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.