Ribavirin Triphosphate Research Articles

Remote Site Control of an Active Site Fidelity Checkpoint in a ViralRNA-dependent RNAPolymerase

The kinetic, thermodynamic, and structural basis for fidelity of nucleic acid polymerases remains controversial. An understanding of viral RNA-dependent RNA polymerase (RdRp) fidelity has become a topic of considerable interest as a result of recent experiments that show that a 2-fold increase in fidelity attenuates viral pathogenesis and a 2-fold decrease in fidelity reduces viral fitness. Here we show that a conformational change step preceding phosphoryl transfer is a key fidelity checkpoint for the poliovirus RdRp (3Dpol). We provide evidence that this conformational change step is orientation of the triphosphate into a conformation suitable for catalysis, suggesting a kinetic and structural model for RdRp fidelity that can be extrapolated to other classes of nucleic acid polymerases. Finally, we show that a site remote from the catalytic center can control this checkpoint, which occurs at the active site. Importantly, similar connections between a remote site and the active site exist in a wide variety of viral RdRps. The capacity for sites remote from the catalytic center to alter fidelity suggests new possibilities for targeting the viral RdRp for antiviral drug development.

Phosphorylation of Ribavirin and Viramidine by Adenosine Kinase and Cytosolic 5′-Nucleotidase II: Implications for Ribavirin Metabolism in Erythrocytes

Many nucleoside analog drugs, such as ribavirin and viramidine, are activated or metabolized in vivo through 5'-phosphorylation. In this report, we determined the steady-state kinetic parameters for 5'-monophosphorylation of ribavirin and viramidine by adenosine kinase. The apparent Km for ribavirin is 540 microM, and k(cat) is 1.8 min-1. Its catalytic efficiency of 3.3 x 10(-3) min-1 . microM-1 is 1,200-fold lower than that of adenosine. In contrast to the common belief that ribavirin is exclusively phosphorylated by adenosine kinase, cytosolic 5'-nucleotidase II was found to catalyze ribavirin phosphorylation in vitro. The reaction is optimally stimulated by the physiological concentration of ATP or 2,3-bisphosphoglycerate. In phosphate-buffered saline plus ATP and 2,3-bisphosphoglycerate, the apparent Km for ribavirin is 88 microM, and k(cat) is 4.0 min-1. These findings suggest that cytosolic 5'-nucleotidase II may be involved in ribavirin phosphorylation in vivo. Like ribavirin, viramidine was found to be phosphorylated by either adenosine kinase or cytosolic 5'-nucleotidase II, albeit with a much lower activity. The catalytic efficiency for viramidine phosphorylation is 10- to 330-fold lower than that of ribavirin, suggesting that other nucleoside kinase(s) may be involved in viramidine phosphorylation in vivo. Both ribavirin and viramidine are not phosphorylated by deoxycytidine kinase and uridine-cytidine kinase. The coincidence of presence of high concentrated 2,3-bisphosphoglycerate in erythrocytes suggests that cytosolic 5'-nucleotidase II could play an important role in phosphorylating ribavirin and contribute to anabolism of ribavirin triphosphate in erythrocytes. Elucidation of ribavirin and viramidine phosphorylation mechanism should shed light on their in vivo metabolism, especially the ribavirin-induced hemolytic anemia in erythrocytes.

Metabolism and antiviral activity of ribavirin

Thirty years after its synthesis, the mechanism of action of ribavirin is still not completely understood. Although much is known about the metabolism and biochemical effects of ribavirin in human cells, there is still much to be learned about the precise mechanism of action of ribavirin with the various viruses. New information about its ability to induce mutations in viral genomes has led to new questions about its mechanism of action. There is considerable evidence that indicates that ribavirin triphosphate (RTP) can interact with the various viral RNA polymerases, and it seems likely that this interaction is important to the mechanism of action of ribavirin. It seems likely that ribavirin will not have one universal mechanism of action, but will inhibit different viruses in different ways. In some cases, inhibition of IMP dehydrogenase may be sufficient for antiviral activity. Whereas, in other cases, inhibition of viral RNA polymerases by RTP may be more important. It is also likely that RTP will interact with the different viral RNA polymerases in different ways leading to different mechanisms of actions. More comprehensive studies are needed that address all aspects of ribavirin metabolism and biochemical actions to gain a thorough understanding of the activity of this agent. Finally, the differences in the metabolism and biochemical actions of ribavirin, selenazofurin, and tiazofurin indicate that small structural changes can have profound effects on biological activity. This observation is well known by investigators familiar with nucleoside analogs, but indicate that one should not assume that agents of similar structure have identical activities.

De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has several distinct biochemical activities, including initiation of RNA synthesis by a de novo mechanism, extension from a primed template, nontemplated nucleotide addition, and synthesis of a recombinant RNA product from two or more noncovalently linked templates (template switch). All of these activities require specific interaction with nucleoside triphosphates (NTPs). Based on the structure of the HCV RdRp bound to NTP (S. Bressanelli, L. Tomei, F. A. Rey, and R. DeFrancesco, J. Virol. 76:3482-3492, 2002), we mutated the amino acid residues that contact the putative initiation GTP and examined the effects on the various activities. Although all mutations retained the ability for primer extension, alanine substitution at R48, R158, R386, R394, or D225 decreased de novo initiation, and two or more mutations abolished de novo initiation. While the prototype enzyme had a K(m) for GTP of 3.5 microM, all of the mutations except one had K(m)s that were three- to sevenfold higher. These results demonstrate that the affected residues are functionally required to interact with the initiation nucleotide. Unexpectedly, many of the mutations also affected the addition of nontemplated nucleotide, indicating that residues in the initiating NTP (NTPi)-binding pocket are required for nontemplated nucleotide additions. Interestingly, mutations in D225 are dramatically affected in template switch, indicating that this residue of the NTPi pocket also interacts with components in the elongation complex. We also examined the interaction of ribavirin triphosphate with the NTPi-binding site.

The Broad Spectrum Antiviral Nucleoside Ribavirin as a Substrate for a Viral RNA Capping Enzyme

The broad spectrum antiviral nucleoside ribavirin displays activity against a variety of RNA and DNA viruses. A number of possible mechanisms have been proposed during the past 30 years to account for the antiviral activity of ribavirin, including the possibility that ribavirin might have a negative effect on the synthesis of the RNA cap structure of viral RNA transcripts. In the present study, we investigated the possibility that ribavirin can directly serve as a substrate for the vaccinia virus RNA capping enzyme. We demonstrate that ribavirin triphosphate can be used as a substrate by the capping enzyme and can form a covalent ribavirin monophosphate-enzyme intermediate reminiscent of the classical GMP-enzyme intermediate. Furthermore, our data indicate that ribavirin monophosphate can be transferred to the diphosphate end of an RNA transcript to form the unusual RpppN structure. Finally, we provide evidence that RNA transcripts that possess ribavirin as the blocking nucleoside are more stable than unblocked transcripts. However, in vitro translation assays indicate that RNA transcripts blocked with ribavirin are not translated efficiently. Our study provides the first biochemical evidences that ribavirin can directly interact with a viral capping enzyme. The ability of a purified RNA capping enzyme to utilize ribavirin as a substrate has not been previously documented and has implications for our understanding of the catalytic mechanisms of RNA capping enzymes. The biological implications of these findings for the proposed ribavirin-mediated inhibition of capping are discussed.

S-adenosylhomocysteine hydrolase, S-adenosylmethionine, S-adenosylhomocysteine: correlations with ribavirin induced anemia

Objective is to speculate on the ribavirin induced anemia by inhibiting S-adenosylhomocysteine (SAH)-hydrolase activity. The major toxicity associated with the use of ribavirin is hemolytic anemia. This adverse effect has been ascribed to the accumulation of ribavirin triphosphate in the erythrocyte, which interferes with erythrocyte function. Ribavirin has been found to inhibit SAH-hydrolase activity in erythrocyte. SAH is further hydrolyzed to adenosine and homocysteine by SAH-hydrolase. The formation of S-adenosylmethionine (SAM) is then demethylated to SAH. SAH, the metabolite of SAM, on the other hand is a powerful inhibitor of methyltransferase enzymes, competing for the SAM binding site. A concurrent decrease in SAM and an increase in SAH levels would inhibit methylation of many tissue components including proteins, DNA, RNA, phospholipids and other small molecules. The enzyme protein carboxyl methyltransferase type II has been recently shown to play a crucial role in the repair of damaged proteins. SAM is the methyl donor of the reaction, and its demethylated product, SAH is the natural inhibitor of this reaction, as well as of most SAM-dependent methylations. The biological function of this transmethylation reaction is related to the repair or degradation of age-damaged proteins. Methyl ester formation in erythrocyte membrane proteins has been used as a marker reaction to tag these abnormal residues and to monitor their increase associated with erythrocyte ageing diseases. Liver disease is complicated by cholesterol deposition in hepatic and extrahepatic membranes. Erythrocyte membrane fluidity has been improved with the administration of SAM and correlated with the cholesterol/phospholipid ratio of the membranes. The levels of SAH-hydrolase activity were also found to undergo a sharp decrease with red cell ageing. The similarity of these alterations with certain morphofunctional characteristics of erythrocyte in some conditions as chronic renal failure, liver disease and hereditary spherocytosis makes it possible to hypothesize that the inhibition of SAH-hydrolase could constitute at least a part of ribavirin induced hemolytic anemia.

S-adenosylhomocysteine hydrolase, S-adenosylmethionine, S-adenosylhomocysteine: correlations with ribavirin induced anemia

Objective is to speculate on the ribavirin induced anemia by inhibiting S-adenosylhomocysteine (SAH)-hydrolase activity. The major toxicity associated with the use of ribavirin is hemolytic anemia. This adverse effect has been ascribed to the accumulation of ribavirin triphosphate in the erythrocyte, which interferes with erythrocyte function. Ribavirin has been found to inhibit SAH-hydrolase activity in erythrocyte. SAH is further hydrolyzed to adenosine and homocysteine by SAH-hydrolase. The formation of S-adenosylmethionine (SAM) is then demethylated to SAH. SAH, the metabolite of SAM, on the other hand is a powerful inhibitor of methyltransferase enzymes, competing for the SAM binding site. A concurrent decrease in SAM and an increase in SAH levels would inhibit methylation of many tissue components including proteins, DNA, RNA, phospholipids and other small molecules. The enzyme protein carboxyl methyltransferase type II has been recently shown to play a crucial role in the repair of damaged proteins. SAM is the methyl donor of the reaction, and its demethylated product, SAH is the natural inhibitor of this reaction, as well as of most SAM-dependent methylations. The biological function of this transmethylation reaction is related to the repair or degradation of age-damaged proteins. Methyl ester formation in erythrocyte membrane proteins has been used as a marker reaction to tag these abnormal residues and to monitor their increase associated with erythrocyte ageing diseases. Liver disease is complicated by cholesterol deposition in hepatic and extrahepatic membranes. Erythrocyte membrane fluidity has been improved with the administration of SAM and correlated with the cholesterol/phospholipid ratio of the membranes. The levels of SAH-hydrolase activity were also found to undergo a sharp decrease with red cell ageing. The similarity of these alterations with certain morphofunctional characteristics of erythrocyte in some conditions as chronic renal failure, liver disease and hereditary spherocytosis makes it possible to hypothesize that the inhibition of SAH-hydrolase could constitute at least a part of ribavirin induced hemolytic anemia.

Initial Binding of the Broad Spectrum Antiviral Nucleoside Ribavirin to the Hepatitis C Virus RNA Polymerase

Ribavirin is a broad spectrum antiviral nucleoside that displays activity against a variety of RNA and DNA viruses. Ribavirin is currently used in combination with interferon-alpha for the treatment of hepatitis C virus (HCV) infection and was recently shown to be directly incorporated by the HCV RNA polymerase into RNA products. This capacity ultimately leads to increased mutation rates and drastically reduces the viral fitness. As a first step toward elucidating the nature of the specific interaction between ribavirin and the HCV polymerase, we have utilized fluorescence spectroscopy to monitor precisely the binding of ribavirin triphosphate (RTP) to the viral polymerase. This spectroscopic approach allowed us to clearly separate the RTP binding activity from the concomitant catalytic steps. We report here the first detailed study of the binding kinetics and thermodynamic parameters involved in the interaction between RTP and an RNA polymerase. We demonstrate that RTP binds to the same active site as nucleotides. Furthermore, we provide evidence that the HCV polymerase cannot only bind to RTP but also to nonphosphorylated ribavirin, albeit with less affinity. By using various combinations of template-primers, we also demonstrate that base pairing is not involved in the initial binding of RTP to the HCV polymerase. Based on the results of circular dichroism and denaturation studies, we show that the RNA polymerase undergoes subtle conformational changes upon the binding of RTP, although the interaction does not significantly modify the stability of the protein. Finally, although metal ions are required for catalytic activity, they are not required for the initial binding of RTP to the polymerase. Such quantitative analyses are of primary importance for the rational design of new ribavirin analogues of potential therapeutic value and provide crucial insights on the interaction between RTP and the HCV RNA polymerase.

Mutagenic and inhibitory effects of ribavirin on hepatitis C virus RNA polymerase.

Crotty et al. recently proposed the primary antiviral action of ribavirin to be that of a potent RNA mutagen [Crotty, S., Maag, D., Arnold, J. J., Zhong, W., Lau, J. Y., Hong, Z., Andino, R., and Cameron, C. E. (2000) Nat. Med. 6, 1375-1379]. Here we investigate the effect of ribavirin triphosphate (RTP) on RNA synthesis catalyzed by a full-length hepatitis C virus (HCV) RNA polymerase in vitro. HCV polymerase can use RTP as a nucleotide substrate in a template-dependent manner, incorporating it opposite a pyrimidine (C or U) template residue, but not a purine (A or G). Kinetic analysis revealed that incorporation of ribavirin monophosphate (RMP) across from C is 3 times more efficient catalytically than that across from U, as determined by the k(cat)/K(m) parameter. The efficiency of RMP incorporation, however, is 50-100 fold lower than that of the natural NMP. RMP incorporation does not lead to termination of RNA chain synthesis, as evidenced by the ability of the polymerase to extend its RNA product many nucleotides beyond the site of RMP incorporation. However, multiple-RMP incorporation at low GTP concentrations induced the formation of stalled elongation complexes, particularly at the template region containing consecutive C residues. Most, but not all, such elongation blocks can be relieved by the re-addition of GTP. When ribavirin is present in the RNA template, pyrimidine (but neither purine nor ribavirin) monophosphate is incorporated opposite ribavirin, but at an exceedingly low catalytic efficiency (200-3000-fold lower) compared to the efficiencies of those templated by A or G. Consequently, the level of RNA synthesis on a ribavirin-containing template is significantly reduced. These findings suggest that ribavirin not only is mutagenic but also interferes with HCV polymerase-mediated RNA synthesis.

Structural analysis of the activation of ribavirin analogs by NDP kinase: comparison with other ribavirin targets.

Ribavirin used in therapies against hepatitis C virus (HCV) is potentially efficient against other viruses but presents a high cytotoxicity. Several ribavirin triphosphate analogs modified on the ribose moiety were synthesized and tested in vitro on the RNA polymerases of HCV, phage T7, and HIV-1 reverse transcriptase. Modified nucleotides with 2'-deoxy, 3'-deoxy, 2',3'-dideoxy, 2',3'-dideoxy-2',3'-dehydro, and 2',3'-epoxy-ribose inhibited the HCV enzyme but not the other two polymerases. They were also analyzed as substrates for nucleoside diphosphate (NDP) kinase, the enzyme responsible for the last step of the cellular activation of antiviral nucleoside analogs. An X-ray structure of NDP kinase complexed with ribavirin triphosphate was determined. It demonstrates that the analog binds as a normal substrate despite the modified base and confirms the crucial role of the 3'-hydroxyl group in the phosphorylation reaction. The 3'-hydroxyl is required for inhibition of the initiation step of RNA synthesis by HCV polymerase, and both sugar hydroxyls must be present to inhibit elongation. The 2'deoxyribavirin is the only derivative efficient in vitro against HCV polymerase and properly activated by NDP kinase.

Conformational restraint is a critical determinant of unnatural nucleotide recognition by protein kinases

This report describes the synthesis of N 4-(benzyl) AICAR triphosphate, a conformationally restrained analogue of N 4-(benzyl) ribavirin triphosphate. Both of these nucleotides were evaluated as phosphodonors for wild-type p38 MAP kinase and T106G p38 MAP kinase, a designed mutant with expanded nucleotide specificity. The conformationally restrained nucleotide, N 4-(benzyl) AICAR triphosphate, is orthogonal to (not accepted as a substrate by) wild-type p38 MAP kinase, in contrast to N 4-(benzyl) ribavirin triphosphate. Furthermore, N 4-(benzyl) AICAR triphosphate, is accepted as a substrate by T106G p38 MAP kinase, in contrast to N 4-(benzyl) ribavirin triphosphate. We hypothesize that the presence of an internal hydrogen bond in N 4-(benzyl) AICAR and its absence in N 4-(benzyl) ribavirin triphosphate is the main determinant for their differing structure–activity relationships.

Synthesis and antiviral evaluation of a mutagenic and non-hydrogen bonding ribonucleoside analogue: 1-beta-D-Ribofuranosyl-3-nitropyrrole.

Synthetic small molecules that promote viral mutagenesis represent a promising new class of antiviral therapeutics. Ribavirin is a broad-spectrum antiviral nucleoside whose antiviral mechanism against RNA viruses likely reflects the ability of this compound to introduce mutations into the viral genome. The mutagenicity of ribavirin results from the incorporation of ribavirin triphosphate opposite both cytidine and uridine in viral RNA. In an effort to identify compounds with mutagenicity greater than that of ribavirin, we synthesized 1-beta-D-ribofuranosyl-3-nitropyrrole (3-NPN) and the corresponding triphosphate (3-NPNTP). These compounds constitute RNA analogues of the known DNA nucleoside 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole. The 3-nitropyrrole pseudobase has been shown to maintain the integrity of DNA duplexes when placed opposite any of the four nucleobases without requiring hydrogen bonding. X-ray crystallography revealed that 3-NPN is structurally similar to ribavirin, and both compounds are substrates for adenosine kinase, an enzyme critical for conversion to the corresponding triphosphate in cells. Whereas ribavirin exhibits antiviral activity against poliovirus in cell culture, 3-NPN lacks this activity. Evaluation of 3-NPNTP utilization by poliovirus RNA-dependent RNA polymerase (RdRP) revealed that 3-NPNTP was not accepted universally. Rather, incorporation was only observed opposite A and U in the template and at a rate 100-fold slower than the rate of incorporation of ribavirin triphosphate. This diminished rate of incorporation into viral RNA likely precludes 3-NPN from functioning as an antiviral agent. These results indicate that hydrogen bonding substituents are critical for efficient incorporation of ribonucleotides into RNA by viral RdRPs, thus providing important considerations for the design of improved mutagenic antiviral nucleosides.

Pleiotropic mechanisms of ribavirin antiviral activities.

Renewed interest in the mechanism of action of ribavirin results from its synergistic enhancement of interferon therapy and the need to develop more efficacious agents to treat hepatitis C virus infection. Since the discovery of ribavirin over 30 years ago by scientists at ICN Pharmaceuticals, many mechanisms of action for ribavirin have been proposed. These include inhibition of host inosine monophosphate dehydrogenase by ribavirin monophosphate, inhibition of viral capping enzymes, inhibition of viral RNA synthesis by ribavirin triphosphate, lethal mutagenesis of viral RNA genomes resulting from promiscuous incorporation of ribavirin triphosphate by the viral RNA polymerase, and modulation of the host immune responses. In this article, we will briefly review the evidence for these mechanisms, emphasizing recent findings. In addition, we will discuss strategies for development of nucleoside analogs that may replace ribavirin in the future.

Hepatitis C Virus RNA-dependent RNA Polymerase (NS5B) as a Mediator of the Antiviral Activity of Ribavirin

Ribavirin is administered in combination with interferon-alpha for treatment of hepatitis C virus (HCV) infection. Recently, we demonstrated that the antiviral activity of ribavirin can result from the ability of a viral RNA polymerase to utilize ribavirin triphosphate and to incorporate this nucleotide with reduced specificity, thereby mutagenizing the genome and decreasing the yield of infectious virus (Crotty, S., Maag, D., Arnold, J. J., Zhong, W., Lau, J. Y., Hong, Z., Andino, R., and Cameron, C. E. (2000) Nat. Med. 6, 1375-1379). In this study, we performed a quantitative analysis of a novel HCV RNA polymerase derivative that is capable of utilizing stably annealed primer-template substrates and exploited this derivative to evaluate whether lethal mutagenesis of the HCV genome is a possible mechanism for the anti-HCV activity of ribavirin. These studies demonstrate HCV RNA polymerase-catalyzed incorporation of ribavirin opposite cytidine and uridine. In addition, we demonstrate that templates containing ribavirin support CMP and UMP incorporation with equivalent efficiency. Surprisingly, templates containing ribavirin can also cause a significant block to RNA elongation. Together, these data suggest that ribavirin can exert a direct effect on HCV replication, which is mediated by the HCV RNA polymerase. We discuss the implications of this work on the development of nucleoside analogs for treatment of HCV infection.

Ribavirin induces error-prone replication of GB virus B in primary tamarin hepatocytes.

GB virus B (GBV-B) is the closest relative of hepatitis C virus (HCV) and is an attractive surrogate model for HCV antiviral studies. GBV-B induces an acute, resolving hepatitis in tamarins. Utilizing primary cultures of tamarin hepatocytes, we have previously developed a tissue culture system that exhibits high levels of GBV-B replication. In this report, we have extended the utility of this system for testing antiviral compounds. Treatment with human interferon provided only a marginal antiviral effect, while poly(I-C) yielded >3 and 4 log units of reduction of cell-associated and secreted viral RNA, respectively. Interestingly, treatment of GBV-B-infected hepatocytes with ribavirin resulted in an approximately 4-log decrease in viral RNA levels. Guanosine blocked the antiviral effect of ribavirin, suggesting that inhibition of IMP dehydrogenase (IMPDH) and reduction of intracellular GTP levels were essential for the antiviral effect. However, mycophenolic acid, another IMPDH inhibitor, had no antiviral effect. Virions harvested from ribavirin-treated cultures exhibited a dramatically reduced specific infectivity. These data suggest that incorporation of ribavirin triphosphate induces error-prone replication with concomitant reduction in infectivity and that reduction of GTP pools may be required for incorporation of ribavirin triphosphate. In contrast to the in vitro studies, no significant reduction in viremia was observed in vivo following treatment of tamarins with ribavirin during acute infection with GBV-B. These findings are consistent with the observation that ribavirin monotherapy for HCV infection decreases liver disease without a significant reduction in viremia. Our data suggest that nucleoside analogues that induce error-prone replication could be an attractive approach for the treatment of HCV infection if administered at sufficient levels to result in efficient incorporation by the viral polymerase.

The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen.

The ribonucleoside analog ribavirin (1-beta-D-ribofuranosyl-1,2, 4-triazole-3-carboxamide) shows antiviral activity against a variety of RNA viruses and is used in combination with interferon-alpha to treat hepatitis C virus infection. Here we show in vitro use of ribavirin triphosphate by a model viral RNA polymerase, poliovirus 3Dpol. Ribavirin incorporation is mutagenic, as it templates incorporation of cytidine and uridine with equal efficiency. Ribavirin reduces infectious poliovirus production to as little as 0. 00001% in cell culture. The antiviral activity of ribavirin correlates directly with its mutagenic activity. These data indicate that ribavirin forces the virus into 'error catastrophe'. Thus, mutagenic ribonucleosides may represent an important class of anti-RNA virus agents.

Towards the Engineering of an Orthogonal Protein Kinase/Nucleotide Triphosphate Pair

Remolding protein/ligand interfaces has led to the development of new tools for the study of biological systems. Such methods allow one to engineer proteins with specificity for designed biological probes such as inhibitors, substrates or small molecule dimerizers. Previous work in our laboratory has resulted in engineered protein kinases with specificity for ATP analogs which are otherwise orthogonal ligands for wild type kinases. Kinase reactions of analog-sensitive mutant kinases in cell lysates using γ 32P labeled ATP analogs allow identification of the direct substrates of the sensitized kinase. As an extension of this methodology, we have designed and evaluated an ATP analog, N 4 (benzyl) ribavirin triphosphate, which may be a suitable phosphodonor for a kinase that does not utilize ATP. Such a modification is likely to be necessary for in vivo kinase substrate labeling experiments where competitive phosphodonors are present in high concentrations.

Hepatitis in haemophilia.

Hepatitis in haemophilia.

Ribavirin uptake by human erythrocytes and the involvement of nitrobenzylthioinosine-sensitive (es)-nucleoside transporters.

1. The major toxicity associated with oral therapy with ribavirin is anaemia, which has been postulated to occur as a result of accumulation of ribavirin triphosphate interfering with erythrocyte respiration. The objective of this study was to determine the mechanism by which ribavirin enters into erythrocytes. 2. Entry into human erythrocytes was examined by measuring influx rates of [3H]-ribavirin alone and with the inhibitor nitrobenzylthioinosine (NBMPR), and by investigating the inhibitory effects of nucleoside and nucleobase permeants on ribavirin transport, by use of inhibitor oil-stop methods. Transport mechanisms were further characterized by assessment of substrates to cause countertransport of ribavirin in preloaded erythrocytes, and by measuring the effects of ribavirin on [3H]-NBMPR binding to erythrocyte membranes. 3. Human erythrocytes had a saturable influx mechanism for ribavirin (Km at 22 degrees C of 440+/-100 microM) which was inhibited by nanomolar concentrations of NBMPR (IC50 0.99+/-0.15 nM). Nucleosides also inhibited the influx of ribavirin (adenosine more effective than uridine) but the nucleobases hypoxanthine and adenine had no effect. In addition, uridine caused the countertransport of ribavirin in human erythrocytes. Entry of ribavirin into horse erythrocytes, a cell type that lacks the NBMPR-sensitive (es) nucleoside transporter, proceeded slowly and via a pathway that was resistant to NBMPR inhibition. Ribavirin was a competitive inhibitor of adenosine influx (mean Ki 0.48+/-0.14 mM) and also inhibited NBMPR binding to erythrocyte membranes (mean Ki 2.2+/-0.39 mM). 4. These data indicate that ribavirin is a transported permeant for the es nucleoside transporter of human erythrocytes. There was no evidence for ribavirin entering cells via a nucleobase transporter.

Analysis of intracellular ribonucleotide pools in a drug‐treated human cancer cell line using capillary zone electrophoresis

AbstractCapillary zone electrophoresis was used to separate and identify intracellular ribonucleotides and nucleotide analogues from ribavirin‐ and guanine‐treated HeLa cells, a human cervical carcinoma cell line. Nine ribonucleotides in HeLa cell extracts were identified and quantitated. In cell extracts, the average ATP/ADP ratio was 6.5 and the average energy charge of the HeLa cells was 0.92. In HeLa cells treated with guanine, the GTP pool increased to 309% of control values. When cells were treated with ribavirin, the GTP pool was reduced to 33% of nondrug treated controls. Ribavirin triphosphate reached levels of 113.25 nmols/107 cells, while ribavirin diphosphate reached levels of 4.96 nmols/107 cells.