The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen (original) (raw)
Related papers
Chembiochem, 2007
Small molecules that mimic natural nucleosides and nucleotides comprise a major class of antiviral agents. A new approach to the design of these compounds focuses on the generation of lethal mutagens: compounds that further accelerate the high rate of viral mutagenesis to confer antiviral effects. By incorporating artificial nucleobases with degenerate base-pairing abilities into viral genomes, lethal mutagens increase viral genomic mutagenesis to intolerable levels during replication, a process termed "error catastrophe", which results in the loss of viral viability. 6] The antiviral drug ribavirin (1) is one such lethal mutagen effective against the RNA viruses poliovirus (PV) and hepatitis C virus. Ribavirin is converted intracellularly to the 5'-triphosphate (RTP), which is a substrate for viral RNA-dependent RNA polymerases (RdRP). By mimicking the natural purines, RTP is misincorporated opposite pyrimidines in the enzyme-bound viral RNA template. The incorporated nucleobase of ribavirin promotes genomic mutatagenesis by templating C and U during subsequent rounds of viral replication; this facilitates error catastrophe and loss of viral viability. As part of our efforts to identify more efficacious antiviral lethal mutagens, we report the synthesis, X-ray structure, and antiviral evaluation of the 5-nitroindole-containing ribonucleoside 3, and incorporation of the related ribonucleotide 5 into RNA by a viral RdRP. These compounds represent RNA analogues of the previously reported "universal" deoxyribonucleoside 2, a compound shown to base pair with all four natural DNA pseudobases. By eliminating strong hydrogen-bond donors/acceptors, and possessing a large aromatic p system, 5-nitroindole 2 stabilizes DNA duplexes by aromatic p-stacking interactions with adjacent DNA bases. The utility of 2 has been demonstrated in applications that range from incorporation into DNA hairpins, primers for PCR and DNA sequencing, detection of single nucleotide polymorphisms, and (pseudobase) incorporation into peptide nucleic acids. We hypothesized that ribonucleoside analogues with universal base-pairing properties might possess enhanced antiviral activity relative to ribavirin (a purine mimic) by accelerating lethal viral mutagenesis. We demonstrate here that 5-nitroindole ribonucleotide 5 is universally incorporated opposite each native RNA base by a viral (poliovirus) RdRP (3D pol ). Although triphosphate 5 becomes incorporated into RNA by poliovirus 3D pol more slowly than ribavirin triphosphate (RTP), 5 represents a much more potent inhibitor of this viral enzyme, and nucleoside 3 exhibits antiviral activity in cell culture.
Biochemistry, 2002
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--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--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.
Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue
Journal of Virology, 2007
Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture, owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses.
The Broad Spectrum Antiviral Nucleoside Ribavirin as a Substrate for a Viral RNA Capping Enzyme
Journal of Biological Chemistry, 2004
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.
Ribavirin's antiviral mechanism of action: lethal mutagenesis?
Journal of Molecular Medicine, 2002
Ribavirin, an antiviral drug discovered in 1972, is interesting and important for three reasons: (a) it exhibits antiviral activity against a broad range of RNA viruses; (b) it is currently used clinically to treat hepatitis C virus infections, respiratory syncytial virus infections, and Lassa fever virus infections; and (c) ribavirin's mechanism of action has remained unclear for many years. Here we recount the history of ribavirin and review recent reports regarding ribavirin's mechanism of action, including our studies demonstrating that ribavirin is an RNA virus mutagen and ribavirin's primary antiviral mechanism of action against a model RNA virus is via lethal mutagenesis of the RNA virus genomes. Implications for the development of improved versions of rib-avirin and for the development of novel antiviral drugs are discussed.
Application of the Phosphoramidate Protide Approach to the Antiviral Drug Ribavirin
Antiviral Research, 2008
We report the application of our phosphoramidate ProTide technology to the ribonucleoside analogue 4′azidouridine to generate novel antiviral agents for the inhibition of hepatitis C virus (HCV). 4′-Azidouridine did not inhibit HCV, although 4′-azidocytidine was a potent inhibitor of HCV replication under similar assay conditions. However 4′-azidouridine triphosphate was a potent inhibitor of RNA synthesis by HCV polymerase, raising the question as to whether our phosphoramidate ProTide approach could effectively deliver 4′-azidouridine monophosphate to HCV replicon cells and unleash the antiviral potential of the triphosphate. Twenty-two phosphoramidates were prepared, including variations in the aryl, ester, and amino acid regions. A number of compounds showed sub-micromolar inhibition of HCV in cell culture without detectable cytotoxicity. These results confirm that phosphoramidate ProTides can deliver monophosphates of ribonucleoside analogues and suggest a potential path to the generation of novel antiviral agents against HCV infection. The generic message is that ProTide synthesis from inactive parent nucleosides may be a warranted drug discovery strategy.
Hepatology, 2000
Ribozymes are catalytic RNA molecules that can be designed to cleave specific RNA sequences. To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection, we designed and synthesized hammerhead ribozymes targeting 15 conserved sites in the 5Ј untranslated region (UTR) of HCV RNA. This region forms an internal ribosome entry site that allows for efficient translation of the HCV polyprotein. The 15 synthetic ribozymes contained modified nucleotides and linkages that stabilize the molecules against nuclease degradation. All 15 ribozymes were tested for their ability to reduce expression in an HCV 5Ј UTR/ luciferase reporter system and for their ability to inhibit replication of an HCV-poliovirus (HCV-PV) chimera. Treatment with several ribozymes resulted in significant downregulation of HCV 5Ј UTR/luciferase reporter expression (range 40% to 80% inhibition, P F .05). Moreover, several ribozymes showed significant inhibition (G90%, P F .001) of chimeric HCV-PV replication. We further show that the inhibitory activity of ribozymes targeting site 195 of HCV RNA exhibits a sequence-specific dose response, requires an active catalytic ribozyme core, and is dependent on the presence of the HCV 5Ј UTR. Treatment with synthetic stabilized anti-HCV ribozymes has the potential to aid patients who are infected with HCV by reducing the viral burden through specific targeting and cleavage of the viral genome. (HEPATOLOGY 2000;31:769-776.)
Mechanisms of action of ribavirin against distinct viruses
Reviews in Medical Virology, 2006
The nucleoside analogue ribavirin has antiviral activity against many distinct viruses both in vitro and in vivo. Five distinct mechanisms have been proposed to explain the antiviral properties of ribavirin. These include both indirect mechanisms (inosine monophosphate dehydrogenase inhibition, immunomodulatory effects) and direct mechanisms (interference with RNA capping, polymerase inhibition, lethal mutagenesis). Recent concerns about bioterrorism have renewed interest in exploring the antiviral activity of ribavirin against unique viruses. In this paper, we review the proposed mechanisms of action with emphasis on recent discoveries, as well as the implications of ribavirin resistance. Evidence exists to support each of the five proposed mechanisms of action, and distinct virus/host combinations may preferentially favour one or more of these mechanisms during antiviral therapy.
Lethal Mutagenesis of RNA Viruses and Approved Drugs with Antiviral Mutagenic Activity
Viruses
In RNA viruses, a small increase in their mutation rates can be sufficient to exceed their threshold of viability. Lethal mutagenesis is a therapeutic strategy based on the use of mutagens, driving viral populations to extinction. Extinction catastrophe can be experimentally induced by promutagenic nucleosides in cell culture models. The loss of HIV infectivity has been observed after passage in 5-hydroxydeoxycytidine or 5,6-dihydro-5-aza-2′-deoxycytidine while producing a two-fold increase in the viral mutation frequency. Among approved nucleoside analogs, experiments with polioviruses and other RNA viruses suggested that ribavirin can be mutagenic, although its mechanism of action is not clear. Favipiravir and molnupiravir exert an antiviral effect through lethal mutagenesis. Both drugs are broad-spectrum antiviral agents active against RNA viruses. Favipiravir incorporates into viral RNA, affecting the G→A and C→U transition rates. Molnupiravir (a prodrug of β-d-N4-hydroxycytidin...