The
pyrimidine acyclonucleoside benzyloxybenzyloxybenzylacyclouridine (
BBBAU) showed growth inhibitory activity against the human
colon cancer HCT-8 cell line with a 50% inhibitory concentration of 55 microM. Unlike its parent compounds,
BBBAU was an extremely weak inhibitor of
uridine phosphorylase. This acyclonucleoside analogue is an inhibitor of
thymidylate synthase (TS) as determined by inhibition of [6-3H]-2'-
deoxyuridine incorporation into
DNA, inhibition of 3H release from [5-3H]-2'-
deoxyuridine, and decrease in both the free and total TS 5'-fluoro-2'-deoxyuridine 5'-monophosphate binding sites. Kinetic analysis revealed that
BBBAUMP, the monophosphate analogue of
BBBAU, is a competitive inhibitor of purified human recombinant TS with a Ki of 8.0 microM.
Nucleoside transport and uptake studies revealed that
BBBAU (30 microM) inhibited the initial rate of transport and the total uptake of
thymidine (25 microM). In contrast, while
BBBAU (30 microM) inhibited the initial rate of transport of
5-fluoro-2'-deoxyuridine (FdUrd, 25 microM), its intracellular accumulation was increased.
BBBAU (10 and 50 microM, respectively) potentiated FdUrd growth inhibition of HCT-8 cells and significantly enhanced the cytotoxic effects of FdUrd (0.3 and 1 microM, respectively) against HCT-8 cells using a clonogenic assay system. This combination resulted in additive inhibitory effects on TS activity resulting in greater depletion of
dTTP pools. Moreover, the incorporation of radiolabeled FdUrd into the
DNA fraction of HCT-8 cells was enhanced. The potential importance of this novel combination for human
colon cancer chemotherapy is discussed.