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Biochemical Screening of Pyrimidine Antimetabolites I. Systems with Oxidative Energy Source* JOSEPHE. STONEANDVANR. POTTER (McArdle Memorial Laboratory, Medical School, University of Wisconsin, Madison, Wis.} Orotic acid (uracil-4-carboxylic acid) has been the livers were quickly excised and placed in a chilled bath of isotonic saline solution. A 20 per cent homogenate in chilled shown under both in vivo and in vitro conditions, 0.25 M sucrose was made with the use of an all-glass Potterin both microorganisms and mammals, to be a pre Elvehjem homogenizer, and Ihis homogenate was centrifuged cursor of the mono-, di-, and triphosphate pyrim- at approximately 600 g for 10 minutes to remove nuclei and idine nucleotides, the pyrimidine coenzymes, and whole cells. Portions of 0.8 ml. of the cytoplasmic liver fraction were also of the pyrimidine moieties in both ribo- and placed in 25-ml. Erlenmeyer flasks, each of which contained deoxyribonucleic acid (5, 6, 11, 12, 13, 15-17). 2.20 ml. of a reaction mixture which had the following com This study was prompted by the current position: progress in the study of nucleic acid metabolism 15.0 Amóles Potassium glutamate and its relationship to tumor growth and metabo 6.0 /«moles Potassium fumarate 15.0 /iiiiole-s lism. The purposes of this research are the Potassium pyruvate 15.0 /¿moles selection of agents as possible components of KHjPO, 9.0 /uñóles MgCl2 sequential (9) or concurrent blocks (4), the clari Ribose-5-phosphate (RSP) 6.0 /¿moles fication of biochemical pathways, and the develop Uridine-5-monophosphate (UMP-5') 1.0 /imoles ment of concepts and technics in a biochemical Orotic acid-6-C" 0.3 /tmoles approach to pharmacological research. Adenosine triphosphate (ATP) 3.0 /uñóles The data presented here represent the results of A sufficient quantity of sucrose to make an isotonicity equiva lent to 0.25 Msucrose. a screening program with a biochemical system A sufficient quantity of water to make a volume of 2.20 ml. which converts orotic acid to the uridine nucleo The pH was adjusted to 7.2-7.4 with 0.2 N KOH. All flasks tides. Normal rat liver cytoplasm furnished the were in duplicate. Drugs were added to the system in a drug: enzyme source, and oxidative substrates furnished orotic acid ratio of 10:1 (3.0 /IMof the drug to 0.3 /IMof the the source of energy for the reactions. A primary orotic acid).2 Whenever possible, the drugs were added in objective of the study was to ascertain which aqueous solution; however, when necessary, acetate solutions drugs could be shown to act upon one or more or propylene glycol solutions were utilized. In these cases, ap steps in the conversion of orotic acid to the uridine propriate control flasks were added. The reaction mixtures were incubated with constant agita nucleotides. tion for 20 minutes at 30°C. The reactions were terminated by MATERIALS AND METHODS It has been demonstrated that under in vitro conditions orotic acid is converted to uridine nucleotides by preparations of normal rat liver (6, 10). The latter observation is the basis of the test system used in this study. The drugs were added to a standardized system, and the con version of orotic acid to the uridine nucleotides was measured and compared with the conversion in appropriate controls. White rats of either sex,1 weighing 150-450 gm., were used in this study. The animals were sacrificed by decapitation, and * This work was supported in part by a grant (No. C2409) from the National Cancer Institute, National Institutes of Health, United States Public Health Service. Received for publication July 10, 1956. the addition of 1.50 ml. of 1.5 N perchloric acid to each flask, which resulted in a final perchloric acid concentration of 0.5 N. The resulting mixture was centrifuged at 600 g for 10 minutes and the pellet discarded. The acid-soluble supernatant liquid was neutralized with 2.0 N KOH with phenol red as an internal indicator, and the neutral solution was then allowed to stand for 10 minutes at —¿10° C. for the precipitation of residual potassium perchlorate. After recentrifugation, the supernatant fluid was placed upon 5-cni. Dowex l (X10) aniónexchange resin columns (14). The Chromatographie columns were submitted to the elution scheme which is summarized in Table 1. Fractions 1, 2, and 3 were not examined and were discarded (see below). Fractions 4, 5, 6, and 7 were read at 260 and 275 1Obtained from the Holtzman-Rolfsmeyer Rat Company, Madison, Wis. 2The orotic acid used in this study was labeled in carbon 6. Its specific activity was 4.1 X 10* counts/min/mg (5.75 /imoles). Approximately 213,000 counts/min were added in 0.3 /imoles added to each flask. 1033 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1956 American Association for Cancer Research. 1034 Cancer Research nan in a Beckman model DU spectrophotometer. The radio activity of the samples was determined by plating a 0.2-ml. aliquot from each tube upon weighed aluminum planchets which were inscribed with a circle 3.8 sq. cm. in area. Approxi mately 0.05 ml. of a 0.1 per cent solution of gelatin was used to spread the solution upon the inscribed area.8 The samples were then dried under a heat lamp, and the determination of radio activity was done in gas-flow proportional counters.4 It should be emphasized that to insure activity of the system all glassware should be washed in sulfurie-nitric acid cleaning solution and glass-distilled water. All reagents should also be prepared with glass-distilled water. Under control conditions, the standard sys tem5 converts an average of 7 per cent (range, 3-12 per cent among all preparations used) of the orotic acid to the uridine nucleotides. The UTP-UTPX6 complex accounts for 80-90 per cent of the orotic acid converted. 4) UMP-5' + ATP ^ UDP + ADP (3). 4a) UDP + ATP ^±UTP + ADP (3). 4b) UTP-> UDPX (UDP conjugates) and UTPXS (3). 5) The ATP renewal is accomplished by oxidative phosphorylation with the Krebs cycle fortified at three points. In testing drugs against the standard system, the following points were taken as criteria of the activity of the system: 1. The total radioactivity in the UTP-UTPX complex is the most reliable index to the effect of the drug upon the conversion of orotic acid to the uridine nucleotides. In this study it has been taken as the major criterion. 2. The quantity of nucleotide in the UTP- TABLE1* SCHEDULE OFELUENTS ANDFRACTIONS COLLECTED Fraction no. 1 2 3 4 5 6 7 Eluent Ml/total fraction No. samples/ fraction Compounds eluted HjO 30 Nucleosides, bases Adenosine monophosphate (AMP), UMP-5', orotic acid 3.0 N formic acid t 150 0.3 N ammonium formate! Orotic acid and ADP 30 Mixed uridine coenzymes (UDPX's) 0.4 N ammonium formate 30 0.7 N ammonium formate Uridine diphosphate (UDP) 30 l. 0 N ammonium formate 30 Uridine triphosphate (UTP) and UTPX 1.25 N ammonium formate ATP 30 * AHsubstances eluted in thé Chromatographie scheme presented in Table 1 were characterized by either chromatography of known samples or reference to previous research done in this laboratory (3). t All formic acid solutions were at pH 2.O. ÃŽ All ammonium formate solutions were at pH 5.O. The known reactions included in the test sys tem may be summarized as follows: 1) R5P + ATP -> PRPP (phosphoribosylpyrophosphate) + AMP (7). 2) Orotic acid + PRPP —¿Â» Orotidylic acid (orotic acid ribotide) + PP (pyrophosphate) (8). 3) Orotidylic acid -> UMP-5' + C02 (8). 3The gelatin solution facilitates spreading of the plated solutions; it also equalizes the crystallization of the ammonium formate present (M. J. Johnson, personal communication). 4After being counted, the plates were again weighed, and correction was made for self-absorption. The assistance of Dr. Charles Heidelberger and his staff in radioactivity determina tions is gratefully acknowledged. 6The first system investigated consisted essentially of the same ox¡dativesubstrates employed in the standard system, including 3.0 jumólesof ATP, a relatively large amount of UMP-5' (6 /¿moles),and a pool of 3.0 /¿molesof orotic acid which contained 0.3 /¿molesof the labeled compound. These substances were incubated with 0.8 ml. of 20 per cent rat liver homogenate for 20 minutes at 30°C. Under these conditions, the conversion of orotic acid to the uridine nucleotides could not be demonstrated; however, under these conditions the UMP-5' pool was almost entirely converted to uridine. The addition of 6.0 /¿moleseach of fructose and ribose-5phosphate to the system outlined above and the substitution of the cytoplasmic fraction of rat liver in the place of the whole homogenate resulted in the production of UTP which had a UTPX fraction is an indication of the transphosphorylase ability of the system in the presence specific activity of 1200 counts/min//*mole. However, this sys tem was unsatisfactory as a test object because of the extreme ly low percentage of orotic acid converted to the uridine nucleotides and because of excessive variation between dupli cate flasks (20-40 per cent). It was found that, with this and all subsequent systems investigated, the major uridine derivative produced was uridine triphosphate (UTP). This fraction con tains small amounts of a uridine nucleotide (Dr. L. Hecht, unpublished data) whose specific activity is about 60 per cent of that of the UTP. This compound has not been completely characterized and is designated as UTPX because it follows UTP on the chromatogram. The radioactivity of the UTPUTPX fraction has been adopted as the major criterion of activity. The second system was further modified by the deletion of the fructose and the reduction of the UMP-5' pool to 1.0 /¿mole. This small amount of UMP-5' serves as a marking pool, and its phosphorylation to UTP-UTPX serves as a measure ment of the transphosphorylative ability of the preparation. Amounts of UMP-5' greater than 1 /¿moledepress the con version of orotic acid into the uridine nucleotides by actual inhibition, aside from the effects due to competition for high energy phosphate and the dilution of the radioactivity. This aspect will be treated fully in a later paper. Ribose-5-phosphate was shown to be present at optimal amounts when used at a level of 2.0 /xmoles/ml. Fructose may be used in place of ribose5-phosphate; however, its inclusion adds a number of addi tional reactions to an already complex system. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1956 American Association for Cancer Research. STONEANDPOTTER—Screeningof Pyrimidine Antimetabolites of the drug. Theoretically, the specific activity of the UTP-UTPX (counts/min/Mmole) can be taken as an indication of the effect of the drug upon oxidative phosphorylation. However, it must be noted that the large ATP pool used in the standard system is sufficient for the transphosphorylation of the UMP-5' pool to UTP.6 Under these condi tions, the UMP-5' pool is converted to UTP re gardless of the effect a drug may have on the 1035 UDP, and UTP-UTPX are good indices of a pri mary effect upon oxidative phosphorylation rather than upon orotic acid metabolism per se. The total radioactivity found in UDP, UDPX's, UMP-5', or in orotic acid itself can be used as an additional criterion of the conversion of orotic acid. However, these fractions (with the exception of the orotic acid fraction) contain relatively little radioactivity and are not so reliable as the UTP TABLE2* DRUGS WHICHDEMONSTRATED No SIGNIFICANT ACTION AGAINST THESTANDARD SYSTEM Compound no. Name 1. 2-Hydroxy-4-amino-S-carbethoxypyrimidine 2. 2-Ammo-4-hydroxy-5-carbethoxypyrimidine 8. 2-Mercapto-4-amino-S-carbethoxypyrimidine 4. 2-Ethoxy-4-hydroxy-5-carbethoxypyrimidine 5. 2-Ethylmercapto-4-chloro-5-carbethoxy pyrimidine 6. l-(D-glucopyranosyl)-carbamylcytosine 7. 2-EthyImercapto-4-ammo-5-4^1orophenoxymethylpyrimidine 8. 2-Ethylsulfonyl-4-amino-S-(2,4-dichlorophenoxymethyl)-pyrimidine 9. 2-Hydroxy-4-amino-5-aminomethylpyrimidine 10. 2,4-Diethoxy-5-carbethoxypyrimidine 11. Na-carbethoxy-L-asparagine 12. Na-carbethoxy-D-asparagine 13. 4-Amino-5-hydroxymethylpyrimidine 14. 5-Methyl-orotic acid 15. 5-Iodo-orotic acid 16. 4-Methyl-uraciI 17. 4-Methyl-2-sulfo-uracil 18. 4-Trifluoromethyl-2-sulfo-uracil 19. 2-Mercapto-orotic acid 20. 5-Amino-orotic acid 21. Thiothymine 22. Bromouracil 23. 4-Oxy-5-(o,p-dichlorophenoxy)-pyrimidine 24. Thiocytosine 25. Thiouracil 26. Azathymine 27. Cytosamine * Drugs nos. 1—15, 28-35, 40, and 46-51 were supplied by Sharpe and Dohme, Inc. Nos. 19, 20, 36-38, and 41 were supplied by Lederle Laboratories. Nos. 21-26, 44, and 45 were supplied by Welcome Research Laboratories. Nos. 27 and 52 were supplied by Eli Lilly and Co. Nos. 16-18, 39, 42, and 43 were supplied by Dr. Charles Heidelberger of this department. energy source of the system. However, in cases of an extreme inhibitory effect of an agent upon the energy source, the absolute amounts of ATP, 6During the course of a later investigation, an experiment was performed in which the following changes were made in the test system employed in this study: The ATP pool was reduced from 3.0 ¿imolesto 0.5 /linole, and the nuclei were washed with chilled 0.25 M sucrose solution and the washings added to the cytoplasmic fraction. The time of incubation was increased to 45 minutes. These modifications resulted in the conversion of 47 per cent (45.6-50.0 per cent) of the orotic acid. Moreover, under these conditions the specific activity of the UTP complex is a valid measurement of the oxidative capacity of the system. It should be noted that this modifica tion allows for a finer differentiation between drug action upon oxidative phosphorylation and upon orotic acid metabolism per se. Such a system, because of its ability to convert a high proportion of orotic acid at low molari! ics to uridine nucleotides, could be used for production of the latter in radioactive form. complex. The UMP-5' fraction is extremely small and is separated only with difficulty from the orotic acid. The orotic acid fraction itself is extremely radioactive, and its net conversion is low. For these reasons, the UMP-5' peak and the orotic acid peak are eluted from the column with 3.0 N formic acid and discarded. RESULTS AND DISCUSSION The drugs which have been tested in this study have been divided into three groups on the basis of their effects upon the test system. Those com pounds which depressed the conversion of orotic acid 0-20 per cent were regarded as demonstrating no significant effect upon orotic acid metabolism (Table 2). Those agents which gave an inhibition of 20-40 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1956 American Association for Cancer Research. 1036 Cancer Research per cent were placed in an intermediate group (Table 3). The drugs which depressed orotic acid conversion 40-100 per cent were selected for further study (Table 4). In general, oxidative phosphorylation is perhaps not the energy source of choice in a biochemical-pharmacological screening system. This source of energy has certain disadvantages, Among these are the rigorous standards of cleanliness which must be observed and the instability of the enzyme system. However, the most serious disadvantage lies in the fact that the energy source may be the most sensitive component of the system. Agents which act upon one or more steps in the oxidative cycle could appear to be inhibitors of reactions in which they actually have no effect. However, in this study oxidative phosphorylation was deliberately chosen as an energy source because of its extreme sensitivity. Under these conditions, it allows for the screening not only of orotic acid antimetabolites but also of agents which may have an effect upon some of the respiratory enzymes. The agents found active against this system were then tested against a more specific system with a glycolytic energy source. The differential screening systems show clearly which agents are active against orotic acid metabolism per se and which are active against oxidative phosphorylation alone, Differential screening with multiple systems can also give information on agents with multiple points of action and could be an aid in the correlation of biochemical and pharmacological phenomena. Programs of this type should also give rise to a dictionary of drugs whose point or points of action are more or less known. It would seem that such a body of knowledge would be invaluable in integrative biochemistry, The thirteen agents which demonstrated some inhibitory capacity can be divided into four chemical and to some extent functional groups, Group 1 consists of the 5-substituted analogs of orotic acid and 5-diazo-uracil. The chloro, bromo, and diazo analogs of orotic acid demonstrated moderate inhibition (average, 45 per cent TABLE 8 DRUGSWHICHDEPRESSEDTHE CONVERSION OF OHOTICACIDBY20-40 PER CENT Compound no. Name 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 2-Ethylmercapto-4-hydroxy-5-carbethoxypyrimidine 2-Ethylrnercapto-4-amino-5-aminomethylpyrimidine 2,4-Diamino-5-carbethoxypyrimidine 2-Mercapto-4-amino-5-hydroxymethylpyrimidine 2-Ethylmercapto-4-amino-5-hydroxymethylpyrimidine D-dihydro-orotic acid 5-Hydroxymethylcytosine Fumarylurea 5-Carboxy-orptic acid 2-Amjno-orotic acid 6-Acetic-erotic acid 4-Trifluoromethyl-uracil TABLE 4* DRUGSWHICHINHIBITEDTHECONVERSION OFOROTICACIDBY40-100 PER CENT UTP-UTPXUTP-UTPX ATP Compound no. Name radioactivity a«per cent of control /¿moles as per cent of control pmoles ns per cent of control 40. 5-Chloro-orotic acid 55 (2) 139 138 41. 5-Bromo-orotic acid 33 (3) 129 131 42. 5-Diazo-orotic acid 47(3) 95 83 43. 5-Diazo-uracil 27(3) 64 57 44. 2,4-Diammo-5-(3',4'-dichlorophenyl)-6-methylpyrimidine 25 (2) 38 27 45. Daraprim 9(2) 14 17 46. 2-Benzylmercapto-4-amino-5-carbethoxypyrimidine 29 (2) 90 93t 47. 2-Ethylmercapto-4-amino-5-chlorornethylpyrimidine 35 (2) 118 84 48. 2-Benzylmercapto-4-araino-5-hydroxymethylpyrimidine 9 (2) 48 60 49. 2-Ethoxy-4-amino-5-carbethoxypyrimidine 17 (2) 73 63 50. 2-Ethylmercapto-4-amino-5-carbethoxypyrimidine 11 (2) 47 43 51. N"-carbethoxy-DL-asparagine 50 (l) 91 52. Amicetin 41 (2) 63 50 * Figures in parentheses give number of experiments. t In one experiment a duplicate flask was extremely aberrant and demonstrated a total count which was 180 per cent of the control levels. This drug is extremely insoluble. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1956 American Association for Cancer Research. STONEANDPOTTER—Screeningof Pyrimidine Antimetabolites of control). This inhibition is accompanied by an actual increase in the amount of UTP-UTPX and in ATP. From these data it would appear that these agents have no inhibitory effect upon oxidative phosphorylation and that the inhibition oc curs in the steps between orotic acid and UMP-5'. The occurrence of abnormal metabolites or the "piling up" of other metabolites could not be demonstrated because of the low net conversion of orotic acid. The inhibition produced by 5-diazouracil would seem to be of a different type be cause of the depression of the amounts of triphosphate which accompany the inhibition. It should be noted that the 5-carboxy, amino, methyl, and iodo analogs of orotic acid were ineffective in this system. The 2-amino and 2-mercapto analogs were likewise ineffective. Group 2 includes Daraprim (2,4-diamino-5-parachlorophenyl-6-ethylpyrimidine) and its analog 2,4-diamino-5- (3',4'-dichlorophenyl) -6-methylpyrimidine. In this system these drugs are marked inhibitors of orotic acid conversion to the uridine nucleotides (average, 17 per cent of control). However, this phenomenon is accompanied by marked depression of the amounts of triphosphate. Thus, it would seem that these drugs have a marked effect upon the energy source of the sys tem. The effect of these drugs upon orotic acid metabolism cannot be ascertained until they are tested against a system whose energy source is unaffected by them. Daraprim has been shown by other workers to be a folie acid antagonist (1). The action shown here may be a corollary of this or may be an additional point of action. Group 3 is composed of the 2,5-substituted 4-amino pyrimidines. The inhibition was accom panied by moderate depression of the amounts of both uridine triphosphate and ATP. In this case, the data derived from a system of this type cannot differentiate between action upon orotic acid metabolism and the energy source. This group of drugs demonstrates the variability between dupli cate flasks and experiments. This phenomenon may be due to variation in solubility with slight variation in pH. These compounds all have an amino group in position 4, and it would seem that a large variety of groups can be used in positions 2 and 5. It is difficult to draw conclusions from these data, because a potentially active compound may be almost completely insoluble. The antibiotic Amicetin (2) produced an aver age inhibition of 41 per cent of the control levels; there was moderate diminution of the amounts of triphosphate. N" carbethoxy-DL-asparagine diminished orotic acid conversion to 50 per cent of the controls. The drugs discussed above were reinvestigated 1037 in a semispecific system which essentially tested orotic acid conversion only. These data will be presented in another publication. SUMMARY Fifty-two pyrimidine derivatives and related compounds have been tested for orotic acid antimetabolite activity against a biochemical system which converts orotic acid to the uridine nucleo tides. Thirteen of these drugs have been demon strated to be active against one or more of the components of the system. REFERENCES 1. FALCO,E. A.; HITCBINGS,G. H.; RÃœSSEL, P. B.; and VANDEBWEBÕT, H. Antimalarials as Antagonists of Purines and Pteroylglutamic Acid. Nature, 164:107-8, 1949. 2. FLTNN,E. H.; HINMAN,J. W.; CARÓN, E. L.; and WOOLF, D. O. The Chemistry of Amicetin, a New Antibiotic. J. Am. Chem. Soc., 76:5867-71,1953. 3. HERBERT, E.; POTTER, V. R.; and TAKAGI, Y. The Phosphorylation of 5'-Uridine Nucleotides by Cell Frac tions from Rat Liver. J. Biol. Chem., 213:923-40, 1955. 4. HITCHINGS,G. H. Purine and Pyrimidine Antagonists. Am. J. Clin. Nutrition, 3:321-27,1955. 5. HUHLBEHT,R. B., and POTTER,V. R. Nucleotide Metabo lism. I. The Conversion of Orotic Acid-6-C14to Uridine Nucleotides. J. Biol. Chem., 209:1-21, 1954. 6. HURLBEBT,R. B., and REICHAHD,P. The Conversion of Orotic Acid to Uridine Nucleotides in Vitro. Acta Chem. Scandinav., 9:251-«2,1955. 7. 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Mono-, Di-, and Triphosphates of Cytidine, Guanosine, and Uridine. J. Biol. Chem., 209:41-54, 1954. 14. SIEKEVITZ,P., and POTTER,V. R. Radioactive Labeling of Intramitochondrial Nucleotides during Oxidative Phos phorylation. J. Biol. Chem., 216:237-55, 1955. 15. WEED,L. L. The Incorporation of Radioactive Orotic Acid into the Nucleic Acid Pyrimidines of Animal and Human Tumors. Cancer Research, 11:470-73, 1951. 16. WEED,L. L., and WILSON,P. W. The Incorporation of C1« Orotic Acid into the Nucleic Acid Pyrimidines in Vitro. J. Biol. Chem., 189:435-41, 1951. 17. •¿â€”. Studies of Pyrimidine Nucleotides with Orotic Acid-2-C" and P».Ibid., 216:745-49, 1953. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1956 American Association for Cancer Research. Biochemical Screening of Pyrimidine Antimetabolites: I. Systems with Oxidative Energy Source Joseph E. Stone and Van R. Potter Cancer Res 1956;16:1033-1037. 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