Synergistic Effects with Inhibitors of De Novo Pyrimidine Synthesis, Acivicin, and A/-(Phosphonacetyl)-I -Aspartic Acid1

Home , CTP synthetase

[CANCER RESEARCH 41, 3419-3423. September 1981] 0008-5472/81 /0041-OOOOS02.00 Synergistic Effects with Inhibitors of de Novo Pyrimidine Synthesis, Acivicin, and A/-(Phosphonacetyl)-i_-aspartic Acid1

Evan Loh and Donald W. Kufe2

Division of Medicine, Sidney Farber Cancer Institute. Harvard Medical School, Boston. Massachusetts 02115

ABSTRACT valions therefore suggest that other steps in nucleic acid pre cursor synthesis are important targets for Acivicin. The effects Acivicin, an inhibitor of L-glutamine-dependent amidotrans- of this agent on deoxyribonucleotide pools have not been ferases, is active against the murine L1210 and P388 leukemia studied and could yield greater insights into the mechanism of models. Cytidine triphosphate synthetase has been proposed action of Acivicin. as the primary target for this agent. Our results demonstrate The initial step in de novo pyrimidine biosynthesis is cata that Acivicin is also an inhibitor of de novo pyrimidine biosyn lyzed by carbamyl phosphate synthetase. A preparation of this thesis. This inhibition results in the depletion of pyrimidine enzyme from Escherichia coli is completely inhibited by 1 mvi deoxyribonucleoside triphosphate pools and explains the effect Acivicin (11). The inhibition of carbamyl phosphate synthetase of this agent on DMA synthesis. Further, Acivicin is synergistic limits the production of carbamyl phosphate which binds to with N-(phosphonacetyl)-L-aspartic acid, another inhibitor of aspartate transcarbamylase. PALA, a transition-state analog of de novo pyrimidine synthesis. The combination of these agents aspartate transcarbamylase, is a competitive inhibitor of car results in a more than additive depletion of deoxycytidine bamyl phosphate (4, 13, 17, 21 ) and therefore might be poten triphosphate pools which may account for their synergism in tiated by Acivicin if carbamyl phosphate synthetase is an im inhibiting cellular growth. Thus, the inhibition of de novo pyrim portant site of action of Acivicin. idine synthesis by Acivicin may be useful in modulating the In this study, we show that Acivicin inhibits de novo pyrimi effects of certain antimetabolites or other inhibitors of this dine biosynthesis, that Acivicin and PALA are synergistic for pathway. inhibitory effects of L1210 growth, and that this synergism is accompanied by a more than additive inhibitory effect on dCTP INTRODUCTION synthesis. Acivicin [L-(aS,5S)-a-amino-3-chloro-4,5-dihydro-5-isoxa- MATERIALS AND METHODS zoleacetic acid] is a fermentation product of Streptomyces sviceus (7, 16, 19). This agent has significant activity in the Chemicals and Drugs murine L1210 and P388 leukemia models (8-10). It is also effective against human breast and lung tumor xenografts in Nucleosides, nucleotides, and orotic acid were purchased from athymic "nude" mice (9, 10). Acivicin inhibits several enzymes Sigma Chemical Co. (St. Louis, Mo.). Acivicin (NSC 163501) was provided by The Upjohn Company (Kalamazoo, Mich.). PALA (NSC which catalyze the transfer of the amide group of L-glutamine 224131) was obtained from the National Cancer Institute. Pyrazofurin (11). The evidence for this mechanism of action is severalfold: was kindly supplied by Eli Lilly and Co. (Indianapolis, Ind.) and by Dr. (a) L1210 growth inhibition by Acivicin is antagonized by E. Cadman (Yale University, New Haven, Conn.). Labeled nucleotides adding L-glutamine to the medium (18); (b) CTP synthetase, a were purchased from Schwarz/Mann (Orangeburg, N. Y.). The Micro- glutamine-dependent amidotransferase, is strongly inhibited by coccus luteus DNA polymerase and the lyophilized noncovalent double- Acivicin (K, = 2.5x10~6 M) (19); and (c) the effects of Acivicin stranded copolymers of poly(deoxyadenylate-deoxythymidylate) and on L1210 ribonucleotide pools are consistent with inhibition of poly(deoxyinosinate-deoxycytidylate) were purchased from Miles-Pen- the glutamine-dependent enzyme, XMP3 aminase (19). tex Laboratories, Kankakee, III. Although Acivicin has been shown to inhibit several enzymes Cell Culture involved in both purine and pyrimidine de novo biosynthesis, L1210 cells were maintained as a suspension culture in Eagle's CTP synthetase has been proposed as a target for Acivicin in modified essential medium supplemented with 10% fetal calf serum in L1210 cells (18). This suggested mechanism is based on the a humidified 5% CO? atmosphere at 37Â°. Cell densities were deter partial reversal (approximately 50%) of the growth-inhibitory mined with a Model ZB-1 Coulter Counter (Coulter Electronics, Hialeah, effect of Acivicin on L1210 cells by exogenous cytidine and Fla.). deoxycytidine. However, guanosine and deoxyguanosine can also reverse Acivicin growth inhibition of L1210 cells by 30%, Measurement of Intracellular Orotate and Orotidine and the combination of all these nucleosides can only reverse Suspension cultures of L1210 cells were exposed to Acivicin, PALA, the inhibition by up to 60%. Furthermore, Acivicin has been pyrazofurin, and various combinations of these agents. Cells were shown to be an inhibitor of DNA synthesis (18). These obser- pelleted at 4Â° and extracted with 1.0 N perchloric acid. The acid- insoluble material was removed by centrifugation, and the acid-soluble ' This work was supported in part by NIH Grant CA-28488 extract was placed in a shaking water bath at 100Â°for 14 min. Orotate ' Recipient of American Cancer Society Junior Faculty Research Award. To and orotidine levels were determined by high-pressure liquid chroma- whom requests for reprints should be addressed, at Division of Medicine. Dana tography using a modification of a procedure developed by Moyer and 1610C. Sidney Farber Cancer Institute, 44 Binney Street. Boston. Mass. 02115. 1The abbreviations used are: XMP. xanthine monophosphate; PALA. W-(phos- Handschumacher (1 7). Chromatography was performed on a Model phonacetylK-aspartic acid; dNTP. deoxyribonucleoside triphosphate. 5000 liquid Chromatograph (Varian Associates, Walnut Creek, Calif.) Received February 5, 1981; accepted June 10, 1981. using a Micropak AX-10 aniÃ³n exchange column with 0.05 M sodium

SEPTEMBER 1981 3419

formate, pH 5.5, run at 1 ml/min. Peaks were identified at 254 nm by triphosphate; and 0 to 60 pmol appropriate limiting substrate. Reac retention times, coinjection with standard solutions, and 280/254 tions were incubated for 60 min for dCTP measurements and 30 min ratios. Free purines elute in less than 5 min, while the retention times for the dATP, dTTP, and dGTP measurements. The reactions were of orotidine and orotate are 15 and 18.7 min, respectively. Concentra terminated by first transferring tubes to an ice water bath and then tions were determined by measurement of peak heights which were adding 0.2 M sodium pyrophosphate, 25 /Â¿gcalfthymus DNA, and 20% found to be directly proportional to the amount injected over the range trichloroacetic acid. Samples were then filtered through Whatman GF/ of 0.5 to 5.0 nmol. C discs and washed with a 5% trichloroacetic acid/1% sodium pyrophosphate solution. The samples were dried and counted in a Beckman Determination of Ribonucleoside Triphosphates liquid scintillation system (Beckman Instruments, Inc., Fullerton, Calif.) L1210 cells were centrifuged at 400 x g for 8 min at 4Â°. The after adding 5 ml Hydrofluor (National Diagnostics, Somerville, N. J.). medium was removed, and the cell pellet was extracted with 1.0 N perchloric acid. The suspension was centrifuged at 4Â°to remove the RESULTS insoluble proteins. The supernatant was then neutralized with KOH, and the resulting Â«CIO* precipitate was removed by centrifugation. The accumulation of orotate and orotidine following pyrazo- The sample was stored at -20Â° and analyzed by high-pressure liquid chromatography within 2 weeks using a Micropak AX-10 aniÃ³n ex furin treatment can be used to measure inhibition of steps in de novo pyrimidine biosynthesis prior to orotidine 5'-mono- change column with 0.4 M sodium phosphate (J. T. Baker Chemical phosphate decarboxylase (17). L1210 cells (106) treated with Co., Phillipsburg, N. J.), pH 3.41, at 1.5 ml/min. In this system, the pyrazofurin (10~6 M) for 20 hr accumulated 800 pmol of oroti retention times were: CTP, 18 min; DTP, 22 min; ATP, 26 min; and GTP, 39 min. dine and 2.0 nmol of orotate (Chart 1A). The concomitant treatment of cells with pyrazofurin (10~6 M) and Acivicin at a Determination of dNTP Pools growth-inhibitory concentration (10~4 M) resulted in the loss of

Preparation of Cell Extracts. Control and drug-treated cells (2 x detectable amounts of orotate and orotidine (Chart 1ÃŸ).This 107) were immediately chilled, washed once with phosphate buffered inhibition of de novo pyrimidine biosynthesis by Acivicin is saline [CaCI2 (100 mg/liter)/KCI (200 mg/liter)/K2H2PO4 (200 mg/ concentration dependent (data not shown). liter)/MgSO4 (59.2 mg/liter)/NaCI (8000 mg/liter)/Na2H2PO4 (1150 Chart 2, left, illustrates the effect of Acivicin alone and in mg/liter)], and extracted with 1.0 N KCICU for 15 min on ice. The combination with PALA on the growth of L1210 cells. PALA, at samples were centrifuged at 500 x g for 10 min at 4Â°, and the a concentration of 4 x 10~6 M, resulted in little additional effect supernatants were adjusted to pH 7.5 with 10 N KOH. The KCICX when combined with Acivicin, while 2 x 10~" M PALA resulted precipitate was removed by centrifugation. The supernatants were stored at -20Â° and assayed for dNTP levels within 1 week. in nearly complete inhibition of growth. Chart 2, right, illustrates the converse experiment using PALA alone and in combination dNTP Assay. The assay of acid-soluble dNTP pools was based on with various concentrations of Acivicin. An isobologram anal the modification of the method of Solter and Handschumacher (20). ysis of these data (Chart 3) demonstrates a synergistic growth- The synthetic heteropolymers poly(deoxyadenylate-deoxythymidylate) and poly(deoxyinosinate-deoxycytidylate) were used as templates for inhibitory interaction between these agents with the resultant M. luteus DNA polymerase. The 140-fjl reactions included: 1 mw Tris- curve concave to the left of the dashed line (1). The open HCI (pH 7.4); 1 mw MgCI?; 1.5 Â¿igsynthetic template; 10 units M. circles in the isobologram were derived from the data shown in luteus DNA polymerase; 150 pmol appropriately labeled nucleoside Chart 2, while the closed circles represent data from other

0.01 A. PYRAZOFURIN

Chart 1. Effect of Acivicin on the accumu lation of orotidine and orotate in pyrazofurin- treated L1210 cells. A. pyrazofurin (10"e M); B, pyrazofurin (10 6 M) and Acivicin (10~4 M). Extracts of L1210 cells were analyzed for orotidine and orotate by high-pressure liquid Â¡0.005 chromatography following 20 hr of drug ex posure. The pyrazofurin-treated cells (106) ac cumulated 800 pmol and 2.0 nmol of orotidine and orotate, respectively.

10 20 30 40 10 Minutes

3420 CANCER RESEARCH VOL. 41

100 -

IO"' IO'0 io-Â« IO"* IO'4 IO'3 UT2

t Acivicin] M [PALA] M Chart 2. Effect of Acivicin and PALA on the growth of L1210 cells in culture. Left, L1210 cells exposed to varying concentrations of Acivicin alone (â€¢)and in combination with PALA at 4 x 10 6 M (â€¢).2 x 10 5 M (A), 4 x 1CT5 M (O), 1 x 10"* M O, and 2 x 10 '' M (A). Right, L1210 cells exposed to varying concentrations of PALA (â€¢),alone and in combination with Acivicin at 1 x 10~7 M (â€¢),2 x 1CT7 M (A), and 3 x 10~* M (O). The results are expressed as the inhibition of cellular growth following 20 hr of drug exposure as compared to control cells grown in the absence of drug.

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I 60 TJ o T Â° 40 o

â€¢ Â»

U PF PF PF PF -t- -t- -t- Acivicin PALA Acivicin Chart 3. Isobologram analysis of combinations of Acivicin and PALA on the inhibition of L1210 cellular growth. The open circles in the isobologram were + derived from Chart 2, while the closed circles represent data from other experi PALA ments performed at additional concentrations of each drug. Chart 4. Effect of Acivicin and PALA on the accumulation of orotidine and orotate in pyrazofurin-treated cells. L1210 cells were exposed to pyrazofurin (10~e M) for 20 hr alone and in combination with Acivicin (2 x 10"' M), PALA (3 experiments performed at additional concentrations of each x 10~5 M), or the combination of these 2 agents. The results are expressed as drug. percentage of control values (mean Â±S.D.) for 6 separate determinations. The The concentrations of Acivicin and PALA resulting in the mean orotidine and orotate levels for the Acivicin/PALA combination were greatest synergism were 2 x 10~7 M and 3 x 10~5 M, respec significantly different (p Â«0.01) from that obtained with either Acivicin or PALA alone. tively. The effect of these drug concentrations on the accu mulation of orotidine and orotate is illustrated in Chart 4. PALA pyrimidine pools with depletions of 25 to 35%, while ATP was exposure resulted in a 48% reduction in accumulation of oro depleted by 20 to 30% and GTP by 7%. A combination of tidine and orotate, while Acivicin alone resulted in approxi Acivicin and PALA was less than additive for each of the mately a 30% reduction. The combination of these drugs ribonucleotide pools except GTP, which was depleted by 65%. inhibited total orotidine and orotate accumulation by 63%. These effects are illustrated in Chart 5. The effects of Acivicin alone and in combination with PALA Chart 6 illustrates the effects of Acivicin and PALA on de on de novo pyrimidine synthesis prompted studies monitoring oxyribonucleotide pools. Acivicin exposure resulted in the de perturbations in ribonucleotide and deoxyribonucleotide pools. pletion of each pool except dGTP which was expanded by over A 20-hr exposure to 2 x 10~7 M Acivicin reduced intracellular 1.5-fold. In contrast, PALA depleted pyrimidine deoxyribonu CTP pools by 25% and DTP pools by 5%. The purine ribonu cleotide pools, while it had no effect on dATP levels. PALA cleotide pools were decreased by 20 to 30%. PALA exposures exposure also resulted in expansion of the dGTP pool. The at 3 x 10~5 M resulted in more pronounced effects on the combination of these 2 agents was more than additive in

SEPTEMBER 1981 3421

effects of Acivicin on ribonucleotide pools are consistent with the inhibition of both CTP synthetase and another L-glutamine- 10080oe dependent enzyme, XMP aminase (19). These 2 enzymes have been proposed as the main sites of activity for Acivicin, based on the finding that cytosine and guanosine significantly reverse the growth-inhibitory activity of this drug. Acivicin, however, is an inhibitor of DMA synthesis (18), which suggests that the fin*20nâ€¢Ã›.010.D.11

â€¢-I-^iIT: phate decarboxylase. In contrast, simultaneous exposure of Acivicin PALA Acivicin cells to pyrazofurin and Acivicin limits this accumulation of + orotate and orotidine in a dose-dependent manner. Thus, ex PALA posure of L1210 cells to 10~4 M Acivicin completely inhibits Chart 5. Effect of Acivicin and PALA on ribonucleoside triphosphate levels in L1210 cells. L1210 cells were treated with Acivicin (2 x 10 ' M) and/or PALA synthesis of orotate and orotidine and confirms that Acivicin is (3 x 10~5 M) for 20 hr, followed by extraction for analysis of ribonucleoside an inhibitor of de novo pyrimidine biosynthesis. The initial step triphosphate pools by high-pressure liquid chromatography. The control values derived for 106 untreated cells were: CTP, 0.91 nmol; ATP, 11.0 nmol; DTP, 1.3 in de novo pyrimidine synthesis is catalyzed by the L-glutamine- nmol; and GTP, 0.42 nmol. The data are expressed as percentage of control dependent enzyme, carbamyl phosphate synthetase, which is value (mean Â±S.D.) for 6 separate determinations. The mean CTP, UTP, and the rate-limiting enzymatic step inhibited by this drug in bac GTP levels for the Acivicin/PALA combination were significantly different (p s terial systems (11). 0.01 ) from that obtained with either Acivicin or PALA alone. The mean ATP levels for the combination and each drug alone were not significantly different. The effect of Acivicin on de novo pyrimidine synthesis sug gested that this agent might interact with other inhibitors of this pathway. Perturbations in carbamyl phosphate levels have 2001600Â£ been shown to modulate PALA inhibition of aspartate transcarbamylase (3, 12, 14). PALA inhibits aspartate transcarbamylase by competing with carbamyl phosphate for binding to this enzyme. Thus, if the production of carbamyl phosphate was limited by inhibition of carbamyl phosphate synthetase, there 1200O should be less competition for the binding of PALA to the active site of aspartate transcarbamylase and a greater inhibition of

* 80400toifcCI&$I;LOT>IiITHIâ€”-rIIÃ¬t:â€¢:;i;::=-: this enzyme. Our isobologram studies demonstrate that Acivi cin and PALA are synergistic in terms of their growth-inhibitory effects. Furthermore, Acivicin and PALA, in combination, inhibit de novo pyrimidine synthesis more than would be expected -; with either drug alone. These observations suggest that Acivi cin is acting at the level of carbamyl phosphate synthetase. Acivicin PALA Acivicin The effect of Acivicin on de novo pyrimidine synthesis was + not particularly reflected by perturbations in ribonucleotide PALA pools. Significant declines were observed in CTP, ATP, and Chart 6. Effect of Acivicin and PALA on deoxyribonucleoside triphosphate levels in cultured L1210 cells. L1210 cells were exposed to Acivicin (2 x 10"' GTP pools, while little, if any, effect was observed for UTP M) and/or PALA (3 x 10~5 M), followed by extraction for analysis of deoxyribo- pools. Previous studies exposing L1210 cells to 5 rtiM Acivicin nucleoside triphosphates by the DMA polymerase assay. Control values for 106 for 4 hr resulted in decreases in CTP and GTP pools and cells were: dCTP. 42 pmol; dTTP, 40 pmol; dATP, 44 pmol; and dGTP. 27 pmol. Results are expressed as percentage of control value (mean Â± S.D.) for 6 increases in UTP and ATP pools (19). The differences between separate determinations. The mean dCTP, dTTP, and dGTP levels for the Acivi results may simply reflect the use of varying drug concentra cin/PALA combination were significantly different (p -s.0.01) from that obtained tions and durations of exposure. Our findings are also consist with either Acivicin or PALA alone. The mean dATP levels for the combination and each drug alone were not significantly different. ent with inhibition of CTP synthetase and XMP aminase, as well as an earlier step in de novo purine synthesis. The effect of Acivicin on deoxyribonucleotide pools was, in contrast, more depleting the dCTP pool and approximately additive in deplet consistent with inhibition of de novo pyrimidine synthesis with ing the dTTP pool. No significant effect was observed for dATP declines in both dCTP and dTTP pools. The dATP pools were levels, and the combination of Acivicin and PALA resulted in also diminished; however, there was expansion of dGTP pools, further expansion of the dGTP pool. and this may reflect lack of utilization. DISCUSSION The effects of PALA on pyrimidine ribonucleotide pools in this study are consistent with those of other investigators and Acivicin has been shown to be a potent inhibitor of CTP are reflected by depletions of CTP and UTP. Similar effects synthetase, as well as other enzymes which catalyze the trans were observed for the deoxyribonucleotides with reductions in fer of the amide group of L-glutamine (11, 19). Further, the dCTP and dTTP. The combination of Acivicin and PALA re-

3422 CANCER RESEARCH VOL 41

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1981 American Association for Cancer Research. Synergism of Acivicin and PALA suited in approximately additive effects in reducing the intra- 5. Grindey. G. B.. and Nichol, C. A. Interaction of drugs inhibiting different steps in the synthesis of DMA. Cancer Res., 32. 527-531, 1972. cellular levels of the pyrimidine ribonucleotides and dTTP, 6. Handschumacher, R. E. Formal discussion: some enzymatic considerations while there was a more than additive depletion of dCTP pools. in combination chemotherapy. Cancer Res., 75. 1541-1543, 1965. The depletion of purine ribonucleotide pools by PALA is in 7. Hanka, L. J., and Dietz, A. U-42,126, a new antimetabolite antibiotic: production, biological activity, and taxonomy of the producing microorgan contrast to previous findings (14, 17). This decrease was also ism. Antimicrob. Agents Chemother., 3: 425-431, 1973. reflected in our studies with the purine deoxyribonucleotide 8. Hanka, L. J., Martin, D. G., and Neil, G. L. U-42,126 (NSC-163501): anew pools which were either unaffected (dATP) or expanded antitumor antimetabolite. Proc. Am. Assoc. Cancer Res., 14: 66, 1973. 9. Houchens, D. P., Ovejera, A. A., Johnson, R. K., Bogden, A. E., and Neil, G. (dGTP). The reason for this discrepancy is unclear. L. Therapy of mouse tumors and human tumor xenografts by the antitumor The kinetics of sequential blockade has been used to explain antibiotic AT-125 (NSC-163501). Proc. Am. Assoc. Cancer Res., ÃŒ9:40, the interactions of drug combinations inhibiting separate en 1978. 10. Houchens, D. P., Ovejera, A. A., Sheridan, M. A.. Johnson, R. K., Bogden. zymes along a synthetic pathway (5, 6). The combination of A. E., and Neil, G. L. Therapy for mouse tumors and human tumor xenografts sequential inhibitors on the pathway for folate synthesis results with the antitumor antibiotic AT-125. Cancer Treat. Rep., 63. 473-476, 1979. in a synergistic interaction (2). Similarly, Acivicin and PALA 1-1. Jayaram, H. M., Cooney, D. A., Ryan, J. A., Neil, G. L., Dion, R. L., and each inhibit de novo pyrimidine biosynthesis and are synergis Bono, V. H. u-(aS,5S)-a-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid tic in their growth-inhibitory effects. This could be explained by (NSC-163501 ): a new amino acid antibiotic with the properties of an antag onist of L-glutamine. Cancer Chemother. Rep., 59: 481 -491, 1975. the more than additive effects of the combination on decreasing 12. Johnson, R. K., Inouye, T., Goldin, A., and Stark, G. R. Antitumor activity of dCTP pools. W-(phosphonacetyl)-L-aspartic acid, a transition-state inhibitor of aspartate Although PALA has significant antitumor activity in animal transcarbamylase. Cancer Res., 36. 2720-2725, 1976. 13. Johnson, R. K., Swyryd, E. A., and Stark, G. R. Effects of A/-(phosphon- tumor models, this agent has been ineffective in the treatment acetyl)-L-aspartate on murine tumors and normal tissues in vivo and in vitro of human cancer. Elevated levels of aspartate transcarbamyl- and the relationship of sensitivity of rate of proliferation and level of aspartate transcarbamylase. Cancer Res., 38. 371-378, 1978. ase or carbamyl phosphate in human tumors could explain 14. Kensler, T. W., Mutter. G., Hankerson, J. G., Reck, L. J., Harley, C., Han, the lack of effectiveness of this agent. Our results are consist N., Ardalan, B., Cysyk, R. L., Johnson, R. K., Jayaram, H. N., and Cooney, ent with the findings that the combination of Acivicin and PALA D. A. Mechanism of resistance of variants of the Lewis lung carcinoma to fV- is effective in the treatment of PALA-resistant tumors (15). (phosphonacetyl)-L-aspartic acid. Cancer Res., 41: 894-904, 1981. 15. Kensler, T. W.. Reck, L. J.. and Cooney, D. A. Therapeutic effects of Acivicin Finally, the results suggest that Acivicin might be a suitable and N-(phosphonacetyl)-L-aspartic acid in a biochemically designed trial candidate for other combination studies with agents such as 1- against a fV-(phosphonacetyl)-L-aspartic acid-resistant variant of the Lewis lung carcinoma. Cancer Res., 41: 905-909, 1981. /8-D-arabinofuranosylcytosine or 5-azacytidine. 16. Martin, D. G.. Duchamp, D. J., and Chidester, C. G. Isolation, structure, and absolute configuration of U-42,126. A novel antitumor antibiotic. Tetrahed ACKNOWLEDGMENTS ron Lett., 27. 2549-2552, 1973. The authors appreciate the helpful suggestions of Dr. R. Johnson and Dr. R. 17. Moyer, J. D., and Handschumacher, R. E. Selective inhibition of pyrimidine synthesis and depletion of nucleotide pools by N-(phosphonacetyl)-L-aspar- E. Handschumacher. The excellent secretarial assistance of Carolyn Klebart is tate. Cancer Res., 39: 3089-3094, 1979. also gratefully acknowledged. 18. Neil, G. L.. Berger, A. E., Bhuyan, B. K., Blowers, C. L., and Kuentzel, S. L. Studies on the biochemical pharmacology of the fermentation-derived anti- REFERENCES tumor agent (5-isoxazoleacetic acid, a-amino-3-chloro-4,5-dihydro) (AT- 125). Adv. Enzyme Regul., / 7: 375-398, 1979. 1. Berenbaum. M. C. Synergy, additivism, and antagonism in immunosuppres- 19. Neil, G. L., Berger, A. E., McPartland, R. P., Grindey, G. B., and Bloch, A. sion: a critical review. Clin. Exp. Immunol.. 28. 1-18. 1977. Biochemical and pharmacological effects of the fermentation-derived anti- 2. Bushby, S. R. M. Combined antibacterial action in vitro of trimethoprim and tumor agent, (aS,5S)-a-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid sulfonamides: the in vitro nature of synergy. Postgrad. Med. J., 45: 10-18. (AT-125). Cancer Res., 39: 852-856. 1979. 1969. 20. Solter, A. W., and Handschumacher. R. E. A rapid quantitative determination 3. Christopherson, R. I., and Jones, M. E. The overall synthesis of L-5,6- of deoxynucleoside triphosphates based on the enzymatic synthesis of DNA. dihydroorotate by multienzyme protein pyr1-3 from hamster cells. J. Biol. Biochim. Biophys. Acta, Ã•74:585-590, 1969. Chem., 255. 11381-11395, 1980. 21. Swyryd, E. A., Seaver, S. S., and Stark, G. R. AMPhosphonacetylH-aspar- 4. Collins, K. D., and Stark, G. R. Aspartate transcarbamylase. Interaction with tate, a potent transition state analog inhibitor of aspartate transcarbamylase, the transition state analog W-{phosphonacetyl)-L-aspartate. J. Biol. Chem., blocks proliferation of mammalian cells in culture. J. Biol. Chem., 249: 246. 6599-6605, 1971. 6945-6950, 1974.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1981 American Association for Cancer Research. Synergistic Effects with Inhibitors of de Novo Pyrimidine Synthesis, Acivicin, and N-(Phosphonacetyl)-l-aspartic Acid

Evan Loh and Donald W. Kufe

Cancer Res 1981;41:3419-3423.

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