodo-1,2,3-triazolonucleoside with terminal alkynes and Minakawa-Matsuda annulation reaction. These modified nucleosides were shown to inhibit MbtA with apparent Ki values ranging from 6.1 to 25 nM and to inhibit Mtb growth under iron-deficient conditions with minimum inhibitory concentrations ranging from 12.5 to >50 μM. PMID:27265685

Etravirine and Rilpivirine Drug Resistance Among HIV-1 Subtype C Infected Children Failing Non-Nucleoside Reverse Transcriptase Inhibitor-Based Regimens in South India.

PubMed

Saravanan, Shanmugam; Kausalya, Bagavathi; Gomathi, Selvamurthi; Sivamalar, Sathasivam; Pachamuthu, Balakrishnan; Selvamuthu, Poongulali; Pradeep, Amrose; Sunil, Solomon; Mothi, Sarvode N; Smith, Davey M; Kantor, Rami

2017-06-01

We have analyzed reverse transcriptase (RT) region of HIV-1 pol gene from 97 HIV-infected children who were identified as failing first-line therapy that included first-generation non-nucleoside RT inhibitors (Nevirapine and Efavirenz) for at least 6 months. We found that 54% and 65% of the children had genotypically predicted resistance to second-generation non-nucleoside RT inhibitors drugs Etravirine (ETR) and Rilpivirine, respectively. These cross-resistance mutations may compromise future NNRTI-based regimens, especially in resource-limited settings. To complement these investigations, we also analyzed the sequences in Stanford database, Monogram weighted score, and DUET weighted score algorithms for ETR susceptibility and found almost perfect agreement between the three algorithms in predicting ETR susceptibility from genotypic data.

Photoelectron and computational studies of the copper-nucleoside anionic complexes, Cu{sup -}(cytidine) and Cu{sup -}(uridine)

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

Li Xiang; Ko, Yeon-Jae; Wang Haopeng

2011-02-07

The copper-nucleoside anions, Cu{sup -}(cytidine) and Cu{sup -}(uridine), have been generated in the gas phase and studied by both experimental (anion photoelectron spectroscopy) and theoretical (density functional calculations) methods. The photoelectron spectra of both systems are dominated by single, intense, and relatively narrow peaks. These peaks are centered at 2.63 and 2.71 eV for Cu{sup -}(cytidine) and Cu{sup -}(uridine), respectively. According to our calculations, Cu{sup -}(cytidine) and Cu{sup -}(uridine) species with these peak center [vertical detachment energy (VDE)] values correspond to structures in which copper atomic anions are bound to the sugar portions of their corresponding nucleosides largely through electrostaticmore » interactions; the observed species are anion-molecule complexes. The combination of experiment and theory also reveal the presence of a slightly higher energy, anion-molecule complex isomer in the case of the Cu{sup -}(cytidine). Furthermore, our calculations found that chemically bond isomers of these species are much more stable than their anion-molecule complex counterparts, but since their calculated VDE values are larger than the photon energy used in these experiments, they were not observed.« less

Ras activation by SOS: Allosteric regulation by altered fluctuation dynamics

PubMed Central

Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen; Christensen, Sune M.; Abel, Steven M.; Iwig, Jeff; Wu, Hung-Jen; Gureasko, Jodi; Rhodes, Christopher; Petit, Rebecca S.; Hansen, Scott D.; Thill, Peter; Yu, Cheng-Han; Stamou, Dimitrios; Chakraborty, Arup K.; Kuriyan, John; Groves, Jay T.

2014-01-01

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras–guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average. PMID:24994643

Hydrogen peroxide yields mechanistic insights into human mRNA capping enzyme function

PubMed Central

Mullen, Nicholas J.

2017-01-01

Capping of nascent RNA polymerase II (Pol II) transcripts is required for gene expression and the first two steps are catalyzed by separate 5′ triphosphatase and guanylyltransferase activities of the human capping enzyme (HCE). The cap is added co-transcriptionally, but how the two activities are coordinated is unclear. Our previous in vitro work has suggested that an unidentified factor modulates the minimum length at which nascent transcripts can be capped. Using the same well-established in vitro system with hydrogen peroxide as a capping inhibitor, we show that this unidentified factor targets the guanylyltransferase activity of HCE. We also uncover the mechanism of HCE inhibition by hydrogen peroxide, and by using mass spectrometry demonstrate that the active site cysteine residue of the HCE triphosphatase domain becomes oxidized. Using recombinant proteins for the two separated HCE domains, we provide evidence that the triphosphatase normally acts on transcripts shorter than can be acted upon by the guanylyltransferase. Our further characterization of the capping reaction dependence on transcript length and its interaction with the unidentified modulator of capping raises the interesting possibility that the capping reaction could be regulated. PMID:29028835

Chaperones are necessary for the expression of catalytically active potato apyrases in prokaryotic cells.

PubMed

Porowińska, Dorota; Czarnecka, Joanna; Komoszyński, Michał

2014-07-01

NTPDases (nucleoside triphosphate diphosphohydrolases) (also called in plants apyrases) hydrolyze nucleoside 5'-tri- and/or diphosphate bonds producing nucleosides di or monophosphate and inorganic phosphate. For years, studies have been carried out to use both plant and animal enzymes for medicine. Therefore, there is a need to develop an efficient method for the quick production of large amounts of homogeneous proteins with high catalytic activity. Expression of proteins in prokaryotic cells is the most common way for the protein production. The aim of our study was to develop a method of expression of potato apyrase (StAPY4, 5, and 6) genes in bacterial cells under conditions that allowed the production of catalytically active form of these enzymes. Apyrase 4 and 6 were overexpressed in BL21-CodonPlus (DE3) bacteria strain but they were accumulated in inclusion bodies, regardless of the culture conditions and induction method. Co-expression of potato apyrases with molecular chaperones allowed the expression of catalytically active apyrase 5. However, its high nucleotidase activity could be toxic for bacteria and is therefore synthesized in small amounts in cells. Our studies show that each protein requires other conditions for maturation and even small differences in amino acid sequence can essentially affect protein folding regardless of presence of chaperones.

Conditional abatement of tissue fibrosis using nucleoside analogs to selectively corrupt DNA replication in transgenic fibroblasts.

PubMed

Iwano, M; Fischer, A; Okada, H; Plieth, D; Xue, C; Danoff, T M; Neilson, E G

2001-02-01

Progressive tissue fibrosis can compromise epithelial function resulting in organ failure. Appreciating evidence suggests that fibroblasts provide fibrogenic collagens during such injury. We further tested this notion by attempting to reduce the physiologic consequences of organ fibrosis through the selective killing of fibroblasts at sites of injury. Here, we report the conditional reduction of tissue fibroblasts using the coding sequence for herpesvirus thymidine kinase (DeltaTK) put under the control of a cell-specific promoter from the gene encoding fibroblast-specific protein 1 (FSP1). Transgenic fibroblasts from mice carrying FSP1.DeltaTK minigenes expressed thymidine kinase concordantly with native FSP1 and, compared to transgenic epithelium, were selectively susceptible to the lethal effects of nucleoside analogs either in culture or during experimental renal fibrosis. The numbers of fibroblasts in fibrogenic kidney tissue were reduced on exposure to nucleoside analogs as was the degree of type I collagen deposition and the extent of fibrosis. Fibroblast reduction following the stress of DNA chain termination highlights the important contribution of cell division during fibrogenesis. Our findings convey a proof of principle regarding the importance of FSP1(+) fibroblasts in fibrosis as well as providing a new approach to treating the relentless scarification of tissue.

Charge-reversal Lipids, Peptide-based Lipids, and Nucleoside-based Lipids for Gene Delivery

PubMed Central

LaManna, Caroline M.; Lusic, Hrvoje; Camplo, Michel; McIntosh, Thomas J.; Barthélémy, Philippe; Grinstaff, Mark W.

2013-01-01

headgroup into the lipid provides a mechanism to affect the binding of the lipid to the nucleic acid, to influence the supramolecular lipoplex structure, and to enhance gene transfection activity. Lastly, we discuss the in-vitro successes we have had when using lipids possessing a nucleoside headgroup to create unique self-assembled structures and to deliver DNA to cells. In this Account, we state our hypotheses and design elements as well as describe the techniques that we have utilized in our research, in order to provide readers with the tools to characterize and engineer new vectors. PMID:22439686

Structures and kinetics for plant nucleoside triphosphate diphosphohydrolases support a domain motion catalytic mechanism.

PubMed

Summers, Emma L; Cumming, Mathew H; Oulavallickal, Tifany; Roberts, Nicholas J; Arcus, Vickery L

2017-08-01

Extracellular nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that hydrolyze extracellular nucleotides to the respective monophosphate nucleotides. In the past 20 years, NTPDases belonging to mammalian, parasitic and prokaryotic domains of life have been discovered, cloned and characterized. We reveal the first structures of NTPDases from the legume plant species Trifolium repens (7WC) and Vigna unguiculata subsp. cylindrica (DbLNP). Four crystal structures of 7WC and DbLNP were determined at resolutions between 1.9 and 2.6 Å. For 7WC, structures were determined for an -apo form (1.89 Å) and with the product AMP (2.15 Å) and adenine and phosphate (1.76 Å) bound. For DbLNP, a structure was solved with phosphate and manganese bound (2.60 Å). Thorough kinetic data and analysis is presented. The structure of 7WC and DbLNP reveals that these NTPDases can adopt two conformations depending on the molecule and co-factor bound in the active site. A central hinge region creates a "butterfly-like" motion of the domains that reduces the width of the inter-domain active site cleft upon molecule binding. This phenomenon has been previously described in Rattus norvegicus and Legionella pneumophila NTPDaseI and Toxoplasma gondii NTPDaseIII suggesting a common catalytic mechanism across the domains of life. © 2017 The Protein Society.

Diaryltriazine non-nucleoside reverse transcriptase inhibitors are potent candidates for pre-exposure prophylaxis in the prevention of sexual HIV transmission.

PubMed

Ariën, Kevin K; Venkatraj, Muthusamy; Michiels, Johan; Joossens, Jurgen; Vereecken, Katleen; Van der Veken, Pieter; Abdellati, Saïd; Cuylaerts, Vicky; Crucitti, Tania; Heyndrickx, Leo; Heeres, Jan; Augustyns, Koen; Lewi, Paul J; Vanham, Guido

2013-09-01

Pre-exposure prophylaxis and topical microbicides are important strategies in the prevention of sexual HIV transmission, especially since partial protection has been shown in proof-of-concept studies. In search of new candidate drugs with an improved toxicity profile and with activity against common non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV, we have synthesized and investigated a library of 60 new diaryltriazine analogues. From this library, 15 compounds were evaluated in depth using a broad armamentarium of in vitro assays that are part of a preclinical testing algorithm for microbicide development. Antiviral activity was assessed in a cell line, and in primary human cells, against both subtype B and subtype C HIV-1 and against viruses resistant to therapeutic NNRTIs and the candidate NNRTI microbicide dapivirine. Toxicity towards primary blood-derived cells, cell lines originating from the female reproductive tract and female genital microflora was also studied. We identified several compounds with highly potent antiviral activity and toxicity profiles that are superior to that of dapivirine. In particular, compound UAMC01398 is an interesting new candidate that warrants further investigation because of its superior toxicity profile and potent activity against dapivirine-resistant viruses.

The MONET trial: darunavir/ritonavir with or without nucleoside analogues, for patients with HIV RNA below 50 copies/ml.

PubMed

Arribas, Jose R; Horban, Andrzej; Gerstoft, Jan; Fätkenheuer, Gerdt; Nelson, Mark; Clumeck, Nathan; Pulido, Federico; Hill, Andrew; van Delft, Yvon; Stark, Thomas; Moecklinghoff, Christiane

2010-01-16

In virologically suppressed patients, darunavir-ritonavir (DRV/r) monotherapy could maintain virological suppression similarly to DRV/r and two nucleosides. Two hundred and fifty-six patients with HIV RNA less than 50 copies/ml for over 24 weeks on current antiretrovirals [non-nucleoside reverse transcriptase inhibitor (NNRTI)-based (43%), or protease inhibitor-based (57%)], switched to DRV/r 800/100 mg once daily, either as monotherapy (n = 127) or with two nucleoside reverse transcriptase inhibitors (NRTIs) (n = 129). Treatment failure was defined as two consecutive HIV RNA levels above 50 copies/ml (TLOVR) by week 48, or switches off study treatment. The trial had 80% power to show noninferiority for the monotherapy arm (delta = -12%). Patients were 81% male and 91% Caucasian, with mean age 44 years, and CD4 cell count of 574 cells/microl. In the primary efficacy analysis, HIV RNA less than 50 copies/ml by week 48 (per protocol) was 86.2 versus 87.8% in the monotherapy and triple therapy arms; by intent-to-treat switch equals failure, efficacy was 84.3 versus 85.3%; by a switch-included analysis, efficacy was 93.5 versus 95.1%: all three comparisons showed noninferior efficacy for DRV/r monotherapy. CD4 cell counts remained stable during the trial in both arms. One patient per arm showed at least one protease inhibitor mutation, and one patient in the triple therapy arm showed an NRTI mutation. Nine patients per arm discontinued randomized treatment for either adverse events or other reasons. No new or unexpected safety signals were detected. In this study for patients with HIV RNA less than 50 copies/ml on other antiretrovirals at baseline, switching to DRV/r monotherapy showed noninferior efficacy versus triple antiretroviral therapy.

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

PubMed Central

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

2009-01-01

Abacavir is one of the most efficacious nucleoside analogues, with a well-characterized inhibitory activity on reverse transcriptase enzymes of retroviral origin, and has been clinically approved for the treatment of AIDS. Recently, Abacavir has been shown to inhibit also the human telomerase activity. Telomerase activity seems to be required in essentially all tumours for the immortalization of a subset of cells, including cancer stem cells. In fact, many cancer cells are dependent on telomerase for their continued replication and therefore telomerase is an attractive target for cancer therapy. Telomerase expression is upregulated in primary primitive neuroectodermal tumours and in the majority of medulloblastomas suggesting that its activation is associated with the development of these diseases. Therefore, we decided to test Abacavir activity on human medulloblastoma cell lines with high telomerase activity. We report that exposure to Abacavir induces a dose-dependent decrease in the proliferation rate of medulloblastoma cells. This is associated with a cell accumulation in the G2/M phase of the cell cycle in the Daoy cell line, and with increased cell death in the D283-MED cell line, and is likely to be dependent on the inhibition of telomerase activity. Interestingly, both cell lines showed features of senescence after Abacavir treatment. Moreover, following Abacavir exposure we detected, by immunofluorescence staining, increased protein expression of the glial marker glial fibrillary acidic protein (GFAP) and the neuronal marker synaptophysin (SYN) in both medulloblastoma cell lines. In conclusion, our results suggest that Abacavir reduces proliferation and induces differentiation of human medulloblastoma cells through the downregulation of telomerase activity. Thus, using Abacavir, alone or in combination with current therapies, might be an effective therapeutic strategy for the treatment of medulloblastoma. PMID:19358275

Nucleoside monophosphorothioates as the new hydrogen sulfide precursors with unique properties.

PubMed

Bełtowski, Jerzy; Guranowski, Andrzej; Jamroz-Wiśniewska, Anna; Korolczuk, Agnieszka; Wojtak, Andrzej

2014-03-01

Hydrogen sulfide (H2S) is the gasotransmitter enzymatically synthesized in mammalian tissues from l-cysteine. H2S donors are considered as the potential drugs for the treatment of cardiovascular, neurological and inflammatory diseases. Recently, it has been demonstrated that synthetic nucleotide analogs, adenosine- and guanosine 5'-monophosphorothioates (AMPS and GMPS) can be converted to H2S and AMP or GMP, respectively, by purified histidine triad nucleotide-binding (Hint) proteins. We examined if AMPS and GMPS can be used as the H2S donors in intact biological systems. H2S production by isolated rat kidney glomeruli was measured by the specific polarographic sensor. H2S production was detected when glomeruli were incubated with AMPS or GMPS and ionotropic purinergic P2X7 receptor/channel agonist, BzATP. More H2S was generated from GMPS than from equimolar amount of AMPS. Nucleoside phosphorothioates together with BzATP relaxed angiotensin II-preconstricted glomeruli. In addition, infusion of AMPS or GMPS together with BzATP into the renal artery increased filtration fraction and glomerular filtration rate but had no effect on renal vascular resistance or renal blood flow. AMPS but not GMPS was converted to adenosine by isolated glomeruli, however, adenosine was not involved in AMPS-induced H2S synthesis because neither adenosine nor specific adenosine receptor agonists had any effect on H2S production. AMPS, but not GMPS, increased phosphorylation level of AMP-stimulated protein kinase (AMPK), but AMPK inhibitor, compound C, had no effect on AMPS-induced H2S production. In conclusion, nucleoside phosphorothioates are converted to H2S which relaxes isolated kidney glomeruli in vitro and increases glomerular filtration rate in vivo. AMPS and GMPS can be used as the H2S donors in experimental studies and possibly also as the H2S-releasing drugs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Movie of the structural changes during a catalytic cycle of nucleoside monophosphate kinases.

PubMed

Vonrhein, C; Schlauderer, G J; Schulz, G E

1995-05-15

There are 17 crystal structures of nucleoside monophosphate kinases known. As expected for kinases, they show large conformational changes upon binding of substrates. These are concentrated in two chain segments, or domains, of 30 and 38 residues that are involved in binding of the substrates N1TP and N2MP (nucleoside tri- and monophosphates with bases N1 and N2), respectively. After aligning the 17 structures on the main parts of their polypeptide chains, two domains in various conformational states were revealed. These states were caused by bound substrate (or analogues) and by crystal-packing forces, and ranged between a 'closed' conformation and a less well defined 'open' conformation. The structures were visually sorted yielding an approximately evenly spaced series of domain states that outlines the closing motions when the substrates bind. The packing forces in the crystals are weak, leaving the natural domain trajectories essentially intact. Packing is necessary, however, to produce stable intermediates. The ordered experimental structures were then recorded as still pictures of a movie and animated to represent the motions of the molecule during a catalytic cycle. The motions were smoothed out by adding interpolated structures to the observed ones. The resulting movies are available through the World Wide Web (http:@bio5.chemie.uni-freiburg.de/ak movie.html). Given the proliferating number of homologous proteins known to exist in different conformational states, it is becoming possible to outline the motions of chain segments and combine them into a movie, which can then represent protein action much more effectively than static pictures alone are able to do.

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

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

Mastrangelo, Eloise; Bollati, Michela; Milani, Mario

2006-08-01

Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed,more » purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup.« less

An indole-linked C8-deoxyguanosine nucleoside acts as a fluorescent reporter of Watson-Crick versus Hoogsteen base pairing.

PubMed

Schlitt, Katherine M; Millen, Andrea L; Wetmore, Stacey D; Manderville, Richard A

2011-03-07

Pyrrole- and indole-linked C(8)-deoxyguanosine nucleosides act as fluorescent reporters of H-bonding specificity. Their fluorescence is quenched upon Watson-Crick H-bonding to dC, while Hoogsteen H-bonding to G enhances emission intensity. The indole-linked probe is ∼ 10-fold brighter and shows promise as a fluorescent reporter of Hoogsteen base pairing.

Nucleoside adducts from the in vitro reaction of benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide or benzo[a]pyrene 4,5-oxide with nucleic acids.

PubMed

Jennette, K W; Jeffrey, A M; Blobstein, S H; Beland, F A; Harvey, R G; Weinstein, I B

1977-03-08

The covalent binding of benzo[a]pyrene 4,5-oxide and benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide isomer I and isomer II to nucleic acids in aqueous acetone solution has been investigated. Benzo[a]pyrene 4,5-oxide reacted preferentially with guanosine residues. On the other hand, benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide isomer I and II reacted extensively with guanosine, adenosine, and cytidine residues. Time course studies showed that the reactivity of isomer I or isomer II with homopolyribonucleotides followed the order poly(G) greater than poly(A) greater than poly(C). Alkaline or enzymatic hydrolysis of the modified nucleic acids and subsequent chromatography on Sephadex LH-20 columns yielded benzo[a]pyrene-nucleotide adducts. These were enzymatically converted to the corresponding nucleosides which were resolved into several distinct components by high-pressure liquid chromatography. Evidence was obtained for the presence of multiple nucleoside adducts of guanosine, adenosine, cytidine, deoxyguanosine, deoxyadenosine, and deoxycytidine. The HPLC profiles of adducts formed with isomer I were different from the corresponding profiles of adducts formed with isomer II. Structural aspects of these nucleoside adducts are discussed.

Purine Restriction Induces Pronounced Translational Upregulation of the NT1 Adenosine/Pyrimidine Nucleoside Transporter in Leishmania major

PubMed Central

Ortiz, Diana; Valdés, Raquel; Sanchez, Marco A.; Hayenga, Johanna; Elya, Carolyn; Detke, Siegfried; Landfear, Scott M.

2010-01-01

Summary Leishmania and other parasitic protozoa are unable to synthesize purines de novo and are reliant upon purine nucleoside and nucleobase transporters to import preformed purines from their hosts. To study the roles of the four purine permeases NT1-NT4 in Leishmania major, null mutants in each transporter gene were prepared and the effect of each gene deletion on purine uptake was monitored. Deletion of the NT3 purine nucleobase transporter gene or both NT3 and the NT2 nucleoside transporter gene resulted in pronounced upregulation of adenosine and uridine uptake mediated by the NT1 permease and also induced up to a 200-fold enhancement in the level of the NT1 protein but not mRNA. A similar level of upregulation of NT1 was achieved in wild type promastigotes that were transferred to medium deficient in purines. Pulse labeling and treatment of cells with the translation inhibitor cycloheximide revealed that control of NT1 expression occurs primarily at the level of translation and not protein turnover. These observations imply the existence of a translational control mechanism that enhances the ability of Leishmania parasites to import essential purines when they are present at limiting concentrations. PMID:20735779

Purine restriction induces pronounced translational upregulation of the NT1 adenosine/pyrimidine nucleoside transporter in Leishmania major.

PubMed

Ortiz, Diana; Valdés, Raquel; Sanchez, Marco A; Hayenga, Johanna; Elya, Carolyn; Detke, Siegfried; Landfear, Scott M

2010-10-01

Leishmania and other parasitic protozoa are unable to synthesize purines de novo and are reliant upon purine nucleoside and nucleobase transporters to import preformed purines from their hosts. To study the roles of the four purine permeases NT1-NT4 in Leishmania major, null mutants in each transporter gene were prepared and the effect of each gene deletion on purine uptake was monitored. Deletion of the NT3 purine nucleobase transporter gene or both NT3 and the NT2 nucleoside transporter gene resulted in pronounced upregulation of adenosine and uridine uptake mediated by the NT1 permease and also induced up to a 200-fold enhancement in the level of the NT1 protein but not mRNA. A similar level of upregulation of NT1 was achieved in wild-type promastigotes that were transferred to medium deficient in purines. Pulse labelling and treatment of cells with the translation inhibitor cycloheximide revealed that control of NT1 expression occurs primarily at the level of translation and not protein turnover. These observations imply the existence of a translational control mechanism that enhances the ability of Leishmania parasites to import essential purines when they are present at limiting concentrations. © 2010 Blackwell Publishing Ltd.

Combinations of Adefovir with Nucleoside Analogs Produce Additive Antiviral Effects against Hepatitis B Virus In Vitro

PubMed Central

Delaney, William E.; Yang, Huiling; Miller, Michael D.; Gibbs, Craig S.; Xiong, Shelly

2004-01-01

Combination therapies may be required for long-term management of some patients chronically infected with hepatitis B virus (HBV). Adefovir is a nucleotide analog that has similar activity against wild-type and lamivudine-resistant HBV. In contrast to lamivudine, clinical resistance to the prodrug adefovir dipivoxil emerges infrequently. Based on its clinical efficacy and low frequency of resistance, adefovir dipivoxil may form an important component of combination regimens. We therefore investigated the in vitro antiviral efficacy of combinations of adefovir with other nucleoside analogs (lamivudine, entecavir, emtricitabine [FTC],and telbivudine [L-dT]) and the nucleotide analog tenofovir. Using a novel stable cell line that expresses high levels of wild-type HBV, we assayed the antiviral activity of each drug alone and in combination with adefovir. All two-drug combinations resulted in greater antiviral effects than treatments with single agents and could be characterized as additive by the Bliss independence model. Analysis using the Loewe additivity model indicated that adefovir exerted additive antiviral effects when combined with lamivudine, FTC, or L-dT and moderately synergistic effects when combined with entecavir or tenofovir. There was no evidence of cytotoxicity with any of the drugs when used alone or in combination at the tested doses. PMID:15388423

Zidovudine Induces Downregulation of Mitochondrial Deoxynucleoside Kinases: Implications for Mitochondrial Toxicity of Antiviral Nucleoside Analogs

PubMed Central

Sun, Ren; Eriksson, Staffan

2014-01-01

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial phosphorylation of deoxynucleosides in the synthesis of the DNA precursors required for mitochondrial DNA (mtDNA) replication and are essential for mitochondrial function. Antiviral nucleosides are known to cause toxic mitochondrial side effects. Here, we examined the effects of 3′-azido-2′,3′-dideoxythymidine (AZT) (zidovudine) on mitochondrial TK2 and dGK levels and found that AZT treatment led to downregulation of mitochondrial TK2 and dGK in U2OS cells, whereas cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) levels were not affected. The AZT effects on mitochondrial TK2 and dGK were similar to those of oxidants (e.g., hydrogen peroxide); therefore, we examined the oxidative effects of AZT. We found a modest increase in cellular reactive oxygen species (ROS) levels in the AZT-treated cells. The addition of uridine to AZT-treated cells reduced ROS levels and protein oxidation and prevented the degradation of mitochondrial TK2 and dGK. In organello studies indicated that the degradation of mitochondrial TK2 and dGK is a mitochondrial event. These results suggest that downregulation of mitochondrial TK2 and dGK may lead to decreased mitochondrial DNA precursor pools and eventually mtDNA depletion, which has significant implications for the regulation of mitochondrial nucleotide biosynthesis and for antiviral therapy using nucleoside analogs. PMID:25182642

Antiviral activities of 2,6-diaminopurine-based acyclic nucleoside phosphonates against herpesviruses: In vitro study results with pseudorabies virus (PrV, SuHV-1).

PubMed

Zouharova, Darina; Lipenska, Ivana; Fojtikova, Martina; Kulich, Pavel; Neca, Jiri; Slany, Michal; Kovarcik, Kamil; Turanek-Knotigova, Pavlina; Hubatka, Frantisek; Celechovska, Hana; Masek, Josef; Koudelka, Stepan; Prochazka, Lubomir; Eyer, Ludek; Plockova, Jana; Bartheldyova, Eliska; Miller, Andrew D; Ruzek, Daniel; Raska, Milan; Janeba, Zlatko; Turanek, Jaroslav

2016-02-29

Pseudorabies virus (PrV), a causative agent of Aujeszky's disease, is deadly to most mammals with the exception of higher primates and men. This disease causes serious economic loses among farm animals, especially pigs, yet many European countries are today claimed to be Aujeszky's disease free because of the discovery of an efficient vaccination for pigs. In reality, the virus is still present in wild boar. Current vaccines are neither suitable for dogs nor are there anti-PrV drugs approved for veterinary use. Therefore, the disease still represents a high threat, particularly for expensive hunting dogs that can come into close contact with infected boars. Here we report on the anti-PrV activities of a series of synthetic diaminopurine-based acyclic nucleoside phosphonate (DAP-ANP) analogues. Initially, all synthetic DAP-ANPs under investigation are shown to exhibit minimal cytotoxicity by MTT and XTT tests (1-100μM range). Thereafter in vitro infection models are established using PrV virus SuHV-1, optimized on PK-15 and RK-13 cell lines. Out of the six DAP-ANP analogues tested, analogue VI functionalized with a cyclopropyl group on the 6-amino position of the purine ring proves the most effective antiviral DAP-ANP analogue against PrV infection, aided by sufficient hydrophobic character to enhance bioavailability to its cellular target viral DNA-polymerase. Four other DAP-ANP analogues with functional groups introduced to the C2'position are shown ineffective against PrV infection, even with favourable hydrophobic properties. Cidofovir(®), a drug approved against various herpesvirus infections, is found to exert only low activity against PrV in these same in vitro models. Copyright © 2016 Elsevier B.V. All rights reserved.

Methylation of ribonucleic acid by the carcinogens dimethyl sulphate, N-methyl-N-nitrosourea and N-methyl-N′-nitro-N-nitrosoguanidine. Comparisons of chemical analyses at the nucleoside and base levels

PubMed Central

Lawley, P. D.; Shah, S. A.

1972-01-01

1. The following methods for hydrolysis of methyl-14C-labelled RNA, and for chromatographic isolation and determination of the products, were investigated: enzymic digestion to nucleosides at pH6 or 8; alkaline hydrolysis and conversion into nucleosides; hydrolysis by acid to pyrimidine nucleotides and purine bases, or completely to bases; chromatography on Dowex 50 (NH4+ form) at pH6 or 8.9, or on Dowex 50 (H+ form), or on Sephadex G-10. 2. The suitability of the various methods for determination of methylation products was assessed. The principal product, 7-methylguanosine, was unstable under the conditions used for determinations of nucleosides. 3- and 7-Methyladenine and 3- and 7-methylguanine are best determined as bases; 1-methyladenine and 3-methylcytosine can be isolated as either nucleosides or bases; O6-methylguanine is unstable under the acid hydrolysis conditions used and can be determined as the nucleoside; 3-methyluracil was detected, but may be derived from methylation of the ionized form of uracil. 3. Differences between the patterns of methylation of RNA and homopolyribonucleotides by the N-methyl-N-nitroso compounds and dimethyl sulphate were found: the nitroso compounds were able to methylate O-6 of guanine, were relatively more reactive at N-7 of adenine and probably at N-3 of guanine, but less reactive at N-1 of adenine, N-3 of cytosine and probably at N-3 of uridine. They probably reacted more with the ribose–phosphate chain, but no products from this were identified. 4. The possible influences of these differences on biological action of the methylating agents is discussed. Nitroso compounds may differ principally in their ability to induce miscoding in the Watson–Crick sense by reaction at O-6 of guanine. Both types of agent may induce miscoding to a lesser extent through methylation at N-3 of guanine; both can methylate N atoms, presumably preventing Watson–Crick hydrogen-bonding. N-Methyl-N-nitrosourea can degrade RNA, possibly

Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

PubMed Central

Thess, Andreas; Grund, Stefanie; Mui, Barbara L; Hope, Michael J; Baumhof, Patrick; Fotin-Mleczek, Mariola; Schlake, Thomas

2015-01-01

Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies. PMID:26050989

Molecular dynamics studies of a hexameric purine nucleoside phosphorylase.

PubMed

Zanchi, Fernando Berton; Caceres, Rafael Andrade; Stabeli, Rodrigo Guerino; de Azevedo, Walter Filgueira

2010-03-01

Purine nucleoside phosphorylase (PNP) (EC.2.4.2.1) is an enzyme that catalyzes the cleavage of N-ribosidic bonds of the purine ribonucleosides and 2-deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate. This enzyme is involved in purine-salvage pathway and has been proposed as a promising target for design and development of antimalarial and antibacterial drugs. Recent elucidation of the three-dimensional structure of PNP by X-ray protein crystallography left open the possibility of structure-based virtual screening initiatives in combination with molecular dynamics simulations focused on identification of potential new antimalarial drugs. Most of the previously published molecular dynamics simulations of PNP were carried out on human PNP, a trimeric PNP. The present article describes for the first time molecular dynamics simulations of hexameric PNP from Plasmodium falciparum (PfPNP). Two systems were simulated in the present work, PfPNP in ligand free form, and in complex with immucillin and sulfate. Based on the dynamical behavior of both systems the main results related to structural stability and protein-drug interactions are discussed.

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

The Pseudomonas aeruginosa PA14 ABC Transporter NppA1A2BCD Is Required for Uptake of Peptidyl Nucleoside Antibiotics.

PubMed

Pletzer, Daniel; Braun, Yvonne; Dubiley, Svetlana; Lafon, Corinne; Köhler, Thilo; Page, Malcolm G P; Mourez, Michael; Severinov, Konstantin; Weingart, Helge

2015-07-01

Analysis of the genome sequence of Pseudomonas aeruginosa PA14 revealed the presence of an operon encoding an ABC-type transporter (NppA1A2BCD) showing homology to the Yej transporter of Escherichia coli. The Yej transporter is involved in the uptake of the peptide-nucleotide antibiotic microcin C, a translation inhibitor that targets the enzyme aspartyl-tRNA synthetase. Furthermore, it was recently shown that the Opp transporter from P. aeruginosa PAO1, which is identical to Npp, is required for uptake of the uridyl peptide antibiotic pacidamycin, which targets the enzyme translocase I (MraY), which is involved in peptidoglycan synthesis. We used several approaches to further explore the substrate specificity of the Npp transporter. Assays of growth in defined minimal medium containing peptides of various lengths and amino acid compositions as sole nitrogen sources, as well as Biolog Phenotype MicroArrays, showed that the Npp transporter is not required for di-, tri-, and oligopeptide uptake. Overexpression of the npp operon increased susceptibility not just to pacidamycin but also to nickel chloride and the peptidyl nucleoside antibiotic blasticidin S. Furthermore, heterologous expression of the npp operon in a yej-deficient mutant of E. coli resulted in increased susceptibility to albomycin, a naturally occurring sideromycin with a peptidyl nucleoside antibiotic. Additionally, heterologous expression showed that microcin C is recognized by the P. aeruginosa Npp system. Overall, these results suggest that the NppA1A2BCD transporter is involved in the uptake of peptidyl nucleoside antibiotics by P. aeruginosa PA14. One of the world's most serious health problems is the rise of antibiotic-resistant bacteria. There is a desperate need to find novel antibiotic therapeutics that either act on new biological targets or are able to bypass known resistance mechanisms. Bacterial ABC transporters play an important role in nutrient uptake from the environment. These uptake

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. Copyright © 2016 King Saud Bin Abdulaziz University for Health Sciences. All rights reserved.

Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.

PubMed

Mishima, Eikan; Jinno, Daisuke; Akiyama, Yasutoshi; Itoh, Kunihiko; Nankumo, Shinnosuke; Shima, Hisato; Kikuchi, Koichi; Takeuchi, Yoichi; Elkordy, Alaa; Suzuki, Takehiro; Niizuma, Kuniyasu; Ito, Sadayoshi; Tomioka, Yoshihisa; Abe, Takaaki

2015-01-01

The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.

Mitochondrial DNA replication, nucleoside reverse-transcriptase inhibitors, and AIDS cardiomyopathy.

PubMed

Lewis, William

2003-01-01

Nucleoside reverse-transcriptase inhibitors (NRTIs) in combination with other antiretrovirals (HAART) are the cornerstones of current AIDS therapy, but extensive use brought mitochondrial side effects to light. Clinical experience, pharmacological, cell, and molecular biological evidence links altered mitochondrial (mt-) DNA replication to the toxicity of NRTIs in many tissues, and conversely, mtDNA replication defects and mtDNA depletion in target tissues are observed. Organ-specific pathological changes or diverse systemic effects result from and are frequently attributed to HAART in which NRTIs are included. The shared features of mtDNA depletion and energy depletion became key observations and related the clinical and in vivo experimental findings to inhibition of mtDNA replication by NRTI triphosphates in vitro. Subsequent to those findings, other observations suggested that mitochondrial energy deprivation is concomitant with or the result of mitochondrial oxidative stress in AIDS (from HIV, for example) or from NRTI therapy itself. Copyright 2003, Elsevier Science (USA)

Inhibition of the AMP-activated protein kinase-α2 accentuates agonist-induced vascular smooth muscle contraction and high blood pressure in mice.

PubMed

Wang, Shuangxi; Liang, Bin; Viollet, Benoit; Zou, Ming-Hui

2011-05-01

The aim of the present study was to determine the effects and molecular mechanisms by which AMP-activated protein kinase (AMPK) regulates smooth muscle contraction and blood pressure in mice. In cultured human vascular smooth muscle cells, we observed that activation of AMPK by 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside inhibited agonist-induced phosphorylation of myosin light chain (MLC) and myosin phosphatase targeting subunit 1 (MYPT1). Conversely, AMPK inhibition with pharmacological or genetic means potentiated agonist-induced the phosphorylation of MLC and MYPT1, whereas it inhibited both Ras homolog gene family member A and Rho-associated kinase activity. In addition, AMPK activation or Rho-associated kinase inhibition with Y27632 abolished agonist-induced phosphorylation of MLC and MYPT1. Gene silencing of p190-guanosine triphosphatase-activating protein abolished the effects of AMPK activation on MLC, MYPT1, and Ras homolog gene family member A in human smooth muscle cells. Ex vivo analyses revealed that agonist-induced contractions of the mesenteric artery and aortas were stronger in both AMPKα1(-/-) and AMPKα2(-/-) knockout mice than in wild-type mice. Inhibition of Rho-associated kinase with Y27632 normalized agonist-induced contractions of AMPKα1(-/-) and AMPKα2(-/-) vessels. AMPKα2(-/-) mice had higher blood pressure along with decreased serine phosphorylation of p190-guanosine triphosphatase-activating protein. Finally, inhibition of the Ras homolog gene family member A/Rho-associated kinase pathway with Y27632, which suppressed MYPT1 and MLC phosphorylation, lowered blood pressure in AMPKα2(-/-) mice. In conclusion, AMPK decreases vascular smooth muscle cell contractility by inhibiting p190-GTP-activating protein-dependent Ras homolog gene family member A activation, indicating that AMPK may be a new therapeutic target in lowering high blood pressure.

Structural analysis of Arabidopsis thaliana nucleoside diphosphate kinase-2 for phytochrome-mediated light signaling.

PubMed

Im, Young Jun; Kim, Jeong-Il; Shen, Yu; Na, Young; Han, Yun-Jeong; Kim, Seong-Hee; Song, Pill-Soon; Eom, Soo Hyun

2004-10-22

In plants, nucleoside diphosphate kinases (NDPKs) play a key role in the signaling of both stress and light. However, little is known about the structural elements involved in their function. Of the three NDPKs (NDPK1-NDPK3) expressed in Arabidopsis thaliana, NDPK2 is involved in phytochrome-mediated signal transduction. In this study, we found that the binding of dNDP or NTP to NDPK2 strengthens the interaction significantly between activated phytochrome and NDPK2. To better understand the structural basis of the phytochrome-NDPK2 interaction, we determined the X-ray structures of NDPK1, NDPK2, and dGTP-bound NDPK2 from A.thaliana at 1.8A, 2.6A, and 2.4A, respectively. The structures showed that nucleotide binding caused a slight conformational change in NDPK2 that was confined to helices alphaA and alpha2. This suggests that the presence of nucleotide in the active site and/or the evoked conformational change contributes to the recognition of NDPK2 by activated phytochrome. In vitro binding assays showed that only NDPK2 interacted specifically with the phytochrome and the C-terminal regulatory domain of phytochrome is involved in the interaction. A domain swap experiment between NDPK1 and NDPK2 showed that the variable C-terminal region of NDPK2 is important for the activation by phytochrome. The structure of Arabidopsis NDPK1 and NDPK2 showed that the isoforms share common electrostatic surfaces at the nucleotide-binding site, but the variable C-terminal regions have distinct electrostatic charge distributions. These findings suggest that the binding of nucleotide to NDPK2 plays a regulatory role in phytochrome signaling and that the C-terminal extension of NDPK2 provides a potential binding surface for the specific interaction with phytochromes.

Bioactive activities of natural products against herpesvirus infection.

PubMed

Son, Myoungki; Lee, Minjung; Sung, Gi-Ho; Lee, Taeho; Shin, Yu Su; Cho, Hyosun; Lieberman, Paul M; Kang, Hyojeung

2013-10-01

More than 90% of adults have been infected with at least one human herpesvirus, which establish long-term latent infection for the life of the host. While anti-viral drugs exist that limit herpesvirus replication, many of these are ineffective against latent infection. Moreover, drug-resistant strains of herpesvirus emerge following chemotherapeutic treatment. For example, resistance to acyclovir and related nucleoside analogues can occur when mutations arise in either HSV thymidine kinase or DNA polymerases. Thus, there exists an unmet medical need to develop new anti-herpesvirus agents with different mechanisms of action. In this Review, we discuss the promise of anti-herpetic substances derived from natural products including extracts and pure compounds from potential herbal medicines. One example is Glycyrrhizic acid isolated from licorice that shows promising antiviral activity towards human gammaherpesviruses. Secondly, we discuss anti-herpetic mechanisms utilized by several natural products in molecular level. While nucleoside analogues inhibit replicating herpesviruses in lytic replication, some natural products can disrupt the herpesvirus latent infection in the host cell. In addition, natural products can stimulate immune responses against herpesviral infection. These findings suggest that natural products could be one of the best choices for development of new treatments for latent herpesvirus infection, and may provide synergistic anti-viral activity when supplemented with nucleoside analogues. Therefore, it is important to identify which natural products are more efficacious anti-herpetic agents, and to understand the molecular mechanism in detail for further advance in the anti-viral therapies.

Gradient enhanced-fluidity liquid hydrophilic interaction chromatography of ribonucleic acid nucleosides and nucleotides: A "green" technique.

PubMed

Beilke, Michael C; Beres, Martin J; Olesik, Susan V

2016-03-04

A "green" hydrophilic interaction liquid chromatography (HILIC) technique for separating the components of mixtures with a broad range of polarities is illustrated using enhanced-fluidity liquid mobile phases. Enhanced-fluidity liquid chromatography (EFLC) involves the addition of liquid CO2 to conventional liquid mobile phases. Decreased mobile phase viscosity and increased analyte diffusivity results when a liquefied gas is dissolved in common liquid mobile phases. The impact of CO2 addition to a methanol:water (MeOH:H2O) mobile phase was studied to optimize HILIC gradient conditions. For the first time a fast separation of 16 ribonucleic acid (RNA) nucleosides/nucleotides was achieved (16min) with greater than 1.3 resolution for all analyte pairs. By using a gradient, the analysis time was reduced by over 100% compared to similar separations conducted under isocratic conditions. The optimal separation using MeOH:H2O:CO2 mobile phases was compared to MeOH:H2O and acetonitrile:water (ACN:H2O) mobile phases. Based on chromatographic performance parameters (efficiency, resolution and speed of analysis) and an assessment of the environmental impact of the mobile phase mixtures, MeOH:H2O:CO2 mixtures are preferred over ACN:H2O or MeOH:H2O mobile phases for the separation of mixtures of RNA nucleosides and nucleotides. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics.

PubMed

Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen; Christensen, Sune M; Abel, Steven M; Iwig, Jeff; Wu, Hung-Jen; Gureasko, Jodi; Rhodes, Christopher; Petit, Rebecca S; Hansen, Scott D; Thill, Peter; Yu, Cheng-Han; Stamou, Dimitrios; Chakraborty, Arup K; Kuriyan, John; Groves, Jay T

2014-07-04

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average. Copyright © 2014, American Association for the Advancement of Science.

Purine nucleoside phosphorylase and the enzymatic antioxidant defense system in breast milk from women with different levels of arsenic exposure.

PubMed

Gaxiola-Robles, Ramón; Labrada-Martagón, Vanessa; Bitzer-Quintero, Oscar Kurt; Zenteno-Savín, Tania; Méndez-Rodríguez, Lía Celina

2015-05-01

Purine nucleoside phosphorylase (PNP) is an ubiquitous enzyme which plays an important role in arsenic (As) detoxification. As is a toxic metalloid present in air, soil and water; is abundant in the environment and is readily transferred along the trophic chain, being found even in human breast milk. Milk is the main nutrient source for the growth and development of neonates. Information on breast milk synthesis and its potential defense mechanism against As toxicity is scarce. In this study, PNP and antioxidant enzymes activities, as well as glutathione (GSH) and total arsenic (TAs) concentrations, were quantified in breast milk samples. PNP, superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR) activities and GSH concentration were determined spectrophotometrically; TAs concentration ([TAs]) was measured by atomic absorption spectrometry. Data suggest an increase in PNP activity (median = 0.034 U mg protein-1) in the presence of TAs (median = 1.16 g L(-1)). To explain the possible association of PNP activity in breast milk with the activity of the antioxidant enzymes as well as with GSH and TAs concentrations, generalized linear models were built. In the adjusted model, GPx and GR activities showed a statistically significant (p<0.01) association with PNP activity. These results may suggest that PNP activity increases in the presence of TAs as part of the detoxification mechanism in breast milk. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

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

Emtricitabine: a once-daily nucleoside reverse transcriptase inhibitor.

PubMed

Modrzejewski, Krysten A; Herman, Ronald A

2004-06-01

To review the pharmacology, virology, pharmacokinetics, safety, and efficacy of the nucleoside reverse transcriptase inhibitor (NRTI) emtricitabine. English-language reports were accessed using MEDLINE (1966-June 2003) and the Iowa Drug Information Service database (1966-June 2003) using emtricitabine and Coviracil as key words. (Coviracil was the proposed trade name for the product prior to approval.) The Internet was also searched using the terms HIV/AIDS conferences, then emtricitabine within the conference proceedings. Abstracts, posters, and oral presentations from scientific conferences, both published and unpublished, were included. Preference was given to published controlled trials. Studies providing a description of the pharmacology, virology, effectiveness, safety, or pharmacokinetics of emtricitabine were used in this review. Emtricitabine is an NRTI used to treat HIV-1 infection. Once-daily administration can decrease pill burden and potentially increase adherence to multidrug HIV therapy. Further, emtricitabine has shown equivalent or improved outcomes compared with lamivudine and stavudine. Emtricitabine is a safe and effective option for HIV-1 infection in adults as part of a multidrug regimen. It may be a better alternative than lamivudine for once-daily therapy because of its extended intracellular half-life and better than lamivudine and stavudine because of a possibly decreased potential for drug resistance.

CONTRASTING BEHAVIOR OF CONFORMATIONALLY LOCKED CARBOCYCLIC NUCLEOSIDES OF ADENOSINE AND CYTIDINE AS SUBSTRATES FOR DEAMINASES

PubMed Central

Schroeder, Gottfried K.; Ludek, Olaf R.; Siddiqui, Maqbool A.; Ezzitouni, Abdallah; Wolfenden, Richard

2010-01-01

In addition to the already known differences between adenosine deaminase (ADA) and cytidine deaminase (CDA) in terms of their tertiary structure, the sphere of Zn+2 coordination, and their reverse stereochemical preference, we present evidence that the enzymes also differ significantly in terms of the North/South conformational preferences for their substrates and the extent to which the lack of the O(4’) oxygen affects the kinetics of the enzymatic deamination of carbocyclic substrates. The carbocyclic nucleoside substrates used in this study have either a flexible cyclopentane ring or a rigid bicyclo[3.1.0]hexane scaffold. PMID:20183605

Identification of a dithiol-dependent nucleoside triphosphate hydrolase in Sarcocystis neurona.

PubMed

Zhang, Deqing; Gaji, Rajshekhar Y; Howe, Daniel K

2006-09-01

A putative nucleoside triphosphate hydrolase (NTPase) gene was identified in a database of expressed sequence tags (ESTs) from the apicomplexan parasite Sarcocystis neurona. Analysis of culture-derived S. neurona merozoites demonstrated a dithiol-dependent NTPase activity, consistent with the presence of a homologue to the TgNTPases of Toxoplasma gondii. A complete cDNA was obtained for the S. neurona gene and the predicted amino acid sequence shared 38% identity with the two TgNTPase isoforms from T. gondii. Based on the obvious homology, the S. neurona protein was designated SnNTP1. The SnNTP1 cDNA encodes a polypeptide of 714 amino acids with a predicted 22-residue signal peptide and an estimated mature molecular mass of 70kDa. Southern blot analysis of the SnNTP1 locus revealed that the gene exists as a single copy in the S. neurona genome, unlike the multiple gene copies that have been observed in T. gondii and Neospora caninum. Analyses of the SnNTP1 protein demonstrated that it is soluble and secreted into the culture medium by extracellular merozoites. Surprisingly, indirect immunofluorescence analysis of intracellular S. neurona revealed apical localisation of SnNTP1 and temporal expression characteristics that are comparable with the microneme protein SnMIC10. The absence of SnNTP1 during much of endopolygeny implies that this protein does not serve a function during intracellular growth and development of S. neurona schizonts. Instead, SnNTP1 may play a role in events that occur during or proximal to merozoite egress from and/or invasion into cells.

VX-222, a non-nucleoside NS5B polymerase inhibitor, in telaprevir-based regimens for genotype 1 hepatitis C virus infection.

PubMed

Di Bisceglie, Adrian M; Sulkowski, Mark; Gane, Ed; Jacobson, Ira M; Nelson, David; DeSouza, Cynthia; Alves, Katia; George, Shelley; Kieffer, Tara; Zhang, Eileen Z; Kauffman, Robert; Asmal, Mohammed; Koziel, Margaret J

2014-07-01

To investigate in this phase 2a study (ZENITH) the safety, tolerability, and antiviral activity of VX-222, a selective, non-nucleoside inhibitor of hepatitis C virus (HCV) NS5B polymerase, combined with various telaprevir-based regimens for treatment of genotype 1 HCV. In total, 152 treatment-naive patients received VX-222+telaprevir ('DUAL' regimen; n=47), with ribavirin ('TRIPLE' regimen; n=46), or with peginterferon+ribavirin ('QUAD' regimen; n=59) for 12 weeks. Patients with detectable HCV RNA at weeks 2 and/or 8 received peginterferon+ribavirin for 24 (DUAL and TRIPLE) or 12 (QUAD) additional weeks. VX-222 (100 or 400 mg twice daily) was well tolerated, with an increased rate of gastrointestinal adverse events observed with the higher dose. Across VX-222 400-mg twice-daily regimens, the QUAD was associated with the highest frequency of grade 3/4 adverse events. The DUAL was discontinued because of high viral breakthrough before week 12. Sustained virologic response (SVR) 24 weeks after end of treatment (SVR24), including patients treated with 12 or 24 additional weeks of peginterferon+ribavirin, was 67% for TRIPLE (VX-222 400 mg twice daily) and 79 and 90% for QUAD (VX-222 100 and 400 mg twice daily, respectively). These results provide valuable information regarding the safety, tolerability, and efficacy of telaprevir combined with a non-nucleoside polymerase inhibitor, as dual therapy or with ribavirin without or with peginterferon. Telaprevir and VX-222, alone or with ribavirin without or with peginterferon, were generally well tolerated, with improved tolerability without peginterferon. SVR24 rates achieved with TRIPLE and QUAD regimens containing telaprevir and VX-222 were comparable to those observed with telaprevir-based therapy.

The cyclohexene ring system as a furanose mimic: synthesis and antiviral activity of both enantiomers of cyclohexenylguanine.

PubMed

Wang, J; Froeyen, M; Hendrix, C; Andrei, G; Snoeck, R; De Clercq, E; Herdewijn, P

2000-02-24

Both enantiomers of cyclohexenylguanine were synthesized in a stereospecific way starting from the same starting material: R-(-)-carvone. Both compounds showed potent and selective anti-herpesvirus activity (HSV-1, HSV-2, VZV, CMV). The binding of both cyclohexene nucleosides in the active site of HSV-1 thymidine kinase was investigated, and a model for the binding of both enantiomers is proposed. The amino acids involved in binding of the optical antipodes are the same, but the interaction energy of both enantiomers is slightly different. This may be attributed to the interaction of the secondary hydroxyl function of the nucleoside analogues with Glu-225. Structural analysis has demonstrated the flexibility of the cyclohexenyl system, and this may be considered as an important conformational characteristic explaining the potent antiviral activity.

Purine derivatives with antituberculosis activity

NASA Astrophysics Data System (ADS)

Gruzdev, D. A.; Musiyak, V. V.; Levit, G. L.; Krasnov, V. P.; Charushin, V. N.

2018-06-01

The review summarizes the data published over the last 10–15 years concerning the key groups of purine derivatives with antituberculosis activity. The structures of purines containing heteroatoms (S, O, N), fragments of heterocycles, amino acids and peptides, in the 6-position, as well as of purine nucleosides are presented. The possible targets for the action of such compounds and structure–activity relationship are discussed. Particular attention is paid to the most active compounds, which are of considerable interest as a basis for the development of efficient antituberculosis drugs. The bibliography includes 99 references.

Novel selective human mitochondrial kinase inhibitors: design, synthesis and enzymatic activity.

PubMed

Ciliberti, Nunzia; Manfredini, Stefano; Angusti, Angela; Durini, Elisa; Solaroli, Nicola; Vertuani, Silvia; Buzzoni, Lisa; Bonache, Maria Cruz; Ben-Shalom, Efrat; Karlsson, Anna; Saada, Ann; Balzarini, Jan

2007-04-15

Selective and effective TK2 inhibitors can be obtained by introduction of bulky lipophilic chains (acyl or alkyl entities) at the 2' position of araT and BVaraU, nucleoside analogues naturally endowed with a low TK2 affinity. These derivatives showed a competitive inhibitory activity against TK2 in micromolar range. BVaraU nucleoside analogues, modified on the 2'-O-acyl chain with a terminal N-Boc amino-group, conserved or increased the inhibitory activity against TK2 (7l and 7m IC(50): 6.4 and 3.8 microM, respectively). The substitution of an ester for a carboxamide moiety at the 2' position of araT afforded a consistent reduction of the inhibitory activity (25, IC(50): 480 microM). On the contrary, modifications at 2'-OH position of araC and araG, have provided inactive derivatives against TK2 and dGK, respectively. The biological activity of a representative compound, 2'-O-decanoyl-BVaraU, was also investigated in normal human fibroblasts and was found to impair mitochondrial function due to TK2 inhibition.

A new fluorescent probe for the equilibrative inhibitor-sensitive nucleoside transporter. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA)-chi 2-fluorescein.

PubMed

Wiley, J S; Brocklebank, A M; Snook, M B; Jamieson, G P; Sawyer, W H; Craik, J D; Cass, C E; Robins, M J; McAdam, D P; Paterson, A R

1991-02-01

The N6-(4-nitrobenzyl) derivative of adenosine is a tight-binding inhibitor of the equilibrative inhibitor-sensitive nucleoside transporter of mammalian cells. A fluorescent ligand for this transporter has been synthesized by allowing an adenosine analogue. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), to react with fluorescein isothiocyanate. The purified adduct had a SAENTA/fluorescein molar ratio of 0.92:1 calculated from its absorption spectrum. The intensity of fluorescent emission from the SAENTA-chi 2-fluorescein adduct was 30% that of fluorescein isothiocyanate (chi 2 is the number of atoms in the linkage between fluorescein and SAENTA). SAENTA-chi 2-fluorescein inhibited the influx of nucleosides into cultured leukaemic cells with an IC50 (total concentration of inhibitor producing 50% inhibition) of 40 nM. The adduct inhibited the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) with half-maximal inhibition at 50-100 nM. Mass Law analysis of the competitive-binding data suggested the presence of two classes of sites for [3H]NBMPR binding, only one of which was accessible to SAENTA-chi 2-fluorescein. Flow cytometry was used to analyse equilibrium binding of SAENTA-chi 2-fluorescein to leukaemic cells and a Kd of 6 nM was obtained. SAENTA-chi 2-fluorescein is a high-affinity ligand for the equilibrative inhibitor-sensitive nucleoside transporter which allows rapid assessment of transport capacity by flow cytometry.

Negative ion electrospray ionization mass spectrometry of nucleoside phosphoramidate monoesters: elucidation of novel rearrangement mechanisms by multistage mass spectrometry incorporating in-source deuterium labelling.

PubMed

Xu, Peng-Xiang; Hu, An-Fu; Hu, Dan; Gao, Xiang; Zhao, Yu-Fen

2008-10-01

Several O-2',3'-isopropylideneuridine-O-5'-phosphoramidate monoesters were synthesized and analyzed by negative ion electrospray ionization tandem mass spectrometry (ESI-MS(n)). Two kinds of novel rearrangement reactions were observed due to the difference in the amino acid in the nucleoside phosphoramidate monoesters, and possible mechanisms were proposed. One involves a five-membered cyclic transition state. The other is formation of a stable five-membered ring intermediate by Michael addition. Results were confirmed by tandem mass spectrometry and isotopically labeled hydrogen atoms. Furthermore, the internal hydrogen exchange between active hydrogen and methyl acrylate in the heated capillary of the mass spectrometer was found. The characteristic fragmentation behavior in ESI-MS may be used to monitor this kind of compounds in the biological metabolism.

A convenient synthesis of 6-amino-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4-one and related 4,6-disubstituted pyrazolopyrimidine nucleosides.

PubMed Central

Cottam, H B; Revankar, G R; Robins, R K

1983-01-01

The glycosylation of 4,6-dichloropyrazolo[3,4-d]pyrimidine and 4-chloro-6-methylthiopyrazolo[3,4-d]pyrimidine via the corresponding trimethylsilyl intermediate and tetra-O-acetyl-beta-D-ribofuranose in the presence of trimethylsilyl triflate as a catalyst, gave selective glycosylation at N1 as the only nucleoside product. The intermediates 4,6-dichloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo [3,4-d]pyrimidine 7 and 4-chloro-6-methylthio-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo [3,4-d]pyrimidine 13 gave new and convenient synthetic routes to the inosine analog 1, the guanosine analog 2, the adenosine analog 3, and the isoguanosine analog 16. Glycosylation of the trimethylsilyl derivative of 6-chloropyrazolo[3,4-d]pyrimidine-4-one unexpectedly gave the N2-glycosyl isomer 20 as the major product. A number of new 4,6-disubstituted pyrazolo[3,4-d]pyrimidine nucleosides were prepared from these glycosyl intermediates. PMID:6835838

[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. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

The Limits of Template-Directed Synthesis with Nucleoside-5'-Phosphoro(2-Methyl) Imidazolides

NASA Technical Reports Server (NTRS)

Hill, Aubrey R., Jr.; Orgel, Leslie E.; Wu, Taifeng

1993-01-01

In earlier work we have shown that C-rich templates containing isolated A, T or G residues and short oligo(G) sequences can be copied effectively using nucleoside-5'-phosphoro(2-methyl)imidazolides as substrates. We now show that isolated A or T residues within an oligo(G) sequence are a complete block to copying and that an isolated C residue is copied inefficiently. Replication is possible only if there are two complementary oligonucleotides each of which acts as a template to facilitate the synthesis of the other. We emphasize the severity of the problems that need to be overcome to make possible non-enzymatic replication in homogeneous aqueous solution. We conclude that an efficient catalyst was involved in the origin of polynucleotide replication.

Mycobacterial Nucleoside Diphosphate Kinase Blocks Phagosome Maturation in Murine Raw 264.7 Macrophages

PubMed Central

Sun, Jim; Wang, Xuetao; Lau, Alice; Liao, Ting-Yu Angela; Bucci, Cecilia; Hmama, Zakaria

2010-01-01

Background Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively. Methodology/Principal Findings Here we show that recombinant Mtb nucleoside diphosphate kinase (Ndk) exhibits GTPase activating protein (GAP) activity towards Rab5 and Rab7. Then, using a model of latex bead phagosomes, we demonstrated that Ndk inhibits phagosome maturation and fusion with lysosomes in murine RAW 264.7 macrophages. Maturation arrest of phagosomes containing Ndk-beads was associated with the inactivation of both Rab5 and Rab7 as evidenced by the lack of recruitment of their respective effectors EEA1 (early endosome antigen 1) and RILP (Rab7-interacting lysosomal protein). Consistent with these findings, macrophage infection with an Ndk knocked-down BCG strain resulted in increased fusion of its phagosome with lysosomes along with decreased survival of the mutant. Conclusion Our findings provide evidence in support of the hypothesis that mycobacterial Ndk is a putative virulence factor that inhibits phagosome maturation and promotes survival of mycobacteria within the macrophage. PMID:20098737

Oxidation of pyrimidine nucleosides and nucleotides by osmium tetroxide

PubMed Central

Burton, K.

1967-01-01

1. Pyrimidine nucleosides such as thymidine, uridine or cytidine are oxidized readily at 0° by osmium tetroxide in ammonium chloride buffer. There is virtually no oxidation in bicarbonate buffer of similar pH. Oxidation of 1-methyluracil yields 5,6-dihydro-4,5,6-trihydroxy-1-methyl-2-pyrimidone. 2. Osmium tetroxide and ammonia react reversibly in aqueous solution to form a yellow 1:1 complex, probably OsO3NH. A second molecule of ammonia must be involved in the oxidation of UMP since the rate of this reaction is approximately proportional to the square of the concentration of unprotonated ammonia. 3. 4-Thiouridine reacts with osmium tetroxide much more rapidly than does uridine. The changes of absorption spectra are different in sodium bicarbonate buffer and in ammonium chloride buffer. They occur faster in the latter buffer and, under suitable conditions, cytidine is a major product. 4. Polyuridylic acid is oxidized readily by ammoniacal osmium tetroxide, but its oxidation is inhibited by polyadenylic acid. Pyrimidines of yeast amino acid-transfer RNA are oxidized more slowly than the corresponding mononucleosides, especially the thymine residues. Appreciable oxidation can occur without change of sedimentation coefficient. PMID:6048808

Reactions of Trimethylsilyl Fluorosulfonyldifluoroacetate with Purine and Pyrimidine Nucleosides

PubMed Central

Rapp, Magdalena; Cai, Xiaohong; Xu, Wei; Dolbier, William R.; Wnuk, Stanislaw F.

2008-01-01

Difluorocarbene, generated from trimethylsilyl fluorosulfonyldifluoroacetate (TFDA), reacts with the uridine and adenosine substrates preferentially at the enolizable amide moiety of the uracil ring and the 6-amino group of the purine ring. 2',3'-Di-O-acetyl-3'-deoxy-3'-methyleneuridine reacts with TFDA to produce 4-O-difluoromethyl product derived from an insertion of difluorocarbene into the 4-hydroxyl group of the enolizable uracil ring. Reaction of the difluorocarbene with the adenosine substrates having the unprotected 6-amino group in the purine ring produced the 6-N-difluoromethyl derivative, while reaction with 6-N-benzoyl protected adenosine analogues gave the difluoromethyl ether product derived from the insertion of difluorocarbene into the enol form of the 6-benzamido group. Treatment of the 6-N-phthaloyl protected adenosine analogues with TFDA resulted in the unexpected one-pot conversion of the imidazole ring of the purine into the corresponding N-difluoromethylthiourea derivatives. Treatment of the suitably protected pyrimidine and purine nucleosides bearing an exomethylene group at carbons 2', 3' or 4' of the sugar rings with TFDA afforded the corresponding spirodifluorocyclopropyl analogues but in low yields. PMID:20160856

Oxidation of pyrimidine nucleosides and nucleotides by osmium tetroxide.

PubMed

Burton, K

1967-08-01

1. Pyrimidine nucleosides such as thymidine, uridine or cytidine are oxidized readily at 0 degrees by osmium tetroxide in ammonium chloride buffer. There is virtually no oxidation in bicarbonate buffer of similar pH. Oxidation of 1-methyluracil yields 5,6-dihydro-4,5,6-trihydroxy-1-methyl-2-pyrimidone. 2. Osmium tetroxide and ammonia react reversibly in aqueous solution to form a yellow 1:1 complex, probably OsO(3)NH. A second molecule of ammonia must be involved in the oxidation of UMP since the rate of this reaction is approximately proportional to the square of the concentration of unprotonated ammonia. 3. 4-Thiouridine reacts with osmium tetroxide much more rapidly than does uridine. The changes of absorption spectra are different in sodium bicarbonate buffer and in ammonium chloride buffer. They occur faster in the latter buffer and, under suitable conditions, cytidine is a major product. 4. Polyuridylic acid is oxidized readily by ammoniacal osmium tetroxide, but its oxidation is inhibited by polyadenylic acid. Pyrimidines of yeast amino acid-transfer RNA are oxidized more slowly than the corresponding mononucleosides, especially the thymine residues. Appreciable oxidation can occur without change of sedimentation coefficient.

Photoelectron spectroscopic study of the hydrated nucleoside anions: Uridine(-)(H(2)O)(n=0-2), cytidine(-)(H(2)O)(n=0-2), and thymidine(-)(H(2)O)(n=0,1).

PubMed

Li, Xiang; Wang, Haopeng; Bowen, Kit H

2010-10-14

The hydrated nucleoside anions, uridine(-)(H(2)O)(n=0-2), cytidine(-)(H(2)O)(n=0-2), and thymidine(-)(H(2)O)(n=0,1), have been prepared in beams and studied by anion photoelectron spectroscopy in order to investigate the effects of a microhydrated environment on parent nucleoside anions. Vertical detachment energies (VDEs) were measured for all eight anions, and from these, estimates were made for five sequential anion hydration energies. Excellent agreement was found between our measured VDE value for thymidine(-)(H(2)O)(1) and its calculated value in the companion article by S. Kim and H. F. Schaefer III.

Photoelectron spectroscopic study of the hydrated nucleoside anions: Uridine-(H2O)n=0-2, cytidine-(H2O)n=0-2, and thymidine-(H2O)n=0,1

NASA Astrophysics Data System (ADS)

Li, Xiang; Wang, Haopeng; Bowen, Kit H.

2010-10-01

The hydrated nucleoside anions, uridine-(H2O)n=0-2, cytidine-(H2O)n=0-2, and thymidine-(H2O)n=0,1, have been prepared in beams and studied by anion photoelectron spectroscopy in order to investigate the effects of a microhydrated environment on parent nucleoside anions. Vertical detachment energies (VDEs) were measured for all eight anions, and from these, estimates were made for five sequential anion hydration energies. Excellent agreement was found between our measured VDE value for thymidine-(H2O)1 and its calculated value in the companion article by S. Kim and H. F. Schaefer III.

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

PubMed

Dao, Doan Y; Seremba, Emmanuel; Ajmera, Veeral; Sanders, Corron; Hynan, Linda S; Lee, William M

2012-05-01

The efficacy of nucleoside(tide) analogues (NA) in the treatment of acute liver failure due to hepatitis B virus (HBV-ALF) remains controversial. We determined retrospectively the impact of NAs in a large cohort of patients with HBV-ALF. The US Acute Liver Failure Study Group, a 23-site registry, prospectively enrolled 1,413 patients with ALF with different etiologies between 1998 and 2008. Of those, 105 patients were identified as HBV-ALF patients, of whom we excluded those without data on NA use or with co-infection with hepatitis C, leaving 85 patients, 43 of whom had received NA treatment. HBV-DNA on admission was quantified by real time polymerase chain reaction. The treated and untreated groups were similar in most respects but differed significantly in regard to higher aminotransferase and bilirubin levels and hepatic coma grades, all being observed in the untreated group. Median duration of NA treatment was 6 days (range, 1-21 days). Overall survival in the NA treated and untreated groups were 61 and 64%, respectively (P = 0.72). Rates of transplant-free survival were 21 and 36% in the treated and untreated groups, respectively (P = 0.42). Multivariate analysis revealed that not using a NA [odds ratio (OR) 4.4, 95% CI 1.1-18.1, P = 0.041], hepatic coma grade I or II [OR 14.4, 95% CI 3.3-62.8, P < 0.001] and prothrombin time (PT) [OR 0.59, 95% CI 0.39-0.89, P = 0.012] were predictors of improved transplant-free survival. Patients who are admitted with established HBV-ALF do not appear to benefit from viral suppression using nucleoside(tide) analogues presumably because of rapid disease evolution and short treatment duration. Despite the lack of benefit, NAs should still be given to transplantation candidates since viral suppression prevents recurrence after grafting.

Inosine triphosphatase polymorphisms and ribavirin pharmacokinetics as determinants of ribavirin-associate anemia in patients receiving standard anti-HCV treatment.

PubMed

DʼAvolio, Antonio; Ciancio, Alessia; Siccardi, Marco; Smedile, Antonina; Baietto, Lorena; Simiele, Marco; Marucco, Diego Aguilar; Cariti, Giuseppe; Calcagno, Andrea; de Requena, Daniel Gonzalez; Sciandra, Mauro; Cusato, Jessica; Troshina, Giulia; Bonora, Stefano; Rizzetto, Mario; Di Perri, Giovanni

2012-04-01

Functional variants of inosine triphosphatase (ITPA) were recently found to protect against ribavirin (RBV)-induced hemolytic anemia. However, no definitive data are yet available on the role of plasma RBV concentrations on hemoglobin (Hb) decrement. Moreover, no data have been published on the possible interplay between these 2 factors. A retrospective analysis included 167 patients. The ITPA variants rs7270101 and rs1127354 were genotyped and tested using the χ test for association with Hb reduction at week 4. We also investigated, using multivariate logistic regression, the impact of RBV plasma exposure on Hb concentrations. Both single nucleotide polymorphisms were associated with Hb decrease. The carrier of at least 1 variant allele in the functional ITPA single nucleotide polymorphisms was associated with a lower decrement of Hb (-1.1 g/dL), as compared with patients without a variant allele (-2.75 g/dL; P = 4.09 × 10). RBV concentrations were not influenced by ITPA genotypes. A cut-off of 2.3 μg/mL of RBV was found to be associated with anemia (area-under-receiver operating characteristic = 0.630, sensitivity = 50.0%, and specificity = 69.5%, P = 0.008). In multivariate logistic regression analyses, the carrier of a variant allele (P = 0.005) and plasma RBV concentrations <2.3 μg/mL (P = 0.016) were independently associated with protection against clinically significant anemia at week 4. Although no direct relationship was found between ITPA polymorphisms and plasma RBV concentrations, both factors were shown to be significantly associated with anemia. A multivariate regression model based on ITPA genetic polymorphisms and RBV trough concentration was developed for predicting the risk of anemia. By relying upon these 2 variables, an individualized management of anemia seems to be feasible in recipients of pegylated interferon-RBV therapy.

On the distributive patterns of ATPase activity and its functional significance in retinae of certain birds.

PubMed

Tewari, H B; Tyagi, H R

1977-01-01

The present study incorporates the details of distribution of adenosine triphosphatase amongst the various constituents of retinae of Passer, Psittacula, Streptopelia and Athene. The outer segments in all the cases are intensely positive for the enzyme. This is the part where the light strikes first and initiates the visual processes. The nuclear layers are also positive for the enzyme activity. It is interesting to note that inner plexiform layers show clear-out demarcations of various sub-synaptic layers in all the birds except Psittacula. The ganglion cells and optic nerve fibres are also positive for the enzyme.

Structure-activity relationship analysis of mitochondrial toxicity caused by antiviral ribonucleoside analogs.

PubMed

Jin, Zhinan; Kinkade, April; Behera, Ishani; Chaudhuri, Shuvam; Tucker, Kathryn; Dyatkina, Natalia; Rajwanshi, Vivek K; Wang, Guangyi; Jekle, Andreas; Smith, David B; Beigelman, Leo; Symons, Julian A; Deval, Jerome

2017-07-01

Recent cases of severe toxicity during clinical trials have been associated with antiviral ribonucleoside analogs (e.g. INX-08189 and balapiravir). Some have hypothesized that the active metabolites of toxic ribonucleoside analogs, the triphosphate forms, inadvertently target human mitochondrial RNA polymerase (POLRMT), thus inhibiting mitochondrial RNA transcription and protein synthesis. Others have proposed that the prodrug moiety released from the ribonucleoside analogs might instead cause toxicity. Here, we report the mitochondrial effects of several clinically relevant and structurally diverse ribonucleoside analogs including NITD-008, T-705 (favipiravir), R1479 (parent nucleoside of balapiravir), PSI-7851 (sofosbuvir), and INX-08189 (BMS-986094). We found that efficient substrates and chain terminators of POLRMT, such as the nucleoside triphosphate forms of R1479, NITD-008, and INX-08189, are likely to cause mitochondrial toxicity in cells, while weaker chain terminators and inhibitors of POLRMT such as T-705 ribonucleoside triphosphate do not elicit strong in vitro mitochondrial effects. Within a fixed 3'-deoxy or 2'-C-methyl ribose scaffold, changing the base moiety of nucleotides did not strongly affect their inhibition constant (K i ) against POLRMT. By swapping the nucleoside and prodrug moieties of PSI-7851 and INX-08189, we demonstrated that the cell-based toxicity of INX-08189 is mainly caused by the nucleoside component of the molecule. Taken together, these results show that diverse 2' or 4' mono-substituted ribonucleoside scaffolds cause mitochondrial toxicity. Given the unpredictable structure-activity relationship of this ribonucleoside liability, we propose a rapid and systematic in vitro screen combining cell-based and biochemical assays to identify the early potential for mitochondrial toxicity. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

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 Central

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

2016-01-01

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 (100 mm × 2.1 mm, 5μm) and isocratic elution with a 10 mM ammonium acetate:acetonitrile with 0.1% formic acid (70:30, v/v) mobile phase over a 5 minute 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 – 400 ng/mL for 5-aza-2′-deoxycytidine, 50 – 10,000 ng/mL for 2′-deoxycytidine, and was 5 – 1,000 ng/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

Discovery of novel dengue virus NS5 methyltransferase non-nucleoside inhibitors by fragment-based drug design.

PubMed

Benmansour, Fatiha; Trist, Iuni; Coutard, Bruno; Decroly, Etienne; Querat, Gilles; Brancale, Andrea; Barral, Karine

2017-01-05

With the aim to help drug discovery against dengue virus (DENV), a fragment-based drug design approach was applied to identify ligands targeting a main component of DENV replication complex: the NS5 AdoMet-dependent mRNA methyltransferase (MTase) domain, playing an essential role in the RNA capping process. Herein, we describe the identification of new inhibitors developed using fragment-based, structure-guided linking and optimization techniques. Thermal-shift assay followed by a fragment-based X-ray crystallographic screening lead to the identification of three fragment hits binding DENV MTase. We considered linking two of them, which bind to proximal sites of the AdoMet binding pocket, in order to improve their potency. X-ray crystallographic structures and computational docking were used to guide the fragment linking, ultimately leading to novel series of non-nucleoside inhibitors of flavivirus MTase, respectively N-phenyl-[(phenylcarbamoyl)amino]benzene-1-sulfonamide and phenyl [(phenylcarbamoyl)amino]benzene-1-sulfonate derivatives, that show a 10-100-fold stronger inhibition of 2'-O-MTase activity compared to the initial fragments. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Functional expression of TcoAT1 reveals it to be a P1-type nucleoside transporter with no capacity for diminazene uptake☆

PubMed Central

Munday, Jane C.; Rojas López, Karla E.; Eze, Anthonius A.; Delespaux, Vincent; Van Den Abbeele, Jan; Rowan, Tim; Barrett, Michael P.; Morrison, Liam J.; de Koning, Harry P.

2013-01-01

It has long been established that the Trypanosoma brucei TbAT1/P2 aminopurine transporter is involved in the uptake of diamidine and arsenical drugs including pentamidine, diminazene aceturate and melarsoprol. Accordingly, it was proposed that the closest Trypanosoma congolense paralogue, TcoAT1, might perform the same function in this parasite, and an apparent correlation between a Single Nucleotide Polymorphism (SNP) in that gene and diminazene tolerance was reported for the strains examined. Here, we report the functional cloning and expression of TcoAT1 and show that in fact it is the syntenic homologue of another T. brucei gene of the same Equilibrative Nucleoside Transporter (ENT) family: TbNT10. The T. congolense genome does not seem to contain a syntenic equivalent to TbAT1. Two TcoAT1 alleles, differentiated by three independent SNPs, were expressed in the T. brucei clone B48, a TbAT1-null strain that further lacks the High Affinity Pentamidine Transporter (HAPT1); TbAT1 was also expressed as a control. The TbAT1 and TcoAT1 transporters were functional and increased sensitivity to cytotoxic nucleoside analogues. However, only TbAT1 increased sensitivity to diamidines and to cymelarsan. Uptake of [3H]-diminazene was detectable only in the B48 cells expressing TbAT1 but not TcoAT1, whereas uptake of [3H]-inosine was increased by both TcoAT1 alleles but not by TbAT1. Uptake of [3H]-adenosine was increased by all three ENT genes. We conclude that TcoAT1 is a P1-type purine nucleoside transporter and the syntenic equivalent to the previously characterised TbNT10; it does not mediate diminazene uptake and is therefore unlikely to play a role in diminazene resistance in T. congolense. PMID:24533295

Metal ion-promoted cleavage of nucleoside diphosphosugars: a model for reactions of phosphodiester bonds in carbohydrates.

PubMed

Dano, Meisa; Elmeranta, Marjukka; Hodgson, David R W; Jaakkola, Juho; Korhonen, Heidi; Mikkola, Satu

2015-12-01

Cleavage of five different nucleoside diphosphosugars has been studied in the presence of Cu(2+) and Zn(2+) complexes. The results show that metal ion catalysts promote the cleavage via intramolecular transesterification whenever a neighbouring HO group can adopt a cis-orientation with respect to the phosphate. The HO group attacks the phosphate and two monophosphate products are formed. If such a nucleophile is not available, Cu(2+) complexes are able to promote a nucleophilic attack of an external nucleophile, e.g. a water molecule or metal ion coordinated HO ligand, on phosphate. With the Zn(2+) complex, this was not observed.

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

PubMed

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

2015-12-12

Chinese jujube (Ziziphus jujuba), a medicinal and edible plant, is widely consumed in Asian countries owing to the remarkable health activities of its fruits. To facilitate selection of the suitable processing method for jujube fruits, in this study their contents of triterpenic acids, nucleosides, nucleobases and saccharides after drying and steaming treatment were determined using ultra-high performance liquid chromatography and high performance liquid chromatography coupled with evaporative light scattering detector methods. The results showed that except for sucrose, the content levels of most analytes were increasing in the jujube fruits during drying treatment at 45 °C. The levels of cyclic nucleotides such as adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, were significantly decreased after the fruits were steamed. Therefore, owing to the bioactivities of these components for human health, the dried fruits would be the better choice as medicinal material or functional food, and dried jujube fruit should not be further steamed.

Efficacy and safety of nucleoside analogues in preventing vertical transmission of the hepatitis B virus from father to infant.

PubMed

Cao, L-H; Zhao, P-L; Liu, Z-M; Sun, S-C; Xu, D-B; Zhang, J-D; Shao, Z-H

2015-12-02

We examined the efficacy and safety of nucleoside analogues in preventing the vertical transmission of hepatitis B virus (HBV) from father to infant. We included 201 patients who visited the liver clinic of our hospital. The patients were positive for HBV surface antigen (HBsAg), HBeAg, anti-HBc, and HBV DNA; 189 patients (94%) had abnormal liver function. In all couples, the fathers were HBV DNA-negative and had normal liver function, and the mothers were anti-HB-positive before pregnancy. The control group comprised 188 couples who visited our hospital during the same time period. The fathers in the control group were positive for HBsAg, HBeAg, anti-HBc, and HBV DNA. The mothers were HBsAg-negative and anti-HBs-positive. No infants in the case group were HBsAg-positive and HBV DNA-positive, and all were anti-HBs-positive, indicating that father to infant HBV vertical transmission was prevented in the case group. In the control group, 147 of 188 newborns (78.2%) were anti-HBs-positive at birth, 28 (14.9%) were HBV DNA-positive, and 19 (10.1%) were HBsAg-positive. A significant difference was observed between the two groups. No statistically significant difference was observed in the gestational age, birth weight, birth length, 1-min and 8-min Apgar score, jaundice, other internal and surgical diseases, delivery mode, and other birth information between the neonates born to couples in the case and control groups; there were no fetal malformations and stillbirths in the two groups. Our results showed that administration of antiretroviral therapy to HBV DNA-positive fathers before pregnancy can cause a decrease in the viral load and prevent father to infant HBV vertical transmission. The use of antiviral nucleoside analogues before pregnancy was safe in fathers, and the fathers who wanted children could continue to use anti-viral therapy. The sample size in our study was small, and further studies with a large sample size and longer follow-up time are required for

Intramolecular CH···O hydrogen bonds in the AI and BI DNA-like conformers of canonical nucleosides and their Watson-Crick pairs. Quantum chemical and AIM analysis.

PubMed

Yurenko, Yevgen P; Zhurakivsky, Roman O; Samijlenko, Svitlana P; Hovorun, Dmytro M

2011-08-01

The aim of this work is to cast some light on the H-bonds in double-stranded DNA in its AI and BI forms. For this purpose, we have performed the MP2 and DFT quantum chemical calculations of the canonical nucleoside conformers, relative to the AI and BI DNA forms, and their Watson-Crick pairs, which were regarded as the simplest models of the double-stranded DNA. Based on the atoms-in-molecules analysis (AIM), five types of the CH···O hydrogen bonds, involving bases and sugar, were detected numerically from 1 to 3 per a conformer: C2'H···O5', C1'H···O2, C6H···O5', C8H···O5', and C6H···O4'. The energy values of H-bonds occupy the range of 2.3-5.6 kcal/mol, surely exceeding the kT value (0.62 kcal/mol). The nucleoside CH···O hydrogen bonds appeared to "survive" turns of bases against the sugar, sometimes in rather large ranges of the angle values, pertinent to certain conformations, which points out to the source of the DNA lability, necessary for the conformational adaptation in processes of its functioning. The calculation of the interactions in the dA·T nucleoside pair gives evidence, that additionally to the N6H···O4 and N1···N3H canonical H-bonds, between the bases adenine and thymine the third one (C2H···O2) is formed, which, though being rather weak (about 1 kcal/mol), satisfies the AIM criteria of H-bonding and may be classified as a true H-bond. The total energy of all the CH···O nontraditional intramolecular H-bonds in DNA nucleoside pairs appeared to be commensurable with the energy of H-bonds between the bases in Watson-Crick pairs, which implies their possible important role in the DNA shaping.

Synthesis of nucleosides and oligonucleotides containing adducts of acrolein and vinyl chloride.

PubMed

Nechev, L V; Harris, C M; Harris, T M

2000-05-01

Vinyl chloride and acrolein are important industrial chemicals. Both form DNA adducts, vinyl chloride after enzymatic oxidation to chlorooxirane and acrolein by direct reaction. Reaction at the N(2) position of guanine is a major pathway. The resulting 2-oxoethyl and 3-oxopropyl adducts cyclize spontaneously to hydroxyethano and hydroxypropano derivatives, respectively. The two cyclic adducts have been detected in DNA exposed to these mutagens. A new method has been developed for the synthesis of deoxyguanosine adducts of chlorooxirane and acrolein, as well as oligonucleotides containing these adducts. Reaction of O(6)-[(trimethylsilyl)ethyl]-2-fluoro-2'-deoxyinosine with the appropriate aminodiol followed by oxidative cleavage of the diol with NaIO(4) gave the adducts in excellent yields. Reaction of oligonucleotides containing the halonucleoside with the aminodiols followed by NaIO(4) efficiently created the nucleosides in the oligonucleotides. Deoxyadenosine adducts were created similarly using 6-chloropurine 9-(2'-deoxyriboside).

Erythrocyte Inosine triphosphatase activity: A potential biomarker for adverse events during combination antiretroviral therapy for HIV

PubMed Central

Bierau, Jörgen; Bakker, Jaap A.; Schippers, Jolanda A.; Lowe, Selwyn H.; Paulussen, Aimée D. C.; van den Bosch, Bianca J. C.; Leers, Mathie P. G.; Hansen, Bettina E.; Verbon, Annelies

2018-01-01

The purine analogues tenofovir and abacavir are precursors of potential substrates for the enzyme Inosine 5’-triphosphate pyrophosphohydrolase (ITPase). Here, we investigated the association of ITPase activity and ITPA genotype with the occurrence of adverse events (AEs) during combination antiretroviral therapy (cART) for human immunodeficiency virus (HIV) infection. In 393 adult HIV-seropositive patients, AEs were defined as events that led to stop of cART regimen. ITPase activity ≥4 mmol IMP/mmol Hb/hour was considered as normal. ITPA genotype was determined by testing two ITPA polymorphisms: c.94C>A (p.Pro32Thr, rs1127354) and c.124+21A>C (rs7270101). Logistic regression analysis determined odds ratios for developing AEs. In tenofovir-containing regimens decreased ITPase activity was associated with less AEs (p = 0.01) and longer regimen duration (p = 0.001). In contrast, in abacavir-containing regimens decreased ITPase activity was associated with more AEs (crude p = 0.02) and increased switching of medication due to AEs (p = 0.03). ITPA genotype wt/wt was significantly associated with an increase in the occurrence of AEs in tenofovir-containing regimens. Decreased ITPase activity seems to be protective against occurrence of AEs in tenofovir-containing cART, while it is associated with an increase in AEs in abacavir-containing regimens. PMID:29329318

Simultaneous separation and determination of eleven nucleosides and bases in beer, herring sperm DNA and RNA soft capsule by high-performance liquid chromatography.

PubMed

Hou, Shengjie; Ding, Mingyu

2010-01-01

A simple and rapid high-performance liquid chromatography method was developed for the determination of eleven nucleosides and bases in beer, herring sperm DNA and RNA soft capsules. The separation was carried out on an Agilent extend-C(18) column with a simple gradient elution of acetonitrile and water as the mobile phase. Good linear relationships between the peak areas and the concentrations of the analytes were obtained. The detection limits for eleven analytes were in the range of 0.007-0.037 mg/L by UV detection at 260 nm. The relative standard deviations (RSDs) of the retention times were in the range of 0.78-1.85% for intra-day and 0.87-1.94% for inter-day, respectively. The RSDs of the peak areas were in the range of 2.71-3.22% for intra-day and 3.03-3.39% for inter-day, respectively. This method has been successfully applied to simultaneous determination of eleven nucleosides and bases in beer, herring sperm DNA and RNA soft capsules with the recoveries in the range of 93.7-108.3%.

Non-nucleoside reverse transcriptase inhibitor efavirenz increases monolayer permeability of human coronary artery endothelial cells

PubMed Central

Jamaluddin, Md Saha; Lin, Peter H.; Yao, Qizhi; Chen, Changyi

2009-01-01

Highly active antiretroviral therapy (HAART) is often associated with endothelial dysfunction and cardiovascular complications. In this study, we determined whether HIV non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) could increase endothelial permeability. Human coronary artery endothelial cells (HCAECs) were treated with EFV (1, 5 and 10 µg/ml) and endothelial permeability was determined by a transwell system with a fluorescence-labeled dextran tracer. HCAECs treated with EFV showed a significant increase of endothelial permeability in a concentration-dependent manner. With real time PCR analysis, EFV significantly reduced the mRNA levels of tight junction proteins claudin-1, occludin, zonula occluden-1 and junctional adhesion molecule-1 compared with controls (P < 0.05). Protein levels of these tight junction molecules were also reduced substantially in the EFV-treated cells by western blot and flow cytometry analyses. In addition, EFV also increased superoxide anion production with dihydroethidium and cellular glutathione assays, while it decreased mitochondrial membrane potential with JC-staining. Antioxidants (ginkgolide B and MnTBAP) effectively blocked EFV-induced endothelial permeability and mitochondrial dysfunction. Furthermore, EFV increased the phosphorylation of MAPK JNK and IκBα, thereby increasing NFκB translocation to the nucleus. Chemical JNK inhibitor and dominant negative mutant JNK and IkBa adenoviruses effectively blocked the effects of EFV on HCAECs. Thus, EFV increases endothelial permeability which may be due to the decrease of tight junction proteins and the increase of superoxide anion. JNK and NFκB activation may be directly involved in the signal transduction pathway of EFV action in HCAECs. PMID:19674747

The development of HEPT-type HIV non-nucleoside reverse transcriptase inhibitors and its implications for DABO family.

PubMed

Chen, Wenmin; Zhan, Peng; Wu, Jingde; Li, Zhenyu; Liu, Xinyong

2012-01-01

1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) was discovered as the first HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) in 1989. The research on HEPT derivatives (HEPTs) has been lasted for more than 20 years and HEPT family is probably the most investigated NNRTI. Extensive molecular modifications on HEPT have led to many highly potent compounds with broad-resistance spectrum and optimal pharmacokinetic profiles. Moreover, X-crystallographic studies of HEPTs/RT complexes revealed the binding mode of HEPTs and the action mechanism of NNRTI, which has greatly facilitated the design of novel NNRTIs. Recently, the development of HEPTs was accelerated by the application of the "follow-on"-based chemical evolution strategies, such as designed multiple ligands (DMLs) and molecular hybridization (MH). Herein, this article will provide an insight into the development of HEPTs, including structural modifications, crystal structure of RT complexed with HEPTs and its structure-activity relationship (SAR). Additionally, this review also covers the emerging HEPT related dual inhibitors and HEPT-pyridinone hybrids, as well as the contributions of HEPTs to the development of dihydro-alkoxy-benzyl-oxopyrimidine (DABO) family, thus highlighting the importance of HEPTs on the development of NNRTIs.

Repriming by PrimPol is critical for DNA replication restart downstream of lesions and chain-terminating nucleosides.

PubMed

Kobayashi, Kaori; Guilliam, Thomas A; Tsuda, Masataka; Yamamoto, Junpei; Bailey, Laura J; Iwai, Shigenori; Takeda, Shunichi; Doherty, Aidan J; Hirota, Kouji

2016-08-02

PrimPol is a DNA damage tolerance enzyme possessing both translesion synthesis (TLS) and primase activities. To uncover its potential role in TLS-mediated IgVλ hypermutation and define its interplay with other TLS polymerases, PrimPol(-/-) and PrimPol(-/-)/Polη(-/-)/Polζ (-/-) gene knockouts were generated in avian cells. Loss of PrimPol had no significant impact on the rate of hypermutation or the mutation spectrum of IgVλ. However, PrimPol(-/-) cells were sensitive to methylmethane sulfonate, suggesting that it may bypass abasic sites at the IgVλ segment by repriming DNA synthesis downstream of these sites. PrimPol(-/-) cells were also sensitive to cisplatin and hydroxyurea, indicating that it assists in maintaining / restarting replication at a variety of lesions. To accurately measure the relative contribution of the TLS and primase activities, we examined DNA damage sensitivity in PrimPol(-/-) cells complemented with polymerase or primase-deficient PrimPol. Polymerase-defective, but not primase-deficient, PrimPol suppresses the hypersensitivity of PrimPol(-/-) cells. This indicates that its primase, rather than TLS activity, is pivotal for DNA damage tolerance. Loss of TLS polymerases, Polη and Polζ has an additive effect on the sensitivity of PrimPol(-/-) cells. Moreover, we found that PrimPol and Polη-Polζ redundantly prevented cell death and facilitated unperturbed cell cycle progression. PrimPol(-/-) cells also exhibited increased sensitivity to a wide variety of chain-terminating nucleoside analogs (CTNAs). PrimPol could perform close-coupled repriming downstream of CTNAs and oxidative damage in vitro. Together, these results indicate that PrimPol's repriming activity plays a central role in reinitiating replication downstream from CTNAs and other specific DNA lesions.

Dysregulation of autophagy in rat liver with mitochondrial DNA depletion induced by the nucleoside analogue zidovudine.

PubMed

Santos-Llamas, Ana; Monte, Maria J; Marin, Jose J G; Perez, Maria J

2018-03-28

The nucleoside reverse transcriptase inhibitor zidovudine (AZT), used in HIV infection treatment, induces mitochondrial DNA (mtDNA) depletion. A cause-effect relationship between mtDNA status alterations and autophagy has been reported. Both events are common in several liver diseases, including hepatocellular carcinoma. Here, we have studied autophagy activation in rat liver with mtDNA depletion induced by AZT administration in drinking water for 35 days. AZT at a concentration of 1 mg/ml, but not 0.5 mg/ml in the drinking water, decreased mtDNA levels in rat liver and extrahepatic tissues. In liver, mtDNA-encoded cytochrome c oxidase 1 protein levels were decreased. Although serum biomarkers of liver and kidney toxicity remained unaltered, β-hydroxybutyrate levels were increased in liver of AZT-treated rats. Moreover, autophagy was dysregulated at two levels: (i) decreased induction signalling of this process as indicated by increases in autophagy inhibitors activity (AKT/mTOR), and absence of changes (Beclin-1, Atg5, Atg7) or decreases (AMPK/ULK1) in the expression/activity of pro-autophagy proteins; and (ii) reduced autophagosome degradation as indicated by decreases in the lysosome abundance (LAMP2 marker) and the transcription factor TFEB controlling lysosome biogenesis. This resulted in increased autophagosome abundance (LC3-II marker) and accumulation of the protein selectively degraded by autophagy p62, and the transcription factor Nrf2 in liver of AZT-treated rats. Nrf2 was activated as indicated by the up-regulation of antioxidant target genes Nqo1 and Hmox-1. In conclusion, rat liver with AZT-induced mtDNA depletion presented dysregulations in autophagosome formation and degradation balance, which results in accumulation of these structures in parenchymal liver cells, favouring hepatocarcinogenesis.

The effect of temperature and anoxia of kidney on the subsequent oxidative phosphorylation of mitochondria

PubMed Central

Rochman, H.; Lathe, G. H.; Levell, M. J.

1967-01-01

1. Kidneys were kept anoxic at 4°, 20° and 38°. Mitochondria were then isolated and their oxidative phosphorylation and respiration were determined. 2. Under all conditions the rate of phosphate esterification was affected to a greater extent, or earlier, than oxygen consumption. 3. Glutamate and succinate were used as substrates. The depression of P/O ratio was greater for glutamate at 4°, and for succinate at 20°. 4. Anoxia abolished the inhibiting effect of fluoride on respiration. 5. Phosphate esterification, after anoxia, was higher in the presence of fluoride than its absence, whereas in control preparations they were the same. 6. The decrease in P/O ratio did not appear to be due to activation of adenosine triphosphatase, as activities of both Mg2+-and dinitrophenol-activated adenosine triphosphatases were decreased after anoxia. PMID:4226526

Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin.

PubMed

Bruntner, C; Lauer, B; Schwarz, W; Möhrle, V; Bormann, C

1999-08-01

Six genes (nikA, nikB, nikD, nikE, nikF, and nikG) from Streptomyces tendae Tü901 were identified by sequencing the region surrounding the nikC gene, which encodes L-lysine 2-aminotransferase, previously shown to catalyze the initial reaction in the biosynthesis of hydroxypyridylhomothreonine, the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin. These genes, together with the nikC gene, span a DNA region of 7.87 kb and are transcribed as a polycistronic mRNA in a growth-phase dependent manner. The sequences of the deduced proteins NikA and NikB exhibit significant similarity to those of acetaldehyde dehydrogenases and 4-hydroxy-2-oxovalerate aldolases, respectively, which are involved in meta-cleavage degradation of aromatic hydrocarbons. The predicted NikD gene product shows sequence similarity to monomeric sarcosine oxidases, and the deduced NikE protein belongs to the superfamily of adenylate-forming enzymes. The nikF gene and the nikG gene encode a cytochrome P450 monooxygenase and a ferredoxin, respectively. Disruption of any of the genes nikA, nikB, nikD, nikE and nikF by insertion of a kanamycin resistance cassette abolished formation of the biologically active nikkomycins I, J, X, and Z. The nikA, nikB, nikD, and nikE mutants accumulated the nucleoside moieties nikkomycins Cx and Cz. In the nikD and nikE mutants nikkomycin production (nikkomycins I, J, X, Z) could be restored by feeding with picolinic acid and hydroxypyridylhomothreonine, respectively. The nikF mutant exclusively produced novel derivatives, nikkomycins Lx and Lz, which contain pyridylhomothreonine as the peptidyl moiety. Our results indicate that the nikA, nikB, nikD, nikE, nikF, and nikG genes, in addition to nikC, function in the biosynthetic pathway leading to hydroxypyridylhomothreonine, the putative activities of each of their products are discussed.

Quercetin derivatives as non-nucleoside inhibitors for dengue polymerase: molecular docking, molecular dynamics simulation, and binding free energy calculation.

PubMed

Anusuya, Shanmugam; Gromiha, M Michael

2017-10-01

Dengue is an important public health problem in tropical and subtropical regions of the world. Neither vaccine nor an antiviral medication is available to treat dengue. This insists the need of drug discovery for dengue. In order to find a potent lead molecule, RNA-dependent RNA polymerase which is essential for dengue viral replication is chosen as a drug target. As Quercetin showed antiviral activity against several viruses, quercetin derivatives developed by combinatorial library synthesis and mined from PubChem databases were screened for a potent anti-dengue viral agent. Our study predicted Quercetin 3-(6″-(E)-p-coumaroylsophoroside)-7-rhamnoside as a dengue polymerase inhibitor. The results were validated by molecular dynamics simulation studies which reveal water bridges and hydrogen bonds as major contributors for the stability of the polymerase-lead complex. Interactions formed by this compound with residues Trp795, Arg792 and Glu351 are found to be essential for the stability of the polymerase-lead complex. Our study demonstrates Quercetin 3-(6″-(E)-p-coumaroylsophoroside)-7-rhamnoside as a potent non-nucleoside inhibitor for dengue polymerase.

TGF-β-induced stromal CYR61 promotes resistance to gemcitabine in pancreatic ductal adenocarcinoma through downregulation of the nucleoside transporters hENT1 and hCNT3.

PubMed

Hesler, Rachel A; Huang, Jennifer J; Starr, Mark D; Treboschi, Victoria M; Bernanke, Alyssa G; Nixon, Andrew B; McCall, Shannon J; White, Rebekah R; Blobe, Gerard C

2016-11-01

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in part due to inherent resistance to chemotherapy, including the first-line drug gemcitabine. Although low expression of the nucleoside transporters hENT1 and hCNT3 that mediate cellular uptake of gemcitabine has been linked to gemcitabine resistance, the mechanisms regulating their expression in the PDAC tumor microenvironment are largely unknown. Here, we report that the matricellular protein cysteine-rich angiogenic inducer 61 (CYR61) negatively regulates the nucleoside transporters hENT1 and hCNT3. CRISPR/Cas9-mediated knockout of CYR61 increased expression of hENT1 and hCNT3, increased cellular uptake of gemcitabine and sensitized PDAC cells to gemcitabine-induced apoptosis. In PDAC patient samples, expression of hENT1 and hCNT3 negatively correlates with expression of CYR61 . We demonstrate that stromal pancreatic stellate cells (PSCs) are a source of CYR61 within the PDAC tumor microenvironment. Transforming growth factor-β (TGF-β) induces the expression of CYR61 in PSCs through canonical TGF-β-ALK5-Smad2/3 signaling. Activation of TGF-β signaling or expression of CYR61 in PSCs promotes resistance to gemcitabine in PDAC cells in an in vitro co-culture assay. Our results identify CYR61 as a TGF-β-induced stromal-derived factor that regulates gemcitabine sensitivity in PDAC and suggest that targeting CYR61 may improve chemotherapy response in PDAC patients. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Incoherent feedforward control governs adaptation of activated ras in a eukaryotic chemotaxis pathway.

PubMed

Takeda, Kosuke; Shao, Danying; Adler, Micha; Charest, Pascale G; Loomis, William F; Levine, Herbert; Groisman, Alex; Rappel, Wouter-Jan; Firtel, Richard A

2012-01-03

Adaptation in signaling systems, during which the output returns to a fixed baseline after a change in the input, often involves negative feedback loops and plays a crucial role in eukaryotic chemotaxis. We determined the dynamical response to a uniform change in chemoattractant concentration of a eukaryotic chemotaxis pathway immediately downstream from G protein-coupled receptors. The response of an activated Ras showed near-perfect adaptation, leading us to attempt to fit the results using mathematical models for the two possible simple network topologies that can provide perfect adaptation. Only the incoherent feedforward network accurately described the experimental results. This analysis revealed that adaptation in this Ras pathway is achieved through the proportional activation of upstream components and not through negative feedback loops. Furthermore, these results are consistent with a local excitation, global inhibition mechanism for gradient sensing, possibly with a Ras guanosine triphosphatase-activating protein acting as a global inhibitor.

Dipyridodiazepinone analogs as human immunodeficiency virus type 1-specific non-nucleoside reverse transcriptase inhibitors: an overview.

PubMed

Lv, M; Xu, H

2010-01-01

According to World Health Organization (WHO)/Joint United Nations Programme on human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) (UNAIDS) Report in 2007, 33.2 million people are living with HIV, 2.5 million ones have been newly infected with HIV, and 2.1 million ones died from AIDS, including 330,000 children. Therefore, HIV/AIDS still remains a public health emergency and a leading cause of mortality worldwide. It is believed that reverse transcriptase (RT) is a crucial enzyme in the life cycle of HIV-1, and thereby RT has been the important drug target for antiretroviral (ARV) chemotherapy against AIDS. To our knowledge, dipyridodiazepinone analogs have been considered as one class of potential non-nucleoside reverse transcriptase inhibitors (NNRTIs), especially the structurally and chemically related nevirapine (Viramune(R)), which was the first NNRTI approved by the U. S. Food and Drug Administration (FDA) for the treatment of HIV-1 infection for adults in 1996 and for children in 1998. This review mainly highlights the progress of synthesis and structure-activity relationship (SAR) of dipyridodiazepinone analogs; in the meantime, the mechanism of action is also presented. It will pave the way for the design and development of novel dipyridodiazepinone analogs as NNRTIs in AIDS chemotherapy in the future.

Naringin Ameliorates HIV-1 Nucleoside Reverse Transcriptase Inhibitors- Induced Mitochondrial Toxicity.

PubMed

Oluwafeyisetan, Adebiyi; Olubunmi, Adebiyi; Peter, Owira

2016-01-01

Mitochondrial reactive oxygen species (ROS) generation and defective oxidative phosphorylation (OXPHOS) have been proposed as possible mechanisms underlying the development of nucleoside reverse transcriptase inhibitors (NRTIs)-induced mitochondrial toxicities. Available options in managing these complications have, so far, produced controversial results, thus necessitating further research into newer agents with promise. Antioxidant and free-radical scavenging effects of naringin, a plant-derived flavonoid, have previously been demonstrated. This study was designed to investigate the effects of naringin on NRTIs-induced mitochondrial toxicity. Wistar rats were randomly divided into Zidovudine (AZT)-only (100 mg/kg body weight BW); AZT+Naringin (100+50 mg/kg BW); AZT+Vitamin E (100+100 mg/kg BW); Stavudine (d4T)- only (50 mg/kg BW); d4T+Naringin (50+50 mg/kg BW); d4T+Vitamin E (50+100 mg/kg BW) and Vehicle (3.0 mL/kg BW)-treated groups, respectively. After 56 days of oral daily dosing, rats were euthanized by halothane overdose, blood collected by cardiac puncture and livers promptly excised for further biochemical and ultrastructural analyses. </p> Results: AZT- or d4T-only caused significant mitochondrial dysfunction and mitochondrial ultrastructural damage compared to controls, while either naringin or vitamin E reversed indices of mitochondrial dysfunction evidenced by significantly reduced mitochondrial malondialdehyde (MDA) and blood lactate concentrations, increased liver manganese superoxide dismutase (MnSOD) activity and upregulate expression of mitochondrial-encoded subunit of electron transport chain (ETC) complex IV protein compared to AZT- or d4T-only treated rats. Furthermore, naringin or vitamin E, respectively, ameliorated mitochondrial damage observed in AZT- or d4T-only treated rats. Naringin ameliorated oxidative stress and NRTI-induced mitochondrial damage and might, therefore, be beneficial in managing toxicities and complications arising

A potent, covalent inhibitor of orotidine 5'-monophosphate decarboxylase with antimalarial activity.

PubMed

Bello, Angelica M; Poduch, Ewa; Fujihashi, Masahiro; Amani, Merhnaz; Li, Yan; Crandall, Ian; Hui, Raymond; Lee, Ping I; Kain, Kevin C; Pai, Emil F; Kotra, Lakshmi P

2007-03-08

Orotidine 5'-monophosphate decarboxylase (ODCase) has evolved to catalyze the decarboxylation of orotidine 5'-monophosphate without any covalent intermediates. Active site residues in ODCase are involved in an extensive hydrogen-bonding network. We discovered that 6-iodouridine 5'-monophosphate (6-iodo-UMP) irreversibly inhibits the catalytic activities of ODCases from Methanobacterium thermoautotrophicum and Plasmodium falciparum. Mass spectral analysis of the enzyme-inhibitor complex confirms covalent attachment of the inhibitor to ODCase accompanied by the loss of two protons and the iodo moiety. The X-ray crystal structure (1.6 A resolution) of the complex of the inhibitor and ODCase clearly shows the covalent bond formation with the active site Lys-72 [corrected] residue. 6-Iodo-UMP inhibits ODCase in a time- and concentration-dependent fashion. 6-Iodouridine, the nucleoside form of 6-iodo-UMP, exhibited potent antiplasmodial activity, with IC50s of 4.4 +/- 1.3 microM and 6.2 +/- 0.7 microM against P. falciparum ItG and 3D7 isolates, respectively. 6-Iodouridine 5'-monophosphate is a novel covalent inhibitor of ODCase, and its nucleoside analogue paves the way to a new class of inhibitors against malaria.

Expression, purification, immunogenicity and protective efficacy of a recombinant nucleoside hydrolase from Leishmania donovani, a vaccine candidate for preventing cutaneous leishmaniasis.

PubMed

McAtee, C Patrick; Seid, Christopher A; Hammond, Molly; Hudspeth, Elissa; Keegan, Brian P; Liu, Zhuyun; Wei, Junfei; Zhan, Bin; Arjona-Sabido, Raul; Cruz-Chan, Vladimir; Dumonteil, Eric; Hotez, Peter J; Bottazzi, Maria Elena

2017-02-01

The nucleoside hydrolase gene from Leishmania donovani was cloned and expressed in Escherichia coli as a full length 36-kDa protein (LdNH36). Following lysis and extraction, the protein was purified by anion exchange and gel filtration chromatography. The purified protein had a molecular mass of approximately 36-kDa and was confirmed to be >99% pure. Using a nucleoside hydrolase assay, the protein was found to exhibit a Km of 741 ± 246 μM. Protein integrity was confirmed by lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE), mass spectrometry (MS), and enzymatic assay. Analysis of antibody levels from immunized mice indicated that LdNH36 alone or in a stable emulsion with the Toll-like receptor-4 ligand glucopyranosyl lipid adjuvant (GLA-SE) as immunostimulant induced high levels of antigen-specific IgG antibodies. The cellular immune response indicated a T h 1 response in mice immunized with LdNH36, but only when formulated with GLA-SE. Mice immunized with the LdNH36 antigen in combination with the GLA-SE adjuvant and challenged with Leishmania mexicana showed significant reductions (>20 fold) in parasite burden, confirming the protective efficacy of this vaccine candidate. Copyright © 2016 Elsevier Inc. All rights reserved.

Substituted cysteine accessibility method (SCAM) analysis of the transport domain of human concentrative nucleoside transporter 3 (hCNT3) and other family members reveals features of structural and functional importance

PubMed Central

Mulinta, Ras; Yao, Sylvia Y. M.; Ng, Amy M. L.; Cass, Carol E.; Young, James D.

2017-01-01

The human SLC28 family of concentrative nucleoside transporter (CNT) proteins has three members: hCNT1, hCNT2, and hCNT3. Na+-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 transports both pyrimidine and purine nucleosides utilizing Na+ and/or H+ electrochemical gradients. Escherichia coli CNT family member NupC resembles hCNT1 in permeant selectivity but is H+-coupled. Using heterologous expression in Xenopus oocytes and the engineered cysteine-less hCNT3 protein hCNT3(C−), substituted cysteine accessibility method analysis with the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate was performed on the transport domain (interfacial helix 2, hairpin 1, putative transmembrane domain (TM) 7, and TM8), as well as TM9 of the scaffold domain of the protein. This systematic scan of the entire C-terminal half of hCNT3(C−) together with parallel studies of the transport domain of wild-type hCNT1 and the corresponding TMs of cysteine-less NupC(C−) yielded results that validate the newly developed structural homology model of CNT membrane architecture for human CNTs, revealed extended conformationally mobile regions within transport-domain TMs, identified pore-lining residues of functional importance, and provided evidence of an emerging novel elevator-type mechanism of transporter function. PMID:28385889

Repeated oral dosing of TAS-102 confers high trifluridine incorporation into DNA and sustained antitumor activity in mouse models

PubMed Central

TANAKA, NOZOMU; SAKAMOTO, KAZUKI; OKABE, HIROYUKI; FUJIOKA, AKIO; YAMAMURA, KEISUKE; NAKAGAWA, FUMIO; NAGASE, HIDEKI; YOKOGAWA, TATSUSHI; OGUCHI, KEI; ISHIDA, KEIJI; OSADA, AKIKO; KAZUNO, HIROMI; YAMADA, YUKARI; MATSUO, KENICHI

2014-01-01

TAS-102 is a novel oral nucleoside antitumor agent containing trifluridine (FTD) and tipiracil hydrochloride (TPI). The compound improves overall survival of colorectal cancer (CRC) patients who are insensitive to standard chemotherapies. FTD possesses direct antitumor activity since it inhibits thymidylate synthase (TS) and is itself incorporated into DNA. However, the precise mechanisms underlying the incorporation into DNA and the inhibition of TS remain unclear. We found that FTD-dependent inhibition of TS was similar to that elicited by fluorodeoxyuridine (FdUrd), another clinically used nucleoside analog. However, washout experiments revealed that FTD-dependent inhibition of TS declined rapidly, whereas FdUrd activity persisted. The incorporation of FTD into DNA was significantly higher than that of other antitumor nucleosides. Additionally, orally administered FTD had increased antitumor activity and was incorporated into DNA more effectively than continuously infused FTD. When TAS-102 was administered, FTD gradually accumulated in tumor cell DNA, in a TPI-independent manner, and significantly delayed tumor growth and prolonged survival, compared to treatment with 5-FU derivatives. TAS-102 reduced the Ki-67-positive cell fraction, and swollen nuclei were observed in treated tumor tissue. The amount of FTD incorporation in DNA and the antitumor activity of TAS-102 in xenograft models were positively and significantly correlated. These results suggest that TAS-102 exerts its antitumor activity predominantly due to its DNA incorporation, rather than as a result of TS inhibition. The persistence of FTD in the DNA of tumor cells treated with TAS-102 may underlie its ability to prolong survival in cancer patients. PMID:25230742

Structure-Activity Relationships of Orotidine-5′-Monophosphate Decarboxylase Inhibitors as Anticancer Agents

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

Bello, A.; Konforte, D; Poduch, E

2009-01-01

A series of 6-substituted and 5-fluoro-6-substituted uridine derivatives were synthesized and evaluated for their potential as anticancer agents. The designed molecules were synthesized from either fully protected uridine or the corresponding 5-fluorouridine derivatives. The mononucleotide derivatives were used for enzyme inhibition investigations against ODCase. Anticancer activities of all the synthesized derivatives were evaluated using the nucleoside forms of the inhibitors. 5-Fluoro-UMP was a very weak inhibitor of ODCase. 6-Azido-5-fluoro and 5-fluoro-6-iodo derivatives are covalent inhibitors of ODCase, and the active site Lys145 residue covalently binds to the ligand after the elimination of the 6-substitution. Among the synthesized nucleoside derivatives, 6-azido-5-fluoro,more » 6-amino-5-fluoro, and 6-carbaldehyde-5-fluoro derivatives showed potent anticancer activities in cell-based assays against various leukemia cell lines. On the basis of the overall profile, 6-azido-5-fluoro and 6-amino-5-fluoro uridine derivatives exhibited potential for further investigations.« less

Regulation of PRPP and nucleoside tri and tetraphosphate pools in Escherichia coli under conditions of nitrogen starvation.

PubMed Central

Villadsen, I S; Michelsen, O

1977-01-01

The ribonucleoside triphosphate, deoxyribonucleoside triphosphate, 3' -diphosphate guanosine 5' -diphosphate (ppGpp), and 5-phosphoribosyl 1-pyrophosphate (PRPP) pools in Escherichia coli B were determined by thin-layer chromatography during changing conditions to ammonium starvation. The intracellular concentrations of all nucleotides were found to change in a well-defined order several minutes before andy observed change in the optical density of the culture. The levels of purine nucleoside triphosphates (adenosine 5' -triphosphate [CTP], dCTP) and uridine nucleotides (uridine 5' -triphosphate, deoxythymidine 5'-triphosphate). The deoxyribonucleotides thus behaved as the ribonucleotides. The levels of ppGpp increased 11-fold after the decrease in uridine nucleotides, when the accumulation of stable ribonucleic acid (RNA) stopped. The level of the nucleotide pool did not stabilize until 30 min after the change in optical density. The pool of dGTP dropped concomitantly with the pool of CTP. The nucleotide precursor PRPP exhibited a transient increase, wtih maximum value of four times the exponential levels at the onset of starvation. Apparently the cell adjusts early to starvation by reducing either the phosphorylating activity or the nucleotide biosynthetic activity. As in other downshift systems, the accumulation of stable RNA stopped before the break in optical density and before the stop in protein accumulation. Cell divisions were quite insensitive to the control mechanisms operating on RNA and protein accumulation under ammonium starvation, since the cells continued to divide for 21 min without any net accumulation of RNA. Images PMID:323222

FLT3 is implicated in cytarabine transport by human equilibrative nucleoside transporter 1 in pediatric acute leukemia.

PubMed

Català, Albert; Pastor-Anglada, Marçal; Caviedes-Cárdenas, Liska; Malatesta, Roberta; Rives, Susana; Vega-García, Nerea; Camós, Mireia; Fernández-Calotti, Paula

2016-08-02

FLT3 abnormalities are negative prognostic markers in acute leukemia. Infant leukemias are a subgroup with frequent MLL (KMT2A) rearrangements, FLT3 overexpression and high sensitivity to cytarabine, but dismal prognosis. Cytarabine is transported into cells by Human Equilibrative Nucleoside Transporter-1 (hENT1, SLC29A1), but the mechanisms that regulate hENT1 in acute leukemia have been scarcely studied.We explored the expression and functional link between FLT3 and main cytarabine transporters in 50 pediatric patients diagnosed with acute lymphoblastic leukemia and MLL rearrangement (ALL-MLL+) and other subtypes of leukemia, and in leukemia cell lines.A significant positive correlation was found between FLT3 and hENT1 expression in patients. Cytarabine uptake into cells was mediated mainly by hENT1, hENT2 and hCNT1. hENT1-mediated uptake of cytarabine was transiently abolished by the FLT3 inhibitor PKC412, and this effect was associated with decreased hENT1 mRNA and protein levels. Noticeably, the cytotoxicity of cytarabine was lower when cells were first exposed to FLT3 inhibitors (PKC412 or AC220), probably due to decreased hENT1 activity, but we observed a higher cytotoxic effect if FLT3 inhibitors were administered after cytarabine.FLT3 regulates hENT1 activity and thereby affects cytarabine cytotoxicity. The sequence of administration of cytarabine and FLT3 inhibitors is important to maintain their efficacy.

Fluorinase: a tool for the synthesis of ¹⁸F-labeled sugars and nucleosides for PET.

PubMed

Onega, Mayca; Winkler, Margit; O'Hagan, David

2009-08-01

There is an increasing interest in the preparation of (18)F-labeled radiopharmaceuticals with potential applications in PET for medicinal imaging. Appropriate synthetic methods require a quick and efficient route in which to incorporate the (18)F into a ligand, due to the relatively short half-life of the (18)F isotope. Enzymatic methods are rare in this area; however, the discovery of a fluorinating enzyme from Streptomyces cattleya (EC 2.5.1.63) has opened up the possibility of the enzymatic synthesis and formation of C-(18)F bonds from the [(18)F]fluoride ion. In this article, the development of enzymatic preparations of (18)F-labeled sugars and nucleosides as potential radiotracers using the fluorinase from S. cattleya for PET applications is reviewed. Enzymatic reactions are not traditional in PET synthesis, but this enzyme has some attractive features. The enzyme is available in an overexpressed form from Escherichia coli and it is relatively stable and can be easily purified and manipulated. Most notably, it utilizes [(18)F] fluoride, the form of the isotope normally generated by the cyclotron and usually in very high specific radioactivity. The disadvantage with the enzyme is that it is substrate specific; however, when the fluorinase is used in combination biotransformations with a second or third enzyme, then a range of radiolabeled nucleosides and ribose sugars can be prepared. The fluorinase enzyme has emerged as a curiosity from biosynthesis studies, but it now has some potential as a new catalyst for (18)F incorporation for PET syntheses. The focus is now on delivering a user-friendly catalyst to the PET synthesis community and establishing a clinical role for some of the (18)F-labeled molecules available using this technology.

8-vinyl-deoxyadenosine, an alternative fluorescent nucleoside analog to 2'-deoxyribosyl-2-aminopurine with improved properties.

PubMed

Ben Gaied, Nouha; Glasser, Nicole; Ramalanjaona, Nick; Beltz, Hervé; Wolff, Philippe; Marquet, Roland; Burger, Alain; Mély, Yves

2005-01-01

We report here the synthesis and the spectroscopic characterization of 8-vinyl-deoxyadenosine (8vdA), a new fluorescent analog of deoxyadenosine. 8vdA was found to absorb and emit in the same wavelength range as 2'-deoxyribosyl-2-aminopurine (2AP), the most frequently used fluorescent nucleoside analog. Though the quantum yield of 8vdA is similar to that of 2AP, its molar absorption coefficient is about twice, enabling a more sensitive detection. Moreover, the fluorescence of 8vdA was found to be sensitive to temperature and solvent but not to pH (around neutrality) or coupling to phosphate groups. Though 8vdA is base sensitive and susceptible to depurination, the corresponding phosphoramidite was successfully prepared and incorporated in oligonucleotides of the type d(CGT TTT XNX TTT TGC) where N = 8vdA and X = A, T or C. The 8vdA-labeled oligonucleotides gave more stable duplexes than the corresponding 2AP-labeled sequences when X = A or T, indicating that 8vdA is less perturbing than 2AP and probably adopts an anti conformation to preserve the Watson-Crick H-bonding. In addition, the quantum yield of 8vdA is significantly higher than 2AP in all tested oligonucleotides in both their single strand and duplex states. The steady-state and time-resolved fluorescence parameters of 8vdA and 2AP were found to depend similarly on the nature of their flanking residues and on base pairing, suggesting that their photophysics are governed by similar mechanisms. Taken together, our data suggest that 8vdA is a non perturbing nucleoside analog that may be used with improved sensitivity for the same applications as 2AP.

Base-Pairing Energies of Protonated Nucleoside Base Pairs of dCyd and m5dCyd: Implications for the Stability of DNA i-Motif Conformations

NASA Astrophysics Data System (ADS)

Yang, Bo; Rodgers, M. T.

2015-08-01

Hypermethylation of cytosine in expanded (CCG)n•(CGG)n trinucleotide repeats results in Fragile X syndrome, the most common cause of inherited mental retardation. The (CCG)n•(CGG)n repeats adopt i-motif conformations that are preferentially stabilized by base-pairing interactions of protonated base pairs of cytosine. Here we investigate the effects of 5-methylation and the sugar moiety on the base-pairing energies (BPEs) of protonated cytosine base pairs by examining protonated nucleoside base pairs of 2'-deoxycytidine (dCyd) and 5-methyl-2'-deoxycytidine (m5dCyd) using threshold collision-induced dissociation techniques. 5-Methylation of a single or both cytosine residues leads to very small change in the BPE. However, the accumulated effect may be dramatic in diseased state trinucleotide repeats where many methylated base pairs may be present. The BPEs of the protonated nucleoside base pairs examined here significantly exceed those of Watson-Crick dGuo•dCyd and neutral dCyd•dCyd base pairs, such that these base-pairing interactions provide the major forces responsible for stabilization of DNA i-motif conformations. Compared with isolated protonated nucleobase pairs of cytosine and 1-methylcytosine, the 2'-deoxyribose sugar produces an effect similar to the 1-methyl substituent, and leads to a slight decrease in the BPE. These results suggest that the base-pairing interactions may be slightly weaker in nucleic acids, but that the extended backbone is likely to exert a relatively small effect on the total BPE. The proton affinity (PA) of m5dCyd is also determined by competitive analysis of the primary dissociation pathways that occur in parallel for the protonated (m5dCyd)H+(dCyd) nucleoside base pair and the absolute PA of dCyd previously reported.

Mimicking phosphorylation of Ser-74 on human deoxycytidine kinase selectively increases catalytic activity for dC and dC analogues.

PubMed

McSorley, Theresa; Ort, Stephan; Hazra, Saugata; Lavie, Arnon; Konrad, Manfred

2008-03-05

Intracellular phosphorylation of dCK on Ser-74 results in increased nucleoside kinase activity. We mimicked this phosphorylation by a Ser-74-Glu mutation in bacterially produced dCK and investigated kinetic parameters using various nucleoside substrates. The S74E mutation increases the k(cat) values 11-fold for dC, and 3-fold for the anti-cancer analogues dFdC and AraC. In contrast, the rate is decreased for the purine substrates. In HEK293 cells, we found that by comparing transiently transfected dCK(S74E)-GFP and wild-type dCK-GFP, mimicking the phosphorylation of Ser-74 has no effect on cellular localisation. We note that phosphorylation may represent a mechanism to enhance the catalytic activity of the relatively slow dCK enzyme.

Mimicking phosphorylation of Ser-74 on human deoxycytidine kinase selectively increases catalytic activity for dC and dC analogues

PubMed Central

McSorley, Theresa; Ort, Stephan; Hazra, Saugata; Lavie, Arnon; Konrad, Manfred

2009-01-01

Intracellular phosphorylation of dCK on Ser-74 results in increased nucleoside kinase activity. We mimicked this phosphorylation by a Ser-74-Glu mutation in bacterially produced dCK and investigated kinetic parameters using various nucleoside substrates. The S74E mutation increases the kcat values 11-fold for dC, and 3-fold for the anti-cancer analogues dFdC and AraC. In contrast, the rate is decreased for the purine substrates. In HEK293 cells, we found that by comparing transiently transfected dCK(S74E)-GFP and wild-type dCK-GFP, mimicking the phosphorylation of Ser-74 has no effect on cellular localisation. We note that phosphorylation may represent a mechanism to enhance the catalytic activity of the relatively slow dCK enzyme. PMID:18258203

Key mediators of intracellular amino acids signaling to mTORC1 activation.

PubMed

Duan, Yehui; Li, Fengna; Tan, Kunrong; Liu, Hongnan; Li, Yinghui; Liu, Yingying; Kong, Xiangfeng; Tang, Yulong; Wu, Guoyao; Yin, Yulong

2015-05-01

Mammalian target of rapamycin complex 1 (mTORC1) is activated by amino acids to promote cell growth via protein synthesis. Specifically, Ras-related guanosine triphosphatases (Rag GTPases) are activated by amino acids, and then translocate mTORC1 to the surface of late endosomes and lysosomes. Ras homolog enriched in brain (Rheb) resides on this surface and directly activates mTORC1. Apart from the presence of intracellular amino acids, Rag GTPases and Rheb, other mediators involved in intracellular amino acid signaling to mTORC1 activation include human vacuolar sorting protein-34 (hVps34) and mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3). Those molecular links between mTORC1 and its mediators form a complicate signaling network that controls cellular growth, proliferation, and metabolism. Moreover, it is speculated that amino acid signaling to mTORC1 may start from the lysosomal lumen. In this review, we discussed the function of these mediators in mTORC1 pathway and how these mediators are regulated by amino acids in details.

Thyroid thermogenesis. Relationships between Na+-dependent respiration and Na+ + K+-adenosine triphosphatase activity in rat skeletal muscle.

PubMed Central

Asano, Y; Liberman, U A; Edelman, I S

1976-01-01

The effect of thyroid status on QO2, QO2 (t) and NaK-ATPase activity was examined in rat skeletal muscle. QO2(t) (i.e. Na+-transport-dependent respiration) was estimated with ouabain or Na+-free media supplemented with K+. In contrast to the effects of ouabain on ion composition, intracellular K+ was maintained at about 125 meq/liter, and intracellular Na+ was almost nil in the Na+-free media. The estimates of QO2(t) were independent of the considerable differences in tissue ion concentrations. The increase in QO2(t) account for 47% of the increase in QO2 in the transition from the hypothyroid to the euthyroid state and 84% of the increase in the transition from the euthyroid to the hyperthyroid state. Surgical thyroidectomy lowered NaK-ATPase activity of the microsomal fraction (expressed per milligram protein) 32%; injections of triodothyronine (T3) increased this activity 75% in initially hypothyroid rats and 26% in initially euthyroid rats. Thyroidectomy was attended by significant falls in serum Ca and Pi concentrations. Administration of T3 resulted in further declines in serum Ca and marked increases in serum Ps concentrations. Similar effects were seen in 131I-treated rats, but the magnitude of the declines in serum Ca were less. The effects of T3 on QO2, QO2(t), and NaK-ATPase activity of skeletal muscle were indistinguishable in the 131I-ablated and surgically thyroidectomized rats. In thyroidectomized or euthyroid rats given repeated doses of T3, QO2(t) and NaA-ATPase activity increased proportionately. In thyroidectomized rats injected with single doses of T3, either 10, 50, or 250 mug/100 g body wt, QO2(t) increased linearly with NaK-ATPase activity. The kinetics of the NaK-ATPase activity was assessed with an ATP-generating system. T3 elicited a significant increase in Vmax with no change in Km for ATP. PMID:130385

[Adenosine triphosphatase activity in the organs of the crab Hemigrapsus sanguineus, acclimated to sea water of different salinity].

PubMed

Busev, V M

1977-01-01

In crabs acclimated to low salinity, the activity of Na, K-ATPase from the gills increases; the activity also increases in the antennal glands after acclimation of the animals to high salinity. The activity of Na, K-ATPase in the abdominal ganglion and in the heart does not depend on the salinity to which crabs had been acclimated. Changes in the activity of Mg-ATPase in the gills and antennal glands associated with acclimation of crabs to sea water with different salinity correspond to those in the activity of Na, K-ATPase.

[Effect of low-intensity 900 MHz frequency electromagnetic radiation on rat liver and blood serum enzyme activities].

PubMed

Nersesova, L S; Petrosian, M S; Gazariants, M G; Mkrtchian, Z S; Meliksetian, G O; Pogosian, L G; Akopian, Zh I

2014-01-01

The comparative analysis of the rat liver and blood serum creatine kinase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and purine nucleoside phosphorylase post-radiation activity levels after a total two-hour long single and fractional exposure of the animals to low-intensity 900 MHz frequency electromagnetic field showed that the most sensitive enzymes to the both schedules of radiation are the liver creatine kinase, as well as the blood serum creatine kinase and alkaline phosphatase. According to the comparative analysis of the dynamics of changes in the activity level of the liver and blood serum creatine kinase, alanine aminotransferase, aspartate aminotransferase and purine nucleoside phosphorylase, both single and fractional radiation schedules do not affect the permeability of a hepatocyte cell membrane, but rather cause changes in their energetic metabolism. The correlation analysis of the post-radiation activity level changes of the investigated enzymes did not reveal a clear relationship between them. The dynamics of post-radiation changes in the activity of investigated enzyme levels following a single and short-term fractional schedules of radiation did not differ essentially.

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

PubMed Central

Lund, Kaleb C.; Wallace, Kendall B.

2008-01-01

Nucleoside analog reverse transcriptase inhibitors (NRTI) 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 μM), AZT monophosphate (150 μM), and 2′,3′-dideoxycytidine (ddC; 1μM) 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 μM) and ddC (1 μM). 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-γ activity; in the case of AZT these observations may provide a mechanism for the observed long-term toxicity with this drug. PMID:17904600

Photocatalysis and the origin of life: synthesis of nucleoside bases from formamide on TiO2(001) single surfaces.

PubMed

Senanayake, S D; Idriss, H

2006-01-31

We report the conversion of a large fraction of formamide (NH(2)CHO) to high-molecular-weight compounds attributed to nucleoside bases on the surface of a TiO(2) (001) single crystal in ultra-high vacuum conditions. If true, we present previously unreported evidence for making biologically relevant molecules from a C1 compound on any single crystal surface in high vacuum and in dry conditions. An UV light of 3.2 eV was necessary to make the reaction. This UV light excites the semiconductor surface but not directly the adsorbed formamide molecules or the reaction products. There thus is no need to use high energy in the form of photons or electrical discharge to make the carbon-carbon and carbon-nitrogen bonds necessary for life. Consequently, the reaction products may accumulate with time and may not be subject to decomposition by the excitation source. The formation of these molecules, by surface reaction of formamide, is proof that some minerals in the form of oxide semiconductors are active materials for making high-molecular-weight organic molecules that may have acted as precursors for biological compounds required for life in the universe.

Nicotinamide riboside, an unusual, non-typical, substrate of purified purine-nucleoside phosphorylases.

PubMed

Wielgus-Kutrowska, B; Kulikowska, E; Wierzchowski, J; Bzowska, A; Shugar, D

1997-01-15

Nicotinamide 1-beta-D-riboside (Nir), the cationic, reducible moiety of the coenzyme NAD+, has been confirmed as an unusual substrate for purified purine-nucleoside phosphorylase (PNP) from a mammalian source (calf spleen). It is also a substrate of the enzyme from Escherichia coli. The Km values at pH 7, 1.48 mM and 0.62 mM, respectively, were 1-2 orders of magnitude higher than for the natural substrate inosine, but the Vmax values were comparable, 96% and 35% that for Ino. The pseudo first-order rate constants, Vmax/Km, were 1.1% and 2.5% for the calf spleen and E. coli enzymes. The aglycon, nicotinamide, was neither a substrate nor an inhibitor of PNP. Nir was a weak inhibitor of inosine phosphorolysis catalyzed by both enzymes, with Ki values close to the Km for its phosphorolysis, consistent with simple competitive inhibition; this was further confirmed by Dixon plots. Phosphorolysis of the fluorescent positively charged substrate 7-methylguanosine was also inhibited in a competitive manner by both Ino and Nir. Phosphorolysis of Nir by both enzymes was inhibited competitively by several specific inhibitors of calf spleen and E. coli PNP, with Ki values similar to those for inhibition of other natural substrates. The pH dependence of the kinetic constants for the phosphorolysis of Nir and of a variety of other substrates, was extensively investigated, particularly in the alkaline pH range, where Nir exhibited abnormally high substrate activity relative to the reduced reaction rates of both enzymes towards other anionic or neutral substrates. The overall results are discussed in relation to present concepts regarding binding and phosphorolysis of substrates by PNP based on crystallographic data of enzyme-inhibitor complexes, and current studies on enzymatic and nonenzymatic mechanisms of the cleavage of the Nir glycosidic bond.

Metabolic Profiles and Free Radical Scavenging Activity of Cordyceps bassiana Fruiting Bodies According to Developmental Stage

PubMed Central

Hyun, Sun-Hee; Lee, Seok-Young; Sung, Gi-Ho; Kim, Seong Hwan; Choi, Hyung-Kyoon

2013-01-01

The metabolic profiles of Cordyceps bassiana according to fruiting body developmental stage were investigated using gas chromatography-mass spectrometry. We were able to detect 62 metabolites, including 48 metabolites from 70% methanol extracts and 14 metabolites from 100% n-hexane extracts. These metabolites were classified as alcohols, amino acids, organic acids, phosphoric acids, purine nucleosides and bases, sugars, saturated fatty acids, unsaturated fatty acids, or fatty amides. Significant changes in metabolite levels were found according to developmental stage. Relative levels of amino acids, purine nucleosides, and sugars were higher in development stage 3 than in the other stages. Among the amino acids, valine, isoleucine, lysine, histidine, glutamine, and aspartic acid, which are associated with ABC transporters and aminoacyl-tRNA biosynthesis, also showed higher levels in stage 3 samples. The free radical scavenging activities, which were significantly higher in stage 3 than in the other stages, showed a positive correlation with purine nucleoside metabolites such as adenosine, guanosine, and inosine. These results not only show metabolic profiles, but also suggest the metabolic pathways associated with fruiting body development stages in cultivated C. bassiana. PMID:24058459

Structural Dynamics Control Allosteric Activation of Cytohesin Family Arf GTPase Exchange Factors

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

Malaby, Andrew W.; Das, Sanchaita; Chakravarthy, Srinivas

Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (GTPase) activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and a membrane-targeting module such as a pleckstrin homology (PH) domain. The catalytic output of cytohesin family Arf GEFs is controlled by autoinhibitory interactions that impede accessibility of the exchange site in the Sec7 domain. These restraints can be relieved through activator Arf-GTP binding to an allosteric site comprising the PH domain and proximal autoinhibitory elements (Sec7-PH linker and C-terminal helix). Small-angle X-ray scattering and negative-stain electron microscopy were used to investigate the structural organization andmore » conformational dynamics of cytohesin-3 (Grp1) in autoinhibited and active states. The results support a model in which hinge dynamics in the autoinhibited state expose the activator site for Arf-GTP binding, while subsequent C-terminal helix unlatching and repositioning unleash conformational entropy in the Sec7-PH linker to drive exposure of the exchange site.« less

Human equilibrative nucleoside transporter 1 (hENT1) levels predict response to gemcitabine in patients with biliary tract cancer (BTC).

PubMed

Santini, Daniele; Schiavon, Gaia; Vincenzi, Bruno; Cass, Carol E; Vasile, Enrico; Manazza, Andrea D; Catalano, Vincenzo; Baldi, Giacomo Giulio; Lai, Raymond; Rizzo, Sergio; Giacobino, Alice; Chiusa, Luigi; Caraglia, Michele; Russo, Antonio; Mackey, John; Falcone, Alfredo; Tonini, Giuseppe

2011-01-01

Translational data suggest that nucleoside transporters, in particular human equilibrative nucleoside transporter 1 (hENT1), play an important role in predicting clinical outcome after gemcitabine chemotherapy for several types of cancer. The aim of this study was to retrospectively determine patients' outcome according to the expression of hENT1 in tumoral cells of patients receiving gemcitabine-based therapy. The immunohistochemistry analysis was performed on samples from thirty-one patients with unresectable biliary tract cancer (BTC) consecutively treated with first line gemcitabine-based regimens. Positive hENT1 staining patients were 21 (67.7%); negative hENT1 staining patients were 10 (32.3%). Statistical analysis revealed no association between baseline characteristics, toxicities and tumor response to gemcitabine and hENT1 levels. In the univariate analysis, HENT1 expression was significantly correlated with time to progression (TTP) (p=0.0394; HR 2.902, 95%CI 1.053-7.996). The median TTP was 6.33 versus 2.83 months, respectively in patients with positive versus negative hENT1 staining. Moreover, patients with positive hENT1 expression showed a longer median overall survival when compared with patients with low hENT1 expression (14 versus 7 months, respectively), but this difference did not reach the statistical significance (p=0.128). Therefore, hENT1 may be a relevant predictive marker of benefit from gemcitabine-based therapies in patients with advanced BTC.

Synthesis and biological properties of 5-(1H-1,2,3-triazol-4-yl)isoxazolidines: a new class of C-nucleosides.

PubMed

Giofrè, Salvatore V; Romeo, Roberto; Carnovale, Caterina; Mancuso, Raffaella; Cirmi, Santa; Navarra, Michele; Garozzo, Adriana; Chiacchio, Maria A

2015-03-24

A novel series of C-nucleosides, featuring the presence of a 1,2,3-triazole ring linked to an isoxazolidine system, has been designed as mimetics of the pyrimidine nucleobases. An antiproliferative effect was observed for compounds 17a and 17b: the growth inhibitory effect reaches the 50% in HepG2 and HT-29 cells and increases up to 56% in the SH-SY5Y cell line after 72 h of incubation at a 100 µM concentration.

8-vinyl-deoxyadenosine, an alternative fluorescent nucleoside analog to 2′-deoxyribosyl-2-aminopurine with improved properties

PubMed Central

Gaied, Nouha Ben; Glasser, Nicole; Ramalanjaona, Nick; Beltz, Hervé; Wolff, Philippe; Marquet, Roland; Burger, Alain; Mély, Yves

2005-01-01

We report here the synthesis and the spectroscopic characterization of 8-vinyl-deoxyadenosine (8vdA), a new fluorescent analog of deoxyadenosine. 8vdA was found to absorb and emit in the same wavelength range as 2′-deoxyribosyl-2-aminopurine (2AP), the most frequently used fluorescent nucleoside analog. Though the quantum yield of 8vdA is similar to that of 2AP, its molar absorption coefficient is about twice, enabling a more sensitive detection. Moreover, the fluorescence of 8vdA was found to be sensitive to temperature and solvent but not to pH (around neutrality) or coupling to phosphate groups. Though 8vdA is base sensitive and susceptible to depurination, the corresponding phosphoramidite was successfully prepared and incorporated in oligonucleotides of the type d(CGT TTT XNX TTT TGC) where N = 8vdA and X = A, T or C. The 8vdA-labeled oligonucleotides gave more stable duplexes than the corresponding 2AP-labeled sequences when X = A or T, indicating that 8vdA is less perturbing than 2AP and probably adopts an anti conformation to preserve the Watson–Crick H-bonding. In addition, the quantum yield of 8vdA is significantly higher than 2AP in all tested oligonucleotides in both their single strand and duplex states. The steady-state and time-resolved fluorescence parameters of 8vdA and 2AP were found to depend similarly on the nature of their flanking residues and on base pairing, suggesting that their photophysics are governed by similar mechanisms. Taken together, our data suggest that 8vdA is a non perturbing nucleoside analog that may be used with improved sensitivity for the same applications as 2AP. PMID:15718302

Quantification of the 2-deoxyribonolactone and nucleoside 5'-aldehyde products of 2-deoxyribose oxidation in DNA and cells by isotope-dilution gas chromatography mass spectrometry: differential effects of gamma-radiation and Fe2+-EDTA.

PubMed

Chan, Wan; Chen, Bingzi; Wang, Lianrong; Taghizadeh, Koli; Demott, Michael S; Dedon, Peter C

2010-05-05

The oxidation of 2-deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC-MS) method for the rigorous quantification of two major 2-deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1'-oxidation and the nucleoside 5'-aldehyde of 5'-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC-MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5'-aldehyde lesions. Further, the well-defined 2-deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin gamma(1)(I), was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5'-aldehyde per 10(6) nt per microM in accord with its established minor 1'- and major 5'-oxidation chemistry. Calicheamicin unexpectedly caused 1'-oxidation at a low level of 10 2-deoxyribonolactone per 10(6) nt per microM in addition to the expected predominance of 5'-oxidation at 560 nucleoside 5'-aldehyde per 10(6) nt per microM. The two hydroxyl radical-mediated DNA oxidants, gamma-radiation and Fe(2+)-EDTA, produced nucleoside 5'-aldehyde at a frequency of 57 per 10(6) nt per Gy (G-value 74 nmol/J) and 3.5 per 10(6) nt per microM, respectively, which amounted to 40% and 35%, respectively, of total 2-deoxyribose oxidation as measured by a plasmid nicking assay. However, gamma-radiation and Fe(2+)-EDTA produced different proportions of 2

A new and efficient synthetic method for 15N3-labeled cytosine nucleosides: Dimroth rearrangement of cytidine N3-oxides.

PubMed

Sako, Magoichi; Kawada, Hiroyoshi

2004-11-12

The treatment of (15)N(4)-labeled cytidine N(3)-oxide and (15)N(4)-labeled 2'-deoxycytidine N(3)-oxide, prepared from the appropriate unprotected uridines in three reaction steps, with benzyl bromide in the presence of excess lithium methoxide allowed the smooth occurrence of their Dimroth rearrangement even under mild conditions leading to the corresponding (15)N(3)-labeled uridine 4-O-benzyloximes which can easily undergo the reductive N-O bond cleavage to give the desirable (15)N(3)-labeled cytosine nucleosides in high total yields.

Preformulation studies of EFdA, a novel nucleoside reverse transcriptase inhibitor for HIV prevention.

PubMed

Zhang, Wei; Parniak, Michael A; Mitsuya, Hiroaki; Sarafianos, Stefan G; Graebing, Phillip W; Rohan, Lisa C

2014-08-01

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a novel nucleoside analog of great interest because of its superior activity against wild-type and multidrug-resistant HIV-1 strains, and favorable safety profiles in vitro and in vivo. The aim of this work was to provide preformulation information of EFdA important for delivery system development. A simple, accurate and specific reverse-phase high performance liquid chromatographic (RP-HPLC) method with UV detection was developed for quantification of EFdA. In addition, physicochemical characterizations including pH solubility profile, octanol/water partition coefficient (Log Po/w), DSC analysis, field emission scanning electron microscopy, and stability studies under various conditions were conducted. EFdA existed in planar or flake shape, with a melting point of ∼130 °C, and had a pH dependent solubility. The log Po/w value of EFdA was -1.19. The compound was stable upon exposure to pH levels from 3 to 9 and showed good stability at elevated temperature (65 °C). In vitro cytotoxicity assessments were performed in two different epithelial cell lines. In cell-based studies, the EFdA selectivity index (50% cytotoxic concentration [CC50] values/50% effective concentration [EC50]) was found to be greater than 1 × 10(3). Permeability studies using cell- and tissue-based models showed that EFdA had an apparent permeability coefficient (Papp) <1 × 10(-6)cm/s and that the paracelluar pathway was the dominant transport route for EFdA. Overall, EFdA possesses favorable characteristics for further formulation development.

Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL

PubMed Central

Tzoneva, Gannie; Garcia, Arianne Perez; Carpenter, Zachary; Khiabanian, Hossein; Tosello, Valeria; Allegretta, Maddalena; Paietta, Elisabeth; Racevskis, Janis; Rowe, Jacob M.; Tallman, Martin S.; Paganin, Maddalena; Basso, Giuseppe; Hof, Jana; Kirschner-Schwabe, Renate; Palomero, Teresa; Rabadan, Raul; Ferrando, Adolfo

2013-01-01

Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric and over 50% of adult ALL patients fail to achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most significant challenge in the treatment of this disease1,2. Using whole exome sequencing, here we identify mutations in the cytosolic 5'-nucleotidase II gene (NT5C2), which encodes a 5'-nucleotidase enzyme responsible for inactivation of nucleoside analog chemotherapy drugs, in 20/103 (19%) relapse T-ALLs and in 1/35 (3%) relapse B-precursor ALLs analyzed. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside analog metabolism in disease progression and chemotherapy resistance in ALL. PMID:23377281

Non-nucleoside building blocks for copper-assisted and copper-free click chemistry for the efficient synthesis of RNA conjugates.

PubMed

Jayaprakash, K N; Peng, Chang Geng; Butler, David; Varghese, Jos P; Maier, Martin A; Rajeev, Kallanthottathil G; Manoharan, Muthiah

2010-12-03

Novel non-nucleoside alkyne monomers compatible with oligonucleotide synthesis were designed, synthesized, and efficiently incorporated into RNA and RNA analogues during solid-phase synthesis. These modifications allowed site-specific conjugation of ligands to the RNA oligonucleotides through copper-assisted (CuAAC) and copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reactions. The SPAAC click reactions of cyclooctyne-oligonucleotides with various classes of azido-functionalized ligands in solution phase and on solid phase were efficient and quantitative and occurred under mild reaction conditions. The SPAAC reaction provides a method for the synthesis of oligonucleotide-ligand conjugates uncontaminated with copper ions.

LTP-triggered cholesterol redistribution activates Cdc42 and drives AMPA receptor synaptic delivery

PubMed Central

Brachet, Anna; Norwood, Stephanie; Brouwers, Jos F.; Palomer, Ernest; Helms, J. Bernd

2015-01-01

Neurotransmitter receptor trafficking during synaptic plasticity requires the concerted action of multiple signaling pathways and the protein transport machinery. However, little is known about the contribution of lipid metabolism during these processes. In this paper, we addressed the question of the role of cholesterol in synaptic changes during long-term potentiation (LTP). We found that N-methyl-d-aspartate–type glutamate receptor (NMDAR) activation during LTP induction leads to a rapid and sustained loss or redistribution of intracellular cholesterol in the neuron. A reduction in cholesterol, in turn, leads to the activation of Cdc42 and the mobilization of GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid–type glutamate receptors (AMPARs) from Rab11-recycling endosomes into the synaptic membrane, leading to synaptic potentiation. This process is accompanied by an increase of NMDAR function and an enhancement of LTP. These results imply that cholesterol acts as a sensor of NMDAR activation and as a trigger of downstream signaling to engage small GTPase (guanosine triphosphatase) activation and AMPAR synaptic delivery during LTP. PMID:25753037

Multiple enzyme activities of flavivirus proteins.

PubMed

Padmanabhan, R; Mueller, N; Reichert, E; Yon, C; Teramoto, T; Kono, Y; Takhampunya, R; Ubol, S; Pattabiraman, N; Falgout, B; Ganesh, V K; Murthy, K

2006-01-01

Dengue viruses (DENV) have 5'-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing. Using a high-throughput protease assay, we screened a small molecule chemical library and identified -200 compounds having > or = 50% inhibition. Moreover, NS3 exhibits RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase activities. The NTPase and the 5'-RTPase activities of NS3 are stimulated by interaction with NS5. Moreover, the conserved, positively charged motif in DENV-2 NS3, 184RKRK, is required for RNA binding and modulates the RNA-dependent enzyme activities of NS3. To study viral replication, a variety of methods are used such as the in vitro RNA-dependent RNA polymerase assays that utilize lysates from DENV-2-infected mosquito or mammalian cells or the purified NS5 along with exogenous short subgenomic viral RNAs or the replicative intracellular membrane-bound viral RNAs as templates. In addition, a cell-based DENV-2 replicon RNA encoding a luciferase reporter is also used to examine the role of cis-acting elements within the 3' UTR and the RKRK motif in viral replication.

Early nucleoside reverse transcriptase inhibitors for the treatment of HIV: a brief history of stavudine (D4T) and its comparison with other dideoxynucleosides.

PubMed

Martin, John C; Hitchcock, Michael J M; De Clercq, Erik; Prusoff, William H

2010-01-01

The occasion of this 25th anniversary issue encouraged us to reminisce about the important history of the discovery of the dideoxynucleoside analogues for the treatment of HIV/AIDS and to chronicle our thoughts about a particular exciting and rewarding period of our scientific careers. Following the identification of the anti-HIV activity of zidovudine (AZT), we participated in the urgent quest to discover optimal treatments of HIV infection and AIDS. A number of previously synthesized nucleoside analogues were comparatively evaluated, and stavudine (D4T) emerged as a promising candidate for development. Following clinical evaluation, D4T became a mainstay of the initial antiretroviral combination therapy, prolonging and saving numerous lives. It has only recently been supplanted by better-tolerated treatments. This article forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, vol. 85, issue 1, 2010. Copyright 2009 Elsevier B.V. All rights reserved.

Cytotoxicity and Antineoplastic Activities of Alkylamines and Their Borane Derivatives

PubMed Central

Tse, Elaine Y.; Muhammad, Rosallah A.

1996-01-01

The alkylamines and their related boron derivatives demonstrated potent cytotoxicity against the growth of murine and human tissue cultured cells. These agents did not necessarily require the boron atom to possess potent cytotoxic action in certain tumor lines. Their ability to suppress tumor cell growth was based on their inhibition of DNA and protein syntheses. DNA synthesis was reduced because purine synthesis was blocked at the enzyme site of IMP dehydrogenase by the agents. In addition ribonucleotide reductase and nucleoside kinase activities were reduced by the agents which would account for the reduced d[NTP] pools. The DNA template or molecule may be a target of the drugs with regard to binding of the drug to nucleoside bases or intercalaction of the drug between DNA base pairs. Only some Of the agents caused DNA fragmentation with reduced DNA viscosity. These effects would contribute to overall cell death afforded by the agents. PMID:18472803

Chemomimesis and Molecular Darwinism in Action: From Abiotic Generation of Nucleobases to Nucleosides and RNA.

PubMed

Saladino, Raffaele; Šponer, Judit E; Šponer, Jiří; Costanzo, Giovanna; Pino, Samanta; Di Mauro, Ernesto

2018-06-20

Molecular Darwinian evolution is an intrinsic property of reacting pools of molecules resulting in the adaptation of the system to changing conditions. It has no a priori aim. From the point of view of the origin of life, Darwinian selection behavior, when spontaneously emerging in the ensembles of molecules composing prebiotic pools, initiates subsequent evolution of increasingly complex and innovative chemical information. On the conservation side, it is a posteriori observed that numerous biological processes are based on prebiotically promptly made compounds, as proposed by the concept of Chemomimesis. Molecular Darwinian evolution and Chemomimesis are principles acting in balanced cooperation in the frame of Systems Chemistry. The one-pot synthesis of nucleosides in radical chemistry conditions is possibly a telling example of the operation of these principles. Other indications of similar cases of molecular evolution can be found among biogenic processes.

A fluorescence study of the molecular interactions of harmane with the nucleobases, their nucleosides and mononucleotides.

PubMed

Balón, M; Muñoz, M A; Carmona, C; Guardado, P; Galán, M

1999-07-19

Fluorescence binding studies of harmane to the elemental components of the nucleic acids were undertaken to investigate the origin of the interaction between the drug and DNA. Most of the tested substrates have been found to induce hypochromism in the absorption spectrum of harmane and to quench its fluorescence. The quenching process induced by the nucleobases and their nucleosides is mainly due to the formation of ground state 1:1 complexes. However, in the case of the mononucleotides a dynamic quenching component is also observed. This quenching component is likely due to the excited state interaction of harmane with the phosphate group of the nucleotides. UV-vis spectral changes and quenching measurements have been used to quantify the ground state association constants of the complexes and the quenching rate constants.

Nucleoside conformation is determined by the electronegativity of the sugar substituent.

PubMed Central

Guschlbauer, W; Jankowski, K

1980-01-01

The proton and 13C NMR spectra of uridine, deoxyuridine and four 2' substituted uridines (dUn, dUz, dUcl and dUfl) are reported. A linear relationship between the electronegativity of the 2'-substituent and the carbon-13 chemical shift of C2' is observed. Taking into account the effect of electronegativity by using the correction proposed by Karplus or by Jankowski, the proton-proton coupling constants have been used to compute the conformational equilibria of the six uridines. It is shown that the contribution of the N form (3'-endo -2'-exo) increases with the electronegativity of the 2' substituent. Thus dUfl contains some 85% N form in solution. - Applying similar corrections to published data in the adenosine series, a similar correlation is observed. This observation, that the most polar substituent pulls the pucker to its side, holds also for 3'-substituted compounds, like cordycepin (3'dAdo) and 3'-deoxy-3'-amino-adenosine. It is suggested that the influence of the electronegativity could be the dominating effect of nucleoside conformations and would also hold for arabinosides and xylosides. This effect should therefore also be the principal force which determines the differences between DNA and RNA. PMID:7433125

A simple and rapid method for the reversible removal of lipids from a membrane-bound enzyme.

PubMed Central

Goodman, S L; Isern de Caldentey, M; Wheeler, K P

1978-01-01

A simple, rapid and reproducible method for the reversible removal of lipids from a membrane-bound enzyme is described. Essentially, a membrane preparation containing (Na+ + K+)-dependent adenosine triphosphatase was extracted with the non-ionic detergent Lubrol WX in the presence of glycerol, and partial separation of protein from lipid was achieved with the use of only two centrifugations. About 74% of the endogenous phospholipid and 79% of the cholesterol were removed, concomitant with a virtually complete loss of ouabain-sensitive adenosine triphosphatase activity, but with retention of 60-100% of the K+-dependent phosphatase activity. The addition of pure phosphatidylserine re-activated the enzyme to more than 80% of the initial activity, and up to 30% of the protein was recovered. Excess of phosphatidylserine could be washed off the enzyme to give a stable 'reconstituted' preparation. The effects of variation in the experimental conditions were examined, and the results are discussed with respect to the possibility of adapting the method to the study of other lipid-dependent enzymes bound to membranes. PMID:147078

The non-nucleoside antiviral, BAY 38-4766, protects against cytomegalovirus (CMV) disease and mortality in immunocompromised guinea pigs

PubMed Central

Schleiss, Mark R.; Bernstein, David I.; McVoy, Michael A.; Stroup, Greg; Bravo, Fernando; Creasy, Blaine; McGregor, Alistair; Henninger, Kristin; Hallenberger, Sabine

2008-01-01

New antiviral drugs are needed for the treatment of cytomegalovirus (CMV) infections, particularly in immunocompromised patients. These studies evaluated the in vitro and in vivo activity of the non-nucleosidic CMV inhibitor, BAY 38-4766, against guinea pig cytomegalovirus (GPCMV). Plaque reduction assays indicated that BAY 38-4766 was active against GPCMV, with an IC50 of 0.5 μM. Yield reduction assays demonstrated an ED90 and ED99 of 0.4 and 0.6 μM, respectively, of BAY 38-4766 against GPCMV. Guinea pigs tolerated oral administration of 50 mg/kg/day of BAY 38-4766 without evidence of biochemical or hematologic toxicity. Plasma concentrations of BAY 38-4766 were high following oral dosing, with a mean peak level at 1-h post-dose of 26.7 mg/ml (n = 6; range, 17.8-35.4). Treatment with BAY 38-4766 reduced both viremia and DNAemia, as determined by a real-time PCR assay, following GPCMV infection of cyclophosphamide-immunosuppressed strain 2 guinea pigs (p < 0.05, Mann-Whitney test). BAY 38-4766 also reduced mortality following lethal GPCMV challenge in immunosuppressed Hartley guinea pigs, from 83% (20/24) in placebo-treated guinea pigs, to 17% (4/24) in BAY 38-4766-treated animals (p < 0.0001, Fisher’s exact test). Mortality differences were accompanied by reduction in DNAemia in Hartley guinea pigs. Based upon its favorable safety, pharmacokinetic, and therapeutic profiles, BAY 38-4766 warrants further investigation in the GPCMV model. PMID:15652969

Molecular adaptability of nucleoside diphosphate kinase b from trypanosomatid parasites: stability, oligomerization and structural determinants of nucleotide binding.

PubMed

Souza, Tatiana A C B; Trindade, Daniel M; Tonoli, Celisa C C; Santos, Camila R; Ward, Richard J; Arni, Raghuvir K; Oliveira, Arthur H C; Murakami, Mário T

2011-07-01

Nucleoside diphosphate kinases play a crucial role in the purine-salvage pathway of trypanosomatid protozoa and have been found in the secretome of Leishmania sp., suggesting a function related to host-cell integrity for the benefit of the parasite. Due to their importance for housekeeping functions in the parasite and by prolonging the life of host cells in infection, they become an attractive target for drug discovery and design. In this work, we describe the first structural characterization of nucleoside diphosphate kinases b from trypanosomatid parasites (tNDKbs) providing insights into their oligomerization, stability and structural determinants for nucleotide binding. Crystallographic studies of LmNDKb when complexed with phosphate, AMP and ADP showed that the crucial hydrogen-bonding residues involved in the nucleotide interaction are fully conserved in tNDKbs. Depending on the nature of the ligand, the nucleotide-binding pocket undergoes conformational changes, which leads to different cavity volumes. SAXS experiments showed that tNDKbs, like other eukaryotic NDKs, form a hexamer in solution and their oligomeric state does not rely on the presence of nucleotides or mimetics. Fluorescence-based thermal-shift assays demonstrated slightly higher stability of tNDKbs compared to human NDKb (HsNDKb), which is in agreement with the fact that tNDKbs are secreted and subjected to variations of temperature in the host cells during infection and disease development. Moreover, tNDKbs were stabilized upon nucleotide binding, whereas HsNDKb was not influenced. Contrasts on the surface electrostatic potential around the nucleotide-binding pocket might be a determinant for nucleotide affinity and protein stability differentiation. All these together demonstrated the molecular adaptation of parasite NDKbs in order to exert their biological functions intra-parasite and when secreted by regulating ATP levels of host cells.

Nucleotides as nucleophiles: Reactions of nucleotides with phosphoimidazolide activated guanosine

NASA Astrophysics Data System (ADS)

Kanavarioti, Anastassia; Rosenbach, Morgan T.; Brian Hurley, T.

1992-07-01

An earlier study of the reaction of phosphoimidazolide activated nucleosides (ImpN) in aqueous phosphate buffers indicated two modes of reaction of the phosphate monoanion and dianion. The first mode is catalysis of the hydrolysis of the P-N bond in ImpN's which leads to imidazole and nucleoside 5'-monophosphate. The second represents a nucleophilic substitution of the imidazole to yield the nucleoside 5'-diphosphate. This earlier study thus served as a model for the reaction of ImpN with nucleoside monophosphates (pN) because the latter can be regarded as phosphate derivatives. In the present study we investigated the reaction of guanosine 5'-phosphate-2-methylimidazolide, 2-MeImpG, in the presence of pN (N=guanosine, adenosine and uridine) in the range 6.9 ≤ pH ≤ 7.7. We observed that pN's do act as nucleophiles to form NppG, and as general base to enhance the hydrolysis of the P-N bond in 2-MeImpG, i.e. pN show the same behavior as inorganic phosphate. The kinetic analysis yields the following rate constants for the dianion pN2-:k {/n pN}=0.17±0.02 M-1 h-1 for nucleophilic attack andk {/h pN}=0.11±0.07 M-1 h-1 for general base catalysis of the hydrolysis. These rate constants which are independent of the nucleobase compare withk p 2=0.415 M-1 h-1 andk_h^{p^2 } =0.217 M-1 h-1 for the reactions of HPO{4/2-}. In addition, this study shows that under conditions where pN presumably form stacks, the reaction mechanism remains unchanged although in quantitative terms stacked pN are somewhat less reactive. Attack by the 2'-OH and 3'-OH groups of the ribose moiety in amounts ≥1% is not observed; this is attributed to the large difference in nucleophilicity in the neutral pH range between the phosphate group and the ribose hydroxyls. This nucleophilicity rank is not altered by stacking.

Surface acidic amino acid of Pseudomonas/Halomonas chimeric nucleoside diphosphate kinase leads effective recovery from heat-denaturation.

PubMed

Tokunaga, Hiroko; Arakawa, Tsutomu; Tokunaga, Masao

2013-07-01

One of the hallmarks of halophilic properties is reversibility of thermal unfolding. A nucleoside diphosphate kinase (NDK) from a moderate halophile Halomonas sp. 593 (HaNDK) follows this behavior. His-tagged chimeric NDK (HisPaHaNDK) consisting of an N-terminal half of a non-halophilic Pseuodomonas aeruginosa NDK (PaNDK) and a Cterminal half of HaNDK loses this reversible property, indicating a critical role of the N-terminal portion of PaNDK in determining the reversibility of the chimeric protein. Various mutations were introduced at Arg45 and Lys61, based on the model NDK structure. It appears that having Glu at position 45 is critical in conferring the thermal reversibility to HisPa- HaNDK chimeric protein.

Metabolic Recruitment and Directed Evolution of Nucleoside Triphosphate Uptake in Escherichia coli.

PubMed

Pezo, Valérie; Hassan, Camille; Louis, Dominique; Sargueil, Bruno; Herdewijn, Piet; Marlière, Philippe

2018-05-18

We report the design and elaboration of a selection protocol for importing a canonical substrate of DNA polymerase, thymidine triphosphate (dTTP) in Escherichia coli. Bacterial strains whose growth depend on dTTP uptake, through the action of an algal plastid transporter expressed from a synthetic gene inserted in the chromosome, were constructed and shown to withstand the simultaneous loss of thymidylate synthase and thymidine kinase. Such thyA tdk dual deletant strains provide an experimental model of tight nutritional containment for preventing dissemination of microbial GMOs. Our strains transported the four canonical dNTPs, in the following order of preference: dCTP > dATP ≥ dGTP > dTTP. Prolonged cultivation under limitation of exogenous dTTP led to the enhancement of dNTP transport by adaptive evolution. We investigated the uptake of dCTP analogues with altered sugar or nucleobase moieties, which were found to cause a loss of cell viability and an increase of mutant frequency, respectively. E. coli strains equipped with nucleoside triphosphate transporters should be instrumental for evolving organisms whose DNA genome is morphed chemically by fully substituting its canonical nucleotide components.

In search of a treatment for HIV--current therapies and the role of non-nucleoside reverse transcriptase inhibitors (NNRTIs).

PubMed

Reynolds, Chevonne; de Koning, Charles B; Pelly, Stephen C; van Otterlo, Willem A L; Bode, Moira L

2012-07-07

The human immunodeficiency virus (HIV) causes AIDS (acquired immune deficiency syndrome), a disease in which the immune system progressively deteriorates, making sufferers vulnerable to all manner of opportunistic infections. Currently, world-wide there are estimated to be 34 million people living with HIV, with the vast majority of these living in sub-Saharan Africa. Therefore, an important research focus is development of new drugs that can be used in the treatment of HIV/AIDS. This review gives an overview of the disease and addresses the drugs currently used for treatment, with specific emphasis on new developments within the class of allosteric non-nucleoside reverse transcriptase inhibitors (NNRTIs).

Structural and Preclinical Studies of Computationally Designed Non-Nucleoside Reverse Transcriptase Inhibitors for Treating HIV infection

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

Kudalkar, Shalley N.; Beloor, Jagadish; Chan, Albert H.

The clinical benefits of HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are hindered by their unsatisfactory pharmacokinetic (PK) properties along with the rapid development of drug-resistant variants. However, the clinical efficacy of these inhibitors can be improved by developing compounds with enhanced pharmacological profiles and heightened antiviral activity. We used computational and structure-guided design to develop two next-generation NNRTI drug candidates, compounds I and II, which are members of a class of catechol diethers. We evaluated the preclinical potential of these compounds in BALB/c mice because of their high solubility (510 µg/ml for compound I and 82.9 µg/ml for compoundmore » II), low cytotoxicity, and enhanced antiviral activity against wild-type (WT) HIV-1 RT and resistant variants. Additionally, crystal structures of compounds I and II with WT RT suggested an optimal binding to the NNRTI binding pocket favoring the high anti-viral potency. A single intraperitoneal dose of compounds I and II exhibited a prolonged serum residence time of 48 hours and concentration maximum (Cmax) of 4000- to 15,000-fold higher than their therapeutic/effective concentrations. These Cmax values were 4- to 15-fold lower than their cytotoxic concentrations observed in MT-2 cells. Compound II showed an enhanced area under the curve (0–last) and decreased plasma clearance over compound I and efavirenz, the standard of care NNRTI. Hence, the overall (PK) profile of compound II was excellent compared with that of compound I and efavirenz. Furthermore, both compounds were very well tolerated in BALB/c mice without any detectable acute toxicity. Taken together, these data suggest that compounds I and II possess improved anti-HIV-1 potency, remarkable in vivo safety, and prolonged in vivo circulation time, suggesting strong potential for further development as new NNRTIs for the potential treatment of HIV infection.« less

Preformulation studies of EFdA, a novel nucleoside reverse transcriptase inhibitor for HIV prevention

PubMed Central

Zhang, Wei; Parniak, Michael A.; Mitsuya, Hiroaki; Sarafianos, Stefan G.; Graebing, Phillip W.; Rohan, Lisa C.

2014-01-01

4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a novel nucleoside analog of great interest because of its superior activity against wild-type and multidrug-resistant HIV-1 strains, and favorable safety profiles in vitro and in vivo. The aim of this work was to provide preformulation information of EFdA important for delivery system development. A simple, accurate and specific reverse-phase high performance liquid chromatographic (RP-HPLC) method with UV detection was developed for quantification of EFdA. In addition, physicochemical characterizations including pH solubility profile, octanol/water partition coefficient (Log Po/w), DSC analysis, field emission scanning electron microscopy, and stability studies under various conditions were conducted. EFdA existed in planar or flake shape, with a melting point of ~130 °C, and had a pH dependent solubility. The log Po/w value of EFdA was −1.19. The compound was stable upon exposure to pH levels from 3 to 9 and showed good stability at elevated temperature (65 °C). In vitro cytotoxicity assessments were performed in two different epithelial cell lines. In cell-based studies, the EFdA selectivity index (50% cytotoxic concentration [CC50] values/50% effective concentration [EC50]) was found to be greater than 1 × 103. Permeability studies using cell- and tissue-based models showed that EFdA had an apparent permeability coefficient (Papp) <1 × 10−6cm/s and that the paracelluar pathway was the dominant transport route for EFdA. Overall, EFdA possesses favorable characteristics for further formulation development. PMID:23841536

The ligand binding mechanism to purine nucleoside phosphorylase elucidated via molecular dynamics and machine learning.

PubMed

Decherchi, Sergio; Berteotti, Anna; Bottegoni, Giovanni; Rocchia, Walter; Cavalli, Andrea

2015-01-27

The study of biomolecular interactions between a drug and its biological target is of paramount importance for the design of novel bioactive compounds. In this paper, we report on the use of molecular dynamics (MD) simulations and machine learning to study the binding mechanism of a transition state analogue (DADMe-immucillin-H) to the purine nucleoside phosphorylase (PNP) enzyme. Microsecond-long MD simulations allow us to observe several binding events, following different dynamical routes and reaching diverse binding configurations. These simulations are used to estimate kinetic and thermodynamic quantities, such as kon and binding free energy, obtaining a good agreement with available experimental data. In addition, we advance a hypothesis for the slow-onset inhibition mechanism of DADMe-immucillin-H against PNP. Combining extensive MD simulations with machine learning algorithms could therefore be a fruitful approach for capturing key aspects of drug-target recognition and binding.

Synthesis of Chromone, Quinolone, and Benzoxazinone Sulfonamide Nucleosides as Conformationally Constrained Inhibitors of Adenylating Enzymes Required for Siderophore Biosynthesis

PubMed Central

Engelhart, Curtis A.; Aldrich, Courtney C.

2013-01-01

MbtA catalyzes the first committed step of mycobactin biosynthesis in Mycobacterium tuberculosis (Mtb) and is responsible for the incorporation of salicylic acid into the mycobactin siderophores. 5′-O-[N-(Salicyl)sulfamoyl]adenosine (Sal-AMS) is an extremely potent nucleoside inhibitor of MbtA that possesses excellent activity against whole-cell Mtb, but suffers from poor bioavailability. In an effort to improve the bioavailability, we have designed four conformationally constrained analogues of Sal-AMS that remove two rotatable bonds and the ionized sulfamate group based on computational and structural studies. Herein we describe the synthesis, biochemical, and microbiological evaluation of chromone-, quinolone-, and benzoxazinone-3-sulfonamide derivatives of Sal-AMS. We developed new chemistry to assemble these three heterocycles from common β-ketosulfonamide intermediates. The synthesis of the chromone- and quinolone-3-sulfonamide intermediates features formylation of a β-ketosulfonamide employing dimethylformamide dimethyl acetal to afford an enaminone that can react intramolecularly with a phenol or intermolecularly with a primary amine via addition-elimination reaction(s). The benzoxazinone-3-sulfonamide was prepared by nitrosation of a β-ketosulfonamide followed by intramolecular nucleophilic aromatic substitution. Mitsunobu coupling of these bicyclic sulfonamides with a protected adenosine derivative followed by global deprotection provides a concise synthesis of the respective inhibitors. PMID:23805993

Simultaneous determination of polysaccharides and 21 nucleosides and amino acids in different tissues of Salvia miltiorrhiza from different areas by UV-visible spectrophotometry and UHPLC with triple quadrupole MS/MS.

PubMed

Xiang, Xiang; Sha, Xiuxiu; Su, Shulan; Zhu, Zhenhua; Guo, Sheng; Yan, Hui; Qian, Dawei; Duan, Jin-Ao

2018-03-01

Salvia miltiorrhiza, a traditional Chinese medicine, is a widely used herbal medicine to treat cardiovascular and cerebrovascular diseases. In this study, ultraviolet (UV)-visible spectrophotometry and ultra-high performance liquid chromatography with triple quadrupole tandem mass spectrometry analytical methods were used for rapid quantification of polysaccharides and 21 nucleosides and amino acids in S. miltiorrhiza to determine 17 samples of different tissues from different areas. Based on the total contents, hierarchical clustering analysis and principal components analysis were performed to classify these samples. The established methods were validated with good linearity, precision, repeatability, stability, and recovery. Chemical analysis revealed a higher content of total analytes in the sample of inflorescence from Nanjing (34.17 mg/g), sample of root and rhizome from Shaanxi (34.13 mg/g) and sample of stem and leaf from Nanjing (31.14 mg/g), respectively, indicating that root and rhizome from Shaanxi and the aerial parts from Nanjing exhibited the highest quality due to their highest content. In addition, contents of nucleosides and amino acids in the aerial parts (14.67 mg/g) were much higher than that in roots and rhizomes (9.17 mg/g). This study suggested that UV-visible spectrophotometry and ultra-high performance liquid chromatography with triple quadrupole tandem mass spectrometry are effective techniques to analyze polysaccharides, nucleosides, and amino acids in plants, and they provided valuable information for the development and utilization value of the aerial parts of S. miltiorrhiza. This analysis would also provide useful information for the quality control of S. miltiorrhiza. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

96 week results from the MONET trial: a randomized comparison of darunavir/ritonavir with versus without nucleoside analogues, for patients with HIV RNA <50 copies/mL at baseline.

PubMed

Clumeck, Nathan; Rieger, Armin; Banhegyi, Denes; Schmidt, Wolfgang; Hill, Andrew; Van Delft, Yvonne; Moecklinghoff, Christiane; Arribas, Jose

2011-08-01

In virologically suppressed patients, switching to darunavir/ritonavir monotherapy could avoid resistance and adverse events from continuing nucleoside analogues. Two hundred and fifty-six patients with HIV RNA <50 copies/mL on current antiretrovirals were switched to darunavir/ritonavir 800/100 mg once daily, either as monotherapy (n = 127) or with two nucleoside analogues (n = 129). Treatment failure was defined as two consecutive HIV RNA levels at least 50 copies/mL by week 96, or discontinuation of study drugs. The trial had 80% power to show non-inferiority (δ = -12%) at week 48. Results Patients were 81% male, 91% Caucasian, and had a median baseline CD4 count of 575 cells/mm(3). There were more patients with hepatitis C co-infection at baseline in the monotherapy arm (18%) compared with the triple therapy arm (12%). In the efficacy analysis, HIV RNA <50 copies/mL by week 96 (per protocol, time to loss of virological response, switch equals failure) was 78% versus 82% in the monotherapy and triple therapy arms [difference -4.2%, 95% confidence interval (CI) -14.3% to +5.8%]; in a switch included analysis, HIV RNA <50 copies/mL was 93% versus 92% (difference +1.6%, 95% CI -5.0% to +8.1%). The percentage of patients with HIV RNA <5 copies/mL (optical density from the sample equal to the negative control) remained constant over time in both treatment arms. Conclusions In the week 96 analysis of the MONotherapy in Europe with TMC114 (MONET) trial, switching to darunavir/ritonavir monotherapy showed non-inferior efficacy to darunavir/ritonavir plus two nucleoside analogues in the switch included and observed failure analyses, but not in the main switch equals failure analysis.

Biological activity of N(4)-boronated derivatives of 2'-deoxycytidine, potential agents for boron-neutron capture therapy.

PubMed

Nizioł, Joanna; Uram, Łukasz; Szuster, Magdalena; Sekuła, Justyna; Ruman, Tomasz

2015-10-01

Boron-neutron capture therapy (BNCT) is a binary anticancer therapy that requires boron compound for nuclear reaction during which high energy alpha particles and lithium nuclei are formed. Unnatural, boron-containing nucleoside with hydrophobic pinacol moiety was investigated as a potential BNCT boron delivery agent. Biological properties of this compound are presented for the first time and prove that boron nucleoside has low cytotoxicity and that observed apoptotic effects suggest alteration of important functions of cancer cells. Mass spectrometry analysis of DNA from cancer cells proved that boron nucleoside is inserted into nucleic acids as a functional nucleotide derivative. NMR studies present very high degree of similarity of natural dG-dC base pair with dG-boron nucleoside system. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effect of nucleoside analogue antimetabolites on the structure of PEO–PPO–PEO micelles investigated by SANS

DOE PAGES

Han, Youngkyu; Zhang, Zhe; Smith, Gregory S.; ...

2017-04-19

In this work, the effect of three nucleoside analogue antimetabolites (5-fluorouracil, floxuridine, and gemcitabine) on the structure of Pluronic L62 copolymer micelles was investigated using small-angle neutron scattering. These antimetabolites used for cancer chemotherapy have analogous molecular structures but different molecular sizes and aqueous solubilities. It was found that the addition of the three antimetabolites slightly reduced the micellar size and aggregation number, and the micellar anisotropy. The added antimetabolites also changed the internal molecular distribution of the micelles as measured by the scattering length densities, resulting in enhanced hydration of the hydrophobic core region of the micelle. The strengthmore » of the effect was found to correlate with the molecular properties of the model drugs, i.e. a larger molecular size and a higher aqueous solubility lead to enhanced hydration of the micellar core.« less

Effect of nucleoside analogue antimetabolites on the structure of PEO–PPO–PEO micelles investigated by SANS

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

Han, Youngkyu; Zhang, Zhe; Smith, Gregory S.

2017-01-01

The effect of three nucleoside analogue antimetabolites (5-fluorouracil, floxuridine, and gemcitabine) on the structure of Pluronic L62 copolymer micelles was investigated using small-angle neutron scattering. These antimetabolites used for cancer chemotherapy have analogous molecular structures but different molecular sizes and aqueous solubilities. It was found that the addition of the three antimetabolites slightly reduced the micellar size and aggregation number, and the micellar anisotropy. The added antimetabolites also changed the internal molecular distribution of the micelles as measured by the scattering length densities, resulting in enhanced hydration of the hydrophobic core region of the micelle. The strength of the effectmore » was found to correlate with the molecular properties of the model drugs, i.e. a larger molecular size and a higher aqueous solubility lead to enhanced hydration of the micellar core.« less

The universally conserved GTPase HflX is an RNA helicase that restores heat-damaged Escherichia coli ribosomes.

PubMed

Dey, Sandip; Biswas, Chiranjit; Sengupta, Jayati

2018-06-21

The ribosome-associated GTPase HflX acts as an antiassociation factor upon binding to the 50S ribosomal subunit during heat stress in Escherichia coli Although HflX is recognized as a guanosine triphosphatase, several studies have shown that the N-terminal domain 1 of HflX is capable of hydrolyzing adenosine triphosphate (ATP), but the functional role of its adenosine triphosphatase (ATPase) activity remains unknown. We demonstrate that E. coli HflX possesses ATP-dependent RNA helicase activity and is capable of unwinding large subunit ribosomal RNA. A cryo-electron microscopy structure of the 50S-HflX complex in the presence of nonhydrolyzable analogues of ATP and guanosine triphosphate hints at a mode of action for the RNA helicase and suggests the linker helical domain may have a determinant role in RNA unwinding. Heat stress results in inactivation of the ribosome, and we show that HflX can restore heat-damaged ribosomes and improve cell survival. © 2018 Dey et al.

Crosstalk between the equilibrative nucleoside transporter ENT2 and alveolar Adora2b adenosine receptors dampens acute lung injury

PubMed Central

Eckle, Tobias; Hughes, Kelly; Ehrentraut, Heidi; Brodsky, Kelley S.; Rosenberger, Peter; Choi, Doo-Sup; Ravid, Katya; Weng, Tingting; Xia, Yang; Blackburn, Michael R.; Eltzschig, Holger K.

2013-01-01

The signaling molecule adenosine has been implicated in attenuating acute lung injury (ALI). Adenosine signaling is terminated by its uptake through equilibrative nucleoside transporters (ENTs). We hypothesized that ENT-dependent adenosine uptake could be targeted to enhance adenosine-mediated lung protection. To address this hypothesis, we exposed mice to high-pressure mechanical ventilation to induce ALI. Initial studies demonstrated time-dependent repression of ENT1 and ENT2 transcript and protein levels during ALI. To examine the contention that ENT repression represents an endogenous adaptive response, we performed functional studies with the ENT inhibitor dipyridamole. Dipyridamole treatment (1 mg/kg; EC50=10 μM) was associated with significant increases in ALI survival time (277 vs. 395 min; P<0.05). Subsequent studies in gene-targeted mice for Ent1 or Ent2 revealed a selective phenotype in Ent2−/− mice, including attenuated pulmonary edema and improved gas exchange during ALI in conjunction with elevated adenosine levels in the bronchoalveolar fluid. Furthermore, studies in genetic models for adenosine receptors implicated the A2B adenosine receptor (Adora2b) in mediating ENT-dependent lung protection. Notably, dipyridamole-dependent attenuation of lung inflammation was abolished in mice with alveolar epithelial Adora2b gene deletion. Our newly identified crosstalk pathway between ENT2 and alveolar epithelial Adora2b in lung protection during ALI opens possibilities for combined therapies targeted to this protein set.—Eckle, T., Hughes, K., Ehrentraut, H., Brodsky, K. S., Rosenberger, P., Choi, D.-S., Ravid, K., Weng, T., Xia, Y., Blackburn, M. R., Eltzschig, H. K. Crosstalk between the equilibrative nucleoside transporter ENT2 and alveolar Adora2b adenosine receptors dampens acute lung injury. PMID:23603835

Meteorite-catalyzed syntheses of nucleosides and of other prebiotic compounds from formamide under proton irradiation

PubMed Central

Saladino, Raffaele; Carota, Eleonora; Botta, Giorgia; Kapralov, Mikhail; Timoshenko, Gennady N.; Rozanov, Alexei Y.; Krasavin, Eugene; Di Mauro, Ernesto

2015-01-01

Liquid formamide has been irradiated by high-energy proton beams in the presence of powdered meteorites, and the products of the catalyzed resulting syntheses were analyzed by mass spectrometry. Relative to the controls (no radiation, or no formamide, or no catalyst), an extremely rich, variegate, and prebiotically relevant panel of compounds was observed. The meteorites tested were representative of the four major classes: iron, stony iron, chondrites, and achondrites. The products obtained were amino acids, carboxylic acids, nucleobases, sugars, and, most notably, four nucleosides: cytidine, uridine, adenosine, and thymidine. In accordance with theoretical studies, the detection of HCN oligomers suggests the occurrence of mechanisms based on the generation of radical cyanide species (CN·) for the synthesis of nucleobases. Given that many of the compounds obtained are key components of extant organisms, these observations contribute to outline plausible exogenous high-energy–based prebiotic scenarios and their possible boundary conditions, as discussed. PMID:25870268

Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase.

PubMed

Isaksen, Geir Villy; Hopmann, Kathrin Helen; Åqvist, Johan; Brandsdal, Bjørn Olav

2016-04-12

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'-deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity.

Supramolecular assemblies of nucleoside functionalized carbon nanotubes: synthesis, film preparation, and properties.

PubMed

Micoli, Alessandra; Turco, Antonio; Araujo-Palomo, Elsie; Encinas, Armando; Quintana, Mildred; Prato, Maurizio

2014-04-25

Nucleoside-functionalized multi-walled carbon nanotubes (N-MWCNTs) were synthesized and characterized. A self-organization process using hydrogen bonding interactions was then used for the fabrication of self-assembled N-MWCNTs films free of stabilizing agents, polymers, or surfactants. Membranes were produced by using a simple water-dispersion-based vacuum-filtration method. Hydrogen-bond recognition was confirmed by analysis with IR spectroscopy and TEM images. Restoration of the electronic conduction properties in the N-MWCNTs membranes was performed by removing the organic portion by thermal treatment under an argon atmosphere to give d-N-MWCNTs. Electrical conductivity and thermal gravimetric analysis (TGA) measurements confirmed the efficiency of the annealing process. Finally, oxidative biodegradation of the films N-MWCNTs and d-N-MWCNTs was performed by using horseradish peroxidase (HRP) and low concentrations of H2 O2 . Our results confirm that functional groups play an important role in the biodegradation of CNT by HRP: N-MWCNTs films were completely biodegraded, whereas for d-N-MWCNTs films no degradation was observed, showing that the pristine CNT undergoes minimal enzyme-catalyzed oxidation This novel methodology offers a straightforward supramolecular strategy for the construction of conductive and biodegradable carbon nanotube films. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Long term expression of Drosophila melanogaster nucleoside kinase in thymidine kinase 2-deficient mice with no lethal effects caused by nucleotide pool imbalances.

PubMed

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

2014-11-21

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. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

BCX4430, a novel nucleoside analog, effectively treats yellow fever in a Hamster model.

PubMed

Julander, Justin G; Bantia, Shanta; Taubenheim, Brian R; Minning, Dena M; Kotian, Pravin; Morrey, John D; Smee, Donald F; Sheridan, William P; Babu, Yarlagadda S

2014-11-01

No effective antiviral therapies are currently available to treat disease after infection with yellow fever virus (YFV). A Syrian golden hamster model of yellow fever (YF) was used to characterize the effect of treatment with BCX4430, a novel adenosine nucleoside analog. Significant improvement in survival was observed after treatment with BCX4430 at 4 mg/kg of body weight per day dosed intraperitoneally (i.p.) twice daily (BID). Treatment with BCX4430 at 12.5 mg/kg/day administered i.p. BID for 7 days offered complete protection from mortality and also resulted in significant improvement of other YF disease parameters, including weight loss, serum alanine aminotransferase levels (6 days postinfection [dpi]), and viremia (4 dpi). In uninfected hamsters, BCX4430 at 200 mg/kg/day administered i.p. BID for 7 days was well tolerated and did not result in mortality or weight loss, suggesting a potentially wide therapeutic index. Treatment with BCX4430 at 12 mg/kg/day i.p. remained effective when administered once daily and for only 4 days. Moreover, BCX4430 dosed at 200 mg/kg/day i.p. BID for 7 days effectively treated YF, even when treatment was delayed up to 4 days after virus challenge, corresponding with peak viral titers in the liver and serum. BCX4430 treatment did not preclude a protective antibody response, as higher neutralizing antibody (nAb) concentrations corresponded with increasing delays of treatment initiation, and greater nAb responses resulted in the protection of animals from a secondary challenge with YFV. In summary, BCX4430 is highly active in a hamster model of YF, even when treatment is initiated at the peak of viral replication. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes*

PubMed Central

Hindman, Ryan; Gollnick, Paul

2016-01-01

Vaccinia virus early genes are transcribed immediately upon infection. Nucleoside triphosphate phosphohydrolase I (NPH I) is an essential component of the early gene transcription complex. NPH I hydrolyzes ATP to release transcripts during transcription termination. The ATPase activity of NPH I requires single-stranded (ss) DNA as a cofactor; however, the source of this cofactor within the transcription complex is not known. Based on available structures of transcription complexes it has been hypothesized that the ssDNA cofactor is obtained from the unpaired non-template strand within the transcription bubble. In vitro transcription on templates that lack portions of the non-template strand within the transcription bubble showed that the upstream portion of the transcription bubble is required for efficient NPH I-mediated transcript release. Complementarity between the template and non-template strands in this region is also required for NPH I-mediated transcript release. This observation complicates locating the source of the ssDNA cofactor within the transcription complex because removal of the non-template strand also disrupts transcription bubble reannealing. Prior studies have shown that ssRNA binds to NPH I, but it does not activate ATPase activity. Chimeric transcription templates with RNA in the non-template strand confirm that the source of the ssDNA cofactor for NPH I is the upstream portion of the non-template strand in the transcription bubble. Consistent with this conclusion we also show that isolated NPH I acts as a 5′ to 3′ translocase on single-stranded DNA. PMID:27189950

MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5.

PubMed

Nezich, Catherine L; Wang, Chunxin; Fogel, Adam I; Youle, Richard J

2015-08-03

The kinase PINK1 and ubiquitin ligase Parkin can regulate the selective elimination of damaged mitochondria through autophagy (mitophagy). Because of the demand on lysosomal function by mitophagy, we investigated a role for the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in this process. We show that during mitophagy TFEB translocates to the nucleus and displays transcriptional activity in a PINK1- and Parkin-dependent manner. MITF and TFE3, homologues of TFEB belonging to the same microphthalmia/transcription factor E (MiT/TFE) family, are similarly regulated during mitophagy. Unlike TFEB translocation after starvation-induced mammalian target of rapamycin complex 1 inhibition, Parkin-mediated TFEB relocalization required Atg9A and Atg5 activity. However, constitutively active Rag guanosine triphosphatases prevented TFEB translocation during mitophagy, suggesting cross talk between these two MiT/TFE activation pathways. Analysis of clustered regularly interspaced short palindromic repeats-generated TFEB/MITF/TFE3/TFEC single, double, and triple knockout cell lines revealed that these proteins partly facilitate Parkin-mediated mitochondrial clearance. These results illuminate a pathway leading to MiT/TFE transcription factor activation, distinct from starvation-induced autophagy, which occurs during mitophagy.

Phytochemical and antimicrobial activities of Himalayan Cordyceps sinensis (Berk.) Sacc.

PubMed

Mamta; Mehrotra, Shubhi; Amitabh; Kirar, Vandana; Vats, Praveen; Nandi, Shoma Paul; Negi, P S; Misra, Kshipra

2015-01-01

This study evaluated the phytochemical and antimicrobial activities and also quantified bioactive nucleoside using high performance thin layer chromatography (HPTLC) of five extracts of Indian Himalayan Cordyceps sinensis prepared with different solvents employing accelerated solvent extraction (ASE) technique. The phytochemical potential of these extracts was quantified in terms of total phenolic and total flavonoid content while antioxidant activities were determined by 1,1-diphenyl-2-pycryl-hydrazyl (DPPH) and 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and ferric-reducing antioxidant power (FRAP) assays. Total reducing power (TRP) was determined by converting iron (III) into iron (II) reduction assay. CS(50%Alc) (15.1 ± 0.67mg/g of dry extract) and CS(100%Alc) (19.3 ± 0.33 mg/g of dry extract) showed highest phenolic and flavonoid content, respectively while CS(Aq) extract showed maximum antioxidant activity and the highest concentration of the three nucleosides (adenine 12.8 ± 0.49 mg/g, adenosine 0.36 ± 0.28 mg/g and uracil 0.14 ± 0.36 mg/g of dry extract) determined by HPTLC. The evaluation of extracts for antimicrobial activity against gram-negative and gram-positive bacterial strains showed CS(25%Alc), CS(75%Alc) and CS(100%Alc) extract to be more effective against E. coli, P. aerugenosa and B. subtilis giving 9, 7 and 6.5 mm of zone of inhibition (ZOI) in 93.75, 93.75 and 45 μg concentration, respectively, whereas CS(Aq) extract showed minimal inhibition against these.

New prodrugs of two pyrimidine acyclic nucleoside phosphonates: Synthesis and antiviral activity.

PubMed

Krečmerová, Marcela; Dračínský, Martin; Snoeck, Robert; Balzarini, Jan; Pomeisl, Karel; Andrei, Graciela

2017-09-01

New 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidine (PMEO-DAPy) and 1-[2-(phosphonomethoxy)ethyl]-5-azacytosine (PME-5-azaC) prodrugs were prepared with a pro-moiety consisting of carbonyloxymethyl esters (POM, POC), alkoxyalkyl esters, amino acid phosphoramidates and/or tyrosine. The activity of the prodrugs was evaluated in vitro against different virus families. None of the synthesized prodrugs demonstrated activity against RNA viruses but some of them proved active against herpesviruses [including herpes simplex virus (HSV), varicella-zoster virus (VZV), and human cytomegalovirus (HCMV)]. The bis(POC) and the bis(amino acid) phosphoramidate prodrugs of PMEO-DAPy inhibited herpesvirus replication at lower doses than the parent compound although the selectivity against HSV and VZV was only slightly improved compared to PMEO-DAPy. The mono-octadecyl ester of PME-5-azaC emerged as the most potent and selective PME-5-azaC prodrug against HSV, VZV and HCMV with EC 50 's of 0.15-1.12µM while PME-5-azaC only had marginal anti-herpesvirus activity. Although the bis(hexadecylamido-l-tyrosyl) and the bis(POM) esters of PME-5-azaC were also very potent anti-herpesvirus drugs, these were less selective than the mono-octadecyl ester prodrug. Copyright © 2017 Elsevier Ltd. All rights reserved.

Nucleotides as nucleophiles: reactions of nucleotides with phosphoimidazolide activated guanosine

NASA Technical Reports Server (NTRS)

Kanavarioti, A.; Rosenbach, M. T.; Hurley, T. B.

1991-01-01

An earlier study of the reaction of phosphoimidazolide activated nucleosides (ImpN) in aqueous phosphate buffers indicated two modes of reaction of the phosphate monoanion and dianion. The first mode is catalysis of the hydrolysis of the P-N bond in ImpN's which leads to imidazole and nucleoside 5'-monophosphate. The second represents a nucleophilic substitution of the imidazole to yield the nucleoside 5'-diphosphate. This earlier study thus served as a model for the reaction of ImpN with nucleoside monophosphates (pN) because the latter can be regarded as phosphate derivatives. In the present study we investigated the reaction of guanosine 5'-phosphate-2-methylimidazolide, 2-MeImpG, in the presence of pN (N = guanosine, adenosine and uridine) in the range 6.9 less than or equal to pH less than or equal to 7.7. We observed that pN's do act as nucleophiles to form NppG, and as general base to enhance the hydrolysis of the P-N bond in 2-MeImpG, i.e. pN show the same behavior as inorganic phosphate. The kinetic analysis yields the following rate constants for the dianion pN2-: knpN = 0.17 +/- 0.02 M-1 h-1 for nucleophilic attack and khpN = 0.11 +/- 0.07 M-1 h-1 for general base catalysis of the hydrolysis. These rate constants which are independent of the nucleobase compare with kp.2 = 0.415 M-1 h-1 and khp2. = 0.217 M-1 h-1 for the reactions of HPO4(2-). In addition, this study shows that under conditions where pN presumably form stacks, the reaction mechanism remains unchanged although in quantitative terms stacked pN are somewhat less reactive. Attack by the 2'-OH and 3'-OH groups of the ribose moiety in amounts greater than or equal to 1% is not observed; this is attributed to the large difference in nucleophilicity in the neutral pH range between the phosphate group and the ribose hydroxyls. This nucleophilicity rank is not altered by stacking.

In vitro transport characteristics of EFdA, a novel nucleoside reverse transcriptase inhibitor using Caco-2 and MDCKII cell monolayers.

PubMed

Zhang, Wei; Parniak, Michael A; Sarafianos, Stefan G; Empey, Philip E; Rohan, Lisa C

2014-06-05

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a novel nucleoside reverse transcriptase inhibitor with a unique mechanism of action and highly potent activity against both wild-type and clinically relevant drug resistant HIV-1 variants. Furthermore, in vivo efficacy and safety evaluations have shown EFdA to be a promising therapeutic candidate for use in the treatment of HIV infection. However, little is known about the pharmacokinetic and biopharmaceutical properties of EFdA. In this study, we evaluated cellular EFdA transport using Caco-2 and Madin-Darby Canine Kidney II (MDCKII) in vitro cell models. Studies using Caco-2 cell monolayers showed that EFdA efflux ratios were >2.0, suggesting that active drug transport mechanisms may play a role in EFdA flux. ABCB1 transporter (PGP1) inhibition was assessed using the acetomethoxy derivate of calcein (calcein-AM) as a fluorescent probe in both wild-type MDCKII and PGP1 overexpressing MDCKII cells. Nonetheless, our data showed that EFdA is not a substrate of PGP1. Additionally, comparative bidirectional flux of EFdA and Lucifer yellow (LY, a well-known paracellular marker) was studied over a range of EFdA concentrations. In MDCKII monolayers, EFdA had an apparent permeability coefficient (Papp) (a-b) of <1×10(-6)cm/s. The Papp values significantly increased in the presence of the paracellular permeability enhancer, indicating that EFdA primarily permeates via the paracellular route. Copyright © 2014 Elsevier B.V. All rights reserved.

Separation of oligopeptides, nucleobases, nucleosides and nucleotides using capillary electrophoresis/electrochromatography with sol-gel modified inner capillary wall.

PubMed

Svobodová, Jana; Kofroňová, Olga; Benada, Oldřich; Král, Vladimír; Mikšík, Ivan

2017-09-29

The aim of this article is to study the modification of an inner capillary wall with sol-gel coating (pure silica sol-gel or silica sol-gel containing porphyrin-brucine conjugate) and determine its influence on the separation process using capillary electrophoresis/electrochromatography method. After modification of the inner capillary surface the separation of analytes was performed using two different phosphate buffers (pH 2.5 and 9.0) and finally the changes in electrophoretic mobilities of various samples were calculated. To confirm that the modification of the inner capillary surface was successful, the parts of the inner surfaces of capillaries were observed using scanning electron microscopy. The analytes used as testing samples were oligopeptides, nucleosides, nucleobases and finally nucleotides. Copyright © 2017 Elsevier B.V. All rights reserved.

Trifluorothymidine resistance is associated with decreased thymidine kinase and equilibrative nucleoside transporter expression or increased secretory phospholipase A2.

PubMed

Temmink, Olaf H; Bijnsdorp, Irene V; Prins, Henk-Jan; Losekoot, Nienke; Adema, Auke D; Smid, Kees; Honeywell, Richard J; Ylstra, Bauke; Eijk, Paul P; Fukushima, Masakazu; Peters, Godefridus J

2010-04-01

Trifluorothymidine (TFT) is part of the novel oral formulation TAS-102, which is currently evaluated in phase II studies. Drug resistance is an important limitation of cancer therapy. The aim of the present study was to induce resistance to TFT in H630 colon cancer cells using two different schedules and to analyze the resistance mechanism. Cells were exposed either continuously or intermittently to TFT, resulting in H630-cTFT and H630-4TFT, respectively. Cells were analyzed for cross-resistance, cell cycle, protein expression, and activity of thymidine phosphorylase (TP), thymidine kinase (TK), thymidylate synthase (TS), equilibrative nucleoside transporter (hENT), gene expression (microarray), and genomic alterations. Both cell lines were cross-resistant to 2'-deoxy-5-fluorouridine (>170-fold). Exposure to IC(75)-TFT increased the S/G(2)-M phase of H630 cells, whereas in the resistant variants, no change was observed. The two main target enzymes TS and TP remained unchanged in both TFT-resistant variants. In H630-4TFT cells, TK protein expression and activity were decreased, resulting in less activated TFT and was most likely the mechanism of TFT resistance. In H630-cTFT cells, hENT mRNA expression was decreased 2- to 3-fold, resulting in a 5- to 10-fold decreased TFT-nucleotide accumulation. Surprisingly, microarray-mRNA analysis revealed a strong increase of secretory phospholipase-A2 (sPLA2; 47-fold), which was also found by reverse transcription-PCR (RT-PCR; 211-fold). sPLA2 inhibition reversed TFT resistance partially. H630-cTFT had many chromosomal aberrations, but the exact role of sPLA2 in TFT resistance remains unclear. Altogether, resistance induction to TFT can lead to different mechanisms of resistance, including decreased TK protein expression and enzyme activity, decreased hENT expression, as well as (phospho)lipid metabolism. Mol Cancer Ther; 9(4); 1047-57. (c)2010 AACR.

Design, synthesis and cellular metabolism study of 4'-selenonucleosides.

PubMed

Yu, Jinha; Sahu, Pramod K; Kim, Gyudong; Qu, Shuhao; Choi, Yoojin; Song, Jayoung; Lee, Sang Kook; Noh, Minsoo; Park, Sunghyouk; Jeong, Lak Shin

2015-01-01

4'-seleno-homonucleosides were synthesized as next-generation nucleosides, and their cellular phosphorylation was studied to confirm the hypothesis that bulky selenium atom can sterically hinder the approach of cellular nucleoside kinase to the 5'-OH for phosphorylation. 4'-seleno-homonucleosides (n = 2), with one-carbon homologation, were synthesized through a tandem seleno-Michael addition-SN2 ring cyclization. LC-MS analysis demonstrated that they were phosphorylated by cellular nucleoside kinases, resulting in anticancer activity. The bulky selenium atom played a key role in deciding the phosphorylation by cellular nucleoside kinases. [Formula: see text].

Neurotrophin Promotes Neurite Outgrowth by Inhibiting Rif GTPase Activation Downstream of MAPKs and PI3K Signaling.

PubMed

Tian, Xiaoxia; Yan, Huijuan; Li, Jiayi; Wu, Shuang; Wang, Junyu; Fan, Lifei

2017-01-13

Members of the well-known semaphorin family of proteins can induce both repulsive and attractive signaling in neural network formation and their cytoskeletal effects are mediated in part by small guanosine 5'-triphosphatase (GTPases). The aim of this study was to investigate the cellular role of Rif GTPase in the neurotrophin-induced neurite outgrowth. By using PC12 cells which are known to cease dividing and begin to show neurite outgrowth responding to nerve growth factor (NGF), we found that semaphorin 6A was as effective as nerve growth factor at stimulating neurite outgrowth in PC12 cells, and that its neurotrophic effect was transmitted through signaling by mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinase (PI3K). We further found that neurotrophin-induced neurite formation in PC12 cells could be partially mediated by inhibition of Rif GTPase activity downstream of MAPKs and PI3K signaling. In conclusion, we newly identified Rif as a regulator of the cytoskeletal rearrangement mediated by semaphorins.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) promotes glioblastoma cell chemotaxis via Lyn activation

PubMed Central

Tran, Nhan L.

2014-01-01

The long-term survival of patients with glioblastoma is compromised by the proclivity for local invasion into the surrounding normal brain, escaping surgical resection and contributing to therapeutic resistance. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell invasion via binding to fibroblast growth factor-inducible 14 (Fn14) and subsequent activation of the Rho guanosine triphosphatase family member Rac1. Here, we demonstrate that TWEAK acts as a chemotactic factor for glioma cells, a potential process for driving cell invasion into the surrounding brain tissue. TWEAK exposure induced the activation of Src family kinases (SFKs), and pharmacologic suppression of SFK activity inhibited TWEAK-induced chemotactic migration. We employed a multiplexed Luminex assay and identified Lyn as a candidate SFK activated by TWEAK. Depletion of Lyn suppressed TWEAK-induced chemotaxis and Rac1 activity. Furthermore, Lyn gene expression levels increase with primary glioma tumor grade and inversely correlate with patient survival. These results show that TWEAK-induced glioma cell chemotaxis is dependent upon Lyn kinase function and, thus, provides opportunities for therapeutic targeting of this deadly disease. PMID:23975833

Highly efficient generation of glutamatergic/cholinergic NT2-derived postmitotic human neurons by short-term treatment with the nucleoside analogue cytosine β-D-arabinofuranoside.

PubMed

González-Burguera, Imanol; Ricobaraza, Ana; Aretxabala, Xabier; Barrondo, Sergio; García del Caño, Gontzal; López de Jesús, Maider; Sallés, Joan

2016-03-01

The human NTERA2/D1 (NT2) cells generate postmitotic neurons (NT2N cells) upon retinoic acid (RA) treatment and are functionally integrated in the host tissue following grafting into the rodent and human brain, thus representing a promising source for neuronal replacement therapy. Yet the major limitations of this model are the lengthy differentiation procedure and its low efficiency, although recent studies suggest that the differentiation process can be shortened to less than 1 week using nucleoside analogues. To explore whether short-term exposure of NT2 cells to the nucleoside analogue cytosine β-d-arabinofuranoside (AraC) could be a suitable method to efficiently generate mature neurons, we conducted a neurochemical and morphometric characterization of AraC-differentiated NT2N (AraC/NT2N) neurons and improved the differentiation efficiency by modifying the cell culture schedule. Moreover, we analyzed the neurotransmitter phenotypes of AraC/NT2N neurons. Cultures obtained by treatment with AraC were highly enriched in postmitotic neurons and essentially composed of dual glutamatergic/cholinergic neurons, which contrasts with the preferential GABAergic phenotype that we found after RA differentiation. Taken together, our results further reinforce the notion NT2 cells are a versatile source of neuronal phenotypes and provide a new encouraging platform for studying mechanisms of neuronal differentiation and for exploring neuronal replacement strategies. Copyright © 2016 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.

Noncanonical RNA Nucleosides as Molecular Fossils of an Early Earth-Generation by Prebiotic Methylations and Carbamoylations.

PubMed

Schneider, Christina; Becker, Sidney; Okamura, Hidenori; Crisp, Antony; Amatov, Tynchtyk; Stadlmeier, Michael; Carell, Thomas

2018-05-14

The RNA-world hypothesis assumes that life on Earth started with small RNA molecules that catalyzed their own formation. Vital to this hypothesis is the need for prebiotic routes towards RNA. Contemporary RNA, however, is not only constructed from the four canonical nucleobases (A, C, G, and U), it also contains many chemically modified (noncanonical) bases. A still open question is whether these noncanonical bases were formed in parallel to the canonical bases (chemical origin) or later, when life demanded higher functional diversity (biological origin). Here we show that isocyanates in combination with sodium nitrite establish methylating and carbamoylating reactivity compatible with early Earth conditions. These reactions lead to the formation of methylated and amino acid modified nucleosides that are still extant. Our data provide a plausible scenario for the chemical origin of certain noncanonical bases, which suggests that they are fossils of an early Earth. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The search for an identification of amino acids, nucleobases and nucleosides in samples returned from Mars

NASA Technical Reports Server (NTRS)

Gehrke, Charles W.; Ponnamperuma, Cyril; Kuo, Kenneth C.; Stalling, David L.; Zumwalt, Robert W.

1988-01-01

The Mars Sample Return mission will provide us with a unique source of material from our solar system; material which could advance our knowledge of the processes of chemical evolution. As has been pointed out, Mars geological investigations based on the Viking datasets have shown that primordial Mars was in many biologically important ways similar to the primordial Earth; the presence of surface liquid water, moderate surface temperatures, and atmosphere of carbon dioxide and nitrogen, and high geothermal heat flow. Indeed, it would seem that conditions on Earth and Mars were fundamentally similar during the first one billion years or so. As has been pointed out, Mars may well contain the best preserved record of the events that transpired on the early planets. Examination of that early record will involve searching for many things, from microfossils to isotopic abundance data. We propose an investigation of the returned Mars samples for biologically important organic compounds, with emphases on amino acids, the purine and pyrimidine bases, and nucleosides.

Determination of nucleoside analog mono-, di-, and tri-phosphates in cellular matrix by solid phase extraction and ultra-sensitive LC-MS/MS detection.

PubMed

Bushman, Lane R; Kiser, Jennifer J; Rower, Joseph E; Klein, Brandon; Zheng, Jia-Hua; Ray, Michelle L; Anderson, Peter L

2011-09-10

An ultra-sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and validated to facilitate the assessment of clinical pharmacokinetics of nucleotide analogs from lysed intracellular matrix. The method utilized a strong anion exchange isolation of mono-(MP), di-(DP), and tri-phosphates (TP) from intracellular matrix. Each fraction was then dephosphorylated to the parent moiety yielding a molar equivalent to the original nucleotide analog intracellular concentration. The analytical portion of the methodology was optimized in specific nucleoside analog centric modes (i.e. tenofovir (TFV) centric, zidovudine (ZDV) centric), which included desalting/concentration by solid phase extraction and detection by LC-MS/MS. Nucleotide analog MP-, DP-, and TP-determined on the TFV centric mode of analysis include TFV, lamivudine (3TC), and emtricitibine (FTC). The quantifiable linear range for TFV was 2.5-2000 fmol/sample, and that for 3TC/FTC was 0.1 200 pmol/sample. Nucleoside analog MP-, DP-, and TP-determined on the ZDV centric mode of analysis included 3TC and ZDV. The quantifiable linear range for 3TC was 0.1 100 pmol/sample, and 5-2000 fmol/sample for ZDV. Stable labeled isotopic internal standards facilitated accuracy and precision in alternative cell matrices, which supported the intended use of the method for MP, DP, and TP determinations in various cell types. The method was successfully applied to clinical research samples generating novel intracellular information for TFV, FTC, ZDV, and 3TC nucleotides. This document outlines method development, validation, and application to clinical research. Copyright © 2011 Elsevier B.V. All rights reserved.

Effect of changes in dietary sodium on active electrolyte transport by erythrocytes at different stages of human pregnancy.