Although the N-6-substituted purine analogues described herein showed more favorable incorporation kinetics than ribavirin (see Table ), they were less effective mutagens based on Guar
frequency. There are a number of possible explanations for this observation. First, ribavirin monophosphate is a known inhibitor of cellular inosine monophosphate dehydrogenase, which should cause a reduction in cellular purine nucleotide pools (most notably GTP). By reducing the concentration of competitor nucleotide, RTP may be able to more effectively compete with the natural nucleotides present in the cell. The ability of the JA nucleosides to influence cellular nucleotide pools by targeting enzymes of cellular nucleotide metabolism has not yet been investigated. Inosine monophosphate dehydrogenase inhibition alone has been demonstrated to cause an antiviral effect and likely contributes to the mutagenic activity of ribavirin (9
Second, the kinetic flux through the cellular nucleotide metabolism pathway may differ between nucleotides. Hence, if ribavirin is more rapidly converted to the corresponding triphosphate by cellular metabolism machinery, it may act as a more potent mutagen due to the higher intracellular concentration of the active metabolite. Finally, the unnatural nucleobases of these analogues may be modified through the action of other cellular metabolic enzymes to yield bases that have diminished or absent ambiguous base-pairing capacity. Such metabolic conversion would reduce the ability of the mutagenic nucleotide to accumulate within the cell.
The differences observed in antiviral activity between nucleosides may also be due to subtle differences in the kinetics of incorporation. In the present study, nucleotide incorporation prior to 60 s was not examined, and thus quantitative information on the rate of incorporation is currently unknown, as in some cases the incorporation had reached completion prior to 60 s. However, we can conclude that JA24, JA28, and JA30 were all incorporated more rapidly than ribavirin in the context of either templating pyrimidine.
Modified nucleosides can often exert activity through inhibition of viral or cellular polymerases by acting as low-efficiency substrates. Low rates of incorporation of nonnatural nucleosides can result in polymerase “stalling” (16
), chain termination, or lowered rates of replication from templates containing modified nucleosides (23
). Thus, the observed antiviral activity may result, at least in part, from a reduction in RNA replication kinetics caused by inhibition of the viral RdRp. However, this effect is likely minor considering the fairly rapid kinetics of incorporation of these analogs relative to ribavirin (Table ).
While the concentrations of nucleoside analogue used in these studies may be considered high for therapeutic application, the proposed mechanism of action for these analogues (i.e., lethal mutagenesis) implies competition with the intracellular pools of natural nucleotides. Particularly, these analogues act as general purine analogues, indicating they would need to compete against the extraordinarily high intracellular concentrations of ATP and GTP (approximately 3 mM total purine nucleotides) (34
). Furthermore, the mutagenic effect we have documented at these concentrations is substantial, and these analogues may in fact provide relevant antiviral effects at lower concentrations, because continuous exposure should lead to accumulation of mutations over multiple rounds of genome replication. With regard to therapeutic relevance, the only clinically employed nucleoside analogue proposed to act via antiviral lethal mutagenesis is ribavirin, which is dosed at extraordinarily high concentrations during treatment (in the range of 1 g per day) and can accumulate to concentrations approaching 0.5 mM in the liver (7
While we have demonstrated that the active nucleoside analogues identified in this work are not efficient substrates for RNR in vitro, conversion to the deoxyribonucleotide may still be possible through nucleotide phosphorolysis, liberating the nucleobase to be available as a substrate for phosphoribosyl transferase and allowing for generation of the deoxyribonucleosides through nucleobase salvage. Furthermore, the ribonucleosides themselves may exert toxicity through inhibition of, or incorporation by, cellular RNA polymerase II or mitochondrial polymerases. Further exploration of the interaction of mutagenic nucleosides with cellular polymerases is necessary to understand the clinical implications of treatment with antiviral lethal mutagens.
Recently, modified bases of JA23 and JA27 were shown to inhibit hepatitis C virus replication when attached to a 2′-C
-methyl ribose (14
). However, those compounds were found not to function as substrates for adenosine kinase, with administration of a cyclic monophosphate prodrug increasing efficacy significantly. While the templating specificity of these 2′-C
-methyl ribonucleotide analogues has not been directly elucidated, some variability in the nucleobase can be tolerated without loss of antiviral activity (8
). Therefore, the use of nucleobases with ambiguous hydrogen bonding properties, such as those described herein, may allow chain termination to be exerted in the context of two or more templating bases. Furthermore, the lack of phosphorylation activity observed by Gunic et al. (14
) may explain, at least in part, the lack of activity of related analogues in the present study.
In summary, we have identified nucleoside analogues which can act as antiviral lethal mutagens against picornaviruses. An increase in the frequency of transition mutations was observed, presumably due to tautomerization of the modified nucleobases leading to two distinct hydrogen bonding acceptor/donor surfaces which can interact favorably with either of the natural pyrimidines. Furthermore, these nucleosides had favorable properties in terms of polymerase incorporation and cellular metabolism and appeared not to be recognized as substrates for mammalian ribonucleotide reductase. Thus, these modified nucleosides represent important lead compounds in the development of antiviral lethal mutagens for clinical use.