Synthesis and antiviral activity of some phosphates of the broad-spectrum antiviral nucleoside, 1-.beta.-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin)

Allen, Lois B.; Boswell, Kay H.; Khwaja, Tasneem A.; Meyer, Rich B.; Sidwell, Robert W.; Witkowski, J. T.; Christensen, Leon F.; Robins, Roland K.

doi:10.1021/jm00206a005

Cited by 42 publications

(21 citation statements)

References 12 publications

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“…Both viruses were purified in sucrose gradients after growth in Chinese hamster ovary cells as described by Stampfer et al (1969). VSV wt was grown at 34 °C for 18 to 20 h, and polR1 was grown at the same temperature for 27 to 30 h. RDP and RTP were a kind gift of Roland K. Robins and Daniel Smith (Allen et al, 1978).…”

Section: Discussionmentioning

confidence: 99%

Altered ATP Function of a Vesicular Stomatitis Virus Mutant Detected by Kinetic Analysis of the Transcriptase Using Phosphorylated Ribavirin

Fernandez-Larsson

1989

Journal of General Virology

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SUMMARYWe have studied the effect of phosphorylated ribavirin on the vesicular stomatitis virus (VSV) in vitro polymerase reaction by analysis of kinetic data obtained by varying the concentration of nucleoside triphosphates. The wild-type VSV had previously shown a competitive inhibition with the four natural nucleoside triphosphates with the use of ribavirin diphosphate (RDP) or ribavirin triphosphate (RTP). In contrast, when RDP (or RTP) was added to a transcription assay system using the polR1 mutant of VSV, a non-competitive or mixed type of inhibition was observed when the concentration of ATP was varied. Our results indicate that polR1 has an altered ATP function in addition to the previously described phenotypic characteristics of this mutant, which include synthesis of readthrough products of the leader/nucleocapsid (N) gene junction and a decreased ATP requirement for transcription. We have also studied CsCl-purified in vitro transcription products by primer-extending leader or N mRNA transcripts and found that the ratio of leader/N mRNA for VSV polR1 (1.3:1) was lower than values obtained previously for wild-type (3.7:1).Vesicular stomatitis virus (VSV) is the prototype rhabdovirus containing a negative-stranded RNA genome 11 kb long. The transcription and replication events of this virus have been studied for several years, but many questions related to these processes and to the switch from the transcriptive to the replicative mode remain unanswered. The VSV polR mutants (Perrault et al., 1983) have unique properties that may help solve the intricacies of transcription and replication. The properties of the polR mutants include synthesis of leader/nucleocapsid (N) gene readthrough transcripts (Perrault et al., 1983), utilization of imido-ATP for transcription initiation (Perrault & McClear, 1984), and an altered ATP utilization , whereas wild-type (wt) VSV has a high ATP requirement as well as an obligatory cleavage of the beta-gamma bond of this nucleotide for transcription (Testa & Banerjee, 1979;Green & Emerson, 1984; Perrault & McClear, 1984). We have used ribavirin to investigate by enzyme kinetic procedures and product analysis the mechanism of VSV mRNA synthesis, in an effort to understand the in vitro transcription of the virus.We have already reported that ribavirin 5'-monophosphate, 5'-diphosphate (RDP) and 5'-triphosphate (RTP) possess a significant direct suppressive effect upon viral polymerase activity. Inhibition by RDP or RTP could be reversed by addition of GTP, CTP and UTP, but not by the addition of GDP or ATP (Toltzis et al., 1988). The effective reversal of inhibition by all nucleoside triphosphates except ATP suggested that the ribavirin molecule does not act only as a guanine analogue as generally believed, but rather may interact with the polymerase complex on a site specific for at least three of the precursors of RNA synthesis. The fact that the effect of phosphorylated ribavirin could not be reversed with the addition of ATP could be explained if ATP had an additional inte...

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Section: Discussionmentioning

confidence: 99%

Altered ATP Function of a Vesicular Stomatitis Virus Mutant Detected by Kinetic Analysis of the Transcriptase Using Phosphorylated Ribavirin

Fernandez-Larsson

1989

Journal of General Virology

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“…Thin-layer chromatography plates (CEL 300 PEI) were purchased from Brinkman Instruments, Inc., Westbury, N.Y. Ribavirin monophosphate (RMP), ribavirin diphosphate (RDP), and ribavirin triphosphate (RTP) were the kind gift of Roland K. Robins and Daniel Smith (1). Phosphorylated ribavirin compounds were stored in the lyophilized form at -20°C and periodically suspended in 0.1 M Tris hydrochloride (pH 8.0); stock solutions were also stored at -20°C and were discarded after 3 to 6 weeks.…”

Section: Methodsmentioning

confidence: 99%

Effect of phosphorylated ribavirin on vesicular stomatitis virus transcription

Toltzis

O’Connell

1988

Antimicrob Agents Chemother

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The effect of phosphorylated ribavirin on a vesicular stomatitis virus (VSV) in vitro tanscription reaction was examined. Viral mRNA synthesized in the presence of the 5' mono-, di-, and triphosphorylated forms of the drug translated with equal efficiencies under the test conditions. However, all three phosphorylated species inhibited VSV transcription. The mono-and diphosphorylated forms of the drug possessed approximately two to three times the inhibitory activity as the triphosphorylated form. Transcripts synthesized in the presence of drug were full length and were absent of incorporated drug. Inhibition by ribavirin 5'-diphosphate could be reversed by the addition of UTP, CTP, and GTP, while the addition of GDP to the reaction did not reverse inhibition. Ribavirin diphosphate was added to a La Crosse virus in vitro transcription assay to determine whether an inhibitory effect could be established in a viral system that was more sensitive to ribavirin than was VSV; it led to profound inhibition of RNA synthesis at concentrations as low as 0.1 ,ug/ml. These data suggest that ribavirin has an effect on the initial steps of transcription by some RNA-dependent RNA polymerases and that this effect may be mediated by several phosphorylated forms.Ribavirin (1-0-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a broad-spectrum antiviral agent whose molecular mode of action remains controversial. The drug was approved by the Food and Drug Administration in 1986 for aerosol use in infants with serious infection due to respiratory syncytial virus. Ribavirin is currently under clinical investigation against a variety of other viral illnesses, including those due to influenza virus and human immunodeficiency virus. However, the drug possesses in vitro activity against a remarkably broad spectrum of viral pathogens, including both DNA and RNA viruses (11), suggesting that its full clinical potential has yet to be realized.Several theories regarding the molecular mode of action of ribavirin have been proposed. One hypothesis states that the drug leads to decreased intracellular pools of GTP, indirectly suppressing viral nucleic acid synthesis (9, 15). A more recent hypothesis proposes that ribavirin therapy of viralinfected cells results in the synthesis of RNA with abnormal or absent 5' cap structures, which in turn leads to inefficient translation of viral transcripts (6). A third hypothesis states that the drug has a direct suppressive effect on viral polymerase activity; however, such activity has been demonstrated only for influenza virus (4,17,18). None of these hypotheses are mutually exclusive, and indeed, they may indicate that ribavirin acts in a complex, multiple-site fashion. In previous experiments (16) in which the effect of ribavirin on vesicular stomatitis virus (VSV) grown in Chinese hamster ovary cells was examined, it was indicated that the drug leads to the synthesis of inefficiently translated viral mRNA, with little demonstrable effect on viral polymerase, lending support to the hypothesis that the dr...

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“…Ribavirin-5 0 -monophosphate was prepared by reaction of ribavirin with phosphorus oxychloride and trimethyl phosphate according to the method of Allen [45]. Purified RMP was converted to ribavirin-5 0 -phosphorimidazolide according to the procedure of Fiume [43], and was then conjugated to hemoglobin.…”

Section: Discussionmentioning

confidence: 99%

Human uridine-cytidine kinase phosphorylation of ribavirin: a convenient method for activation of ribavirin for conjugation to proteins

Jukić

Duvnjak

et al. 2007

J Biomed Sci

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Ribavirin is a synthetic nucleoside analog that is used for the treatment of hepatitis C virus (HCV) infection. Its primary toxicity is hemolytic anemia, which sometimes necessitates dose reduction or discontinuation of therapy. Selective delivery of ribavirin into liver cells would be desirable to enhance its antiviral activity and avoid systemic side effects. One approach to liver-specific targeting is conjugation of the ribavirin with asialoglycoprotein that is taken up specifically by liver cells. Human uridine-cytidine kinase-1 (UCK-1) was used for ribavirin phosphorylation to its monophosphate form. 1-Ethyl-3-diisopropylaminocarbodiimide (EDC) was used as a coupling agent. The best results were obtained using direct conjugation protocol with a molar ratio of 6.5 ribavirin monophosphate (RMP) molecules per one asialoorosomucoid (AsOR) molecule. Our findings show that ribavirin is a potential substrate of UCK-1, and RMP formed could be chemically coupled to AsOR to form a conjugate for liver specific targeting.

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Synthesis and antiviral activity of some phosphates of the broad-spectrum antiviral nucleoside, 1-.beta.-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin)

Cited by 42 publications

References 12 publications

Altered ATP Function of a Vesicular Stomatitis Virus Mutant Detected by Kinetic Analysis of the Transcriptase Using Phosphorylated Ribavirin

Altered ATP Function of a Vesicular Stomatitis Virus Mutant Detected by Kinetic Analysis of the Transcriptase Using Phosphorylated Ribavirin

Effect of phosphorylated ribavirin on vesicular stomatitis virus transcription

Human uridine-cytidine kinase phosphorylation of ribavirin: a convenient method for activation of ribavirin for conjugation to proteins

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