Identification of the antimetabolite of L-alanosine, L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide, in tumors and assessment of its inhibition of adenylosuccinate synthetase

Abstract

The conjugate of L-alanosine [L-2-amino-3-(N-hydroxyN-nitrosamino)propionic acid] and 5-amino-4-imidazolecarboxylic acid ribonucleotide has been synthesized in good yield by enzymatic means, using partially purified chicken liver 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (EC 6.3.2.6). The chromatographic behavior of this molecule was characterized, as was its ability to inhibit adenylosuccinate synthetase, an enzyme long considered to be the locus of action of the drug. The K_i of-L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide versus a partially purified adenylosuccinate synthetase from the L5178Y/AR leukemia of C57BL × DBA/2 F₁ (hereafter called BD2F₁) mice was 0.228 μm, whereas the K_i of L-alanosine was 57.23 mm. Administration of 50 μCi of dl-[1-¹⁴C]alanosine along with unlabeled L-alanosine (500 mg/kg) to BD2F1 mice bearing s.c. nodules of Leukemia L5178Y/AR resulted in the accumulation in tumors of a material with properties compatible with those of L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide. It coeluted with L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide in the high-resolution chromatographic system used, was Bratton-Marshall positive, and inhibited adenylosuccinate synthetase strongly. In tumor nodules 2 hr after dosage, the concentration of this compound approximated 70 μm. Under the same circumstances, the intratumoral concentration of L-alanosine was found to be 440 μm. At this concentration, the antibiotic itself exerts only a marginal inhibition of leukemic adenylosuccinate synthetase. In ancillary studies, it was shown for the first time in vivo that the parenteral administration of L-alanosine reduces the specific activity of intratumoral adenylosuccinate synthetase by 70% and depresses the synthesis of DNA to an equivalent or greater extent; adenine but not hypoxanthine (both at 250 mg/kg) was able to reverse the latter inhibition. No effect on purine salvage enzymes was exerted by L-alanosine. Viewed in concert, these experiments establish that the adduct of L-alanosine with 5-amino-4-imidazolecarboxylic acid is formed by neoplastic cells in vivo and that this anabolite is most probably responsible for the inhibition of adenylosuccinate synthetase and, in turn, for the diminished synthesis of DNA seen after a therapeutic dose of L-alanosine.