Synthesis of a Universal 5‐Nitroindole Ribonucleotide and Incorporation into RNA by a Viral RNA‐Dependent RNA Polymerase

Abstract

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: [1,2] compounds that further accelerate the high rate of viral mutagenesis [3,4] 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. [5, 6] The antiviral drug ribavirin (1) is one such lethal mutagen effective against the RNA viruses A C H T U N G T R E N N U N G poliovirus (PV) [7] and hepatitis C virus. [8] 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. [7,[9][10][11] 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. [12,13] 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. [14,15] The utility of 2 has been demonstrated in applications that range from incorporation into DNA hairpins, [16,17] primers for PCR and DNA sequencing, [13,18,19] detection of single nucleotide polymorphisms, [20,21] and (pseudobase) incorporation into peptide nucleic acids.[22]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.5-Nitroindole ribonucleoside 3 and phosphorylated analogues 5-7 were synthesized as shown in Scheme 1. Commercially available 9 was chlorinated with TiCl 4

Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue

“…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.…”

“…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.…”

Structure(s), function(s), and inhibition of the RNA-dependent RNA polymerase of noroviruses

“…The antiviral activity of 5-nitrocytidine was first described against poliovirus and coxsackievirus B3, demonstrating greater inhibition potency than the control drug ribavirin (Harki et al, 2007). In the same study, 5-nitrocytidine triphosphate (NCT) inhibited poliovirus RdRp activity.…”

Arenaviruses and Lethal Mutagenesis. Prospects for New Ribavirin-based Interventions

“…Mutagenesis can provide an advantage over conventional non-mutagenic inhibitors because mutagenized genomes may suppress the most infective subpopulations of genomes coexisting in the same quasispecies, as discussed in the next section. A number of base and nucleoside analogues are currently under development as potential new antiviral mutagenic agents [44,45,46,47,48,49,50]. …”