Tautomerism provides a molecular explanation for the mutagenic properties of the anti-HIV nucleoside 5-aza-5,6-dihydro-2'-deoxycytidine

Abstract

Viral lethal mutagenesis is a strategy whereby the innate immune system or mutagenic pool nucleotides increase the error rate of viral replication above the error catastrophe limit. Lethal mutagenesis has been proposed as a mechanism for several antiviral compounds, including the drug candidate 5-aza-5,6-dihydro-2'-deoxycytidine (KP1212), which causes A-to-G and G-to-A mutations in the HIV genome, both in tissue culture and in HIV positive patients undergoing KP1212 monotherapy. This work explored the molecular mechanism(s) underlying the mutagenicity of KP1212, and specifically whether tautomerism, a previously proposed hypothesis, could explain the biological consequences of this nucleoside analog. Establishing tautomerism of nucleic acid bases under physiological conditions has been challenging because of the lack of sensitive methods. This study investigated tautomerism using an array of spectroscopic, theoretical, and chemical biology approaches. Variable temperature NMR and 2D infrared spectroscopic methods demonstrated that KP1212 existed as a broad ensemble of interconverting tautomers, among which enolic forms dominated. The mutagenic properties of KP1212 were determined empirically by in vitro and in vivo replication of a single-stranded vector containing a single KP1212. It was found that KP1212 paired with both A (10%) and G (90%), which is in accord with clinical observations. Moreover, this mutation frequency is sufficient for pushing a viral population over its error catastrophe limit, as observed before in cell culture studies. Finally, a model is proposed that correlates the mutagenicity of KP1212 with its tautomeric distribution in solution.

Keywords: KP1461; site-specific mutagenesis; spectral deconvolution; viral decay acceleration; viral extinction.

Fig. 1.

Schematic presentation of KP1212's mutagenic effect on viruses. (A) KP1212 exists as an array of different tautomeric forms, whereas cytosine almost exclusively exists as one form, the canonical keto-amino tautomer. (B) The deoxynucleotide analog of KP1212 is incorporated by viral polymerases, causing G-to-A and A-to-G mutations during viral replication. KP1212 is a poor substrate for human polymerases, which provides selectivity in its action against the virus. The progressive acquisition of mutations in the viral genome leads to viral population collapse.

Fig. 2.

In vivo and in vitro demonstration of the promiscuous base-pairing properties of KP1212. (A) A 16mer oligonucleotide containing KP1212 (or controls) at a specific site was chemically synthesized and ligated into the genome of an M13 bacteriophage. (B) The KP1212-containing M13 genomes were replicated within E. coli cells. Progeny were analyzed to characterize the amount and the type of nucleic acid base placed opposite the lesion during replication. The relative reduction in lesion vs. nonlesion competitor progeny formation was used as an estimate of the extent to which KP1212 and m3C inhibited DNA replication. (C) The KP1212-containing M13 genomes were used as templates for in vitro polymerase extension assays, carried out at 72 °C, using the high-fidelity DNA polymerase PfuTurbo. SD of the measurements is ∼1% (n = 3). (D) Bypass efficiency (CRAB assay) of KP1212, m3C, C in HK81 (AlkB⁺), and HK82 (AlkB⁻) E. coli cells. m3C and undamaged C were used as controls. Genomes were made and normalized to one another before being combined with a competitor genome. Each mixture was transformed into the corresponding cell strain in triplicate, and bypass efficiency was calculated by using the undamaged C genome as 100% bypass, with error bars representing one SD (n = 3). (E) Mutagenesis (REAP assay) of KP1212, m3C, and C in HK82 (AlkB⁻) E. coli cells. m3C, undamaged C, and an approximately equimolar mixture of genomes carrying unmodified G/A/T/C bases at the site of inquiry (denoted as GATC) were used as controls. Genomes containing the lesions of interest were transfected into E. coli in triplicate. The percentage of G, A, T, and C incorporated opposite the lesion site reveals the base-pairing preference of the lesions, with error bars representing one SD (n = 3).

Fig. 3.

Variable temperature ¹H NMR spectra of KP1212 in DMF-d₇ (20 °C to −60 °C). The peaks corresponding to the active protons on the base portion of KP1212 are labeled as i–vi at −60 °C and i′–iii′ at 20 °C.

Fig. 4.

NMR studies demonstrate the existence of different tautomeric forms of KP1212. (A) Structures of the five possible tautomeric forms of KP1212. The active protons (a–o) on the nucleobase portion of the molecule are designated with different colors to indicate their chemical environment (type): blue (imino), purple (amido), red (enol), and green (amino). (B) The ¹H NMR spectrum of KP1212 in DMF-d₇ at −50 °C (5.5–12.0 ppm), from Fig. 3. The peaks from the active protons on the nucleobase portion are labeled as i–vi and their corresponding areas are indicated. According to their chemical shifts, the type of the active protons on the KP1212 nucleobase that contribute to each peak is indicated. (C) Schematic of the deconvolution process of the ¹H NMR spectrum of KP1212 at −50 °C depicting how the active proton peaks corresponding to each tautomer contribute to the overall spectrum. Each of the six peaks identified in the NMR spectrum in B, denoted i–vi, is schematically represented as the bottom trace (in blue). To indicate each tautomer’s respective contributions to the six peaks, schematic representations of the NMR signals of the active protons of each tautomer are shown (black traces). Each peak is labeled with a color coded letter (a–o), which corresponds to the active protons labeled in section A. (D) Mathematical analysis of NMR spectrum using matrix algebra to calculate relative distribution of tautomers. The elements of matrix A represent the number of active protons from each tautomer (columns) that contribute to each of the six NMR peaks (rows). The matrix X elements are the unknown variables, which represent the relative amounts of each tautomer. Matrix B contains the areas corresponding to each peak in the NMR spectrum at −50 °C. Linear equations were generated from the matrix equation A × X = B. Solving the system of linear equations yielded values for the unknowns, which provided the relative distribution of individual tautomers of KP1212.

Fig. 5.

IR studies demonstrate the existence of different tautomeric forms of KP1212. (A) Variable temperature FTIR spectra of KP1212 in deuterated 0.5 M phosphate buffer pD = 7.9 taken at various temperatures (10 °C–80 °C). The black arrow indicates the temperature-dependent increase of the keto-carbonyl stretch. (B) 2D IR spectrum of KP1212 under the same solvent condition as FTIR at 20 °C.

Fig. 6.

A mechanistic model explaining the mutagenesis of KP1212 by the distinct base-pairing preferences of its different tautomers. KP1212 tautomers are paired with purine bases (G or A), by maximizing the number of possible hydrogen bonds between the bases. Tautomers 1, 2Z, 4, and 5E have a canonical Watson–Crick face and therefore are proposed to pair exclusively with either G or A. The remaining tautomers are proposed to pair either in wobble position (2E, 3, and 5Z) or involving a syn-conformer of KP1212 (3 and 5Z).