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Fialuridine and its metabolites inhibit DNA polymerase gamma at sites of multiple adjacent analog incorporation,
W Lewis1, E S Levine, B Griniuviene
1Department of Pathology and Laboratory Medicine, College of Medicine, University of Cincinnati, OH 45267-0529, USA.
Abstract:
The thymidine analog fialuridine deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) was toxic in trials for chronic hepatitis B infection. One mechanism postulated that defective mtDNA replication was mediated through inhibition of DNA polymerase-gamma (DNA pol-gamma), by FIAU triphosphate (FIALTP) or by triphosphates of FIAU metabolites. Inhibition kinetics and primer-extension analyses determined biochemical mechanisms of FIAU, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) -5-methyluracil (FAU), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil triphosphate (TP) inhibition of DNA pol-gamma. dTMP incorporation by DNA pol-gamma was inhibited competitively by FIAUTP, FMAUTP, and FAUTP (K1=0.015, 0.03, and 1.0 microM, respectively). By using oliginucleotide template-primers. DNA pol-gamma incorporated each analog into DNA opposite a single adenosine efficiently without effects on DNA chain elongation. Incorporation of multiple adjacent analogs at positions of consecutive adenosines dramatically impaired chain elongation by DNA pol-gamma. Effects of FIAU, FMAU, and FAU on HepG2 cell mmtDNA abundance and ultrastructure were determined. After 14 days, mtDNA decreased by 30% with 20 microM FIAU or 20 microM FMAU and decreased less than 10% with 100 microM FAU. FIAU and FMAU disrupted mitochondria and caused accumulation of intracytoplasmic lipid droplets. Biochemical and cell biological findings suggest that FIAU and its metabolites inhibit mtDNA replication, most likely at positions of adenosine tracts, leading to decreased mtDNA and mitochondrial ultrastructural defects.
Insights
Fialuridine (FIAU) and its metabolites inhibit mitochondrial DNA replication by targeting DNA polymerase-gamma, particularly at adenosine tracts. This leads to reduced mtDNA levels and mitochondrial damage, explaining FIAU
Area of Science:
- Biochemistry
- Molecular Biology
- Hepatology
Background:
- Fialuridine (FIAU), a thymidine analog, exhibited toxicity in chronic hepatitis B trials.
- A proposed mechanism involved inhibition of mitochondrial DNA polymerase-gamma (DNA pol-gamma) by FIAU metabolites.
- Understanding the precise biochemical interactions is crucial for explaining FIAU's cellular toxicity.
Purpose of the Study:
- To elucidate the biochemical mechanisms by which FIAU, FMAU, and FAU inhibit DNA pol-gamma.
- To investigate the effects of these compounds on mitochondrial DNA (mtDNA) abundance and cellular ultrastructure.
Main Methods:
- Inhibition kinetics and primer-extension assays were used to study DNA pol-gamma inhibition.
- Oligonucleotide template-primers assessed analog incorporation and chain elongation.
- HepG2 cells were treated to evaluate mtDNA levels and mitochondrial morphology.
Main Results:
- FIAU triphosphate (FIATUP), FMAU triphosphate (FMAUTP), and FAU triphosphate (FAUTP) competitively inhibited dTMP incorporation by DNA pol-gamma.
- DNA pol-gamma efficiently incorporated single analogs opposite adenosine residues but chain elongation was impaired by multiple adjacent analogs.
- FIAU and FMAU significantly reduced mtDNA abundance by 30% and disrupted mitochondrial ultrastructure, unlike FAU.
Conclusions:
- FIAU and its metabolites inhibit mtDNA replication, likely by interfering with DNA pol-gamma at adenosine-rich regions.
- Impaired mtDNA replication leads to decreased mtDNA levels and mitochondrial damage, contributing to FIAU's observed toxicity.
- These findings provide a biochemical basis for the mitochondrial toxicity associated with FIAU and related compounds.