[CANCER RESEARCH 33, 2867 2871, November 1973] Inhibition of Furine Nucleotide Metabolism by 6-Methylthiopurine Ribonucleoside and Structurally Related Compounds1 Geraldine D. Shantz,2 Camilla M. Smith, Lydia J. Fontanelle, Herbert K. F. Lau/ and J. Frank Henderson5 University of Alberta Cancer Research Unit (McEachern Laboratory) and Department of Biochemistry. Edmonton T6G 2E1, Alberta, Canada SUMMARY lich ascites tumor cells has been studied. In addition, biochemical effects of a number of other methylthiopurine 6-Methylthiopurine ribonucleoside was found to inhibit derivatives and analogs are also reported. nucleotide formation from hypoxanthine and aminoimida- zole carboxamide in Ehrlich ascites tumor cells in vitro and to inhibit inosinate dehydrogenase activity in intact cells. MATERIALS AND METHODS These effects are produced at higher drug concentrations 6MeMPR-3H (2564 mCi/mmole) was purchased from than required to inhibit purine biosynthesis de novo. New England Nuclear Corp. (Dorval, Quebec, Canada), 4-Methylthio-7-|8-D-ribofuranosyl pyrrolo[2,3-i/]pyrimi- AIC-2-14C (24.2 mCi/mmole) was from Calbiochem (Los dine also inhibited nucleotide formation and inosinate Angeles, Calif.), and 6MeMPR was from Sigma Chemical dehydrogenase activity, and several other methylthio purine Co. (St. Louis, Mo.). Fischer's medium, RPMI Medium ribonucleoside analogs or derivatives also inhibited one or 1620, and fetal calf serum were obtained from Grand Island another of these processes. Biological Co. (Berkeley, Calif.). Sources of most other materials used have been reported previously (6). INTRODUCTION The following compounds were provided by the Drug Evaluation Branch, Drug Research and Development, Na Before a specific biochemical effect of a drug can be tional Cancer Institute: 9-ethyl-6-(methylthio)purine (NSC identified as its mechanism of growth inhibition, as many 14576), 6-methylthio-9-phenyl purine (NSC 26294), 6- areas of metabolism as possible should be studied in order methylthio-9-(tetrahydro-2-pyranyl)purine (NSC 33184), 2- to detect drug effects which might previously have gone amino-9-benzyl-6-(methylthio)purine (NSC 42380), 6- unrecognized. Thus the best known and most thoroughly methylthio-9-(tetrahydro-2-furyl)purine (NSC 44579), 9-(2- studied biochemical effect of 6MeMPR6 ribonucleoside is hydroxyethyl)-6-(methylthio)purine (NSC 55464), 6-meth- inhibition of PP-ribose-P amidotransferase (EC 2.4.2.14) ylthio-9-(tetrahydro-2-thienyl)purine (NSC 56937), 6-meth- (10, 14, 21), the 1st enzyme of the pathway of purine ylthio-9-^-D-xylofuranosyl purine (NSC 95103), 4-meth- biosynthesis de novo. Although evidence has been presented ylthio-l-0-n-ribofuranosyl imidazol[4,5-c]pyridine (NSC that supports the view that inhibition of this pathway is the 95965), 4-methylthio-7-/3-D-ribofuranosyl pyrrolo[2,3-</]- basis of the growth inhibitory activity of this drug (1, 20), pyrimidine (NSC 105826), 6-methylthio-9-(6-[3,5-dihy- few studies of possible effects of 6MeMPR on other droxy-2-hydroxymethyl]dioxane)purine (NSC 111702), pathways of purine metabolism have been conducted. The 5-bromo-4-(methylthio)-7-j8-i)-ribofuranosyl pyrrolo[2,3- report of Hill (13) that 6MeMPR inhibits the incorporation ¿Ipyrimidine (NSC 113942), 4-methylthio-7-/3-r>-ribo- of radioactive adenine, guanine, and hypoxanthine into furanosyl pyrrolo[2,3-d]pyrimidine-5-carbonitrile (NSC nucleotides and nucleic acids in Adenocarcinoma 755 cells, 116098), 6-methylthio-7-|8-[)-ribofuranosyl purine (NSC however, suggests the need for further studies of the 122326), 7-methylthio-3-/8-D-ribofuranosyl pyrazolo[4,3- biochemical effects of this purine antimetabolite. In this i/]pyrimidine (NSC 124166), and 6-methylthio-9-(2-amino- paper the possibility that 6MeMPR affects the reactions of 2-deoxy-0-D-ribofuranosyl)punne (NSC 131605). purine ribonucleotide synthesis and interconversion in Ehr- Methods for the propagation and incubation of Ehrlich ' This work was supported by the National Cancer Institute of Canada. ascites tumor cells have been reported (6). Human leukemia "Present address: Department of Microbiology, Hershey Medical RPMI 6410 cells (obtained from Associated Biomedic Center, Hershey, Pa. 'Present address: Department of Pharmacology, Idaho State Univer Systems, Buffalo, N. Y.) were grown in RPMI Medium sity, Pocatello, Idaho. 1620 containing 10% fetal calf serum, 100 ¿jgstreptomycin 4 Present address: Department of Biochemistry, State University of per ml, and 100 units penicillin per ml. New York, Buffalo, N. Y. Procedures for separation and measurement of radioac "To whom requests for reprints should be addressed. "The abbreviations used are: 6MeMPR, 6-methylthiopurine ribonu tivity in purine bases, ribonucleosides, and ribonucleotides cleoside: PP-ribose-P, phosphoribosyl pyrophosphate; A 1C, aminoimida- following incubation of cells with radioactive purines have zole carboxamide. RPMI, Roswell Park Memorial Institute. been described (6, 22). Results presented are measurements Received March 5, 1973; accepted July 27, 1973. of the amounts of radioactivity in each metabolite, ex- NOVEMBER 1973 2867 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1973 American Association for Cancer Research. Shantz, Smith, Fontenelle, Lau, and Henderson Table 2 pressed in nmoles/g, wet weight of cells, rather than the Effect ofbMeMPR on inosinate dehydrogenase activity total amount of each metabolite. Average values from Ehrlich ascites tumor cells were incubated as described in Table I with 50 MM hypoxanthine-14C, 50 uu adenine-"C, or 50 /ÕM samples in 2 experiments are reported. AIC-"C. Radioactive metabolites (nmoles/g) RESULTS Xanthosine Furine Ribonucleotide Synthesis. Table 1 shows the monophos effects of a range of concentrations of 6MeMPR on phate nucleotide synthesis from radioactive adenine, guanine, GMP + Inosine hypoxanthine, and AIC in Ehrlich ascites tumor cells 6MeMPR GDP + Xantho- + hypo- Hypo- Precursor (MM) GTP sine xanthine xanthine incubated in Fischer's medium buffered with phosphate. It t may be seen that 6MeMPR inhibited nucleotide formation HypoxanthineAdenineAIC02050100100002050[0002050100350356338282176113110997.969.819718514656.324.122.017.211.875.666.161.250.014517017625717.324.375.9123264267294352 from hypoxanthine-I4C by 37% at 1000 MMand from AIC by 32% at 100 MM.Little or no effect of 6MeMPR was observed when radioactive adenine and guanine were used as precursors. Inosinate Dehydrogenase. Measurements of radioactivity in individual metabolites suggested that 6MeMPR also inhibits one of the reactions of purine ribonucleotide interconversion, inosinate dehydrogenase (EC 1.2.1.14). Data for radioactivity in products of this reaction (Xantho- sine monophosphate + GMP + GDP + GTP and xantho- sine) and products of IMP catabolism (hypoxanthine plus inosine or inosine only when hypoxanthine is used as pre cursor) are presented in Table 2. It may be seen that 6MeMPR inhibited the conversion of radioactive hypox 250 anthine, adenine, and AIC to Xanthosine monophosphate _ + GMP + GDP + GTP and to xanthosine, while causing o E an increase in the formation of hypoxanthine plus inosine or e inosine alone; concentrations of radioactive IMP were within the normal range (10 to 50 nmoles/g) under all of these conditions. The potency of 6MeMPR on inosinate I 150 dehydrogenase was greater than on total nucleotide syn Q- l/l thesis from hypoxanthine but varied with the precursor O I and medium used. No other effects of 6MeMPR on purine Q_ ribonucleotide synthesis, interconversion, and catabolism were noted. 6MeM PR-phosphate Concentration. The concentrations ee. 0. 50 of 6MeMPR required to inhibit inosinate dehydrogenase and nucleotide synthesis from hypoxanthine were higher than those required to inhibit purine biosynthesis de novo in 50 100 Table 1 Effect ofoMeMPR on purine ribonucleotide synthesis óMeMPR (/¿M) Ehrlich ascites tumor cells, 2% by volume, were incubated with various Chart 1. 6MeMPR-phosphate synthesis. Ehrlich ascites tumor cells concentrations of 6 MeMPR for 20 min at 37°with shaking in 0.1 ml of were incubated as described in Table l in Krebs-Ringer medium containing Fischer's medium containing 25 m.\i phosphate and an air atmosphere. 25 mMphosphate (D), Fischer's medium containing 25 mMphosphate (A), Hypoxanthine-"C (50 MM).final concentration; adenine-"C (50 ^M); or Fischer's medium containing 15 mM sodium bicarbonate (O). After 20 guanine-"C (50 /IM): or AIC-"C (50 MM)were then added and in min, 6MeMPR-3H was added and the incubation was continued for 30 cubation continued for 60 min. min. Each figure represents the mean of separate analyses of 4 flasks in 2 Total nucleotides (nmoles/g) at 6MeMPR experiments. concentration (/¿M)below cells (1, 3, 7, 10, 12, 17, 18), and experiments were Precursor 20 50 100 1000 conducted to measure the intracellular concentrations of Hypoxanthine 6MeMPR-phosphate (the presumed active drug metabolite) AdenineGuanineAIC1758218119041370175921592099109817672137199610861622201519679371104in cells incubated with a range of initial extracellular concentrations of 6MeMPR. These data are shown in Chart 1. It is apparent that nucleotide formation from 6MeMPR 2868 CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1973 American Association for Cancer Research. 6-MeMPR was the same in all 3 media used. It was also shown that 6MeMPR-phosphate concentration is virtually constant during the periods of measurement of purine metabolism used in the previous