Inhibition of Dihydroorotase in Pyrimidine Biosynthesis"

LLOYDH. SMITH,JR., MARGARETSULLIVAN,FAITHA. BAKER, ANDELIZABETHFREDERICK

(Department of Medicine and the Huntington Memorial Laboratorien, Massachusetts General Hospital and Harvard Medical School, Boston, Mays.)

SUMMARY Five structural analogs of carbamylaspartic acidâ€”carbamylglutamic acid, carbamylcysteic acid, carbaniylcysteinesulfinic acid, carbamylasparagine, and o-ureido- (J-methyl-succinic acidâ€”have been studied as inhibitors of dihydroorotase. The in hibitory activities of these analogs varied considerably in the three in vitro assay sys tems utilizedâ€”E. coli B extracts, rat liver homogenates, and human leukocyte soni cates. The inhibition observed was found to be competitive. Some of the biochemical characteristics of the conversion of carbamylaspartic acid to orotic acid in human leukocyte sonicates were studied. In normal circulating leukocytes this two-step con version was found to be of reproducible activity. The effectiveness of carbamylaspartic acid analogs as in vivoinhibitors of nucleic acid synthesis or as carcinostatic agents has vet to be evaluated.

In the search for agents of potential antineo- enzyme has added significance in that it seems to plastic usefulness, attention has always been di be the main focus of feedback control of pyrimidine rected toward inhibitors of nucleic acid biosynthe synthesis by product competitive inhibition (25). sis. Nucleic acids are major constituents of genes The roles of carbamylphosphate in the urea cycle and viruses, entities which are prominent in most and of aspartate in a wide variety of reactions dis conceptual schemes of neoplasia. This approach courage attempts to develop competitive inhibi has been validated by the temporary effectiveness tors of pyrimidine synthesis based on analogs of of certain antineoplastic compounds which seem these substrates. The product of aspartate car- to have as their mode of action an inhibition of bamyltransferase is carbamylaspartate, the first purine or pyrimidine nucleotide synthesis. This compound unique to pyrimidine synthesis. This limited success has been achieved despite the failure paper describes the synthesis and in vitro testing to demonstrate an exploitable qualitative differ of several simple analogs of carbamylaspartate, ence between normal and neoplastic nucleic acid which are shown to inhibit competitively its con metabolism (19). version to dihydroorotate. In the past relatively little attention has been directed to the development of agents to inhibit MATERIALS AND METHODS the earlier steps of pyrimidine synthesis, despite Syntheses.â€”The carbamyl derivatives of L-cys- the success of such an approach in the inhibition teic acid, L-asparagine, and L-glutamic acid were of purine synthesis. The first enzyme unique to prepared by condensation with cyanate essentially pyrimidine synthesis is aspartate carbamyltrans- as described by Nyc and Mitchell (10). Carbamyl- cysteicacidâ€”m.p. (uncorrected) = 167Â°-167.5Â°C. ferase, which catalyzes the irreversible carbamyla- (d.); calculated for CJIgOeX-A: C 22.63, H 3.77, tion of aspartate by carbamylphosphate (15). This N 13.20, S 15.13; found: C 22.85, H 3.87, N 13.12, * This study was supported by research grants A-1606 and S 14.69. Carbamylasparagineâ€”m.p., 158Â°-159Â°C.; A-1606 (C-2)from the Institute of Arthritis and Metabolic Dis reported 157Â°C. (12). Carbamylglutamic acidâ€” eases, National Institutes of Health, United States Public m.p., 158Â°-159Â°C;reported, 160Â°C.(2). L-Cysteine- Health Service. This is Publication Xo. 1001 of the Harvard Cancer Com sulfinic acid was made as described by Toenuies and mission. Lavine (22) with perbenzoic acid oxidation (21). Received for publication January la, 1960. Carbamvlcvsteinesulfinic acid was then formed as 1059

above, purified by gradient elution from a Dowex appearing in 20-26 column volumes. It was con 1-C1column with spot testing for the ureide group centrated from small Dowex-1-formate columns (11), and recrystallized from a small amount of and assayed in duplicate at 240 m/u as previously warm water; m.p., 99Â°-104Â°C.(d.); calculated for described (20). Duplicate aliquots were counted CiHgOsNÃ„H-Ã¼O:C 22.41, H 4.67, N 13.07, at infinite thinness on glass planchets with a Nu S 14.96; found: C 22.89, H 4.84, X 12.93, S 14.24. clear Chicago gas-flow counter with micromil DL-a-Ureido-j3-methylsuccinic acid was synthe window. From the specific activity of the isolated sized from 5-methylorotic acid, purchased from carrier the synthesis of DHO was calculated by the Nutritional Biochemical Corporation, by the standard carrier technic. catalytic hydrogÃ©nation with rhodium-alumina Rat liver homogenates (1:10 in 0.25 Msucrose) (4) followed by hydrolysis in 2 N KOH. Potassium or sonicates of isolated human leukocytes from was removed with a Doewex 50-H+ column, and patients with leukemia, isolated as previously de following vacuum distillation of the eluate the scribed (20), were used to study the conversion of product was recrystallized from water; m.p., 176Â°- CAA-C14to orotic acid (OA). The incubation was 177Â°C. (d.), calculated for CMioO^S^: C 37.89, carried out in a volume of 1.0 ml. for 1 hour at pH H 5.26, N 14.74; found: C 37.75, H 5.54, N 15.05. 7.4 (phosphate 0.3 M) with CAA-C14, IO"3 M, and ^Tiether this compound has a threo or erythro analog concentrations as noted. After the addition configuration is not known. In the calculations to of 5 AmÃ³lesOA as a carrier, the reaction was follow it has been assumed that only the L-form stopped with perchloric acid and the product is active as a competitive inhibitor. L-Carbamyl- isolated, purified, and analyzed for specific ac aspartic acid-C14(CAA-C14)labeled in the ureide C tivity (20). All reactions were studied with con was prepared from L-asparatate and cyanate-C14 stant shaking at 37Â°C. (10) with specific activity of 3.9 X IO5 counts/ min//jmole and was measured by the Koritz and RESULTS Cohen procedure (7). Cyanate-C14 was synthesized Characteristics of the assay systems.â€”The con by the fusion of potassium carbonate and urea-C14 version of carbamylaspartic acid to orotic acid in (16), which was purchased from the New England rat liver homogenates has been previously studied Nuclear Corporation. Non-labeled dihydroorotic by Wu and Wilson (24). In confirmation of their acid (DHO) was prepared by the method of Miller, observations on this two-enzyme system (dihydro Gordon, and Englehardt (9). orotase and dihydroorotic dehydrogenase), there Methods of assay.â€”Escherichiacoli B was cul was enzyme saturation at 1.0-1.5 X 10~3M CAÃ• tured as described by Yates and Pardee (26), har and a broad pH maximum over a range of 6.5-8.0. vested, washed, and extracts were prepared by The reaction was linear with time for 3 hours. sonication for 10 minutes using a Raytheon 10 kc. Some of the characteristics of this enzyme system Oscillator (model DF 101). The sonicate was cen- in sonicates of human leukocytes are presented in trifuged at 10,000 X g for 10 minutes, and the Chart 1. The results are comparable to those found supernatant cell-free extract was stored at â€”10Â°C. in rat liver, although enzyme saturation occurred until used as the source of dihydroorotase. En at 0.5 X 10~3MCAÃ•,and there was a more clearly zymatic activity was measured by the conversion defined pH maximum at approximately pH 7.8. of carbamylaspartate-C14 to dihydroorotate-C14, The biochemical characteristics of dihydrooro which was then isolated and analyzed by the car tase solubilized from E. coli B sonicates are pre rier technic. The incubation mixture consisted of sented in Chart 2. The calculated Km of 5.5 X E. coli supernatant with about 0.15 mg. N; acetate 10~4Mconfirms that of 5.3 X 10~4M reported by buffer, pH 5.5, 0.2 M; EDTA .01 M; and CAA-C14 Yates and Pardee (26). The pH optimum was and analogs as noted in a final volume of 1.0 ml. After incubation for 30 minutes at 30Â°C., 10 found to be 5.0, and the reaction was linear for only 30 minutes. The assay procedures which were AmÃ³lesofcarrier nonlabeled DHO was added and the reaction stopped with cold perchloric acid. developed, based on these studies, have been sum After centrifugation the supernatant was carefully marized under the experimental procedure. formal synthesis of orotic acid in leukocytes.â€” neutralized with KOH with magnetic stirring and constant monitoring of pH (because of the lability In previous studies reproducible levels of dihydro of DHO in alkaline solution). After being chilled orotase and dihydroorotic dehydrogenase activi the supernatant was added to a Dowex-1-formate ties have been described in sonicates of isolated column (1 X 10 cm.), washed, and eluted with human leukocytes (20). In these studies the hy sodium formate buffer, pH 3.2, 0.035 M.DHO was drolysis of DHO-C14 to CAA and the oxidation of detected by the alkali-dependent ureide spot test, DHO-C14 to OA were used to measure these en-

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1960 American Association for Cancer Research. SMITH et al.â€”Inhibition of Dihydroorotase 1061 zymes, respectively. It seemed of interest to deter of human leukocytes are shown in Table 1. mine the reproducibility of the over-all conversion Carbamylglutamic acid, carbamylcysteic acid, and of CAA-C14 to OA in normal leukocytes. Using the a-ureido-/3-methylsuccinic acid were more active assay system described above (but with Ringer's in the rat liver system than were carbamylcys- bicarbonate buffer, which was found to give some teinesulfinic acid and carbamylasparagine. It is of what greater activity) the leukocytes from eleven interest that all the analogs, with the exception of normal subjects were studied. Orotic acid synthe carbamylasparagine, were much less effective in sis was found to be 19.1 Â±3.5 m/umoles OA/108 inhibiting the enzyme preparations from human leukocytes/hr. leukocytes than those from rat liver homogenates. Inhibition of orotic acid synthesis.â€”The struc This discrepancy was documented on a number of tural analogs of carbamylaspartate did not affect occasions. dihydroorotic dehydrogenase activity, as meas Inhibition of dihydroorotic acid synthesis.â€”In ured by the oxidation of dihydroorotate-C14 to the soluble enzyme preparation from E. coli B orotate-C14. The over-all conversion of CAA-C14 studies were carried out on the kinetics of the in to orotate was therefore used as a convenient sur hibition of dihydroorotase by the three most active vey system of the in vitro inhibitory effect of the analogs as judged by the rat liver homogenate sys analogs on the synthesis of dihydroorotate by di- tem. Chart 4 illustrates that a-ureido-/3-methyl- hydroorotase. Chart 3 illustrates the progressive succinic acid is a competitive inhibitor with a cal inhibition of orotate synthesis in a rat liver culated KÃŒof1.7 X 10~5 M. In similar studies the homogenate with increasing concentrations of KÃŒof Carbamylglutamic acid was found to be L-carbamylglutamate. Comparative studies of the 3.3 X 10~3 M and that of carbamylcysteic acid, activities of the five analogs in inhibiting orotate 1.4 X 10~2 M, when measured in the bacterial synthesis in rat liver homogenates and in sonicates extract.

234567 CARBAMYLASPARTKACID tlO'4M TIME IN HOURS \ Â¡2

8.6 9.0 30 60 LEUKOCYTES i IO6 CHART1.â€”Substrate concentration curve, time curve, pH human leukocytes. Experimental conditions have been listedL curve, and enzyme concentration curve for the conversion of under "Materials and Methods." carbarnylaspartie acid-C14to orotic acid in sonicates of isolated

DISCUSSION compounds interfere with all the known functions Among the classes of compounds which have of tetrahydrofolic acid in the metabolism of one- proved to be of greatest effectiveness in the inhi carbon particles (6). In purine synthesis this con bition of purine biosynthesis in vitro and in car- cerns the introduction of carbon atoms at positions cinostasis in vivoare several which have their main 2 and 8, i.e., steps prior to the completion of ring loci of action early in the sequence of reactions formation. Similarly, azaserine and 6-diazo-5- leading to the final purine moiety. The antifolic oxo-L-norleucine (DON), competitive inhibitors of

1000 2000 3000 3O 40 50 GO

I/S ILITCPS/MOLf CAAI TIME IN MINUTES

CHARTi.â€”Lineweaver-Burke plot of the substrate concen acid-C14to dihydroorotic acid in extracts of E. coli K. Kxperi- tration curve, the time curve, the pH curve, and the enzyme mental conditions have been listed under "Materiuls and â€¢concentrationcurve for the cyclization of carharaylaspartic Methods." Enzyme preparation contained 0.85 mj{. \ per ml.

glutamine metabolism, also affect purine synthesis at an early stage, primarily the formation of formyl- glycineamidineribotide (8). More recently 6-mercaptopurine has been found to function as a feed back inhibitor of earlier steps in purine synthesis (3), as one of its several demonstrated mechanisms of action. In contrast most of the inhibitors of pyrimidine metabolism which have been developed represent derivatives based on the completed pyrimidine ring or alterations of the pyrimidine ring per se (especially the "aza" compounds). Com 2 i e e io L-CAPBAMYL GLUTAMATC t pounds formed by substitution at position 5 in the CHART3.â€”Inhibition of orotic acid synthesis from car- pyrimidine ring have been especially active, nota bamylaspartic acid-Cu (10~3M) in a rat liver homogenate by bly the fluoro derivatives of uracil and orotic acid increasing concentrations of 1-carbamylglutamate. Experi (5). It is of interest that fluoroorotic acid and mental conditions as listed under "Materials and Methods." 5-fluorocytosine, in analogy to 6-mercaptopurine,

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1960 American Association for Cancer Research. SMITH et ai.â€”Inhibition of Dihydroorotase 106!5 have also been recently shown to have some ac chosen for carbamylation as potential competitive tivity as feedback inhibitors of earlier steps in inhibitors of reactions involving carbamylaspar- pyrimidine synthesis.1 In general little attention tate. Cysteic acid and glutamic acid have been has been directed toward the development of previously shown to inhibit aspartic acid metabo-

TABLE 1 INHIBITIONOFOROTICACIDSYNTHESISINRATLIVERHOMOGENATESAND HUMANLEUKOCYTESONICATESBYCARBAMYLASPARTATEANALOGS*

RAT LIVEK HCMAN U.TKOCYTK8 INHIBITOR Cose.(XIO-' M)024610024fi8OA(m/imoles)28.516cent)0425668022445161OA(m/imoles)33.627.624.418.014.314cent)01827460091332 1. Carbamylglutamic acid 512 59.020

2. Carbamylcysteic acid 516.011.510 513.012

08.0Inhibition(per 49.7Inhibition(per

a-Ureido-0-methyIsuc- cinic acid 012481025.519.016.514.510.0025liÃ³436117.218.515

813 .212 90082325

4. Carbamylcysteinesul- finic acid 0 55 0 9.1 0 2 45 18 9 (I 0 5 41 25 75 18 10 33 40 fi. M 23 58 6.7

5. Carbamylasparagine 0 82 0 10.2 0 2 71 14 9.1 11 4 62 24 8.5 16 6 54 34 6.9 32 1(1 6.3 38 15 38 64 * Reaction conditions are described under the section on "Materials and Methods.' CAA-C14, IO"3 M in all experiments. OA = orotic acid synthesized. agents to inhibit the enzymatic steps prior to com pletion of the pyrimidine ringâ€”aspartate car- bamyltransferase, dihydroorotase, or dihydroorotic dehydrogenase. For reasons previously men tioned competitive inhibition of aspartate car- bamyltransferase would seem to be impractical in the mammalian system. It is of interest that carbamylphosphate itself may be a growth inhibitor of bacteria under certain conditions (13). At tempts were, therefore, made to study certain carbamylated analogs of aspartate for inhibitory activity in the conversion of carbamylaspartate to clihydroorotate by dihydroorotase. I/S ILITCRS/MOLC C/Sil CHART 4.â€”Competitive inhibition of the synthesis of di- Several structural analogs of aspartic acid were hydrooroticacid (DHO) from carbamylaspartic acid-C14 (CAA) 1L. H. Smith, Jr., and M. Sullivan, Feedback Inhibition by by o-ureido-|S-methylaspartic acid (3 X 10~5 M) in extracts of E. filli B. Experimental conditions are listed under "Materials Fluorinated Pyrimidines. Submitted to Biochim. et Biophys. ocia 39:554-547, 1960. and Methods."

lism in microorganisms (14). Cysteinesulfinic inhibition was competitive in type. At present in acid, a probable intermediate in the biological oxi formation is not available concerning the intra- dation of cysteine to cysteic acid (18), and as- cellular concentration of carbamylaspartate or the paragine were chosen for structural similarity to passage of these analogs across the intact cell aspartic acid. 0-Methyl-aspartic acid has been en- membrane. These preliminary in vitro studies must zymatically formed from mesaconate and ammo be extended to in vivosystems before the effective nia in cell-free extracts of Clostridium tetanomor- ness of carbamylaspartate analogs as antineoplas- phum (1). It was selected for carbamylation tic agents can be evaluated. (through a different synthetic sequence) as an ex ample of a ^-substituted aspartate analog. Other REFERENCES aspartic acid analogs, especially /3-hydroxyaspar- 1. BARKER,H. S.; SMYTH,R.D; WAWSZKIEWICZ,E.J.; LEE, tic acid and diaminosuccinic acid, may be worthy M. N.; and WILSON,R. M. Enzymatic Preparation and Characterization of an a-L-/3-methylaspartic Acid. Arch. of similar carbamylation because of their previous Biochem. & Biophys., 78:408-76, 1958. ly demonstrated inhibitory effect on aspartate 2. COHEN,P. P., and GRISOLIA,S.The Role of Carbamyl-L- metabolism (17). glutamic Acid in the Enzymatic Synthesis of Citrulline The five analogs were tested in vitro in three dif from Ornithine. J. Biol. Cheni., 182:747-61, 1950. ferent systemsâ€”E. cÃ²li B extracts, rat liver 3. GOTS,J. S., and GOLLUB,E. G. Furine Analogs as Feed back Inhibitors. Proc. Soc. Exper. Biol. & Med., 101:641- homogenates, and sonicates of human leukocytes. 43, 1959. The inhibitory activities of the analogs varied con 4. GKEEN,M., and COHEN,S. S. Studies on the Biosynthesis siderably depending upon the system being of Bacterial and Viral Pyrmidines. II. Dihydrouracil and studied. In general, carbamylglutamic acid, car- Dihydrothymine Nucleosides. J. Biol. Chem., 226:397-407, bamylcysteic acid, and a-ureido-/3-methylsuccinic 1957. 5. HEIDELBERGER,C.;CHAUDHURI,N.K.; DANNEBERO,P.; acid were approximately equal to inhibitory ac MOOREN,D.; GRIESBACH,L.; DUSCHINSKY,R.; SCHNIT tivity in rat liver. a-Ureido-/3-methylsuccinic acid ZER,R. J.; PLEVEN,E.; and SCHEINER,J.Flurinated Pyrim- was by far the most active of the three when tested idines, a New Class of Tumor-inhibitory Compounds. on bacterial dihydroorotase, with a calculated A", Nature, 179:663-66, 1957. of 1.7 X 10~6 M. With the possible exception of 6. HUENNEKENS,F, M., and OSBORN,M. J. Folie Acid Co- enzymes and One-Carbon Metabolism. In: F. F. NORO carbamylasparagine, the analogs were much less (ed.), Advances in Enzymology, 21:369-446. New York: active when measured in human leukocyte soni Interscience Publishers, Inc., 1959. cates than in rat liver homogenates. It has been 7. KORITZ,S. B., and COHEN,P. P. Colorimetrie Determina tion of Carbamylamino Acids and Related Compounds. previously shown that leukocyte dihydroorotase J. Biol. Chem., 209:145-50, 1954. activity approximates that of rat liver on a weight 8. LEVENBERO,B.;MELNICK,I.; and BUCHANAN,J.M. Bio basis, whereas dihydroorotic dehydrogenase ac synthesis of the Purines. The Effect of Aza-L-serine and tivity in the leukocyte is only ^ of that in liver 6-diazo-5-oxo-L-norleucine on Inosinic Acid Biosynthesis de (20). If the concentration of dihydroorotic de nom. 3. Biol. Chem., 225:163-76, 1957. hydrogenase is more rate-limiting in the leukocyte 9. MILLER,C. S.; GORDON,J.T.; and ENGELHARDT,E.L. Di hydroorotic Acid. J. Am. Chem. Soc., 76:6086-87, 1953. than in rat liver, a given reduction in dihydro 10. NYC,J. F., and MITCHELL,H. K. Synthesis of Orotic Acid orotic acid synthesis from carbamylaspartic acid from Aspartic Acid. J. Am. Chem. Soc., 69:1382-84, 1947. (caused by the analog) would be expected to exert 11. PHILLIPS,D. M. P. Chroinatography of the N-carbamoyl- a smaller effect on the over-all conversion of car amino Acids (Hydantoic Acids). Biochim. et Biophys. bamylaspartic acid to orotic acid. The data at acta, 13:560-63, 1954. hand do not finally determine whether this is the 12. PIUTTI,A. Ein Neues Asparagin. Ber., 19:1691-95, 1886. 13. RAVEL,J. M.; ESTES,J. M.; MOLLENHAUER,B.F.; and true explanation of the differences observed in in SHIVE,W. Some Inhibitory Properties of Carbamylphos- hibitory activities with these two test systems. phate. J. Biol. Chem., 229:93-99, 1957. Intact human leukocytes have been previously 14. RAVEL,J. M.; FELSING,B.; and SHIVE,W. Comparative utilized by Winzler and his colleagues in a number Inhibitory Effects of Glutamic and Cysteic Acids on Aspar of studies to test the effectiveness of various anti- tic Acid Utilization. Arch. Biochem. & Biophys., 64:541- metabolites on nucleic acid synthesis (23). 48, 1955. 15. 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18. SINGER,T. P., and KEARNEY,E. B. Enzymatic Pathways Sons, Inc., 1953. in the Degradation of Sulfur-containing Amino Acids. In: 22. TOENNIES,G.,and LAVINE,T. F. The Oxidation of Cystine W. D. MCELROYand H. B. GLASS(eds.), Amino Acid in Non-aqueous Media. V. Isolation of a Disulfoxide of Metabolism, pp. 558-590. Baltimore: The Johns Hopkins L-cystine. J. Biol. Chem., 113:571-82, 1936. Press, 1955. 23. WINZLER,R. J. Differences in Nucleic Acid Metabolism 19. SKIPPER,H. S., and BENNETT,L. L., JR. Biochemistry of between Normal and Leukemic Human Leukocytes. Ann. Cancer. In: J. M. LrCK (ed.), Ann. Rev. Biochem., 27: N.Y. Acad. Sc., 75:37-44, 1958. 137-66. Palo Alto: Annual Reviews, Inc., 1958. 24. WIT,R., and WILSON,D. \V. Studies on the Biosynthesis â€¢20.SMITH,L. H., JR., and BAKER,F. A. Pyrimidine Metabo of Orotic Acid. J. Biol. Chem., 223:195-205, 1956. lism in Man. I. The Biosynthesis of Orotic Acid. J. Clin. 25. YATES,R. A., and PARDEE,A. B. Control of Pyrimidine Invest., 38:798-809, 1959. Biosynthesis in Escherichia coli by a Feedback Mech al. SWERN,D. Epoxidation and Hydroxylation of Ethylenic anism. J. Biol. Chem., 221:757-70, 1956. Compounds with Organic Peracids. In: R. ADAMS(ed.). 26. . Pyrmidine Biosynthesis in Escherichia coli. Ibid., Organic Reactions, 7:378-433. Xew York: John Wiley & pp. 743-56.

Lloyd H. Smith, Jr., Margaret Sullivan, Faith A. Baker, et al.

Cancer Res 1960;20:1059-1065.

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