[CANCER RESEARCH 40, 4390-4397, December 1980] 0008-5472/80/0040-OOOOS02.00 Identification of the Antimetabolite of L-Alanosine, i_-Alanosyl-5-Amino-4- Imidazolecarboxylic Acid Ribonucleotide, in Tumors and Assessment of Its Inhibition of Adenylosuccinate Synthetase Anil K. Tyagi and David A. Cooney Biochemistry Section. Laboratory of Toxicology. Division of Cancer Treatment. National Cancer Institute. NIH. Bethesda. Maryland 20205 ABSTRACT The conjugate of L-alanosine [L-2-amino-3-(/V-hydroxy/V-ni- trosamino)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-A/-succinocarboxamide ribonucleotide synthetase (EC 6.3.2.6). The Chromatographie 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¡of-L-alanosyl-5-amino-4-imidazole- carboxylic acid ribonucleotide versus a partially purified ade nylosuccinate synthetase from the L5178Y/AR leukemia of C57BL x DBA/2 F, (hereafter called BD2F,) mice was 0.228 /IM, whereas the K, of L-alanosine was 57.23 mw. Administra tion of 50 ¡iC\of DL-{1-t'tC]alanosine along with unlabeled L- alanosine (500 mg/kg) to BD2Ft 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-alano- syl-5-amino-4-imidazolecarboxylic acid ribonucleotide. It coe- luted with L-alanosyl-5-amino-4-imidazolecarboxylic acid ri bonucleotide in the high-resolution Chromatographie system used, was Bratton-Marshall positive, and inhibited adenylosuc cinate synthetase strongly. In tumor nodules 2 hr after dosage, the concentration of this compound approximated 70 /¿M. Un der the same circumstances, the intratumoral concentration of L-alanosine was found to be 440 fiM. 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 adenylo succinate synthetase by 70% and depresses the synthesis of DNA to an equivalent or greater extent; adenine but not hypo- xanthine (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 adduci of L-alanosine with 5-amino-4-imidazolecarbox- ylic 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. INTRODUCTION Although it has been postulated repeatedly that the active metabolite of antitumor agent L-alanosine [L-2-amino-3-(A/-hy- droxy, A/-nitrosamino)propionic acid] is the adduci of the anli- biolic wilh AICOR1 (Chart 1), allempls to demonstrate this molecule in tumors have, thus far, met with failure (10, 19). However, because its identificalion is cenlral lo any explanalion of Ihe mechanism of aclion of L-alanosine, we have undertaken addilional, more comprehensive studies on the in vivo formation of L-alanosyl-AICOR. Leukemia L5178Y/AR, growing as s.c. nodules in the flanks of C57BL x DBA/2 F, (hereafter called BD2Fi) mice, has been used for these investigations because of its clear sensitivily lo L-alanosine. It will be demonstraled lhat Ihe concentration of unaltered L- alanosine in such tumors is inadequate lo explain Ihe biochem ical effecls of the drug, and that, under the experimental conditions used, a compound wilh chromalographic and chro- mogenic properties of the pulalive adduci of L-alanosine wilh AICOR is, in fact, present in the neoplasms at a concentration which ought to engender lotal inhibition of the target enzyme, adenylosuccinate synthetase. Ancillary experiments will also be described which fortify this conclusion and quantify the exlenl to which L-alanosine, after its anabolism, inhibits the formation of adenylosuccinic acid in vivo, thereby restricling the concentralion of adenine nucleolides required for the syn thesis of DNA in the actively dividing cells of this leukemia. MATERIALS AND METHODS L-Alanoslne (NSC 153353) was obtained from the Drug Research and Development Branch, National Cancer Institute, Belhesda, Md. DL-[1-14C]Alanosine (specific activity, 7.1 mCi/ mmol) was supplied by Stanford Research Institute, Menlo Park, Calif. This radioactive alanosine was purified before use as described earlier (1 ). L-[4-14C]Aspartic acid (specific aclivily, 19.5 mCi/mmol) was oblained from Amersham/Searle Corp., Arlingfon Heighls, III. Adenosine, hypoxanlhine, ribose 1-phos- phale, GTP, and IMP were purchased from Sigma Chemical Co., St. Louis, Mo. All other reagenls were of Ihe highesl purily available. Scalable polypropylene test tubes (1600 /il), producÃ-s of Brinkmann Inslrumenls, Inc., Weslbury, N. Y., were used for all enzyme assays (6). Animals. Male BD2F, mice on an ad libitum diel of Purina mouse chow were used during the course of these studies. Preparation of Homogenates. BD2F, mice bearing 10-day- old L5178Y/AR s.c. tumors were killed by cervical dislocalion; Received March 10. 1980; accepted June 3, 1980. ' The abbreviations used are: AICOR, 5-amino-4-imidazolecarboxylic acid ribonucleotide; L-alanosyl-AICOR, L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide; IMP. inosine 5'-monophosphate; DTT, dithiothreitol; TCA.trichloroacetic acid; PRPP, 5-phosphorylribose 1-pyrophosphate; HEPES, 4-{2-hydroxyethyl)-1-piperazineethanesulfonic acid; SAICAR, 5-amino-4-imidaz- ole-A/-succinocarboxamide ribonucleotide. 4390 CANCER RESEARCH VOL. 40 Research. on December 14, 2021. © 1980 American Association for Cancer cancerres.aacrjournals.org Downloaded from