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.

Pyrimidine metabolism in Tritrichomonas foetus.

PubMed Central

Wang, C C; Verham, R; Tzeng, S F; Aldritt, S; Cheng, H W

1983-01-01

The anaerobic parasitic protozoa Tritrichomonas foetus is found incapable of de novo pyrimidine biosynthesis by its failure to incorporate bicarbonate, aspartate, or orotate into pyrimidine nucleotides or nucleic acids. Uracil phosphoribosyltransferase in the cytoplasm provides the major pyrimidine salvage for the parasite. Exogenous uridine and cytidine are mostly converted to uracil by uridine phosphorylase and cytidine deaminase in T. foetus prior to incorporation. T. foetus cannot incorporate labels from exogenous uracil or uridine into DNA; it has no detectable dihydrofolate reductase or thymidylate synthetase and is resistant to methotrexate, pyrimethamine, trimethoprim, and 5-bromovinyldeoxyuridine at millimolar concentrations. It has an enzyme thymidine phosphotransferase in cellular fraction pelleting at 100,000 X g that can convert exogenous thymidine to TMP via a phosphate donor such as p-nitrophenyl phosphate or nucleoside 5'-monophosphate. Thymidine salvage in T. foetus is thus totally dissociated from other pyrimidine salvage. PMID:6573672

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.

Mineral catalysis of the formation of the phosphodiester bond in aqueous solution - The possible role of montmorillonite clays

NASA Technical Reports Server (NTRS)

Ferris, James P.; Ertem, Gozen; KAMALUDDIN; Agarwal, Vipin; Hua, Lu Lin

1989-01-01

The possible role of montmorillonite clays in the spontaneous formation on the primitive earth of the phosphodiester bond in the presence of water was investigated in experiments measuring the binding of various nucleosides and nucleotides with Na(+)-montmorillonite 22A and the reactions of these compounds with a water-soluble carbodiimide. It was found that, at neutral pH, adenine derivatives bind stronger than the corresponding uracil derivatives, consistent with the protonation of the adenine by the acidic clay surface and a cationic binding of the protonated ring to the anionic clay surface. The reaction of the 5-prime-AMP with carbodiimide resulted in the formation of 2-prime,5-prime-pApA (18.9 percent), 3-prime,5-prime-pApA (11 percent), and AppA (4.8 percent). The yields of these oligomers obtained when poly(U) was used in place of the clay were 15.5 percent, 3.7 percent, and 14.9 percent AppA, respectively.

Effect of pH and temperature on the stability of UV-induced repairable pyrimidine hydrates in DNA.

PubMed

O'Donnell, R E; Boorstein, R J; Cunningham, R P; Teebor, G W

1994-08-23

UV irradiation of cytosine yields 6-hydroxy-5,6-dihydrocytosine (cytosine hydrate) whether the cytosine is in solution as base, nucleoside, or nucleotide or on the DNA backbone. Cytosine hydrate decomposes by elimination of water, yielding cytosine, or by irreversible deamination, yielding uracil hydrate, which, in turn, decomposes by dehydration yielding uracil. To determine how pH and temperature affect these decomposition reactions, alternating poly(dG-[3H]dC) copolymer was irradiated at 254 nm and incubated under different conditions of pH and temperature. The cytosine hydrate and uracil hydrate content of the DNA was determined by the use of Escherichia coli endonuclease III, which releases pyrimidine hydrates from DNA by virtue of its DNA glycosylase activity. Uracil content was determined by using uracil-DNA glycosylase. The rate of decomposition of cytosine hydrate to cytosine was determined at 4 temperatures at pH 3.1, 5.4, and 7.4. The Ea was determined from the rates by using the Arrhenius equation and proved to be the same at pH 5.4 and 7.4, although the decomposition rate at pH 5.4 was faster at all temperatures. At pH 3.1, the Ea was reduced. These results suggest that the dehydration reaction is affected by two discrete protonations, most probably of the N-3 and the OH group of C-6 of cytosine hydrate. The deamination of cytosine hydrate to uracil hydrate was maximal at pH 3.1 at all temperatures. The doubly protonated cytosine hydrate probably is the common intermediate for both competing decomposition reactions, explaining why cytosine hydrate is prone to deamination at acid pH.(ABSTRACT TRUNCATED AT 250 WORDS)

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 through phosphotriester formation, but this mechanism is not proven. PMID:4673570

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.

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.

Bacterial-derived uracil as a modulator of mucosal immunity and gut-microbe homeostasis in Drosophila.

PubMed

Lee, Kyung-Ah; Kim, Sung-Hee; Kim, Eun-Kyoung; Ha, Eun-Mi; You, Hyejin; Kim, Boram; Kim, Min-Ji; Kwon, Youngjoo; Ryu, Ji-Hwan; Lee, Won-Jae

2013-05-09

All metazoan guts are subjected to immunologically unique conditions in which an efficient antimicrobial system operates to eliminate pathogens while tolerating symbiotic commensal microbiota. However, the molecular mechanisms controlling this process are only partially understood. Here, we show that bacterial-derived uracil acts as a ligand for dual oxidase (DUOX)-dependent reactive oxygen species generation in Drosophila gut and that the uracil production in bacteria causes inflammation in the gut. The acute and controlled uracil-induced immune response is required for efficient elimination of bacteria, intestinal cell repair, and host survival during infection of nonresident species. Among resident gut microbiota, uracil production is absent in symbionts, allowing harmonious colonization without DUOX activation, whereas uracil release from opportunistic pathobionts provokes chronic inflammation. These results reveal that bacteria with distinct abilities to activate uracil-induced gut inflammation, in terms of intensity and duration, act as critical factors that determine homeostasis or pathogenesis in gut-microbe interactions. Copyright © 2013 Elsevier Inc. All rights reserved.

Ubiquitous water-soluble molecules in aquatic plant exudates determine specific insect attraction.

PubMed

Sérandour, Julien; Reynaud, Stéphane; Willison, John; Patouraux, Joëlle; Gaude, Thierry; Ravanel, Patrick; Lempérière, Guy; Raveton, Muriel

2008-10-08

Plants produce semio-chemicals that directly influence insect attraction and/or repulsion. Generally, this attraction is closely associated with herbivory and has been studied mainly under atmospheric conditions. On the other hand, the relationship between aquatic plants and insects has been little studied. To determine whether the roots of aquatic macrophytes release attractive chemical mixtures into the water, we studied the behaviour of mosquito larvae using olfactory experiments with root exudates. After testing the attraction on Culex and Aedes mosquito larvae, we chose to work with Coquillettidia species, which have a complex behaviour in nature and need to be attached to plant roots in order to obtain oxygen. This relationship is non-destructive and can be described as commensal behaviour. Commonly found compounds seemed to be involved in insect attraction since root exudates from different plants were all attractive. Moreover, chemical analysis allowed us to identify a certain number of commonly found, highly water-soluble, low-molecular-weight compounds, several of which (glycerol, uracil, thymine, uridine, thymidine) were able to induce attraction when tested individually but at concentrations substantially higher than those found in nature. However, our principal findings demonstrated that these compounds appeared to act synergistically, since a mixture of these five compounds attracted larvae at natural concentrations (0.7 nM glycerol, <0.5 nM uracil, 0.6 nM thymine, 2.8 nM uridine, 86 nM thymidine), much lower than those found for each compound tested individually. These results provide strong evidence that a mixture of polyols (glycerol), pyrimidines (uracil, thymine), and nucleosides (uridine, thymidine) functions as an efficient attractive signal in nature for Coquillettidia larvae. We therefore show for the first time, that such commonly found compounds may play an important role in plant-insect relationships in aquatic eco-systems.

Nutrition beyond nutrition: plausibility of immunotrophic nutrition for space travel.

PubMed

Kulkarni, A D; Yamauchi, K; Hales, N W; Ramesh, V; Ramesh, G T; Sundaresan, A; Andrassy, R J; Pellis, N R

2002-06-01

Microgravity has adverse effects on the immune system. We examined the effects of supplemental dietary nucleotides on immune function in ground-based in vivo anti-orthostatic tail-suspended (AOS) mice and in vitro (bioreactor-BIO) analogs of microgravity. BALB/c mice were divided into the following three groups: group housed, single isolation, and AOS. Mice were fed either control chow or chow supplemented with RNA or uracil. Immune function was assessed by in vivo popliteal lymph node proliferation (PLN), in vitro PHA-stimulated proliferation of splenocytes and cytokine production. BIO splenocytes were cultured in vitro with/without PHA, a nucleoside-nucleotide mixture (NS/NT) or uridine. The cell proliferation and scanning electron microscopic examination for cells were carried out. PLN response was significantly suppressed in AOS mice (P<0.05) and was restored by RNA and uracil diets. Splenocytes from AOS mice had decreased phytohemagglutinin (PHA)-stimulated proliferation, decreased IL-2 and IFN-gamma cytokine levels (P<0.05). These responses were restored by RNA and uracil diets. In BIO cultures, PHA response was suppressed significantly, and uridine and NS/NT restored the proliferative responses. Scanning electron microscopic analysis of cells cultured in BIO revealed cells with pinched, distorted and eroded membranes. Nucleotide supplementation especially uridine restored normal activated cell surface appearance and ruffling. In the microgravity analog environment of AOS and BIO, supplemental nucleotides and especially uracil/uridine have up-regulating and immunoprotective effects with potential as a countermeasure to the observed immune dysfunction in true microgravity.

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

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

Uridine homeostatic disorder leads to DNA damage and tumorigenesis.

PubMed

Cao, Zhe; Ma, Jun; Chen, Xinchun; Zhou, Boping; Cai, Chuan; Huang, Dan; Zhang, Xuewen; Cao, Deliang

2016-03-28

Uridine is a natural nucleoside precursor of uridine monophosphate in organisms and thus is considered to be safe and is used in a wide range of clinical settings. The far-reaching effects of pharmacological uridine have long been neglected. Here, we report that the homeostatic disorder of uridine is carcinogenic. Targeted disruption (-/-) of murine uridine phosphorylase (UPase) disrupted the homeostasis of uridine and increased spontaneous tumorigenesis by more than 3-fold. Multiple tumors (e.g., lymphoma, hepatoma and lung adenoma) occurred simultaneously in some UPase deficient mice, but not in wild-type mice raised under the same conditions. In the tissue from UPase -/- mice, the 2'-deoxyuridine,5'-triphosphate (dUTP) levels and uracil DNA were increased and p53 was activated with an increased phospho-Ser18 p53 level. Exposing cell lines (e.g., MCF-7, RKO, HCT-8 and NCI-H460) to uridine (10 or 30 µM) led to uracil DNA damage and p53 activation, which in turn triggered the DNA damage response. In these cells, phospho-ATM, phospho-CHK2, and phospho-γH2AX were increased by uridine. These data suggest that uridine homeostatic disorder leads to uracil DNA damage and that pharmacological uridine may be carcinogenic. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Scaffold hopping: exploration of acetanilide-containing uracil analogues as potential NNRTIs.

PubMed

Babkov, Denis A; Valuev-Elliston, Vladimir T; Paramonova, Maria P; Ozerov, Alexander A; Ivanov, Alexander V; Chizhov, Alexander O; Khandazhinskaya, Anastasia L; Kochetkov, Sergey N; Balzarini, Jan; Daelemans, Dirk; Pannecouque, Christophe; Seley-Radtke, Katherine L; Novikov, Mikhail S

2015-03-01

In order to identify novel nonnucleoside inhibitors of HIV-1 reverse transcriptase two series of amide-containing uracil derivatives were designed as hybrids of two scaffolds of previously reported inhibitors. Subsequent biological evaluation confirmed acetamide uracil derivatives 15a-k as selective micromolar NNRTIs with a first generation-like resistance profile. Molecular modeling of the most active compounds 15c and 15i was employed to provide insight on their inhibitory properties and direct future design efforts. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters*

PubMed Central

Di Noia, Maria Antonietta; Todisco, Simona; Cirigliano, Angela; Rinaldi, Teresa; Agrimi, Gennaro; Iacobazzi, Vito; Palmieri, Ferdinando

2014-01-01

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown. PMID:25320081

From the Primitive Atmosphere to the Prebiotic Soup to the Pre-RNA World

NASA Technical Reports Server (NTRS)

Miller, Stanley L.

1996-01-01

Organic compounds would have been produced in an earth's atmosphere that was reducing. The soup would contain amino and hydroxy acids, together with smaller amounts of purines and pyrimidines. The presence' of sugars is less likely, although they can be produced by the formose reaction from formaldehyde. However, the prebiotic synthesis of RNA has not been demonstrated. One problem is that ribose is not produced selectively over other pentoses and hexoses, except under special conditions. The second problem is that ribose is unstable, with a half-life at pH7 and 100 C of 73 minutes (44 years at 0 C). Other sugars are similarly unstable. Another problem is that there is no efficient prebiotic synthesis of polyphosphates, nor the glycosidic bond of nucleosides. This suggests that there may have been an informational macromolecule that preceded RNA. The RNA world refers to the time when RNA carried both the genetic information and the catalytic activity, and was subsequently converted to the DNA/protein world when protein synthesis began. Preceeding the RNA world was the Pre-RNA world, where a backbone different from ribose phosphate was used, and the bases may have been different from adenine, uracil, guanine and cytosine. We have shown recently that cytosine and uracil can be synthesized efficiently under prebiotic conditions using a dried lagoon model instead of the usual dilute ocean hypothesis. In addition, we have shown that uracil adds formaldehyde efficiently to give 5- hydroxymethyl uracil, which in turn adds various nucleophiles to give uracil analogs of most of the amino acids that occur in proteins. For example, the ammonia, guanidine and imidazole adducts from the analogs of lysine, arginine and histidine. This suggests that the catalytic potential of RNA may have been much more extensive than previously assumed. The major problem is finding out what was the precursor to the ribose phosphate backbone. This will be the key to developing prebiotic self-replicating systems.

Establishment of uracil auxotrophic dikaryotic strains of Lentinula edodes by crossbreeding.

PubMed

Zhou, Chenli; Xi, Liping; Mao, Wenjun; Wan, Jianing; Li, Yan; Wang, Ying; Bao, Dapeng

2017-03-01

The uracil auxotrophic monokaryotic strain 423-9 of Lentinula edodes was crossed with nine monokaryons (cro2-2-9, W66-1, xd2-3-2, QingKe 20A, 241-1-1, 9015-1, L66-2, 241-1-2, and Qing 23A) derived from wild type strains of L. edodes . Nine dikaryotic hybrids were established from these crosses. These hybrids were fruited and 496 single spore isolates were obtained. Among these single spore isolates, 166 were identified as monokaryons under a microscope. We screened these monokaryons on selective medium and obtained 19 uracil auxotrophic monokaryons. By using the Monkaryon-monkaryon crossing method among the uracil auxotrophic monokaryons, 56 uracil auxotrophic dikaryotic strains were established on selective medium. These dikaryotic strains were unable to grow on minimal medium without uracil and exhibited slow growth rates on PDA plates compared to the wild type strain. The uracil auxotrophic dikaryotic strains also showed more vigorous growth on sawdust cultivation medium containing uracil than that without uracil. The fruiting tests showed that they formed normal fruiting bodies on the sawdust medium containing uracil. The results show that the uracil auxotrophic dikaryotic strain of L. edodes could be produced by mating, and will provide a valuable resource for future genetic studies and for spawn protection and identification.

De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis.

PubMed

Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

2012-04-01

Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified.

Spectroscopic study of uracil, 1-methyluracil and 1-methyl-4-thiouracil: Hydrogen bond interactions in crystals and ab-initio molecular dynamics

NASA Astrophysics Data System (ADS)

Brela, Mateusz Z.; Boczar, Marek; Malec, Leszek M.; Wójcik, Marek J.; Nakajima, Takahito

2018-05-01

Hydrogen bond networks in uracil, 1-methyluracil and 1-methyl-4-thiouracil were studied by ab initio molecular dynamics as well as analysis of the orbital interactions. The power spectra calculated by ab initio molecular dynamics for atoms involved in hydrogen bonds were analyzed. We calculated spectra by using anharmonic approximation based on the autocorrelation function of the atom positions obtained from the Born-Oppenheimer simulations. Our results show the differences between hydrogen bond networks in uracil and its methylated derivatives. The studied methylated derivatives, 1-methyluracil as well as 1-methyl-4-thiouracil, form dimeric structures in the crystal phase, while uracil does not form that kind of structures. The presence of sulfur atom instead oxygen atom reflects weakness of the hydrogen bonds that build dimers.

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.

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.

Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells.

PubMed

Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R; Hosmane, Nina N; Capoferri, Adam A; Bruner, Katherine M; Pollack, Ross A; Zhang, Hao; Drummond, Michael Bradley; Siliciano, Janet M; Siliciano, Robert; Stivers, James T

2016-09-20

We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection.

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

Spectroscopic study of uracil, 1-methyluracil and 1-methyl-4-thiouracil: Hydrogen bond interactions in crystals and ab-initio molecular dynamics.

PubMed

Brela, Mateusz Z; Boczar, Marek; Malec, Leszek M; Wójcik, Marek J; Nakajima, Takahito

2018-05-15

Hydrogen bond networks in uracil, 1-methyluracil and 1-methyl-4-thiouracil were studied by ab initio molecular dynamics as well as analysis of the orbital interactions. The power spectra calculated by ab initio molecular dynamics for atoms involved in hydrogen bonds were analyzed. We calculated spectra by using anharmonic approximation based on the autocorrelation function of the atom positions obtained from the Born-Oppenheimer simulations. Our results show the differences between hydrogen bond networks in uracil and its methylated derivatives. The studied methylated derivatives, 1-methyluracil as well as 1-methyl-4-thiouracil, form dimeric structures in the crystal phase, while uracil does not form that kind of structures. The presence of sulfur atom instead oxygen atom reflects weakness of the hydrogen bonds that build dimers. Copyright © 2018 Elsevier B.V. All rights reserved.

The human SLC25A33 and SLC25A36 genes of solute carrier family 25 encode two mitochondrial pyrimidine nucleotide transporters.

PubMed

Di Noia, Maria Antonietta; Todisco, Simona; Cirigliano, Angela; Rinaldi, Teresa; Agrimi, Gennaro; Iacobazzi, Vito; Palmieri, Ferdinando

2014-11-28

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Stabilizing effect of propionic acid derivative of anthraquinone--polyamine conjugate incorporated into α-β chimeric oligonucleotides on the alternate-stranded triple helix.

PubMed

Moriguchi, Tomohisa; Azam, A T M Zafrul; Shinozuka, Kazuo

2011-06-15

Two types of anthraquinone conjugates were synthesized as non-nucleosidic oligonucleotide components. These include an anthraquinone derivative conjugated with 2,2-bis(hydroxymethyl)propionic acid and an anthraquinone--polyamine derivative conjugated with 2,2-bis(hydroxymethyl)propionic acid. The conjugates were successfully incorporated into the "linking-region" of the α-β chimeric oligonucleotides via phosphoramidite method as non-nucleosidic backbone units. The resultant novel α-β chimeric oligonucleotides possessed two diastereomers that were generated by the introduction of the anthraquinone conjugate with a stereogenic carbon atom. The isomers were successfully separated by a reversed-phase HPLC. UV-melting experiments revealed that both stereoisomers formed a substantially stable alternate-strand triple helix, irrespective of the stereochemistry of the incorporated non-nucleosidic backbone unit. However, the enhancing effect on thermal stability depended on the length of the alkyl linker connecting anthraquinone moiety and the propionic acid moiety. The sequence discrimination ability of the chimeric oligonucleotides toward mismatch target duplex was also examined. The T(m) values of the triplexes containing the mismatch target were substantially lower than the T(m) values of those containing the full-match target. The thermodynamic parameters (ΔH°, ΔS°, and ΔG°) required for the dissociation of the triplexes into the third strand and target duplex were also measured.

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.

De Novo Pyrimidine Nucleotide Synthesis Mainly Occurs outside of Plastids, but a Previously Undiscovered Nucleobase Importer Provides Substrates for the Essential Salvage Pathway in Arabidopsis[W

PubMed Central

Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

2012-01-01

Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified. PMID:22474184

Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells

PubMed Central

Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R; Hosmane, Nina N; Capoferri, Adam A; Bruner, Katherine M; Pollack, Ross A; Zhang, Hao; Drummond, Michael Bradley; Siliciano, Janet M; Siliciano, Robert; Stivers, James T

2016-01-01

We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection. DOI: http://dx.doi.org/10.7554/eLife.18447.001 PMID:27644592

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.

Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

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

Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert

Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

DOE PAGES

Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; ...

2015-06-02

Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

Oligonucleotide synthesis catalyzed by the Zn/2+/ ion

NASA Technical Reports Server (NTRS)

Sawai, H.; Orgel, L. E.

1975-01-01

Results of experiments are reported in which Zn(2+) ion catalyzed the formation of oligonucleotides from nucleoside phosphorimidazolides in aqueous solution, even in the absence of a template. Specifically, the imidazolides (ImpU or ImpA) polymerized to form ImpApA, and pApA, pApApA, and pApApApA, or the analogous uracil compounds. In addition, the expected hydrolysis products of the hydrolysis of ImpA were formed (pA, imidazole). Judging from the ratio of pA(n) over pA (with and without zinc ion), this ion increased the efficiency of phosphodiester-bond formation by up to 10 times. Possible mechanisms for the reaction are tentatively proposed.

Interplay Between Capsule Expression and Uracil Metabolism in Streptococcus pneumoniae D39

PubMed Central

Carvalho, Sandra M.; Kloosterman, Tomas G.; Manzoor, Irfan; Caldas, José; Vinga, Susana; Martinussen, Jan; Saraiva, Lígia M.; Kuipers, Oscar P.; Neves, Ana R.

2018-01-01

Pyrimidine nucleotides play an important role in the biosynthesis of activated nucleotide sugars (NDP-sugars). NDP-sugars are the precursors of structural polysaccharides in bacteria, including capsule, which is a major virulence factor of the human pathogen S. pneumoniae. In this work, we identified a spontaneous non-reversible mutant of strain D39 that displayed a non-producing capsule phenotype. Whole-genome sequencing analysis of this mutant revealed several non-synonymous single base modifications, including in genes of the de novo synthesis of pyrimidines and in the −10 box of capsule operon promoter (Pcps). By directed mutagenesis we showed that the point mutation in Pcps was solely responsible for the drastic decrease in capsule expression. We also demonstrated that D39 subjected to uracil deprivation shows increased biomass and decreased Pcps activity and capsule amounts. Importantly, Pcps expression is further decreased by mutating the first gene of the de novo synthesis of pyrimidines, carA. In contrast, the absence of uracil from the culture medium showed no effect on the spontaneous mutant strain. Co-cultivation of the wild-type and the mutant strain indicated a competitive advantage of the spontaneous mutant (non-producing capsule) in medium devoid of uracil. We propose a model in that uracil may act as a signal for the production of different capsule amounts in S. pneumoniae. PMID:29599757

Interplay Between Capsule Expression and Uracil Metabolism in Streptococcus pneumoniae D39.

PubMed

Carvalho, Sandra M; Kloosterman, Tomas G; Manzoor, Irfan; Caldas, José; Vinga, Susana; Martinussen, Jan; Saraiva, Lígia M; Kuipers, Oscar P; Neves, Ana R

2018-01-01

Pyrimidine nucleotides play an important role in the biosynthesis of activated nucleotide sugars (NDP-sugars). NDP-sugars are the precursors of structural polysaccharides in bacteria, including capsule, which is a major virulence factor of the human pathogen S. pneumoniae . In this work, we identified a spontaneous non-reversible mutant of strain D39 that displayed a non-producing capsule phenotype. Whole-genome sequencing analysis of this mutant revealed several non-synonymous single base modifications, including in genes of the de novo synthesis of pyrimidines and in the -10 box of capsule operon promoter (P cps ). By directed mutagenesis we showed that the point mutation in P cps was solely responsible for the drastic decrease in capsule expression. We also demonstrated that D39 subjected to uracil deprivation shows increased biomass and decreased P cps activity and capsule amounts. Importantly, P cps expression is further decreased by mutating the first gene of the de novo synthesis of pyrimidines, carA . In contrast, the absence of uracil from the culture medium showed no effect on the spontaneous mutant strain. Co-cultivation of the wild-type and the mutant strain indicated a competitive advantage of the spontaneous mutant (non-producing capsule) in medium devoid of uracil. We propose a model in that uracil may act as a signal for the production of different capsule amounts in S. pneumoniae .

Rigid amphipathic fusion inhibitors demonstrate antiviral activity against African swine fever virus.

PubMed

Hakobyan, Astghik; Galindo, Inmaculada; Nañez, Almudena; Arabyan, Erik; Karalyan, Zaven; Chistov, Alexey A; Streshnev, Philipp P; Korshun, Vladimir A; Alonso, Covadonga; Zakaryan, Hovakim

2018-01-01

Rigid amphipathic fusion inhibitors (RAFIs) are a family of nucleoside derivatives that inhibit the infectivity of several enveloped viruses by interacting with virion envelope lipids and inhibiting fusion between viral and cellular membranes. Here we tested the antiviral activity of two RAFIs, 5-(Perylen-3-ylethynyl)-arabino-uridine (aUY11) and 5-(Perylen-3-ylethynyl)uracil-1-acetic acid (cm1UY11) against African swine fever virus (ASFV), for which no effective vaccine is available. Both compounds displayed a potent, dose-dependent inhibitory effect on ASFV infection in Vero cells. The major antiviral effect was observed when aUY11 and cm1UY11 were added at early stages of infection and maintained during the complete viral cycle. Furthermore, virucidal assay revealed a significant extracellular anti-ASFV activity for both compounds. We also found decrease in the synthesis of early and late viral proteins in Vero cells treated with cm1UY11. Finally, the inhibitory effect of aUY11 and cm1UY11 on ASFV infection in porcine alveolar macrophages was confirmed. Overall, our study has identified novel anti-ASFV compounds with potential for future therapeutic developments.

Enhanced Photolysis of Nucleic Acid Monomers by Pyrophosphate in the Simulated Primitive Soup

NASA Astrophysics Data System (ADS)

Kongjiang, Wang; Zhifang, Chai; Xianming, Pan

1999-05-01

In our simulation of the photochemistry of the primitive soup, it was found that yield of chromophore loss of some nucleic acid bases, nucleosides and nucleotides [NA] undergoing ultraviolet radiation by medium pressure mercury lamp [MPML] was enhanced by pyrophosphate and triphosphate whether O2 is present or not. The yield of chromophore loss of guanosine, uracil, 5' CMP, and the derivatives of adenine and thymine was observed to rise with the increase of molar concentration of pyrophosphate in N2-saturated systems. In air-saturated samples, increase in yield of chromophore loss was observed when the concentration of pyrophosphate reaches 5×10-4 M, relative yield of chromophore loss (CLrel) coming to maximum in the range from 0.01 to 0.1 M, followed by a slight decline with the further increase of pyrophosphate concentration. The enhancement of photolysis of NA by pyrophosphate was due to the interaction between NA and pyrophosphate radicals photoionized by UV quanta of wavelength less than about 210 nm in the emission spectrum of a MPML. The relevance of this phenomenon to the origin of life has been discussed as well.

Iron Sequestration in Microbiota Biofilms As A Novel Strategy for Treating Inflammatory Bowel Disease.

PubMed

Motta, Jean-Paul; Allain, Thibault; Green-Harrison, Luke E; Groves, Ryan A; Feener, Troy; Ramay, Hena; Beck, Paul L; Lewis, Ian A; Wallace, John L; Buret, Andre G

2018-06-08

Significant alterations of intestinal microbiota and anemia are hallmarks of inflammatory bowel disease (IBD). It is widely accepted that iron is a key nutrient for pathogenic bacteria, but little is known about its impact on microbiota associated with IBD. We used a model device to grow human mucosa-associated microbiota in its physiological anaerobic biofilm phenotype. Compared to microbiota from healthy donors, microbiota from IBD patients generate biofilms ex vivo that were larger in size and cell numbers, contained higher intracellular iron concentrations, and exhibited heightened virulence in a model of human intestinal epithelia in vitro and in the nematode Caenorhabditis elegans. We also describe an unexpected iron-scavenging property for an experimental hydrogen sulfide-releasing derivative of mesalamine. The findings demonstrate that this new drug reduces the virulence of IBD microbiota biofilms through a direct reduction of microbial iron intake and without affecting bacteria survival or species composition within the microbiota. Metabolomic analyses indicate that this drug reduces the intake of purine nucleosides (guanosine), increases the secretion of metabolite markers of purine catabolism (urate and hypoxanthine), and reduces the secretion of uracil (a pyrimidine nucleobase) in complex multispecies human biofilms. These findings demonstrate a new pathogenic mechanism for dysbiotic microbiota in IBD and characterize a novel mode of action for a class of mesalamine derivatives. Together, these observations pave the way towards a new therapeutic strategy for treatment of patients with IBD.

[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.

Biological evaluation of some uracil derivatives as potent glutathione reductase inhibitors

NASA Astrophysics Data System (ADS)

Güney, Murat; Ekinci, Deniz; Ćavdar, Huseyin; Şentürk, Murat; Zilbeyaz, Kani

2016-04-01

Discovery of glutathione reductase (GR) inhibitors has become very popular recently due to antimalarial and anticancer activities. In this study, GR inhibitory capacities of some uracil derivatives (UDCs) (1-4) were reported. Some commercially available molecules (5-6) were also tested for comparison reasons. The novel UDCs were obtained in high yields using simple chemical procedures and exhibited much potent inhibitory activities against GR at low nanomolar concentrations with IC50 values ranging from 2.68 to 166.6 nM as compared with well-known agents.

The Photochemistry of Pyrimidine in Pure H2O Ice Subjected to Different Radiation Environments and the Formation of Uracil

NASA Technical Reports Server (NTRS)

Nuevo, M.; Chen, Y.-J.; Materese. C. K..; Hu, W.-J.; Qiu, J.-M.; Wu, S.-R.; Fung, H.-S.; Sandford, S. A.; Chu, C.-C.; Yih, T.-S.;

Attempted nonenzymatic template-directed oligomerizations on a polyadenylic acid template: implications for the nature of the first genetic material

NASA Technical Reports Server (NTRS)

Stribling, R.; Miller, S. L.

1991-01-01

Previous attempts to produce nonenzymatic template-directed oligomerizations of activated pyrimidines on polypurine templates have been unsuccessful. The only efficient reactions are those where the template is composed primarily of pyrimidines, especially cytosine. Because molecular evolution requires that a synthesized daughter polynucleotide be capable of acting as a template for the synthesis of the original polynucleotide, the one-way replication achieved thus far is inadequate to initiate an evolving system. Several uracil analogs were used in this investigation in order to search for possible replacements for uracil. The monomers used in this investigation were the imidazolides of UMP, xanthosine 5'-monophosphate, the bis-monophosphates of the acyclic nucleosides of uracil, and 2,4-quinazolinedione. The concentrations of various salts, buffers, pH, and temperature were among the different variables investigated in attempts to find conditions that would permit template-directed oligomerizations. Although the different monomers in this study demonstrated varying abilities to form very short oligomers, we were unable to detect any enhancement of this oligomerization that could be attributed to the poly(A) template. Although special conditions might be found that would allow purine-rich templates to work, these reactions cannot be considered robust. The results of our experiments suggest that pyrimidines were not part of the original replicating system on the primitive Earth. It has already been shown that ribose is an unlikely component of the first replicating systems, and we now suggest that phosphate was absent as well. This is due to the low solubility of phosphate in the present ocean (3 x 10(-6) M), as well as the difficulty of prebiotic activation of phosphates.

Identification of proton-pump inhibitor drugs that inhibit Trichomonas vaginalis uridine nucleoside ribohydrolase.

PubMed

Shea, Tara A; Burburan, Paola J; Matubia, Vivian N; Ramcharan, Sandy S; Rosario, Irving; Parkin, David W; Stockman, Brian J

2014-02-15

Trichomonas vaginalis continues to be a major health problem with drug-resistant strains increasing in prevalence. Novel antitrichomonal agents that are mechanistically distinct from current therapies are needed. The NIH Clinical Compound Collection was screened to find inhibitors of the uridine ribohydrolase enzyme required by the parasite to scavenge uracil for its growth. The proton-pump inhibitors omeprazole, pantoprazole, and rabeprazole were identified as inhibitors of this enzyme, with IC50 values ranging from 0.3 to 14.5 μM. This suggests a molecular mechanism for the in vitro antitrichomonal activity of these proton-pump inhibitors, and may provide important insights toward structure-based drug design. Copyright © 2014 Elsevier Ltd. All rights reserved.

Glycal Formation in Crystals of Uridine Phosphorylase

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

Paul, Debamita; O’Leary, Sen E.; Rajashankar, Kanagalaghatta

2010-06-22

Uridine phosphorylase is a key enzyme in the pyrimidine salvage pathway. This enzyme catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate (or 2{prime}-deoxyuridine to 2{prime}-deoxyribose 1-phosphate). Here we report the structure of hexameric Escherichia coli uridine phosphorylase treated with 5-fluorouridine and sulfate and dimeric bovine uridine phosphorylase treated with 5-fluoro-2{prime}-deoxyuridine or uridine, plus sulfate. In each case the electron density shows three separate species corresponding to the pyrimidine base, sulfate, and a ribosyl species, which can be modeled as a glycal. In the structures of the glycal complexes, the fluorouracil O2 atom is appropriately positioned to actmore » as the base required for glycal formation via deprotonation at C2{prime}. Crystals of bovine uridine phosphorylase treated with 2{prime}-deoxyuridine and sulfate show intact nucleoside. NMR time course studies demonstrate that uridine phosphorylase can catalyze the hydrolysis of the fluorinated nucleosides in the absence of phosphate or sulfate, without the release of intermediates or enzyme inactivation. These results add a previously unencountered mechanistic motif to the body of information on glycal formation by enzymes catalyzing the cleavage of glycosyl bonds.« less

Simultaneous determination of nucleosides and their bases in Cordyceps sinensis and its substitutes by matrix solid-phase dispersion extraction and HPLC.

PubMed

Wang, Zhibing; Li, Na; Wang, Min; Wang, Yue; Du, Lin; Ji, Xiaofeng; Yu, Aimin; Zhang, Hanqi; Qiu, Fangping

2013-07-01

Nine nucleosides and nucleobases, including uracil, adenine, thymine, uridine, adenosine, thymidine, cytidine, guanosine, and cordycepin in natural Cordyceps sinensis, cultured Cordyceps mycelia, and Cordyceps fruiting bodies were extracted by matrix solid-phase dispersion (MSPD) and determined by HPLC. The experimental conditions for the MSPD extraction were optimized. Florisil was used as dispersant, petroleum ether as washing solvent, and methanol as elution solvent. The Florisil-to-sample ratio was selected to be 4:1 and no additional clean-up sorbent was needed. The calibration curves had good linear relationships (r > 0.9997). The LOD and LOQ were in the range of 12～79 and 41～265 ng/mL, respectively. The intra- and interday precision were lower than 8.3%. The recoveries were between 61.5 and 93.2%. The present method consumed less sample compared with ultrasonic extraction and heating reflux extraction (HRE). The extraction yields obtained by using the present method are much higher than those obtained by UE and comparable to those obtained by HRE. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Peptide nucleic acids rather than RNA may have been the first genetic molecule

NASA Technical Reports Server (NTRS)

Nelson, K. E.; Levy, M.; Miller, S. L.

2000-01-01

Numerous problems exist with the current thinking of RNA as the first genetic material. No plausible prebiotic processes have yet been demonstrated to produce the nucleosides or nucleotides or for efficient two-way nonenzymatic replication. Peptide nucleic acid (PNA) is a promising precursor to RNA, consisting of N-(2-aminoethyl)glycine (AEG) and the adenine, uracil, guanine, and cytosine-N-acetic acids. However, PNA has not yet been demonstrated to be prebiotic. We show here that AEG is produced directly in electric discharge reactions from CH(4), N(2), NH(3), and H(2)O. Electric discharges also produce ethylenediamine, as do NH(4)CN polymerizations. AEG is produced from the robust Strecker synthesis with ethylenediamine. The NH(4)CN polymerization in the presence of glycine leads to the adenine and guanine-N(9)-acetic acids, and the cytosine and uracil-N(1)-acetic acids are produced in high yield from the reaction of cyanoacetaldehyde with hydantoic acid, rather than urea. Preliminary experiments suggest that AEG may polymerize rapidly at 100 degrees C to give the polypeptide backbone of PNA. The ease of synthesis of the components of PNA and possibility of polymerization of AEG reinforce the possibility that PNA may have been the first genetic material.

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.

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 .

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.

Dancing with chemical formulae of antivirals: a personal account.

PubMed

De Clercq, Erik

2013-09-15

A chemical structure is a joy forever, and this is how I perceived the chemical structures of a number of antiviral compounds with which I have been personally acquainted over the past 3 decades: (1) amino acid esters of acyclovir (i.e. valaciclovir); (2) 5-substituted 2'-deoxyuridines (i.e. brivudin); (3) 2',3'-dideoxynucleoside analogues (i.e. stavudine); (4) acyclic nucleoside phosphonates (ANPs) (i.e. cidofovir, adefovir); (5) tenofovir disoproxil fumarate (TDF) and drug combinations therewith; (6) tenofovir alafenamide (TAF, GS-7340), a new phosphonoamidate prodrug of tenofovir; (7) pro-prodrugs of PMEG (i.e. GS-9191 and GS-9219); (8) new ANPs: O-DAPy and 5-aza-C phosphonates; (9) non-nucleoside reverse transcriptase inhibitors (NNRTIs): HEPT and TIBO derivatives; and (10) bicyclam derivatives (i.e. AMD3100). Copyright © 2013 Elsevier Inc. All rights reserved.

Exploration of acetanilide derivatives of 1-(ω-phenoxyalkyl)uracils as novel inhibitors of Hepatitis C Virus replication

PubMed Central

Magri, Andrea; Ozerov, Alexander A.; Tunitskaya, Vera L.; Valuev-Elliston, Vladimir T.; Wahid, Ahmed; Pirisi, Mario; Simmonds, Peter; Ivanov, Alexander V.; Novikov, Mikhail S.; Patel, Arvind H.

2016-01-01

Hepatitis C Virus (HCV) is a major public health problem worldwide. While highly efficacious directly-acting antiviral agents have been developed in recent years, their high costs and relative inaccessibility make their use limited. Here, we describe new 1-(ω-phenoxyalkyl)uracils bearing acetanilide fragment in 3 position of pyrimidine ring as potential antiviral drugs against HCV. Using a combination of various biochemical assays and in vitro virus infection and replication models, we show that our compounds are able to significantly reduce viral genomic replication, independently of virus genotype, with their IC50 values in the nanomolar range. We also demonstrate that our compounds can block de novo RNA synthesis and that effect is dependent on a chemical structure of the compounds. A detailed structure-activity relationship revealed that the most active compounds were the N3-substituted uracil derivatives containing 6-(4-bromophenoxy)hexyl or 8-(4-bromophenoxy)octyl fragment at N1 position. PMID:27406141

Exploration of acetanilide derivatives of 1-(ω-phenoxyalkyl)uracils as novel inhibitors of Hepatitis C Virus replication.

PubMed

Magri, Andrea; Ozerov, Alexander A; Tunitskaya, Vera L; Valuev-Elliston, Vladimir T; Wahid, Ahmed; Pirisi, Mario; Simmonds, Peter; Ivanov, Alexander V; Novikov, Mikhail S; Patel, Arvind H

2016-07-12

Hepatitis C Virus (HCV) is a major public health problem worldwide. While highly efficacious directly-acting antiviral agents have been developed in recent years, their high costs and relative inaccessibility make their use limited. Here, we describe new 1-(ω-phenoxyalkyl)uracils bearing acetanilide fragment in 3 position of pyrimidine ring as potential antiviral drugs against HCV. Using a combination of various biochemical assays and in vitro virus infection and replication models, we show that our compounds are able to significantly reduce viral genomic replication, independently of virus genotype, with their IC50 values in the nanomolar range. We also demonstrate that our compounds can block de novo RNA synthesis and that effect is dependent on a chemical structure of the compounds. A detailed structure-activity relationship revealed that the most active compounds were the N(3)-substituted uracil derivatives containing 6-(4-bromophenoxy)hexyl or 8-(4-bromophenoxy)octyl fragment at N(1) position.

Pyrimidine homoribonucleosides: synthesis, solution conformation, and some biological properties.

PubMed

Lassota, P; Kuśmierek, J T; Stolarski, R; Shugar, D

1987-05-01

Conversion of uridine and cytidine to their 5'-O-tosyl derivatives, followed by cyanation with tetraethylammonium cyanide, reduction and deamination, led to isolation of the hitherto unknown homouridine (1-(5'-deoxy-beta-D-allofuranosyl)uracil) and homocytidine (1-(5'-deoxy-beta-D-allofuranosyl)cytosine), analogues of uridine and cytidine in which the exocyclic 5'-CH2OH chain is extended by one carbon to CH2CH2OH. Homocytidine was also phosphorylated to its 6'-phosphate and 6'-pyrophosphate analogues. In addition, it was converted, via its 2,2'-anhydro derivative, to arahomocytidine, an analogue of the chemotherapeutically active araC. The structures of all the foregoing were established by various criteria, including 1H and 13C NMR spectroscopy, both of which were also applied to analyses of the solution conformations of the various compounds, particularly as regards the conformations of the exocyclic chains. The behaviour of the homo analogues was examined in several enzymatic systems. Homocytidine was a feeble substrate, without inhibitory properties, of E. coli cytidine deaminase. Homocytidine was an excellent substrate for wheat shoot nucleoside phosphotransferase; while homouridine was a good substrate for E. coli uridine phosphorylase. Although homoCMP was neither a substrate, nor an inhibitor, of snake venom 5'-nucleotidase, homoCDP was a potent inhibitor of this enzyme (Ki approximately 6 microM). HomoCDP was not a substrate for M. luteus polynucleotide phosphorylase. None of the compounds exhibited significant activity vs herpes simplex virus type 1, or cytotoxic activity in several mammalian cell lines.

Supramolecular polymeric chemosensor for biomedical applications: design and synthesis of a luminescent zinc metallopolymer as a chemosensor for adenine detection.

PubMed

Chow, Cheuk-Fai

2012-11-01

Adenine is an important bio-molecule that plays many crucial roles in food safety and biomedical diagnostics. Differentiating adenine from a mixture of adenosine and other nucleic bases (guanine, thymine, cytosine, and uracil) is particularly important for both biological and clinical applications. A neutral Zn(II) metallosupramolecular polymer based on acyl hydrazone derived coordination centres (P1) were generated through self-assembly polymerization. It is a linear coordination polymer that behaves like self-standing film. The synthesis, (1)H-NMR characterization, and spectroscopic properties of this supramolecular material are reported. P1 was found to be a chemosensor specific to adenine, with a luminescent enhancement. The binding properties of P1 with common nucleic bases and nucleosides reveal that this supramolecular polymer is very selective to adenine molecules (~20 to 420 times more selectivity than other nucleic bases). The formation constant (K) of P1 to adenine was found to be log K = 4.10 ± 0.02. This polymeric chemosensor produces a specific response to adenine down to 90 ppb. Spectrofluorimetric and (1)H-NMR titration studies showed that the P1 polymer allows each Zn(II) coordination centre to bind to two adenine molecules through hydrogen bonding with their imine and hydrazone protons.

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.

Modified RS-LAMP assay and use of lateral flow devices for rapid detection of Leifsonia xyli subsp. xyli.

PubMed

Naidoo, N; Ghai, M; Moodley, K; Mkize, L; Martin, L; McFarlane, S; Rutherford, S

2017-12-01

Ratoon stunt (RS) caused by bacterium Leifsonia xyli subsp. xyli (Lxx) results in substantial yield losses in sugarcane (Saccharum sp. L. hybrid). Since RS does not produce reliable symptoms in the field, laboratory-based techniques are necessary for detection. Loop-mediated isothermal amplification (LAMP) assay overcomes the limitations of laboratory-based techniques which are costly, time consuming and cannot be used for near-field detection. A sensitive LAMP assay was developed to detect Lxx at 65°C in 30 min. However, carry-over contamination affected the reliability of the assay. In the present study, contaminants were successfully eliminated by incorporation of uracil nucleoside glycosylase (1 U μl -1 ) into the LAMP assay and incubation for 10 min at 37°C. To avoid the use of colorimetric reagents, lateral flow devices were successfully used for the detection of LAMP products and were equally sensitive to detection by agarose gel electrophoresis. The use of exudate from leaf sheath discs as an alternate template for the LAMP assay was found to be less sensitive than xylem sap. The preprepared master mix could be stored for up to 4 months at -20°C without any reduction in performance. These changes make the assay suitable for near-field detection in laboratories with basic facilities. This study presents a modified loop-mediated isothermal amplification (LAMP) assay for the detection of Leifsonia xyli subsp. xyli. Modifications include incorporation of uracil nucleoside glycosylase to eliminate carry-over contamination and substitution of colorimetric detection for the use of lateral flow devices. LAMP master mix was preprepared and was stably stored up to 4 months at -20°C. Sugarcane leaf sheaths worked well as a substitute to xylem sap as template, although the sensitivity was lower. These modifications allow the assay to be conducted without contamination concerns and reduction in set up time, making it ideal for near-field diagnosis. © 2017 The Society for Applied Microbiology.

Functional and genetic evidence that nucleoside transport is highly conserved in Leishmania species: Implications for pyrimidine-based chemotherapy.

PubMed

Alzahrani, Khalid J H; Ali, Juma A M; Eze, Anthonius A; Looi, Wan Limm; Tagoe, Daniel N A; Creek, Darren J; Barrett, Michael P; de Koning, Harry P

2017-08-01

Leishmania pyrimidine salvage is replete with opportunities for therapeutic intervention with enzyme inhibitors or antimetabolites. Their uptake into cells depends upon specific transporters; therefore it is essential to establish whether various Leishmania species possess similar pyrimidine transporters capable of drug uptake. Here, we report a comprehensive characterization of pyrimidine transport in L. major and L. mexicana. In both species, two transporters for uridine/adenosine were detected, one of which also transported uracil and the antimetabolites 5-fluoruracil (5-FU) and 5F,2'deoxyuridine (5F,2'dUrd), and was designated uridine-uracil transporter 1 (UUT1); the other transporter mediated uptake of adenosine, uridine, 5F,2'dUrd and thymidine and was designated Nucleoside Transporter 1 (NT1). To verify the reported L. donovani model of two NT1-like genes encoding uridine/adenosine transporters, and an NT2 gene encoding an inosine transporter, we cloned the corresponding L. major and L. mexicana genes, expressing each in T. brucei. Consistent with the L. donovani reports, the NT1-like genes of either species mediated the adenosine-sensitive uptake of [ 3 H]-uridine but not of [ 3 H]-inosine. Conversely, the NT2-like genes mediated uptake of [ 3 H]-inosine but not [ 3 H]-uridine. Among pyrimidine antimetabolites tested, 5-FU and 5F,2'dUrd were the most effective antileishmanials; resistance to both analogs was induced in L. major and L. mexicana. In each case it was found that the resistant cells had lost the transport capacity for the inducing drug. Metabolomics analysis found that the mechanism of action of 5-FU and 5F-2'dUrd was similar in both Leishmania species, with major changes in deoxynucleotide metabolism. We conclude that the pyrimidine salvage system is highly conserved in Leishmania species - essential information for the development of pyrimidine-based chemotherapy. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

attempted prebiotic synthesis of pseudouridine

NASA Astrophysics Data System (ADS)

DWORKIN, JASON P.

1997-08-01

Pseudouridine is a modified base found in all tRNA and rRNA. Hence, it is reasonable to think that pseudouridine was important in the early evolution, if not the origin, of life. Since uracil reacts rapidly with formaldehyde and other aldehydes at the C-5 position, it is plausible that pseudouridine could be synthesized in a similar way by the reaction of the C-5 of uracil with the C-1 of ribose. The determining factor is whether the ribose could react with the uracil faster than ribose decomposes. However, both rates are determined by the amount of free aldehyde in the ribose. Various plausible prebiotic reactions were investigated and none showed pseudouridine above the detection limit (<0.01%). Only unreacted uracil and ribose decomposition products could be observed. Thus the rate of addition of ribose to uracil is much slower than the decomposition of ribose under any reasonable prebiotic conditions. Unless efficient non-biological catalysts for any of these reactions exist, pseudouridine would not have been synthesized to any significant extent without the use of biologically produced enzymes.

Attempted prebiotic synthesis of pseudouridine

NASA Technical Reports Server (NTRS)

Dworkin, J. P.; Miller, S. L. (Principal Investigator)

1997-01-01

Pseudouridine is a modified base found in all tRNA and rRNA. Hence, it is reasonable to think that pseudouridine was important in the early evolution, if not the origin, of life. Since uracil reacts rapidly with formaldehyde and other aldehydes at the C-5 position, it is plausible that pseudouridine could be synthesized in a similar way by the reaction of the C-5 of uracil with the C-1 of ribose. The determining factor is whether the ribose could react with the uracil faster than ribose decomposes. However, both rates are determined by the amount of free aldehyde in the ribose. Various plausible prebiotic reactions were investigated and none showed pseudouridine above the detection limit (<0.01%). Only unreacted uracil and ribose decomposition products could be observed. Thus the rate of addition of ribose to uracil is much slower than the decomposition of ribose under any reasonable prebiotic conditions. Unless efficient non-biological catalysts for any of these reactions exist, pseudouridine would not have been synthesized to any significant extent without the use of biologically produced enzymes.

Poxvirus uracil-DNA glycosylase-An unusual member of the family I uracil-DNA glycosylases: Poxvirus Uracil-DNA Glycosylase

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

Schormann, Norbert; Zhukovskaya, Natalia; Bedwell, Gregory

We report that uracil-DNA glycosylases are ubiquitous enzymes, which play a key role repairing damages in DNA and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. Within the superfamily of uracil-DNA glycosylases family I enzymes or UNGs are specific for recognizing and removing uracil from DNA. These enzymes feature conserved structural folds, active site residues and use common motifs for DNA binding, uracil recognition and catalysis. Within this family the enzymes of poxviruses are unique and most remarkable in terms of amino acid sequences, characteristic motifs and more importantly for their novel non-enzymaticmore » function in DNA replication. UNG of vaccinia virus, also known as D4, is the most extensively characterized UNG of the poxvirus family. D4 forms an unusual heterodimeric processivity factor by attaching to a poxvirus-specific protein A20, which also binds to the DNA polymerase E9 and recruits other proteins necessary for replication. D4 is thus integrated in the DNA polymerase complex, and its DNA-binding and DNA scanning abilities couple DNA processivity and DNA base excision repair at the replication fork. In conclusion, the adaptations necessary for taking on the new function are reflected in the amino acid sequence and the three-dimensional structure of D4. We provide an overview of the current state of the knowledge on the structure-function relationship of D4.« less

Conditionally Pathogenic Gut Microbes Promote Larval Growth by Increasing Redox-Dependent Fat Storage in High-Sugar Diet-Fed Drosophila.

PubMed

Whon, Tae Woong; Shin, Na-Ri; Jung, Mi-Ja; Hyun, Dong-Wook; Kim, Hyun Sik; Kim, Pil Soo; Bae, Jin-Woo

2017-12-01

Changes in the composition of the gut microbiota contribute to the development of obesity and subsequent complications that are associated with metabolic syndrome. However, the role of increased numbers of certain bacterial species during the progress of obesity and factor(s) controlling the community structure of gut microbiota remain unclear. Here, we demonstrate the inter-relationship between Drosophila melanogaster and their resident gut microbiota under chronic high-sugar diet (HSD) conditions. Chronic feeding of an HSD to Drosophila resulted in a predominance of resident uracil-secreting bacteria in the gut. Axenic insects mono-associated with uracil-secreting bacteria or supplemented with uracil under HSD conditions promoted larval development. Redox signaling induced by bacterial uracil promoted larval growth by regulating sugar and lipid metabolism via activation of p38a mitogen-activated protein kinase. The present study identified a new redox-dependent mechanism by which uracil-secreting bacteria (previously regarded as opportunistic pathobionts) protect the host from metabolic perturbation under chronic HSD conditions. These results illustrate how Drosophila and gut microbes form a symbiotic relationship under stress conditions, and changes in the gut microbiota play an important role in alleviating deleterious diet-derived effects such as hyperglycemia. Antioxid. Redox Signal. 27, 1361-1380.

Prognostic factors of pathologic stage IB non-small cell lung cancer.

PubMed

Yano, Motoki; Sasaki, Hidefumi; Moriyama, Satoru; Kawano, Osamu; Hikosaka, Yu; Fujii, Yoshitaka

2011-01-01

In pathologic IB (pIB) non-small cell lung cancer, especially in adenocarcinoma, adjuvant chemotherapy with uracil-tegafur is widely recognized as being effective. The aim of this study was to determine the prognostic factors of pIB disease. Sixty patients who were diagnosed with pIB disease between 2004 and 2007 were retrospectively analyzed. Of 60 patients, 22 (36.7%) opted for surgery plus adjuvant chemotherapy with uracil-tegafur, whereas 38 (63.3%) opted for surgery only. The oral administration dose of uracil-tegafur was 400 mg/body. Compliance of adjuvant chemotherapy with uracil-tegafur was 65.5% in 12 months, 57.3% in 24 months. Adjuvant chemotherapy was interrupted in 11 patients because of the recurrence of disease in 3 patients and adverse reaction in 8 patients. Anorexia was the most common adverse reaction. The larger tumor diameter (5 cm<) and p2 pleural invasion were the worse prognostic factors in disease free survival in a univariate analysis and a multivariate analysis (hazard ratio = 0.26 and 0.25; p = 0.028 and 0.032, respectively). The prognosis of the patients with pleural invasion and a tumor diameter >5 cm was poor, and these, partly support the forthcoming classification.

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.

Ligand Binding Phenomena that Pertain to the Metabolic Function of Renalase

PubMed Central

Beaupre, Brett A.; Roman, Joseph V.; Hoag, Matthew R.; Meneely, Kathleen M.; Silvaggi, Nicholas R.; Lamb, Audrey L.; Moran, Graham R.

2017-01-01

Renalase catalyzes the oxidation of isomers of β-NAD(P)H that carry the hydride in the 2 or 6 positions of the nicotinamide base to form β-NAD(P)+. This activity is thought to alleviate inhibition of multiple β-NAD(P)-dependent enzymes of primary and secondary metabolism by these isomers. Here we present evidence for a variety of ligand binding phenomena relevant to the function of renalase. We offer evidence of the potential for primary metabolism inhibition with structures of malate dehydrogenase and lactate dehydrogenase bound to the 6-dihydroNAD isomer. The previously observed preference of renalase from Pseudomonas for NAD-derived substrates over those derived from NADP is accounted for by the structure of the enzyme in complex with NADPH. We also show that nicotinamide nucleosides and mononucloetides reduced in the 2- and 6-positions are renalase substrates, but bind weakly. A seven-fold enhancement of acquisition (kred/Kd) for 6-dihydronicotinamide riboside was observed for human renalase in the presence of ADP. However, generally the addition of complement ligands, ADP for mononucloetide or AMP for nucleoside substrates, did not enhance the reductive half-reaction. Non-substrate nicotinamide nucleosides or nucleotides bind weakly suggesting that only β-NADH and β-NADPH compete with dinucleotide substrates for access to the active site. PMID:27769837

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.

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.

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.

Transition state analysis of Trypanosoma cruzi uridine phosphorylase-catalyzed arsenolysis of uridine

PubMed Central

Silva, Rafael G.; Vetticatt, Mathew J.; Merino, Emilio F.; Cassera, Maria B.; Schramm, Vern L.

2011-01-01

Uridine phosphorylase catalyzes the reversible phosphorolysis of uridine and 2′-deoxyuridine to generate uracil and (2-deoxy)ribose 1-phosphate, an important step in the pyrimidine salvage pathway. The coding sequence annotated as a putative nucleoside phosphorylase in the Trypanosoma cruzi genome was overexpressed in Escherichia coli, purified to homogeneity, and shown to be a homodimeric uridine phosphorylase, with similar specificity for uridine and 2′-deoxyuridine, and undetectable activity towards thymidine and purine nucleosides. Competitive kinetic isotope effects (KIEs) were measured and corrected for a forward commitment factor using arsenate as the nucleophile. The intrinsic KIEs are: 1′-14C = 1.103, 1,3-15N2 = 1.034, 3-15N = 1.004, 1-15N = 1.030, 1′-3H = 1.132, 2′-2H = 1.086 and 5′-3H2 = 1.041 for this reaction. Density functional theory was employed to quantitatively interpret the KIEs in terms of transition state structure and geometry. Matching of experimental KIEs to proposed transition state structures suggests an almost synchronous, SN2-like transition state model, in which the ribosyl moiety possesses significant bond order to both nucleophile and leaving group. Natural bond orbital analysis allowed a comparison of the charge distribution pattern between the ground state and the transition state model. PMID:21599004

Pharmacokinetics of antiretroviral drugs in anatomical sanctuary sites: the male and female genital tract.

PubMed

Else, Laura J; Taylor, Stephen; Back, David J; Khoo, Saye H

2011-01-01

HIV resides within anatomical 'sanctuary sites', where local drug exposure and viral dynamics may differ significantly from the systemic compartment. Suboptimal antiretroviral concentrations in the genital tract may result in compartmentalized viral replication, selection of resistant mutations and possible re-entry of wild-type/resistant virus into the systemic circulation. Therefore, achieving adequate antiretroviral exposure in the genital tract has implications for the prevention of sexual and vertical transmission of HIV. Penetration of antiretrovirals in the genital tract is expressed by accumulation ratios derived from the measurement of drug concentrations in time-matched seminal plasma/cervicovaginal fluid and plasma samples. Penetration varies by gender and may be drug (as opposed to class) specific with high interindividual variability. Concentrations in seminal plasma are highest for nucleoside analogues and lowest for protease inhibitors and efavirenz. Seminal accumulation of newer agents, raltegravir and maraviroc, is moderate (rank order of accumulation is nucleoside/nucleotide reverse transcriptase inhibitors [lamivudine/zidovudine/tenofovir/didanosine > stavudine/abacavir] > raltegravir > indinavir/maraviroc/nevirapine > efavirenz/protease inhibitors [amprenavir/atazanavir/darunavir > lopinavir/ritonavir > saquinavir] > enfuvirtide). In the female genital tract, the nucleoside analogues exhibit high accumulation ratios, whereas protease inhibitors have limited penetration; however, substantial variability exists between individuals and study centres. Second generation non-nucleoside reverse transcriptase inhibitor etravirine, and maraviroc and raltegravir, demonstrate effective accumulation in cervicovaginal secretions (rank order of accumulation is nucleoside/nucleotide reverse transcriptase inhibitor [zidovudine/lamivudine/didanosine > emtricitabine/tenofovir] > indinavir > maraviroc/raltegravir/darunavir/etravirine > nevirapine/abacavir > protease inhibitors [amprenavir/atazanavir/ritonavir] > lopinavir/stavudine/efavirenz > saquinavir).

Mung bean nuclease: mode of action and specificity vs synthetic esters of 3′-nucleotides

PubMed Central

Kole, R.; Sierakowska, Halina; Szemplińska, Halina; Shugar, D.

1974-01-01

Mung bean nuclease hydrolyzes synthetic esters of 3′-nucleotides to nucleosides and phosphate esters; esters of 2′-nucleotides, and 2′→ 5′ internucleotide linkages, are resistant. Esters of ribonucleotides are cleaved at 100-fold the rate for deoxyribonucleotides, the increased rate being due to presence of the 2′-hydroxyl and not to differences in conformation. Introduction of a 5′-substituent leads to a 3-fold increase in rate. The rates of hydrolysis vary up to 10-fold with the nature of the base, in the order adenine > hypoxanthine > uracil; and up to 6-fold with the nature of the ester radical. This form of cleavage of esters of 3′-nucleotides is also characteristic for nuclease-3′-nucleotidase activities from potato tubers and wheat, suggesting that one type of enzyme is responsible for all these activities. PMID:10793750

Gas-phase study on uridine: Conformation and X-ray photofragmentation

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

Itälä, Eero, E-mail: ersita@utu.fi; Kooser, Kuno; Levola, Helena

2015-05-21

Fragmentation of RNA nucleoside uridine, induced by carbon 1s core ionization, has been studied. The measurements by combined electron and ion spectroscopy have been performed in gas phase utilizing synchrotron radiation. As uridine is a combination of d-ribose and uracil, which have been studied earlier with the same method, this study also considers the effect of chemical environment and the relevant functional groups. Furthermore, since in core ionization the initial core hole is always highly localized, charge migration prior to fragmentation has been studied here. This study also demonstrates the destructive nature of core ionization as in most cases themore » C 1s ionization of uridine leads to concerted explosions producing only small fragments with masses ≤43 amu. In addition to fragmentation patterns, we found out that upon evaporation the sugar part of the uridine molecule attains hexagonal form.« less

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

Syntheses of nicotinamide riboside and derivatives: effective agents for increasing nicotinamide adenine dinucleotide concentrations in mammalian cells.

PubMed

Yang, Tianle; Chan, Noel Yan-Ki; Sauve, Anthony A

2007-12-27

A new two-step methodology achieves stereoselective synthesis of beta-nicotinamide riboside and a series of related amide, ester, and acid nucleosides. Compounds were prepared through a triacetylated-nicotinate ester nucleoside, via coupling of either ethylnicotinate or phenylnicotinate with 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose. Nicotinamide riboside, nicotinic acid riboside, O-ethylnicotinate riboside, O-methylnicotinate riboside, and several N-alkyl derivatives increased NAD+ concentrations from 1.2-2.7-fold in several mammalian cell lines. These findings establish bioavailability and potent effects of these nucleosides in stimulating the increase of NAD+ concentrations in mammalian cells.

Efficacy of Antiretroviral Agents against Murine Replication-Competent Retrovirus Infection in Human Cells

PubMed Central

Powell, Sharon K.; Artlip, Moria; Kaloss, Michele; Brazinski, Scott; Lyons, Russette; McGarrity, Gerard J.; Otto, Edward

1999-01-01

Retroviral vectors for gene therapy are designed to minimize the occurrence of replication-competent retrovirus (RCR); nonetheless, it is possible that a vector-derived RCR could establish an infection in a patient. Since the efficacy of antiretroviral agents can be impacted by interactions between virus, host cell, and drug, five commonly used antiretroviral drugs were evaluated for their abilities to inhibit the replication of a murine leukemia virus (MLV)-derived RCR in human cells. The results obtained indicate that the combination of nucleoside analogs zidovudine and dideoxyinosine with the protease inhibitor indinavir effectively inhibits MLV-derived RCR replication in three human cell lines. In addition, MLV-derived RCR was found to be inherently resistant to the nucleoside analogs lamivudine and stavudine, suggesting that mutations conferring resistance to nucleoside analogs in human immunodeficiency virus type 1 have the same effect even in an alternative viral backbone. PMID:10482636

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 ribosylation. Absent from the enzymatic and chemical glycosylations is the natural pattern of N3 ribosylation, verified by comparison of spectroscopic and chromatographic properties with an authentic standard synthesized by an unambiguous route. PMID:25521390

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.

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.

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.

Ligand binding phenomena that pertain to the metabolic function of renalase.

PubMed

Beaupre, Brett A; Roman, Joseph V; Hoag, Matthew R; Meneely, Kathleen M; Silvaggi, Nicholas R; Lamb, Audrey L; Moran, Graham R

2016-12-15

Renalase catalyzes the oxidation of isomers of β-NAD(P)H that carry the hydride in the 2 or 6 positions of the nicotinamide base to form β-NAD(P) + . This activity is thought to alleviate inhibition of multiple β-NAD(P)-dependent enzymes of primary and secondary metabolism by these isomers. Here we present evidence for a variety of ligand binding phenomena relevant to the function of renalase. We offer evidence of the potential for primary metabolism inhibition with structures of malate dehydrogenase and lactate dehydrogenase bound to the 6-dihydroNAD isomer. The previously observed preference of renalase from Pseudomonas for NAD-derived substrates over those derived from NADP is accounted for by the structure of the enzyme in complex with NADPH. We also show that nicotinamide nucleosides and mononucleotides reduced in the 2- and 6-positions are renalase substrates, but bind weakly. A seven-fold enhancement of acquisition (k red /K d ) for 6-dihydronicotinamide riboside was observed for human renalase in the presence of ADP. However, generally the addition of complement ligands, AMP for mononucleotide or ADP for nucleoside substrates, did not enhance the reductive half-reaction. Non-substrate nicotinamide nucleosides or nucleotides bind weakly suggesting that only β-NADH and β-NADPH compete with dinucleotide substrates for access to the active site. Copyright © 2016 Elsevier Inc. All rights reserved.

Triazole nucleoside derivatives bearing aryl functionalities on the nucleobases show antiviral and anticancer activity.

PubMed

Xia, Yi; Qu, Fanqi; Peng, Ling

2010-08-01

Synthetic nucleoside mimics are important candidates in the searing for antiviral and anticancer drugs. Ribavirin, the first antiviral nucleoside drug, is unique in its antiviral activity with mutilple modes of action, which are mainly due to its special triazole heterocycle as nucleobase. Additionally, introducing aromatic functionalities to the nucleobase is able to confer novel mechanisms of action for nucleoside mimics. With the aim to combine the special characteristics of unnatural triazole heterocycles with those of the appended aromatic groups on the nucleobases, novel 1,2,4-triazole nucleoside analogs bearing aromatic moieties were designed and developed. The present short review summarizes the molecular design, chemical synthesis and biological activity of these triazole nucleoside analogs. Indeed, the discovery of antiviral and anticancer activities shown by these triazole nucleosides as well as the new mechanism underlying the biological activity by one of the anticancer leads has validated the rationale for molecular design and impacted us to further explore the concept with the aim of developing structurally novel nucleoside drug candidates with new modes of action.

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.

Mineral catalysis of the formation of the phosphodiester bond in aqueous solution: The possible role of montmorillonite clays

NASA Astrophysics Data System (ADS)

Ferris, James P.; Ertem, Gözen; Kamaluddin; Agarwal, Vipin; Hua, Lu Lin

The binding of adenosine to Na+-montmorillonite 22A is greater than 5'-AMP, at neutral pH. Adenine derivatives bind more strongly to the clay than the corresponding uracil derivatives. These data are consistent with the protonation of the adenine by the acidic clay surface and a cationic binding of the protonated ring to the anionic clay surface. Other forces must be operative in the binding of uracil derivatives to the clay since the uracil ring system is not basic. The reaction of the 5'-AMP with water soluble carbodiimide in the presence of Na+-montmorillonite results in the formation of 2',5'-pApA (18.9%), 3',5'-pApA (11%), and AppA (4.8%). When poly(U) is used in place of the clay the product yields are 2',5',-pApA (15.5%), 3',5'-pApA (3.7%) and AppA (14.9%). The cyclic nucleotide, c(pA)2 is also formed when poly(U) is used. AppA is the principal reaction product when neither clay nor poly(U) is present in the reaction mixture. When 2'-deoxy-5'-AMP reacts with carbodiimide in the presence of Na+-montmorillonite 22A the products are dpApA (4.8%), dAppApA (4.5%) and dAppA (17.4%). Cyclic 3',5'-dAMP is the main product (14%) of the reaction of 2'-deoxy-3'-AMP.

A multi-functional guanine derivative for studying the DNA G-quadruplex structure.

PubMed

Ishizuka, Takumi; Zhao, Pei-Yan; Bao, Hong-Liang; Xu, Yan

2017-10-23

In the present study, we developed a multi-functional guanine derivative, 8F G, as a G-quadruplex stabilizer, a fluorescent probe for the detection of G-quadruplex formation, and a 19 F sensor for the observation of the G-quadruplex. We demonstrate that the functional nucleoside bearing a 3,5-bis(trifluoromethyl)benzene group at the 8-position of guanine stabilizes the DNA G-quadruplex structure and fluoresces following the G-quadruplex formation. Furthermore, we show that the functional sensor can be used to directly observe DNA G-quadruplexes by 19 F-NMR in living cells. To our knowledge, this is the first study showing that the nucleoside derivative simultaneously allows for three kinds of functions at a single G-quadruplex DNA. Our results suggest that the multi-functional nucleoside derivative can be broadly used for studying the G-quadruplex structure and serves as a powerful tool for examining the molecular basis of G-quadruplex formation in vitro and in living cells.

Escherichia coli-Derived Uracil Increases the Antibacterial Activity and Growth Rate of Lactobacillus plantarum.

PubMed

Ha, Eun-Mi

2016-05-28

Lactobacillus plantarum (L. plantarum) is a representative probiotic. In particular, L. plantarum is the first commensal bacterium to colonize the intestine of infants. For this reason, the initial settlement of L. plantarum can play an important role in determining an infant's health as well as their eventual health status as an adult. In addition, L. plantarum combats pathogenic infections (such as Escherichia coli (E. coli), one of the early pathogenic colonizers in an unhealthy infant gut) by secreting antimicrobial substances. The aim of this research was to determine how L. plantarum combats E. coli infection and why it is a representative probiotic in the intestine. Consequently, this research observed that E. coli releases uracil. L. plantarum specifically recognizes E. coli-derived uracil, which increases the growth rate and production of antimicrobial substance of L. plantarum. In addition, through the inhibitory activity test, this study postulates that the antimicrobial substance is a protein and can be considered a bacteriocin-like substance. Therefore, this research assumes that L. plantarum exerts its antibacterial ability by recognizing E. coli and increasing its growth rate as a result, and this phenomenon could be one of the reasons for L. plantarum settling in the intestine of infants as a beneficial bacterium.

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.

Multi-temperature study of potassium uridine-5'-monophosphate: electron density distribution and anharmonic motion modelling.

PubMed

Jarzembska, Katarzyna N; Řlepokura, Katarzyna; Kamiński, Radosław; Gutmann, Matthias J; Dominiak, Paulina M; Woźniak, Krzysztof

2017-08-01

Uridine, a nucleoside formed of a uracil fragment attached to a ribose ring via a β-N1-glycosidic bond, is one of the four basic components of ribonucleic acid. Here a new anhydrous structure and experimental charge density distribution analysis of a uridine-5'-monophosphate potassium salt, K(UMPH), is reported. The studied case constitutes the very first structure of a 5'-nucleotide potassium salt according to the Cambridge Structural Database. The excellent crystal quality allowed the collection of charge density data at various temperatures, i.e. 10, 100, 200 and 300 K on one single crystal. Crystal structure and charge density data were analysed thoroughly in the context of related literature-reported examples. Detailed analysis of the charge density distribution revealed elevated anharmonic motion of part of the uracil ring moiety relatively weakly interacting with the neighbouring species. The effect was manifested by alternate positive and negative residual density patterns observed for these atoms, which `disappear' at low temperature. It also occurred that the potassium cation, quite uniformly coordinated by seven O atoms from all molecular fragments of the UMPH - anion, including the O atom from the ribofuranose ring, can be treated as spherical in the charge density model which was supported by theoretical calculations. Apart from the predominant electrostatic interactions, four relatively strong hydrogen bond types further support the stability of the crystal structure. This results in a compact and quite uniform structure (in all directions) of the studied crystal, as opposed to similar cases with layered architecture reported in the literature.

Combining H/D exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase

PubMed Central

Roberts, Victoria A.; Pique, Michael E.; Hsu, Simon; Li, Sheng; Slupphaug, Geir; Rambo, Robert P.; Jamison, Jonathan W.; Liu, Tong; Lee, Jun H.; Tainer, John A.; Ten Eyck, Lynn F.; Woods, Virgil L.

2012-01-01

X-ray crystallography provides excellent structural data on protein–DNA interfaces, but crystallographic complexes typically contain only small fragments of large DNA molecules. We present a new approach that can use longer DNA substrates and reveal new protein–DNA interactions even in extensively studied systems. Our approach combines rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). DXMS identifies solvent-exposed protein surfaces; docking is used to create a 3-dimensional model of the protein–DNA interaction. We investigated the enzyme uracil-DNA glycosylase (UNG), which detects and cleaves uracil from DNA. UNG was incubated with a 30 bp DNA fragment containing a single uracil, giving the complex with the abasic DNA product. Compared with free UNG, the UNG–DNA complex showed increased solvent protection at the UNG active site and at two regions outside the active site: residues 210–220 and 251–264. Computational docking also identified these two DNA-binding surfaces, but neither shows DNA contact in UNG–DNA crystallographic structures. Our results can be explained by separation of the two DNA strands on one side of the active site. These non-sequence-specific DNA-binding surfaces may aid local uracil search, contribute to binding the abasic DNA product and help present the DNA product to APE-1, the next enzyme on the DNA-repair pathway. PMID:22492624

Organic Heat Stabilizers for Polyvinyl Chloride (PVC): A Synergistic Behavior of Eugenol and Uracil Derivative

NASA Astrophysics Data System (ADS)

Asawakosinchai, Aran; Jubsilp, Chanchira; Mora, Phattarin; Rimdusit, Sarawut

2017-10-01

Recycling ability, mechanical, and thermal properties of PVC stabilized with organic heat stabilizers, i.e., uracil (DAU) and eugenol were investigated to substitute PVCs stabilized with commercial lead, Ca/Zn, and organic-based stabilizer for PVC pipe production. PVC stabilized with the DAU and the eugenol can be processable at 30 °C lower than that of the PVC stabilized with commercial heat stabilizers. The most remarkable short-term thermal stability belonged to the PVC stabilized with the DAU, and its original color can be maintained at least up to 3 processing cycles. Synergistic behavior in thermal stability of the PVC mixed with DAU and eugenol at mass ratios of 1.5:1.5 was observed. Mechanical properties of DAU- and eugenol-stabilized PVC were higher than the samples with other heat stabilizers. Glass transition temperature of the PVC stabilized with all heat stabilizers was determined to be 99 °C with the exception of the value of 89 °C for eugenol-stabilized PVC. Therefore, the DAU and the eugenol showed high potential to be used as an organic heat stabilizer for PVC because of their non-toxic and good heat resistance properties.

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.

Cost-Effectiveness of the Third-Agent Class in Treatment-Naive Human Immunodeficiency Virus-Infected Patients in Portugal

PubMed Central

Aragão, Filipa; Vera, José; Vaz Pinto, Inês

2012-01-01

Introduction Current Portuguese HIV treatment guidelines recommend initiating antiretroviral therapy with a regimen composed of two Nucleoside Reverse Transcriptase Inhibitors plus one Non-nucleoside Reverse Transcriptase Inhibitor (2NRTI+NNRTI) or two Nucleoside Reverse Transcriptase Inhibitors plus one boosted protease inhibitor (2NRTI+PI/r). Given the lower daily cost of NNRTI as the third agent when compared to the average daily costs of PI/r, it is relevant to estimate the long term impact of each treatment option in the Portuguese context. Methods We developed a microsimulation discrete events model for cost-effectiveness analysis of HIV treatment, simulating individual paths from ART initiation to death. Four driving forces determine the course of events: CD4+ cell count, viral load, resistance and adherence. Distributions of time to event are conditional to individuals’ characteristics and past history. Time to event was modeled using parametric survival analysis using Stata 11®. Disease progression was structured according to therapy lines and the model was parameterized with cohort Portuguese observational data. All resources were valued at 2009 prices. The National Health Service’s perspective was assumed considering a lifetime horizon and a 5% annual discount rate. Results In this analysis, initiating therapy with two Nucleoside Reverse Transcriptase Inhibitors plus one Non-nucleoside Reverse Transcriptase Inhibitor reduces the average number of switches by 17%, saves 19.573€ per individual and increases life expectancy by 1.7 months showing to be a dominant strategy in 57% of the simulations when compared to two Nucleoside Reverse Transcriptase Inhibitors plus one boosted protease inhibitor. Conclusion This study suggests that, when clinically valid, initiating therapy with two Nucleoside Reverse Transcriptase Inhibitors plus one Non-nucleoside Reverse Transcriptase Inhibitor is a cost-saving strategy and equally effective when compared to two Nucleoside Reverse Transcriptase Inhibitors plus one boosted protease inhibitor as the first regimen. PMID:23028618

Probing the interaction of archaeal DNA polymerases with deaminated bases using X-ray crystallography and non-hydrogen bonding isosteric base analogues†

PubMed Central

Killelea, Tom; Ghosh, Samantak; Tan, Samuel S.; Heslop, Pauline; Firbank, Susan; Kool, Eric T.; Connolly, Bernard A.

2010-01-01

Archaeal family-B DNA polymerases stall replication on encountering the pro-mutagenic bases uracil and hypoxanthine. This publication describes an X-ray crystal structure of Thermococcus gorgonarius polymerase in complex with a DNA containing hypoxanthine in the single-stranded region of the template, two bases ahead of the primer-template junction. Full details of the specific recognition of hypoxanthine are revealed, allowing a comparison with published data that describes uracil binding. The two bases are recognized by the same pocket, in the N-terminal domain, and make very similar protein-DNA interactions. Specificity for hypoxanthine (and uracil) arises from a combination of polymerase-base hydrogen bonds and shape fit between the deaminated bases and the pocket. The structure with hypoxanthine at the +2 position explains the stimulation of the polymerase 3′-5′ proof reading exonuclease, observed with deaminated bases at this location. A β hairpin element, involved in partitioning the primer strand between the polymerase and exonuclease active sites, inserts between the two template bases at the extreme end of the double stranded DNA. This denatures the two complementary primer bases and directs the resulting 3′ single-stranded extension towards the exonuclease active site. Finally the relative importance of hydrogen bonding and shape fit in determining selectivity for deaminated bases has been examined using non-polar isosteres. Affinity for both 2,4 difluorobenzene and fluorobenzimidazole, non-hydrogen bonding shape mimics of uracil and hypoxanthine respectively, is strongly diminished, suggesting polar protein-base contacts are important. However, residual interaction with 2,4 difluorobenzene is seen, confirming a role for shape recognition. PMID:20527806

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.

Antimalarial and antimicrobial activities of 8-Aminoquinoline-Uracils metal complexes

PubMed Central

Phopin, Kamonrat; Sinthupoom, Nujarin; Treeratanapiboon, Lertyot; Kunwittaya, Sarun; Prachayasittikul, Supaluk; Ruchirawat, Somsak; Prachayasittikul, Virapong

2016-01-01

8-Aminoquinoline (8AQ) derivatives have been reported to have antimalarial, anticancer, and antioxidant activities. This study investigated the potency of 8AQ-5-substituted (iodo and nitro) uracils metal (Mn, Cu, Ni) complexes (1-6) as antimalarial and antimicrobial agents. Interestingly, all of these metal complexes (1-6) showed fair antimalarial activities. Moreover, Cu complexes 2 (8AQ-Cu-5Iu) and 5 (8AQ-Cu-5Nu) exerted antimicrobial activities against Gram-negative bacteria including P. shigelloides and S. dysenteriae. The results reveal application of 8AQ and its metal complexes as potential compounds to be further developed as novel antimalarial and antibacterial agents. PMID:27103894

Antimalarial and antimicrobial activities of 8-Aminoquinoline-Uracils metal complexes.

PubMed

Phopin, Kamonrat; Sinthupoom, Nujarin; Treeratanapiboon, Lertyot; Kunwittaya, Sarun; Prachayasittikul, Supaluk; Ruchirawat, Somsak; Prachayasittikul, Virapong

2016-01-01

8-Aminoquinoline (8AQ) derivatives have been reported to have antimalarial, anticancer, and antioxidant activities. This study investigated the potency of 8AQ-5-substituted (iodo and nitro) uracils metal (Mn, Cu, Ni) complexes (1-6) as antimalarial and antimicrobial agents. Interestingly, all of these metal complexes (1-6) showed fair antimalarial activities. Moreover, Cu complexes 2 (8AQ-Cu-5Iu) and 5 (8AQ-Cu-5Nu) exerted antimicrobial activities against Gram-negative bacteria including P. shigelloides and S. dysenteriae. The results reveal application of 8AQ and its metal complexes as potential compounds to be further developed as novel antimalarial and antibacterial agents.

The UNG2 Arg88Cys variant abrogates RPA-mediated recruitment of UNG2 to single-stranded DNA.

PubMed

Torseth, Kathrin; Doseth, Berit; Hagen, Lars; Olaisen, Camilla; Liabakk, Nina-Beate; Græsmann, Heidi; Durandy, Anne; Otterlei, Marit; Krokan, Hans E; Kavli, Bodil; Slupphaug, Geir

2012-06-01

In human cell nuclei, UNG2 is the major uracil-DNA glycosylase initiating DNA base excision repair of uracil. In activated B cells it has an additional role in facilitating mutagenic processing of AID-induced uracil at Ig loci and UNG-deficient patients develop hyper-IgM syndrome characterized by impaired class-switch recombination and disturbed somatic hypermutation. How UNG2 is recruited to either error-free or mutagenic uracil processing remains obscure, but likely involves regulated interactions with other proteins. The UNG2 N-terminal domain contains binding motifs for both proliferating cell nuclear antigen (PCNA) and replication protein A (RPA), but the relative contribution of these interactions to genomic uracil processing is not understood. Interestingly, a heterozygous germline single-nucleotide variant leading to Arg88Cys (R88C) substitution in the RPA-interaction motif of UNG2 has been observed in humans, but with unknown functional relevance. Here we demonstrate that UNG2-R88C protein is expressed from the variant allele in a lymphoblastoid cell line derived from a heterozygous germ line carrier. Enzyme activity as well as localization in replication foci of UNG2-R88C was similar to that of WT. However, binding to RPA was essentially abolished by the R88C substitution, whereas binding to PCNA was unaffected. Moreover, we show that disruption of the PCNA-binding motif impaired recruitment of UNG2 to S-phase replication foci, demonstrating that PCNA is a major factor for recruitment of UNG2 to unperturbed replication forks. Conversely, in cells treated with hydroxyurea, RPA mediated recruitment of UNG2 to stalled replication forks independently of functional PCNA binding. Modulation of PCNA- versus RPA-binding may thus constitute a functional switch for UNG2 in cells subsequent to genotoxic stress and potentially also during the processing of uracil at the immunoglobulin locus in antigen-stimulated B cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Nucleobases and corresponding nucleosides display potent antiviral activities against dengue virus possibly through viral lethal mutagenesis.

PubMed

Qiu, Li; Patterson, Steven E; Bonnac, Laurent F; Geraghty, Robert J

2018-04-01

Dengue virus affects millions of people worldwide each year. To date, there is no drug for the treatment of dengue-associated disease. Nucleosides are effective antivirals and work by inhibiting the accurate replication of the viral genome. Nucleobases offer a cheaper alternative to nucleosides for broad antiviral applications. Metabolic activation of nucleobases involves condensation with 5-phosphoribosyl-1-pyrophosphate to give the corresponding nucleoside-5'-monophosphate. This could provide an alternative to phosphorylation of a nucleoside, a step that is often rate limiting and inefficient in activation of nucleosides. We evaluated more than 30 nucleobases and corresponding nucleosides for their antiviral activity against dengue virus. Five nucleobases and two nucleosides were found to induce potent antiviral effects not previously described. Our studies further revealed that nucleobases were usually more active with a better tissue culture therapeutic index than their corresponding nucleosides. The development of viral lethal mutagenesis, an antiviral approach that takes into account the quasispecies behavior of RNA viruses, represents an exciting prospect not yet studied in the context of dengue replication. Passage of the virus in the presence of the nucleobase 3a (T-1105) and corresponding nucleoside 3b (T-1106), favipiravir derivatives, induced an increase in apparent mutations, indicating lethal mutagenesis as a possible antiviral mechanism. A more concerted and widespread screening of nucleobase libraries is a very promising approach to identify dengue virus inhibitors including those that may act as viral mutagens.

Indolylarylsulfones carrying a heterocyclic tail as very potent and broad spectrum HIV-1 non-nucleoside reverse transcriptase inhibitors.

PubMed

Famiglini, Valeria; La Regina, Giuseppe; Coluccia, Antonio; Pelliccia, Sveva; Brancale, Andrea; Maga, Giovanni; Crespan, Emmanuele; Badia, Roger; Riveira-Muñoz, Eva; Esté, José A; Ferretti, Rosella; Cirilli, Roberto; Zamperini, Claudio; Botta, Maurizio; Schols, Dominique; Limongelli, Vittorio; Agostino, Bruno; Novellino, Ettore; Silvestri, Romano

2014-12-11

We synthesized new indolylarylsulfone (IAS) derivatives carrying a heterocyclic tail at the indole-2-carboxamide nitrogen as potential anti-HIV/AIDS agents. Several new IASs yielded EC50 values <1.0 nM against HIV-1 WT and mutant strains in MT-4 cells. The (R)-11 enantiomer proved to be exceptionally potent against the whole viral panel; in the reverse transcriptase (RT) screening assay, it was remarkably superior to NVP and EFV and comparable to ETV. The binding poses were consistent with the one previously described for the IAS non-nucleoside reverse transcriptase inhibitors. Docking studies showed that the methyl group of (R)-11 points toward the cleft created by the K103N mutation, different from the corresponding group of (S)-11. By calculating the solvent-accessible surface, we observed that the exposed area of RT in complex with (S)-11 was larger than the area of the (R)-11 complex. Compounds 6 and 16 and enantiomer (R)-11 represent novel robust lead compounds of the IAS class.

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.

A structural determinant in the uracil DNA glycosylase superfamily for the removal of uracil from adenine/uracil base pairs

PubMed Central

Lee, Dong-Hoon; Liu, Yinling; Lee, Hyun-Wook; Xia, Bo; Brice, Allyn R.; Park, Sung-Hyun; Balduf, Hunter; Dominy, Brian N.; Cao, Weiguo

2015-01-01

The uracil DNA glycosylase superfamily consists of several distinct families. Family 2 mismatch-specific uracil DNA glycosylase (MUG) from Escherichia coli is known to exhibit glycosylase activity on three mismatched base pairs, T/U, G/U and C/U. Family 1 uracil N-glycosylase (UNG) from E. coli is an extremely efficient enzyme that can remove uracil from any uracil-containing base pairs including the A/U base pair. Here, we report the identification of an important structural determinant that underlies the functional difference between MUG and UNG. Substitution of a Lys residue at position 68 with Asn in MUG not only accelerates the removal of uracil from mismatched base pairs but also enables the enzyme to gain catalytic activity on A/U base pairs. Binding and kinetic analysis demonstrate that the MUG-K68N substitution results in enhanced ground state binding and transition state interactions. Molecular modeling reveals that MUG-K68N, UNG-N123 and family 5 Thermus thermophiles UDGb-A111N can form bidentate hydrogen bonds with the N3 and O4 moieties of the uracil base. Genetic analysis indicates the gain of function for A/U base pairs allows the MUG-K68N mutant to remove uracil incorporated into the genome during DNA replication. The implications of this study in the origin of life are discussed. PMID:25550433

Probing the interaction of archaeal DNA polymerases with deaminated bases using X-ray crystallography and non-hydrogen bonding isosteric base analogues.

PubMed

Killelea, Tom; Ghosh, Samantak; Tan, Samuel S; Heslop, Pauline; Firbank, Susan J; Kool, Eric T; Connolly, Bernard A

2010-07-13

Archaeal family-B DNA polymerases stall replication on encountering the pro-mutagenic bases uracil and hypoxanthine. This publication describes an X-ray crystal structure of Thermococcus gorgonarius polymerase in complex with a DNA containing hypoxanthine in the single-stranded region of the template, two bases ahead of the primer-template junction. Full details of the specific recognition of hypoxanthine are revealed, allowing a comparison with published data that describe uracil binding. The two bases are recognized by the same pocket, in the N-terminal domain, and make very similar protein-DNA interactions. Specificity for hypoxanthine (and uracil) arises from a combination of polymerase-base hydrogen bonds and shape fit between the deaminated bases and the pocket. The structure with hypoxanthine at position 2 explains the stimulation of the polymerase 3'-5' proofreading exonuclease, observed with deaminated bases at this location. A beta-hairpin element, involved in partitioning the primer strand between the polymerase and exonuclease active sites, inserts between the two template bases at the extreme end of the double-stranded DNA. This denatures the two complementary primer bases and directs the resulting 3' single-stranded extension toward the exonuclease active site. Finally, the relative importance of hydrogen bonding and shape fit in determining selectivity for deaminated bases has been examined using nonpolar isosteres. Affinity for both 2,4-difluorobenzene and fluorobenzimidazole, non-hydrogen bonding shape mimics of uracil and hypoxanthine, respectively, is strongly diminished, suggesting polar protein-base contacts are important. However, residual interaction with 2,4-difluorobenzene is seen, confirming a role for shape recognition.

Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

PubMed Central

Roosild, Tarmo P.; Castronovo, Samantha

2010-01-01

Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 Å resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an “induced-fit” association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer's and Parkinson's, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications. PMID:20856879

3D-QSAR CoMFA of a series of DABO derivatives as HIV-1 reverse transcriptase non-nucleoside inhibitors.

PubMed

de Brito, Monique Araújo; Rodrigues, Carlos Rangel; Cirino, José Jair Vianna; de Alencastro, Ricardo Bicca; Castro, Helena Carla; Albuquerque, Magaly Girão

2008-08-01

A series of 74 dihydroalkoxybenzyloxopyrimidines (DABOs), a class of highly potent non-nucleoside reverse transcriptase inhibitors (NNRTIs), was retrieved from the literature and studied by comparative molecular field analysis (CoMFA) in order to derive three-dimensional quantitative structure-activity relationship (3D-QSAR) models. The CoMFA study has been performed with a training set of 59 compounds, testing three alignments and four charge schemes (DFT, HF, AM1, and PM3) and using defaults probe atom (Csp (3), +1 charge), cutoffs (30 kcal.mol (-1) for both steric and electrostatic fields), and grid distance (2.0 A). The best model ( N = 59), derived from Alignment 1 and PM3 charges, shows q (2) = 0.691, SE cv = 0.475, optimum number of components = 6, r (2) = 0.930, SEE = 0.226, and F-value = 115.544. The steric and electrostatic contributions for the best model were 43.2% and 56.8%, respectively. The external predictive ability (r (2) pred = 0.918) of the resultant best model was evaluated using a test set of 15 compounds. In order to design more potent DABO analogues as anti-HIV/AIDS agents, attention should be taken in order to select a substituent for the 4-oxopyrimidine ring, since, as revealed by the best CoMFA model, there are a steric restriction at the C2-position, a electron-rich group restriction at the C6-position ( para-substituent of the 6-benzyl group), and a steric allowed region at the C5-position.

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.

Synthesis and triplex forming properties of pyrimidine derivative containing extended functionality.

PubMed

Gianolio, D A; McLaughlin, L W

1999-08-01

Two pyrimidine nucleosides have been synthesized containing extended hydrogen bonding functionality. In one case the side chain is based upon semicarbazide and in the second monoacetylated carbohydrazide was employed. DNA sequences could be prepared using both analogue nucleosides in a reverse coupling protocol, and provided that the normal capping step was eliminated and that the iodine-based oxidizing solution was replaced with one based upon 10-camphorsulfonyl oxaziridine. Both derivatives exhibited moderate effects in targeting selectively C-G base pairs embedded within a polypurine target sequence.

Growth inhibition of Saccharomyces cerevisiae by the immunosuppressant leflunomide is due to the inhibition of uracil uptake via Fur4p.

PubMed

Fujimura, H

1998-10-01

The immunosuppressant leflunomide inhibits cytokine-stimulated proliferation of lymphoid cells in vitro and also inhibits the growth of the eukaryotic microorganism Saccharomyces cerevisiae. To elucidate the molecular mechanism of action of the drug, two yeast genes which suppress the anti-proliferative effect when present in multiple copies were cloned and designated MLF1 and MLF2 for multicopy suppressor of leflunomide sensitivity. DNA sequencing analysis revealed that the MLF1 gene is identical to the FUR4 gene, which encodes a uracil permease and functions to import uracil efficiently. The MLF2 was found to be identical to the URA3 gene. Excess exogenous uracil also overcomes the anti-proliferative effect of leflunomide on yeast cells. Uracil prototrophy also conferred resistance to leflunomide. Uracil uptake was inhibited by leflunomide. Thus, the growth inhibition by leflunomide seen in a S. cerevisiae ura3 auxotroph is due to the inhibition of the entry of exogenous uracil via the Fur4 uracil permease.

Nucleobases and Prebiotic Molecules in Organic Residues Produced from the Ultraviolet Photo-Irradiation of Pyrimidine in NH3 and H2O+NH3 Ices

NASA Technical Reports Server (NTRS)

Nuevo, Michel; Milam, Stefanie N.; Sandford, Scott

2012-01-01

Although not yet identified in the interstellar medium (ISM), N-heterocycles including nucleobases the information subunits of DNA and RNA are present in carbonaceous chondrites, which indicates that molecules of biological interest can be formed in non-terrestrial environments via abiotic pathways. Recent laboratory experiments and ab-initio calculations have already shown that the irradiation of pyrimidine in pure H2O ices leads to the formation of a suite of oxidized pyrimidine derivatives, including the nucleobase uracil. In the present work, NH3:pyrimidine and H2O:NH3:pyrimidine ice mixtures with different relative proportions were irradiated with UV photons under astrophysically relevant conditions. Liquid- and gas-chromatography analysis of the resulting organic residues has led to the detection of the nucleobases uracil and cytosine, as well as other species of prebiotic interest such as urea and small amino acids. The presence of these molecules in organic residues formed under abiotic conditions supports scenarios in which extraterrestrial organics that formed in space and were subsequently delivered to telluric planets via comets and meteorites could have contributed to the inventory of molecules that triggered the first biological reactions on their surfaces.

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.

HIV-1 RT Inhibitors with a Novel Mechanism of Action: NNRTIs that Compete with the Nucleotide Substrate

PubMed Central

Maga, Giovanni; Radi, Marco; Gerard, Marie-Aline; Botta, Maurizio; Ennifar, Eric

2010-01-01

HIV-1 reverse transcriptase (RT) inhibitors currently used in antiretroviral therapy can be divided into two classes: (i) nucleoside analog RT inhibitors (NRTIs), which compete with natural nucleoside substrates and act as terminators of proviral DNA synthesis, and (ii) non-nucleoside RT inhibitors (NNRTIs), which bind to a hydrophobic pocket close to the RT active site. In spite of the efficiency of NRTIs and NNRTIs, the rapid emergence of multidrug-resistant mutations requires the development of new RT inhibitors with an alternative mechanism of action. Recently, several studies reported the discovery of novel non-nucleoside inhibitors with a distinct mechanism of action. Unlike classical NNRTIs, they compete with the nucleotide substrate, thus forming a new class of RT inhibitors: nucleotide-competing RT inhibitors (NcRTIs). In this review, we discuss current progress in the understanding of the peculiar behavior of these compounds. PMID:21994659

Uracil-DNA Glycosylase in Base Excision Repair and Adaptive Immunity

PubMed Central

Doseth, Berit; Visnes, Torkild; Wallenius, Anders; Ericsson, Ida; Sarno, Antonio; Pettersen, Henrik Sahlin; Flatberg, Arnar; Catterall, Tara; Slupphaug, Geir; Krokan, Hans E.; Kavli, Bodil

2011-01-01

Genomic uracil is a DNA lesion but also an essential key intermediate in adaptive immunity. In B cells, activation-induced cytidine deaminase deaminates cytosine to uracil (U:G mispairs) in Ig genes to initiate antibody maturation. Uracil-DNA glycosylases (UDGs) such as uracil N-glycosylase (UNG), single strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), and thymine-DNA glycosylase remove uracil from DNA. Gene-targeted mouse models are extensively used to investigate the role of these enzymes in DNA repair and Ig diversification. However, possible species differences in uracil processing in humans and mice are yet not established. To address this, we analyzed UDG activities and quantities in human and mouse cell lines and in splenic B cells from Ung+/+ and Ung−/− backcrossed mice. Interestingly, human cells displayed ∼15-fold higher total uracil excision capacity due to higher levels of UNG. In contrast, SMUG1 activity was ∼8-fold higher in mouse cells, constituting ∼50% of the total U:G excision activity compared with less than 1% in human cells. In activated B cells, both UNG and SMUG1 activities were at levels comparable with those measured for mouse cell lines. Moreover, SMUG1 activity per cell was not down-regulated after activation. We therefore suggest that SMUG1 may work as a weak backup activity for UNG2 during class switch recombination in Ung−/− mice. Our results reveal significant species differences in genomic uracil processing. These findings should be taken into account when mouse models are used in studies of uracil DNA repair and adaptive immunity. PMID:21454529

[Quantitative analysis of nucleotide mixtures with terahertz time domain spectroscopy].

PubMed

Zhang, Zeng-yan; Xiao, Ti-qiao; Zhao, Hong-wei; Yu, Xiao-han; Xi, Zai-jun; Xu, Hong-jie

2008-09-01

Adenosine, thymidine, guanosine, cytidine and uridine form the building blocks of ribose nucleic acid (RNA) and deoxyribose nucleic acid (DNA). Nucleosides and their derivants are all have biological activities. Some of them can be used as medicine directly or as materials to synthesize other medicines. It is meaningful to detect the component and content in nucleosides mixtures. In the present paper, components and contents of the mixtures of adenosine, thymidine, guanosine, cytidine and uridine were analyzed. THz absorption spectra of pure nucleosides were set as standard spectra. The mixture's absorption spectra were analyzed by linear regression with non-negative constraint to identify the components and their relative content in the mixtures. The experimental and analyzing results show that it is simple and effective to get the components and their relative percentage in the mixtures by terahertz time domain spectroscopy with a relative error less than 10%. Component which is absent could be excluded exactly by this method, and the error sources were also analyzed. All the experiments and analysis confirms that this method is of no damage or contamination to the sample. This means that it will be a simple, effective and new method in biochemical materials analysis, which extends the application field of THz-TDS.

Intratumoral gene expression of 5-fluorouracil pharmacokinetics-related enzymes in stage I and II non-small cell lung cancer patients treated with uracil-tegafur after surgery: a prospective multi-institutional study in Japan.

PubMed

Eguchi, Keisuke; Oyama, Takahiko; Tajima, Atsushi; Abiko, Tomohiro; Sawafuji, Makoto; Horio, Hirotoshi; Hashizume, Toshinori; Matsutani, Noriyuki; Kato, Ryoichi; Nakayama, Mitsuo; Kawamura, Masafumi; Kobayashi, Koichi

2015-01-01

This investigation was conducted to assess the use of the intratumoral mRNA expression levels of nucleic acid-metabolizing enzymes as biomarkers of adjuvant chemotherapy for non-small cell lung cancer (NSCLC) using uracil-tegafur in a multi-institutional prospective study. 236 patients with a completely resected NSCLC (adenocarcinoma and squamous cell carcinoma) of pathological stage IA (maximum tumor diameter of 2 cm or greater), IB, and II tumors were given a dose of 250 mg of uracil-tegafur per square meter of body surface area per day orally for two years after surgery. Intratumoral mRNA levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), and thymidine phosphorylase (TP) genes relative to an internal standard, β-actin, were determined using laser-capture microdissection and fluorescence-based real time PCR detection systems. Among 5-FU target enzymes, TS was the only one that showed a significant difference in the level of gene expression between the high and low gene expression groups, for both disease-free survival (DFS) and overall survival (OS), when patients were divided according to median values; 5-year DFS rates in high/low TS gene expression were 60.4% and 72.6%, respectively (p=0.050), 5-year OS rates were 78.1% and 88.6%, respectively (p=0.011). Cox's proportional hazard model indicated that the pathological stage and TS gene expression level were independent values for predicting DFS. The TS gene expression level was shown to be an independent predictive factor for DFS in stage I and II NSCLC patients who were treated with uracil-tegafur following surgery. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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.

Uracil Accumulation and Mutagenesis Dominated by Cytosine Deamination in CpG Dinucleotides in Mice Lacking UNG and SMUG1

DOE PAGES

Alsøe, Lene; Sarno, Antonio; Carracedo, Sergio; ...

2017-08-03

Both a DNA lesion and an intermediate for antibody maturation, uracil is primarily processed by base excision repair (BER), either initiated by uracil-DNA glycosylase (UNG) or by single-strand selective monofunctional uracil DNA glycosylase (SMUG1). The relative in vivo contributions of each glycosylase remain elusive. To assess the impact of SMUG1 deficiency, we measured uracil and 5-hydroxymethyluracil, another SMUG1 substrate, in Smug1 -/ - mice. Here, we found that 5-hydroxymethyluracil accumulated in Smug1 -/ - tissues and correlated with 5-hydroxymethylcytosine levels. The highest increase was found in brain, which contained about 26-fold higher genomic 5-hydroxymethyluracil levels than the wild type. Smug1more » -/ - mice did not accumulate uracil in their genome and Ung -/ - mice showed slightly elevated uracil levels. Contrastingly, Ung -/ -Smug1 -/ - mice showed a synergistic increase in uracil levels with up to 25-fold higher uracil levels than wild type. Whole genome sequencing of UNG/SMUG1-deficient tumours revealed that combined UNG and SMUG1 deficiency leads to the accumulation of mutations, primarily C to T transitions within CpG sequences. This unexpected sequence bias suggests that CpG dinucleotides are intrinsically more mutation prone. In conclusion, we showed that SMUG1 efficiently prevent genomic uracil accumulation, even in the presence of UNG, and identified mutational signatures associated with combined UNG and SMUG1 deficiency.« less

Uracil Accumulation and Mutagenesis Dominated by Cytosine Deamination in CpG Dinucleotides in Mice Lacking UNG and SMUG1

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

Alsøe, Lene; Sarno, Antonio; Carracedo, Sergio

Both a DNA lesion and an intermediate for antibody maturation, uracil is primarily processed by base excision repair (BER), either initiated by uracil-DNA glycosylase (UNG) or by single-strand selective monofunctional uracil DNA glycosylase (SMUG1). The relative in vivo contributions of each glycosylase remain elusive. To assess the impact of SMUG1 deficiency, we measured uracil and 5-hydroxymethyluracil, another SMUG1 substrate, in Smug1 -/ - mice. Here, we found that 5-hydroxymethyluracil accumulated in Smug1 -/ - tissues and correlated with 5-hydroxymethylcytosine levels. The highest increase was found in brain, which contained about 26-fold higher genomic 5-hydroxymethyluracil levels than the wild type. Smug1more » -/ - mice did not accumulate uracil in their genome and Ung -/ - mice showed slightly elevated uracil levels. Contrastingly, Ung -/ -Smug1 -/ - mice showed a synergistic increase in uracil levels with up to 25-fold higher uracil levels than wild type. Whole genome sequencing of UNG/SMUG1-deficient tumours revealed that combined UNG and SMUG1 deficiency leads to the accumulation of mutations, primarily C to T transitions within CpG sequences. This unexpected sequence bias suggests that CpG dinucleotides are intrinsically more mutation prone. In conclusion, we showed that SMUG1 efficiently prevent genomic uracil accumulation, even in the presence of UNG, and identified mutational signatures associated with combined UNG and SMUG1 deficiency.« less

Primary fragmentation pathways of gas phase [M(uracil-H)(uracil)]+ complexes (M=Zn, Cu, Ni, Co, Fe, Mn, Cd, Pd , Mg, Ca, Sr, Ba, and Pb): loss of uracil versus HNCO.

PubMed

Ali, Osama Y; Randell, Nicholas M; Fridgen, Travis D

2012-04-23

Complexes formed between metal dications, the conjugate base of uracil, and uracil are investigated by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Positive-ion electrospray spectra show that [M(Ura-H)(Ura)](+) (M=Zn, Cu, Ni, Co, Fe, Mn, Cd, Pd, Mg, Ca, Sr, Ba, or Pb) is the most abundant ion even at low concentrations of uracil. SORI-CID experiments show that the main primary decomposition pathway for all [M(Ura-H)(Ura)](+) , except where M=Ca, Sr, Ba, or Pb, is the loss of HNCO. Under the same SORI-CID conditions, when M is Ca, Sr, Ba, or Pb, [M(Ura-H)(Ura)](+) are shown to lose a molecule of uracil. Similar results were observed under infrared multiple-photon dissociation excitation conditions, except that [Ca(Ura-H)(Ura)](+) was found to lose HNCO as the primary fragmentation product. The binding energies between neutral uracil and [M(Ura-H)](+) (M=Zn, Cu, Ni, Fe, Cd, Pd ,Mg, Ca, Sr Ba, or Pb) are calculated by means of electronic-structure calculations. The differences in the uracil binding energies between complexes which lose uracil and those which lose HNCO are consistent with the experimentally observed differences in fragmentation pathways. A size dependence in the binding energies suggests that the interaction between uracil and [M(Ura-H)](+) is ion-dipole complexation and the experimental evidence presented supports this. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural basis for non-competitive product inhibition in human thymidine phosphorylase: implications for drug design

PubMed Central

Omari, Kamel EL; Bronckaers, Annelies; Liekens, Sandra; Pérez-Pérez, Maria-Jésus; Balzarini, Jan; Stammers, David K.

2006-01-01

HTP (human thymidine phosphorylase), also known as PD-ECGF (platelet-derived endothelial cell growth factor) or gliostatin, has an important role in nucleoside metabolism. HTP is implicated in angiogenesis and apoptosis and therefore is a prime target for drug design, including antitumour therapies. An HTP structure in a closed conformation complexed with an inhibitor has previously been solved. Earlier kinetic studies revealed an ordered release of thymine followed by ribose phosphate and product inhibition by both ligands. We have determined the structure of HTP from crystals grown in the presence of thymidine, which, surprisingly, resulted in bound thymine with HTP in a closed dead-end com-plex. Thus thymine appears to be able to reassociate with HTP after its initial ordered release before ribose phosphate and induces the closed conformation, hence explaining the mechanism of non-competitive product inhibition. In the active site in one of the four HTP molecules within the crystal asymmetric unit, additional electron density is present. This density has not been previously seen in any pyrimidine nucleoside phosphorylase and it defines a subsite that may be exploitable in drug design. Finally, because our crystals did not require proteolysed HTP to grow, the structure reveals a loop (residues 406–415), disordered in the previous HTP structure. This loop extends across the active-site cleft and appears to stabilize the dimer interface and the closed conformation by hydrogen-bonding. The present study will assist in the design of HTP inhibitors that could lead to drugs for anti-angiogenesis as well as for the potentiation of other nucleoside drugs. PMID:16803458

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

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.

Effects of seven chemicals on DNA damage in the rat urinary bladder: a comet assay study.

PubMed

Wada, Kunio; Yoshida, Toshinori; Takahashi, Naofumi; Matsumoto, Kyomu

2014-07-15

The in vivo comet assay has been used for the evaluation of DNA damage and repair in various tissues of rodents. However, it can give false-positive results due to non-specific DNA damage associated with cell death. In this study, we examined whether the in vivo comet assay can distinguish between genotoxic and non-genotoxic DNA damage in urinary bladder cells, by using the following seven chemicals related to urinary bladder carcinogenesis in rodents: N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), glycidol, 2,2-bis(bromomethyl)-1,3-propanediol (BMP), 2-nitroanisole (2-NA), benzyl isothiocyanate (BITC), uracil, and melamine. BBN, glycidol, BMP, and 2-NA are known to be Ames test-positive and they are expected to produce DNA damage in the absence of cytotoxicity. BITC, uracil, and melamine are Ames test-negative with metabolic activation but have the potential to induce non-specific DNA damage due to cytotoxicity. The test chemicals were administered orally to male Sprague-Dawley rats (five per group) for each of two consecutive days. Urinary bladders were sampled 3h after the second administration and urothelial cells were analyzed by the comet assay and subjected to histopathological examination to evaluate cytotoxicity. In the urinary bladders of rats treated with BBN, glycidol, and BMP, DNA damage was detected. In contrast, 2-NA induced neither DNA damage nor cytotoxicity. The non-genotoxic chemicals (BITC, uracil, and melamine) did not induce DNA damage in the urinary bladders under conditions where some histopathological changes were observed. The results indicate that the comet assay could distinguish between genotoxic and non-genotoxic chemicals and that no false-positive responses were obtained. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Synthesis and biological properties of novel 2-aminopyrimidin-4(3H)-ones highly potent against HIV-1 mutant strains.

PubMed

Mai, Antonello; Artico, Marino; Rotili, Dante; Tarantino, Domenico; Clotet-Codina, Imma; Armand-Ugón, Mercedes; Ragno, Rino; Simeoni, Silvia; Sbardella, Gianluca; Nawrozkij, Maxim B; Samuele, Alberta; Maga, Giovanni; Esté, José A

2007-11-01

Following the disclosure of dihydro-alkoxy-, dihydro-alkylthio-, and dihydro-alkylamino-benzyl-oxopyrimidines (DABOs, S-DABOs, and NH-DABOs) as potent and selective anti-HIV-1 agents belonging to the non-nucleoside reverse transcriptase inhibitor (NNRTI) class, we report here the synthesis and biological evaluation of a novel series of DABOs bearing a N,N-disubstituted amino group or a cyclic amine at the pyrimidine-C2 position, a hydrogen atom or a small alkyl group at C5 and/or at the benzylic position, and the favorable 2,6-difluorobenzyl moiety at the C6 position (F2-N,N-DABOs). The new compounds were highly active up to the subnanomolar level against both wt HIV-1 and the Y181C mutant and at the submicromolar to nanomolar range against the K103N and Y188L mutant strains. Such derivatives were more potent than S-DABOs, NH-DABOs, and nevirapine and efavirenz were chosen as reference drugs. The higher inhibitor adaptability to the HIV-1 RT non-nucleoside binding site (NNBS) may account for the higher inhibitory effect exerted by the new molecules against the mutated RTs.

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.

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.

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.

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.

Compartmentalized self-replication (CSR) selection of Thermococcus litoralis Sh1B DNA polymerase for diminished uracil binding.

PubMed

Tubeleviciute, Agne; Skirgaila, Remigijus

2010-08-01

The thermostable archaeal DNA polymerase Sh1B from Thermococcus litoralis has a typical uracil-binding pocket, which in nature plays an essential role in preventing the accumulation of mutations caused by cytosine deamination to uracil and subsequent G-C base pair transition to A-T during the genomic DNA replication. The uracil-binding pocket recognizes and binds uracil base in a template strand trapping the polymerase. Since DNA replication stops, the repair systems have a chance to correct the promutagenic event. Archaeal family B DNA polymerases are employed in various PCR applications. Contrary to nature, in PCR the uracil-binding property of archaeal polymerases is disadvantageous and results in decreased DNA amplification yields and lowered sensitivity. Furthermore, in diagnostics qPCR, RT-qPCR and end-point PCR are performed using dNTP mixtures, where dTTP is partially or fully replaced by dUTP. Uracil-DNA glycosylase treatment and subsequent heating of the samples is used to degrade the DNA containing uracil and prevent carryover contamination, which is the main concern in diagnostic laboratories. A thermostable archaeal DNA polymerase with the abolished uracil binding would be a highly desirable and commercially interesting product. An attempt to disable uracil binding in DNA polymerase Sh1B from T. litoralis by generating site-specific mutants did not yield satisfactory results. However, a combination of random mutagenesis of the whole polymerase gene and compartmentalized self-replication was successfully used to select variants of thermostable Sh1B polymerase capable of performing PCR with dUTP instead of dTTP.

Recognition of the pro-mutagenic base uracil by family B DNA polymerases from archaea.

PubMed

Shuttleworth, Gillian; Fogg, Mark J; Kurpiewski, Michael R; Jen-Jacobson, Linda; Connolly, Bernard A

2004-03-26

Archaeal family B DNA polymerases contain a specialised pocket that binds tightly to template-strand uracil, causing the stalling of DNA replication. The mechanism of this unique "template-strand proof-reading" has been studied using equilibrium binding measurements, DNA footprinting, van't Hoff analysis and calorimetry. Binding assays have shown that the polymerase preferentially binds to uracil in single as opposed to double-stranded DNA. Tightest binding is observed using primer-templates that contain uracil four bases in front of the primer-template junction, corresponding to the observed stalling position. Ethylation interference analysis of primer-templates shows that the two phosphates, immediately flanking the uracil (NpUpN), are important for binding; contacts are also made to phosphates in the primer-strand. Microcalorimetry and van't Hoff analysis have given a fuller understanding of the thermodynamic parameters involved in uracil recognition. All the results are consistent with a "read-ahead" mechanism, in which the replicating polymerase scans the template, ahead of the replication fork, for the presence of uracil and halts polymerisation on detecting this base. Post-stalling events, serving to eliminate uracil, await full elucidation.

Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya.

PubMed

Wardle, Josephine; Burgers, Peter M J; Cann, Isaac K O; Darley, Kate; Heslop, Pauline; Johansson, Erik; Lin, Li-Jung; McGlynn, Peter; Sanvoisin, Jonathan; Stith, Carrie M; Connolly, Bernard A

2008-02-01

Family B DNA polymerases from archaea such as Pyrococcus furiosus, which live at temperatures approximately 100 degrees C, specifically recognize uracil in DNA templates and stall replication in response to this base. Here it is demonstrated that interaction with uracil is not restricted to hyperthermophilic archaea and that the polymerase from mesophilic Methanosarcina acetivorans shows identical behaviour. The family B DNA polymerases replicate the genomes of archaea, one of the three fundamental domains of life. This publication further shows that the DNA replicating polymerases from the other two domains, bacteria (polymerase III) and eukaryotes (polymerases delta and epsilon for nuclear DNA and polymerase gamma for mitochondrial) are also unable to recognize uracil. Uracil occurs in DNA as a result of deamination of cytosine, either in G:C base-pairs or, more rapidly, in single stranded regions produced, for example, during replication. The resulting G:U mis-pairs/single stranded uracils are promutagenic and, unless repaired, give rise to G:C to A:T transitions in 50% of the progeny. The confinement of uracil recognition to polymerases of the archaeal domain is discussed in terms of the DNA repair pathways necessary for the elimination of uracil.

Effects of microsolvation on uracil and its radical anion: uracil(H2O)n (n = 1-5).

PubMed

Kim, Sunghwan; Schaefer, Henry F

2006-10-14

Microsolvation effects on the stabilities of uracil and its anion have been investigated by explicitly considering the structures of complexes of uracil with up to five water molecules at the B3LYPDZP++ level of theory. For all five systems, the global minimum of the neutral cluster has a different equilibrium geometry from that of the radical anion. Both the vertical detachment energy (VDE) and adiabatic electron affinity (AEA) of uracil are predicted to increase gradually with the number of hydrating molecules, qualitatively consistent with experimental results from a photodetachment-photoelectron spectroscopy study [J. Schiedt et al., Chem. Phys. 239, 511 (1998)]. The trend in the AEAs implies that while the conventional valence radical anion of uracil is only marginally bound in the gas phase, it will form a stable anion in aqueous solution. The gas-phase AEA of uracil (0.24 eV) was higher than that of thymine by 0.04 eV and this gap was not significantly affected by microsolvation. The largest AEA is that predicted for uracil(H2O)5, namely, 0.96 eV. The VDEs range from 0.76 to 1.78 eV.

Nucleic acids--genes, drugs, molecular lego and more.

PubMed

Häner, Robert

2010-01-01

Chemically modified nucleic acids find widespread use as tools in research, as diagnostic reagents and even as pharmaceutical compounds. On the background of antisense research and development, the synthesis and evaluation of modified oligonucleotides was intensively pursued in the early to mid nineties in corporate research of former Ciba. Most of these efforts concentrated on the development of sugar and/or backbone-modified derivatives for pharmaceutical applications. Additionally, oligonucleotide metal conjugates were investigated with the goal to develop artificial ribonucleases. Since the turn of the millennium also the potential of non-nucleosidic and non-hydrogen bonding building blocks has increasingly been recognized. Such derivatives possess unique properties that may have an impact in the fields of materials and genetic research. In this brief account, we take a personal look back on some past as well as some recent results.

Hydrogen abstraction from deoxyribose by a neighboring 3'-uracil peroxyl radical.

PubMed

Schyman, Patric; Eriksson, Leif A; Laaksonen, Aatto

2009-05-07

Theoretical examination of the reactivity of the uracil-5-peroxyl radical when abstracting a hydrogen atom from a neighboring 5'-deoxyribose in 5'-ApU-5-peroxyl-3' has been performed using density functional theory with the MPWB1K functional. Halogenated uracils are often used as radiosensitizers in DNA since the reactive uracil-5-yl radical is formed upon radiation and is known to create strand break and alkali-labile sites. Under aerobic conditions, such as in the cell, it has been proposed that the uracil-5-peroxyl radical is formed and would be the damaging agent. Our results show low reactivity for the uracil-5-peroxyl radical, determined by calculating the activation and reaction energies for the plausible hydrogen abstraction sites C1', C2', and C3' of the neighboring 5'-deoxyribose. These findings support the hypothesis that hydrogen abstraction primarily occurs by the uracil-5-yl radical, also under aerobic conditions, prior to formation of the peroxyl radical.

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

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.

Formation of Nucleobases from the UV Irradiation of Pyrimidine in Astrophysical Ice Analogs

NASA Technical Reports Server (NTRS)

Sandford, Scott A.; Nuevo, Michel; Materese, Christopher K.

2014-01-01

Nucleobases are the informational subunits of DNA and RNA. They consist of Nheterocycles that belong to either the pyrimidine-base group (uracil, cytosine, and thymine) or the purinebase group (adenine and guanine). Several nucleobases, mostly purine bases, have been detected in meteorites [1-3], with isotopic signatures consistent with an extraterrestrial origin [4]. Uracil is the only pyrimidine-base compound formally reported in meteorites [2], though the presence of cytosine cannot be ruled out [5,6]. However, the actual process by which the uracil was made and the reasons for the non-detection of thymine in meteorites have yet to be fully explained. Although no N-heterocycles have ever been observed in the ISM [7,8], the positions of the 6.2-µm interstellar emission features suggest a population of such molecules is likely to be present [9]. In this work we study the formation of pyrimidine-based molecules, including the three nucleobases uracil, cytosine, and thymine from the ultraviolet (UV) irradiation of pyrimidine in ices consisting of several combinations of H(sub2)O, NH(sub3), CH(sub3)OH, and CH(sub4) at low temperature, in order to simulate the astrophysical conditions under which prebiotic species may be formed in the interstellar medium, in the protosolar nebula, and on icy bodies of the Solar System.

Error-free versus mutagenic processing of genomic uracil--relevance to cancer.

PubMed

Krokan, Hans E; Sætrom, Pål; Aas, Per Arne; Pettersen, Henrik Sahlin; Kavli, Bodil; Slupphaug, Geir

2014-07-01

Genomic uracil is normally processed essentially error-free by base excision repair (BER), with mismatch repair (MMR) as an apparent backup for U:G mismatches. Nuclear uracil-DNA glycosylase UNG2 is the major enzyme initiating BER of uracil of U:A pairs as well as U:G mismatches. Deficiency in UNG2 results in several-fold increases in genomic uracil in mammalian cells. Thus, the alternative uracil-removing glycosylases, SMUG1, TDG and MBD4 cannot efficiently complement UNG2-deficiency. A major function of SMUG1 is probably to remove 5-hydroxymethyluracil from DNA with general back-up for UNG2 as a minor function. TDG and MBD4 remove deamination products U or T mismatched to G in CpG/mCpG contexts, but may have equally or more important functions in development, epigenetics and gene regulation. Genomic uracil was previously thought to arise only from spontaneous cytosine deamination and incorporation of dUMP, generating U:G mismatches and U:A pairs, respectively. However, the identification of activation-induced cytidine deaminase (AID) and other APOBEC family members as DNA-cytosine deaminases has spurred renewed interest in the processing of genomic uracil. Importantly, AID triggers the adaptive immune response involving error-prone processing of U:G mismatches, but also contributes to B-cell lymphomagenesis. Furthermore, mutational signatures in a substantial fraction of other human cancers are consistent with APOBEC-induced mutagenesis, with U:G mismatches as prime suspects. Mutations can be caused by replicative polymerases copying uracil in U:G mismatches, or by translesion polymerases that insert incorrect bases opposite abasic sites after uracil-removal. In addition, kataegis, localized hypermutations in one strand in the vicinity of genomic rearrangements, requires APOBEC protein, UNG2 and translesion polymerase REV1. What mechanisms govern error-free versus error prone processing of uracil in DNA remains unclear. In conclusion, genomic uracil is an essential intermediate in adaptive immunity and innate antiviral responses, but may also be a fundamental cause of a wide range of malignancies. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Physiologically based pharmacokinetic modelling of the three-step metabolism of pyrimidine using C-uracil as an in vivo probe.

PubMed

Ito, Suminobu; Kawamura, Takeshi; Inada, Makoto; Inoue, Yoshiharu; Hirao, Yukihiro; Koga, Toshihisa; Kunizaki, Jun-ichi; Shimizu, Takefumi; Sato, Hitoshi

2005-12-01

Approximately 80% of uracil is excreted as beta-alanine, ammonia and CO2 via three sequential reactions. The activity of the first enzyme in this scheme, dihydropyrimidine dehydrogenase (DPD), is reported to be the key determinant of the cytotoxicity and side-effects of 5-fluorouracil. The aim of the present study was to re-evaluate the pharmacokinetics of uracil and its metabolites using a sensitive assay and based on a newly developed, physiologically based pharmacokinetic (PBPK) model. [2-(13)C]Uracil was orally administrated to 12 healthy males at escalating doses of 50, 100 and 200 mg, and the concentrations of [2-(13)C]uracil, [2-(13)C]5,6-dihydrouracil and beta-ureidopropionic acid (ureido-(13)C) in plasma and urine and (13)CO2 in breath were measured by liquid chromatography-tandem mass spectrometry and gas chromatograph-isotope ratio mass spectrometry, respectively. The pharmacokinetics of [2-(13)C]uracil were nonlinear. The elimination half-life of [2-(13)C]5,6-dihydrouracil was 0.9-1.4 h, whereas that of [2-(13)C]uracil was 0.2-0.3 h. The AUC of [2-(13)C]5,6-dihydrouracil was 1.9-3.1 times greater than that of [2-(13)C]uracil, whereas that of ureido-(13)C was 0.13-0.23 times smaller. The pharmacokinetics of (13)CO2 in expired air were linear and the recovery of (13)CO2 was approximately 80% of the dose. The renal clearance of [2-(13)C]uracil was negligible. A PBPK model to describe (13)CO2 exhalation after orally administered [2-(13)C]uracil was successfully developed. Using [2-(13)C]uracil as a probe, this model could be useful in identifying DPD-deficient patients at risk of 5-fluorouracil toxicity.

DNA translocation by human uracil DNA glycosylase: the case of single-stranded DNA and clustered uracils.

PubMed

Schonhoft, Joseph D; Stivers, James T

2013-04-16

Human uracil DNA glycosylase (hUNG) plays a central role in DNA repair and programmed mutagenesis of Ig genes, requiring it to act on sparsely or densely spaced uracil bases located in a variety of contexts, including U/A and U/G base pairs, and potentially uracils within single-stranded DNA (ssDNA). An interesting question is whether the facilitated search mode of hUNG, which includes both DNA sliding and hopping, changes in these different contexts. Here we find that hUNG uses an enhanced local search mode when it acts on uracils in ssDNA, and also, in a context where uracils are densely clustered in duplex DNA. In the context of ssDNA, hUNG performs an enhanced local search by sliding with a mean sliding length larger than that of double-stranded DNA (dsDNA). In the context of duplex DNA, insertion of high-affinity abasic product sites between two uracil lesions serves to significantly extend the apparent sliding length on dsDNA from 4 to 20 bp and, in some cases, leads to directionally biased 3' → 5' sliding. The presence of intervening abasic product sites mimics the situation where hUNG acts iteratively on densely spaced uracils. The findings suggest that intervening product sites serve to increase the amount of time the enzyme remains associated with DNA as compared to nonspecific DNA, which in turn increases the likelihood of sliding as opposed to falling off the DNA. These findings illustrate how the search mechanism of hUNG is not predetermined but, instead, depends on the context in which the uracils are located.

Discovery of piperidin-4-yl-aminopyrimidine derivatives as potent non-nucleoside HIV-1 reverse transcriptase inhibitors.

PubMed

Wan, Zheng-Yong; Yao, Jin; Tao, Yuan; Mao, Tian-Qi; Wang, Xin-Long; Lu, Yi-Pei; Wang, Hai-Feng; Yin, Hong; Wu, Yan; Chen, Fen-Er; De Clercq, Erik; Daelemans, Dirk; Pannecouque, Christophe

2015-06-05

A novel series of piperidin-4-yl-aminopyrimidine derivatives were designed fusing the pharmacophore templates of etravirine-VRX-480773 hybrids our group previously described and piperidine-linked aminopyrimidines. Most compounds displayed significantly improved activity against wild-type HIV-1 with EC50 values in single-digit nanomolar concentrations compared to etravirine-VRX-480773 hybrids. Selected compounds were also evaluated for activity against reverse transcriptase, and had lower IC50 values than that of nevirapine. The improved potency observed in this in vitro model of HIV RNA replication partly validates the mechanism by which this class of allosteric pyrimidine derivatives inhibits reverse transcriptase, and represents a remarkable step forward in the development of AIDS therapeutics. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

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

Grossman, L.

Two uracil photoproducts are formed when polyuridylic acid (poly U) is irradiated with ultraviolet light. A molecule of water may add at the 4,-5 double bond of the uracil moieties as a result of irradiation and these may be reconverted to uracil by base-catalyzed dehydration. The other photoproduct formed is a uracil-uracil dimer, which reverts to uracil by reirradiation at lower wavelengths of ultraviolet light. The effects of irradiated poly U were studied iu the amino acid incorporating system in which dehydration and photoreversal of the irradiated poly U separated some of the ultraviolet effects. It was concluded that themore » water adduct is responsible for the coding transition of C 14-phenylalanine to C 14-serine, and the formation of dimer results in the loss of the incorporation of C 14-phenylalanine, which is not replaced by any other amino acid.« less

Synthesis of 5-(1,2,3-triazol-4-yl)-2'-deoxyuridines by a click chemistry approach: stacking of triazoles in the major groove gives increased nucleic acid duplex stability.

PubMed

Kocalka, Petr; Andersen, Nicolai K; Jensen, Frank; Nielsen, Poul

2007-11-23

A general protocol for converting alkyl and aryl halides into azides and for converting these in situ into 1,4-disubstituted triazoles was applied with 5-ethynyl-2'-deoxyuridine. This afforded three modified 2'-deoxyuridine analogues with either unsubstituted or 1-phenyl-/1-benzyl-substituted triazoles in their 5-positions. Modelling demonstrates coplanarity of the two heteroaromatic rings, and UV spectroscopy showed the uracil pK(a) values to be almost unchanged. The three nucleosides were introduced into nonamer oligonucleotides by phosphoramidite chemistry. The heteroaromatic triazoles became positioned in the major grooves of the short dsDNA and DNA-RNA duplexes. While single modifications led to decreased duplex stability, the stacking of four consecutive modifications led to enhanced duplex stability, especially for DNA-RNA duplexes. The duplex structures were studied by CD spectroscopy and molecular dynamics simulations, which supported the conjecture that the duplex stabilizing effect is due to efficient stacking of the heteroaromatic triazoles.

Novel Method for Simultaneous Quantification of Phenotypic Resistance to Maturation, Protease, Reverse Transcriptase, and Integrase HIV Inhibitors Based on 3′Gag(p2/p7/p1/p6)/PR/RT/INT-Recombinant Viruses: a Useful Tool in the Multitarget Era of Antiretroviral Therapy▿†

PubMed Central

Weber, Jan; Vazquez, Ana C.; Winner, Dane; Rose, Justine D.; Wylie, Doug; Rhea, Ariel M.; Henry, Kenneth; Pappas, Jennifer; Wright, Alison; Mohamed, Nizar; Gibson, Richard; Rodriguez, Benigno; Soriano, Vicente; King, Kevin; Arts, Eric J.; Olivo, Paul D.; Quiñones-Mateu, Miguel E.

2011-01-01

Twenty-six antiretroviral drugs (ARVs), targeting five different steps in the life cycle of the human immunodeficiency virus type 1 (HIV-1), have been approved for the treatment of HIV-1 infection. Accordingly, HIV-1 phenotypic assays based on common cloning technology currently employ three, or possibly four, different recombinant viruses. Here, we describe a system to assess HIV-1 resistance to all drugs targeting the three viral enzymes as well as viral assembly using a single patient-derived, chimeric virus. Patient-derived p2-INT (gag-p2/NCp7/p1/p6/pol-PR/RT/IN) products were PCR amplified as a single fragment (3,428 bp) or two overlapping fragments (1,657 bp and 2,002 bp) and then recombined into a vector containing a near-full-length HIV-1 genome with the Saccharomyces cerevisiae uracil biosynthesis gene (URA3) replacing the 3,428 bp p2-INT segment (Dudley et al., Biotechniques 46:458–467, 2009). P2-INT-recombinant viruses were employed in drug susceptibility assays to test the activity of protease (PI), nucleoside/nucleotide reverse transcriptase (NRTI), nonnucleoside reverse transcriptase (NNRTI), and integrase strand-transfer (INSTI) inhibitors. Using a single standardized test (ViralARTS HIV), this new technology permits the rapid and automated quantification of phenotypic resistance for all known and candidate antiretroviral drugs targeting all viral enzymes (PR, RT, including polymerase and RNase H activities, and IN), some of the current and potential assembly inhibitors, and any drug targeting Pol or Gag precursor cleavage sites (relevant for PI and maturation inhibitors) This novel assay may be instrumental (i) in the development and clinical assessment of novel ARV drugs and (ii) to monitor patients failing prior complex treatment regimens. PMID:21628544

Sorption of organic molecules on surfaces of a microporous polymer adsorbent modified with different quantities of uracil

NASA Astrophysics Data System (ADS)

Gus'kov, V. Yu.; Ganieva, A. G.; Kudasheva, F. Kh.

2016-11-01

The sorption of organic molecules on the surfaces of a number of adsorbents based on a microporous copolymer of styrene and divinylbenzene modified with different quantities of uracil is studied by means of inverse gas chromatography at infinite dilution. Samples containing 10-6, 10-5, 10-4, 10-3, 10-2, and 0.5 × 10‒1 weight parts of uracil (the pC of uracil ranges from 1.3 to 6) are studied. The contributions from different intermolecular interactions to the Helmholtz energy of sorption are calculated via the linear free energy relationship. It is found that as the concentration of uracil on the surface of the polymer adsorbent grows, the contributions from different intermolecular interactions and the conventional polarity of the surface have a bend at pC = 3, due probably to the formation of a supramolecular structure of uracil. Based on the obtained results, it is concluded that the formation of the supramolecular structure of uracil on the surface of the polymer adsorbent starts when pC < 3.

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 ).

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.

1H-1,2,3-triazole-tethered uracil-ferrocene and uracil-ferrocenylchalcone conjugates: Synthesis and antitubercular evaluation.

PubMed

Singh, Amandeep; Biot, Christophe; Viljoen, Albertus; Dupont, Christian; Kremer, Laurent; Kumar, Kewal; Kumar, Vipan

2017-06-01

Copper-catalyzed azide-alkyne [3 + 2] cycloaddition has been utilized for preparing a series of 1H-1,2,3-triazoles with the purpose of probing structure-activity relationships among a uracil-ferrocene-triazole conjugate family. The antitubercular evaluation studies revealed an improvement in activity with the introduction of a ferrocene nucleus among N-alkylazido-uracil precursors, with a preference for a bromo-substituent along with moderate chain lengths of n = 2-6. The reported protocol is a successful approach for integrating uracil-ferrocene-chalcone functionalities tethered via 1H-1,2,3-triazole rings with apparent physicochemical stability. © 2016 John Wiley & Sons A/S.

Semisynthesis, Characterization and Evaluation of New Adenosine Derivatives as Antiproliferative Agents.

PubMed

Valdés Zurita, Francisco; Brown Vega, Nelson; Gutiérrez Cabrera, Margarita

2018-05-08

We describe the semisynthesis and biological effects of adenosine derivatives, which were anticipated to function as agonists for the A₃ receptor. Molecular docking was used to select candidate compounds. Fifteen nucleoside derivatives were obtained through nucleophilic substitutions of the N ⁶-position of the nucleoside precursor 6-chloropurine riboside by amines of different origin. All compounds were purified by column chromatography and further characterized by spectroscopic and spectrometric techniques, showing moderate yield. These molecules were then evaluated for their antiproliferative activity in human gastric cancer cells expressing the A₃ receptor. We found that the compounds obtained have antiproliferative activity and that new structural modifications can enhance their biological activity. The ADME (Absorption, Distribution, Metabolism and Excretion) properties of the most active compounds were also evaluated theoretically.

[Polymethylene derivatives of nucleic bases with omega-functional groups: VII. Cytotoxicity in the series of N-(2-oxocyclohexyl)-omega-oxoalkyl substituted purines and pyrimidines].

PubMed

Komissarov, V V; Volgareva, G M; Ol'shanskaia, Ia S; Chernyshova, M E; Zavalishina, L E; Frank, G A; Shtil', A A; Kritsyn, A M

2009-01-01

New polymethylene derivatives of nucleic bases with a beta-diketo function in the omega-position were obtained by alkylation of uracil, thymine, cytosine, hypoxanthine, adenine, and N(2)-isobutyryl guanine with 2-omega-chloroal-kanoyl)cyclohexanones. The physical and chemical characteristics of the compounds synthesized and their effect on the K562 and HCT116 tumor cell lines were studied.

Effects of microsolvation on uracil and its radical anion: Uracil.(H2O)n (n=1-5)

NASA Astrophysics Data System (ADS)

Kim, Sunghwan; Schaefer, Henry F.

2006-10-01

Microsolvation effects on the stabilities of uracil and its anion have been investigated by explicitly considering the structures of complexes of uracil with up to five water molecules at the B3LYP /DZP++ level of theory. For all five systems, the global minimum of the neutral cluster has a different equilibrium geometry from that of the radical anion. Both the vertical detachment energy (VDE) and adiabatic electron affinity (AEA) of uracil are predicted to increase gradually with the number of hydrating molecules, qualitatively consistent with experimental results from a photodetachment-photoelectron spectroscopy study [J. Schiedt et al., Chem. Phys. 239, 511 (1998)]. The trend in the AEAs implies that while the conventional valence radical anion of uracil is only marginally bound in the gas phase, it will form a stable anion in aqueous solution. The gas-phase AEA of uracil (0.24eV) was higher than that of thymine by 0.04eV and this gap was not significantly affected by microsolvation. The largest AEA is that predicted for uracil•(H2O)5, namely, 0.96eV. The VDEs range from 0.76to1.78eV.

Electrophilic 5-Substituted Uracils as Potential Radiosensitizers: A Density Functional Theory Study.

PubMed

Makurat, Samanta; Chomicz-Mańka, Lidia; Rak, Janusz

2016-08-18

Although 5-bromo-2'-deoxyuridine (5BrdU) possesses significant radiosensitizing power in vitro, clinical studies do not confirm any advantages of radiotherapy employing 5BrdU. This situation calls for a continuous search for efficient radiosensitizers. Using the proposed mechanism of radiosensitization by 5BrdU, we propose a series of 5-substituted uracils, XYU, that should undergo efficient dissociative electron attachment. The DFT-calculated thermodynamic and kinetic data concerning the XYU degradations induced by electron addition suggests that some of the scrutinized derivatives have much better characteristics than 5BrdU itself. Synthesis of these promising candidates for radiosensitizers, followed by studies of their radiosensitizing properties in DNA context, and ultimately in cancer cells, are further steps to confirm their potential applicability in anticancer treatment. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular modeling studies of 1,4-dihydro-4-oxoquinoline ribonucleosides with anti-HSV-1 activity

NASA Astrophysics Data System (ADS)

Yoneda, Julliane Diniz; Albuquerque, Magaly Girão; Leal, Kátia Zaccur; Seidl, Peter Rudolf; de Alencastro, Ricardo Bicca

2011-12-01

Eight human herpes viruses ( e.g., herpes simplex, varicella-zoster, Epstein-Barr, cytomegalovirus, Kaposi's sarcoma) are responsible for several diseases from sub-clinic manifestations to fatal infections, mostly in immunocompromised patients. The major limitations of the currently available antiviral drug therapy are drug resistance, host toxicity, and narrow spectrum of activity. However, some non-nucleoside 1,4-dihydro-4-oxoquinoline derivatives ( e.g., PNU-183792) [4] shows broad spectrum antiviral activity. We have developed molecular modeling studies, including molecular docking and molecular dynamics simulations, based on a model proposed by Liu and co-workers [14] in order to understand the mechanism of action of a 6-chloro substituted 1,4-dihydro-4-oxoquinoline ribonucleoside, synthesized by the synthetic group, which showed anti-HSV-1 activity [9]. The molecular docking simulations confirmed the Liu's model showing that the ligand needs to dislocate template residues from the active site in order to interact with the viral DNA polymerase enzyme, reinforcing that the interaction with the Val823 residue is pivotal for the inhibitory activity of non-nucleoside 1,4-dihydro-4-oxoquinoline derivatives, such as PNU-183792, with the HSV-1. The molecular dynamics simulations showed that the 6-chloro-benzyl group of PNU-183792 maintains its interaction with residues of the HSV-1 DNA polymerase hydrophobic pocket, considered important according to the Liu's model, and also showed that the methyl group bounded to the nitrogen atom from PNU-183792 is probably contributing to a push-pull effect with the carbonyl group.

Prebiotic synthesis of 5-substituted uracils: a bridge between the RNA world and the DNA-protein world.

PubMed

Robertson, M P; Miller, S L

1995-05-05

Under prebiotic conditions, formaldehyde adds to uracil at the C-5 position to produce 5-hydroxymethyluracil with favorable rates and equilibria. Hydroxymethyluracil adds a variety of nucleophiles, such as ammonia, glycine, guanidine, hydrogen sulfide, hydrogen cyanide, imidazole, indole, and phenol, to give 5-substituted uracils with the side chains of most of the 20 amino acids in proteins. These reactions are sufficiently robust that, if uracil had been present on the primitive Earth, then these substituted uracils would also have been present. The ribozymes of the RNA world would have included many of the functional groups found in proteins today, and their catalytic activities may have been considerably greater than presently assumed.

Uracil in formic acid hydrolysates of deoxyribonucleic acid

PubMed Central

Schein, Arnold H.

1966-01-01

1. When DNA is hydrolysed with formic acid for 30min. at 175° and the hydrolysate is chromatographed on paper with propan-2-ol–2n-hydrochloric acid, in addition to expected ultraviolet-absorbing spots corresponding to guanine, adenine, cytosine and thymine, an ultraviolet-absorbing region with RF similar to that of uracil can be detected. Uracil was separated from this region and identified by its spectra in acid and alkali, and by its RF in several solvent systems. 2. Cytosine, deoxyribocytidine and deoxyribocytidylic acid similarly treated with formic acid all yielded uracil, as did a mixture of deoxyribonucleotides. 3. Approx. 4% of deoxyribonucleotide cytosine was converted into uracil by the formic acid treatment. ImagesFig. 1. PMID:5949371

Prebiotic synthesis of 5-substituted uracils: a bridge between the RNA world and the DNA-protein world

NASA Technical Reports Server (NTRS)

Robertson, M. P.; Miller, S. L.

1995-01-01

Under prebiotic conditions, formaldehyde adds to uracil at the C-5 position to produce 5-hydroxymethyluracil with favorable rates and equilibria. Hydroxymethyluracil adds a variety of nucleophiles, such as ammonia, glycine, guanidine, hydrogen sulfide, hydrogen cyanide, imidazole, indole, and phenol, to give 5-substituted uracils with the side chains of most of the 20 amino acids in proteins. These reactions are sufficiently robust that, if uracil had been present on the primitive Earth, then these substituted uracils would also have been present. The ribozymes of the RNA world would have included many of the functional groups found in proteins today, and their catalytic activities may have been considerably greater than presently assumed.

Arylazolylthioacetanilide. Part 11: design, synthesis and biological evaluation of 1,2,4-triazole thioacetanilide derivatives as novel non-nucleoside HIV-1 reverse transcriptase inhibitors.

PubMed

Li, Zhenyu; Cao, Yuan; Zhan, Peng; Pannecouque, Christophe; Balzarini, Jan; Clercq, Erik De; Shen, Yuemao; Liu, Xinyong

2013-11-01

A series of novel 1,2,4-triazole thioacetanilide derivatives has been designed, synthesized and evaluated for their anti-HIV activities in MT-4 cells. Half of these compounds showed moderate to potent activities against wild-type HIV-1 with an EC50 ranging from 38.0 μM to 4.08 µM. Among them, 2-(4-(2-fluorobenzyl)-5-isopropyl-4H-1,2,4-triazol- 3-ylthio)-N-(2-nitrophenyl)acetamide 7d was identified as the most promising compound (EC50 = 4.26 µM, SI = 49). However, no compound was active against HIV-2. The preliminary structure-activity relationships among the newly synthesized congeners are discussed.

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.

The methyl- and aza-substituent effects on nonradiative decay mechanisms of uracil in water: a transient absorption study in the UV region.

PubMed

Hua, XinZhong; Hua, LinQiang; Liu, XiaoJun

2016-05-18

The nonradiative decay dynamics of photo-excited uracil (Ura) and its derivatives, i.e., thymine (5-methyluracil, Thy), 6-methyluracil (6-MU) and 6-azauracil (6-AU) in water, has been studied using a femtosecond transient absorption method. The molecules are populated in the lowest (1)ππ* state by a pump pulse at 266 nm, and a broadband continuum in the deep UV region is then employed as the probe. The extension of the continuous UV probe down to 250 nm enables us to investigate comprehensively the population dynamics of the ground states for those molecules and to uncover the substituent effects on nonradiative decay dynamics of uracil. Vibrational cooling in the ground states of Ura, Thy and 6-MU has been directly observed for the first time, providing solid evidence of the ultrafast (1)ππ* → S0 decay. In combination with the ground state bleaching signals, it is consolidated that their lowest (1)ππ* state decays via two parallel pathways, i.e., (1)ππ* → S0 and (1)ππ* → (1)nπ*. Moreover, the contribution of the (1)ππ* → (1)nπ* channel is found to be much smaller for Thy or 6-MU than for Ura. Different from methyl-substitution, the initial (1)ππ* state of the aza-substituent 6-AU decays primarily to the (1)nπ* state, while the (1)ππ* → S0 channel can be negligible. Our study provides a comprehensive understanding of the substituent effects on the excited-state dynamics of uracil in water.

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

Synthesis and properties of 2'-O-methyl-4'-thioRNA.

PubMed

Takahashi, Mayumi; Inoue, Naonori; Minakawa, Noriaki; Matsuda, Akira

2005-01-01

In this presentation, we will discuss the synthesis and properties of 2'-O-methyl-4'-thioRNA, an RNA molecule consisting of 2'-O-methyl-4'-thionucleosides. We first synthesized 2'-O-methyl-4'-thiouridine and -cytidine derivatives via 2,2'-O-anhydro-4'-thiouridine. The RNA consisting of 2'-O-methyl-4'-thiopyrimidine nucleosides and 2'-O-methylpurine nucleosides, 2'-OMe-4'-thioRNA, was synthesized on a DNA synthesizer according to the standard phosphoramidite protocol.

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.

DIFFERENTIAL ROLE OF BASE EXCISION REPAIR PROTEINS IN MEDIATING CISPLATIN CYTOTOXICITY

PubMed Central

Sawant, Akshada; Floyd, Ashley M.; Dangeti, Mohan; Lei, Wen; Sobol, Robert W.; Patrick, Steve M.

2017-01-01

Interstrand crosslinks (ICLs) are covalent lesions formed by cisplatin. The mechanism for the processing and removal of ICLs by DNA repair proteins involves nucleotide excision repair (NER), homologous recombination (HR) and fanconi anemia (FA) pathways. In this report, we monitored the processing of a flanking uracil adjacent to a cisplatin ICL by the proteins involved in the base excision repair (BER) pathway. Using a combination of extracts, purified proteins, inhibitors, functional assays and cell culture studies, we determined the specific BER proteins required for processing a DNA substrate with a uracil adjacent to a cisplatin ICL. Uracil DNA glycosylase (UNG) is the primary glycosylase responsible for the removal of uracils adjacent to cisplatin ICLs, whereas other uracil glycosylases can process uracils in the context of undamaged DNA. Repair of the uracil adjacent to cisplatin ICLs proceeds through the classical BER pathway, highlighting the importance of specific proteins in this redundant pathway. Removal of uracil is followed by the generation of an abasic site and subsequent cleavage by AP endonuclease 1 (APE1). Inhibition of either the repair or redox domain of APE1 gives rise to cisplatin resistance. Inhibition of the lyase domain of Polymerase β (Polβ) does not influence cisplatin cytotoxicity. In addition, lack of XRCC1 leads to increased DNA damage and results in increased cisplatin cytotoxicity. Our results indicate that BER activation at cisplatin ICLs influences crosslink repair and modulates cisplatin cytotoxicity via specific UNG, APE1 and Polβ polymerase functions. PMID:28110804

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.

Photochemistry of Pyrimidine in Astrophysical Ices: Formation of Nucleobases and Other Prebiotic Species

NASA Technical Reports Server (NTRS)

Nuevo, Michel; Sandford, Scott A.; Materese, Christopher K.; Milam, Stefanie N.

2012-01-01

Nucleobases are N-heterocycles that are the informational subunits of DNA and RNA. They are divided into two molecular groups: pyrimidine bases (uracil, cytosine, and thymine) and purine bases (adenine and guanine). Nucleobases have been detected in meteorites, and their extraterrestrial origin confirmed by isotopic measurements. Although no N-heterocycles have ever been observed in the ISM, the positions of the 6.2- m interstellar emission features suggest a population of such molecules is likely to be present. However, laboratory experiments have shown that the ultraviolet (UV) irradiation of pyrimidine in ices of astrophysical relevance such as H2O, NH3, CH3OH, CH4, CO, or combinations of these at low temperature (less than or equal to 20 K) leads to the formation of several pyrimidine derivatives including the nucleobases uracil and cytosine, as well as precursors such as 4(3H)-pyrimidone and 4-aminopyrimidine. Quantum calculations on the formation of 4(3H)-pyrimidone and uracil from the irradiation of pyrimidine in pure H2O ices are in agreement with their experimental formation pathways.10 In those residues, other species of prebiotic interest such as urea as well as the amino acids glycine and alanine could also be identified. However, only very small amounts of pyrimidine derivatives containing CH3 groups could be detected, suggesting that the addition of methyl groups to pyrimidine is not an efficient process. For this reason, the nucleobase thymine was not observed in any of the samples. In this work, we study the formation of nucleobases and other photo-products of prebiotic interest from the UV irradiation of pyrimidine in ices containing H2O, NH3, CH3OH, and CO, mixed in astrophysical proportions.

Purification, crystallization and preliminary X-ray analysis of uracil-DNA glycosylase from Sulfolobus tokodaii strain 7

PubMed Central

Kawai, Akito; Higuchi, Shigesada; Tsunoda, Masaru; Nakamura, Kazuo T.; Miyamoto, Shuichi

2012-01-01

Uracil-DNA glycosylase (UDG) specifically removes uracil from DNA by catalyzing hydrolysis of the N-glycosidic bond, thereby initiating the base-excision repair pathway. Although a number of UDG structures have been determined, the structure of archaeal UDG remains unknown. In this study, a deletion mutant of UDG isolated from Sulfolobus tokodaii strain 7 (stoUDGΔ) and stoUDGΔ complexed with uracil were crystallized and analyzed by X-ray crystallography. The crystals were found to belong to the orthorhombic space group P212121, with unit-cell parameters a = 52.2, b = 52.3, c = 74.7 Å and a = 52.1, b = 52.2, c = 74.1 Å for apo stoUDGΔ and stoUDGΔ complexed with uracil, respectively. PMID:22949205

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.

Opinion: uracil DNA glycosylase (UNG) plays distinct and non-canonical roles in somatic hypermutation and class switch recombination.

PubMed

Yousif, Ashraf S; Stanlie, Andre; Begum, Nasim A; Honjo, Tasuku

2014-10-01

Activation-induced cytidine deaminase (AID) is essential to class switch recombination (CSR) and somatic hypermutation (SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair complex, is required for CSR. The role of UNG in CSR and SHM is extremely controversial. AID deficiency in mice abolishes both CSR and SHM, while UNG-deficient mice have drastically reduced CSR but augmented SHM raising a possibility of differential functions of UNG in CSR and SHM. Interestingly, UNG has been associated with a CSR-specific repair adapter protein Brd4, which interacts with acetyl histone 4, γH2AX and 53BP1 to promote non-homologous end joining during CSR. A non-canonical scaffold function of UNG, but not the catalytic activity, can be attributed to the recruitment of essential repair proteins associated with the error-free repair during SHM, and the end joining during CSR. © The Japanese Society for Immunology. 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Differential role of base excision repair proteins in mediating cisplatin cytotoxicity.

PubMed

Sawant, Akshada; Floyd, Ashley M; Dangeti, Mohan; Lei, Wen; Sobol, Robert W; Patrick, Steve M

2017-03-01

Interstrand crosslinks (ICLs) are covalent lesions formed by cisplatin. The mechanism for the processing and removal of ICLs by DNA repair proteins involves nucleotide excision repair (NER), homologous recombination (HR) and fanconi anemia (FA) pathways. In this report, we monitored the processing of a flanking uracil adjacent to a cisplatin ICL by the proteins involved in the base excision repair (BER) pathway. Using a combination of extracts, purified proteins, inhibitors, functional assays and cell culture studies, we determined the specific BER proteins required for processing a DNA substrate with a uracil adjacent to a cisplatin ICL. Uracil DNA glycosylase (UNG) is the primary glycosylase responsible for the removal of uracils adjacent to cisplatin ICLs, whereas other uracil glycosylases can process uracils in the context of undamaged DNA. Repair of the uracil adjacent to cisplatin ICLs proceeds through the classical BER pathway, highlighting the importance of specific proteins in this redundant pathway. Removal of uracil is followed by the generation of an abasic site and subsequent cleavage by AP endonuclease 1 (APE1). Inhibition of either the repair or redox domain of APE1 gives rise to cisplatin resistance. Inhibition of the lyase domain of Polymerase β (Polβ) does not influence cisplatin cytotoxicity. In addition, lack of XRCC1 leads to increased DNA damage and results in increased cisplatin cytotoxicity. Our results indicate that BER activation at cisplatin ICLs influences crosslink repair and modulates cisplatin cytotoxicity via specific UNG, APE1 and Polβ polymerase functions. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficacy of tegafur-uracil in advanced urothelial cancer patients after the treatment failure of platinum-based chemotherapy.

PubMed

Maolake, Aerken; Izumi, Kouji; Takahashi, Rie; Itai, Shingo; Machioka, Kazuaki; Yaegashi, Hiroshi; Nohara, Takahiro; Kitagawa, Yasuhide; Kadono, Yoshifumi; Konaka, Hiroyuki; Mizokami, Atsushi; Namiki, Mikio

2015-03-01

Platinum-based chemotherapy is the first-line treatment for advanced urinary tract urothelial cancers. However, the optimal second-line treatment is unclear. Although tegafur-uracil is sometimes used for advanced urothelial cancer patients after the treatment failure of platinum-based chemotherapy, there is little evidence regarding its use as a second-line treatment. Advanced urothelial cancer patients previously treated with platinum-based chemotherapy were retrospectively analyzed. Overall survival (OS) was compared between patients with and without tegafur-uracil treatment. Thirty-one patients (27 and 4 patients with and without tegafur-uracil treatment, respectively) were analyzed. OS from the last day of the final chemotherapy course was better in patients with tegafur-uracil treatment than in those without (p<0.001, 358 and 66.5 days of the median survival time, respectively). Tegafur-uracil may be a candidate for the secondary treatment of advanced urothelial cancer patients after the treatment failure of platinum-based chemotherapy. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Unravelling the potential of a new uracil phosphoribosyltransferase (UPRT) from Arabidopsis thaliana in sensitizing HeLa cells towards 5-fluorouracil.

PubMed

Narayanan, Sharmila; Sanpui, Pallab; Sahoo, Lingaraj; Ghosh, Siddhartha Sankar

2016-10-01

In silico studies with uracil phosphoribosyltransferase from Arabidopsis thaliana (AtUPRT) revealed its lower binding energies for uracil and 5-fluorouracil (5-FU) as compared to those of bacterial UPRT indicating the prospective of AtUPRT in gene therapy implications. Hence, AtUPRT was cloned and stably expressed in cervical cancer cells (HeLa) to investigate the effect of prodrug 5-FU on these transfected cancer cells. The treatment of AtUPRT-expressing HeLa (HeLa-UPP) cells with 5-FU for 72h resulted in significant decrease in cell viability. Moreover, 5-FU was observed to induce apoptosis and perturb mitochondrial membrane potential in HeLa-UPP cells. While cell cycle analysis revealed significant S-phase arrest as a result of 5-FU treatment in HeLa-UPP cells, quantitative gene expression analysis demonstrated simultaneous upregulation of important cell cycle related genes, cyclin D1 and p21. The survival fractions of non-transfected, vector-transfected and AtUPRT-transfected HeLa cells, following 5-FU treatment, were calculated to be 0.425, 0.366 and 0.227, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Synthesis and biological evaluation of 3-(2-aminoethyl) uracil derivatives as gonadotropin-releasing hormone (GnRH) receptor antagonists.

PubMed

Kim, Seon-Mi; Lee, Minhee; Lee, So Young; Lee, Soo-Min; Kim, Eun Jeong; Kim, Jae Sun; Ann, Jihyae; Lee, Jiyoun; Lee, Jeewoo

2018-02-10

We investigated a series of uracil analogues by introducing various substituents on the phenyl ring of the N-3 aminoethyl side chain and evaluated their antagonistic activity against human gonadotropin-releasing hormone (GnRH) receptors. Analogues with substituents at the ortho or meta position demonstrated potent in vitro antagonistic activity. Specifically, the introduction of a 2-OMe group enhanced nuclear factor of activated T-cells (NFAT) inhibition up to 6-fold compared to the unsubstituted analogue. We identified compound 12c as a highly potent GnRH antagonist with moderate CYP inhibition. Compound 12c showed potent and prolonged LH suppression after a single dose was orally administered in castrated monkeys compared to a known antagonist, Elagolix. We believe that our SAR study offers useful insights to design GnRH antagonists as a potential treatment option for endometriosis. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Anaerobic biodegradation of halogenated and nonhalogenated N-, s-, and o-heterocyclic compounds in aquifer slurries

USGS Publications Warehouse

Adrian, Neal R.; Suflita, Joseph M.

1994-01-01

The fate of several halogenated and nonhalogenated heterocyclic compounds in anoxic aquifer slurries was investigated Substrate depletion and methane formation were monitored in serum bottle incubations by HPLC and GC, respectively Pyridine, pyrimidine, thiophene, and furan were not mineralized following an 11-month incubation, but the corresponding carboxylated or oxygenated compounds were That is, >74% of the theoretically expected amount of methane was recovered from nicotinic acid, uracil, or 2-furoic acid Chlorinated derivatives, like 2 chloro- or 6-chloronicotinic acid, as well as 4 chloro- and 5-chlorouracil resisted mineralization However, 5-bromouracil was reductively dehalogenated to stoichiometric amounts of uracil, whereas 2-chloropyrimidine was metabolized to a more polar unidentified compound that resisted further anaerobic biodegradation Microorganisms acclimated to 5-bromouracil were unable to transform 4 chloro or 5 chlorouracil These findings illustrate how the structure of heterocyclic contaminants influences their susceptibility to anaerobic decay

Nevirapine resistance mutation at codon 181 of the HIV-1 reverse transcriptase confers stavudine resistance by increasing nucleotide substrate discrimination and phosphorolytic activity.

PubMed

Blanca, Giuseppina; Baldanti, Fausto; Paolucci, Stefania; Skoblov, Alexander Yu; Victorova, Lyubov; Hübscher, Ulrich; Gerna, Giuseppe; Spadari, Silvio; Maga, Giovanni

2003-05-02

Recombinant HIV-1 reverse transcriptase (RT) carrying non-nucleoside inhibitors (NNRTIs) resistance mutation at codon 181 showed reduced incorporation and high efficiency of phosphorolytic removal of stavudine, a nucleoside RT inhibitor. These results reveal a new mechanism for cross-resistance between different classes of HIV-1 RT inhibitors.

Comparative absorption spectroscopy involving 4f-4f transitions to explore the kinetics of simultaneous coordination of uracil with Nd(III) and Zn(II) and its associated thermodynamics

NASA Astrophysics Data System (ADS)

Victory Devi, Ch.; Rajmuhon Singh, N.

2011-10-01

The interaction of uracil with Nd(III) has been explored in presence and absence of Zn(II) using the comparative absorption spectroscopy involving the 4f-4f transitions in different solvents. The complexation of uracil with Nd(III) is indicated by the change in intensity of 4f-4f bands expressing in terms of significant change in oscillator strength and Judd-Ofelt parameters. Intensification of this bands became more prominent in presence of Zn(II) suggesting the stimulative effect of Zn(II) towards the complexation of Nd(III) with uracil. Other spectral parameters namely Slator-Condon ( Fk's), nephelauxetic effect ( β), bonding ( b1/2) and percent covalency ( δ) parameters are computed to correlate their simultaneous binding of metal ions with uracil. The sensitivities of the observed 4f-4f transitions towards the minor coordination changes around Nd(III) has been used to monitor the simultaneous coordination of uracil with Nd(III) and Zn(II). The variation of intensities (oscillator strengths and Judd-Ofelt parameters) of 4f-4f bands during the complexation has helped in following the heterobimetallic complexation of uracil. Rate of complexation with respect to hypersensitive transition was evaluated. Energy of activation and thermodynamic parameters for the complexation reaction were also determined.

Single nucleotide polymorphisms in uracil-processing genes, intake of one-carbon nutrients and breast cancer risk

USDA-ARS?s Scientific Manuscript database

Background/Objectives: The misincorporation of uracil into DNA leads to genomic instability. In a previous study, some of us identified four common single nucleotide polymorphisms (SNPs) in uracil-processing genes (rs2029166 and rs7296239 in SMUG1, rs34259 in UNG and rs4775748 in DUT) that were asso...

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 biochemical activity indicate potential use of C. nidus in pharmaceutical applications. PMID:28636672

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 biochemical activity indicate potential use of C. nidus in pharmaceutical applications.

Generation of a Uracil Auxotroph Strain of the Probiotic Yeast Saccharomyces boulardii as a Host for the Recombinant Protein Production

PubMed Central

Hamedi, Hassan; Misaghi, Ali; Modarressi, Mohammad Hossein; Salehi, Taghi Zahraei; Khorasanizadeh, Dorsa; Khalaj, Vahid

2013-01-01

Background Saccharomyces boulardii (S. boulardii) is the best known probiotic yeast. The genetic engineering of this probiotic strain requires the availability of appropriate mutants to accept various gene constructs carrying different selection markers. As the auxotrophy selection markers are under focus, we have generated a ura3 auxotroph mutant of S. boulardii for use in further genetic manipulations. Methods Classical UV mutagenesis was used for the generation of auxotroph mutants. The mutants were selected in the presence of 5-FOA (5-Fluoroorotic acid), uracil and uridine. Uracil auxotrophy phenotype was confirmed by the ability of mutants to grow in the presence of uracil and the lack of growth in the absence of this compound. To test whether the uracil auxotrophy phenotype is due to the inactivation of URA3, the mutants were transformed with a plasmid carrying the gene. An in vitro assay was used for the analysis of acid and bile resistance capacity of these mutants. Results Three mutants were found to be ura3 auxotroph as they were able to grow only in the presence of uracil. When the URA3 gene was added, these mutants were able to grow normally in the absence of uracil. Further in vitro analysis showed that the acid and bile resistance capacity of one of these mutants is intact and similar to the wild type. Conclusion A uracil auxotroph mutant of the probiotic yeast, S. boulardii, was generated and characterized. This auxotroph strain may have potential applications in the production and delivery of the recombinant pharmacuetics into the intestinal lumen. PMID:23626874

Mitochondrial inhibition of uracil-DNA glycosylase is not mutagenic

PubMed Central

Kachhap, Sushant; Singh, Keshav K

2004-01-01

Background Uracil DNA glycosylase (UDG) plays a major role in repair of uracil formed due to deamination of cytosine. UDG in human cells is present in both the nucleus and mitochondrial compartments. Although, UDG's role in the nucleus is well established its role in mitochondria is less clear. Results In order to identify UDG's role in the mitochondria we expressed UGI (uracil glycosylase inhibitor) a natural inhibitor of UDG in the mitochondria. Our studies suggest that inhibition of UDG by UGI in the mitochondria does not lead to either spontaneous or induced mutations in mtDNA. Our studies also suggest that UGI expression has no affect on cellular growth or cytochrome c-oxidase activity. Conclusions These results suggest that human cell mitochondria contain alternatives glycosylase (s) that may function as back up DNA repair protein (s) that repair uracil in the mitochondria. PMID:15574194

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 structurally closely related alpha-substituted cyclic nitrosamines. In part or whole, this may be due to methodological differences in the conduct of the present and previous reports.

Isolation, crystallization and preliminary crystallographic analysis of Salmonella typhimurium uridine phosphorylase crystallized with 2,2′-anhydrouridine

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

Timofeev, Vladimir I.; Lashkov, Alexander A.; Gabdoulkhakov, Azat G.

2007-10-01

S. typhimurium uridine phosphorylase has been isolated and crystallized in the presence of ligand. Uridine phosphorylase (UPh; EC 2.4.2.3) is a member of the pyrimidine nucleoside phosphorylase family of enzymes which catalyzes the phosphorolytic cleavage of the C—N glycoside bond of uridine, with the formation of ribose 1-phosphate and uracil. This enzyme has been shown to be important in the activation and catabolism of fluoropyrimidines. Modulation of its enzymatic activity may affect the therapeutic efficacy of chemotherapeutic agents. The structural investigation of the bacterial uridine phosphorylases, both unliganded and complexed with substrate/product analogues and inhibitors, may help in understanding themore » catalytic mechanism of the phosphorolytic cleavage of uridine. Salmonella typhimurium uridine phosphorylase has been crystallized with 2,2′-anhydrouridine. X-ray diffraction data were collected to 2.15 Å. Preliminary analysis of the diffraction data indicates that the crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 88.52, b = 123.98, c = 133.52 Å. The solvent content is 45.51%, assuming the presence of one hexamer molecule per asymmetric unit.« less

Electron-impact excitation of gas-phase uracil

NASA Astrophysics Data System (ADS)

Chernyshova, I. V.; Kontros, J. E.; Markush, P. P.; Borovik, A. A.; Shpenik, O. B.

2012-11-01

The low lying excited states of uracil have been studied using electron energy-loss spectroscopy. In addition to the dipole allowed transitions to the singlet states, the two lowest triplet states are also observed. In the uracil molecule, the singlet electronic states have been found, being blue-shifted by about 0.5 eV as compared to the UV-absorption results.

Correlated Mutation in the Evolution of Catalysis in Uracil DNA Glycosylase Superfamily

NASA Astrophysics Data System (ADS)

Xia, Bo; Liu, Yinling; Guevara, Jose; Li, Jing; Jilich, Celeste; Yang, Ye; Wang, Liangjiang; Dominy, Brian N.; Cao, Weiguo

2017-04-01

Enzymes in Uracil DNA glycosylase (UDG) superfamily are essential for the removal of uracil. Family 4 UDGa is a robust uracil DNA glycosylase that only acts on double-stranded and single-stranded uracil-containing DNA. Based on mutational, kinetic and modeling analyses, a catalytic mechanism involving leaving group stabilization by H155 in motif 2 and water coordination by N89 in motif 3 is proposed. Mutual Information analysis identifies a complexed correlated mutation network including a strong correlation in the EG doublet in motif 1 of family 4 UDGa and in the QD doublet in motif 1 of family 1 UNG. Conversion of EG doublet in family 4 Thermus thermophilus UDGa to QD doublet increases the catalytic efficiency by over one hundred-fold and seventeen-fold over the E41Q and G42D single mutation, respectively, rectifying the strong correlation in the doublet. Molecular dynamics simulations suggest that the correlated mutations in the doublet in motif 1 position the catalytic H155 in motif 2 to stabilize the leaving uracilate anion. The integrated approach has important implications in studying enzyme evolution and protein structure and function.

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.

Stereoselective synthesis of the 5'-hydroxy-5'-phosphonate derivatives of cytidine and cytosine arabinoside.

PubMed

Chen, Xuemei; Wiemer, Andrew J; Hohl, Raymond J; Wiemer, David F

2002-12-27

Both the (R)- and (S)-5'-hydroxy 5'-phosphonate derivatives of cytidine and cytosine arabinoside (ara-C) have been prepared via phosphite addition or a Lewis acid mediated hydrophosphonylation of appropriately protected 5'-nucleoside aldehydes. Phosphite addition to a cytosine aldehyde protected as the 2',3'-acetonide gave predominately the 5'R isomer, while phosphite addition to the corresponding 2',3'-bis TBS derivative favored the 5'S stereochemistry. In contrast, phosphite addition to the 2',3'-bis TBS protected aldehyde derived from ara-C gave only the 5'R adduct. However, TiCl(4)-mediated hydrophosphonylation of the same ara-C aldehyde favored the 5'S stereoisomer by a 2:1 ratio. Once all four of the diastereomers were in hand, the stereochemistry of these compounds could be assigned based on their spectral data or that obtained from their O-methyl mandelate derivatives. After hydrolysis of the phosphonate esters and various protecting groups, the four alpha-hydroxy phosphonic acids were tested for their ability to serve as substrates for the enzyme nucleoside monophosphate kinase and for their toxicity to K562 cells.

Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs.

PubMed

Girelli Zubani, Giulia; Zivojnovic, Marija; De Smet, Annie; Albagli-Curiel, Olivier; Huetz, François; Weill, Jean-Claude; Reynaud, Claude-Agnès; Storck, Sébastien

2017-04-03

During somatic hypermutation (SHM) of immunoglobulin genes, uracils introduced by activation-induced cytidine deaminase are processed by uracil-DNA glycosylase (UNG) and mismatch repair (MMR) pathways to generate mutations at G-C and A-T base pairs, respectively. Paradoxically, the MMR-nicking complex Pms2/Mlh1 is apparently dispensable for A-T mutagenesis. Thus, how detection of U:G mismatches is translated into the single-strand nick required for error-prone synthesis is an open question. One model proposed that UNG could cooperate with MMR by excising a second uracil in the vicinity of the U:G mismatch, but it failed to explain the low impact of UNG inactivation on A-T mutagenesis. In this study, we show that uracils generated in the G1 phase in B cells can generate equal proportions of A-T and G-C mutations, which suggests that UNG and MMR can operate within the same time frame during SHM. Furthermore, we show that Ung -/- Pms2 -/- mice display a 50% reduction in mutations at A-T base pairs and that most remaining mutations at A-T bases depend on two additional uracil glycosylases, thymine-DNA glycosylase and SMUG1. These results demonstrate that Pms2/Mlh1 and multiple uracil glycosylases act jointly, each one with a distinct strand bias, to enlarge the immunoglobulin gene mutation spectrum from G-C to A-T bases. © 2017 Girelli Zubani et al.

Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs

PubMed Central

De Smet, Annie; Albagli-Curiel, Olivier; Huetz, François; Weill, Jean-Claude

2017-01-01

During somatic hypermutation (SHM) of immunoglobulin genes, uracils introduced by activation-induced cytidine deaminase are processed by uracil-DNA glycosylase (UNG) and mismatch repair (MMR) pathways to generate mutations at G-C and A-T base pairs, respectively. Paradoxically, the MMR-nicking complex Pms2/Mlh1 is apparently dispensable for A-T mutagenesis. Thus, how detection of U:G mismatches is translated into the single-strand nick required for error-prone synthesis is an open question. One model proposed that UNG could cooperate with MMR by excising a second uracil in the vicinity of the U:G mismatch, but it failed to explain the low impact of UNG inactivation on A-T mutagenesis. In this study, we show that uracils generated in the G1 phase in B cells can generate equal proportions of A-T and G-C mutations, which suggests that UNG and MMR can operate within the same time frame during SHM. Furthermore, we show that Ung−/−Pms2−/− mice display a 50% reduction in mutations at A-T base pairs and that most remaining mutations at A-T bases depend on two additional uracil glycosylases, thymine-DNA glycosylase and SMUG1. These results demonstrate that Pms2/Mlh1 and multiple uracil glycosylases act jointly, each one with a distinct strand bias, to enlarge the immunoglobulin gene mutation spectrum from G-C to A-T bases. PMID:28283534

Structure-Activity Analysis of Biased Agonism at the Human Adenosine A3 Receptor

PubMed Central

Baltos, Jo-Anne; Paoletta, Silvia; Nguyen, Anh T. N.; Gregory, Karen J.; Tosh, Dilip K.; Christopoulos, Arthur; Jacobson, Kenneth A.

2016-01-01

Biased agonism at G protein–coupled receptors (GPCRs) has significant implications for current drug discovery, but molecular determinants that govern ligand bias remain largely unknown. The adenosine A3 GPCR (A3AR) is a potential therapeutic target for various conditions, including cancer, inflammation, and ischemia, but for which biased agonism remains largely unexplored. We now report the generation of bias “fingerprints” for prototypical ribose containing A3AR agonists and rigidified (N)-methanocarba 5′-N-methyluronamide nucleoside derivatives with regard to their ability to mediate different signaling pathways. Relative to the reference prototypical agonist IB-MECA, (N)-methanocarba 5′-N-methyluronamide nucleoside derivatives with significant N6 or C2 modifications, including elongated aryl-ethynyl groups, exhibited biased agonism. Significant positive correlation was observed between the C2 substituent length (in Å) and bias toward cell survival. Molecular modeling suggests that extended C2 substituents on (N)-methanocarba 5′-N-methyluronamide nucleosides promote a progressive outward shift of the A3AR transmembrane domain 2, which may contribute to the subset of A3AR conformations stabilized on biased agonist binding. PMID:27136943

Production of uracil from methane by a newly isolated Methylomonas sp. SW1.

PubMed

Kim, Sangwoo; Lee, Wangjun; Song, Insu; Kwon, Yuhyun; Yun, Seokhun; Park, Soohyun; Cho, Sukhyeong; Oh, Byung-Keun; Oh, Han Bin; Lee, Jinwon

2016-12-20

Methane is an abundant, inexpensive one-carbon feedstock and one of the most powerful greenhouse gases. Because it does not compete with food demand, it is considered a promising carbon feedstock for the production of valuable products using methanotrophic bacteria. Here, we isolated a novel methanotrophic bacterium, Methylomonas sp. SW1, from a sewage sample obtained from Wonju City Water Supply Drainage Center, Republic of Korea. The conditions for uracil production by Methylomonas sp. SW1, such as Cu 2+ concentration and temperature were investigated and optimized. As a result, Methylomonas sp. SW1 produced uracil from methane as a sole carbon source with a titer of 2.1mg/L in 84h without genetic engineering under the optimized condition. The results in this study demonstrate the feasibility of using Methylomonas sp. SW1 for the production of uracil from methane. This is the first report of uracil production from gas feedstock by methanotrophic bacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

6-[(Dimethyl­amino)methyl­ene­amino]-1,3-dimethyl­pyrimidine-2,4(1H,3H)-dione dihydrate

PubMed Central

Das, Subrata; Saikia, Binoy K.; Sridhar, B.; Thakur, Ashim J.

2008-01-01

Uracil, the pyrimidine nucleobase, which combined with adenine forms one of the major motifs present in the biopolymer RNA, is also involved in the self-assembly of RNA. In the title compound, C9H14N4O2·2H2O, the asymmetric unit contains one dimethyl­amino­uracil group and two water mol­ecules. The plane of the N=C—NMe2 side chain is inclined at 27.6 (5)° to the plane of the uracil ring. Both water mol­ecules form O—H⋯O hydrogen bonds with the carbonyl O atoms of the uracil group. Additional water–water hydrogen-bond inter­actions are also observed in the crystal structure. The O—H⋯O hydrogen bonds lead to the formation of a two-dimensional hydrogen-bonded network cage consisting of two dimethyl­amino­uracil groups and six water mol­ecules. PMID:21201655

Probing chemical space of tick-borne encephalitis virus reproduction inhibitors with organoselenium compounds.

PubMed

Orlov, Alexey A; Eletskaya, Anastasia A; Frolov, Konstantin A; Golinets, Anastasia D; Palyulin, Vladimir A; Krivokolysko, Sergey G; Kozlovskaya, Liubov I; Dotsenko, Victor V; Osolodkin, Dmitry I

2018-06-01

Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is the leading cause of arboviral neuroinfections in Europe. Only a few classes of the nucleoside and non-nucleoside inhibitors were investigated against TBEV reproduction. Paving the way to previously unexplored areas of anti-TBEV chemical space, we assessed the inhibition of TBEV reproduction in the plaque reduction assay by various compounds derived from cyanothioacetamide and cyanoselenoacetamide. Compounds from seven classes, including 4-(alkylthio)-2-aryl-3-azaspiro[5.5]undec-4-ene-1,1,5-tricarbonitriles, 3-arylamino-2-(selenazol-2-yl)acrylonitriles, ethyl 6-(alkylseleno)-5-cyano-2-oxo-1,2-dihydropyridine-3-carboxylates, 6-(alkylseleno)-2-oxo-1,4,5,6-tetrahydropyridine-3-carbonitriles, 2-(alkylseleno)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carbonitriles, 8-selenoxo-3,5,7,11-tetraazatricyclo[7.3.1.0 2,7 ]tridec-2-ene-1,9-dicarbonitriles, and selenolo[2,3-b]quinolines, inhibited TBEV reproduction with EC 50 values in the micromolar range while showing moderate cytotoxicity and no inhibition of enterovirus reproduction. Thus, new scaffolds with promising anti-TBEV activity were found. © 2018 Deutsche Pharmazeutische Gesellschaft.

New branched DNA constructs.

PubMed

Chandra, Madhavaiah; Keller, Sascha; Gloeckner, Christian; Bornemann, Benjamin; Marx, Andreas

2007-01-01

The Watson-Crick base pairing of DNA is an advantageous phenomenon that can be exploited when using DNA as a scaffold for directed self-organization of nanometer-sized objects. Several reports have appeared in the literature that describe the generation of branched DNA (bDNA) with variable numbers of arms that self-assembles into predesigned architectures. These bDNA units are generated by using cleverly designed rigid crossover DNA molecules. Alternatively, bDNA can be generated by using synthetic branch points derived from either nucleoside or non-nucleoside building blocks. Branched DNA has scarcely been explored for use in nanotechnology or from self-assembling perspectives. Herein, we wish to report our results for the synthesis, characterization, and assembling properties of asymmetrical bDNA molecules that are able to generate linear and circular bDNA constructs. Our strategy for the generation of bDNA is based on a branching point that makes use of a novel protecting-group strategy. The bDNA units were generated by means of automated DNA synthesis methods and were used to generate novel objects by employing chemical and biological techniques. The entities generated might be useful building blocks for DNA-based nanobiotechnology.

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

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.

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.

The Relative Reactivity of Deoxyribose and Ribose: Did DNA Come Before RNA?

NASA Technical Reports Server (NTRS)

Dworkin, Jason P.; Miller, Stanley L.

1995-01-01

If it is assumed that there was a precursor to the ribose-phosphate backbone of RNA in the preRNA world (such as peptide nucleic acid), then the entry of various sugars into the genetic material may be related to the stability and non-enzymatic reactivity of the aldose. The rate of decomposition of 2-deoxyribose has been determined to be 1/3 that of ribose. In addition we have measured the amount of free aldehyde by H-1 and C-13 NMR and find that it has approximately 0.15% free aldehyde compared to 0.05% for ribose at 25 C. This suggests that deoxyribose would be significantly more reactive with early bases in the absence of enzymes. This is confirmed by urazole and deoxyribose reacting to form the deoxynucleoside 45 times faster as 25 C than urazole reacts with ribose to form the Ribonucleoside. Urazole is a potential precursor of uracil and is a plausible prebiotic compound which reacts with aldoses to form nucleosides. Thus the non-enzymatic reactivity of deoxyribose would favor its early use over ribose until enzymes could change the relative reactivities. Most of the reasons that RNA is presumed to have come before DNA are extrapolations back from contemporary metabolism (e.g. the abundance of ribose based coenzymes, the biosynthesis of histidine, deoxyribonucleotides are synthesized from ribonucleotides, etc.). It is very difficult to reconstruct biochemical pathways much before the last common ancestor, and it is even more difficult to do more than guess at the biochemistry of very early self-replicating systems. Thus we believe that these reasons are not compelling and that the non-enzymatic chemistry may be more important than enzymatic pathways for constructing the earliest of biochemical pathways. While the RNA world has been discussed at great length, there has not been an exploration of the transition out of the RNA world. We have constructed many possible schemes of genetic takeover events from preRNA to modern DNA, RNA, protein system which could generate the RNA metabolic fossils we see today.

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.

Constitutional self-organization of adenine-uracil-derived hybrid materials.

PubMed

Arnal-Hérault, Carole; Barboiu, Mihai; Pasc, Andreea; Michau, Mathieu; Perriat, Pascal; van der Lee, Arie

2007-01-01

The alkoxysilane nucleobase adenine (A) and uracil (U) precursors described in this paper generate in solution a complex library of hydrogen-bonded aggregates, which can be expressed in the solid state as discrete higher oligomers. The different interconverting outputs that nucleobases may form by oligomerization define a dynamic polyfunctional diversity that may be "extracted selectively" in solid state by sol-gel transcription, under the intrinsic stability of the system. After the sol-gel process, unique constitutional preference for specific geometries in hybrid materials is consistent with a preferential arrangement of nucleobase systems, favoring the self-assembly by the Hoogsteen geometry. FTIR and NMR spectroscopy and X-ray powder diffraction experiments demonstrate the formation of self-organized hybrid supramolecular materials. Electron microscopy reveals the micrometric platelike morphology of the hybrid materials. The M(A-U) hybrid material is nanostructured in ordered circular domains of 5 nm in diameter of alternative light and dark rows with an one-dimensional periodicity of 3.5 A.

Excited-state properties of nucleic acid components

NASA Astrophysics Data System (ADS)

Salet, C.; Bensasson, R. V.; Becker, R. S.

1981-12-01

Measurements were made of the fluorescence and phosphorescence spectra and lifetimes, and also of the absorption spectra, lifetimes, extinction coefficients, and quantum yields of the T1 lower triplet states of thymine, uracil, their N, N'-dimethyl derivatives, thymidine, thymidine monophosphate, uridine, and uridine monophosphate in various solvents at 300 °K. The influence of the solvent on the quantum yield of the T1 state of nucleic acid components is discussed.

TD-DFT investigation of the magnetic circular dichroism spectra of some purine and pyrimidine bases of nucleic acids.

PubMed

Fahleson, Tobias; Kauczor, Joanna; Norman, Patrick; Santoro, Fabrizio; Improta, Roberto; Coriani, Sonia

2015-05-28

We present a computational study of the magnetic circular dichroism (MCD) spectra in the 200-300 nm wavelength region of purine and its derivative hypoxanthine, as well as of the pyrimidine bases of nucleic acids uracil, thymine, and cytosine, using the B3LYP and CAM-B3LYP functionals. Solvent effects are investigated within the polarizable continuum model and by inclusion of explicit water molecules. In general, the computed spectra are found to be in good agreement with the experimental ones, apart from some overall blue shifts. Both the pseudo-A term shape of the MCD spectra of the purines and the B term shape of the spectra of pyrimidine bases are reproduced. Our calculations also correctly reproduce the reversed phase of the MCD bands in purine compared to that of its derivatives present in nucleic acids. Solvent effects are sizable and system specific, but they do not in general alter the qualitative shape of the spectra. The bands are dominated by the bright π → π* transitions, and our calculations in solution nicely reproduce their energy differences, improving the estimates obtained in the gas phase. Shoulders are predicted for purine and uracil due to n → π* excitations, but they are too weak to be observed in the experiment.

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.

Time-resolved radiation chemistry: Dynamics of electron attachment to uracil following UV excitation of iodide-uracil complexes

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

King, Sarah B.; Yandell, Margaret A.; Stephansen, Anne B.

Electron attachment to uracil was investigated by applying time-resolved photoelectron imaging to iodide-uracil (I{sup –}U) complexes. In these studies, an ultraviolet pump pulse initiated charge transfer from the iodide to the uracil, and the resulting dynamics of the uracil temporary negative ion were probed. Five different excitation energies were used, 4.00 eV, 4.07 eV, 4.14 eV, 4.21 eV, and 4.66 eV. At the four lowest excitation energies, which lie near the vertical detachment energy of the I{sup –}U complex (4.11 eV), signatures of both the dipole bound (DB) as well as the valence bound (VB) anion of uracil were observed.more » In contrast, only the VB anion was observed at 4.66 eV, in agreement with previous experiments in this higher energy range. The early-time dynamics of both states were highly excitation energy dependent. The rise time of the DB anion signal was ∼250 fs at 4.00 eV and 4.07 eV, ∼120 fs at 4.14 eV and cross-correlation limited at 4.21 eV. The VB anion rise time also changed with excitation energy, ranging from 200 to 300 fs for excitation energies 4.00–4.21 eV, to a cross-correlation limited time at 4.66 eV. The results suggest that the DB state acts as a “doorway” state to the VB anion at 4.00–4.21 eV, while direct attachment to the VB anion occurs at 4.66 eV.« less

Insight on specificity of uracil permeases of the NAT/NCS2 family from analysis of the transporter encoded in the pyrimidine utilization operon of Escherichia coli.

PubMed

Botou, Maria; Lazou, Panayiota; Papakostas, Konstantinos; Lambrinidis, George; Evangelidis, Thomas; Mikros, Emmanuel; Frillingos, Stathis

2018-04-01

The uracil permease UraA of Escherichia coli is a structurally known prototype for the ubiquitous Nucleobase-Ascorbate Transporter (NAT) or Nucleobase-Cation Symporter-2 (NCS2) family and represents a well-defined subgroup of bacterial homologs that remain functionally unstudied. Here, we analyze four of these homologs, including RutG of E. coli which shares 35% identity with UraA and is encoded in the catabolic rut (pyrimidine utilization) operon. Using amplified expression in E. coli K-12, we show that RutG is a high-affinity permease for uracil, thymine and, at low efficiency, xanthine and recognizes also 5-fluorouracil and oxypurinol. In contrast, UraA and the homologs from Acinetobacter calcoaceticus and Aeromonas veronii are permeases specific for uracil and 5-fluorouracil. Molecular docking indicates that thymine is hindered from binding to UraA by a highly conserved Phe residue which is absent in RutG. Site-directed replacement of this Phe with Ala in the three uracil-specific homologs allows high-affinity recognition and/or transport of thymine, emulating the RutG profile. Furthermore, all RutG orthologs from enterobacteria retain an Ala at this position, implying that they can use both uracil and thymine and, possibly, xanthine as substrates and provide the bacterial cell with a range of catabolizable nucleobases. © 2018 John Wiley & Sons Ltd.

Studies toward the oxidative and reductive activation of C-S bonds in 2'-S-aryl-2'-thiouridine derivatives.

PubMed

Rayala, Ramanjaneyulu; Giuglio-Tonolo, Alain; Broggi, Julie; Terme, Thierry; Vanelle, Patrice; Theard, Patricia; Médebielle, Maurice; Wnuk, Stanislaw F

2016-04-21

Studies directed toward the oxidative and reductive desulfurization of readily available 2'- S -aryl-2'-thiouridine derivatives were investigated with the prospect to functionalize the C2'-position of nucleosides. The oxidative desulfurization-difluorination strategy was successful on 2-(arylthio)alkanoate surrogates, while extension of the combination of oxidants and fluoride sources was not an efficient fluorination protocol when applied to 2'- S -aryl-2'-thiouridine derivatives, resulting mainly in C5-halogenation of the pyrimidine ring and C2'-monofluorination without desulfurization. Cyclic voltammetry of 2'-arylsulfonyl-2'-deoxyuridines and their 2'-fluorinated analogues showed that cleavage of the arylsulfone moiety could occur, although at relatively high cathodic potentials. While reductive-desulfonylation of 2'-arylsulfonyl-2'-deoxyuridines with organic electron donors (OEDs) gave predominantly base-induced furan type products, chemical (OED) and electrochemical reductive-desulfonylation of the α-fluorosulfone derivatives yielded the 2'-deoxy-2'-fluorouridine and 2',3'-didehydro-2',3'-dideoxy-2'-fluorouridine derivatives. These results provided good evidence of the generation of a C2'-anion through carbon-sulfur bond cleavage, opening new horizons for the reductive-functionalization approaches in nucleosides.

Studies toward the oxidative and reductive activation of C-S bonds in 2'-S-aryl-2'-thiouridine derivatives

PubMed Central

Rayala, Ramanjaneyulu; Giuglio-Tonolo, Alain; Broggi, Julie; Terme, Thierry; Vanelle, Patrice; Theard, Patricia; Médebielle, Maurice; Wnuk, Stanislaw F.

2016-01-01

Studies directed toward the oxidative and reductive desulfurization of readily available 2'-S-aryl-2'-thiouridine derivatives were investigated with the prospect to functionalize the C2'-position of nucleosides. The oxidative desulfurization-difluorination strategy was successful on 2-(arylthio)alkanoate surrogates, while extension of the combination of oxidants and fluoride sources was not an efficient fluorination protocol when applied to 2'-S-aryl-2'-thiouridine derivatives, resulting mainly in C5-halogenation of the pyrimidine ring and C2'-monofluorination without desulfurization. Cyclic voltammetry of 2'-arylsulfonyl-2'-deoxyuridines and their 2'-fluorinated analogues showed that cleavage of the arylsulfone moiety could occur, although at relatively high cathodic potentials. While reductive-desulfonylation of 2'-arylsulfonyl-2'-deoxyuridines with organic electron donors (OEDs) gave predominantly base-induced furan type products, chemical (OED) and electrochemical reductive-desulfonylation of the α-fluorosulfone derivatives yielded the 2'-deoxy-2'-fluorouridine and 2',3'-didehydro-2',3'-dideoxy-2'-fluorouridine derivatives. These results provided good evidence of the generation of a C2'-anion through carbon-sulfur bond cleavage, opening new horizons for the reductive-functionalization approaches in nucleosides. PMID:27019535

Bacterial uracil modulates Drosophila DUOX-dependent gut immunity via Hedgehog-induced signaling endosomes.

PubMed

Lee, Kyung-Ah; Kim, Boram; Bhin, Jinhyuk; Kim, Do Hun; You, Hyejin; Kim, Eun-Kyoung; Kim, Sung-Hee; Ryu, Ji-Hwan; Hwang, Daehee; Lee, Won-Jae

2015-02-11

Genetic studies in Drosophila have demonstrated that generation of microbicidal reactive oxygen species (ROS) through the NADPH dual oxidase (DUOX) is a first line of defense in the gut epithelia. Bacterial uracil acts as DUOX-activating ligand through poorly understood mechanisms. Here, we show that the Hedgehog (Hh) signaling pathway modulates uracil-induced DUOX activation. Uracil-induced Hh signaling is required for intestinal expression of the calcium-dependent cell adhesion molecule Cadherin 99C (Cad99C) and subsequent Cad99C-dependent formation of endosomes. These endosomes play essential roles in uracil-induced ROS production by acting as signaling platforms for PLCβ/PKC/Ca2+-dependent DUOX activation. Animals with impaired Hh signaling exhibit abolished Cad99C-dependent endosome formation and reduced DUOX activity, resulting in high mortality during enteric infection. Importantly, endosome formation, DUOX activation, and normal host survival are restored by genetic reintroduction of Cad99C into enterocytes, demonstrating the important role for Hh signaling in host resistance to enteric infection. Copyright © 2015 Elsevier Inc. All rights reserved.

Hydrogen bond formation between the naturally modified nucleobase and phosphate backbone

PubMed Central

Sheng, Jia; Zhang, Wen; Hassan, Abdalla E. A.; Gan, Jianhua; Soares, Alexei S.; Geng, Song; Ren, Yi; Huang, Zhen

2012-01-01

Natural RNAs, especially tRNAs, are extensively modified to tailor structure and function diversities. Uracil is the most modified nucleobase among all natural nucleobases. Interestingly, >76% of uracil modifications are located on its 5-position. We have investigated the natural 5-methoxy (5-O-CH3) modification of uracil in the context of A-form oligonucleotide duplex. Our X-ray crystal structure indicates first a H-bond formation between the uracil 5-O-CH3 and its 5′-phosphate. This novel H-bond is not observed when the oxygen of 5-O-CH3 is replaced with a larger atom (selenium or sulfur). The 5-O-CH3 modification does not cause significant structure and stability alterations. Moreover, our computational study is consistent with the experimental observation. The investigation on the uracil 5-position demonstrates the importance of this RNA modification at the atomic level. Our finding suggests a general interaction between the nucleobase and backbone and reveals a plausible function of the tRNA 5-O-CH3 modification, which might potentially rigidify the local conformation and facilitates translation. PMID:22641848

Inosine and 2'-deoxyinosine and their synthetic analogues: lipophilicity in the relation to their retention in reversed-phase liquid chromatography and the stability characteristics.

PubMed

Novotny, L; Abdel-Hamid, M; Hamza, H

2000-12-01

The purines and among them inosine synthetic nucleoside derivatives and analogues belong to a group of compounds to which the attention is being paid because of their biological activities. Relationships of their various parameters are being investigated because of their effect on biological (antineoplastic, virostatic, immunosuppressive) properties. Hydrophobicity parameters expressed as the logarithm of the partition coefficient (log P) and the capacity factor k' for naturally occurring inosine, 2'-deoxyinosine, 2'-deoxyadenosine and 2'-deoxyguanosine and for inosine synthetic analogues 5'-deoxyinosine, 5'-chloro-5'-deoxyinosine and 2',3'-dideoxyinosine were measured. The effect of methanol percentage in the mobile phase and its pH on the retention of the studied compounds in a reversed-phase system was also examined. There was a good correlation between the lipophilicity expressed as log P and capacity factor k'. It was also determined that dissociation has a marginal effect on capacity factor k' in this group of nucleoside derivatives as the k' values were almost unchanged at various pH of the mobile phase used. The stability of the all investigated compounds was investigated in basic, neutral and acidic conditions. The values of the reaction constant k1 were calculated and effects of nucleoside structural characteristic on stability are discussed.

Hypochlorite-induced damage to DNA, RNA, and polynucleotides: formation of chloramines and nitrogen-centered radicals.

PubMed

Hawkins, Clare L; Davies, Michael J

2002-01-01

Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is a key bactericidal agent, but can also damage host tissue. As there is a strong link between chronic inflammation and some cancers, we have investigated HOCl damage to DNA, RNA, and polynucleotides. Reaction of HOCl with these materials is shown to yield multiple semistable chloramines (RNHCl/RR'NCl), which are the major initial products, and account for 50-95% of the added HOCl. These chloramines decay by thermal and metal-ion catalyzed processes, to give nucleoside-derived, nitrogen-centered, radicals. The latter have been characterized by EPR spin trapping. The propensity for radical formation with polynucleotides is cytidine > adenosine = guanosine > uridine = thymidine. The rates of decay, and yield of radicals formed, are dependent on the nature of the nucleobase on which they are formed, with chloramines formed from ring heterocyclic amine groups being less stable than those formed on exocyclic amines (RNH2 groups). Evidence is presented for chlorine transfer from the former, kinetically favored, sites to the more thermodynamically favored exocyclic amines. EPR experiments have also provided evidence for the rapid addition of pyrimidine-derived nitrogen-centered radicals to other nucleobases to give dimers and the oxidation of DNA by radicals derived from preformed nucleoside chloramines. Direct reaction of HOCl with plasmid DNA gives rise to single- and double-strand breaks via chloramine-mediated reactions. Preformed nucleoside chloramines also induce plasmid cleavage, though this only occurs to a significant extent with unstable thymidine- and uridine-derived chloramines, where radical formation is rapid. Overall the data rationalize the preferential formation of chlorinated 2'-deoxycytidine and 2'-deoxyadenosine in DNA and suggest that DNA damage induced by HOCl, and preformed chloramines, occurs at sequence-specific sites.

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.

Effect of molecular environment on the vibrational dynamics of pyrimidine bases as analysed by NIS, optical spectroscopy and quantum mechanical force fields

NASA Astrophysics Data System (ADS)

Ghomi, M.; Aamouche, A.; Cadioli, B.; Berthier, G.; Grajcar, L.; Baron, M. H.

1997-06-01

A complete set of vibrational spectra, obtained from several spectroscopic techniques, i.e. neutron inelastic scattering (NIS), Raman scattering and infrared absorption (IR), has been used in order to assign the vibrational modes of pyrimidine bases (uracil, thymine, cytosine) and their N-deuterated species. The spectra of solid and aqueous samples allowed us to analyse the effects of hydrogen bonding in crystal and in solution. In a first step, to assign the observed vibrational modes, we have resorted to harmonic quantum mechanical force field, calculated at SCF + MP2 level using double-zeta 6-31G and D95V basis sets with non-standard exponents for d-orbital polarisation functions. In order to improve the agreement between the experimental results obtained in condensed phases and the calculated ones based on isolated molecules, the molecular force field has been scaled. In a second step, to estimate the effect of intermolecular interactions on the vibrational dynamics of pyrimidine bases, we have undertaken additional calculations with the density functional theory (DFT) method using B3LYP functionals and polarised 6-31G basis sets. Two theoretical models have been considered: 1. a uracil embedded in a dielectric continuum ( ɛ = 78), and 2. a uracil H-bonded to two water molecules (through N1 and N3 atoms).

Response of biological uv dosimeters to the simulated extraterrestrial uv radiation

NASA Astrophysics Data System (ADS)

Bérces, A.; Rontó, G.; Kerékgyártó, T.; Kovács, G.; Lammer, H.

In the Laboratory polycrystalline uracil thin layer and bacteriophage T7 detectors have been developed for UV dosimetry on the EarthSs surface. Exponential response of the uracil polycrystal has been detected both by absorption spectroscopy and measurements of the refractive index under the influence of terrestrial solar radiation or using UV-C sources. In UV biological dosimetry the UV dose scale is additive starting at a value of zero according to the definition of CIE (Technical Report TC-6-18). The biological dose can be defined by a measured end-effect. In our dosimeters (phage T7 and uracil dosimeter) exposed to natural (terrestrial) UV radiation the proportion of pyrimidin photoproducts among the total photoproducts is smaller than 0.1 and the linear correlation between the biological and physical dose is higher than 0.9. According to the experimental data this linear relationship is often not valid. We observed that UV radiation did not only induce dimerisation but shorter wavelengths caused monomerisation of pyrimidin dimers. Performing the irradiation in oxygen free environment and using a Deuterium lamp as UV source, we could increase monomerisation against dimerisation thus the DNA-based dosimetrySs additivity rule is not fulfilled in these conditions. In this study we will demonstrate those non-linear experiments which constitute the basis of our biological experiments on the International Space Station.

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).

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.

Indolylarylsulfones as HIV-1 non-nucleoside reverse transcriptase inhibitors: new cyclic substituents at indole-2-carboxamide.

PubMed

La Regina, Giuseppe; Coluccia, Antonio; Brancale, Andrea; Piscitelli, Francesco; Gatti, Valerio; Maga, Giovanni; Samuele, Alberta; Pannecouque, Christophe; Schols, Dominique; Balzarini, Jan; Novellino, Ettore; Silvestri, Romano

2011-03-24

New indolylarylsulfone derivatives bearing cyclic substituents at indole-2-carboxamide linked through a methylene/ethylene spacer were potent inhibitors of the WT HIV-1 replication in CEM and PBMC cells with inhibitory concentrations in the low nanomolar range. Against the mutant L100I and K103N RT HIV-1 strains in MT-4 cells, compounds 20, 24-26, 36, and 40 showed antiviral potency superior to that of NVP and EFV. Against these mutant strains, derivatives 20, 24-26, and 40 were equipotent to ETV. Molecular docking experiments on this novel series of IAS analogues have also suggested that the H-bond interaction between the nitrogen atom in the carboxamide chain of IAS and Glu138:B is important in the binding of these compounds. These results are in accordance with the experimental data obtained on the WT and on the mutant HIV-1 strains tested.

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

Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells.

PubMed

Carta, M C; Mattana, A; Camici, M; Allegrini, S; Tozzi, M G; Sgarrella, F

2001-10-03

Uptake and catabolism of purine nucleosides have been commonly considered as means to salvage the purine ring for nucleic acid synthesis, usually neglecting the destiny of the pentose moiety. With the aim to ascertain if deoxyribose derived from exogenous DNA can be utilised as a carbon and energy source, we studied the catabolism of exogenous deoxyinosine in a cell line derived from human amnion epithelium (WISH). Intact WISH cells catabolise deoxyinosine by conversion into hypoxanthine. The nucleoside enters the cell through a nitrobenzylthioinosine-insensitive equilibrative transport. Deoxyinosine undergoes a phosphorolytic cleavage inside the cell. The purine base diffuses back to the external medium, while the phosphorylated pentose moiety can be further catabolised to glycolysis and citric acid cycle intermediates. Our results indicate that the catabolism of the deoxynucleoside can be considered mainly as a means to meet the carbon and energy requirements of growing cells.

Specificity and Catalytic Mechanism in Family 5 Uracil DNA Glycosylase*

PubMed Central

Xia, Bo; Liu, Yinling; Li, Wei; Brice, Allyn R.; Dominy, Brian N.; Cao, Weiguo

2014-01-01

UDGb belongs to family 5 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that family 5 UDGb from Thermus thermophilus HB8 is not only a uracil DNA glycosyase acting on G/U, T/U, C/U, and A/U base pairs, but also a hypoxanthine DNA glycosylase acting on G/I, T/I, and A/I base pairs and a xanthine DNA glycosylase acting on all double-stranded and single-stranded xanthine-containing DNA. Analysis of potentials of mean force indicates that the tendency of hypoxanthine base flipping follows the order of G/I > T/I, A/I > C/I, matching the trend of hypoxanthine DNA glycosylase activity observed in vitro. Genetic analysis indicates that family 5 UDGb can also act as an enzyme to remove uracil incorporated into DNA through the existence of dUTP in the nucleotide pool. Mutational analysis coupled with molecular modeling and molecular dynamics analysis reveals that although hydrogen bonding to O2 of uracil underlies the UDG activity in a dissociative fashion, Tth UDGb relies on multiple catalytic residues to facilitate its excision of hypoxanthine and xanthine. This study underscores the structural and functional diversity in the UDG superfamily. PMID:24838246

A Critical Review on Clinical Application of Separation Techniques for Selective Recognition of Uracil and 5-Fluorouracil.

PubMed

Pandey, Khushaboo; Dubey, Rama Shankar; Prasad, Bhim Bali

2016-03-01

The most important objectives that are frequently found in bio-analytical chemistry involve applying tools to relevant medical/biological problems and refining these applications. Developing a reliable sample preparation step, for the medical and biological fields is another primary objective in analytical chemistry, in order to extract and isolate the analytes of interest from complex biological matrices. Since, main inborn errors of metabolism (IEM) diagnosable through uracil analysis and the therapeutic monitoring of toxic 5-fluoruracil (an important anti-cancerous drug) in dihydropyrimidine dehydrogenase deficient patients, require an ultra-sensitive, reproducible, selective, and accurate analytical techniques for their measurements. Therefore, keeping in view, the diagnostic value of uracil and 5-fluoruracil measurements, this article refines several analytical techniques involved in selective recognition and quantification of uracil and 5-fluoruracil from biological and pharmaceutical samples. The prospective study revealed that implementation of molecularly imprinted polymer as a solid-phase material for sample preparation and preconcentration of uracil and 5-fluoruracil had proven to be effective as it could obviates problems related to tedious separation techniques, owing to protein binding and drastic interferences, from the complex matrices in real samples such as blood plasma, serum samples.

Synthesis of 5'-deoxy-5'-nucleosideacetic acid derivatives

NASA Technical Reports Server (NTRS)

Harada, Kazuo; Orgel, Leslie E.

1990-01-01

Several new 5'-deoxy-5'-nucleosideacetic acid derivatives have been synthesized by the reactions of alkoxycarbonylmethylene triphenylphosphoranes with nucleoside 5'-aldehydes. The oligomerization of adenine derivatives IIa, IIIa, IV, V and guanine derivatives IIc and IIIc in aqueous solution was studied using a water-soluble carbodiimide as a condensing agent. It is found that the saturated acid (IV) tends to cyclize to the lactone, while IIa and unsaturated acids (IIIa and V) oligomerized efficiently, especially in the presence of poly (U) as a template.

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

Combined effects of π-π stacking and hydrogen bonding on the (N1) acidity of uracil and hydrolysis of 2'-deoxyuridine.

PubMed

Kellie, Jennifer L; Navarro-Whyte, Lex; Carvey, Matthew T; Wetmore, Stacey D

2012-03-01

M06-2X/6-31+G(d,p) is used to study the simultaneous effects of π-π stacking interactions with phenylalanine (modeled as benzene) and hydrogen bonding with small molecules (HF, H(2)O, and NH(3)) on the N1 acidity of uracil and the hydrolytic deglycosylation of 2'-deoxyuridine (dU) (facilitated by fully (OH(-)) or partially (HCOO(-)···H(2)O) activated water). When phenylalanine is complexed with isolated uracil, the proton affinity of all acceptor sites significantly increases (by up to 28 kJ mol(-1)), while the N1 acidity slightly decreases (by ~6 kJ mol(-1)). When small molecules are hydrogen bound to uracil, addition of the phenylalanine ring can increase or decrease the acidity of uracil depending on the number and nature (acidity) of the molecules bound. Furthermore, a strong correlation between the effects of π-π stacking on the acidity of U and the dU deglycosylation reaction energetics is found, where the hydrolysis barrier can increase or decrease depending on the nature and number of small molecules bound, the nucleophile considered (which dictates the negative charge on U in the transition state), and the polarity of the (bulk) environment. These findings emphasize that the catalytic (or anticatalytic) role of the active-site aromatic amino acid residues is highly dependent on the situation under consideration. In the case of uracil-DNA glycosylase (UNG), which catalyzes the hydrolytic excision of uracil from DNA, the type of discrete hydrogen-bonding interactions with U, the nature of the nucleophile, and the anticipated weak, nonpolar environment in the active site suggest that phenylalanine will be slightly anticatalytic in the chemical step, and therefore experimentally observed contributions to catalysis may entirely result from associated structural changes that occur prior to deglycosylation.

Analysis of uracil phosphoribosyltransferase expression in Mycobacterium tuberculosis and evaluation of upp knockout strain in infected mice.

PubMed

Villela, Anne Drumond; Pham, Ha; Jones, Victoria; Grzegorzewicz, Anna E; Rodrigues-Junior, Valnês da Silva; Campos, Maria Martha; Basso, Luiz Augusto; Jackson, Mary; Santos, Diógenes Santiago

2017-02-01

The upp (Rv3309c)-encoded uracil phosphoribosyltransferase from Mycobacterium tuberculosis (MtUPRT) converts uracil and 5-phosphoribosyl-α-1-pyrophosphate into pyrophosphate and uridine 5΄-monophosphate, the precursor of all pyrimidine nucleotides. A M. tuberculosis knockout strain for upp gene was generated by allelic replacement. Knockout and complemented strains were validated by a functional assay of uracil incorporation. A basal level of MtUPRT expression is shown to be independent of either growth medium used, addition of bases, or oxygen presence/absence. The upp disruption does not affect M. tuberculosis growth in Middlebrook 7H9 medium, and it is not required for M. tuberculosis virulence in a mouse model of infection. Thus, MtUPRT is unlikely to be a good target for drugs against M. tuberculosis. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Increased postischemic brain injury in mice deficient in uracil-DNA glycosylase

PubMed Central

Endres, Matthias; Biniszkiewicz, Detlev; Sobol, Robert W.; Harms, Christoph; Ahmadi, Michael; Lipski, Andreas; Katchanov, Juri; Mergenthaler, Philipp; Dirnagl, Ulrich; Wilson, Samuel H.; Meisel, Andreas; Jaenisch, Rudolf

2004-01-01

Uracil-DNA glycosylase (UNG) is involved in base excision repair of aberrant uracil residues in nuclear and mitochondrial DNA. Ung knockout mice generated by gene targeting are viable, fertile, and phenotypically normal and have regular mutation rates. However, when exposed to a nitric oxide donor, Ung–/– fibroblasts show an increase in the uracil/cytosine ratio in the genome and augmented cell death. After combined oxygen-glucose deprivation, Ung–/– primary cortical neurons have increased vulnerability to cell death, which is associated with early mitochondrial dysfunction. In vivo, UNG expression and activity are low in brains of naive WT mice but increase significantly after reversible middle cerebral artery occlusion and reperfusion. Moreover, major increases in infarct size are observed in Ung–/– mice compared with littermate control mice. In conclusion, our results provide compelling evidence that UNG is of major importance for tissue repair after brain ischemia. PMID:15199406

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.

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-tumor activity of cisplatin, temsirolimus, bortezomib, bendamustine, cyclophosphamide and rituximab remained comparable between the two cohorts. Conclusions Acquired resistance of MCL cells to araC is associated with downregulation of DCK, enzyme of the nucleotide salvage pathway responsible for the first phosphorylation (=activation) of most nucleoside analogs used in anti-cancer therapy. The data suggest that nucleoside analogs should not be used in the therapy of MCL patients, who relapse after failure of araC-based therapies. PMID:24972933

Uracil misincorporation into DNA and folic acid supplementation

USDA-ARS?s Scientific Manuscript database

BACKGROUND: Folate deficiency decreases thymidylate synthesis from deoxyuridylate, which results in an imbalance of deoxyribonucleotide that may lead to excessive uracil misincorporation (UrMis) into DNA during replication and repair. OBJECTIVE: We evaluated the relation between UrMis in different ...

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.

A robust, sensitive assay for genomic uracil determination by LC/MS/MS reveals lower levels than previously reported.

PubMed

Galashevskaya, Anastasia; Sarno, Antonio; Vågbø, Cathrine B; Aas, Per A; Hagen, Lars; Slupphaug, Geir; Krokan, Hans E

2013-09-01

Considerable progress has been made in understanding the origins of genomic uracil and its role in genome stability and host defense; however, the main question concerning the basal level of uracil in DNA remains disputed. Results from assays designed to quantify genomic uracil vary by almost three orders of magnitude. To address the issues leading to this inconsistency, we explored possible shortcomings with existing methods and developed a sensitive LC/MS/MS-based method for the absolute quantification of genomic 2'-deoxyuridine (dUrd). To this end, DNA was enzymatically hydrolyzed to 2'-deoxyribonucleosides and dUrd was purified in a preparative HPLC step and analyzed by LC/MS/MS. The standard curve was linear over four orders of magnitude with a quantification limit of 5 fmol dUrd. Control samples demonstrated high inter-experimental accuracy (94.3%) and precision (CV 9.7%). An alternative method that employed UNG2 to excise uracil from DNA for LC/MS/MS analysis gave similar results, but the intra-assay variability was significantly greater. We quantified genomic dUrd in Ung(+/+) and Ung(-/-) mouse embryonic fibroblasts and human lymphoblastoid cell lines carrying UNG mutations. DNA-dUrd is 5-fold higher in Ung(-/-) than in Ung(+/+) fibroblasts and 11-fold higher in UNG2 dysfunctional than in UNG2 functional lymphoblastoid cells. We report approximately 400-600 dUrd per human or murine genome in repair-proficient cells, which is lower than results using other methods and suggests that genomic uracil levels may have previously been overestimated. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Biological UV dosimeters in simulated space irradiation conditions

NASA Astrophysics Data System (ADS)

Rontó, G.; Bérces, A.; Fekete, A.; Kovács, G.; Lammer, H.

For the measurement of the harmful biological effect of solar UV radiation bacteriophage T7 and polycrystalline uracil dosimeters were used. For terrestrial dosimetric purposes bacteriophage T7 has been applied in solution, while uracil in the form of thin layers. For space irradiation dosimetry the uracil, phage T7-DNA and bacteriophage T7 thin layer samples were prepared in vacuum tightly closed sandwich forms covered either by calciumfluoride or quartz windows. The experimental conditions tested correspond to the conditions planned in the EXPOSE facility: the samples were surrounded by nitrogen atmosphere at various humidities, their vacuum stability was tested in the vacuum chamber of the Institute of Space Research,, Graz. All kinds of the thin film samples have been stored in an atmosphere containing Nitrogen and Hidrogen, in quality control no change in the structure of them has been found. To attenuate the high extraterrestrial irradiance neutral filters of 0.5 and 1.0 optical densities have been tested. Irradiation of the samples has been performed with various UV sources: solar simulator, low pressure Mercury lamp, Deuterium lamp. Dose-effect functions have been determined using for the evaluation spectrophotometry in the characteristic UV range, HPLC of photoproducts, PCR of two different primer sequences of phage T7-DNA. Photoproduct formation kinetics was followed by the saturation level of uracil thin layer. Attenuation ability of the neutral filters was controlled with low pressure Mercury lamp by the exposure necessary for saturation of uracil dosimeters. A three and tenfold increase in the exposure was found respectively, while the influence of spectral composition of the irradiation source was tested using Deuterium lamp supplied with Ca F2 and quartz filters respectively. A doubling of the irradiance was necessary for the saturation of uracil with quartz filter.

Extracellular palladium-catalysed dealkylation of 5-fluoro-1-propargyl-uracil as a bioorthogonally activated prodrug approach

PubMed Central

Weiss, Jason T.; Dawson, John C.; Macleod, Kenneth G.; Rybski, Witold; Fraser, Craig; Torres-Sánchez, Carmen; Patton, E. Elizabeth; Bradley, Mark; Carragher, Neil O.; Unciti-Broceta, Asier

2014-01-01

A bioorthogonal organometallic reaction is a biocompatible transformation undergone by a synthetic material exclusively through the mediation of a non-biotic metal source; a selective process used to label biomolecules and activate probes in biological environs. Here we report the in vitro bioorthogonal generation of 5-fluorouracil from a biologically inert precursor by heterogeneous Pd0 catalysis. Although independently harmless, combined treatment of 5-fluoro-1-propargyl-uracil and Pd0-functionalized resins exhibits comparable antiproliferative properties to the unmodified drug in colorectal and pancreatic cancer cells. Live-cell imaging and immunoassay studies demonstrate that the cytotoxic activity of the prodrug/Pd0-resin combination is due to the in situ generation of 5-fluorouracil. Pd0-resins can be carefully implanted in the yolk sac of zebrafish embryos and display excellent biocompatibility and local catalytic activity. The in vitro efficacy shown by this masking/activation strategy underlines its potential to develop a bioorthogonally activated prodrug approach and supports further in vivo investigations. PMID:24522696

Transformation by complementation of a uracil auxotroph of the hyper lignin-degrading basidiomycete Phanerochaete sordida YK-624.

PubMed

Yamagishi, Kenji; Kimura, Toshiyuki; Oita, Sigeru; Sugiura, Tatsuki; Hirai, Hirofumi

2007-10-01

Phanerochaete sordida YK-624 is a hyper lignin-degrading basidiomycete possessing greater ligninolytic selectivity than either P. chrysosporium or Trametes versicolor. To construct a gene transformation system for P. sordida YK-624, uracil auxotrophic mutants were generated using a combination of ultraviolet (UV) radiation and 5-fluoroorotate resistance as a selection scheme. An uracil auxotrophic strain (UV-64) was transformed into a uracil prototroph using the marker plasmid pPsURA5 containing the orotate phosphoribosyltransferase gene from P. sordida YK-624. This system generated approximately 50 stable transformants using 2 x 10(7) protoplasts. Southern blot analysis demonstrated that the transformed pPsURA5 was ectopically integrated into the chromosomal DNA of all transformants. The enhanced green fluorescent protein (EGFP) gene was also introduced into UV-64. The transformed EGFP was expressed in the co-transformants driven by P. sordida glyceraldehyde-3-phosphate dehydrogenase gene promoter and terminator regions.

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.

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.

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 methylenetetrahydrofolate reductase C677T mutation induces cell-specific changes in genomic DNA methylation and uracil misincorporation: A possible molecular basis for the site-specific cancer risk modification

PubMed Central

Sohn, Kyoung-Jin; Jang, Hyeran; Campan, Mihaela; Weisenberger, Daniel J.; Dickhout, Jeffrey; Wang, Yi-Cheng; Cho, Robert C.; Yates, Zoe; Lucock, Mark; Chiang, En-Pei; Austin, Richard C.; Choi, Sang-Woon; Laird, Peter W.; Kim, Young-In

2009-01-01

The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with a decreased risk of colon cancer while it may increase the risk of breast cancer. This polymorphism is associated with changes in intracellular folate cofactors, which may affect DNA methylation and synthesis via altered one-carbon transfer reactions. We investigated the effect of this mutation on DNA methylation and uracil misincorporation and its interaction with exogenous folate in further modulating these biomarkers of one-carbon transfer reactions in an in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA-MB-435 breast adenocarcinoma cells. In HCT116 cells, the MTHFR 677T mutation was associated with significantly increased genomic DNA methylation when folate supply was adequate or high; however, in the setting of folate insufficiency, this mutation was associated with significantly decreased genomic DNA methylation. In contrast, in MDA-MB-435 cells, the MTHFR 677T mutation was associated with significantly decreased genomic DNA methylation when folate supply was adequate or high and with no effect when folate supply was low. The MTHFR 677T mutation was associated with a nonsignificant trend toward decreased and increased uracil misincorporation in HCT116 and MDA-MB-435 cells, respectively. Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR 677T mutation in cells derived from the target organs of interest, thus providing a plausible cellular mechanism that may partly explain the site-specific modification of colon and breast cancer risks associated with the MTHFR C677T mutation. PMID:19123462

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.

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

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

[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.

Clay catalysis of oligonucleotide formation: kinetics of the reaction of the 5'-phosphorimidazolides of nucleotides with the non-basic heterocycles uracil and hypoxanthine

NASA Technical Reports Server (NTRS)

Kawamura, K.; Ferris, J. P.

1999-01-01

The montmorillonite clay catalyzed condensation of activated monocleotides to oligomers of RNA is a possible first step in the formation of the proposed RNA world. The rate constants for the condensation of the phosphorimidazolide of adenosine were measured previously and these studies have been extended to the phosphorimidazolides of inosine and uridine in the present work to determine of substitution of neutral heterocycles for the basic adenine ring changes the reaction rate or regioselectivity. The oligomerization reactions of the 5'-phosphoromidazolides of uridine (ImpU) and inosine (ImpI) on montmorillonite yield oligo(U)s and oligo(I)s as long as heptamers. The rate constants for oligonucleotide formation were determined by measuring the rates of formation of the oligomers by HPLC. Both the apparent rate constants in the reaction mixture and the rate constants on the clay surface were calculated using the partition coefficients of the oligomers between the aqueous and clay phases. The rate constants for trimer formation are much greater than those dimer synthesis but there was little difference in the rate constants for the formation of trimers and higher oligomers. The overall rates of oligomerization of the phosphorimidazolides of purine and pyrimidine nucleosides in the presence of montmorillonite clay are the same suggesting that RNA formed on the primitive Earth could have contained a variety of heterocyclic bases. The rate constants for oligomerization of pyrimidine nucleotides on the clay surface are significantly higher than those of purine nucleotides since the pyrimidine nucleotides bind less strongly to the clay than do the purine nucleotides. The differences in the binding is probably due to Van der Waals interactions between the purine bases and the clay surface. Differences in the basicity of the heterocyclic ring in the nucleotide have little effect on the oligomerization process.

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.

Conformational analysis of a quinolonic ribonucleoside with anti-HSV-1 activity

NASA Astrophysics Data System (ADS)

Yoneda, Julliane D.; Velloso, Marcia Helena R.; Leal, Kátia Z.; Azeredo, Rodrigo B. de V.; Sugiura, Makiko; Albuquerque, Magaly G.; Santos, Fernanda da C.; Souza, Maria Cecília B. V. de; Cunha, Anna Claudia; Seidl, Peter R.; Alencastro, Ricardo B. de; Ferreira, Vitor F.

2011-01-01

The infections caused by the Herpes Simplex Virus are one of the most common sources of diseases in adults and several natural nucleoside analogues are currently used in the treatment of these infections. In vitro tests of a series of quinolonic ribonucleosides derivatives synthesized by part of our group indicated that some of them have antiviral activity against HSV-1. The conformational analysis of bioactive compounds is extremely important in order to better understand their chemical structures and biological activity. In this work, we have carried out a nuclear relaxation NMR study of 6-Me ribonucleoside derivative in order to determine if the syn or anti conformation is preferential. The NMR analysis permits the determination of inter-atomic distances by using techniques which are based on nuclear relaxation and related phenomena. Those techniques are non-selective longitudinal or spin-lattice relaxation rates and NULL pulse sequence, which allow the determination of distances between pairs of hydrogen atoms. The results of NMR studies were compared with those obtained by molecular modeling.

3'-O-Substituted 5-(perylen-3-ylethynyl)-2'-deoxyuridines as tick-borne encephalitis virus reproduction inhibitors.

PubMed

Proskurin, Gleb V; Orlov, Alexey A; Brylev, Vladimir A; Kozlovskaya, Liubov I; Chistov, Alexey A; Karganova, Galina G; Palyulin, Vladimir A; Osolodkin, Dmitry I; Korshun, Vladimir A; Aralov, Andrey V

2018-05-26

A series of analogues of potent antiviral perylene nucleoside dUY11 with methylthiomethyl (MTM), azidomethyl (AZM) and HO-C 1-4 -alkyl-1,2,3-triazol-1,4-diyl groups at 3'-O-position as well as the two products of copper-free alkyne-azide cycloaddition of the AZM derivative were prepared and evaluated against tick-borne encephalitis virus (TBEV). Four compounds (4, 6, 8a, 8b) showed EC 50  ≤ 10 nM, thus appearing the most potent TBEV inhibitors to date. Moreover, these nucleosides have higher lipophilicity (clogP) and increased solubility in aq. DMSO vs. parent compound dUY11. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

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

Pyrimidine Biosynthesis Is Not an Essential Function for Trypanosoma brucei Bloodstream Forms

PubMed Central

Munday, Jane C.; Donachie, Anne; Morrison, Liam J.; de Koning, Harry P.

2013-01-01

Background African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host, but it is unknown whether either process is essential to the parasite. Methodology/Principal Findings Pyrimidine requirements for growth were investigated using strictly pyrimidine-free media, with or without single added pyrimidine sources. Growth rates of wild-type bloodstream form Trypanosoma brucei brucei were unchanged in pyrimidine-free medium. The essentiality of the de novo pyrimidine biosynthesis pathway was studied by knocking out the PYR6-5 locus that produces a fusion product of orotate phosphoribosyltransferase (OPRT) and Orotidine Monophosphate Decarboxylase (OMPDCase). The pyrimidine auxotroph was dependent on a suitable extracellular pyrimidine source. Pyrimidine starvation was rapidly lethal and non-reversible, causing incomplete DNA content in new cells. The phenotype could be rescued by addition of uracil; supplementation with uridine, 2′deoxyuridine, and cytidine allowed a diminished growth rate and density. PYR6-5−/− trypanosomes were more sensitive to pyrimidine antimetabolites and displayed increased uracil transport rates and uridine phosphorylase activity. Pyrimidine auxotrophs were able to infect mice although the infection developed much more slowly than infection with the parental, prototrophic trypanosome line. Conclusions/Significance Pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal. PMID:23505454

An uracil-linked hydroxyflavone probe for the recognition of ATP

PubMed Central

Bojtár, Márton; Janzsó-Berend, Péter Zoltán; Mester, Dávid; Hessz, Dóra; Kállay, Mihály; Kubinyi, Miklós

2018-01-01

Background: Nucleotides are essential molecules in living systems due to their paramount importance in various physiological processes. In the past years, numerous attempts were made to selectively recognize and detect these analytes, especially ATP using small-molecule fluorescent chemosensors. Despite the various solutions, the selective detection of ATP is still challenging due to the structural similarity of various nucleotides. In this paper, we report the conjugation of a uracil nucleobase to the known 4’-dimethylamino-hydroxyflavone fluorophore. Results: The complexation of this scaffold with ATP is already known. The complex is held together by stacking and electrostatic interactions. To achieve multi-point recognition, we designed the uracil-appended version of this probe to include complementary base-pairing interactions. The theoretical calculations revealed the availability of multiple complex structures. The synthesis was performed using click chemistry and the nucleotide recognition properties of the probe were evaluated using fluorescence spectroscopy. Conclusions: The first, uracil-containing fluorescent ATP probe based on a hydroxyflavone fluorophore was synthesized and evaluated. A selective complexation with ATP was observed and a ratiometric response in the excitation spectrum. PMID:29719572

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

Ombitasvir, Paritaprevir, Ritonavir, and Dasabuvir

MedlinePlus

... so that the medication will have a greater effect. Dasabuvir is a non-nucleoside NS5B polymerase inhibitor. ... the medication, and whether you experience severe side effects. Do not stop taking ombitasvir, paritaprevir, ritonavir, and ...

Prebiotic Synthesis of Diaminopyrimidine and Thiocytosine

NASA Technical Reports Server (NTRS)

Robertson, Michael P.; Levy, Matthew; Miller, Stanley L.

1996-01-01

The reaction of guanidine hydrochloride with cyanoacetaldehyde gives high yields (40-85%) of 2,4-diaminopyrimidine under the concentrated conditions of a drying lagoon model of prebiotic synthesis, in contrast to the low yields previously obtained under more dilute conditions. The prebiotic source of cyanoacetaldehyde, cyanoacetylene, is produced from electric discharges under reducing conditions. The effect of pH and concentration of guanidine hydrochloride on the rate of synthesis and yield of diaminopyrimidine were investigated, as well as the hydrolysis of diaminopyrimidine to cytosine, isocytosine, and uracil. Thiourea also reacts with cyanoacetaldehyde to give 2-thiocytosine, but the pyrimidine yields are much lower than with guanidine hydrochloride or urea. Thiocytosine hydrolyzes to thiouracil and cytosine and then to uracil. This synthesis would have been a significant prebiotic source of 2-thiopyrimidines and 5-substituted derivatives of thiouracil, many of which occur in tRNA. The applicability of these results to the drying lagoon model of prebiotic synthesis was tested by dry-down experiments where dilute solutions of cyanoacetaldehyde, guanidine hydrochloride, and 0.5 M NaCl were evaporated over varying periods of time. The yields of diaminopyrimidine varied from 1 to 7%. These results show that drying lagoons and beaches may have been major sites of prebiotic syntheses.

Cytosine to uracil conversion through hydrolytic deamination of cytidine monophosphate hydroxy-alkylated on the amino group: a liquid chromatography--electrospray ionization--mass spectrometry investigation.

PubMed

Losito, I; Angelico, R; Introna, B; Ceglie, A; Palmisano, F

2012-10-01

A novel pathway for cytosine to uracil conversion performed in a micellar environment, leading to the generation of uridine monophosphate (UMP), was evidenced during the alkylation reaction of cytidine monophosphate (CMP) by dodecyl epoxide. Liquid chromatography-electrospray ionization - ion trap - mass spectrometry was used to separate and identify the reaction products and to follow their formation over time. The detection of hydroxy-amino-dodecane, concurrently with free UMP, in the reaction mixture suggested that, among the various alkyl-derivatives formed, CMP alkylated on the amino group of cytosine could undergo tautomerization to an imine and hydrolytic deamination, generating UMP. Interestingly, no evidence for this peculiar conversion pathway was obtained when guanosine monophosphate (GMP), the complementary ribonucleotide of CMP, was also present in the reaction mixture, due to the fact that NH(2)-alkylated CMP was not formed in this case. The last finding emphasized the role played by CMP-GMP molecular interactions, mediated by a micellar environment, in hindering the alkylation reaction at the level of the cytosine amino group. Copyright © 2012 John Wiley & Sons, Ltd.

An unconventional family 1 uracil DNA glycosylase in Nitratifractor salsuginis.

PubMed

Li, Jing; Chen, Ran; Yang, Ye; Zhang, Zhemin; Fang, Guang-Chen; Xie, Wei; Cao, Weiguo

2017-12-01

The uracil DNA glycosylase superfamily consists of at least six families with a diverse specificity toward DNA base damage. Family 1 uracil N-glycosylase (UNG) exhibits exclusive specificity on uracil-containing DNA. Here, we report a family 1 UNG homolog from Nitratifractor salsuginis with distinct biochemical features that differentiate it from conventional family 1 UNGs. Globally, the crystal structure of N. salsuginisUNG shows a few additional secondary structural elements. Biochemical and enzyme kinetic analysis, coupled with structural determination, molecular modeling, and molecular dynamics simulations, shows that N. salsuginisUNG contains a salt bridge network that plays an important role in DNA backbone interactions. Disruption of the amino acid residues involved in the salt bridges greatly impedes the enzymatic activity. A tyrosine residue in motif 1 (GQDPY) is one of the distinct sequence features setting family 1 UNG apart from other families. The crystal structure of Y81G mutant indicates that several subtle changes may account for its inactivity. Unlike the conventional family 1 UNG enzymes, N. salsuginisUNG is not inhibited by Ugi, a potent inhibitor specific for family 1 UNG. This study underscores the diversity of paths that a uracil DNA glycosylase may take to acquire its unique structural and biochemical properties during evolution. Structure data are available in the PDB under accession numbers 5X3G and 5X3H. © 2017 Federation of European Biochemical Societies.

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-deoxyribonolactone at 7% and 24% of total 2-deoxyribose oxidation, respectively, with frequencies of 10 lesions per 10(6) nt per Gy (G-value, 13 nmol/J) and 2.4 lesions per 10(6) nt per microM. Studies in TK6 human lymphoblastoid cells, in which the analytical data were corrected for losses sustained during DNA isolation, revealed background levels of 2-deoxyribonolactone and nucleoside 5'-aldehyde of 9.7 and 73 lesions per 10(6) nt, respectively. Gamma-irradiation of the cells caused increases of 0.045 and 0.22 lesions per 10(6) nt per Gy, respectively, which represents a approximately 250-fold quenching effect of the cellular environment similar to that observed in previous studies. The proportions of the various 2-deoxyribose oxidation products generated by gamma-radiation are similar for purified DNA and cells. These results are consistent with solvent exposure as a major determinant of hydroxyl radical reactivity with 2-deoxyribose in DNA, but the large differences between gamma-radiation and Fe(2+)-EDTA suggest that factors other than hydroxyl radical reactivity govern DNA oxidation chemistry.

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.

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. PMID:29765475

Synthesis and dynamic 1H NMR study of pyrazolo substituted pyrrolo[2,3-d]pyrimidines via a regioselective heterocyclization

NASA Astrophysics Data System (ADS)

Bayat, Mohammad; Nasri, Shima

2018-02-01

A new series of pyrrolo[2,3-d]pyrimidine derivatives substituted with pyrazolone were designed and prepared, by the three-component reaction of pyrazolone derivatives, arylglyoxal and 6-aminouracil derivatives in ethanol at reflux. The direction of heterocyclization has confirmed and the structure of final products were identified spectroscopically (IR, 1H- and 13C-NMR, and EI-MS). The significant advantages of this protocol include simplicity, regioselectivity, existence of numerous hydrogen bonding possibilities in product, good yields and catalyst-free approach. When the uracil is 6-amino-1,3-dimethyluracil, the product exists as two tautomers at room temperature. The dynamic NMR effects are observed in the 1H NMR spectra. The calculated free-energy of activation (ΔG≠) for prototropic tautomerism is about 68 ± 2 kJ mol-1.

Sofosbuvir, Velpatasvir, and Voxilaprevir

MedlinePlus

... voxilaprevir is used to treat chronic (long-term) hepatitis C infection (swelling of the liver caused by a virus) in adults who have already received other HCV treatments. Sofosbuvir is a non-nucleoside NS5B polymerase inhibitor. ...

Visual and light scattering spectrometric method for the detection of melamine using uracil 5‧-triphosphate sodium modified gold nanoparticles

NASA Astrophysics Data System (ADS)

Liang, Lijiao; Zhen, Shujun; Huang, Chengzhi

2017-02-01

A highly selective method was presented for colorimetric determination of melamine using uracil 5‧-triphosphate sodium modified gold nanoparticles (UTP-Au NPs) in this paper. Specific hydrogen-bonding interaction between uracil base (U) and melamine resulted in the aggregation of AuNPs, displaying variations of localized surface plasmon resonance (LSPR) features such as color change from red to blue and enhanced localized surface plasmon resonance light scattering (LSPR-LS) signals. Accordingly, the concentration of melamine could be quantified based on naked eye or a spectrometric method. This method was simple, inexpensive, environmental friendly and highly selective, which has been successfully used for the detection of melamine in pretreated liquid milk products with high recoveries.

Biological samples on the ISS-EXPOSE facility for the ROSE/PUR experiment

NASA Astrophysics Data System (ADS)

Rontó, Gy.; Bérces, A.; Fekete, A.; Kerékgyártó, T.; Lammer, H.; Kargl, G.; Kömle, N. I.

2002-11-01

Three types of samples, bacteriophage T7, isolated phage DNA, polycrystalline uracil thin films were prepared in sandwich form that were closed vacuum-tightly with inert gaseous environment. The response of the samples to the following selected space environmental conditions was investigated: temperature (-20 - +40°C), vacuum, short wavelength UV irradiation. Uracil thin layer samples proved to be insensitive for temperature. In the vacuum chamber the structure of the samples in sandwich arrangement did not change. Irradiation with germicidal and Deuterium lamps caused a decrease in the optical density of the uracil layers and the decrease showed a saturation tendency in both cases. The dose-effect curves at germicidal lamp possess a saturation level at a lower optical density than at Deuterium.

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

The activity of thymidine phosphorylase obtained from human uterine leiomyomas and studied in the presence of pyrimidine derivatives.

PubMed

Miszczak-Zaborska, E; Woźniak, K

1997-01-01

Partially purified samples of thymidine phosphorylase were obtained from four preparations of human uterine leiomyomas and uteri using the method of Yoshimura et al. (1990), Biochim. Biophys. Acta 1034, 107-113. Among the studied twelve pyrimidine derivatives, 5-bromouracil, 5-nitrouracil, 5-fluorouracil, 6-aminouracil, 4, 6-dihydroxy-5-nitropyrimidine are competitive inhibitors, while allyloxymethylthymine is an uncompetitive inhibitor of thymidine phosphorylase activity, 6-benzyl-2-thiouracil inhibits the activity of the enzyme in a mixed way. The most potent inhibitor of the thymidine phosphorylase activity is 5-bromouracil and uracil the weakest one. Stronger inhibition of these compounds on the activity of thymidine phosphorylase was found in uterine leiomyomas than in uteri.

Utilization of 2,6-diaminopurine by Salmonella typhimurium.

PubMed Central

Garber, B B; Gots, J S

1980-01-01

The pathway for the utilization of 2,6-diaminopurine (DAP) as an exogenous purine source in Salmonella typhimurium was examined. In strains able to use DAP as a purine source, mutant derivatives lacking either purine nucleoside phosphorylase or adenosine deaminase activity lost the ability to do so. The implied pathway of DAP utilization was via its conversion to DAP ribonucleoside by purine nucleoside phosphorylase, followed by deamination to guanosine by adenosine deaminase. Guanosine can then enter the established purine salvage pathways. In the course of defining this pathway, purine auxotrophs able to utilize DAP as sole purine source were isolated and partially characterized. These mutants fell into several classes, including (i) strains that only required an exogenous source of guanine nucleotides (e.g., guaA and guaB strains); (ii) strains that had a purF genetic lesion (i.e., were defective in alpha-5-phosphoribosyl 1-pyrophosphate amidotransferase activity); and (iii) strains that had constitutive levels of purine nucleoside phosphorylase. Selection among purine auxotrophs blocked in the de novo synthesis of inosine 5'-monophosphate, for efficient growth on DAP as sole source of purine nucleotides, readily yielded mutants which were defective in the regulation of their deoxyribonucleoside-catabolizing enzymes (e.g., deoR mutants). PMID:6782081

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.

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

PubMed

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

2016-05-01

Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final accumulation of the nucleoside. The transcript levels of the five TvNTPDases gene sequences were analyzed by qRT-PCR and the highest gene expressions were found for TvNTPDase 2 and 4. The extracellular guanosine uptake was observed as (13C)GTP nucleotide into parasite DNA and it was lower than that observed for adenosine, labeled as (13C)ATP. These findings indicate the T. vaginalis preference for adenosine uptake and the accumulation of guanosine in the extracellular milieu, corroborating with HPLC data. Our data demonstrate, for the first time, the cascade of guanine nucleotides in T. vaginalis and open possibilities on the study of guanine-related purines other than the classical intracellular activity of G proteins for signal transduction. Copyright © 2016 Elsevier B.V. All rights reserved.

A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities.

PubMed

Wu, Yushu; Yan, Ping; Xu, Xiaowen; Jiang, Wei

2016-03-07

Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL(-1) for UDG and 0.11 U mL(-1) for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses.

Structure-Function Relationship of a Plant NCS1 Member – Homology Modeling and Mutagenesis Identified Residues Critical for Substrate Specificity of PLUTO, a Nucleobase Transporter from Arabidopsis

PubMed Central

Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M. Joanne; Möhlmann, Torsten

2014-01-01

Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. PMID:24621654

Structure-function relationship of a plant NCS1 member--homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis.

PubMed

Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M Joanne; Möhlmann, Torsten

2014-01-01

Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.

Conformational change of adenosine deaminase during ligand-exchange in a crystal.

PubMed

Kinoshita, Takayoshi; Tada, Toshiji; Nakanishi, Isao

2008-08-15

Adenosine deaminase (ADA) perpetuates chronic inflammation by degrading extracellular adenosine which is toxic for lymphocytes. ADA has two distinct conformations: open form and closed form. From the crystal structures with various ligands, the non-nucleoside type inhibitors bind to the active site occupying the critical water-binding-position and sustain the open form of apo-ADA. In contrast, substrate mimics do not occupy the critical position, and induce the large conformational change to the closed form. However, it is difficult to predict the binding of (+)-erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), as it possesses characteristic parts of both the substrate and the non-nucleoside inhibitors. The crystal structure shows that EHNA binds to the open form through a novel recognition of the adenine base accompanying conformational change from the closed form of the PR-ADA complex in crystalline state.

Nucleobases and other Prebiotic Species from the Ultraviolet Irradiation of Pyrimidine in Astrophysical Ices

NASA Technical Reports Server (NTRS)

Sandford, S. A.; Nuevo, M.; Materese, C. K.; Milam, S. N.

2012-01-01

Nucleobases are N-heterocycles that are the informational subunits of DNA and RNA, and are divided into two families: pyrimidine bases (uracil, cytosine, and thymine) and purine bases (adenine and guanine). Nucleobases have been detected in meteorites and their extraterrestrial origin confirmed by isotope measurement. Although no Nheterocycles have ever been observed in the ISM, the positions of the 6.2-m interstellar emission features suggest a population of such molecules is likely to be present. In this work we study the formation of pyrimidine-based molecules, including nucleobases, as well as other species of prebiotic interest, from the ultraviolet (UV) irradiation of pyrimidine in combinations of H2O, NH3, CH3OH, and CH4 ices at low temperature, in order to simulate the astrophysical conditions under which prebiotic species may be formed in the interstellar medium and icy bodies of the Solar System. Experimental: Gas mixtures are prepared in a glass mixing line (background pressure approx. 10(exp -6)-10(exp -5) mbar). Relative proportions between mixture components are determined by their partial pressures. Gas mixtures are then deposited on an aluminum foil attached to a cold finger (15-20 K) and simultaneously irradiated with an H2 lamp emitting UV photons (Lyman and a continuum at approx.160 nm). After irradiation samples are warmed to room temperature, at which time the remaining residues are recovered to be analyzed with liquid and gas chromatographies. Results: These experiments showed that the UV irradiation of pyrimidine mixed in these ices at low temperature leads to the formation of several photoproducts derived from pyrimidine, including the nucleobases uracil and cytosine, as well as their precursors 4(3H)-pyrimidone and 4-aminopyrimidine (Fig. 1). Theoretical quantum calculations on the formation of 4(3H)-pyrimidone and uracil from the irradiation of pyrimidine in pure H2O ices are in agreement with their experimental formation pathways. In those residues, other species of prebiotic interest such as urea and the amino acids glycine and alanine could also be identified. However, no pyrimidine derivatives containing CH3 groups, including the third nucleobase thymine, could be identified, suggesting that the addition of methyl groups to pyrimidine is not an efficient process.

Folate supplementation differently affects uracil content in DNA in the mouse colon and liver

USDA-ARS?s Scientific Manuscript database

High folate intake may increase the risk of cancer, especially in the elderly. The present study examined the effects of ageing and dietary folate on uracil misincorporation into DNA, which has a mutagenic effect, in the mouse colon and liver. Old (18 months; n 42) and young (4 months; n 42) male C5...

Ultramicroelectrode Sensors and Detectors. Considerations of the Stability, Sensitivity, Reproducibility, and Mechanism of Ion Transport in Gas Phase Chromatography and in High Performance Liquid Phase Chromatography

DTIC Science & Technology

1988-07-15

solvents were used. For high performance liquid chromatographic studies, the DNA bases thymine, adenine, cytocine, uracil, and guanine (Aldrich...this experiment. The DNA bases guanine, adenine, cytocine, uracil, and thymine were detected for a gradient elution of a mixture of the bases in a

Alternative bases in the RNA world: the prebiotic synthesis of urazole and its ribosides

NASA Technical Reports Server (NTRS)

Kolb, V. M.; Dworkin, J. P.; Miller, S. L.

1994-01-01

Urazole is a five-membered heterocyclic compound which is isosteric with uracil's hydrogen-bonding segment. Urazole reacts spontaneoulsy with ribose (and other aldoses) to give a mixture of four ribosides: alpha and beta pyranosides and furanosides. This reaction occurs in aqueous solution at mild temperatures. Thermodynamic and kinetic parameters for the reaction of urazole with ribose were determined. In contrast, uracil is completely unreactive with ribose under these conditions. Urazole's unusual reactivity is ascribed to the hydrazine portion of the molecule. Urazole can be synthesized from biuret and hydrazine under prebiotic conditions. The prebiotic synthesis of guanazole, which is isosteric in part to diaminopyrimidine and cytosine, is accomplished from dicyandiamide and hydrazine. Kinetic parameters for both prebiotic reactions were measured. Urazole and guanazole are transparent in the UV, which would be a favorable property in the absence of an ozone layer on the early Earth. Urazole makes hydrogen bonds with adenine in DMSO similar to those of uracil, as established by H NMR. All of these properties make urazole an attractive potential precursor to uracil and guanazole a potential precursor to cytosine in the RNA or pre-RNA world.

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.

8-Oxo-7,8-dihydroadenine and 8-Oxo-7,8-dihydroadenosine-Chemistry, Structure, and Function in RNA and Their Presence in Natural Products and Potential Drug Derivatives.

PubMed

Choi, Yu Jung; Chang, Stephanie J; Gibala, Krzysztof S; Resendiz, Marino J E

2017-05-17

A description and history of the role that 8-oxo-7,8-dihydroadenine (8-oxoAde) and 8-oxo-7,8-dihydroadenosine (8-oxoA) have in various fields has been compiled. This Review focusses on 1) the formation of this oxidatively generated modification in RNA, its interactions with other biopolymers, and its potential role in the development/progression of disease; 2) the independent synthesis and incorporation of this modified nucleoside into oligonucleotides of RNA to display the progress that has been made in establishing its behavior in biologically relevant systems; 3) reported synthetic routes, which date back to 1890, along with the progress that has been made in the total synthesis of the nucleobase, nucleoside, and their corresponding derivatives; and 4) the isolation, total synthesis, and biological activity of natural products containing these moieties as the backbone. The current state of research regarding this oxidatively generated lesion as well as its importance in the context of RNA, natural products, and potential as drug derivatives is illustrated using all available examples reported to date. © 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.

Tautomerism in cytosine and uracil: an experimental and theoretical core level spectroscopic study.

PubMed

Feyer, Vitaliy; Plekan, Oksana; Richter, Robert; Coreno, Marcello; Vall-llosera, Gemma; Prince, Kevin C; Trofimov, Alexander B; Zaytseva, Irina L; Moskovskaya, Tatyana E; Gromov, Evgeniy V; Schirmer, Jochen

2009-05-14

The O, N, and C 1s core level photoemission spectra of the nucleobases cytosine and uracil have been measured in the vapor phase, and the results have been interpreted via theoretical calculations. Our calculations accurately predict the relative binding energies of the core level features observed in the experimental photoemission results and provide a full assignment. In agreement with previous work, a single tautomer of uracil is populated at 405 K, giving rise to relatively simple spectra. At 450 K, three tautomers of cytosine, one of which may consist of two rotamers, are identified, and their populations are determined. This resolves inconsistencies between recent laser studies of this molecule in which the rare imino-oxo tautomer was not observed and older microwave spectra in which it was reported.

Synthesis and antimicrobial activity of new 1-[(tetrazol-5-yl)methyl] indole derivatives, their 1,2,4-triazole thioglycosides and acyclic analogs.

PubMed

El-Sayed, Weal A; Abdel Megeid, Randa E; Abbas, Hebat-Allah S

2011-07-01

New 1-[(tetrazol-5-yl)methyl]indole derivatives, their acyclic nucleoside analogs and the corresponding glycoside derivatives were synthesized. Furthermore, the [)(1,2,4-triazol-3-yl)methyl])-2H-tetrazole derivative as well as the corresponding thioglucoside were prepared. The synthesized compounds were tested for their antimicrobial activity against Aspergillus Niger, Penicillium sp, Candida albican, Bacillus subtilis, Streptococcus lacti, Escherichia coli, Pseudomonas sp., and streptomyces sp. Compounds 3, 5 and 19b exhibited potent antibacterial activity and compounds 4, 5 and 10 exhibited high activities against the tested fungi compared with fusidic acid.

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.

Adenosine, but not guanosine, protects vaginal epithelial cells from Trichomonas vaginalis cytotoxicity.

PubMed

Menezes, Camila Braz; Frasson, Amanda Piccoli; Meirelles, Lucia Collares; Tasca, Tiana

2017-02-01

Trichomonas vaginalis causes the most common non-viral sexually transmitted disease worldwide. The cytoadherence and cytotoxicity upon the vaginal epithelial cells are crucial for the infection. Extracellular nucleotides are released during cell damage and, along with their nucleosides, can activate purinoceptors. The opposing effects of nucleotides versus nucleosides are regulated by ectonucleotidases. Herein we evaluated the hemolysis and cytolysis induced by T. vaginalis, as well as the extracellular nucleotide hydrolysis along with the effects mediated by nucleotides and nucleosides on cytotoxicity. In addition, the gene expression of purinoceptors in host cells was determined. The hemolysis and cytolysis exerted by all T. vaginalis isolates presented positive Pearson correlation. All T. vaginalis isolates were able to hydrolyze nucleotides, showing higher NTPDase than ecto-5'-nucleotidase activity. The most cytotoxic isolate, TV-LACM6, hydrolyzes ATP, GTP with more efficiency than AMP and GMP. The vaginal epithelial cell line (HMVII) expressed the genes for all subtypes of P1, P2X and P2Y receptors. Finally, when nucleotides and nucleosides were tested, the cytotoxic effect elicited by TV-LACM6 was increased with nucleotides. In contrast, the cytotoxicity was reversed by adenosine in presence of EHNA, but not by guanosine, contributing to the understanding of the purinergic signaling role on T. vaginalis cytotoxicity. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

[HIV drug resistance in ART-experienced patients in Cali, Colombia, 2008-2010].

PubMed

Martínez-Cajas, Jorge L; Mueses-Marín, Héctor F; Galindo-Orrego, Pablo; Agudelo, Juan F; Galindo-Quintero, Jaime

2013-01-01

Little has been published in Colombia on HIV drug resistance in patients taking antiretroviral treatment (ART). Currently, the Colombian guidelines do not recommend the use of genotypic antiretroviral resistance tests (GART) for treatment-naive patients or for those experiencing a first therapeutic failure. To determine the frequency of relevant resistance mutations and the degree of susceptibility/ resistance of HIV to antiretroviral drugs (ARVs) in ART-experienced patients. A non-random sample of 170 ART-experienced HIV patients with virologic failure and who underwent GART was recruited. A study of HIV drug resistance was carried out in two groups of patients: one group that underwent early GART and the other group that received late GART testing. The most frequent type of resistance affected the non-nucleoside class (76%). The late-GART group had higher risk of nucleoside analog and protease inhibitor drug resistance, a higher number of resistance mutations and more complex mutational profiles than the early-GART group. A high cross resistance level (30%) was found in the nucleoside analog class. The least affected medications were tenofovir and darunavir. Our results suggest that performing GART late is associated with levels of ARV resistance that could restrict the use of an important number of essential ARV in subsequent regimens. There is a need to revise the current recommendations to include GART prior to start of treatment and after the first virologic failure.

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,…

Enabling techniques in the search for new antibiotics: Combinatorial biosynthesis of sugar-containing antibiotics.

PubMed

Park, Je Won; Nam, Sang-Jip; Yoon, Yeo Joon

2017-06-15

Nature has a talent for inventing a vast number of natural products, including hybrids generated by blending different scaffolds, resulting in a myriad of bioactive chemical entities. Herein, we review the highlights and recent trends (2010-2016) in the combinatorial biosynthesis of sugar-containing antibiotics where nature's structural diversification capabilities are exploited to enable the creation of new anti-infective and anti-proliferative drugs. In this review, we describe the modern combinatorial biosynthetic approaches for polyketide synthase-derived complex and aromatic polyketides, non-ribosomal peptide synthetase-directed lipo-/glycopeptides, aminoglycosides, nucleoside antibiotics, and alkaloids, along with their therapeutic potential. Finally, we present the feasible nexus between combinatorial biosynthesis, systems biology, and synthetic biology as a toolbox to provide new antibiotics that will be indispensable in the post-antibiotic era. Copyright © 2016 Elsevier Inc. All rights reserved.

Synthesis and biological evaluation of 2-thioxopyrimidin-4(1H)-one derivatives as potential non-nucleoside HIV-1 reverse transcriptase inhibitors.

PubMed

Khalifa, Nagy M; Al-Omar, Mohamed A

2014-11-12

A series of new 5-allyl-6-benzylpyrimidin-4(3H)-ones bearing different substituents at the C-2 position of the pyrimidine core have been synthesized and evaluated for their in vitro activities against human immunodeficiency virus type 1 (HIV-1) in the human T-lymphotropic type (MT-4 cell cultures). The majority of the title compounds showed moderate to good activities against HIV-1. Amongst them, 5-allyl-6-benzyl-2-(3-hydroxypropylthio)pyrimidin-4(3H)-one analogue 11c exhibited the most potent anti-HIV-1 activity (IC50 0.32 µM). The biological testing results clearly indicated that the substitution at C-2 position of the pyrimidine ring could increase the anti-HIV-1 reverse transcriptase (RT) activity.

Comparative effectiveness of efavirenz-based antiretroviral regimens in resource-limited settings

PubMed Central

Castillo-Mancilla, Jose R; Campbell, Thomas B

2012-01-01

Efavirenz (EFV) is a non-nucleoside widely used as first-line therapy for HIV-1 infection. Most of the research available on EFV comes from trials performed in industrialized countries and only a few studies have evaluated EFV in resource-limited settings (RLSs). In this article, we present a systematic review of the available randomized-controlled trials performed in RLSs that have compared EFV with other antiretrovirals, such as nevirapine and protease inhibitors. The data derived from these studies show that both EFV and nevirapine are adequate first-line therapy options for HIV-1 infection in RLSs, even in patients with concomitant tuberculosis. However, EFV may show a slight benefit in terms of toxicity and adverse events. By contrast, the data comparing EFV versus protease inhibitors is contradictory and further studies may be required to elucidate these discrepancies. PMID:22707879

Synthesis and Biological Evaluation of 2-Thioxopyrimidin-4(1H)-one Derivatives as Potential Non-Nucleoside HIV-1 Reverse Transcriptase Inhibitors

PubMed Central

Khalifa, Nagy M.; Al-Omar, Mohamed A.

2014-01-01

A series of new 5-allyl-6-benzylpyrimidin-4(3H)-ones bearing different substituents at the C-2 position of the pyrimidine core have been synthesized and evaluated for their in vitro activities against human immunodeficiency virus type 1 (HIV-1) in the human T-lymphotropic type (MT-4 cell cultures). The majority of the title compounds showed moderate to good activities against HIV-1. Amongst them, 5-allyl-6-benzyl-2-(3-hydroxypropylthio)pyrimidin-4(3H)-one analogue 11c exhibited the most potent anti-HIV-1 activity (IC50 0.32 µM). The biological testing results clearly indicated that the substitution at C-2 position of the pyrimidine ring could increase the anti-HIV-1 reverse transcriptase (RT) activity. PMID:25397597

Long-term efficiency of mesenchymal stromal cell-mediated CD-MSC/5FC therapy in human melanoma xenograft model.

PubMed

Kucerova, L; Skolekova, S; Demkova, L; Bohovic, R; Matuskova, M

2014-10-01

Mesenchymal stromal cells (MSC) can be exploited as cellular delivery vehicles for the enzymes converting non-toxic prodrugs to toxic substances. Because of their inherent chemoresistance, they exert potent bystander and antitumor effect. Here we show that the human adipose tissue-derived MSC expressing fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CD-MSC) in combination with 5-fluorocytosine (5FC) mediated a long-term tumor-free survival in the 83.3% of tumor-bearing animals. CD-MSC/5FC treatment induced cytotoxicity against model human melanoma cells EGFP-A375. Only 4% of the therapeutic CD-MSC cells eliminated >98.5% of the tumor cells in vitro. Long-term tumor-free survival was confirmed in 15 out of the 18 animals. However, repeatedly used CD-MSC/5FC therapeutic regimen generated more aggressive and metastatic variant of the melanoma cells EGFP-A375/Rel3. These cells derived from the refractory xenotransplants exhibited increased resistance to the CD-MSC/5FC treatment, altered cell adhesion, migration, tumorigenic and metastatic properties. However, long-term curative effect was achieved by the augmentation of the CD-MSC/5FC regimen along with the inhibition of c-Met/hepatocyte growth factor signaling axis in this aggressive melanoma derivative. In summary, the CD-MSC/5FC regimen can be regarded as a very effective antitumor approach to achieve long-term tumor-free survival as demonstrated on a mouse model of aggressive human melanoma xenografts.

[Thyroid dysfunction in adults infected by human immunodeficiency virus].

PubMed

Abelleira, Erika; De Cross, Graciela A; Pitoia, Fabián

2014-01-01

Patients infected with human immunodeficiency virus (HIV) have a higher prevalence of thyroid dysfunction when compared with the general population. The most frequently observed manifestations are euthyroid sick syndrome, Graves' disease and subclinical hypothyroidism. The relationship between the use of highly active antiretroviral therapy and the increased prevalence of thyroid dysfunction has been demonstrated in several series of patients. Grave's disease is recognized as a consequence of immune restitution syndrome. Besides, several studies have suggested an association between hypothyroidism and the use of nucleoside reverse transcriptase inhibitors, particularly stavudine and non-nucleoside reverse transcriptase inhibitors such as efavirenz. Further studies could provide additional evidence of the need for routine assessment of thyroid function in HIV-infected patients.

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.

Characterization of the Adsorption of Nucleic Acid Bases onto Ferrihydrite via Fourier Transform Infrared and Surface-Enhanced Raman Spectroscopy and X-ray Diffractometry.

PubMed

Canhisares-Filho, José E; Carneiro, Cristine E A; de Santana, Henrique; Urbano, Alexandre; da Costa, Antonio C S; Zaia, Cássia T B V; Zaia, Dimas A M

2015-09-01

Minerals could have played an important role in concentration, protection, and polymerization of biomolecules. Although iron is the fourth most abundant element in Earth's crust, there are few works in the literature that describe the use of iron oxide-hydroxide in prebiotic chemistry experiments. In the present work, the interaction of adenine, thymine, and uracil with ferrihydrite was studied under conditions that resemble those of prebiotic Earth. At acidic pH, anions in artificial seawater decreased the pH at the point of zero charge (pHpzc) of ferrihydrite; and at basic pH, cations increased the pHpzc. The adsorption of nucleic acid bases onto ferrihydrite followed the order adenine > uracil > thymine. Adenine adsorption peaked at neutral pH; however, for thymine and uracil, adsorption increased with increasing pH. Electrostatic interactions did not appear to play an important role on the adsorption of nucleic acid bases onto ferrihydrite. Adenine adsorption onto ferrihydrite was higher in distilled water compared to artificial seawater. After ferrihydrite was mixed with artificial seawaters or nucleic acid bases, X-ray diffractograms and Fourier transform infrared spectra did not show any change. Surface-enhanced Raman spectroscopy showed that the interaction of adenine with ferrihydrite was not pH-dependent. In contrast, the interactions of thymine and uracil with ferrihydrite were pH-dependent such that, at basic pH, thymine and uracil lay flat on the surface of ferrihydrite, and at acidic pH, thymine and uracil were perpendicular to the surface. Ferrihydrite adsorbed much more adenine than thymine; thus adenine would have been better protected against degradation by hydrolysis or UV radiation on prebiotic Earth.

Excision Repair-Initiated Enzyme-Assisted Bicyclic Cascade Signal Amplification for Ultrasensitive Detection of Uracil-DNA Glycosylase.

PubMed

Wang, Li-Juan; Ren, Ming; Zhang, Qianyi; Tang, Bo; Zhang, Chun-Yang

2017-04-18

Uracil-DNA glycosylase (UDG) is an important base excision repair (BER) enzyme responsible for the repair of uracil-induced DNA lesion and the maintenance of genomic integrity, while the aberrant expression of UDG is associated with a variety of cancers. Thus, the accurate detection of UDG activity is essential to biomedical research and clinical diagnosis. Here, we develop a fluorescent method for ultrasensitive detection of UDG activity using excision repair-initiated enzyme-assisted bicyclic cascade signal amplification. This assay involves (1) UDG-actuated uracil-excision repair, (2) excision repair-initiated nicking enzyme-mediated isothermal exponential amplification, (3) ribonuclease H (RNase H)-induced hydrolysis of signal probes for generating fluorescence signal. The presence of UDG enables the removal of uracil from U·A pairs and generates an apurinic/apyrimidinic (AP) site. Endonuclease IV (Endo IV) subsequently cleaves the AP site, resulting in the break of DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate isothermal exponential amplification, producing a large number of triggers. The resultant trigger may selectively hybridize with the signal probe which is modified with FAM and BHQ1, forming a RNA-DNA heterogeneous duplex. The subsequent hydrolysis of RNA-DNA duplex by RNase H leads to the generation of fluorescence signal. This assay exhibits ultrahigh sensitivity with a detection limit of 0.0001 U/mL, and it can even measure UDG activity at the single-cell level. Moreover, this method can be applied for the measurement of kinetic parameters and the screening of inhibitors, thereby providing a powerful tool for DNA repair enzyme-related biomedical research and clinical diagnosis.

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

[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.

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.

Methods for detection of methyl-CpG dinucleotides

DOEpatents

Dunn, John J

2013-11-26

The invention provides methods for enriching methyl-CpG sequences from a DNA sample. The method makes use of conversion of cytosine residues to uracil under conditions in which methyl-cytosine residues are preserved. Additional methods of the invention enable to preservation of the context of me-CpG dinucleotides. The invention also provides a recombinant, full length and substantially pure McrA protein (rMcrA) for binding and isolation of DNA fragments containing the sequence 5'-C.sup.MeCpGG-3'. Methods for making and using the rMcrA protein, and derivatives thereof are provided.

Methods for detection of methyl-CpG dinucleotides

DOEpatents

Dunn, John J.

2013-01-29

The invention provides methods for enriching methyl-CpG sequences from a DNA sample. The method makes use of conversion of cytosine residues to uracil under conditions in which methyl-cytosine residues are preserved. Additional methods of the invention enable to preservation of the context of me-CpG dinucleotides. The invention also provides a recombinant, full length and substantially pure McrA protein (rMcrA) for binding and isolation of DNA fragments containing the sequence 5'-C.sup.MeCpGG-3'. Methods for making and using the rMcrA protein, and derivatives thereof are provided.

Methods for detection of methyl-CpG dinucleotides

DOEpatents

Dunn, John J.

2012-09-11

The invention provides methods for enriching methyl-CpG sequences from a DNA sample. The method makes use of conversion of cytosine residues to uracil under conditions in which methyl-cytosine residues are preserved. Additional methods of the invention enable to preservation of the context of me-CpG dinucleotides. The invention also provides a recombinant, full length and substantially pure McrA protein (rMcrA) for binding and isolation of DNA fragments containing the sequence 5'-C.sup.MeCpGG-3'. Methods for making and using the rMcrA protein, and derivatives thereof are provided.

Electronic structure of uracil-like nucleobases adsorbed on Si(001): uracil, thymine and 5-fluorouracil

NASA Astrophysics Data System (ADS)

Molteni, Elena; Onida, Giovanni; Cappellini, Giancarlo

2016-04-01

We study the electronic properties of the Si(001):Uracil, Si(001):Thymine, and Si(001):5-Fluorouracil systems, focusing on the Si dimer-bridging configuration with adsorption governed by carbonyl groups. While the overall structural and electronic properties are similar, with small differences due to chemical substitutions, much larger effects on the surface band dispersion and bandgap show up as a function of the molecular orientation with respect to the surface. An off-normal orientation of the molecular planes is favored, showing larger bandgap and lower total energy than the upright position. We also analyze the localization of gap-edge occupied and unoccupied surface states. Supplementary material in the form of one pdf file available from the Journal web page at http://dx.doi.org/10.1140/epjb/e2016-70011-1

Electron-induced hydrogen loss in uracil in a water cluster environment

NASA Astrophysics Data System (ADS)

Smyth, M.; Kohanoff, J.; Fabrikant, I. I.

2014-05-01

Low-energy electron-impact hydrogen loss due to dissociative electron attachment (DEA) to the uracil and thymine molecules in a water cluster environment is investigated theoretically. Only the A'-resonance contribution, describing the near-threshold behavior of DEA, is incorporated. Calculations are based on the nonlocal complex potential theory and the multiple scattering theory, and are performed for a model target with basic properties of uracil and thymine, surrounded by five water molecules. The DEA cross section is strongly enhanced when the attaching molecule is embedded in a water cluster. This growth is due to two effects: the increase of the resonance lifetime and the negative shift in the resonance position due to interaction of the intermediate negative ion with the surrounding water molecules. A similar effect was earlier found in DEA to chlorofluorocarbons.

Mechanisms controlling nucleic acid-sensing Toll-like receptors.

PubMed

Miyake, Kensuke; Shibata, Takuma; Ohto, Umeharu; Shimizu, Toshiyuki; Saitoh, Shin-Ichiroh; Fukui, Ryutaro; Murakami, Yusuke

2018-03-08

Nucleic acid (NA)-sensing Toll-like receptors (TLRs) respond to DNA/RNA derived from pathogens and dead cells. Structural studies have revealed a variety of molecular mechanisms by which TLRs sense NAs. Double-stranded RNA and single-stranded DNA directly bind to TLR3 and TLR9, respectively, whereas TLR7 and TLR8 bind to nucleosides and oligoribonucleotides derived from RNAs. Activation of ligand-bound TLRs is influenced by the functional status of TLRs. Proteolytic cleavage of NA-sensing TLRs enables ligand-dependent TLR dimerization. Trafficking of ligand-activated TLRs in endosomal and lysosomal compartments is requisite for production of type I interferons. Activation of NA-sensing TLRs is required for the control of viruses such as herpes simplex virus and endogenous retroviruses. On the other hand, excessive activation of NA-sensing TLRs drives disease progression in a variety of inflammatory diseases including systemic lupus erythematosus, heart failure, arthritis and non-alcoholic steatohepatitis. NA-sensing TLRs are targets for therapeutic intervention in these diseases. We here focus on our recent progresses in our understanding of NA-sensing TLRs.

Enzymatic cleavage of uracil-containing single-stranded DNA linkers for the efficient release of affinity-selected circulating tumor cells.

PubMed

Nair, Soumya V; Witek, Małgorzata A; Jackson, Joshua M; Lindell, Maria A M; Hunsucker, Sally A; Sapp, Travis; Perry, Caroline E; Hupert, Mateusz L; Bae-Jump, Victoria; Gehrig, Paola A; Wysham, Weiya Z; Armistead, Paul M; Voorhees, Peter; Soper, Steven A

2015-02-21

We report a novel strategy to enzymatically release affinity-selected cells, such as circulating tumor cells (CTCs), from surfaces with high efficiency (∼90%) while maintaining cell viability (>85%). The strategy utilizes single-stranded DNAs that link a capture antibody to the surfaces of a CTC selection device. The DNA linkers contain a uracil residue that can be cleaved.

Aging of Escherichia coli

PubMed Central

Clifton, C. E.

1966-01-01

Clifton, C. E. (Stanford University, Stanford, Calif.). Aging of Escherichia coli. J. Bacteriol. 92:905–912. 1966.—The rates of endogenous and exogenous (glucose) respiration decreased much more rapidly than did the viable count during the first 24 hr of aging of washed, C14-labeled cells of Escherichia coli K-12 suspended in a basal salt medium devoid of ammonium salts. The rates of decrease of respiration and of death approached each other as the age of the cells increased, but death was not the only factor involved in decreased respiratory activity of the suspensions. The greatest decrease in cellular contents with aging was noted in the ribonucleic acid fraction, of which the ribose appeared to be oxidized, while uracil accumulated in the suspension medium. The viable count and respiratory activities remained higher in glucose-fed than in nonfed suspensions. Proline-labeled cells fed glucose tended to lose more of their proline and to convert more proline into C14O2 than in unfed controls. On the other hand, uracil-labeled cells fed glucose retained more of the uracil than did nonfed cells, but glucose elicited some oxidation of uracil. An exogenous energy source such as glucose aided in the maintenance of a population, but it was not the only factor needed for such maintenance. PMID:5332874

Product release mechanism and the complete enzyme catalysis cycle in yeast cytosine deaminase (yCD): A computational study.

PubMed

Zhao, Yuan; She, Nai; Zhang, Xin; Wang, Chaojie; Mo, Yirong

2017-08-01

Yeast cytosine deaminase (yCD) is critical in gene-directed enzyme prodrug therapy as it catalyzes the hydrolytic deamination of cytosine. The product (uracil) release process is considered as rate-limiting in the whole enzymatic catalysis and includes the cleavage of the uracil-metal bond and the delivery of free uracil out of the reactive site. Herein extensive combined random acceleration molecular dynamics (RAMD) and molecular dynamics (MD) simulations coupled with the umbrella sampling technique have been performed to study the product transport mechanism. Five channels have been identified, and the thermodynamic and dynamic characterizations for the two most favorable channels have been determined and analyzed. The free energy barrier for the most beneficial pathway is about 13kcal/mol and mainly results from the cleavage of hydrogen bonds between the ligand uracil and surrounding residues Asn51, Glu64, and Asp155. The conjugated rings of Phe114 and Trp152 play gating and guiding roles in the product delivery via π⋯π van der Waals interactions with the product. Finally, the full cycle of the enzymatic catalysis has been determined, making the whole process computationally more precise. Copyright © 2017 Elsevier B.V. All rights reserved.

Photoelectron spectroscopy of the 6-azauracil anion.

PubMed

Chen, Jing; Buonaugurio, Angela; Dolgounitcheva, Olga; Zakrzewski, V G; Bowen, Kit H; Ortiz, J V

2013-02-14

We report the photoelectron spectrum of the 6-azauracil anion. The spectrum is dominated by a broad band exhibiting a maximum at an electron binding energy (EBE) of 1.2 eV. This spectral pattern is indicative of a valence anion. Our calculations were carried out using ab initio electron propagator and other many-body methods. Comparison of the anion and corresponding neutral of 6-azauracil with those of uracil shows that substituting a nitrogen atom for C-H at the C6 position of uracil gives rise to significant changes in the electronic structure of 6-azauracil versus that of uracil. The adiabatic electron affinity (AEA) of the canonical 6-azauracil tautomer is substantially larger than that of canonical uracil. Among the five tautomeric, 6-azauracil anions studied computationally, the canonical structure was found to be the most stable. The vertical detachment energies (VDE) of the canonical, valence-bound anion of 6-azauracil and its closest "very-rare" tautomer have been calculated. Electron propagator calculations on the canonical anion yield a VDE value that is in close agreement with the experimentally determined VDE value of 1.2 eV. The AEA value of 6-azauracil, assessed at the CCSD(T) level of theory to be 0.5 eV, corresponds with the EBE value of the onset of the experimental spectrum.

Anti-Wrinkle and Anti-Inflammatory Effects of Active Garlic Components and the Inhibition of MMPs via NF-κB Signaling

PubMed Central

Kim, So Ra; Jung, Yu Ri; An, Hye Jin; Kim, Dae Hyun; Jang, Eun Ji; Choi, Yeon Ja; Moon, Kyoung Mi; Park, Min Hi; Park, Chan Hum; Chung, Ki Wung; Bae, Ha Ram; Choi, Yung Whan; Kim, Nam Deuk; Chung, Hae Young

2013-01-01

Skin aging is a multisystem degenerative process caused by several factors, such as, UV irradiation, stress, and smoke. Furthermore, wrinkle formation is a striking feature of photoaging and is associated with oxidative stress and inflammatory response. In the present study, we investigated whether caffeic acid, S-allyl cysteine, and uracil, which were isolated from garlic, modulate UVB-induced wrinkle formation and effect the expression of matrix-metalloproteinase (MMP) and NF-κB signaling. The results obtained showed that all three compounds significantly inhibited the degradation of type І procollagen and the expressions of MMPs in vivo and attenuated the histological collagen fiber disorder and oxidative stress in vivo. Furthermore, caffeic acid and S-allyl cysteine were found to decrease oxidative stress and inflammation by modulating the activities of NF-κB and AP-1, and uracil exhibited an indirect anti-oxidant effect by suppressing cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expressions levels and downregulating transcriptional factors. These results suggest that the anti-wrinkle effects of caffeic acid, S-allyl cysteine, and uracil are due to anti-oxidant and/or anti-inflammatory effects. Summarizing, caffeic acid, S-allyl cysteine, and uracil inhibited UVB-induced wrinkle formation by modulating MMP via NF-κB signaling. PMID:24066081

5-Fluorouracil-resistant strain of Methanobacterium thermoautotrophicum.

PubMed

Nagle, D P; Teal, R; Eisenbraun, A

1987-09-01

Growth of Methanobacterium thermoautotrophicum Marburg is inhibited by the pyrimidine, 5-fluorouracil (FU). It was shown previously that methanogenesis is not inhibited to the same extent as growth. A spontaneously occurring FU-resistant strain (RTAE-1) was isolated from a culture of strain Marburg. The growth of both strains was inhibited by 5-fluorodeoxyuridine but not 5-fluorocytosine, and the wild type was more susceptible to inhibition by 5-azauracil and 6-azauracil than was strain RTAE-1. The cellular targets for the pyrimidine analogs are not known. When the accumulation of 14C-labeled uracil or FU by the two strains was compared, the wild type took up 15-fold more radiolabel per cell than did the FU-resistant strain. In the wild type, radiolabel from uracil was incorporated into the soluble pool, RNA, and DNA. The metabolism of uracil appeared to involve a uracil phosphoribosyltransferase activity. Strain Marburg extracts contained this enzyme, whereas FU-resistant strain RTAE-1 extracts had less than 1/10 as much activity. Although it is possible that a change in permeability to the compounds plays a role in the stable resistance of strain RTAE-1, the fact that it lacks the ability to metabolize pyrimidines to nucleotides is sufficient to account for its phenotype.

5-Fluorouracil-resistant strain of Methanobacterium thermoautotrophicum.

PubMed Central

Nagle, D P; Teal, R; Eisenbraun, A

1987-01-01

Growth of Methanobacterium thermoautotrophicum Marburg is inhibited by the pyrimidine, 5-fluorouracil (FU). It was shown previously that methanogenesis is not inhibited to the same extent as growth. A spontaneously occurring FU-resistant strain (RTAE-1) was isolated from a culture of strain Marburg. The growth of both strains was inhibited by 5-fluorodeoxyuridine but not 5-fluorocytosine, and the wild type was more susceptible to inhibition by 5-azauracil and 6-azauracil than was strain RTAE-1. The cellular targets for the pyrimidine analogs are not known. When the accumulation of 14C-labeled uracil or FU by the two strains was compared, the wild type took up 15-fold more radiolabel per cell than did the FU-resistant strain. In the wild type, radiolabel from uracil was incorporated into the soluble pool, RNA, and DNA. The metabolism of uracil appeared to involve a uracil phosphoribosyltransferase activity. Strain Marburg extracts contained this enzyme, whereas FU-resistant strain RTAE-1 extracts had less than 1/10 as much activity. Although it is possible that a change in permeability to the compounds plays a role in the stable resistance of strain RTAE-1, the fact that it lacks the ability to metabolize pyrimidines to nucleotides is sufficient to account for its phenotype. PMID:3624203

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.

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.

Optimizing Associative Experimental Design for Protein Crystallization Screening

PubMed Central

Dinç, Imren; Pusey, Marc L.; Aygün, Ramazan S.

2016-01-01

The goal of protein crystallization screening is the determination of the main factors of importance to crystallizing the protein under investigation. One of the major issues about determining these factors is that screening is often expanded to many hundreds or thousands of conditions to maximize combinatorial chemical space coverage for maximizing the chances of a successful (crystalline) outcome. In this paper, we propose an experimental design method called “Associative Experimental Design (AED)” and an optimization method includes eliminating prohibited combinations and prioritizing reagents based on AED analysis of results from protein crystallization experiments. AED generates candidate cocktails based on these initial screening results. These results are analyzed to determine those screening factors in chemical space that are most likely to lead to higher scoring outcomes, crystals. We have tested AED on three proteins derived from the hyperthermophile Thermococcus thioreducens, and we applied an optimization method to these proteins. Our AED method generated novel cocktails (count provided in parentheses) leading to crystals for three proteins as follows: Nucleoside diphosphate kinase (4), HAD superfamily hydrolase (2), Nucleoside kinase (1). After getting promising results, we have tested our optimization method on four different proteins. The AED method with optimization yielded 4, 3, and 20 crystalline conditions for holo Human Transferrin, archaeal exosome protein, and Nucleoside diphosphate kinase, respectively. PMID:26955046

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.

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

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.

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.

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.

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 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

Creation of a Long-Acting Nanoformulated 2′,3′-Dideoxy-3′-Thiacytidine

PubMed Central

Guo, Dongwei; Zhou, Tian; Araínga, Mariluz; Palandri, Diana; Gautam, Nagsen; Bronich, Tatiana; Alnouti, Yazen; McMillan, JoEllyn; Edagwa, Benson

2017-01-01

Background: Antiretroviral drug discovery and formulation design will facilitate viral clearance in infectious reservoirs. Although progress has been realized for selected hydrophobic integrase and nonnucleoside reverse transcriptase inhibitors, limited success has been seen to date with hydrophilic nucleosides. To overcome these limitations, hydrophobic long-acting drug nanoparticles were created for the commonly used nucleoside reverse transcriptase inhibitor, lamivudine (2′,3′-dideoxy-3′-thiacytidine, 3TC). Methods: A 2-step synthesis created a slow-release long-acting hydrophobic 3TC. Conjugation of 3TC to a fatty acid created a myristoylated prodrug which was encased into a folate-decorated poloxamer 407. Both in vitro antiretroviral efficacy in human monocyte-derived macrophages and pharmacokinetic profiles in mice were evaluated for the decorated nanoformulated drug. Results: A stable drug formulation was produced by poloxamer encasement that improved monocyte–macrophage uptake, antiretroviral activities, and drug pharmacokinetic profiles over native drug formulations. Conclusions: Sustained release of long-acting antiretroviral therapy is a new therapeutic frontier for HIV/AIDS. 3TC depot formation in monocyte-derived macrophages can be facilitated through stable subcellular internalization and slow drug release. PMID:27559685

Crosslinking of Chitosan with Dialdehyde Derivatives of Nucleosides and Nucleotides. Mechanism and Comparison with Glutaraldehyde.

PubMed

Mikhailov, Sergey N; Zakharova, Alexandra N; Drenichev, Mikhail S; Ershov, Andrey V; Kasatkina, Mariya A; Vladimirov, Leonid V; Novikov, Valentin V; Kildeeva, Natalia R

2016-01-01

In medical and pharmaceutical applications, chitosan is used as a component of hydrogels-macromolecular networks swollen in water. Chemical hydrogels are formed by covalent links between the crosslinking reagents and amino functionalities of chitosan. To date, the most commonly used chitosan crosslinkers are dialdehydes, such as glutaraldehyde (GA). We have developed novel GA like crosslinkers with additional functional groups-dialdehyde derivatives of uridine (oUrd) and nucleotides (oUMP and oAMP)-leading to chitosan-based biomaterials with new properties. The process of chitosan crosslinking was investigated in details and compared to crosslinking with GA. The rates of crosslinking with oUMP, oAMP, and GA were essentially the same, though much higher than in the case of oUrd. The remarkable difference in the crosslinking properties of nucleoside and nucleotide dialdehydes can be clearly attributed to the presence of the phosphate group in nucleotides that participates in the gelation process through ionic interactions with the amino groups of chitosan. Using NMR spectroscopy, we have not observed the formation of aldimine bonds. It can be concluded that the real number of crosslinks needed to cause gelation of chitosan chains may be less than 1%.

Reversible sol-to-gel transformation of uracil gelators: specific colorimetric and fluorimetric sensor for fluoride ions.

PubMed

Xing, Ling-Bao; Yang, Bing; Wang, Xiao-Jun; Wang, Jiu-Ju; Chen, Bin; Wu, Qianhong; Peng, Hui-Xing; Zhang, Li-Ping; Tung, Chen-Ho; Wu, Li-Zhu

2013-03-05

A new type of anthracene organogelator based on uracil was obtained using organic aromatic solvents, cyclohexane, DMSO, ethanol, and ethyl acetate. It was further characterized by field-emission scanning electron microscopy and transmission electron microscopy. Specifically, the resulting organogels were demonstrated to be promising colorimetric and fluorescent sensors toward fluoride ions with high sensitivity and selectivity, accompanying the disruption of the gelators. Spectroscopic study and (1)H NMR titration experiment revealed that the deprotonation of the hydrogen atom on the N position of uracil moiety by fluoride ions is responsible for the recognition events, evidenced by immediate transformation from the sol phase to the gel state upon adding a small amount of a proton solvent, methanol. The process is reversible, with zero loss in sensing activity and sol-to-gel transformation ability even after five runs.

The study of interaction between PFOA/PFOS and uracil by topology quality and spectroscopic analysis

NASA Astrophysics Data System (ADS)

Xu, Hui-Ying; Zhu, Jian-Qing; Wang, Wei; Xu, Xiao-Lu; Lu, Yin

2014-02-01

It has been established that perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) can be considered as emerging persistent organic pollutants. In recent years, there was increasing distribution of PFOA/PFOS in environmental systems, and accumulation and toxic effects of PFOA/PFOS in human body. In this paper, quantum chemistry methods were employed to study the interaction between perfluorinated organic pollutants and base (uracil). The results showed that there were four stable binding modes between the two perfluorinated compounds with uracil, especially the second mode which caused the most detrimental physiological functional response. NBO analysis showed that reactive hydrogen in the two perfluorinated compounds had the greatest effect on the hydrogen bond. The nature of the hydrogen bond formed between the two perfluorinated compounds and base was investigated using the AIM theory. The changes of spectroscopic properties in complexes were analyzed by IR and NMR spectra.

A molecular-field-based similarity study of non-nucleoside HIV-1 reverse transcriptase inhibitors

NASA Astrophysics Data System (ADS)

Mestres, Jordi; Rohrer, Douglas C.; Maggiora, Gerald M.

1999-01-01

This article describes a molecular-field-based similarity method for aligning molecules by matching their steric and electrostatic fields and an application of the method to the alignment of three structurally diverse non-nucleoside HIV-1 reverse transcriptase inhibitors. A brief description of the method, as implemented in the program MIMIC, is presented, including a discussion of pairwise and multi-molecule similarity-based matching. The application provides an example that illustrates how relative binding orientations of molecules can be determined in the absence of detailed structural information on their target protein. In the particular system studied here, availability of the X-ray crystal structures of the respective ligand-protein complexes provides a means for constructing an 'experimental model' of the relative binding orientations of the three inhibitors. The experimental model is derived by using MIMIC to align the steric fields of the three protein P66 subunit main chains, producing an overlay with a 1.41 Å average rms distance between the corresponding Cα's in the three chains. The inter-chain residue similarities for the backbone structures show that the main-chain conformations are conserved in the region of the inhibitor-binding site, with the major deviations located primarily in the 'finger' and RNase H regions. The resulting inhibitor structure overlay provides an experimental-based model that can be used to evaluate the quality of the direct a priori inhibitor alignment obtained using MIMIC. It is found that the 'best' pairwise alignments do not always correspond to the experimental model alignments. Therefore, simply combining the best pairwise alignments will not necessarily produce the optimal multi-molecule alignment. However, the best simultaneous three-molecule alignment was found to reproduce the experimental inhibitor alignment model. A pairwise consistency index has been derived which gauges the quality of combining the pairwise alignments and aids in efficiently forming the optimal multi-molecule alignment analysis. Two post-alignment procedures are described that provide information on feature-based and field-based pharmacophoric patterns. The former corresponds to traditional pharmacophore models and is derived from the contribution of individual atoms to the total similarity. The latter is based on molecular regions rather than atoms and is constructed by computing the percent contribution to the similarity of individual points in a regular lattice surrounding the molecules, which when contoured and colored visually depict regions of highly conserved similarity. A discussion of how the information provided by each of the procedures is useful in drug design is also presented.

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.

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

The mycotoxin patulin reacts with DNA bases with and without previous conjugation to GSH: implication for related α,β-unsaturated carbonyl compounds?

PubMed

Pfenning, Carolin; Esch, Harald L; Fliege, Ralph; Lehmann, Leane

2016-02-01

The α,β-unsaturated carbonyl group is recognized as alert for mutagenicity, attributed to (1) its direct reaction with DNA, counteractable by glutathione (GSH), and (2) oxidative stress caused indirectly by GSH depletion. Accordingly, the α,β,γ,δ-unsaturated lactone patulin (PAT), a mycotoxin detected in fruits and products derived thereof, is known to induce gene, chromosome, and genome mutations in vitro, its mutagenicity correlating inversely with intracellular GSH levels. Thus, the reactivity of PAT against DNA bases and nucleosides in the absence and presence of GSH and glutathione S-transferases (GSTs) was investigated under cell-free conditions using HPLC mass spectrometry techniques for identification of reaction products. Adduct formation with all four nucleobases as well as with purine base nucleosides occurred even in the presence of GSH, revealing several adducts of PAT, mono- and disubstituted with nucleobases/nucleosides as well as novel GSH-PAT adducts. In addition, novel mixed GSH-PAT-nucleobase adducts were observed. These adducts exhibited a ketohexanoic acid-type structure of the PAT molecule, C6 substituted with GSH and linking C1 of PAT with nitrogens of nucleobases/nucleosides via an amide bond. Formation of GSH-PAT-adenine adducts was not prevented by GSTs, and excess of GSH needed to reduce their formation was higher than for PAT-adenine adducts. The formation of mixed GSH-DNA base adducts has not been described for PAT or any other α,β-unsaturated carbonyl before, although the reaction mechanism seems to be applicable to a variety of α,β-unsaturated carbonyls occurring in food and in the environment.

Electron configuration and hydrogen-bonding pattern in several thymine and uracil analogues studied by 1H-14N NQDR and DFT/QTAIM.

PubMed

Seliger, Janez; Žagar, Veselko; Latosińska, Magdalena; Latosińska, Jolanta Natalia

2012-08-02

Some thio- and aza-derivatives of natural nucleobases uracil and thymine: 2-thiouracil, 4-thiouracil, 6-methyl-2-thiouracil, 6-azauracil, and 6-aza-2-thiothymine have been studied experimentally in solid state by (1)H-(14)N NMR-NQR double resonance (NQDR) and theoretically by the Density Functional Theory (DFT)/Quantum Theory of Atoms in Molecules (QTAIM). The (14)N resonance frequencies have been measured at 173 and 295 K and assigned to particular nitrogen sites (-N═ and -NH-). The temperature factor has been found negligible. The changes in the molecular skeletons, electric charge distribution, intermolecular interactions pattern, and molecular aggregations caused by oxygen replacement with sulfur and carbon replacement with nitrogen are discussed in detail. Correlations between all the principal components of the (14)N quadrupole coupling tensor have been found helpful in the search for the experimental (14)N NQR frequencies, their assignment to a particular nitrogen positions and estimation of the strength of the inter- and intramolecular interactions. The variation in the NQR parameters have been mainly related to the variation in the population of π-electron orbital. For thiouracil derivatives a general trend is that the stronger the hydrogen bond is, the lower is the asymmetry parameter, while for thymine and 6-aza-2-thiotymine, the opposite relation holds. Differences in correlations of the principal components of the (14)N quadrupole coupling tensor at the amino and iminonitrogen positions in heterocyclic rings are discussed. The effect of C→H and C→N substitution at the amino nitrogen position and C→N substitution at the iminonitrogen position on the quadrupole coupling tensor is analyzed. This study also demonstrates the advantages of combining NQR and DFT/QTAIM to predict an unsolved crystalline structure of 4-thiouracil.

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.

Design, enantiopure synthesis, and biological evaluation of novel iso-D-2',3'-dideoxy-3'-fluorothianucleoside derivatives as a bioisostere of lamivudine.

PubMed

Kim, Kyung Ran; Park, Ah-Young; Moon, Hyung Ryong; Chun, Moon Woo; Jeong, Lak Shin

2007-01-01

Novel iso D-2',3'-dideoxythianucleoside derivatives 1-3 were designed and asymmetrically synthesized to search for new anti-HIV agents. Final compounds 1-3 were evaluated against a variety of viruses including HIV-1 and 2. Only cytosine analog 3 showed a potent anti-VSV activity (EC(50) = 9.43 microg/mL). This result implies that iso 2',3'-dideoxy sugar templates might play a role of a sugar surrogate of nucleosides for the development of anti-RNA virus agent.

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

Structural determinants for the inhibitory ligands of orotidine-5′-monophosphate decarboxylase

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

Meza-Avina, Maria Elena; Wei, Lianhu; Liu, Yan

2010-06-14

In recent years, orotidine-5{prime}-monophosphate decarboxylase (ODCase) has gained renewed attention as a drug target. As a part of continuing efforts to design novel inhibitors of ODCase, we undertook a comprehensive study of potent, structurally diverse ligands of ODCase and analyzed their structural interactions in the active site of ODCase. These ligands comprise of pyrazole or pyrimidine nucleotides including the mononucleotide derivatives of pyrazofurin, barbiturate ribonucleoside, and 5-cyanouridine, as well as, in a computational approach, 1,4-dihydropyridine-based non-nucleoside inhibitors such as nifedipine and nimodipine. All these ligands bind in the active site of ODCase exhibiting distinct interactions paving the way to designmore » novel inhibitors against this interesting enzyme. We propose an empirical model for the ligand structure for rational modifications in new drug design and potentially new lead structures.« less

Natural Products as New Treatment Options for Trichomoniasis: A Molecular Docking Investigation.

PubMed

Setzer, Mary Snow; Byler, Kendall G; Ogungbe, Ifedayo Victor; Setzer, William N

2017-01-27

Trichomoniasis, caused by the parasitic protozoan Trichomonas vaginalis, is the most common non-viral sexually-transmitted disease, and there can be severe complications from trichomoniasis. Antibiotic resistance in T. vaginalis is increasing, but there are currently no alternatives treatment options. There is a need to discover and develop new chemotherapeutic alternatives. Plant-derived natural products have long served as sources for new medicinal agents, as well as new leads for drug discovery and development. In this work, we have carried out an in silico screening of 952 antiprotozoal phytochemicals with specific protein drug targets of T. vaginalis. A total of 42 compounds showed remarkable docking properties to T. vaginalis methionine gamma-lyase (TvMGL) and to T. vaginalis purine nucleoside phosphorylase (TvPNP). The most promising ligands were polyphenolic compounds, and several of these showed docking properties superior to either co-crystallized ligands or synthetic enzyme inhibitors.

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.

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).

Favipiravir elicits antiviral mutagenesis during virus replication in vivo.

PubMed

Arias, Armando; Thorne, Lucy; Goodfellow, Ian

2014-10-21

Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses.

The Molecular Basis of the Response to Radiation

DTIC Science & Technology

1999-07-01

S. cerevisiae and S. pombe ). After PCR amplification of human cDNA libraries as described below, PCR products are analyzed on 4% NuSieve agarose...media lacking uracil as well as counterselected against on media containing uracil and 0.1% 5-fluoroorotic acid (5FOA). Induction of the LacZ gene...evolutionarily distant species (S. cerevisiae andS. pombe ) to develop degenerate PCR based primers. For example, a fission yeast homolog of RAD9 named rhp9 was

A new and efficient approach for construction of uridine/uracil auxotrophic mutants in the filamentous fungus Aspergillus oryzae using Agrobacterium tumefaciens-mediated transformation.

PubMed

Nguyen, Khuyen Thi; Ho, Quynh Ngoc; Do, Loc Thi Binh Xuan; Mai, Linh Thi Dam; Pham, Duc-Ngoc; Tran, Huyen Thi Thanh; Le, Diep Hong; Nguyen, Huy Quang; Tran, Van-Tuan

2017-06-01

Aspergillus oryzae is a filamentous fungus widely used in food industry and as a microbial cell factory for recombinant protein production. Due to the inherent resistance of A. oryzae to common antifungal compounds, genetic transformation of this mold usually requires auxotrophic mutants. In this study, we show that Agrobacterium tumefaciens-mediated transformation (ATMT) method is very efficient for deletion of the pyrG gene in different Aspergillus oryzae wild-type strains to generate uridine/uracil auxotrophic mutants. Our data indicated that all the obtained uridine/uracil auxotrophic transformants, which are 5- fluoroorotic acid (5-FOA) resistant, exist as the pyrG deletion mutants. Using these auxotrophic mutants and the pyrG selectable marker for genetic transformation via A. tumefaciens, we could get about 1060 transformants per 10 6 fungal spores. In addition, these A. oryzae mutants were also used successfully for expression of the DsRed fluorescent reporter gene under control of the A. oryzae amyB promoter by the ATMT method, which resulted in obvious red transformants on agar plates. Our work provides a new and effective approach for constructing the uridine/uracil auxotrophic mutants in the importantly industrial fungus A. oryzae. This strategy appears to be applicable to other filamentous fungi to develop similar genetic transformation systems based on auxotrophic/nutritional markers for food-grade recombinant applications.

Apurinic/Apyrimidinic Endonuclease 1 Is the Essential Nuclease during Immunoglobulin Class Switch Recombination

PubMed Central

Masani, Shahnaz; Han, Li

2013-01-01

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID) that catalyzes numerous DNA cytosine deaminations within switch regions. The resulting uracils are processed by uracil base excision and/or mismatch repair enzymes that ultimately generate switch region DNA double-strand breaks (DSBs). Uracil glycosylase 2 (UNG2) is required for CSR, most likely by removing uracils to generate abasic sites. Although it is presumed that the apurinic/apyrimidinic endonuclease 1 (APE1) generates DNA strand incisions (a prerequisite for CSR) at these abasic sites, a direct test of the requirement for APE1 in CSR has been difficult because of the embryonic lethality of APE1 ablation in mice. Here, we report the successful deletion of the APE1 gene in a mouse B cell line (CH12F3) capable of robust CSR in vitro. In contrast to the general assumption that APE1 is essential for cellular viability, deletion of APE1 in CH12F3 cells has no apparent effect on cell viability or growth. Moreover, CSR in APE1-null CH12F3 cells is drastically reduced, providing direct evidence for an essential role for APE1 in switch region cleavage and CSR. Finally, deletion of AP endonuclease 2 (APE2) has no effect on CSR in either APE1-proficient or -deficient cells. PMID:23382073

Ab Initio Anharmonic Analysis of Vibrational Spectra of Uracil Using the Numerical-Analytic Implementation of Operator Van Vleck Perturbation Theory.

PubMed

Krasnoshchekov, Sergey V; Vogt, Natalja; Stepanov, Nikolay F

2015-06-25

The numerical-analytic implementation of the operator version of the canonical Van Vleck second-order vibrational perturbation theory (CVPT2) is employed for a purely ab initio prediction and interpretation of the infrared (IR) and Raman anharmonic spectra of a medium-size molecule of the diketo tautomer of uracil (2,4(1H,3H)-pyrimidinedione), which has high biological importance as one of the four RNA nucleobases. A nonempirical, semidiagonal quartic potential energy surface (PES) expressed in normal coordinates was evaluated at the MP2/cc-pVTZ level of theory. The quality of the PES was improved by replacing the harmonic frequencies with the "best" estimated CCSD(T)-based values taken from the literature. The theoretical method is enhanced by an accurate treatment of multiple Fermi and Darling-Dennison resonances with evaluation of the corresponding resonance constants W and K (CVPT2+WK method). A prediction of the anharmonic frequencies as well as IR and Raman intensities was used for a detailed interpretation of the experimental spectra of uracil. Very good agreement between predicted and observed vibrational frequencies has been achieved (RMSD ∼4.5 cm(-1)). The model employed gave a theoretically robust treatment of the multiple resonances in the 1680-1790 cm(-1) region. Our new analysis gives the most reliable reassignments of IR and Raman spectra of uracil available to date.

Butyrate influences intracellular levels of adenine and adenine derivatives in the fungus Penicillium restrictum.

PubMed

Zutz, Christoph; Chiang, Yi Ming; Faehnrich, Bettina; Bacher, Markus; Hellinger, Roland; Kluger, Bernhard; Wagner, Martin; Strauss, Joseph; Rychli, Kathrin

2017-04-01

Butyrate, a small fatty acid, has an important role in the colon of ruminants and mammalians including the inhibition of inflammation and the regulation of cell proliferation. There is also growing evidence that butyrate is influencing the histone structure in mammalian cells by inhibition of histone deacetylation. Butyrate shows furthermore an antimicrobial activity against fungi, yeast and bacteria, which is linked to its toxicity at a high concentration. In fungi there are indications that butyrate induces the production of secondary metabolites potentially via inhibition of histone deacetylases. However, information about the influence of butyrate on growth, primary metabolite production and metabolism, besides lipid catabolism, in fungi is scarce. We have identified the filamentous fungus Penicillium (P.) restrictum as a susceptible target for butyrate treatment in an antimicrobial activity screen. The antimicrobial activity was detected only in the mycelium of the butyrate treated culture. We investigated the effect of butyrate ranging from low (0.001mM) to high (30mM), potentially toxic, concentrations on biomass and antimicrobial activity. Butyrate at high concentrations (3 and 30mM) significantly reduced the fungal biomass. In contrast P. restrictum treated with 0.03mM of butyrate showed the highest antimicrobial activity. We isolated three antimicrobial active compounds, active against Staphylococcus aureus, from P. restrictum cellular extracts treated with butyrate: adenine, its derivate hypoxanthine and the nucleoside derivate adenosine. Production of all three compounds was increased at low butyrate concentrations. Furthermore we found that butyrate influences the intracellular level of the adenine nucleoside derivate cAMP, an important signalling molecule in fungi and various organisms. In conclusion butyrate treatment increases the intracellular levels of adenine and its respective derivatives. Copyright © 2017 Elsevier GmbH. All rights reserved.

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.

Comparative Biochemistry and Metabolism

DTIC Science & Technology

1978-12-01

pyrimidines). When interest includes labile pyrimidine derivatives, the DNA is hydrolyzed enzymatically; 5 mg DNA is dis- solved in water containing 20 j...Individual labeled pyrimidine nucleosides from animals so treated have been isolated but not yet identified. The DNA is hydrolyzed enzymatically to... hydrolyzed and chromatographically separated into pyrimidine oligonucleotides and free purine bases. At a dose of 60 mg hydrazine/kg body weight (LDO.0O

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.

Polymerization of amino acids containing nucleotide bases

NASA Technical Reports Server (NTRS)

Ben Cheikh, Azzouz; Orgel, Leslie E.

1990-01-01

The nucleoamino acids 1-(3'-amino,3'-carboxypropyl)uracil (3) and 9-(3'-amino,3'-carboxypropyl)adenine (4) have been prepared as (L)-en-antiomers and as racemic mixtures. When 3 or 4 is suspended in water and treated with N,N'-carbon-yldiimidazole, peptides are formed in good yield. The products formed from the (L)-enantiomers are hydrolyzed to the monomeric amino acids by pronase. Attempts to improve the efficiency of these oligomerizations by including a polyuridylate template in the reaction mixture were not successful. Similarly, oligomers derived from the (L)-enantiomer of 3 did not act as templates to facilitate the oligomerization of 4.

In silico studies to explore the mutagenic ability of 5-halo/oxy/li-oxy-uracil bases with guanine of DNA base pairs.

PubMed

Jana, Kalyanashis; Ganguly, Bishwajit

2014-10-16

DNA nucleobases are reactive in nature and undergo modifications by deamination, oxidation, alkylation, or hydrolysis processes. Many such modified bases are susceptible to mutagenesis when formed in cellular DNA. The mutagenesis can occur by mispairing with DNA nucleobases by a DNA polymerase during replication. We have performed a study of mispairing of DNA bases with unnatural bases computationally. 5-Halo uracils have been studied as mispairs in mutagenesis; however, the reports on their different forms are scarce in the literature. The stability of mispairs with keto form, enol form, and ionized form of 5-halo-uracil has been computed with the M06-2X/6-31+G** level of theory. The enol form of 5-halo-uracil showed remarkable stability toward DNA mispair compared to the corresponding keto and ionized forms. (F)U-G mispair showed the highest stability in the series and (Halo)(U(enol/ionized)-G mispair interactions energies are more stable than the natural G-C basepair of DNA. To enhance the stability of DNA mispairs, we have introduced the hydroxyl group in the place of halogen atoms, which provides additional hydrogen-bonding interactions in the system while forming the 5-membered ring. The study has been further extended with lithiated 5-hydroxymethyl-uracil to stabilize the DNA mispair. (CH2OLi)U(ionized)-G mispair has shown the highest stability (ΔG = -32.4 kcal/mol) with multi O-Li interactions. AIM (atoms in molecules) and EDA (energy decomposition analysis) analysis has been performed to examine the nature of noncovalent interactions in such mispairs. EDA analysis has shown that electrostatic energy mainly contributes toward the interaction energy of mispairs. The higher stability achieved in these studied mispairs can play a pivotal role in the mutagenesis and can help to attain the mutation for many desired biological processes.

A phase II study of preoperative chemoradiation with tegafur-uracil plus leucovorin for locally advanced rectal cancer with pharmacogenetic analysis.

PubMed

Kim, Sun Young; Baek, Ji Yeon; Oh, Jae Hwan; Park, Sung Chan; Sohn, Dae Kyung; Kim, Min Ju; Chang, Hee Jin; Kong, Sun-Young; Kim, Dae Yong

2017-03-27

This study aimed to evaluate the efficacy of a high dose of oral tegafur-uracil (400 mg/m 2 ) plus leucovorin with preoperative chemoradiation of locally advanced rectal cancer and to explore the impact of polymorphisms of cytochrome P 2A6 (CYP2A6), uridine monophosphate synthetase (UMPS), and ATP-binding cassette B1 (ABCB1) on clinical outcome. Patients with cT3 or cT4 rectal cancer were enrolled and were given tegafur-uracil 400 mg/m 2 /day and leucovorin 90 mg/m 2 /day for 7 days a week during preoperative chemoradiation (50.4 Gy/28 fractions) in this phase II trial. Primary endpoint was pathologic complete response rate, and the secondary endpoint was to explore the association between clinical outcomes and genetic polymorphisms CYP2A6 (*4, *7, *9 and *10), UMPS G638C, and three ABCB1 genotypes (C1236T, C3435T, and G2677T). Ninety-one patients were given study treatment, and 90 underwent surgery. Pathologic complete response was noted in 10 patients (11.1%). There was no grade 4 or 5 toxicity; 20 (22.0%) experienced grade 3 toxicities, including diarrhea (10, 11.0%), abdominal pain (2, 2.2%), and anemia (2, 2.2%). Relapse-free survival and overall survival at 5 years were 88.6% and 94.2%, respectively. Patients with the UMPS 638 CC genotype experienced significantly more frequent grade 2 or 3 diarrhea (p for trend = 0.018). Preoperative chemoradiation with tegafur-uracil 400 mg/m 2 /day with leucovorin was feasible, but did not meet the expected pathologic complete response rate. The UMPS 638 CC genotype might be a candidate biomarker predicting toxicity in patients receiving tegafur-uracil/leucovorin-based preoperative chemoradiation for locally advanced rectal cancer. ISRCTN11812525 , registered on 25 July 2016. Retrospectively registered.

Design, discovery, modelling, synthesis, and biological evaluation of novel and small, low toxicity s-triazine derivatives as HIV-1 non-nucleoside reverse transcriptase inhibitors.

PubMed

Viira, Birgit; Selyutina, Anastasia; García-Sosa, Alfonso T; Karonen, Maarit; Sinkkonen, Jari; Merits, Andres; Maran, Uko

2016-06-01

A set of top-ranked compounds from a multi-objective in silico screen was experimentally tested for toxicity and the ability to inhibit the activity of HIV-1 reverse transcriptase (RT) in cell-free assay and in cell-based assay using HIV-1 based virus-like particles. Detailed analysis of a commercial sample that indicated specific inhibition of HIV-1 reverse transcription revealed that a minor component that was structurally similar to that of the main compound was responsible for the strongest inhibition. As a result, novel s-triazine derivatives were proposed, modelled, discovered, and synthesised, and their antiviral activity and cellular toxicity were tested. Compounds 18a and 18b were found to be efficient HIV-1 RT inhibitors, with an IC50 of 5.6±1.1μM and 0.16±0.05μM in a cell-based assay using infectious HIV-1, respectively. Compound 18b also had no detectable toxicity for different human cell lines. Their binding mode and interactions with the RT suggest that there was strong and adaptable binding in a tight (NNRTI) hydrophobic pocket. In summary, this iterative study produced structural clues and led to a group of non-toxic, novel compounds to inhibit HIV-RT with up to nanomolar potency. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthesis, antiviral evaluation and molecular docking studies of N4-aryl substituted/unsubstituted thiosemicarbazones derived from 1-indanones as potent anti-bovine viral diarrhea virus agents.

PubMed

Soraires Santacruz, María C; Fabiani, Matías; Castro, Eliana F; Cavallaro, Lucía V; Finkielsztein, Liliana M

2017-08-01

A series of N 4 -arylsubstituted thiosemicarbazones derived from 1-indanones and a set of compounds lacking such substitution in the N 4 position of the thiosemicarbazone moiety were synthesized and evaluated for their anti-bovine viral diarrhea virus (BVDV) activity. Among these, derivatives 2 and 15 displayed high activity (EC 50 =2.7±0.4 and 0.7±0.1µM, respectively) as inhibitors of BVDV replication. Novel key structural features related to the anti-BVDV activity were identified by structure-activity relationship (SAR) analysis. In a previous study, the thiosemicarbazone of 5,6-dimethoxy-1-indanone (5,6-TSC) was characterized as a non-nucleoside inhibitor (NNI) of the BVDV RNA-dependent RNA polymerase. In the present work, cross-resistance assays were performed with the most active compounds. Such studies were carried out on 5,6-TSC resistant BVDV (BVDV-TSC r T1) carrying mutations in the viral polymerase. This BVDV mutant was also resistant to compound 15. Molecular docking studies and MM/PBSA calculations were performed to assess the most active derivatives at the 5,6-TSC viral polymerase binding site. The differences in the interaction pattern and the binding affinity of derivative 15 either to the wild type or BVDV-TSC r T1 polymerase were key factors to define the mode of action of this compound. Copyright © 2017 Elsevier Ltd. All rights reserved.

Growth and sporulation of a pyrimidine spore color mutant of Sordaria fimicola.

PubMed

el-Ani, A S

1967-04-07

A nonautonomous spore color mutant of Sordaria fimicola is a pyrimidine auxotroph that produces hyaline nonviable ascospores. Uracil, uridine, and cytidine are more effective growth factors than cytosine and thymine and, in high concentrations, render the mutant self-fertile by inducing the ascospores to resume development and maturation. Crosses with the unlinked arginine non-autonomus spore color mutant st-59 yielded the double mutant st-59 pyr that requires both arginine and a pyrimidine for growth, which indicates a lack of suppression of the pyrimidine requirement by the arginine locus.

Synthesis and biological activity of chloroethyl pyrimidine nucleosides.

PubMed

Colombeau, Ludovic; Teste, Karine; Hadj-Bouazza, Amel; Chaleix, Vincent; Zerrouki, Rachida; Kraemer, Michel; Catherine, Odile Sainte

2008-02-01

The synthesis and biological activity of chloroethyl pyrimidine nucleosides is presented. One of these new nucleosides analogues significantly inhibited cell proliferation, migration and invasion as tested in vitro on the A431 vulvar epidermal carcinoma cell line.

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.

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.

Hydrolysis of N3-methyl-2'-deoxycytidine: model compound for reactivity of protonated cytosine residues in DNA.

PubMed

Sowers, L C; Sedwick, W D; Shaw, B R

1989-11-01

Protonation of cytosine residues at physiological pH may occur in DNA as a consequence of both alkylation and aberrant base-pair formation. When cytosine derivatives are protonated, they undergo hydrolysis reactions at elevated rates and can either deaminate to form the corresponding uracil derivatives or depyrimidinate generating abasic sites. The kinetic parameters for reaction of protonated cytosine are derived by studying the hydrolysis of N3-methyl-2'-deoxycytidine (m3dC), a cytosine analogue which is predominantly protonated at physiological pH. Both deamination and depyrimidimation reaction rates are shown to be linearly dependent upon the fraction of protonated molecules. We present here thermodynamic parameters which allow determination of hydrolysis rates of m3dC as functions of pH and temperature. Protonation of cytosine residues in DNA, as induced by aberrant base-pair formation or base modification, may accelerate the rate of both deamination and depyrimidation up to several thousand-fold under physiological conditions.

Synthesis of 4'-Selenoribonucleosides, the Building Blocks of 4'-SelenoRNA, Using a Hypervalent Iodine.

PubMed

Saito-Tarashima, Noriko; Ota, Masashi; Minakawa, Noriaki

2017-09-18

Herein is described a detailed protocol for the synthesis of 4'-selenoribonucleoside derivatives that involves the use of a hypervalent iodine species. These derivatives are versatile units for the preparation of 4'-selenoRNA. Large-scale synthesis of a 4-selenosugar starting from D-ribose is achieved in eight steps, including a final chromatographic purification. The resulting 4-selenosugar is then subjected to the one-pot Pummerer-like reaction using hypervalent iodine in the presence of silylated nucleobases. The reaction with silylated uracil affords the desired 4'-selenouridine derivatives with excellent β-selectivity and in good yield. Conversely, when purine nucleobases are used in the Pummerer-like reaction, N7 4'-selenoribonucleoside isomers are obtained alongside the desired N9 isomers. However, the undesired N7 isomers can be converted to the desired N9 ones under acidic conditions. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Post-transcriptional modifications in the small subunit ribosomal RNA from Thermotoga maritima, including presence of a novel modified cytidine

PubMed Central

Guymon, Rebecca; Pomerantz, Steven C.; Ison, J. Nicholas; Crain, Pamela F.; McCloskey, James A.

2007-01-01

Post-transcriptional modifications of RNA are nearly ubiquitous in the principal RNAs involved in translation. However, in the case of rRNA the functional roles of modification are far less established than for tRNA, and are subject to less knowledge in terms of specific nucleoside identities and their sequence locations. Post-transcriptional modifications have been studied in the SSU rRNA from Thermotoga maritima (optimal growth 80°C), one of the most deeply branched organisms in the Eubacterial phylogenetic tree. A total of 10 different modified nucleosides were found, the greatest number reported for bacterial SSU rRNA, occupying a net of ∼14 sequence sites, compared with a similar number of sites recently reported for Thermus thermophilus and 11 for Escherichia coli. The relatively large number of modifications in Thermotoga offers modest support for the notion that thermophile rRNAs are more extensively modified than those from mesophiles. Seven of the Thermotoga modified sites are identical (location and identity) to those in E. coli. An unusual derivative of cytidine was found, designated N-330 (M r 330.117), and was sequenced to position 1404 in the decoding region of the rRNA. It was unexpectedly found to be identical to an earlier reported nucleoside of unknown structure at the same location in the SSU RNA of the archaeal mesophile Haloferax volcanii. PMID:17255199

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.

Dietary nucleotides prevent decrease in cellular immunity in ground-based microgravity analog

NASA Technical Reports Server (NTRS)

Yamauchi, Keiko; Hales, Nathan W.; Robinson, Sandra M.; Niehoff, Michael L.; Ramesh, Vani; Pellis, Neal R.; Kulkarni, Anil D.

2002-01-01

Microgravity and stress of spaceflights result in immune dysfunction. The role of nutrition, especially nucleotide supplementation, has become an area of intensive research and significant interest in immunomodulation for maintenance of cellular immune responses. The studies presented here evaluate the plausibility of administering nucleotides to obviate immune dysfunction in an Earth-based in vivo analog of microgravity as studied in anti-orthostatic tail suspension (AOS) of mice. Mice were divided into three housing groups: group, isolation, and AOS. Mice were fed either control chow diet (CD), or RNA-, adenine-, or uracil-supplemented CD for the 1-wk duration of the experiments. In AOS mice, supplemental nucleotides significantly increased in vivo lymph node proliferation and ex vivo lymphoproliferation response to alloantigen and mitogens, respectively, and interleukin-2 and interferon-gamma production. A lower corticosterone level was observed in uracil-supplemented CD compared with CD. These results suggest that exogenous nucleotide supplementation, especially uracil, of normal diet is beneficial in the maintenance and restoration of the immune response during the microgravity analog conditions.

The Methanothermobacter thermautotrophicus ExoIII homologue Mth212 is a DNA uridine endonuclease

PubMed Central

Georg, Jens; Schomacher, Lars; Chong, James P. J.; Majerník, Alan I.; Raabe, Monika; Urlaub, Henning; Müller, Sabine; Ciirdaeva, Elena; Kramer, Wilfried; Fritz, Hans-Joachim

2006-01-01

The genome of Methanothermobacter thermautotrophicus, as a hitherto unique case, is apparently devoid of genes coding for general uracil DNA glycosylases, the universal mediators of base excision repair following hydrolytic deamination of DNA cytosine residues. We have now identified protein Mth212, a member of the ExoIII family of nucleases, as a possible initiator of DNA uracil repair in this organism. This enzyme, in addition to bearing all the enzymological hallmarks of an ExoIII homologue, is a DNA uridine endonuclease (U-endo) that nicks double-stranded DNA at the 5′-side of a 2′-d-uridine residue, irrespective of the nature of the opposing nucleotide. This type of activity has not been described before; it is absent from the ExoIII homologues of Escherichia coli, Homo sapiens and Methanosarcina mazei, all of which are equipped with uracil DNA repair glycosylases. The U-endo activity of Mth212 is served by the same catalytic center as its AP-endo activity. PMID:17012282

Incorporation of Exogenous Purines and Pyrimidines by Methanococcus voltae and Isolation of Analog-Resistant Mutants

PubMed Central

Bowen, Timothy L.; Whitman, William B.

1987-01-01

Methanococcus voltae incorporated exogenous adenine, guanine, hypoxanthine, and uracil, but not thymine. Growth of M. voltae was also sensitive to purine and pyrimidine analogs. Of the 20 analogs tested, 12 were inhibitory at 1 mg/ml. The most effective inhibitors were purine analogs with endocyclic substitutions. Nucleoside analogs and analogs with exocyclic substitutions or additions were less effective. Four purine analogs, 8-aza-2,6-diaminopurine, 8-azaguanine, 8-azahypoxanthine, and 6-mercaptopurine and one pyrimidine analog, 6-azauracil, were especially toxic. The MICs were 20, 0.5, 2.0, 80, and 10 μg/ml, respectively. Spontaneous resistance mutants were isolated for these five analogs. The MICs for these mutants were 20.5, 8.2, >65, >41, and 20.5 mg/ml, respectively. These concentrations far exceeded the solubilities of the analogs and represented an increase in resistance of at least three orders of magnitude. In addition to demonstrating cross resistance to several of the analogs, four of these mutants lost the ability to incorporate exogenous bases. These appeared to be mutations in the salvage pathways for purines and pyrimidines. In contrast, the mutant resistant to 6-mercaptopurine was not defective in purine uptake. Instead, it degraded 6-mercaptopurine. In the presence or absence of high concentrations of the analogs, the growth rates of the resistant mutants were no less than one-half of the growth rate of the wild type in the absence of the analog. The high level of resistance and rapid growth are very desirable properties for the application of the mutants in genetic experiments. PMID:16347408

Analysis of the High-Performance Liquid Chromatography Fingerprints and Quantitative Analysis of Multicomponents by Single Marker of Products of Fermented Cordyceps sinensis

PubMed Central

Guan, Yong-mei; Jin, Chen; Zhu, Wei-feng; Yang, Ming

2018-01-01

Fermented Cordyceps sinensis, the succedaneum of Cordyceps sinensis which is extracted and separated from Cordyceps sinensis by artificial fermentation, is commonly used in eastern Asia in clinical treatments due to its health benefit. In this paper, a new strategy for differentiating and comprehensively evaluating the quality of products of fermented Cordyceps sinensis has been established, based on high-performance liquid chromatography (HPLC) fingerprint analysis combined with similar analysis (SA), hierarchical cluster analysis (HCA), and the quantitative analysis of multicomponents by single marker (QAMS). Ten common peaks were collected and analysed using SA, HCA, and QAMS. These methods indicated that 30 fermented Cordyceps sinensis samples could be categorized into two groups by HCA. Five peaks were identified as uracil, uridine, adenine, guanosine, and adenosine, and according to the results from the diode array detector, which can be used to confirm peak purity, the purities of these compounds were greater than 990. Adenosine was chosen as the internal reference substance. The relative correction factors (RCF) between adenosine and the other four nucleosides were calculated and investigated using the QAMS method. Meanwhile, the accuracy of the QAMS method was confirmed by comparing the results of that method with those of an external standard method with cosines of the angles between the groups. No significant difference between the two methods was observed. In conclusion, the method established herein was efficient, successful in identifying the products of fermented Cordyceps sinensis, and scientifically valid to be applicable in the systematic quality control of fermented Cordyceps sinensis products. PMID:29850373

Analysis of the High-Performance Liquid Chromatography Fingerprints and Quantitative Analysis of Multicomponents by Single Marker of Products of Fermented Cordyceps sinensis.

PubMed

Chen, Li-Hua; Wu, Yao; Guan, Yong-Mei; Jin, Chen; Zhu, Wei-Feng; Yang, Ming

2018-01-01

Fermented Cordyceps sinensis , the succedaneum of Cordyceps sinensis which is extracted and separated from Cordyceps sinensis by artificial fermentation, is commonly used in eastern Asia in clinical treatments due to its health benefit. In this paper, a new strategy for differentiating and comprehensively evaluating the quality of products of fermented Cordyceps sinensis has been established, based on high-performance liquid chromatography (HPLC) fingerprint analysis combined with similar analysis (SA), hierarchical cluster analysis (HCA), and the quantitative analysis of multicomponents by single marker (QAMS). Ten common peaks were collected and analysed using SA, HCA, and QAMS. These methods indicated that 30 fermented Cordyceps sinensis samples could be categorized into two groups by HCA. Five peaks were identified as uracil, uridine, adenine, guanosine, and adenosine, and according to the results from the diode array detector, which can be used to confirm peak purity, the purities of these compounds were greater than 990. Adenosine was chosen as the internal reference substance. The relative correction factors (RCF) between adenosine and the other four nucleosides were calculated and investigated using the QAMS method. Meanwhile, the accuracy of the QAMS method was confirmed by comparing the results of that method with those of an external standard method with cosines of the angles between the groups. No significant difference between the two methods was observed. In conclusion, the method established herein was efficient, successful in identifying the products of fermented Cordyceps sinensis , and scientifically valid to be applicable in the systematic quality control of fermented Cordyceps sinensis products.

Mechanisms for the Formation of Thymine Under Astrophysical Conditions and Implications for the Origin of Life

PubMed Central

Bera, Partha P.; Nuevo, Michel; Materese, Christopher K.; Sandford, Scott A.; Lee, Timothy J.

2018-01-01

Nucleobases are the carriers of the genetic information in RNA and DNA for all life on Earth. Their presence in meteorites clearly indicates that compounds of biological importance can form via non-biological processes in extraterrestrial environments. Recent experimental studies have shown that the pyrimidine-based nucleobases uracil and cytosine can be easily formed from the ultraviolet irradiation of pyrimidine in H2O-rich ice mixtures that simulate astrophysical processes. In contrast, thymine, which is found only in DNA, is more difficult to form under the same experimental conditions, as its formation usually requires a higher photon dose. Earlier quantum chemical studies confirmed that the reaction pathways were favorable provided that several H2O molecules surrounded the reactants. However, the present quantum chemical study shows that the formation of thymine is limited because of the inefficiency of the methylation of pyrimidine and its oxidized derivatives in an H2O ice, as supported by the laboratory studies. Our results constrain the formation of thymine in astrophysical environments and thus the inventory of organic molecules delivered to the early Earth, and have implications for the role of thymine and DNA in the origin of life. PMID:27083722

Mechanisms for the formation of thymine under astrophysical conditions and implications for the origin of life

NASA Astrophysics Data System (ADS)

Bera, Partha P.; Nuevo, Michel; Materese, Christopher K.; Sandford, Scott A.; Lee, Timothy J.

2016-04-01

Nucleobases are the carriers of the genetic information in ribonucleic acid and deoxyribonucleic acid (DNA) for all life on Earth. Their presence in meteorites clearly indicates that compounds of biological importance can form via non-biological processes in extraterrestrial environments. Recent experimental studies have shown that the pyrimidine-based nucleobases uracil and cytosine can be easily formed from the ultraviolet irradiation of pyrimidine in H2O-rich ice mixtures that simulate astrophysical processes. In contrast, thymine, which is found only in DNA, is more difficult to form under the same experimental conditions, as its formation usually requires a higher photon dose. Earlier quantum chemical studies confirmed that the reaction pathways were favorable provided that several H2O molecules surrounded the reactants. However, the present quantum chemical study shows that the formation of thymine is limited because of the inefficiency of the methylation of pyrimidine and its oxidized derivatives in an H2O ice, as supported by the laboratory studies. Our results constrain the formation of thymine in astrophysical environments and thus the inventory of organic molecules delivered to the early Earth and have implications for the role of thymine and DNA in the origin of life.

Retrospective analysis of an oral combination of dexamethasone, uracil plus tegafur and cyclophosphamide for hormone-refractory prostate cancer.

PubMed

Hatano, Koji; Nonomura, Norio; Nishimura, Kazuo; Kawashima, Atsunari; Mukai, Masatoshi; Nagahara, Akira; Nakai, Yasutomo; Nakayama, Masashi; Takayama, Hitoshi; Tsujimura, Akira; Okuyama, Akihiko

2011-02-01

To evaluate the clinical utility of an oral combination of dexamethasone, uracil plus tegafur and cyclophosphamide as a treatment for patients with hormone-refractory prostate cancer. Fifty-seven patients with hormone-refractory prostate cancer were treated with an oral administration of dexamethasone (1.0 mg/day), uracil plus tegafur (400 mg/day) and cyclophosphamide (100 mg/day). The median patient age was 71 years. Sixteen patients had symptomatic bone metastasis, 31 had asymptomatic bone metastasis and 8 showed lymph node metastasis. Eight patients presented with only biochemical progression as evaluated by serum prostate-specific antigen levels. Thirty-six (63%) of 57 patients demonstrated a ≥50% decline in serum prostate-specific antigen levels. The median time to prostate-specific antigen progression was 7.2 months. In patients with a prostate-specific antigen decline of ≥50%, the median time to progression was 13.3 months. With respect to pre-treatment markers, the duration of response to initial hormonal treatment was associated with the time to prostate-specific antigen progression. In 11 of 16 (69%) patients who complained of bone pain, the pain improved and became stable in 5 of those patients (31%). Most adverse events were mild and only three (5%) patients showed neutropenia of Grade 3 or higher. The combination of dexamethasone, uracil plus tegafur and cyclophosphamide is an effective and well tolerated regimen for hormone-refractory prostate cancer. To evaluate the survival benefits, further randomized studies are required.

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.

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.

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.

The S-acyl-2-thioethyl pronucleotide approach applied to acyclovir: part I. Synthesis and in vitro anti-hepatitis B virus activity of bis(S-acyl-2-thioethyl)phosphotriester derivatives of acyclovir.

PubMed

Périgaud, C; Gosselin, G; Girardet, J L; Korba, B E; Imbach, J L

1999-01-01

The synthesis and in vitro anti-hepatitis B virus (HBV) activity of two mononucleoside phosphotriester derivatives of acyclovir incorporating S-acyl-2-thioethyl (SATE) groups are reported. In contrast to the parent nucleoside, the described phosphotriesters emerged as potent and selective inhibitors of HBV replication in HepG2.2.15 cells. This result can be attributed to the unique cellular metabolism of the SATE pronucleotides giving rise to the delivery to acyclovir 5'-monophosphate inside the infected cells. Moreover, the in vitro anti-HBV activities of one of these bis(SATE)phosphotriesters and of (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (lamivudine, 3TC) were compared alone and in combination. Analysis of the combination data indicates that 3TC and the studied SATE pronucleotide of acyclovir exhibited strong synergistic interactions. The present study provides an example where the use of a pronucleotide approach extends the antiviral spectrum of a nucleoside analogue. Given the potency of SATE pronucleotides of acyclovir against HBV in HepG2.2.15 cells, further studies including animal experiments seem warranted to evaluate the potential of these compounds as anti-HBV agents.

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

On the gas phase fragmentation of protonated uracil: a statistical perspective.

PubMed

Rossich Molina, Estefanía; Salpin, Jean-Yves; Spezia, Riccardo; Martínez-Núñez, Emilio

2016-06-01

The potential energy surface of protonated uracil has been explored by an automated transition state search procedure, resulting in the finding of 1398 stationary points and 751 reactive channels, which can be categorized into isomerizations between pairs of isomers, unimolecular fragmentations and bimolecular reactions. The use of statistical Rice-Ramsperger-Kassel-Marcus (RRKM) theory and Kinetic Monte Carlo (KMC) simulations allowed us to determine the relative abundances of each fragmentation channel as a function of the ion's internal energy. The KMC/RRKM product abundances are compared with novel mass spectrometry (MS) experiments in the collision energy range 1-6 eV. To facilitate the comparison between theory and experiments, further dynamics simulations are carried out to determine the fraction of collision energy converted into the ion's internal energy. The KMC simulations show that the major fragmentation channels are isocyanic acid and ammonia losses, in good agreement with experiments. The third predominant channel is water loss according to both theory and experiments, although the abundance obtained in the KMC simulations is very low, suggesting that non-statistical dynamics might play an important role in this channel. Isocyanic acid (HNCOH(+)) is also an important product in the KMC simulations, although its abundance is only significant at internal energies not accessible in the MS experiments.

AXM mutagenesis: an efficient means for the production of libraries for directed evolution of proteins.

PubMed

Holland, Erika G; Buhr, Diane L; Acca, Felicity E; Alderman, Dawn; Bovat, Kristin; Busygina, Valeria; Kay, Brian K; Weiner, Michael P; Kiss, Margaret M

2013-08-30

Affinity maturation is an important part of the recombinant antibody development process. There are several well-established approaches for generating libraries of mutated antibody genes for affinity maturation, but these approaches are generally too laborious or expensive to allow high-throughput, parallel processing of multiple antibodies. Here, we describe a scalable approach that enables the generation of libraries with greater than 10(8) clones from a single Escherichia coli transformation. In our method, a mutated DNA fragment is produced using PCR conditions that promote nucleotide misincorporation into newly synthesized DNA. In the PCR reaction, one of the primers contains at least three phosphorothioate linkages at its 5' end, and treatment of the PCR product with a 5' to 3' exonuclease is used to preferentially remove the strand synthesized with the non-modified primer, resulting in a single-stranded DNA fragment. This fragment then serves as a megaprimer to prime DNA synthesis on a uracilated, circular, single-stranded template in a Kunkel-like mutagenesis reaction that biases nucleotide base-changes between the megaprimer and uracilated DNA sequence in favor of the in vitro synthesized megaprimer. This method eliminates the inefficient subcloning steps that are normally required for the construction of affinity maturation libraries from randomly mutagenized antibody genes. Copyright © 2013. Published by Elsevier B.V.

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

Anti-HIV drugs: 25 compounds approved within 25 years after the discovery of HIV.

PubMed

De Clercq, Erik

2009-04-01

In 2008, 25 years after the human immunodeficiency virus (HIV) was discovered as the then tentative aetiological agent of acquired immune deficiency syndrome (AIDS), exactly 25 anti-HIV compounds have been formally approved for clinical use in the treatment of AIDS. These compounds fall into six categories: nucleoside reverse transcriptase inhibitors (NRTIs: zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir and emtricitabine); nucleotide reverse transcriptase inhibitors (NtRTIs: tenofovir); non-nucleoside reverse transcriptase inhibitors (NNRTIs: nevirapine, delavirdine, efavirenz and etravirine); protease inhibitors (PIs: saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir and darunavir); cell entry inhibitors [fusion inhibitors (FIs: enfuvirtide) and co-receptor inhibitors (CRIs: maraviroc)]; and integrase inhibitors (INIs: raltegravir). These compounds should be used in drug combination regimens to achieve the highest possible benefit, tolerability and compliance and to diminish the risk of resistance development.

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].

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

Approved Antiviral Drugs over the Past 50 Years

PubMed Central

2016-01-01

SUMMARY Since the first antiviral drug, idoxuridine, was approved in 1963, 90 antiviral drugs categorized into 13 functional groups have been formally approved for the treatment of the following 9 human infectious diseases: (i) HIV infections (protease inhibitors, integrase inhibitors, entry inhibitors, nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and acyclic nucleoside phosphonate analogues), (ii) hepatitis B virus (HBV) infections (lamivudine, interferons, nucleoside analogues, and acyclic nucleoside phosphonate analogues), (iii) hepatitis C virus (HCV) infections (ribavirin, interferons, NS3/4A protease inhibitors, NS5A inhibitors, and NS5B polymerase inhibitors), (iv) herpesvirus infections (5-substituted 2′-deoxyuridine analogues, entry inhibitors, nucleoside analogues, pyrophosphate analogues, and acyclic guanosine analogues), (v) influenza virus infections (ribavirin, matrix 2 protein inhibitors, RNA polymerase inhibitors, and neuraminidase inhibitors), (vi) human cytomegalovirus infections (acyclic guanosine analogues, acyclic nucleoside phosphonate analogues, pyrophosphate analogues, and oligonucleotides), (vii) varicella-zoster virus infections (acyclic guanosine analogues, nucleoside analogues, 5-substituted 2′-deoxyuridine analogues, and antibodies), (viii) respiratory syncytial virus infections (ribavirin and antibodies), and (ix) external anogenital warts caused by human papillomavirus infections (imiquimod, sinecatechins, and podofilox). Here, we present for the first time a comprehensive overview of antiviral drugs approved over the past 50 years, shedding light on the development of effective antiviral treatments against current and emerging infectious diseases worldwide. PMID:27281742

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.

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

Fatty acid carbon is essential for dNTP synthesis in endothelial cells

PubMed Central

Missiaen, Rindert; Queiroz, Karla CS; Borgers, Gitte; Elia, Ilaria; Zecchin, Annalisa; Cantelmo, Anna Rita; Christen, Stefan; Goveia, Jermaine; Heggermont, Ward; Goddé, Lucica; Vinckier, Stefan; Van Veldhoven, Paul P.; Eelen, Guy; Schoonjans, Luc; Gerhardt, Holger; Dewerchin, Mieke; Baes, Myriam; De Bock, Katrien; Ghesquière, Bart; Lunt, Sophia Y.; Fendt, Sarah-Maria; Carmeliet, Peter

2015-01-01

The metabolism of endothelial cells (ECs) during vessel sprouting remains poorly studied. Here, we report that endothelial loss of CPT1a, a rate-limiting enzyme of fatty acid oxidation (FAO), caused vascular sprouting defects due to impaired proliferation, not migration of ECs. Reduction of FAO in ECs did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labeling studies in control ECs showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1a silencing reduced these processes and depleted EC stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1a-silenced ECs. Finally, CPT1 blockade inhibited pathological ocular angiogenesis, suggesting a novel strategy for blocking angiogenesis. PMID:25830893

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.

Synthesis and photochemical behavior of the tetrazolo tautomer of 2-azido-4-pyrimidinone-2'-deoxyriboside.

PubMed

Gourdain, Stéphanie; Petermann, Christian; Martinez, Agathe; Harakat, Dominique; Clivio, Pascale

2011-03-18

The 2-azido analogue of 2'-deoxyuridine was prepared in three steps from 2'-deoxy-2-thiouridine. The sulfur atom of the 2-thio nucleoside was methylated and then displaced by hydrazine to furnish the corresponding 2-hydrazino derivative. After diazotization, the 2-azido compound that exists as its tetrazolo tautomer was obtained. Upon UV irradiation in aqueous solution, the title compound led to isocytosine.

NS5B RNA dependent RNA polymerase inhibitors: the promising approach to treat hepatitis C virus infections.

PubMed

Deore, R R; Chern, J-W

2010-01-01

Hepatitis C virus (HCV), a causative agent for non-A and non-B hepatitis, has infected approximately 3% of world's population. The current treatment option of ribavirin in combination with pegylated interferon possesses lower sustained virological response rates, and has serious disadvantages. Unfortunately, no prophylactic vaccine has been approved yet. Therefore, there is an unmet clinical need for more effective and safe anti-HCV drugs. HCV NS5B RNA dependent RNA polymerase is currently pursued as the most popular target to develop safe anti-HCV agents, as it is not expressed in uninfected cells. More than 25 pharmaceutical companies and some research groups have developed ≈50 structurally diverse scaffolds to inhibit NS5B. Here we provide comprehensive account of the drug development process of these scaffolds. NS5B polymerase inhibitors have been broadly classified in nucleoside and non nucleoside inhibitors and are sub classified according to their mechanism of action and structural diversities. With some additional considerations about the inhibitor bound NS5B enzyme X-ray crystal structure information and pharmacological aspects of the inhibitors, this review summarizes the lead identification, structure activity relationship (SAR) studies leading to the most potent NS5B inhibitors with subgenomic replicon activity.

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

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.

Curious discoveries in antiviral drug development: the role of serendipity.

PubMed

De Clercq, Erik

2015-07-01

Antiviral drug development has often followed a curious meandrous route, guided by serendipity rather than rationality. This will be illustrated by ten examples. The polyanionic compounds (i) polyethylene alanine (PEA) and (ii) suramin were designed as an antiviral agent (PEA) or known as an antitrypanosomal agent (suramin), before they emerged as, respectively, a depilatory agent, or reverse transcriptase inhibitor. The 2',3'-dideoxynucleosides (ddNs analogues) (iii) have been (and are still) used in the "Sanger" DNA sequencing technique, although they are now commercialized as nucleoside reverse transcriptase inhibitors (NRTIs) in the treatment of HIV infections. (E)-5-(2-Bromovinyl)-2'-deoxyuridine (iv) was discovered as a selective anti-herpes simplex virus compound and is now primarily used for the treatment of varicella-zoster virus infections. The prototype of the acyclic nucleoside phosphonates (ANPs), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], (v) was never commercialized, although it gave rise to several marketed products (cidofovir, adefovir, and tenofovir). 1-[2-(Hydroxyethoxy)methyl]-6-(phenylthio)thymine (vi) and TIBO (tetrahydroimidazo[4,5,1-jk][1,4-benzodiazepin-2(1H)]-one and -thione) (vii) paved the way to a number of compounds (i.e., nevirapine, delavirdine, etravirine, and rilpivirine), which are now collectively called non-NRTIs. The bicyclam AMD3100 (viii) was originally described as an anti-HIV agent before it became later marketed as a stem cell mobilizer. The S-adenosylhomocysteine hydrolase inhibitors (ix), while active against a broad range of (-)RNA viruses and poxviruses may be particularly effective against Ebola virus, and for (x) the O-ANP derivatives, the potential application range encompasses virtually all DNA viruses. © 2015 Wiley Periodicals, Inc.

Extracellular ATP is Differentially Metabolized on Papillary Thyroid Carcinoma Cells Surface in Comparison to Normal Cells.

PubMed

Bertoni, Ana Paula Santin; de Campos, Rafael Paschoal; Tsao, Marisa; Braganhol, Elizandra; Furlanetto, Tania Weber; Wink, Márcia Rosângela

2018-02-17

The incidence of differentiated thyroid cancer has been increasing. Nevertheless, its molecular mechanisms are not well understood. In recent years, extracellular nucleotides and nucleosides have emerged as important modulators of tumor microenvironment. Extracellular ATP is mainly hydrolyzed by NTPDase1/CD39 and NTPDase2/CD39L1, generating AMP, which is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, a possible promoter of tumor growth and metastasis. There are no studies evaluating the expression and functionality of these ectonucleotidases on normal or tumor-derived thyroid cells. Thus, we investigated the ability of thyroid cancer cells to hydrolyze extracellular ATP generating adenosine, and the expression of ecto-enzymes, as compared to normal cells. We found that normal thyroid derived cells presented a higher ability to hydrolyze ATP and higher mRNA levels for ENTDP1-2, when compared to papillary thyroid carcinoma (PTC) derived cells, which had a higher ability to hydrolyze AMP and expressed CD73 mRNA and protein at higher levels. In addition, adenosine induced an increase in proliferation and migration in PTC derived cells, whose effect was blocked by APCP, a non-hydrolysable ADP analogue, which is an inhibitor of CD73. Taken together, these results showed that thyroid follicular cells have a functional purinergic signaling. The higher expression of CD73 in PTC derived cells might favor the accumulation of extracellular adenosine in the tumor microenvironment, which could promote tumor progression. Therefore, as already shown for other tumors, the purinergic signaling should be considered a potential target for thyroid cancer management and treatment.

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.

Crystal structure of NTPDase2 in complex with the sulfoanthraquinone inhibitor PSB-071.

PubMed

Zebisch, Matthias; Baqi, Younis; Schäfer, Petra; Müller, Christa E; Sträter, Norbert

2014-03-01

In many vertebrate tissues CD39-like ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) act in concert with ecto-5'-nucleotidase (e5NT, CD73) to convert extracellular ATP to adenosine. Extracellular ATP is a cytotoxic, pro-inflammatory signalling molecule whereas its product adenosine constitutes a universal and potent immune suppressor. Interference with these ectonucleotidases by use of small molecule inhibitors or inhibitory antibodies appears to be an effective strategy to enhance anti-tumour immunity and suppress neoangiogenesis. Here we present the first crystal structures of an NTPDase catalytic ectodomain in complex with the Reactive Blue 2 (RB2)-derived inhibitor PSB-071. In both of the two crystal forms presented the inhibitor binds as a sandwich of two molecules at the nucleoside binding site. One of the molecules is well defined in its orientation. Specific hydrogen bonds are formed between the sulfonyl group and the nucleoside binding loop. The methylphenyl side chain functionality that improved NTPDase2-specificity is sandwiched between R245 and R394, the latter of which is exclusively found in NTPDase2. The second molecule exhibits great in-plane rotational freedom and could not be modelled in a specific orientation. In addition to this structural insight into NTPDase inhibition, the observation of the putative membrane interaction loop (MIL) in two different conformations related by a 10° rotation identifies the MIL as a dynamic section of NTPDases that is potentially involved in regulation of catalysis. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

An Insight into the Environmental Effects of the Pocket of the Active Site of the Enzyme. Ab initio ONIOM-Molecular Dynamics (MD) Study on Cytosine Deaminase

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

Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

2008-02-01

We applied the ONIOM-molecular dynamics (MD) method to cytosine deaminase to examine the environmental effects of the amino acid residues in the pocket of the active site on the substrate taking account of their thermal motion. The ab initio ONIOM-MD simulations show that the substrate uracil is strongly perturbed by the amino acid residue Ile33, which sandwiches the uracil with His62, through the steric contact due to the thermal motion. As a result, the magnitude of the thermal oscillation of the potential energy and structure of the substrate uracil significantly increases. TM and MA were partly supported by grants frommore » the Ministry of Education, Culture, Sports, Science and Technology of Japan.MD was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, and by the Office of Biological and Environmental Research of the U.S. Department of Energy DOE. Battelle operates Pacific Northwest National Laboratory for DOE.« less

In Situ Biodosimetric Experiment for Space Applications

NASA Astrophysics Data System (ADS)

Goldschmidt, Gergely; Kovaliczky, Éva; Szabó, József; Rontó, Györgyi; Bérces, Attila

2012-06-01

This paper presents the principles and application of DNA based biological UV dosimeters, as developed by Research Group for Biophysics (RGB). These dosimeters are used for assessing the biological hazard of living systems on the Earth's surface and in different waters (rivers, lakes, seas, etc.). The UV dosimetry system has also been used in the space. In dosimeters a bacterial virus, bacteriophage T7 and polycrystalline uracil thin layers have been used as biological detectors. On the Earth's surface the UV radiation induces dimer formation in phage T7 and in the uracil detector, which was evaluated by loss of viability of the phage particles and by the decrease of the characteristic optical density (OD) of uracil thin layers. Recently the development of human space activities has also increased the need to measure the biological effect of extraterrestrial solar radiation, too. The evaluation of the space samples occurred on ground, thus only the starting and the final state were taken into account. A new improved, automated method is presented below which makes data collection more efficient and also makes the dynamics of the process observable.

1-Benzyl-2-(1H-indol-3-yl)-5-oxo-pyrrolidine-2-carbonitrile.

PubMed

Tamazyan, Rafael; Armen, Ayvazyan; Ashot, Martirosyan; Sahak, Gasparyan; Schinazi, Raymond

2008-01-04

In the title compound, C(20)H(17)N(3)O, a potential anti-human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse-transcriptase inhibitor, the pyrrolidine ring has an envelope conformation. In the crystal structure, adjacent mol-ecules are connected into infinite chains via an N-H⋯O hydrogen bond.

Synthesis of 4-amino-5-H-2,3-dihydroisothiazole-1,1-dioxide ring systems on sugar templates via carbanion-mediated sulfonamide intramolecular cyclization reactions (CSIC protocols) of glyco-alpha-sulfonamidonitriles.

PubMed

Domínguez, Laura; van Nhien, Albert Nguyen; Tomassi, Cyrille; Len, Christophe; Postel, Denis; Marco-Contelles, José

2004-02-06

The carbanion-mediated sulfonate intramolecular cyclizations (CSIC protocols) of glyco-alpha-sulfonamidonitriles derived from readily available monosaccharides have been extensively investigated using potassium carbonate, cesium carbonate, n-BuLi, and LDA as bases. As a result, a series of enantiomerically pure spiro(4-amino-5-H-2,3-dihydroisothiazole-1,1-dioxide) derivatives have been prepared efficiently and isolated in good yield. The synthesis of these new bicyclic systems is key to accessing a novel range of aza analogues of TSAO nucleosides (ATSAOs).

Gas-liquid chromatography in lunar organic analysis.

NASA Technical Reports Server (NTRS)

Gehrke, C. W.

1972-01-01

Gas-liquid chromatography (GLC) is a powerful and sensitive method for the separation and detection of organic compounds at nanogram levels. The primary requirement for successful analyses is that the compounds of interest must be volatile under the chromatographic conditions employed. Nonvolatile organic compounds must be converted to volatile derivatives prior to analysis. The derivatives of choice must be both amenable to chromatographic separation and be relatively stable. The condition of volatility necessitates the development of efficient derivatization reactions for important groups of compounds as amino acids, carbohydrates, nucleosides, etc. Trimethylsilylation and trifluoroacetylation represent specific areas of recent prominence. Some relevant practical aspects of GLC are discussed.

Synthesis of a mixed-model stationary phase derived from glutamine for HPLC separation of structurally different biologically active compounds: HILIC and reversed-phase applications.

PubMed

Aral, Tarık; Aral, Hayriye; Ziyadanoğulları, Berrin; Ziyadanoğulları, Recep

2015-01-01

A novel mixed-mode stationary phase was synthesised starting from N-Boc-glutamine, aniline and spherical silica gel (4 µm, 60 Å). The prepared stationary phase was characterized by IR and elemental analysis. The new stationary phase bears an embedded amide group into phenyl ring, highly polar a terminal amide group and non-polar groups (phenyl and alkyl groups). At first, this new mixed-mode stationary phase was used for HILIC separation of four nucleotides and five nucleosides. The effects of different separation conditions, such as pH value, mobile phase and temperature, on the separation process were investigated. The optimum separation for nucleotides was achieved using HILIC isocratic elution with aqueous mobile phase and acetonitrile with 20°C column temperature. Under these conditions, the four nucleotides could be separated and detected at 265 nm within 14 min. Five nucleosides were separated under HILIC isocratic elution with aqueous mobile phase containing pH=3.25 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and detected at 265 nm within 14 min. Chromatographic parameters as retention factor, selectivity, theoretical plate number and peak asymmetry factor were calculated for the effect of temperature and water content in mobile phase on the separation process. The new column was also tested for nucleotides and nucleosides mixture and six analytes were separated in 10min. The chromatographic behaviours of these polar analytes on the new mixed-model stationary phase were compared with those of HILIC columns under similar conditions. Further, phytohormones and phenolic compounds were separated in order to see influence of the new stationary phase in reverse phase conditions. Eleven plant phytohormones were separated within 13 min using RP-HPLC gradient elution with aqueous mobile phase containing pH=2.5 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and detected at 230 or 278 nm. The best separation conditions for seven phenolic compounds was also achieved using reversed-phase HPLC gradient elution with aqueous mobile phase containing pH=2.5 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and seven phenolic compounds could be separated and detected at 230 nm within 16 min. Copyright © 2014 Elsevier B.V. All rights reserved.

Functional expression and characterization of a purine nucleobase transporter gene from Leishmania major.

PubMed

Sanchez, Marco A; Tryon, Rob; Pierce, Steven; Vasudevan, Gayatri; Landfear, Scott M

2004-01-01

Leishmania major, like all the other kinetoplastid protozoa, are unable to synthesize purines and rely on purine nucleobase and nucleoside acquisition across the parasite plasma membrane by specific permeases. Although, several genes have been cloned that encode nucleoside transporters in Leishmania and Trypanosoma brucei, much less progress has been made on nucleobase transporters, especially at the molecular level. The studies reported here have cloned and expressed the first gene for a L. major nucleobase transporter, designated LmaNT3. The LmaNT3 permease shows 33% identity to L. donovani nucleoside transporter 1.1 (LdNT1.1) and is, thus, a member of the equilibrative nucleoside transporter (ENT) family. ENT family members identified to date are nucleoside transporters, some of which also transport one or several nucleobases. Functional expression studies in Xenopus laevis oocytes revealed that LmaNT3 mediates high levels of uptake of hypoxanthine, xanthine, adenine and guanine. Moreover, LmaNT3 is an high affinity transporter with K(m) values for hypoxanthine, xanthine, adenine and guanine of 16.5 +/- 1.5, 8.5 +/- 0.6, 8.5 +/- 1.1, and 8.8 +/- 4.0 microM, respectively. LmaNT3 is, thus, the first member of the ENT family identified in any organism that functions as a nucleobase rather than nucleoside or nucleoside/nucleobase transporter.

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

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...

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…

Study of an efficient conversion of 1,3-dimethyl-5-(Arylazo)-6-Amino-Uracils to 1,3-dimethyl-8-(Aryl)-Azapurin-2,6-Diones

NASA Astrophysics Data System (ADS)

Debnath, Diptanu; Purkayastha, Atanu; Kirillov, Alexander; Ganguly, Rakesh; Misra, Tarun Kumar

2017-12-01

6-Aminouracils have extensively been used as precursors for synthesizing numerous uracil derivatives of biological and pharmaceutical significance. This study describes an application of 1,3-dimethyl-5-(arylazo)-6-aminouracils (Uazo: Uazo1-Uazo4, precursors) for an efficient synthesis of a series of 8-substituted-azapurins (AP), namely 1,3-dimethyl-8-(aryl)-azapurin-2,6-diones (aryl = p-HC6H4 (AP1), -MeC6H4 (AP2), sbnd ClC6H4 (AP3), and sbnd SO2NH2C6H4 (AP4)) following an oxidation method in the presence of copper (II) nitrate and in alkaline medium. The obtained compounds were isolated in good yields as crystalline air-stable products and have been fully characterized in the solution by UV-vis and NMR spectroscopy, as well as in the solid state by FT-IR spectroscopy, elemental analysis, and single-crystal X-ray diffraction (for AP2 and AP4). UV-vis study evidences that the conversion of the 6-aminouracil precursors occurs via an intermediate, Cu(II)-complex and a plausible mechanism for the formation of AP1-AP4 has been proposed. Unlike AP2 the crystal structure of AP4 reveals the formation of interdigitated 1D H-bonded chains that has been topologically classified within the 2C1 type. The 1H NMR spectra of the products have proton signals that completely devoid of hydrazone (sbnd NHsbnd) and imine (=NH) signals of their parent Uazo derivatives, thus confirming their full conversion and a stability of the AP1-AP4 in solution. The excitation and emission spectra of AP1-AP4 were also recorded in solution, revealing electronic transitions between similar vibrational energy levels of S0 (singlet ground state) and S1 (singlet first excited state).

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

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.

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

HIV-1 reverse transcriptase and antiviral drug resistance. Part 2.

PubMed

Das, Kalyan; Arnold, Eddy

2013-04-01

Structures of RT and its complexes combined with biochemical and clinical data help in illuminating the molecular mechanisms of different drug-resistance mutations. The NRTI drugs that are used in combinations have different primary mutation sites. RT mutations that confer resistance to one drug can be hypersensitive to another RT drug. Structure of an RT-DNA-nevirapine complex revealed how NNRTI binding forbids RT from forming a polymerase competent complex. Collective knowledge about various mechanisms of drug resistance by RT has broader implications for understanding and targeting drug resistance in general. In Part 1, we discussed the role of RT in developing HIV-1 drug resistance, structural and functional states of RT, and the nucleoside/nucleotide analog (NRTI) and non-nucleoside (NNRTI) drugs used in treating HIV-1 infections. In this part, we discuss structural understanding of various mechanisms by which RT confers antiviral drug resistance. Copyright © 2013 Elsevier B.V. All rights reserved.

The history of antiretrovirals: key discoveries over the past 25 years.

PubMed

De Clercq, Erik

2009-09-01

Within 25 years after zidovudine (3'-azido-2',3'-dideoxythymidine, AZT) was first described as an inhibitor of HIV replication, 25 anti-HIV drugs have been formally approved for clinical use in the treatment of HIV infections: seven nucleoside reverse transcriptase inhibitors (NRTIs): zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir and emtricitabine; one nucleotide reverse transcriptase inhibitor (NtRTI): tenofovir [in its oral prodrug form: tenofovir disoproxil fumarate (TDF)]; four non-nucleoside reverse transcriptase inhibitors (NNRTIs): nevirapine, delavirdine, efavirenz and etravirine; ten protease inhibitors (PIs): saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir and darunavir; one fusion inhibitor (FI): enfuvirtide; one co-receptor inhibitor (CRI): maraviroc and one integrase inhibitor (INI): raltegravir. These compounds are used in various drug combination (some at fixed dose) regimens so as to achieve the highest possible benefit and tolerability, and to diminish the risk of virus-drug resistance development. (c) 2009 John Wiley & Sons, Ltd.

Molecular design, synthesis and physical properties of novel Cytisine-derivatives - Experimental and theoretical study

NASA Astrophysics Data System (ADS)

Ivanova, Bojidarka; Spiteller, Michael

2013-02-01

The paper presented a comprehensive theoretical and experimental study on the molecular drugs-design, synthesis, isolation, physical spectroscopic and mass spectrometric elucidation of novel functionalization derivatives of Cytisine (Cyt), using nucleosidic residues. Since these alkaloids have established biochemical profile, related the binding affinity of the nicotinic acetylcholine receptors (nAChRs), particularly α7 sub-type, the presented correlation between the molecular structure and properties allowed to evaluated the highlights of the biochemical hypothesises related the Schizophrenia. The anticancer activity of α7 subtype agonists and the crucial role of the nucleoside-based medications in the cancer therapy provided opportunity for further study on the biochemical relationship between Schizophrenia and few kinds of cancers, which has been hypothesized recently. The physical electronic absorptions (EAs), circular dichroic (CD) and Raman spectroscopic (RS) properties as well as mass spectrometric (MS) data, obtained using electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) methods under the positive single (MS) and tandem (MS/MS) modes of operation are discussed. Taking into account reports on a fatal intoxication of Cyt, the presented data would be of interest in the field of forensic chemistry, through development of highly selective and sensitive analytical protocols. Quantum chemical method is used to predict the physical properties of the isolated alkaloids, their affinity to the receptor loop and gas-phase stabilized species, observed mass spectrometrically.

Quantitative analysis of DNA methylation in the promoter region of the methylguanine-O(6) -DNA-methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis.

PubMed

Goedecke, Simon; Mühlisch, Jörg; Hempel, Georg; Frühwald, Michael C; Wünsch, Bernhard

2015-12-01

Along with histone modifications, RNA interference and delayed replication timing, DNA methylation belongs to the key processes in epigenetic regulation of gene expression. Therefore, reliable information about the methylation level of particular DNA fragments is of major interest. Herein the methylation level at two positions of the promoter region of the gene methylguanine-O(6) -DNA-Methyltransferase (MGMT) was investigated. Previously, it was demonstrated that the epigenetic status of this DNA region correlates with response to alkylating anticancer agents. An automated CGE method with LIF detection was established to separate the six DNA fragments resulting from combined bisulfite restriction analysis of the methylated and non-methylated MGMT promoter. In COBRA, the DNA was treated with bisulfite converting cytosine into uracil. During PCR uracil pairs with adenine, which changes the original recognition site of the restriction enzyme Taql. Artificial probes generated by mixing appropriate amounts of DNA after bisulfite treatment and PCR amplification were used for validation of the method. The methylation levels of these samples could be determined with high accuracy and precision. DNA samples prepared by mixing the corresponding clones first and then performing PCR amplification led to non-linear correlation between the corrected peak areas and the methylation levels. This effect is explained by slightly different PCR amplification of DNA with different sequences present in the mixture. The superiority of CGE over PAGE was clearly demonstrated. Finally, the established method was used to analyze the methylation levels of human brain tumor tissue samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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.

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.

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.

The Photosynthesis and Photo-Stability of Nucleic Acids in Prebiotic Extraterrestrial Environments

PubMed Central

Sandford, Scott A.; Bera, Partha P.; Lee, Timothy J.; Materese, Christopher K.; Nuevo, Michel

2017-01-01

Laboratory experiments have shown that the UV photo-irradiation of low-temperature ices of astrophysical interest leads to the formation of organic molecules, including molecules important for biology such as amino acids, quinones, and amphiphiles. When pyrimidine is introduced in these ices, the products of irradiation include the nucleobases uracil, cytosine, and thymine, the informational sub-units of DNA and RNA, as well as some of their isomers. The formation of these compounds, which has been studied both experimentally and theoretically, requires a succession of additions of OH, NH2, and CH3 groups to pyrimidine. Results show that H2O ice plays key roles in the formation of the nucleobases, as an oxidant, as a matrix in which reactions can take place, and as a catalyst that assists proton abstraction from intermiediate compounds. As H2O is also the most abundant icy component in most cold astrophysical environments, it probably plays the same roles in space for the formation of biologically relevant compounds. Results also show that although the formation of uracil and cytosine from pyrimidine in ices is fairly straightforward, the formation of thymine is not. This is mostly due to the fact that methylation is a limiting step for its formation, particularly in H2O-rich ices, where methylation must competes with oxidation. The relative inefficiency of the abiotic formation of thymine to that of uracil and cytosine, coupled with the fact that thymine has not been detected in meteorites are not inconsistent with the RNA world hypothesis. Indeed, a lack of abiotically produced thymine delivered to the early Earth may have forced the choice for an RNA world, in which only uracil and cytosine are needed, but not thymine. PMID:24500331

Toehold-mediated strand displacement reaction-dependent fluorescent strategy for sensitive detection of uracil-DNA glycosylase activity.

PubMed

Wu, Yushu; Wang, Lei; Jiang, Wei

2017-03-15

Sensitive detection of uracil-DNA glycosylase (UDG) activity is beneficial for evaluating the repairing process of DNA lesions. Here, toehold-mediated strand displacement reaction (TSDR)-dependent fluorescent strategy was constructed for sensitive detection of UDG activity. A single-stranded DNA (ssDNA) probe with two uracil bases and a trigger sequence were designed. A hairpin probe with toehold domain was designed, and a reporter probe was also designed. Under the action of UDG, two uracil bases were removed from ssDNA probe, generating apurinic/apyrimidinic (AP) sites. Then, the AP sites could inhibit the TSDR between ssDNA probe and hairpin probe, leaving the trigger sequence in ssDNA probe still free. Subsequently, the trigger sequence was annealed with the reporter probe, initiating the polymerization and nicking amplification reaction. As a result, numerous G-quadruplex (G4) structures were formed, which could bind with N-methyl-mesoporphyrin IX (NMM) to generate enhanced fluorescent signal. In the absence of UDG, the ssDNA probe could hybridize with the toehold domain of the hairpin probe to initiate TSDR, blocking the trigger sequence, and then the subsequent amplification reaction would not occur. The proposed strategy was successfully implemented for detecting UDG activity with a detection limit of 2.7×10 -5 U/mL. Moreover, the strategy could distinguish UDG well from other interference enzymes. Furthermore, the strategy was also applied for detecting UDG activity in HeLa cells lysate with low effect of cellular components. These results indicated that the proposed strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of base-excision repair in the treatment of childhood acute lymphoblastic leukaemia with 6-mercaptopurine and high doses of methotrexate.

PubMed

Stanczyk, M; Sliwinski, T; Trelinska, J; Cuchra, M; Markiewicz, L; Dziki, L; Bieniek, A; Bielecka-Kowalska, A; Kowalski, M; Pastorczak, A; Szemraj, J; Mlynarski, W; Majsterek, I

2012-01-24

Methotrexate (MTX) and 6-mercaptopurine (6MP) are the most commonly used drugs in the therapy of childhood acute lymphoblastic leukaemia (ALL). The main genotoxic effect of MTX resulting from inhibition of thymidylate synthase is mis-incorporation of uracil into DNA, which is considered essential for the effectiveness of the Protocol M in ALL IC BFM 2002/EURO LB 2002 regimens. In this study, we investigated the level of basal and induced DNA damage as well as the effectiveness of DNA repair in lymphocytes of children with ALL at four time-points during therapy with MTX and 6MP. To assess DNA damage and the efficacy of DNA repair we used the modified alkaline comet assay with uracil DNA glycosylase (Udg) and endonuclease III (EndoIII). In addition, we examined the induction of apoptosis in the lymphocytes of the patients during treatment. Finally, we compared the activity of base-excision repair (BER), involved in removal of both uracil and oxidized bases from DNA in lymphocytes of children with ALL and lymphocytes of healthy children. BER efficiency was estimated in an in vitro assay with cellular extracts and plasmid substrates of heteroduplex DNA with an AP-site. Our results indicate that there is a significant decrease in the efficacy of DNA repair associated with an increased level of uracil in DNA and induction of apoptosis during therapy. Moreover, it was found that the BER capacity was decreased in the lymphocytes of ALL patients in contrast to that in lymphocytes of healthy children. Thus, we suggest that an impairment of the BER pathway may play a role in the pathogenesis and therapy of childhood ALL. © 2011 Elsevier B.V. All rights reserved.

Molecular Dynamics Simulations of the Bacterial UraA H+-Uracil Symporter in Lipid Bilayers Reveal a Closed State and a Selective Interaction with Cardiolipin

PubMed Central

Kalli, Antreas C.; Sansom, Mark S. P.; Reithmeier, Reinhart A. F.

2015-01-01

The Escherichia coli UraA H+-uracil symporter is a member of the nucleobase/ascorbate transporter (NAT) family of proteins, and is responsible for the proton-driven uptake of uracil. Multiscale molecular dynamics simulations of the UraA symporter in phospholipid bilayers consisting of: 1) 1-palmitoyl 2-oleoyl-phosphatidylcholine (POPC); 2) 1-palmitoyl 2-oleoyl-phosphatidylethanolamine (POPE); and 3) a mixture of 75% POPE, 20% 1-palmitoyl 2-oleoyl-phosphatidylglycerol (POPG); and 5% 1-palmitoyl 2-oleoyl-diphosphatidylglycerol/cardiolipin (CL) to mimic the lipid composition of the bacterial inner membrane, were performed using the MARTINI coarse-grained force field to self-assemble lipids around the crystal structure of this membrane transport protein, followed by atomistic simulations. The overall fold of the protein in lipid bilayers remained similar to the crystal structure in detergent on the timescale of our simulations. Simulations were performed in the absence of uracil, and resulted in a closed state of the transporter, due to relative movement of the gate and core domains. Anionic lipids, including POPG and especially CL, were found to associate with UraA, involving interactions between specific basic residues in loop regions and phosphate oxygens of the CL head group. In particular, three CL binding sites were identified on UraA: two in the inner leaflet and a single site in the outer leaflet. Mutation of basic residues in the binding sites resulted in the loss of CL binding in the simulations. CL may play a role as a “proton trap” that channels protons to and from this transporter within CL-enriched areas of the inner bacterial membrane. PMID:25729859

Photosynthesis and photo-stability of nucleic acids in prebiotic extraterrestrial environments.

PubMed

Sandford, Scott A; Bera, Partha P; Lee, Timothy J; Materese, Christopher K; Nuevo, Michel

2015-01-01

Laboratory experiments have shown that the UV photo-irradiation of low-temperature ices of astrophysical interest leads to the formation of organic molecules, including molecules important for biology such as amino acids, quinones, and amphiphiles. When pyrimidine is introduced into these ices, the products of irradiation include the nucleobases uracil, cytosine, and thymine, the informational sub-units of DNA and RNA, as well as some of their isomers. The formation of these compounds, which has been studied both experimentally and theoretically, requires a succession of additions of OH, NH₂, and CH₃groups to pyrimidine. Results show that H₂O ice plays key roles in the formation of the nucleobases, as an oxidant, as a matrix in which reactions can take place, and as a catalyst that assists proton abstraction from intermediate compounds. As H₂O is also the most abundant icy component in most cold astrophysical environments, it probably plays the same roles in space in the formation of biologically relevant compounds. Results also show that although the formation of uracil and cytosine from pyrimidine in ices is fairly straightforward, the formation of thymine is not. This is mostly due to the fact that methylation is a limiting step for its formation, particularly in H₂O-rich ices, where methylation must compete with oxidation. The relative inefficiency of the abiotic formation of thymine to that of uracil and cytosine, together with the fact that thymine has not been detected in meteorites, are not inconsistent with the RNA world hypothesis. Indeed, a lack of abiotically produced thymine delivered to the early Earth may have forced the choice for an RNA world, in which only uracil and cytosine are needed, but not thymine.

Pyrimidine Salvage in Trypanosoma brucei Bloodstream Forms and the Trypanocidal Action of Halogenated Pyrimidiness

PubMed Central

Ali, Juma A. M.; Creek, Darren J.; Burgess, Karl; Allison, Harriet C.; Field, Mark C.; Mäser, Pascal; De Koning, Harry P.

2016-01-01

African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host. However, uptake of pyrimidines in bloodstream form trypanosomes has not been investigated, making it difficult to judge the relative importance of salvage and synthesis or to design a pyrimidine-based chemotherapy. Detailed characterization of pyrimidine transport activities in bloodstream form Trypanosoma brucei brucei found that these cells express a high-affinity uracil transporter (designated TbU3) that is clearly distinct from the procyclic pyrimidine transporters. This transporter had low affinity for uridine and 2′deoxyuridine and was the sole pyrimidine transporter expressed in these cells. In addition, thymidine was taken up inefficiently through a P1-type nucleoside transporter. Of importance, the anticancer drug 5-fluorouracil was an excellent substrate for TbU3, and several 5-fluoropyrimidine analogs were investigated for uptake and trypanocidal activity; 5F-orotic acid, 5F-2′deoxyuridine displayed activity in the low micromolar range. The metabolism and mode of action of these analogs was determined using metabolomic assessments of T. brucei clonal lines adapted to high levels of these pyrimidine analogs, and of the sensitive parental strains. The analysis showed that 5-fluorouracil is incorporated into a large number of metabolites but likely exerts toxicity through incorporation into RNA. 5F-2′dUrd and 5F-2′dCtd are not incorporated into nucleic acids but act as prodrugs by inhibiting thymidylate synthase as 5F-dUMP. We present the most complete model of pyrimidine salvage in T. brucei to date, supported by genome-wide profiling of the predicted pyrimidine biosynthesis and conversion enzymes. PMID:23188714

1-Benzyl-2-(1H-indol-3-yl)-5-oxo­pyrrolidine-2-carbonitrile

PubMed Central

Tamazyan, Rafael; Armen, Ayvazyan; Ashot, Martirosyan; Sahak, Gasparyan; Schinazi, Raymond

2008-01-01

In the title compound, C20H17N3O, a potential anti-human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse-transcriptase inhibitor, the pyrrolidine ring has an envelope conformation. In the crystal structure, adjacent mol­ecules are connected into infinite chains via an N—H⋯O hydrogen bond. PMID:21201400

Pharmacophore Identification, Molecular Docking, Virtual Screening, and In Silico ADME Studies of Non-Nucleoside Reverse Transcriptase Inhibitors.

PubMed

Pirhadi, Somayeh; Ghasemi, Jahan B

2012-12-01

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have gained a definitive place due to their unique antiviral potency, high specificity and low toxicity in antiretroviral combination therapies used to treat HIV. In this study, chemical feature based pharmacophore models of different classes of NNRT inhibitors of HIV-1 have been developed. The best HypoRefine pharmacophore model, Hypo 1, which has the best correlation coefficient (0.95) and the lowest RMS (0.97), contains two hydrogen bond acceptors, one hydrophobic and one ring aromatic feature, as well as four excluded volumes. Hypo 1 was further validated by test set and Fischer validation method. The best pharmacophore model was then utilized as a 3D search query to perform a virtual screening to retrieve potential inhibitors. The hit compounds were subsequently subjected to filtering by Lipinski's rule of five and docking studies by Libdock and Gold methods to refine the retrieved hits. Finally, 7 top ranked compounds based on Gold score fitness function were subjected to in silico ADME studies to investigate for compliance with the standard ranges. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Discovery of 3-{5-[(6-Amino-1H-pyrazolo[3,4-b]pyridine-3-yl)methoxy]-2-chlorophenoxy}-5-chlorobenzonitrile (MK-4965): A Potent, Orally Bioavailable HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitor with Improved Potency against Key Mutant Viruses

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

Tucker, Thomas J.; Sisko, John T.; Tynebor, Robert M.

2009-07-10

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been shown to be a key component of highly active antiretroviral therapy (HAART). The use of NNRTIs has become part of standard combination antiviral therapies producing clinical outcomes with efficacy comparable to other antiviral regimens. There is, however, a critical issue with the emergence of clinical resistance, and a need has arisen for novel NNRTIs with a broad spectrum of activity against key HIV-1 RT mutations. Using a combination of traditional medicinal chemistry/SAR analyses, crystallography, and molecular modeling, we have designed and synthesized a series of novel, highly potent NNRTIs that possess broad spectrummore » antiviral activity and good pharmacokinetic profiles. Further refinement of key compounds in this series to optimize physical properties and pharmacokinetics has resulted in the identification of 8e (MK-4965), which has high levels of potency against wild-type and key mutant viruses, excellent oral bioavailability and overall pharmacokinetics, and a clean ancillary profile.« less

Reactive Derivatives of Nucleic Acids and Their Components as Affinity Reagents

NASA Astrophysics Data System (ADS)

Knorre, Dmitrii G.; Vlasov, Valentin V.

1985-09-01

The review is devoted to derivatives of nucleic acids and their components — nucleotides, nucleoside triphosphates, and oligonucleotides carrying reactive groups. Such derivatives are important tools for the investigation of protein-nucleic acid interactions and the functional topography of complex protein and nucleoprotein structures and can give rise to the prospect of being able to influence in a highly selective manner living organisms, including the nucleic acids and the nucleoproteins of the genetic apparatus. The review considers the principal groups of such reagents, the methods of their synthesis, and their properties which determine the possibility of their use for the selective (affinity) modification of biopolymers. The general principles of the construction of affinity reagents and their applications are analysed in relation to nucleotide affinity reagents. The bibliography includes 121 references.

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 that confer both indole and benzimidazole nucleoside resistance (D344E and A355T). PMID:25348532

Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea.

PubMed

Kasai, Yuki; Oshima, Kohei; Ikeda, Fukiko; Abe, Jun; Yoshimitsu, Yuya; Harayama, Shigeaki

2015-01-01

Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea. In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura(+)) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator. A self-cloning-based positive selection system for the genetic transformation of P. ellipsoidea was developed. Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.

New one-pot synthesis of spiro[furo[2,3-d]pyrimidine-6,5'-pyrimidine]pentaones and their sulfur analogues.

PubMed

Jalilzadeh, Mohammad; Noroozi Pesyan, Nader; Rezaee, Fereshteh; Rastgar, Saeed; Hosseini, Yaser; Sahin, Ertan

2011-08-01

Reaction of barbituric acid (BA), 1,3-dimethyl barbituric acid (DMBA) and 2-thiobarbituric acid (TBA) with cyanogen bromide and various aldehydes in presence of triethylamine afforded a new class of heterocyclic stable 5-alkyl and/or 5-aryl-1H, 1'H-spiro[furo[2,3-d]pyrimidine-6,5'-pyrimidine]2,2',4,4',6'(3H,3'H,5H)-pentaones which are dimeric forms of barbiturate (uracil and thiouracil derivatives) at 0 °C to ambient temperatures. Structure elucidation is proved by X-ray crystallography, (1)H NMR, (13)C NMR, FT-IR, CHN and mass analyses techniques. Mechanisms of the formations are discussed.

Formation of Nucleobases from the UV Irradiation of Pyrimidine in Interstellar Ice Analogs

NASA Technical Reports Server (NTRS)

Milam, Stefanie N.; Nuevo, Michel; Sandford, Scott A.; Elsila, Jamie E.; Dworkin, Jason P.

2010-01-01

Previous laboratory simulations showed that complex molecules, including prebiotic compounds/can be formed under interstellar conditions from the vacuum UV irradiation of interstellar ice analogs containing H2O, CO, NH3 etc. Although some complex prebiotic species have not been confirmed In the interstellar medium, they are known to be present in meteorites. Nucleobases, the building blocks of DNA and RNA, have also been detected in meteorites. Here, we present a study of the formation of pyrimidine-based compounds from the UV irradiation of pyrimidine in H2O- and/or NH3-ices at 20-30 K, Our results show that various derivatives, induding the nucleobases uracil and cytosine, are formed under these conditions.

Mechanisms for the formation of thymine under astrophysical conditions and implications for the origin of life

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

Bera, Partha P., E-mail: Partha.P.Bera@nasa.gov, E-mail: Timothy.J.Lee@nasa.gov; Nuevo, Michel; Materese, Christopher K.

Nucleobases are the carriers of the genetic information in ribonucleic acid and deoxyribonucleic acid (DNA) for all life on Earth. Their presence in meteorites clearly indicates that compounds of biological importance can form via non-biological processes in extraterrestrial environments. Recent experimental studies have shown that the pyrimidine-based nucleobases uracil and cytosine can be easily formed from the ultraviolet irradiation of pyrimidine in H{sub 2}O-rich ice mixtures that simulate astrophysical processes. In contrast, thymine, which is found only in DNA, is more difficult to form under the same experimental conditions, as its formation usually requires a higher photon dose. Earlier quantummore » chemical studies confirmed that the reaction pathways were favorable provided that several H{sub 2}O molecules surrounded the reactants. However, the present quantum chemical study shows that the formation of thymine is limited because of the inefficiency of the methylation of pyrimidine and its oxidized derivatives in an H{sub 2}O ice, as supported by the laboratory studies. Our results constrain the formation of thymine in astrophysical environments and thus the inventory of organic molecules delivered to the early Earth and have implications for the role of thymine and DNA in the origin of life.« less

Mechanisms for the Formations of the Thymine Under Astrophysical Conditions and Implications for the Origin of Life

NASA Technical Reports Server (NTRS)

Bera, Partha P.; Nuevo, Michel; Materese, Christopher K.; Sandford, Scott A.; Lee, Timothy J.

2016-01-01

Nucleobases are the carriers of the genetic information in ribonucleic acid and deoxyribonucleic acid (DNA) for all life on Earth. Their presence in meteorites clearly indicates that compounds of biological importance can form via non-biological processes in extraterrestrial environments. Recent experimental studies have shown that the pyrimidine-based nucleobases uracil and cytosine can be easily formed from the ultraviolet irradiation of pyrimidine in H2O-rich ice mixtures that simulate astrophysical processes. In contrast, thymine, which is found only in DNA, is more difficult to form under the same experimental conditions, as its formation usually requires a higher photon dose. Earlier quantum chemical studies confirmed that the reaction pathways were favorable provided that several H2O molecules surrounded the reactants. However, the present quantum chemical study shows that the formation of thymine is limited because of the inefficiency of the methylation of pyrimidine and its oxidized derivatives in an H2O ice, as supported by the laboratory studies. Our results constrain the formation of thymine in astrophysical environments and thus the inventory of organic molecules delivered to the early Earth and have implications for the role of thymine and DNA in the origin of life.

The discovery of novel diarylpyri(mi)dine derivatives with high level activity against a wide variety of HIV-1 strains as well as against HIV-2.

PubMed

Lu, Xueyi; Yang, Jiapei; Kang, Dongwei; Gao, Ping; Daelemans, Dirk; De Clercq, Erik; Pannecouque, Christophe; Zhan, Peng; Liu, Xinyong

2018-05-01

By means of structure-based molecular hybridization strategy, a series of novel diarylpyri(mi)dine derivatives targeting the entrance channel of HIV-1 reverse transcriptase (RT) were designed, synthesized and evaluated as potent non-nucleoside reverse transcriptase inhibitors (NNRTIs). Encouragingly, all the tested compounds showed good activities against wild-type (WT) HIV-1 (IIIB) with EC 50 in the range of 1.36 nM-29 nM, which is much better than those of nevirapine (NVP, EC 50  = 125.42 nM) and azidothymidine (AZT, EC 50  = 11.36 nM). Remarkably, these compounds also displayed effective activity against the most of the single and double-mutated HIV-1 strains with low EC 50 values, which is comparable to the control drugs. Besides, these compounds were also exhibited favorable enzymatic inhibitory activity. Moreover, preliminary structure-activity relationships (SARs) and molecular modeling study were investigated and discussed in detail. Unexpectedly, four diarylpyrimidines yielded moderate anti-HIV-2 activities. To our knowledge, this is rarely reported that diarylpyrimidine-based NNRTIs have potent activity against both HIV-1 and HIV-2 in cell culture. Copyright © 2018 Elsevier Ltd. All rights reserved.

Exploiting the anti-HIV 6-desfluoroquinolones to design multiple ligands.

PubMed

Sancineto, Luca; Iraci, Nunzio; Barreca, Maria Letizia; Massari, Serena; Manfroni, Giuseppe; Corazza, Gianmarco; Cecchetti, Violetta; Marcello, Alessandro; Daelemans, Dirk; Pannecouque, Christophe; Tabarrini, Oriana

2014-09-01

It is getting clearer that many drugs effective in different therapeutic areas act on multiple rather than single targets. The application of polypharmacology concepts might have numerous advantages especially for disease such as HIV/AIDS, where the rapid emergence of resistance requires a complex combination of more than one drug. In this paper, we have designed three hybrid molecules combining WM5, a quinolone derivative we previously identified as HIV Tat-mediated transcription (TMT) inhibitor, with the tricyclic core of nevirapine and BILR 355BS (BILR) non-nucleoside reverse transcriptase inhibitors (NNRTIs) to investigate whether it could be possible to obtain molecules acting on both transcription steps of the HIV replicative cycle. One among the three designed multiple ligands, reached this goal. Indeed, compound 1 inhibited both TMT and reverse transcriptase (RT) activity. Unexpectedly, while the anti-TMT activity exerted by compound 1 resulted into a selective inhibition of HIV-1 reactivation from latently infected OM10.1 cells, the anti-RT properties shown by all of the synthesized compounds did not translate into an anti-HIV activity in acutely infected cells. Thus, we have herein produced the proof of concept that the design of dual TMT-RT inhibitors is indeed possible, but optimization efforts are needed to obtain more potent derivatives. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

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%.

Stability of the resistance to the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone, a non-nucleoside polymerase inhibitor of bovine viral diarrhea virus.

PubMed

Castro, Eliana F; Campos, Rodolfo H; Cavallaro, Lucía V

2014-01-01

Bovine viral diarrhea virus (BVDV) is the prototype Pestivirus. BVDV infection is distributed worldwide and causes serious problems for the livestock industry. The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC) is a non-nucleoside polymerase inhibitor (NNI) of BVDV. All TSC-resistant BVDV variants (BVDV-TSCr T1-5) present an N264D mutation in the NS5B gene (RdRp) whereas the variant BVDV-TSCr T1 also presents an NS5B A392E mutation. In the present study, we carried out twenty passages of BVDV-TSCr T1-5 in MDBK cells in the absence of TSC to evaluate the stability of the resistance. The viral populations obtained (BVDV R1-5) remained resistant to the antiviral compound and conserved the mutations in NS5B associated with this phenotype. Along the passages, BVDV R2, R3 and R5 presented a delay in the production of cytopathic effect that correlated with a decrease in cell apoptosis and intracellular accumulation of viral RNA. The complete genome sequences that encode for NS2 to NS5B, Npro and Erns were analyzed. Additional mutations were detected in the NS5B of BVDV R1, R3 and R4. In both BVDV R2 and R3, most of the mutations found were localized in NS5A, whereas in BVDV R5, the only mutation fixed was NS5A V177A. These results suggest that mutations in NS5A could alter BVDV cytopathogenicity. In conclusion, the stability of the resistance to TSC may be due to the fixation of different compensatory mutations in each BVDV-TSCr. During their replication in a TSC-free medium, some virus populations presented a kind of interaction with the host cell that resembled a persistent infection: decreased cytopathogenicity and viral genome synthesis. This is the first report on the stability of antiviral resistance and on the evolution of NNI-resistant BVDV variants. The results obtained for BVDV-TSCr could also be applied for other NNIs.

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

Barc, B.; Ryszka, M.; Spurrell, J.

Multi-photon ionization (MPI) of the RNA base uracil has been studied in the wavelength range 220–270 nm, coinciding with excitation to the S{sub 2}(ππ*) state. A fragment ion at m/z = 84 was produced by 2-photon absorption at wavelengths ≤232 nm and assigned to C{sub 3}H{sub 4}N{sub 2}O{sup +} following CO abstraction. This ion has not been observed in alternative dissociative ionization processes (notably electron impact) and its threshold is close to recent calculations of the minimum activation energy for a ring opening conical intersection to a σ(n-π)π* closed shell state. Moreover, the predicted ring opening transition leaves a COmore » group at one end of the isomer, apparently vulnerable to abstraction. An MPI mass spectrum of uracil-water clusters is presented for the first time and compared with an equivalent dry measurement. Hydration enhances certain fragment ion pathways (particularly C{sub 3}H{sub 3}NO{sup +}) but represses C{sub 3}H{sub 4}N{sub 2}O{sup +} production. This indicates that hydrogen bonding to water stabilizes uracil with respect to neutral excited-state ring opening.« less

XRCC1 suppresses somatic hypermutation and promotes alternative nonhomologous end joining in Igh genes.

PubMed

Saribasak, Huseyin; Maul, Robert W; Cao, Zheng; McClure, Rhonda L; Yang, William; McNeill, Daniel R; Wilson, David M; Gearhart, Patricia J

2011-10-24

Activation-induced deaminase (AID) deaminates cytosine to uracil in immunoglobulin genes. Uracils in DNA can be recognized by uracil DNA glycosylase and abasic endonuclease to produce single-strand breaks. The breaks are repaired either faithfully by DNA base excision repair (BER) or mutagenically to produce somatic hypermutation (SHM) and class switch recombination (CSR). To unravel the interplay between repair and mutagenesis, we decreased the level of x-ray cross-complementing 1 (XRCC1), a scaffold protein involved in BER. Mice heterozygous for XRCC1 showed a significant increase in the frequencies of SHM in Igh variable regions in Peyer's patch cells, and of double-strand breaks in the switch regions during CSR. Although the frequency of CSR was normal in Xrcc1(+/-) splenic B cells, the length of microhomology at the switch junctions decreased, suggesting that XRCC1 also participates in alternative nonhomologous end joining. Furthermore, Xrcc1(+/-) B cells had reduced Igh/c-myc translocations during CSR, supporting a role for XRCC1 in microhomology-mediated joining. Our results imply that AID-induced single-strand breaks in Igh variable and switch regions become substrates simultaneously for BER and mutagenesis pathways.

Effects of Hypoxanthine Substitution in Peptide Nucleic Acids Targeting KRAS2 Oncogenic mRNA Molecules: Theory and Experiment

PubMed Central

Sanders, Jeffrey M.; Wampole, Matthew E.; Chen, Chang-Po; Sethi, Dalip; Singh, Amrita; Dupradeau, François-Yves; Wang, Fan; Gray, Brian D.; Thakur, Mathew L.; Wickstrom, Eric

2013-01-01

Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multi-mutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick basepairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA-PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA-PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition. PMID:23972113

[Over-expression of uracil DNA glycosylase 2 (UNG2) enhances the resistance to oxidative damage in HepG2 cells].

PubMed

Cao, Liyan; Cheng, Shan; Du, Juan; Guo, Yanhai; Huang, Xiaofeng

2017-04-01

Objective To investigate the uracil glycosidic enzyme activity of uracil DNA glycosylase 2 (UNG2) and study the role of UNG2 in the resistance of antioxidant stress of HepG2 cells. Methods The UNG2-expressing vector was built. Western blotting was used to detect the expression of UNG2. Immunofluorescence staining was performed to observe the cellular location of UNG2. Oligonucleotide was used as substrate for the determination of the UNG2 glycosidic enzyme activity. H 2 O 2 toxicity assay was done to study the function of UNG2 in the antioxidant resistance of hepatocellular carcinoma HepG2 cells. Results UNG2 was successfully over-expressed in HEK293FT cells, and UNG2 was found to be mainly located in nucleus. Enzyme activity assay showed that UNG2 had significant oligonucleotide dU glycosidic enzyme activity. H 2 O 2 toxicity assay showed that over-expressed UNG2 could remarkably increase the survival of HepG2 cells after exposed to H 2 O 2 . Conclusion UNG2 possesses specific DNA glycosidic enzyme activity, and it can protect HepG2 cells against oxidative stress damage.

Immunochemical characterization of the anti-RNA antibodies found in scleroderma and systemic lupus erythematosus. II. Reactivity with hsa-coupled, uridine-containing, monophosphoric ribodinucleotides.

PubMed Central

Alarcón-Segovia, D; Fishbein, E; Estrada-Parra, S; García-Ortigoza, E

1976-01-01

Sera from patients with scleroderma have been found to have anti-RNA antibodies which react with human serum albumin (HSA)-coupled uridine and uridine monophosphate (UMP) and are inhibited by uracil, uridine and UMP. Scleroderma sera react uniformly with 5'-polyuridylic acid (poly(U)) and fail to react with polyadenylic, polyuridylic acid poly(A) - poly(U)) which is also indicative of their uracil specificity. Anti-RNA antibodies found in systemic lupus erythematosus (SLE) are immunochemically different from those found in scleroderma in that, instead of being uniformly specific to uracil, they are markedly heterogeneous and may react with uracil, uridine and/or UMP. SLE sera frequently react with poly(A) - poly(U), indicating also their ability to recognize the double helical structure of double-stranded RNA. Thirty-seven scleroderma and thirty-four SLE sera from as many patients with either of these conditions were tested against HSA-coupled, uridine-containing monophosphoric dinucleotides in an attempt to characterize further their anti-RNA antibodies. Scleroderma sera were found to react primarily with dinucleotides in which uridine was the base proximal to the carrier protein and, except for sera that also contained antibodies to adenosine which reacted with UpA, they failed to react with dinucleotides in which uridine was in a terminal position only. Reaction with dinucleotides in which uridine was proximal to the carrier protein could be inhibited by uracil but not by the corresponding terminal base. Some lupus sera were found to react with both dinucleotides that contain the same bases in opposite sequence, e.g. ApU and UpA, while others were found to react with only one of the sequences. They were also found to react more frequently with dinucleotides in which HSA was coupled to a base other than uridine, suggesting that the reaction is primarily due to anti-DNA antibodies. Because immunization with dinucleotides coupled to protein prepared by the same method we have used, yields higher specificity to the base attached to the carrier protein, our findings suggest that, in scleroderma, a single event, akin to that of immunization with a purified antigen, gives rise to the anti-RNA antibodies, whereas in systemic lupus erythematosus there is a considerably wider immunological aberration. PMID:1082854

Structure determination of uracil-DNA N-glycosylase from Deinococcus radiodurans in complex with DNA.

PubMed

Pedersen, Hege Lynum; Johnson, Kenneth A; McVey, Colin E; Leiros, Ingar; Moe, Elin

2015-10-01

Uracil-DNA N-glycosylase (UNG) is a DNA-repair enzyme in the base-excision repair (BER) pathway which removes uracil from DNA. Here, the crystal structure of UNG from the extremophilic bacterium Deinococcus radiodurans (DrUNG) in complex with DNA is reported at a resolution of 1.35 Å. Prior to the crystallization experiments, the affinity between DrUNG and different DNA oligonucleotides was tested by electrophoretic mobility shift assays (EMSAs). As a result of this analysis, two 16 nt double-stranded DNAs were chosen for the co-crystallization experiments, one of which (16 nt AU) resulted in well diffracting crystals. The DNA in the co-crystal structure contained an abasic site (substrate product) flipped into the active site of the enzyme, with no uracil in the active-site pocket. Despite the high resolution, it was not possible to fit all of the terminal nucleotides of the DNA complex into electron density owing to disorder caused by a lack of stabilizing interactions. However, the DNA which was in contact with the enzyme, close to the active site, was well ordered and allowed detailed analysis of the enzyme-DNA interaction. The complex revealed that the interaction between DrUNG and DNA is similar to that in the previously determined crystal structure of human UNG (hUNG) in complex with DNA [Slupphaug et al. (1996). Nature (London), 384, 87-92]. Substitutions in a (here defined) variable part of the leucine loop result in a shorter loop (eight residues instead of nine) in DrUNG compared with hUNG; regardless of this, it seems to fulfil its role and generate a stabilizing force with the minor groove upon flipping out of the damaged base into the active site. The structure also provides a rationale for the previously observed high catalytic efficiency of DrUNG caused by high substrate affinity by demonstrating an increased number of long-range electrostatic interactions between the enzyme and the DNA. Interestingly, specific interactions between residues in the N-terminus of a symmetry-related molecule and the complementary DNA strand facing away from the active site were also observed which seem to stabilize the enzyme-DNA complex. However, the significance of this observation remains to be investigated. The results provide new insights into the current knowledge about DNA damage recognition and repair by uracil-DNA glycosylases.

Distinct Purine Distribution in Carbonaceous Chondrites

NASA Technical Reports Server (NTRS)

Callahan, Michael P.; Smith, Karen E.; Cleaves, Henderson J.; Ruzicka, Josef; Stern, Jennifer C.; Glavin, Daniel P.; House, Christopher H.; Dworkin, Jason P.

2011-01-01

Carbonaceous chondrite meteorites are known to contain a diverse suite of organic compounds, many of which are essential components of biochemistry. Amino acids, which are the monomers of proteins, have been extensively studied in such meteorites (e.g. Botta and Bada 2002; Pizzarello et aI., 2006). The origin of amino acids in meteorites has been firmly established as extraterrestrial based on their detection typically as racemic mixtures of amino acids, the presence of many non-protein amino acids, and non-terrestrial values for compound-specific deuterium, carbon, and nitrogen isotopic measurements. In contrast to amino acids, nucleobases in meteorites have been far less studied. Nucleobases are substituted one-ring (pyrimidine) or two-ring (purine) nitrogen heterocyclic compounds and serve as the information carriers of nucleic acids and in numerous coenzymes. All of the purines (adenine, guanine, hypoxanthine, and xanthine) and pyrimidines (uracil) previously reported in meteorites are biologically common and could be interpreted as the result of terrestrial contamination (e.g. van del' Velden and Schwartz, 1974.) Unlike other meteoritic organics, there have been no observations of stochastic molecular diversity of purines and pyrimidines in meteorites, which has been a criterion for establishing extraterrestrial origin. Maltins et al. (2008) performed compound-specific stable carbon isotope measurements for uracil and xanthine in the Murchison meteorite. They assigned a non-terrestrial origin for these nucleobases; however, the possibility that interfering indigenous molecules (e.g. carboxylic acids) contributed to the 13C-enriched isotope values for these nucleobases cannot be completely ruled out. Thus, the origin of these meteoritic nucleobases has never been established unequivocally. Here we report on our investigation of extracts of II different carbonaceous chondrites covering various petrographic types (Cl, CM, and CR) and degrees of aqueous alteration (l, 2, and 3) and one ureilite. Analysis via liquid chromatography coupled with electrospray triple-stage mass spectrometry or orbitrap mass spectrometry employed a targeted approach for analysis focused on the five canonical RNA/DNA nucleobases as well as 14 non-canonical pyrimidines and purines, which have bcen observed under plausible prebiotic reactions.

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

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

Synthesis and biological evaluation of 5-substituted derivatives of the potent antiherpes agent (north)-methanocarbathymine.

PubMed

Russ, Pamela; Schelling, Pierre; Scapozza, Leonardo; Folkers, Gerd; Clercq, Erik De; Marquez, Victor E

2003-11-06

The conformationally locked nucleoside, (north)-methanocarbathymine (1a), is a potent and selective anti-herpes agent effective against herpes simplex type 1 (HSV1) and type 2 (HSV2) viruses. Hereby, we report on the synthesis and biological evaluation of a small set of 5-substituted pyrimidine nucleosides belonging to the same class of bicyclo[3.1.0]hexane nucleosides. Both the 5-bromovinyl (4) and the 5-bromo analogue (3) appeared to be exclusive substrates of HSV1 thymidine kinase (TK), contrasting with the 5-iodo analogue (2), which was significantly phosphorylated by the human cytosolic TK. The binding affinity constant and catalytic turnover for HSV1 TK were measured to assess the influence of the substitution on these parameters. In the plaque reduction and cytotoxicity assays, the 5-bromo analogue (3) showed good activity against HSV1 and HSV2 with less general toxicity than 1a. Against varicella-zoster virus (VZV), the north-locked 5-bromovinyl analogue (4) proved to be as potent as its conformationally unlocked 2'-deoxyriboside equivalent BVDU. The three compounds were also tested in vitro as prodrugs used in a gene therapy context on three osteosarcoma cell lines, either deficient in TK (TK(-)), nontransduced, or stably transduced with HSV1 TK. The 5-iodo compound (2, CC(50) 25 +/- 7 microM) was more efficient than ganciclovir (GCV, CC(50) 75 +/- 35 microM) in inhibiting growth of HSV1-TK transfected cells and less inhibitory than GCV toward TK(-) cells, whereas compound 3 inhibited transfected and nontransfected cell lines in a relatively similar dose-dependent manner.

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.

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.

2[prime] and 3[prime] Carboranyl uridines and their diethyl ether adducts

DOEpatents

Soloway, A.H.; Barth, R.F.; Anisuzzaman, A.K.; Alam, F.; Tjarks, W.

1992-12-15

A process is described for preparing carboranyl uridine nucleoside compounds and their diethyl ether adducts, which exhibit a tenfold increase in boron content over prior art boron containing nucleoside compounds. The carboranyl uridine nucleoside compounds exhibit enhanced lipophilicity and hydrophilic properties adequate to enable solvation in aqueous media for subsequent incorporation of the compounds in methods for boron neutron capture therapy in mammalian tumor cells. No Drawings

2' and 3' Carboranyl uridines and their diethyl ether adducts

DOEpatents

Soloway, Albert H.; Barth, Rolf F.; Anisuzzaman, Abul K.; Alam, Fazlul; Tjarks, Werner

1992-01-01

There is disclosed a process for preparing carboranyl uridine nucleoside compounds and their diethyl ether adducts, which exhibit a tenfold increase in boron content over prior art boron containing nucleoside compounds. Said carboranyl uridine nucleoside compounds exhibit enhanced lipophilicity and hydrophilic properties adequate to enable solvation in aqueous media for subsequent incorporation of said compounds in methods for boron neutron capture therapy in mammalian tumor cells.

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

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.

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

Structual Effects of Cytidine 2^' Ribose Modifications as Determined by Irmpd Action Spectroscopy

NASA Astrophysics Data System (ADS)

Hamlow, Lucas; He, Chenchen; Fan, Lin; Wu, Ranran; Yang, Bo; Rodgers, M. T.; Berden, Giel; Oomens, J.

2015-06-01

Modified nucleosides, both naturally occurring and synthetic play an important role in understanding and manipulating RNA and DNA. Naturally occurring modified nucleosides are commonly found in functionally important regions of RNA and also affect antibiotic resistance or sensitivity. Synthetic modifications of nucleosides such as fluorinated and arabinosyl nucleosides have found uses as anti-virals and chemotherapy agents. Understanding the effect that modifications have on structure and glycosidic bond stability may lend insight into the functions of these modified nucleosides. Modifications such as the naturally occurring 2^'-O-methylation and the synthetic 2^'-fluorination are believed to help stabilize the nucleoside through the glycosidic bond stability and intramolecular hydrogen bonding. Changing the sugar from ribose to arabinose alters the stereochemistry at the 2^' position and thus shifts the 3D orientation of the 2^'-hydroxyl group, which also affects intramolecular hydrogen bonding and glycosidic bond stability. The structures of 2^'-deoxy-2^'-fluorocytidine, 2^'-O-methylcytidine and cytosine arabinoside are examined in the current work by measuring the infrared spectra in the IR fingerprint region using infrared multiple photon dissociation (IRMPD) action spectroscopy. The structures accessed in the experiments were determined via comparison of the measured IRMPD action spectra to the theoretical linear IR spectra determined by density functional theory and molecular modeling for the stable low-energy structures. Although glycosidic bond stability cannot be quantitatively determined from this data, complementary TCID studies will establish the effect of these modifications. Comparison of these modified nucleosides with their RNA and DNA analogues will help elucidate differences in their intrinsic chemistry.

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.

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

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.

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

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

Thermoadaptation-Directed Enzyme Evolution in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426

PubMed Central

Kobayashi, Jyumpei; Wada, Keisuke; Furukawa, Megumi; Doi, Katsumi

2014-01-01

Thermostability is an important property of enzymes utilized for practical applications because it allows long-term storage and use as catalysts. In this study, we constructed an error-prone strain of the thermophile Geobacillus kaustophilus HTA426 and investigated thermoadaptation-directed enzyme evolution using the strain. A mutation frequency assay using the antibiotics rifampin and streptomycin revealed that G. kaustophilus had substantially higher mutability than Escherichia coli and Bacillus subtilis. The predominant mutations in G. kaustophilus were A · T→G · C and C · G→T · A transitions, implying that the high mutability of G. kaustophilus was attributable in part to high-temperature-associated DNA damage during growth. Among the genes that may be involved in DNA repair in G. kaustophilus, deletions of the mutSL, mutY, ung, and mfd genes markedly enhanced mutability. These genes were subsequently deleted to construct an error-prone thermophile that showed much higher (700- to 9,000-fold) mutability than the parent strain. The error-prone strain was auxotrophic for uracil owing to the fact that the strain was deficient in the intrinsic pyrF gene. Although the strain harboring Bacillus subtilis pyrF was also essentially auxotrophic, cells became prototrophic after 2 days of culture under uracil starvation, generating B. subtilis PyrF variants with an enhanced half-denaturation temperature of >10°C. These data suggest that this error-prone strain is a promising host for thermoadaptation-directed evolution to generate thermostable variants from thermolabile enzymes. PMID:25326311

Thermoadaptation-directed enzyme evolution in an error-prone thermophile derived from Geobacillus kaustophilus HTA426.

PubMed

Suzuki, Hirokazu; Kobayashi, Jyumpei; Wada, Keisuke; Furukawa, Megumi; Doi, Katsumi

2015-01-01

Thermostability is an important property of enzymes utilized for practical applications because it allows long-term storage and use as catalysts. In this study, we constructed an error-prone strain of the thermophile Geobacillus kaustophilus HTA426 and investigated thermoadaptation-directed enzyme evolution using the strain. A mutation frequency assay using the antibiotics rifampin and streptomycin revealed that G. kaustophilus had substantially higher mutability than Escherichia coli and Bacillus subtilis. The predominant mutations in G. kaustophilus were A · T→G · C and C · G→T · A transitions, implying that the high mutability of G. kaustophilus was attributable in part to high-temperature-associated DNA damage during growth. Among the genes that may be involved in DNA repair in G. kaustophilus, deletions of the mutSL, mutY, ung, and mfd genes markedly enhanced mutability. These genes were subsequently deleted to construct an error-prone thermophile that showed much higher (700- to 9,000-fold) mutability than the parent strain. The error-prone strain was auxotrophic for uracil owing to the fact that the strain was deficient in the intrinsic pyrF gene. Although the strain harboring Bacillus subtilis pyrF was also essentially auxotrophic, cells became prototrophic after 2 days of culture under uracil starvation, generating B. subtilis PyrF variants with an enhanced half-denaturation temperature of >10°C. These data suggest that this error-prone strain is a promising host for thermoadaptation-directed evolution to generate thermostable variants from thermolabile enzymes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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.

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.

Structure-activity relationship studies on quinoxalin-2(1H)-one derivatives containing thiazol-2-amine against hepatitis C virus leading to the discovery of BH6870.

PubMed

Zhong, Qi-Fei; Liu, Rui; Liu, Gang

2015-11-01

Chronic hepatitis C virus infection represents a serious global public health problem, typically resulting in fibrosis, cirrhosis, and ultimately hepatocellular carcinoma. Based on our previous discovery of lead compound 2 (Liu et al. J Med Chem 54:5747-5768, 2011), 35 new quinoxalinone derivatives were explored in this study. Outline of the structure-activity relationships (SARs) revealed that compound BH6870 (36) showed high anti-HCV potency ([Formula: see text]) and a good cell safety index (SI [Formula: see text]). SARs analysis indicated that quinoxalin-2(1H)-one containing a 4-aryl-substituted thiazol-2-amine moiety was optimal for antiviral activity. Introducing a hydrogen-bond acceptor (such as ester or amide group) at the C-3 position of quinoxalin-2(1H)-one was beneficial for the antiviral potency, and especially, N,N-disubstituted amide was far superior to N-monosubstituted amide. Incorporation of more than one halogen (fluorine or chlorine atom) or a strong electron-withdrawing group on the benzene ring of the thiazole-phenyl moiety might reduce electron atmosphere density further and resulted in a dramatical loss of activity. The NH-group of the lactam moiety was clearly required for anti-HCV activity. Design and synthesis of quinoxalin-2(1H)-one derivatives as new non-nucleoside small-molecule HCV inhibitors. BH6870 (36), showing higher antiviral potency and a good cell safety index, was identified.

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.

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.

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.

The future of pre-exposure prophylaxis (PrEP) for human immunodeficiency virus (HIV) infection.

PubMed

Özdener, Ayşe Elif; Park, Tae Eun; Kalabalik, Julie; Gupta, Rachna

2017-05-01

People at high risk for HIV acquisition should be offered pre-exposure prophylaxis (PrEP). Tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC) is currently the only medication recommended for pre-exposure prophylaxis (PrEP) by the Centers for Disease Control and Prevention (CDC) in people at high risk for HIV acquisition. This article will review medications currently under investigation and the future landscape of PrEP therapy. Areas covered: This article will review clinical trials that have investigated nontraditional regimens of TDF/FTC, antiretroviral agents from different drug classes such as integrase strand transfer inhibitors (INSTI), nucleoside reverse transcriptase inhibitors (NRTI), and non-nucleoside reverse transcriptase inhibitors (NNRTI) as potential PrEP therapies. Expert commentary: Currently, there are several investigational drugs in the pipeline for PrEP against HIV infection. Increased utilization of PrEP therapy depends on provider identification of people at high risk for HIV transmission. Advances in PrEP development will expand options and access for people and reduce the risk of HIV acquisition.

HIV type-1 genotypic resistance profiles in vertically infected patients from Argentina reveal an association between K103N+L100I and L74V mutations.

PubMed

Aulicino, Paula C; Rocco, Carlos A; Mecikovsky, Debora; Bologna, Rosa; Mangano, Andrea; Sen, Luisa

2010-01-01

Patterns and pathways of HIV type-1 (HIV-1) antiretroviral (ARV) drug resistance-associated mutations in clinical isolates are conditioned by ARV history and factors such as viral subtype and fitness. Our aim was to analyse the frequency and association of ARV drug resistance mutations in a group of long-term vertically infected patients from Argentina. Plasma samples from 71 patients (38 children and 33 adolescents) were collected for genotypic HIV-1 ARV resistance testing during the period between February 2006 and October 2008. Statistically significant pairwise associations between ARV resistance mutations in pol, as well as associations between mutations and drug exposure, were identified using Fisher's exact tests with Bonferroni and false discovery rate corrections. Phylogenetic analyses were performed for subtype assignment. In protease (PR), resistance-associated mutations M46I/L, I54M/L/V/A/S and V82A/F/T/S/M/I were associated with each other and with minor mutations at codons 10, 24 and 71. Mutations V82A/F/T/S/M/I were primarily selected by the administration of ritonavir (RTV) in an historical ARV regimen. In reverse transcriptase, thymidine analogue mutation (TAM)1 profile was more common than TAM2. The non-nucleoside K103N+L100I mutations were observed at high frequency (15.5%) and were significantly associated with the nucleoside mutation L74V in BF recombinants. Associations of mutations at PR sites reflect the frequent use of RTV at an early time in this group of patients and convergent resistance mechanisms driven by the high exposure to protease inhibitors, as well as local HIV-1 diversity. The results provide clinical evidence of a molecular interaction between K103N+L100I and L74V mutations at the reverse transcriptase gene in vivo, limiting the future use of second-generation non-nucleoside reverse transcriptase inhibitors such as etravirine.

High Levels of Transmitted HIV Drug Resistance in a Study in Papua New Guinea.

PubMed

Lavu, Evelyn; Kave, Ellan; Mosoro, Euodia; Markby, Jessica; Aleksic, Eman; Gare, Janet; Elsum, Imogen A; Nano, Gideon; Kaima, Petronia; Dala, Nick; Gurung, Anup; Bertagnolio, Silvia; Crowe, Suzanne M; Myatt, Mark; Hearps, Anna C; Jordan, Michael R

2017-01-01

Papua New Guinea is a Pacific Island nation of 7.3 million people with an estimated HIV prevalence of 0.8%. ART initiation and monitoring are guided by clinical staging and CD4 cell counts, when available. Little is known about levels of transmitted HIV drug resistance in recently infected individuals in Papua New Guinea. Surveillance of transmitted HIV drug resistance in a total of 123 individuals recently infected with HIV and aged less than 30 years was implemented in Port Moresby (n = 62) and Mount Hagen (n = 61) during the period May 2013-April 2014. HIV drug resistance testing was performed using dried blood spots. Transmitted HIV drug resistance was defined by the presence of one or more drug resistance mutations as defined by the World Health Organization surveillance drug resistance mutations list. The prevalence of non-nucleoside reverse transcriptase inhibitor transmitted HIV drug resistance was 16.1% (95% CI 8.8%-27.4%) and 8.2% (95% CI 3.2%-18.2%) in Port Moresby and Mount Hagen, respectively. The prevalence of nucleoside reverse transcriptase inhibitor transmitted HIV drug resistance was 3.2% (95% CI 0.2%-11.7%) and 3.3% (95% CI 0.2%-11.8%) in Port Moresby and Mount Hagen, respectively. No protease inhibitor transmitted HIV drug resistance was observed. The level of non-nucleoside reverse transcriptase inhibitor drug resistance in antiretroviral drug naïve individuals recently infected with HIV in Port Moresby is amongst the highest reported globally. This alarming level of transmitted HIV drug resistance in a young sexually active population threatens to limit the on-going effective use of NNRTIs as a component of first-line ART in Papua New Guinea. To support the choice of nationally recommended first-line antiretroviral therapy, representative surveillance of HIV drug resistance among antiretroviral therapy initiators in Papua New Guinea should be urgently implemented.

Non-covalent and coordination interactions in Cu(II) systems with uridine, uridine 5'-monophosphate and triamine or tetramine as biogenic amine analogues in aqueous solutions.

PubMed

Łomozik, Lechosław; Jastrzab, Renata

2003-10-01

Reactions of metallation and non-covalent interactions have been studied in ternary systems of Cu(II) ions with uridine, uridine 5'-monophosphate and diamines or triamines. It has been found that in metal-free systems the reaction centres of the nucleoside with the polyamine are the donor nitrogen atoms N(3) and protonated -NH(x) groups of the amines. In comparison to systems with adenosine or cytidine, the pH range of complex formation is shifted towards higher values. It is a consequence of significantly higher basicity of uridine and in agreement with the ion-ion, ion-dipole interaction model assumed. Formation of molecular complexes of uridine 5'-monophosphate with polyamines at a low pH is the result of activity of the phosphate group which plays the role of a negatively charged reaction site. Non-covalent interactions interfere in processes of bioligand metallation. Centres of weak interactions are simultaneously binding sites of metal ions. In protonated Cu(Urd)(PA)H(x) complexes, coordination has been found to involve the N(3) atom from the nucleoside and two donor nitrogen atoms from the polyamine (PA). In the heteroligand species Cu(Urd)(PA), despite deprotonation of all amine groups, one of these groups is located outside the inner coordination sphere. In complexes with uridine-5'-monophosphate, the phosphate group is active in metallation. Moreover, in certain coordination compounds this group is engaged in non-covalent interactions with PA molecules, despite binding Cu ions, as has been shown on the basis of equilibrium and spectral studies.

Facile construction of substituted pyrimido[4,5-d]pyrimidones by transformation of enaminouracil

PubMed Central

Hamama, Wafaa S.; Ismail, Mohamed A.; Al-Saman, Hanaa A.; Zoorob, Hanafi H.

2012-01-01

The reaction of 6-amino-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (1) as a binucleophile with primary aromatic or heterocyclic amines and formaldehyde or aromatic (heterocyclic) aldehydes in a molar ratio (1:1:2) gave the pyrimido[4,5-d]pyrimidin-2,4-dione ring systems 2–5. Treatment of 1 with diamines and formalin in molar ratio (2:1:4) gave the bis-pyrimido[4,5-d]pyrimidin-2,4-diones 6–8. Furthermore, substituted pyrimido[4,5-d]pyrimidin-2,4-diones with uracil derivative 11 or spiro indole 16 were synthesized. Synthesis of pyrimido[4,5-d]pyrimidin-2,4-diones with different substitution at C-5 and C-7 was achieved to give 13 and 18, respectively. PMID:25685408

The metabolism of isocytidine in Escherichia coli

PubMed Central

Doskočil, J.; Holý, A.; Filip, J.

1974-01-01

Intact cells and cell-free extracts of E. coli convert isocytidine to isocytosine and uracil. The radioactive label of 5-[3H]isocytidine is incorporated into RNA and, DNA of growing bacteria at a rate equal to about 1.4% of that of cytidine under similar conditions; the radioactivity is found in uridylic, cytidylic and 2′-deoxythymidylic acids, while less than 0.4% of incorporated radioactive material might be due to possible incorporation of intact isocytidine. Uridine phosphorylase and cytidine deaminase apparently do not participate in the metabolic conversion of isocytidine. 5-[3H]isocytidine was prepared by platinum-catalyzed dehalogenation of 5-bromoisocytidine in the presence of tritium. The 5-bromo derivative was obtained from 2′,3′-0- -isopropylideneisocytidine by N-bromsuccinimide bromination followed by acidic hydrolysis. PMID:10793683

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.

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.

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.

Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy

PubMed Central

Pukáncsik, Mária; Orbán, Ágnes; Nagy, Kinga; Matsuo, Koichi; Gekko, Kunihiko; Maurin, Damien; Hart, Darren; Kézsmárki, István; Vertessy, Beata G.

2016-01-01

A novel uracil-DNA degrading protein factor (termed UDE) was identified in Drosophila melanogaster with no significant structural and functional homology to other uracil-DNA binding or processing factors. Determination of the 3D structure of UDE is excepted to provide key information on the description of the molecular mechanism of action of UDE catalysis, as well as in general uracil-recognition and nuclease action. Towards this long-term aim, the random library ESPRIT technology was applied to the novel protein UDE to overcome problems in identifying soluble expressing constructs given the absence of precise information on domain content and arrangement. Nine constructs of UDE were chosen to decipher structural and functional relationships. Vacuum ultraviolet circular dichroism (VUVCD) spectroscopy was performed to define the secondary structure content and location within UDE and its truncated variants. The quantitative analysis demonstrated exclusive α-helical content for the full-length protein, which is preserved in the truncated constructs. Arrangement of α-helical bundles within the truncated protein segments suggested new domain boundaries which differ from the conserved motifs determined by sequence-based alignment of UDE homologues. Here we demonstrate that the combination of ESPRIT and VUVCD spectroscopy provides a new structural description of UDE and confirms that the truncated constructs are useful for further detailed functional studies. PMID:27273007

The PUR Experiment on the EXPOSE-R facility: biological dosimetry of solar extraterrestrial UV radiation

NASA Astrophysics Data System (ADS)

Bérces, A.; Egyeki, M.; Fekete, A.; Horneck, G.; Kovács, G.; Panitz, C.

2015-01-01

The aim of our experiment Phage and Uracil Response was to extend the use of bacteriophage T7 and uracil biological dosimeters for measuring the biologically effective ultraviolet (UV) dose in the harsh extraterrestrial radiation conditions. The biological detectors were exposed in vacuum-tightly cases in the European Space Agency (ESA) astrobiological exposure facility attached to the external platform of Zvezda (EXPOSE-R). EXPOSE-R took off to the International Space Station (ISS) in November 2008 and was installed on the External platform of the Russian module Zvezda of the ISS in March 2009. Our goal was to determine the dose-effect relation for the formation of photoproducts (i.e. damage to phage DNA and uracil, respectively). The extraterrestrial solar UV radiation ranges over the whole spectrum from vacuum-UV (λ<200 nm) to UVA (315 nm<λ<400 nm), which causes photolesions (photoproducts) in the nucleic acids/their components either by photoionization or excitation. However, these wavelengths cause not only photolesions but in a wavelength-dependent efficiency the reversion of some photolesions, too. Our biological detectors measured in situ conditions the resultant of both reactions induced by the extraterrestrial UV radiation. From this aspect the role of the photoreversion in the extension of the biological UV dosimetry are discussed.

Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

PubMed Central

Huovinen, Tuomas; Brockmann, Eeva-Christine; Akter, Sultana; Perez-Gamarra, Susan; Ylä-Pelto, Jani; Liu, Yuan; Lamminmäki, Urpo

2012-01-01

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material. PMID:22355397