We examined the cytotoxicity, biochemical effects and metabolism of 4-memethoxy-8-(β-d-ribofuranosylamino)pyrimido[5,4-d] pyrimidine (MRPP), a synthetic nucleoside inhibitor of phosphoribosylpyrophosphate synthetase, in HCT 116 human colorectal cancer cells. A 4-hr exposure to 1 and 10 μM MRPP inhibited cell growth over a 72-hr period by 76 and 89%, and inhibited clonogenic capacity by 36 and 65%, respectively. MRPP was avidly metabolized to the 5′-monophosphate derivative (MRPP-MP), and MRPP-MP formation increased with increasing MRPP exposure (μM·hr). MRPP-MP was stable, and the intracellular half-life was in excess of 48 hr. A 4-hr exposure to 10 μM MRPP resulted in significant decreases in ATP, UTP, GTP, CTP, dATP, dTTP, and PRPP pools. Near maximal ribonucleotide triphosphate depletion was achieved with ≥24 μM·hr MRPP, and growth inhibition as a function of MRPP μM·hr closely reflected the biochemical effects. Ribonucleotide triphosphate pools remained depleted for up to 48 hr after drug removal, apparently as a consequence of the prolonged retention of MRPP-MP. MRPP (10 μM) inhibited the salvage of [3H]guanine, [3H]-adenine and [3H]guanosine, and concurrent exposure to MRPP and either 100 μM adenine, hypoxanthine, or guanine did not reverse ATP or GTP depletion. Concurrent exposure to 10 μM MRPP and either 10 μM adenosine, uridine or thymidine was accompanied by repletion of ATP, UTP, and dTTP pools, respectively, but depletion of other nucleotide pools was not corrected. In contrast, 10 μM guanosine did not correct GTP depletion in the presence of MRPP. The combination of 10 μM each of thymidine, uridine, adenosine and guanosine during and following a 24-hr exposure to MRPP provided partial protection against 0.1 or 1 μM MRPP, but did not affect the cytotoxicity associated with 10 μM MRPP. MRPP is a novel antimetabolite that inhibits both de novo and salvage pathways for purine synthesis and de novo pyrimidine synthesis.
- experimental therapeutics
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