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Published on: September 5, 2016
Evaluation of anti-HIV-1 mutagenic nucleoside analogues
Valérie Vivet-Boudou1, Catherine Isel2, Yazan El Safadi3
1From the Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 67084 Strasbourg Cedex and v.vivet@ibmc-cnrs.unistra.fr.
Abstract:
Because of their high mutation rates, RNA viruses and retroviruses replicate close to the threshold of viability. Their existence as quasi-species has pioneered the concept of "lethal mutagenesis" that prompted us to synthesize pyrimidine nucleoside analogues with antiviral activity in cell culture consistent with an accumulation of deleterious mutations in the HIV-1 genome. However, testing all potentially mutagenic compounds in cell-based assays is tedious and costly. Here, we describe two simple in vitro biophysical/biochemical assays that allow prediction of the mutagenic potential of deoxyribonucleoside analogues. The first assay compares the thermal stabilities of matched and mismatched base pairs in DNA duplexes containing or not the nucleoside analogues as follows. A promising candidate should display a small destabilization of the matched base pair compared with the natural nucleoside and the smallest gap possible between the stabilities of the matched and mismatched base pairs. From this assay, we predicted that two of our compounds, 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine, should be mutagenic. The second in vitro reverse transcription assay assesses DNA synthesis opposite nucleoside analogues inserted into a template strand and subsequent extension of the newly synthesized base pairs. Once again, only 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine are predicted to be efficient mutagens. The predictive potential of our fast and easy first line screens was confirmed by detailed analysis of the mutation spectrum induced by the compounds in cell culture because only compounds 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine were found to increase the mutation frequency by 3.1- and 3.4-fold, respectively.
Insights
Developing novel antiviral therapies, researchers identified two deoxyribonucleoside analogues, 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine, as potent mutagens. These compounds effectively increase mutation frequency, offering a promising strategy for lethal mutagenesis against viruses like HIV-1.
Area of Science:
- Biochemistry
- Virology
- Medicinal Chemistry
Background:
- RNA viruses and retroviruses exhibit high mutation rates, operating near their viability threshold.
- The concept of "lethal mutagenesis" emerged from studying quasi-species, leading to the synthesis of pyrimidine nucleoside analogues for antiviral applications.
- Existing cell-based assays for mutagenic compounds are time-consuming and expensive.
Purpose of the Study:
- To develop rapid and cost-effective in vitro assays for predicting the mutagenic potential of deoxyribonucleoside analogues.
- To identify specific nucleoside analogues with potential for inducing lethal mutagenesis in viral genomes, such as HIV-1.
Main Methods:
- A biophysical assay evaluating the thermal stability of DNA duplexes with and without nucleoside analogues to assess base pairing.
- A biochemical reverse transcription assay measuring DNA synthesis opposite incorporated nucleoside analogues in a template strand.
- In vitro and cell culture analyses to confirm mutagenic predictions and quantify mutation frequency increases.
Main Results:
- The thermal stability assay predicted that 5-hydroxymethyl-2 ahydro-deoxyuridine and 5-hydroxymethyl-2 ahydro-deoxycytidine would be mutagenic due to minimal destabilization of matched base pairs and a small stability gap.
- The reverse transcription assay corroborated these predictions, identifying the same two compounds as efficient mutagens.
- Cell culture studies confirmed that 5-hydroxymethyl-2 ahydro-deoxyuridine and 5-hydroxymethyl-2 ahydro-deoxycytidine significantly increased HIV-1 mutation frequency by 3.1- and 3.4-fold, respectively.
Conclusions:
- Two simple in vitro assays can accurately predict the mutagenic potential of deoxyribonucleoside analogues.
- 5-hydroxymethyl-2 ahydro-deoxyuridine and 5-hydroxymethyl-2 ahydro-deoxycytidine are identified as promising candidates for lethal mutagenesis strategies against viruses.
- These assays offer a faster and more economical alternative to cell-based screening for antiviral drug development.
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