Evaluation of anti-HIV-1 mutagenic nucleoside analogues.
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.
From the Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 67084 Strasbourg Cedex and v.vivet@ibmc-cnrs.unistra.fr.
Valérie
V
Vivet-Boudou
From the Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 67084 Strasbourg Cedex and c.isel@ibmc-cnrs.unistra.fr.
Catherine
C
Isel
From the Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 67084 Strasbourg Cedex and.
Yazan
Y
El Safadi
From the Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 67084 Strasbourg Cedex and.
Redmond P
RP
Smyth
the Unité INSERM 748, Université de Strasbourg, Institut de Virologie, 67000 Strasbourg, France.
Géraldine
G
Laumond
the Unité INSERM 748, Université de Strasbourg, Institut de Virologie, 67000 Strasbourg, France.
Christiane
C
Moog
From the Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 67084 Strasbourg Cedex and.
Jean-Christophe
JC
Paillart
From the Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 67084 Strasbourg Cedex and r.marquet@ibmc-cnrs.unistra.fr.
Roland
R
Marquet
D019380:Anti-HIV Agents / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D020137:Base Pair Mismatch; D020029:Base Pairing; D001483:Base Sequence; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D015195:Drug Design; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D057166:High-Throughput Screening Assays / Q000191:economics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009153:Mutagens / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D009691:Nucleic Acid Denaturation; D011237:Predictive Value of Tests; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D048348:Reverse Transcription; D013816:Thermodynamics; D013936:Thymidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013997:Time Factors
C540838:5-hydroxymethyl-2'-deoxycytidine; D019380:Anti-HIV Agents; D009153:Mutagens; D018894:Reverse Transcriptase Inhibitors; D003841:Deoxycytidine; C018080:5-hydroxymethyl-2'-deoxyuridine; D054303:HIV Reverse Transcriptase; D013936:Thymidine
Antiviral Agent; Human Immunodeficiency Virus (HIV); Lethal; Mutagenesis; Nucleoside/Nucleotide Analogue; Reverse Transcription