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. 1992 Apr;36(4):704–711. doi: 10.1128/aac.36.4.704

Molecular targets of 5-fluoroorotate in the human malaria parasite, Plasmodium falciparum.

P K Rathod 1, N P Leffers 1, R D Young 1
PMCID: PMC189360  PMID: 1503432

Abstract

5-Fluoroorotate is known to have potent antimalarial activity against chloroquine-susceptible as well as chloroquine-resistant clones of Plasmodium falciparum. It was hypothesized that this activity was mediated through synthesis of 5-fluoro-2'-deoxyuridylate, an inactivator of thymidylate synthase, or through incorporation of 5-fluoropyrimidine residues into nucleic acids. Treatment of P. falciparum in culture with 100 nM 5-fluoroorotate resulted in rapid inactivation of malarial thymidylate synthase activity. A 50% loss of thymidylate synthase activity as well as a 50% decrease in parasite proliferation were seen with 5 nM 5-fluoroorotate. Dihydrofolate reductase activity, which resides on the same bifunctional protein as thymidylate synthase, was not affected by 5-fluoroorotate treatment. Incubation of malarial parasites with 3 to 10 microM radioactive 5-fluoroorotic acid for 48 h resulted in significant incorporation of radioactivity into the RNA fraction of P. falciparum; approximately 9% of the uridine residues were substituted with 5-fluorouridine. However, compared with the 50% inhibitory concentrations of 5-fluoroorotate, a 1,000-fold higher concentration of the pyrimidine analog was required to see significant modification of RNA molecules. Results of these studies are consistent with the hypothesis that thymidylate synthase is the primary target of 5-fluoroorotate in malarial parasites.

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Selected References

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  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Bzik D. J., Li W. B., Horii T., Inselburg J. Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8360–8364. doi: 10.1073/pnas.84.23.8360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen G. X., Zolg J. W. Purification of the bifunctional thymidylate synthase-dihydrofolate reductase complex from the human malaria parasite Plasmodium falciparum. Mol Pharmacol. 1987 Dec;32(6):723–730. [PubMed] [Google Scholar]
  4. Clyde D. F. Recent trends in the epidemiology and control of malaria. Epidemiol Rev. 1987;9:219–243. doi: 10.1093/oxfordjournals.epirev.a036303. [DOI] [PubMed] [Google Scholar]
  5. Coderre J. A., Beverley S. M., Schimke R. T., Santi D. V. Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2132–2136. doi: 10.1073/pnas.80.8.2132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Danenberg P. V., Danenberg K. D. Effect of 5, 10-methylenetetrahydrofolate on the dissociation of 5-fluoro-2'-deoxyuridylate from thymidylate synthetase: evidence for an ordered mechanism. Biochemistry. 1978 Sep 19;17(19):4018–4024. doi: 10.1021/bi00612a022. [DOI] [PubMed] [Google Scholar]
  7. Danenberg P. V., Heidelberger C., Mulkins M. A., Peterson A. R. The incorporation of 5-fluoro-2'-deoxyuridine into DNA of mammalian tumor cells. Biochem Biophys Res Commun. 1981 Sep 30;102(2):654–658. doi: 10.1016/s0006-291x(81)80182-9. [DOI] [PubMed] [Google Scholar]
  8. Danenberg P. V., Lockshin A. Fluorinated pyrimidines as tight-binding inhibitors of thymidylate synthetase. Pharmacol Ther. 1981;13(1):69–90. doi: 10.1016/0163-7258(81)90068-1. [DOI] [PubMed] [Google Scholar]
  9. Desjardins R. E., Canfield C. J., Haynes J. D., Chulay J. D. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother. 1979 Dec;16(6):710–718. doi: 10.1128/aac.16.6.710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Evans R. M., Laskin J. D., Hakala M. T. Assessment of growth-limiting events caused by 5-fluorouracil in mouse cells and in human cells. Cancer Res. 1980 Nov;40(11):4113–4122. [PubMed] [Google Scholar]
  11. Fernandes D. J., Cranford S. K. A method for the determination of total, free, and 5-fluorodeoxyuridylate-bound thymidylate synthase in cell extracts. Anal Biochem. 1984 Nov 1;142(2):378–385. doi: 10.1016/0003-2697(84)90480-9. [DOI] [PubMed] [Google Scholar]
  12. Furuichi Y., Miura K. A blocked structure at the 5' terminus of mRNA from cytoplasmic polyhedrosis virus. Nature. 1975 Jan 31;253(5490):374–375. doi: 10.1038/253374a0. [DOI] [PubMed] [Google Scholar]
  13. GAREN A., LEVINTHAL C. A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. I. Purification and characterization of alkaline phosphatase. Biochim Biophys Acta. 1960 Mar 11;38:470–483. doi: 10.1016/0006-3002(60)91282-8. [DOI] [PubMed] [Google Scholar]
  14. Garrett C. E., Coderre J. A., Meek T. D., Garvey E. P., Claman D. M., Beverley S. M., Santi D. V. A bifunctional thymidylate synthetase-dihydrofolate reductase in protozoa. Mol Biochem Parasitol. 1984 Apr;11:257–265. doi: 10.1016/0166-6851(84)90070-7. [DOI] [PubMed] [Google Scholar]
  15. Gero A. M., O'Sullivan W. J. Purines and pyrimidines in malarial parasites. Blood Cells. 1990;16(2-3):467–498. [PubMed] [Google Scholar]
  16. Gilles H. M. Malaria--an overview. J Infect. 1989 Jan;18(1):11–23. doi: 10.1016/s0163-4453(89)93517-2. [DOI] [PubMed] [Google Scholar]
  17. Gleason M. K., Fraenkel-Conrat H. Biological consequences of incorporation of 5-fluorocytidine in the RNA of 5-fluorouracil-treated eukaryotic cells. Proc Natl Acad Sci U S A. 1976 May;73(5):1528–1531. doi: 10.1073/pnas.73.5.1528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gutteridge W. E., Trigg P. I. Incorporation of radioactive precursors into DNA and RNA of Plasmodium knowlesi in vitro. J Protozool. 1970 Feb;17(1):89–96. doi: 10.1111/j.1550-7408.1970.tb05163.x. [DOI] [PubMed] [Google Scholar]
  19. Gómez Z. M., Rathod P. K. Antimalarial activity of a combination of 5-fluoroorotate and uridine in mice. Antimicrob Agents Chemother. 1990 Jul;34(7):1371–1375. doi: 10.1128/aac.34.7.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Haynes J. D., Diggs C. L., Hines F. A., Desjardins R. E. Culture of human malaria parasites Plasmodium falciparum. Nature. 1976 Oct 28;263(5580):767–769. doi: 10.1038/263767a0. [DOI] [PubMed] [Google Scholar]
  21. Heidelberger C., Danenberg P. V., Moran R. G. Fluorinated pyrimidines and their nucleosides. Adv Enzymol Relat Areas Mol Biol. 1983;54:58–119. [PubMed] [Google Scholar]
  22. Houghton J. A., Weiss K. D., Williams L. G., Torrance P. M., Houghton P. J. Relationship between 5-fluoro-2'-deoxyuridylate, 2'-deoxyuridylate, and thymidylate synthase activity subsequent to 5-fluorouracil administration, in xenografts of human colon adenocarcinomas. Biochem Pharmacol. 1986 Apr 15;35(8):1351–1358. doi: 10.1016/0006-2952(86)90281-9. [DOI] [PubMed] [Google Scholar]
  23. Houghton P. J., Germain G. S., Hazelton B. J., Pennington J. W., Houghton J. A. Mutants of human colon adenocarcinoma, selected for thymidylate synthase deficiency. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1377–1381. doi: 10.1073/pnas.86.4.1377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Krogstad D. J., Herwaldt B. L. Chemoprophylaxis and treatment of malaria. N Engl J Med. 1988 Dec 8;319(23):1538–1540. doi: 10.1056/NEJM198812083192308. [DOI] [PubMed] [Google Scholar]
  25. Kufe D. W., Major P. P., Egan E. M., Loh E. 5-Fluoro-2'-deoxyuridine incorporation in L1210 DNA. J Biol Chem. 1981 Sep 10;256(17):8885–8888. [PubMed] [Google Scholar]
  26. McCormick G. J., Canfield C. J., Willet G. P. In vitro antimalarial activity of nucleic acid precursor analogues in the simian malaria Plasmodium knowlesi. Antimicrob Agents Chemother. 1974 Jul;6(1):16–21. doi: 10.1128/aac.6.1.16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Meek T. D., Garvey E. P., Santi D. V. Purification and characterization of the bifunctional thymidylate synthetase-dihydrofolate reductase from methotrexate-resistant Leishmania tropica. Biochemistry. 1985 Jan 29;24(3):678–686. doi: 10.1021/bi00324a021. [DOI] [PubMed] [Google Scholar]
  28. Miura K., Watanabe K., Sugiura M. 5'-Terminal nucleotide sequences of the double-stranded RNA of silkworm cytoplasmic polyhedrosis virus. J Mol Biol. 1974 Jun 15;86(1):31–48. doi: 10.1016/s0022-2836(74)80005-7. [DOI] [PubMed] [Google Scholar]
  29. Oduola A. M., Weatherly N. F., Bowdre J. H., Desjardins R. E. Plasmodium falciparum: cloning by single-erythrocyte micromanipulation and heterogeneity in vitro. Exp Parasitol. 1988 Jun;66(1):86–95. doi: 10.1016/0014-4894(88)90053-7. [DOI] [PubMed] [Google Scholar]
  30. Parker W. B., Cheng Y. C. Metabolism and mechanism of action of 5-fluorouracil. Pharmacol Ther. 1990;48(3):381–395. doi: 10.1016/0163-7258(90)90056-8. [DOI] [PubMed] [Google Scholar]
  31. Payne D. Spread of chloroquine resistance in Plasmodium falciparum. Parasitol Today. 1987 Aug;3(8):241–246. doi: 10.1016/0169-4758(87)90147-5. [DOI] [PubMed] [Google Scholar]
  32. Peters W. The problem of drug resistance in malaria. Parasitology. 1985 Apr;90(Pt 4):705–715. doi: 10.1017/s003118200005232x. [DOI] [PubMed] [Google Scholar]
  33. Pogolotti A. L., Jr, Nolan P. A., Santi D. V. Methods for the complete analysis of 5-fluorouracil metabolites in cell extracts. Anal Biochem. 1981 Oct;117(1):178–186. doi: 10.1016/0003-2697(81)90708-9. [DOI] [PubMed] [Google Scholar]
  34. Queen S. A., Jagt D. L., Reyes P. In vitro susceptibilities of Plasmodium falciparum to compounds which inhibit nucleotide metabolism. Antimicrob Agents Chemother. 1990 Jul;34(7):1393–1398. doi: 10.1128/aac.34.7.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rathod P. K., Gomez Z. M. Plasmodium yoelii: oral delivery of 5-fluoroorotate to treat malaria in mice. Exp Parasitol. 1991 Nov;73(4):512–514. doi: 10.1016/0014-4894(91)90075-8. [DOI] [PubMed] [Google Scholar]
  36. Rathod P. K., Khatri A., Hubbert T., Milhous W. K. Selective activity of 5-fluoroorotic acid against Plasmodium falciparum in vitro. Antimicrob Agents Chemother. 1989 Jul;33(7):1090–1094. doi: 10.1128/aac.33.7.1090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rathod P. K., Khatri A. Synthesis and antiproliferative activity of threo-5-fluoro-L-dihydroorotate. J Biol Chem. 1990 Aug 25;265(24):14242–14249. [PubMed] [Google Scholar]
  38. Reyes P., Rathod P. K., Sanchez D. J., Mrema J. E., Rieckmann K. H., Heidrich H. G. Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum. Mol Biochem Parasitol. 1982 May;5(5):275–290. doi: 10.1016/0166-6851(82)90035-4. [DOI] [PubMed] [Google Scholar]
  39. Rieckmann K. H. Falciparum malaria: the urgent need for safe and effective drugs. Annu Rev Med. 1983;34:321–335. doi: 10.1146/annurev.me.34.020183.001541. [DOI] [PubMed] [Google Scholar]
  40. Santi D. V., McHenry C. S., Perriard E. R. A filter assay for thymidylate synthetase using 5-fluoro-2'-deoxyuridylate as an active site titrant. Biochemistry. 1974 Jan 29;13(3):467–470. doi: 10.1021/bi00700a011. [DOI] [PubMed] [Google Scholar]
  41. Santi D. V., McHenry C. S., Sommer H. Mechanism of interaction of thymidylate synthetase with 5-fluorodeoxyuridylate. Biochemistry. 1974 Jan 29;13(3):471–481. doi: 10.1021/bi00700a012. [DOI] [PubMed] [Google Scholar]
  42. Sherman I. W. Biochemistry of Plasmodium (malarial parasites). Microbiol Rev. 1979 Dec;43(4):453–495. doi: 10.1128/mr.43.4.453-495.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Siddiqui W. A., Kramer K., Richard-Crum S. M. In vitro cultivation and partial purification of Plasmodium falciparum antigen suitable for vaccination studies in Aotus monkeys. J Parasitol. 1978 Feb;64(1):168–169. [PubMed] [Google Scholar]
  44. Sirawaraporn W., Sirawaraporn R., Cowman A. F., Yuthavong Y., Santi D. V. Heterologous expression of active thymidylate synthase-dihydrofolate reductase from Plasmodium falciparum. Biochemistry. 1990 Dec 4;29(48):10779–10785. doi: 10.1021/bi00500a009. [DOI] [PubMed] [Google Scholar]
  45. Spears C. P., Shahinian A. H., Moran R. G., Heidelberger C., Corbett T. H. In vivo kinetics of thymidylate synthetase inhibition of 5-fluorouracil-sensitive and -resistant murine colon adenocarcinomas. Cancer Res. 1982 Feb;42(2):450–456. [PubMed] [Google Scholar]
  46. Trager W., Jensen J. B. Human malaria parasites in continuous culture. Science. 1976 Aug 20;193(4254):673–675. doi: 10.1126/science.781840. [DOI] [PubMed] [Google Scholar]
  47. Washtien W. L., Santi D. V. Assay of intracellular free and macromolecular-bound metabolites of 5-fluorodeoxyuridine and 5-fluorouracil. Cancer Res. 1979 Sep;39(9):3397–3404. [PubMed] [Google Scholar]
  48. Wyler D. J. Malaria--resurgence, resistance, and research. (First of two parts). N Engl J Med. 1983 Apr 14;308(15):875–878. doi: 10.1056/NEJM198304143081505. [DOI] [PubMed] [Google Scholar]

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