The aminopyrimidopyrimidine
nucleoside 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]
pyrimidine (APP), which was previously shown to possess experimental antitumor and
antiviral activity, was metabolized within WI-L2 human lymphoblastoid cells to a derivative identified as the beta-D-
ribonucleotide (
APP-MP). In a subline of WI-L2 cells deficient in
adenosine kinase, this metabolite was not formed and APP was not cytotoxic, suggesting that APP is converted by
adenosine kinase to its 5'-monophosphate. Because no evidence of di- or triphosphates was seen, the monophosphate appeared to be the active species. Treatment of WI-L2 or L1210 cells with APP (10 microM) for 30 min caused extensive depletion of both
purine and
pyrimidine ribonucleotides.
Purine and
pyrimidine deoxyribonucleotides were also depleted. Cells were not protected from the cytotoxicity of APP by
hypoxanthine plus
uridine, but
uridine plus
adenosine plus 2-deoxycoformycin gave considerable protection. This result was consistent with
APP-MP acting as an inhibitor of 5-phosphoribosyl-1-pyrophosphate (
PRPP) synthetase, a hypothesis that was confirmed by preparing
PRPP synthetase from
Novikoff hepatoma cells;
APP-MP was a noncompetitive inhibitor, with a Ki of 0.43 mM.
APP-MP was found to accumulate in APP-treated cells to a concentration of almost 3 mM. The relevance of
PRPP synthetase inhibition to the cytotoxic mechanism of APP is indicated by the fact that depletion of the PRPP pool was seen as early as 15 min
after treatment, before any change was apparent in cellular levels of
ATP or
UTP.
DNA synthesis was markedly suppressed within 30 min of APP treatment of WI-L2 cells, and a lesser degree of inhibition of
RNA synthesis was apparent after 45 min.