Inhibition of 5-phosphoribosyl-1-pyrophosphate synthetase by the monophosphate metabolite of 4-amino-8-(beta-D- ribofuranosylamino)pyrimido[5,4-d]pyrimidine: a novel mechanism for antitumor activity

DW Fry, TJ Boritzki, RC Jackson, PD Cook and WR Leopold

Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Co., Ann Arbor, Michigan 48105.

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.

Volume 44, Issue 2, pp. 479-485, 08/01/1993
Copyright © 1993 by American Society for Pharmacology and Experimental Therapeutics