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PubMed1993

Nucleoside transport in Crithidia luciliae.

Nucleoside transport was evaluated in the trypanosomatid Crithidia luciliae by a rapid sampling technique. C. luciliae was shown to possess two independent nucleoside transporters, one which transported adenosine, deoxyadenosine, tubercidin, sangivamycin and the pyrimidine nucleoside thymidine, while the second was specific for guanosine, inosine and deoxyguanosine. The rapid influx occurred by a process of facilitated transport. The apparent Km values for adenosine and guanosine were 9.34 +/- 1.30 and 10.6 +/- 2.60 microM, respectively. The pyrimidine nucleoside thymidine was transported at a rate approximately 50% lower than the purine nucleosides, whilst uridine, deoxyuridine and deoxycytidine were not transported. The optical isomer, L-adenosine entered the organism by simple diffusion rather than by facilitated transport. In contrast to mammalian cells, neither of the nucleoside transporters in C. luciliae were inhibited by nitrobenzylthioinosine, dilazep, or dipyridamole, potent inhibitors of nucleoside transport in mammalian cells, whilst p-chloromercuribenzoate sulphonate inhibited both nucleoside transporters in C. luciliae.

Animals↗
PubMed1987

Two high affinity nucleoside transporters in Leishmania donovani.

A rapid sampling kinetic technique has been used to evaluate the nucleoside transport functions of Leishmania donovani. The results indicated that L. donovani promastigotes possessed two independent purine nucleoside transporters with nonoverlapping substrate specificity. The first transported inosine, guanosine, and their analogs, while the second carried adenosine, analogs of adenosine, and the pyrimidine nucleosides, uridine, cytidine, and thymidine. The apparent Km values of the two nucleoside permeases for their purine nucleoside substrates were extraordinarily low, in the micromolar range. The organisms were capable of concentrating purine nucleosides from the medium and converting them to the nucleotide level with great efficiency and rapidity. Inosine and adenosine transport could be distinguished by different sensitivities to sulfhydryl reagents, suggesting structural differences between the two transporters. Finally, the two nucleoside transport systems of L. donovani were virtually refractory to inhibition by 4-nitrobenzylthioinosine and dipyridamole, two potent inhibitors of nucleoside entry into mammalian cells.

Adenosine↗
PubMed2004

Structural and biochemical effects of human immunodeficiency virus mutants resistant to non-nucleoside reverse transcriptase inhibitors.

Non-nucleoside reverse transcriptase inhibitors are potent, highly specific, noncompetitive inhibitors of the Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. They are used commonly as part of potent antiretroviral regimens for the treatment of HIV-1 infection, either in combination with nucleoside analogs, protease inhibitors, or both. A major limitation to the success of non-nucleoside inhibitors is the rapid emergence of HIV-1 variants resistant to these drugs. These drug-resistant variants contain one or more mutations in the non-nucleoside inhibitor binding pocket of reverse transcriptase. This review summarizes the effects that these mutations have on non-nucleoside inhibitor binding, reverse transcriptase structure, HIV-1 replication, and the ability of reverse transcriptase to catalyze DNA polymerization and RNase H cleavage. In addition, studies are summarized evaluating important interactions between mutations conferring resistance to non-nucleoside inhibitors and those conferring resistance to nucleoside analogs. The studies summarized in this review provide important insights into potentially useful approaches to minimize the development of HIV-1 resistant to non-nucleoside inhibitors.

Codon↗
PubMed2005

MALDI-TOF MS analysis of urinary nucleosides.

As RNA turnover seems to be impaired in cancer patients, modified nucleosides have been evaluated as potential tumor markers. Modified nucleosides are mainly formed post-transcriptionally in tRNA, set free during RNA metabolism, and excreted in urine. Especially methylated nucleosides play an important role, as their levels are higher in urine from cancer patients. For structural elucidation of known and unknown nucleosides from urine samples of cancer patients, MALDI-TOF MS and MALDI-PSD were used for the first time. This technique generally ensures high sensitivity, mass resolution, and accuracy. In our analytical approach we prepurified nucleosides from urine by affinity chromatography and subsequently separated them by semipreparative high performance liquid chromatography. The different fractions were collected separately and analyzed by MALDI-TOF MS and PSD-MALDI using a mixture of six low molecular weight calibrants for internal or external calibration. The molecular totals formulas based on a mass accuracy of 10 ppm and below were calculated and a systematic data base search was performed. The inherent problem of the MALDI-technique, the reduced sensitivity for low molecular weight substances caused by matrix suppression effects, was reduced by our technique. We identified several nucleosides in urine, which were previously identified via retention times and UV spectra of standards after HPLC analysis. Eight further nucleosides were observed. This work demonstrates for the first time the potential of MALDI-TOF and PSD-MALDI in combination with semipreparative HPLC for assignment of nucleosides in urine. The particularly high mass accuracy of this mass spectrometric method provides opportunities for identifying unknown compounds.

Aged↗
PubMed2006

Nucleoside transporters: from scavengers to novel therapeutic targets.

Hydrophilic purine and pyrimidine nucleosides rely on specialized carrier proteins for their membrane translocation. The recent identification of two gene families encoding equilibrative and concentrative nucleoside transporters in mammals and other organisms has provided the essential breakthrough to a more complete understanding of the biological significance of nucleoside transport. Although nucleoside salvage is a primary function of these proteins, recent data indicate functions beyond metabolic recycling. In brain and spinal cord, for example, nucleoside transporters have the potential to regulate synaptic levels of neuroactive purines such as adenosine and, thereby, indirectly modulate physiological processes through G-protein-coupled purine P1 receptors. As described in this review, recent research indicates novel putative functions for CNS nucleoside transporters in sleep, arousal, drug and alcohol addiction, nociception and analgesia. The therapeutic use of nucleoside analogue drugs and nucleoside transporter inhibitors in viral, neoplastic, cardiovascular and infectious disease is also described.

Animals↗
PubMed2000

Stimulation of nucleoside efflux and inhibition of adenosine kinase by A1 adenosine receptor activation.

Adenosine is produced intracellularly during conditions of metabolic stress and is an endogenous agonist for four subtypes of G-protein linked receptors. Nucleoside transporters are membrane-bound carrier proteins that transfer adenosine, and other nucleosides, across biological membranes. We investigated whether adenosine receptor activation could modulate transporter-mediated adenosine efflux from metabolically stressed cells. DDT1 MF-2 smooth muscle cells were incubated with 10 microM [3H]adenine to label adenine nucleotide pools. Metabolic stress with the glycolytic inhibitor iodoacetic acid (1AA, 5 mM) increased tritium release by 63% (P < 0.01), relative to cells treated with buffer alone. The IAA-induced increase was blocked by the nucleoside transport inhibitor nitrobenzylthioinosine (1 microM), indicating that the increased tritium release was primarily a purine nucleoside. HPLC verified this to be [3H]adenosine. The adenosine A1 receptor selective agonist N6-cyclohexyladenosine (CHA, 300 nM) increased the release of [3H]purine nucleoside induced by IAA treatment by 39% (P < 0.05). This increase was blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (10 microM). Treatment of cells with UTP (100 microM), histamine (100 microM), or phorbol-12-myristate-13-acetate (PMA, 10 microM) also increased [3H]purine nucleoside release. The protein kinase C inhibitor chelerythrine chloride (500 nM) inhibited the increase in [3H]purine nucleoside efflux induced by CHA or PMA treatment. The adenosine kinase activity of cells treated with CHA or PMA was found to be decreased significantly compared with buffer-treated cells. These data indicated that adenosine A1 receptor activation increased nucleoside efflux from metabolically stressed DDT1 MF-2 cells by a PKC-dependent inhibition of adenosine kinase activity.

Adenosine↗
PubMed2001

Increased activity of membrane-associated nucleoside diphosphate kinase and inhibition of cAMP synthesis in failing human myocardium.

OBJECTIVE: Chronic heart failure is associated with a decreased responsiveness of the heart to beta-adrenergic receptor agonists. We recently demonstrated a receptor-independent activation of G proteins and modulation of cardiac adenylyl cyclase activity by sarcolemmal membrane-associated nucleoside diphosphate kinase. We wondered whether changes in the activity of nucleoside diphosphate kinase occur in heart failure and contribute to or compensate for the impairment in myocardial receptor-mediated cAMP generation. METHODS: Sarcolemmal membranes were purified from non-failing and failing human left ventricular myocardium. The protein level and activity of nucleoside diphosphate kinase were quantified. The influence of nucleoside diphosphate kinase on adenylyl cyclase activity was determined by measuring the effect of GDP on adenylyl cyclase activity in the absence and presence of nucleoside diphosphate kinase inhibitors. RESULTS: The amount and activity of nucleoside diphosphate kinase in sarcolemmal membranes from failing hearts (n=13) were increased 3- to 4-fold compared to levels in membranes from non-failing myocardium (n=5). This increase in sarcolemmal nucleoside diphosphate kinase activity resulted in a 50% inhibition of adenylyl cyclase activity over a range of GDP and ATP concentrations. CONCLUSION: The amount and activity of nucleoside diphosphate kinase are increased in sarcolemmal membranes of failing human myocardium, resulting in a substantial receptor-independent inhibition of adenylyl cyclase activity.

5'-Nucleotidase↗
PubMed1998

Transport and mode of action of nucleoside derivatives used in chemical and antiviral therapies.

Nucleoside analogues used in cancer and anti-viral therapies interfere with nucleotide metabolism and DNA replication, thus inducing their pharmacological effects. A long-awaited goal in the understanding of the pharmacological properties of these molecules, that is the molecular characterization of nucleoside plasma-membrane transporters, has been achieved very recently. These carrier proteins are encoded by at least two gene families and new isoforms remain to be identified. Direct demonstration of translocation of these drugs by nucleoside transporters has already been provided and most of them can inhibit natural nucleoside transport, probably in a competitive manner. The expression of these genes is clearly tissue-specific and might depend on the differentiated status of a cell. This is relevant because the sensitivity of a cell to a drug can depend on the type of nucleoside carrier expressed, and the drug itself might modulate nucleoside carrier expression. In this article, Marçal Pastor-Anglada, Antonio Felipe and Javier Casado discuss recent studies on the regulation of nucleoside carrier expression and of the molecular determinants of substrate specificity. Better knowledge of these will contribute to an improved design of therapies based on nucleoside derivatives.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Potential inhibitors of S-adenosylmethionine-dependent methyltransferases. 9. 2',3'-Dialdehyde derivatives of carbocyclic purine nucleosides as inhibitors of S-adenosylhomocysteine hydrolase.

A series of purine (e.g., adenine, N6-methyladenine, 8-azaadenine, 3-deazaadenine) carbocyclic nucleosides, nucleoside 2',3'-dialdehydes, and nucleoside 2',3'-diols were synthesized as potential inhibitors of bovine liver S-adenosyl-L-homocysteine (AdoHcy) hydrolase (EC 3.3.1.1) and as potential inhibitors of vaccinia virus replication. The 2',3'-dialdehydes were prepared by periodate oxidation of the corresponding carbocyclic nucleosides. Reduction of the intermediate dialdehydes with sodium borohydride afforded the corresponding 2',3'-diols. Of the nucleosides tested, the most potent inhibitors of AdoHcy hydrolase were the adenine analogue (Ki = 110 +/- 38 nM) and the 3-deazaadenine analogue (Ki = 4 +/- 0.9 nM), which were reversible, competitive inhibitors. In contrast, the 2',3'-dialdehydes produced irreversible inhibition of AdoHcy hydrolase, resulting in incorporation of two to four molecules of the dialdehyde per molecule (tetramer) of the enzyme. On the basis of an Ackermann-Potter analysis, the following "apparent" Ki values were determined for the 2',3'-dialdehydes: adenine analogue, 61 nM; 8-azaadenine analogue, 57.5 nM; and 3-deazaadenine analogue, 32 nM. The nucleoside 2',3'-diols were substantially less effective as inhibitors of AdoHcy hydrolase, requiring millimolar concentrations to achieve significant inhibition. When tested for their ability to inhibit vaccinia virus replication, several carbocyclic nucleosides (e.g., adenine and 3-deazaadenine analogues) and several nucleoside 2',3'-dialdehydes (e.g., adenine, N6-methyladenine, 8-azaadenine, and 3-deazaadenine analogues) exhibited good antiviral effects. A good correlation existed between a compound's inhibitory effects on AdoHcy hydrolase and its antiviral effects, suggesting that the inhibition of viral replication is caused by inhibition of a critical methylation reaction, e.g., methylation of the 5'-cap of viral mRNA.

Adenosylhomocysteinase↗
PubMed1985

Nucleoside transport in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. Differences in sensitivity to nitrobenzylthioinosine and thiol reagents.

The characteristics of nucleoside transport were examined in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. In Walker 256 cells the initial rates of uridine, thymidine and adenosine uptake were insensitive to the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR) (1 microM), but were partially inhibited by dipyridamole (10 microM), another inhibitor of nucleoside transport. In contrast, the transport of these nucleosides in S49 cells was completely blocked by both inhibitors. Nucleoside transport in Walker 256 and S49 cells also differed in its sensitivity to the thiol reagent p-chloromercuribenzenesulphonate (pCMBS). Uridine transport in Walker 256 cells was inhibited by pCMBS with an IC50 (concentration producing 50% inhibition) of less than 25 microM, and inhibition was readily reversed by beta-mercaptoethanol. In S49 cells uridine transport was only inhibited at much higher concentrations of pCMBS (IC50 approximately equal to 300 microM). In other respects nucleoside transport in Walker 256 and S49 cells were quite similar. The Km and Vmax. values for uridine transport were nearly identical, and the transporters of both cell lines appeared to accept a broad range of nucleosides as substrates. Uridine transport in Walker 256 cells was non-concentrative and did not require an energy source. These studies demonstrate that nucleoside uptake in Walker 256 cells is mediated by a facilitated-diffusion mechanism which differs markedly from that of S49 cells in its sensitivity to the transport inhibitor NBMPR and the thiol reagent pCMBS.

4-Chloromercuribenzenesulfonate↗
PubMedDate not supplied

Correlation of nucleoside and nucleobase transporter gene expression with antimetabolite drug cytotoxicity.

Antimetabolite drugs that inhibit nucleic acid metabolism are widely used in cancer chemotherapy. Nucleoside and nucleobase transporters are important for the cellular uptake of nucleic acids and their corresponding anticancer analogue drugs. Thus, these transporters may play a role both in antimetabolite drug sensitivity, by mediating the uptake of nucleoside analogues, and in antimetabolite drug resistance, by mediating the uptake of endogenous nucleosides that may rescue cells from toxicity. Therefore, we examined the relation of the expression of nucleoside and nucleobase transporters to antimetabolite cytotoxicity. We measured the RNA levels of all eight known nucleoside and nucleobase transporters in 50 cell lines included in the National Cancer Institute's Anticancer Drug Screen panel. RNA levels of concentrative nucleoside transporters (CNTs), equilibrative nucleoside transporters (ENTs) and nucleobase transporters (NCBTs) were determined by quantitative RT-PCR using real-time fluorescence acquisition. This method was validated by measuring the expression of the MDR1 gene, and correlating our results with independently determined measurements of MDR1 RNA levels and protein function in these cell lines. We then correlated the pattern of RNA levels to the pattern of cytotoxicity of anticancer drugs in the NCI drug screen database using the COMPARE analysis. Several hypothesized relations between transporter gene expression and cytotoxicity, based upon known interactions between certain nucleoside analogues and transporter proteins, were not observed, suggesting that expression of individual transporters may not be a significant determinant of the cytotoxicity of these drugs. The most closely correlated drug cytotoxicity patterns to transporter gene expression patterns (where increased expression corresponds to increase sensitivity) included those between CNT1 and O6-methylguanine and between ENT2 and hydroxyurea. We also observed that p53 status influenced correlations between ENT1 transporter gene RNA levels and sensitivity to the drugs tiazafurin, AZQ and 3-deazauridine. One of three drugs identified by correlation of cytotoxicity patterns with ENT1 RNA levels, 3-deazauridine, inhibited uptake of the classic ENT1 substrate uridine, demonstrating a physical interaction between an identified drug and the transporter. These studies demonstrate that it is possible to correlate genetic information to functional databases to determine the influence of transport gene expression on drug sensitivity and to identify transporter-drug interactions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Phosphorylation of pyrimidine deoxynucleoside analog diphosphates: selective phosphorylation of L-nucleoside analog diphosphates by 3-phosphoglycerate kinase.

D-Nucleoside analogs, which are in the natural configuration, as well as the L-nucleoside analogs, are clinically relevant antiviral and anticancer agents. Metabolism of L-nucleoside analog diphosphates to the triphosphates, however, remains unexplored. Studies with recombinant nm23-H1 and -H2 isoforms indicated that L-nucleoside analog diphosphates were not phosphorylated by their nucleoside diphosphate kinase (NDPK) activity. Therefore, roles of creatine kinase, 3-phosphoglycerate kinase, and pyruvate kinase were evaluated using preparations from commercial sources and human HepG2 cells. Phosphorylation of L-OddC, L-SddC, L-Fd4C, L-FMAU, and L-ddC were compared with D-deoxynucleoside analogs, AraC, dFdC, and D-FMAU, and D-dideoxynucleoside analogs, ddC and d4T. Results based on preparations from HepG2 cells showed that L-nucleoside analog diphosphates were selectively phosphorylated by 3-phosphoglycerate kinase, whereas, D-deoxynucleoside analog diphosphates were phosphorylated by NDPK. Interestingly, ddCDP and d4TDP were substrates for creatine kinase, but were not phosphorylated by NDPK. In conclusion, it is proposed that specificity of the phosphorylating enzymes toward the nucleoside analog diphosphates is dependent on the configuration of the analog (L or D) and the presence or absence of 3'-hydroxyl group in the sugar moiety. The enzymatic process of phosphorylation of L- and D-nucleoside analog diphosphates is different in cells.

Adenosine Diphosphate↗
PubMed1994

Uptake, transport and metabolism of exogenous nucleosides in intestinal epithelial cell cultures.

The uptake and transport of nucleosides (adenosine, cytidine, guanosine, thymidine, uridine and inosine) were examined in intestinal cell lines (Caco-2 and IEC-6). Well-differentiated Caco-2 cells took up significantly more uridine and thymidine than less-differentiated cells over 24 h. These differences were reflected in the resulting trichloroacetic acid-soluble (nucleotide, nucleoside and nucleobase) pool sizes rather than in differences in nucleic acid incorporation. IEC-6 cells have smaller soluble pools than Caco-2 cells, and the uptake of nucleosides over 60 min reflected this difference. Caco-2 cells transported significantly more thymidine, cytidine, guanosine, adenosine and inosine into acid-soluble pools than IEC-6 cells. Caco-2 cells were grown on filters to examine the transcellular transport of nucleosides and to examine the chemical form in which nucleosides appeared at the basolateral aspect of the cell monolayer. The pattern of transport varied for individual nucleosides. The transport of cytidine and guanosine was significantly greater in the apical-to-basolateral direction than in the opposite direction over 2 h. No purine nucleoside was transported intact across the Caco-2 cell monolayer in either direction, with the majority of the transported material appearing as nucleobases. Nucleobases also represented the main metabolites of pyrimidines transported. However, some cytidine and uridine were detectable, but each constituted < 20% of transported material. Nucleosides are therefore transported by enterocytes but metabolized during transport.

Adenosine↗
PubMed1979

Effect of nucleosides on interferon production and development of antiviral state induced by poly I.poly C.

Effect of nucleosides both on induction of antiviral state in chick embryo cells (CEC) or rabbit kidney cells (RK13) and on interferon production in RK13 or mouse fibroblast cells (L cells) by polyriboinosinic-polyribocytidylic acid (poly I.poly C) was studied. Addition of inosine or a fifty-fifty mixture of inosine and uridine at a final concentration of 0.1 mM to 10 mM to a growth medium enhanced development of antiviral state in CEC. The nucleoside effect was also observed in RK13 at 0.1 mM but not at a concentration higher than 1 mM. Interferon production in RK13 by superinduction (sequential treatment with metabolic inhibitors after exposure to poly I.poly C) was enhanced 1.5- to 4.0-fold by addition of the nucleoside mixture to the growth medium. When RK13 was pretreated with 10 units per ml of interferon and then superinduced by inhibitors, the enhancing effect of nucleosides on interferon production was not observed. Interferon production in L cells was potentiated a little by addition of 1 mM of the nucleoside mixture to the growth medium. The effect of nucleoside was not observed when the nucleosides were added after exposure to poly I.poly C. The nucleoside effect may be applicable for production of high titered interferon.

Animals↗
PubMed1986

Incomplete nucleoside transport deficiency with increased hypoxanthine transport capability in mutant T-lymphoblastoid cells.

From a mutagenized population of wild-type mouse (S49) T-lymphoma cells, a clone, 80-5D2, was isolated in a single step by virtue of its ability to survive in 80 nM 5-fluorouridine. Unlike previously isolated nucleoside transport-deficient cell lines (A. Cohen, B. Ullman, and D. W. Martin, Jr., J. Biol. Chem. 254:112-116, 1979), 80-5D2 cells were only slightly less sensitive to growth inhibition by a variety of cytotoxic nucleosides and were capable of proliferating in hypoxanthine-amethopterin-thymidine-containing medium. The molecular basis for the phenotype of 80-5D2 cells was incomplete deficiency in the ability of the mutant cells to translocate nucleosides across the plasma membrane. Interestingly, mutant cells were more capable than wild-type cells of transporting the nucleobase hypoxanthine. Residual transport of adenosine into 80-5D2 cells was just as sensitive to inhibition by nucleosides and more sensitive to inhibition by hypoxanthine than that in wild-type cells, indicating that the phenomena of ligand binding and translocation can be uncoupled genetically. The 80-5D2 cells lacked cell surface binding sites for the potent inhibitor of nucleoside transport p-nitrobenzylthioinosine (NBMPR) and, consequently, were largely resistant to the physiological effects of NBMPR. However, the altered transporter retained its sensitivity to dipyridamole, another inhibitor of nucleoside transport. The biochemical phenotype of the 80-5D2 cell line supports the hypothesis that the determinants that comprise the nucleoside carrier site, the hypoxanthine carrier site, the NBMPR binding site, and the dipyridamole binding site of the nucleoside transport function of mouse S49 cells are genetically distinguishable.

Animals↗
PubMed1983

Nucleoside diphosphokinase and cell cycle control in the fission yeast Schizosaccharomyces pombe.

Centrifugal elutriation was used to prepare synchronous cultures of Schizosaccharomyces pombe. Nucleoside diphosphokinase activity was measured throughout the cell cycle. In the wild-type strain (972) nucleoside diphosphokinase activity doubled in a stepwise fashion. The midpoint of the rise in enzyme activity was at 0.65 of a cycle, 0.29 of a cycle before the next S phase. Synchronous cultures of the mutant wee 1-6 were also prepared. In this strain S phase is delayed, occurring about 0.3 cycle later than in the wild-type. In wee 1-6 the midpoint of the stepwise doubling in nucleoside diphosphokinase activity occurred at 0.084; showing that the rise in enzyme activity is also delayed. Addition of cycloheximide to an exponentially growing culture caused an immediate inhibition of protein synthesis, yet nucleoside diphosphokinase activity continued to increase exponentially for a further 300 min. This indicates that the stepwise doubling of nucleoside diphosphokinase activity during the cell cycle is not achieved by a simple control on protein synthesis. Two temperature-sensitive cdc- mutants were also used: cdc2-33, a mutant whose single genetic lesion results in the twin defects of a loss of mitotic control and a loss of commitment to the cell cycle; and cdc 10-129, which has a defect in DNA replication. In both mutants a temperature shift-up of an asynchronously growing culture from the permissive (25 degrees C) to the restrictive temperature (36.5 degrees C) results in a rapid inhibition of DNA replication. In both mutants nucleoside diphosphokinase continues to increase exponentially. Therefore, although nucleoside diphosphokinase is required for DNA replication, apparently DNA replication is not required for an increase in nucleoside diphosphokinase activity.

Ascomycota↗
PubMed2002

Trends in the design of nucleoside analogues as anti-HIV drugs.

HIV infection remains incurable although several anti-HIV drugs have been identified and developed. Among these the nucleoside analogues were and remain in the forefront of anti-HIV chemotherapeutic regimens. Most of these nucleoside analogues are modified mainly in the sugar moiety. In general, they lack a free hydroxy group at the 3'-position. Consequently, they cannot participate in the formation of a 3',5'-phosphodiester linkage, which renders the nucleoside 5'-triphosphates of such nucleoside analogues effective anti-HIV agents. Preventing the formation of 3',5'-phosphodiester linkages leads to inhibition of the viral DNA strand elongation and ultimately chain termination. The phosphorylation of nucleoside analogues is a key factor in their efficacy as anti-HIV agents. Efficient phosphorylation depends largely on the structure of the nucleoside. Therefore, modification of the structure of nucleoside analogues are sought to enhance their phosphorylation and ultimately, effective inhibition of the HIV reverse transcriptase. This review is concerned with the trends in the design of nucleoside analogues as anti-HIV agents.

Anti-HIV Agents↗
PubMedDate not supplied

Molecular cloning, functional expression and chromosomal localization of a cDNA encoding a human Na+/nucleoside cotransporter (hCNT2) selective for purine nucleosides and uridine.

Two Na(+)-dependent nucleoside transporters implicated in adenosine and uridine transport in mammalian cells are distinguished functionally on the basis of substrate specificity: CNT1 is selective for pyrimidine nucleosides but also transports adenosine; CNT2 (also termed SPNT) is selective for purine nucleosides but also transports uridine. Both proteins belong to a gene family that includes the NupC proton/nucleoside symporter of E. coli. cDNAs encoding members of the CNT family have been isolated from rat tissues (jejunum, brain, liver; rCNT1 and rCNT2/SPNT) and, most recently, human kidney (hCNT1 and hSPNT1). Here, the molecular cloning and functional characterization of a CNT2/SPNT-type transporter from human small intestine are described. The encoded 658-residue protein (hCNT2 in the nomenclature) had the same predicted amino acid sequence as human kidney hSPNT1, except for a polymorphism at residue 75 (Arg substituted by Ser), and was 83 and 72% identical to rCNT2 and hCNT1, respectively. Sequence differences between hCNT2 and rCNT2 were greatest at the N-terminus. In Xenopus oocytes, recombinant hCNT2 exhibited the functional characteristics of a Na(+)-dependent nucleoside transporter with selectivity for adenosine, other purine nucleosides and uridine (adenosine and uridine K(m) app values 8 and 40 microM, respectively). hCNT2 transcripts were found in kidney and small intestine but, unlike rCNT2, were not detected in liver. Deoxyadenosine, which undergoes net renal secretion in humans, was less readily transported than adenosine. hCNT2 also mediated small, but significant, fluxes of the antiviral purine nucleoside analogue 2',3'-dideoxyinosine. hCNT2 is, therefore potentially involved in both the intestinal absorption and renal handling of purine nucleosides (including adenosine), uridine and purine nucleoside drugs. The gene encoding hCNT2 was mapped to chromosome 15q15.

Adenosine↗