INHIBITION OF 5-PHOSPHORIBOSYL-1-PYROPHOSPHATE SYNTHETASE BY THE MONOPHOSPHATE METABOLITE OF ETA-D-RIBOFURANOSYLAMINO)PYRIMIDO[5,4-D]PYRIMIDINE - A NOVEL MECHANISM FOR ANTITUMOR-ACTIVITY

The aminopyrimidopyrimidine nucleoside ta-D-ribofur-anosylamino)pyrimi do[5,4-d]pyrimidine (APP), which was previously shown to possess exper imental antitumor and antiviral activity, was metabolized within WI-L2 human lymphoblastoid cells to a derivative identified as the beta-D-r ibonucleotide (APP-MP). In a subline of WI-L2 cells deficient in adeno sine kinase, this metabolite was not formed and APP was not cytotoxic, suggesting that APP is converted by adenosine kinase to its 5'-monoph osphate. Because no evidence of di- or triphosphates was seen, the mon ophosphate appeared to be the active species. Treatment of WI-L2 or Ll 21 0 cells with APP (10 mum) for 30 min caused extensive depletion of both purine and pyrimidine ribonucleotides. Purine and pyrimidine deo xyribonucleotides were also depleted. Cells were not protected from th e cytotoxicity of APP by hypoxanthine plus uridine, but uridine plus a denosine plus 2-deoxycoformycin gave considerable protection. This res ult was consistent with APP-MP acting as an inhibitor of 5-phosphoribo syl-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 K(i) 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 s een as early as 15 min after treatment, before any change was apparent in cellular levels of ATP or UTP. DNA synthesis was markedly suppress ed within 30 min of APP treatment of WI-L2 cells, and a lesser degree of inhibition of RNA synthesis was apparent after 45 min.