Synthesis and Antiviral Evaluation of a Mutagenic and Non-Hydrogen Bonding Ribonucleoside Analogue:  1-β-<scp>d</scp>-Ribofuranosyl-3-nitropyrrole

Harki, Daniel A.; Graci, Jason D.; Korneeva, Victoria S.; Ghosh, Saikat; Hong, Zhi; Cameron, Craig Ε.; Peterson, Blake R.

doi:10.1021/bi026120w

Cited by 41 publications

(48 citation statements)

References 58 publications

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“…Despite this, previous attempts to design novel mutagenic nucleotide analogues that are capable of inducing mutation in RNA viruses have had only limited success (17)(18)(19)39). In this study, we have demonstrated that rPTP is an efficient and ambiguous substrate of a viral RNA-dependent RNA polymerase.…”

Section: Discussionmentioning

confidence: 75%

“…Cells were maintained at 37°C in a 5% CO 2 atmosphere. Antiviral activity assays were performed as previously described (19). A guanidine resistance assay was performed as previously described (7).…”

Section: Methodsmentioning

confidence: 99%

“…We have previously investigated the use of "universal" bases with minimal hydrogen bonding potential as lethal mutagens of PV (17,19). Here we investigate the antiviral and mutagenic potential of a nucleoside analogue with the capacity to stably base pair with two natural nucleobases.…”

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confidence: 99%

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Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue

Graci¹,

Harki

Korneeva³

et al. 2007

J Virol

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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.

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Section: Discussionmentioning

confidence: 75%

Section: Methodsmentioning

confidence: 99%

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Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue

Graci¹,

Harki

Korneeva³

et al. 2007

J Virol

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“…While both nucleotides reduced the specific infectivity of viral RNA, the responses observed for PV and CVB3 were identical. PMP substitution sufficient for a 10-fold reduction in PV-specific HeLa S3 cells (1 ϫ 10 5 in 24-well plates) were pretreated with ribavirin for 1 h prior to infection with either PV or CVB3 at an MOI of 0.01 (A) or 5 (B) as previously described (17). Cells were incubated with ribavirin at the indicated concentration until all cells demonstrated a cytopathic effect or for 6 days, at which time virus was collected and titers were determined using HeLa S3 monolayers.…”

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confidence: 99%

Mutational Robustness of an RNA Virus Influences Sensitivity to Lethal Mutagenesis

Graci

Gnädig

Galarraga

et al. 2012

J Virol

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The ability to extinguish a viral population of fixed reproductive capacity by causing small changes in the mutation rate is referred to as lethal mutagenesis and is a corollary of population genetics theory. Here we show that coxsackievirus B3 (CVB3) exhibits reduced mutational robustness relative to poliovirus, manifesting in enhanced sensitivity of CVB3 to lethal mutagens that is dependent on the size of the viral population. We suggest that mutational robustness may be a useful measure of the sensitivity of a virus to lethal mutagenesis. RNA virus genomes are replicated at mutation rates orders of magnitude higher than DNA genomes (10, 27) and exist as heterogeneous populations of genetically distinct yet related genomes. Attempts to define these populations have relied upon population genetics theory (5,6,34,35); unfortunately, little empirical evidence exists validating these theories in vivo (30).Population genetics theory predicts a log-linear relationship between the genomic mutation rate and the fecundity (average number of progeny generated per infectious cycle) needed to avoid extinction and defines a theoretical "extinction threshold" (6). A corollary of this theory is lethal mutagenesis, the ability to drive a viral population to extinction by increasing the error frequency (13).A high mutation rate can negatively affect viral fitness and may drive a population to extinction. However, the deleterious effects of a high mutation rate may differ between viruses depending on their mutational robustness, i.e., the constancy of a phenotype in the face of deleterious mutations (26). A more robust population is able to minimize the deleterious effects of a high mutation rate by opting not to maximize fitness, a phenomenon termed "survival of the flattest" (35). Previous studies have suggested that the distributions of mutational effects between DNA and RNA virus genomes are similar (25). Therefore, the mutational robustness of a virus might be expected to be similar to that of closely related viruses.We investigated the mutational robustness of two closely related enteroviruses, poliovirus (PV) and coxsackievirus B3 (CVB3), which employ similar replication strategies and genome organizations and exhibit 59% polyprotein identity (74% similarity) as shown by BLAST analysis (GenBank accession numbers Q5UEA2 and P03300). To compare the characteristics of mutational robustness of these two viruses, we examined the response of PV (Mahoney) and of CVB3 (CVB3/0) to increasing concentrations of the mutagenic nucleoside ribavirin (Fig. 1A). Infection was performed under conditions that included a low multiplicity of infection (MOI) so that the cumulative effect of increased mutation frequency could be observed over two or more replicative cycles. CVB3 was observed to be considerably more sensitive than PV to ribavirin treatment at 0.5 mM or greater. Dramatically increased sensitivity was not observed under conditions of high MOI (Fig. 1B), consistent with the deleterious effect of mutation being reversed by populati...

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“…To examine the biological activity of a triphosphate prepared by this method, we investigated the incorporation of 6-methylpurine ribonucleoside triphosphate (25) into RNA by RNA-dependent RNA polymerase from poliovirus (3D pol ). The cognate ribonucleoside, an antiviral and anticancer agent with activity against RNA viruses, lacks a clearly defined mechanism of action.…”

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confidence: 99%

One-Pot Synthesis of Nucleoside 5′-Triphosphates from Nucleoside 5′-H-Phosphonates

Sun

Edathil

et al. 2008

Org. Lett.

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Nucleoside 5′-triphosphates (NTPs) play key roles in biology and medicine. However, these compounds are notoriously difficult to synthesize. We describe a one-pot method to prepare NTPs from nucleoside 5′-H-phosphonate monoesters via pyridinium phosphoramidates, and we used this approach to synthesize ATP, UTP, GTP, CTP, ribavirin-TP, and 6-methylpurine ribonucleoside-TP (6MePTP). Poliovirus RNA-dependent RNA polymerase efficiently employed 6MePTP as a substrate, suggesting that the cognate nucleoside, a poorly understood antiviral agent, may damage viral RNA.Nucleoside 5′-triphosphates (NTPs) are critical mediators of myriad biological processes including DNA replication, transcription, and translation. Correspondingly, synthetic mimics of NTPs have been widely used as molecular probes and biological assay components and represent active metabolites of certain drugs such as the antiviral agent ribavirin. Despite their importance in biology and medicine, the diversity of commercially available NTPs is limited because these compounds are often difficult to prepare and isolate in pure form. 1 Traditional approaches for the synthesis of NTPs include the "one-pot, three-step" method and the method of Ludwig and Eckstein. 2,3 Although these strategies work well for some substrates, others are plagued by low yields and difficulties in purification. 4 More recently, Ahmadibeni reported 5 a solid-phase route to NTPs, and Borch reported 6 a method for the preparation of activated phosphoramidates that can be converted to NTPs by reaction with tris(tetra-n-butylammonium) hydrogen pyrophosphate. The final coupling step employed by Borch was found to proceed in remarkably high yield, but the required four-step synthesis of We hypothesized that nucleoside 5′-H-phosphonates might provide novel and more readily synthesized precursors to NTPs. This hypothesis was based on reported syntheses of phosphates, phosphoramidates, and other phosphate derivatives from these precursors. [7][8][9][10] After conversion to silyl phosphites with TMSCl, H-phosphonate monoesters can be oxidized by elemental iodine and other reagents to generate electrophilic intermediates. 11,12 These intermediates are known to react with a variety of nucleophiles to afford addition products, but surprisingly, this strategy has not been previously investigated for the synthesis of NTPs.We report here a one-pot approach for the synthesis of NTPs from nucleoside 5′-Hphosphonate monoesters, relatively stable compounds that can be easily prepared from 2′,3′-O-isopropylidene-protected nucleosides. 13 We demonstrate that fully deprotected ribonucleoside 5′-H-phosphonate monoesters can be converted in situ to pyridinium phosphoramidate intermediates. Upon addition of nucleophilic tris(tetra-n-butylammonium) hydrogen pyrophosphate, NTPs can be isolated by use of a two-step purification protocol.As shown in Scheme 1, starting with the known 2′,3′-O-isopropylidene-protected nucleosides 1-6, 13-15 phosphitylation 16 with salicyl phosphorochlorodite or PCl 3 provi...

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Synthesis and Antiviral Evaluation of a Mutagenic and Non-Hydrogen Bonding Ribonucleoside Analogue: 1-β-d-Ribofuranosyl-3-nitropyrrole

Cited by 41 publications

References 58 publications

Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue

Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue

Mutational Robustness of an RNA Virus Influences Sensitivity to Lethal Mutagenesis

One-Pot Synthesis of Nucleoside 5′-Triphosphates from Nucleoside 5′-H-Phosphonates

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