Mammalian DNA Polymerases .alpha., .beta., .gamma., .delta., and .epsilon. Incorporate Fialuridine (FIAU) Monophosphate into DNA and Are Inhibited Competitively by FIAU Triphosphate

Eli Lilly, Indianapolis, Indiana, United States
Biochemistry (Impact Factor: 3.02). 01/1995; 33(48):14620-4. DOI: 10.1021/bi00252a030
Source: PubMed


Fialuridine [FIAU, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5- iodouridine] was used in clinical trials for chronic hepatitis B virus infection and was extremely toxic. Evidence suggested targets of FIAU toxicity included mitochondria, but toxic mechanisms were unclear. Since FIAU is a thymidine analog, we reasoned that triphosphorylated FIAU (FIAUTP) could be incorporated into mitochondrial DNA by DNA pol-gamma and into genomic DNA by DNA polymerases alpha, beta, delta, and epsilon. All five purified mammalian DNA polymerases incorporated FIAUMP into the nascent DNA chain during in vitro DNA synthesis. When FIAUTP was substituted for dTTP, oligonucleotide products were generated efficiently by DNA pol-gamma and were similar to those generated in the presence of the four normal dNTPs. In contrast, oligonucleotide products generated by the four nuclear DNA polymerases in the presence of FIAUTP were significantly reduced in length relative to those generated in the presence of dTTP. In parallel kinetic assays, FIAUTP competitively inhibited the accumulation of radiolabeled dTTP into DNA by DNA pol-gamma. The Ki with DNA pol-gamma was 0.04 microM, the lowest Ki among the mammalian DNA polymerases. Competition between FIAUTP and dTTP and the relative ease of accumulation of FIAUMP in mitochondrial DNA by DNA pol-gamma in vitro together may relate to clinical FIAU toxicity.

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    • "Most of the NRTI-induced mitochondrial toxicity results from the activity of the mtDNA polymerase g (pol g), which incorporates the nucleoside analogs into mtDNA, leading to chain termination and inhibition of mtDNA replication. Furthermore, pol g is unique among the cellular replicative DNA polymerases in that it is also highly sensitive to inhibition by NRTIs [Parker et al., 1991; Copeland et al., 1992; Hart et al., 1992; Huang et al., 1992; Nickel et al., 1992; Lewis et al., 1994; Martin et al., 1994; Eriksson et al., 1995; Johnson et al., 2001; Lim and Copeland, 2001]. Inhibition of the exonuclease activity of pol g is mediated at least in part by AZT-monophosphate at concentrations known to occur within cells [Lim and Copeland , 2001]. "
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    • " The study of the interaction between dideoxynucleotides and human DNA polymerases shows no affinity of these nucleo - tides for the a or the e species , a low but detectable in - teraction with the b - polymerase ( Ono et al . , 1989 ; Yarchoan et al . , 1989 ) and a significant inhibitory activity against g - polymerase ( Brinkman et al . 1998 ; Lewis et al . , 1994 ; Lewis Table 1 Current NRTI regimen therapies to reduce mother - to - child transmission"
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    ABSTRACT: In the last 10 years, zidovudine (AZT) has become the main prophylactic therapy against vertical HIV-1 transmission. AIDS Clinical Trials Group (ACTG) 076 have demonstrated that the administration of AZT to HIV-infected women during their third trimester of pregnancy, trough labor and given orally to babies for 6 weeks, reduced by two-thirds the rate of vertical infection. Although the rapid diffusion of this regimen into clinical practice together with the implementation of HIV counseling and testing practices have dramatically reduced the vertical transmission rate in the US and Western Europe, there is a growing concern on the adverse effects of antiretroviral therapy on the fetus and the newborn. In fact, even though shorter regimen therapies that are less complex and expensive to implement in poor countries have been demonstrated as effective as ACTG 076 regimen, the distribution of the risk of vertical transmission in the developing countries is still very high. Consequently, a large number of unborns will be a candidate to developmental exposure to antiretroviral agents. To date, data on the transplacental mutagenicity, carcinogenicity and mitochondrial dysfunction induced by developmental exposure to AZT have been reported in several animal models. Furthermore, one study reported severe yet few human cases of cardiomyopathy and neurological disease likely associated with mitochondrial dysfunction in uninfected infants of seropositive mothers perinatally exposed to AZT. For all of these reasons, many investigations have been focusing on the assessment of the potential adverse effects of nucleoside reverse transcriptase (RT) inhibitors (NRTI) administration during development. A survey of the main results derived from clinical and animal studies is reported here, focusing on those neurobehavioral studies that have been looking for specific and/or aspecific changes in the nervous system induced by NRTI exposure in utero.
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    • "This suggests that the NRTI monophosphate is incorporated into mtDNA. FIAU, FIAC (1-[2-deoxy-2-fluoro-␤-D- arabinofuranosyl]-5-iodocytosine), FMAU (1-[2-deoxy-2- fluoro-␤-D-arabinofuranosyl]-5-methyluracil), and FEAU (1- [2-deoxy-2-fluoro-␤-D-arabinofuranosyl]-5-ethyluracil) each demonstrated efficacy in viral disease models (Fourel et al, 1990), and many of their triphosphates inhibit mammalian DNA pol-␥ in vitro (Lewis et al, 1994a). With these agents, competition with the native nucleotide and NRTI at the nucleotide binding site of DNA pol-␥ appears to be a critical event. "

    Full-text · Article · Jul 2001 · Laboratory Investigation
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