5-Modified-2′-dU and 2′-dC as Mutagenic Anti HIV-1 Proliferation Agents: Synthesis and Activity

Abstract

With the goal of limiting HIV-1 proliferation by increasing the mutation rate of the viral genome, we synthesized a series of pyrimidine nucleoside analogues modified in position 5 of the aglycone moiety but unmodified on the sugar part. The synthetic strategies allow us to prepare the targeted compounds directly from commercially available nucleosides. All compounds were tested for their ability to reduce HIV-1 proliferation in cell culture. Two of them (5-hydroxymethyl-2'-dU (1c) and 5-hydroxymethyl-2'-dC (2c)) displayed a moderate antiviral activity in single passage experiments. The same two compounds plus two additional ones (5-carbamoyl-2'-dU (1a) and 5-carbamoylmethyl-2'-dU (1b)) were potent inhibitors of HIV-1 RT activity in serial passage assays, in which they induced a progressive loss of HIV-1 replication. In addition, viruses collected after seven passages in the presence of 1c and 2c replicated very poorly after withdrawal of these compounds, consistent with the accumulation of deleterious mutations in the HIV-1 genome.

Evaluation of Anti-HIV-1 Mutagenic Nucleoside Analogues

“…Among a series of eight nucleoside analogues, we previously showed that two of them, hm 5 -dU (compound 3) and hm 5 -dC (compound 6) ( Fig. 1), were able to progressively inhibit HIV-1 replication, most likely by causing lethal mutagenesis (58). Here, we compared six of these compounds, including the two mutagenic nucleosides, using thermal denaturation of short DNA duplexes and extension of a primer annealed to a modified template.…”

“…As a control, we have also prepared one analogue modified on position 3 (Watson-Crick position) of the base moiety by a carbamoylmethyl group. Interestingly, some of these compounds displayed antiviral activity in cell culture, and two of them displayed antiviral activity in cell culture in a way that was consistent with an accumulation of deleterious mutations in the HIV-1 genome (58).…”

“…Sequencing of the HIV-1 pol Gene after Treatment with Mutagenic Nucleosides-Serial HIV-1 passage experiments in the presence of compounds 1-6 or in the presence of DMSO (as control) were described previously (58). After the first incubation with the nucleoside analogues (passage 0) and at the end of the procedure (passage 8), total cellular DNA was extracted and purified using the DNeasy tissue kit (Qiagen).…”

“…Cell Culture-Our previous study has shown that serial passages of HIV-1 in the presence of compounds 1-3 and 6 resulted in a progressive loss of viral replication, which was almost complete after eight passages with compounds 2, 3, and 6, and very significant with compound 1 (58). When viruses passaged seven times in the presence of nucleoside analogues were collected and subsequently cultured in the absence of inhibitor, viruses passaged in the presence of compounds 3 and 6 did not replicate, as expected for lethal mutagenesis (58).…”

“…Based on that idea, we have synthesized several pyrimidine nucleoside analogues that are modified exclusively on position 5 of the base moiety by carbamoyl, carbamoylmethyl, hydroxyl or hydroxymethyl groups to remain potential substrates for HIV-1 RT after their conversion into triphosphates, to base pair with at least two natural nucleosides, and ultimately to increase the error rate in the proviral DNA (58). Accordingly, O'Neil et al (12) established that the contribution of RT in HIV-1 mutagenesis is about 2-fold higher than the contribution of host cell polymerases.…”