Skip to main content
NCBI home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1996 Jul;40(7):1628–1632. doi: 10.1128/aac.40.7.1628

Kinetics of Plasmodium falciparum thymidylate synthase: interactions with high-affinity metabolites of 5-fluoroorotate and D1694.

M Hekmat-Nejad 1, P K Rathod 1
PMCID: PMC163385  PMID: 8807052

Abstract

Consistent with a proposed mechanism for the potent antimalarial activity of 5-fluoroorotate, 5-fluoro-2'-deoxyuridylate inhibited Plasmodium falciparum thymidylate synthase with a Ki of 2 nM. Steady-state kinetics revealed no significant differences between malarial and mammalian thymidylate synthases. Thus, additional biochemical parameters must underlie the selective antimalarial activity of 5-fluoroorotate. A polyglutamylated folate analog, D1694-(glu)4, was also a potent inhibitor of malarial thymidylate synthase (Kis = 1.5 nM).

Full Text

The Full Text of this article is available as a PDF (282.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Banerjee C. K., Bennett L. L., Jr, Brockman R. W., Sani B. P., Temple C., Jr A convenient procedure for purification of thymidylate synthase from L1210 cells. Anal Biochem. 1982 Apr;121(2):275–280. doi: 10.1016/0003-2697(82)90479-1. [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. Carreras C. W., Santi D. V. The catalytic mechanism and structure of thymidylate synthase. Annu Rev Biochem. 1995;64:721–762. doi: 10.1146/annurev.bi.64.070195.003445. [DOI] [PubMed] [Google Scholar]
  4. 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]
  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., 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]
  8. Davisson V. J., Sirawaraporn W., Santi D. V. Expression of human thymidylate synthase in Escherichia coli. J Biol Chem. 1989 Jun 5;264(16):9145–9148. [PubMed] [Google Scholar]
  9. Dolnick B. J., Cheng Y. c. Human thymidylate synthetase derived from blast cells of patients with acture myelocytic leukemia. Purification and chracterization. J Biol Chem. 1977 Nov 10;252(21):7697–7703. [PubMed] [Google Scholar]
  10. Ferone R., Burchall J. J., Hitchings G. H. Plasmodium berghei dihydrofolate reductase. Isolation, properties, and inhibition by antifolates. Mol Pharmacol. 1969 Jan;5(1):49–59. [PubMed] [Google Scholar]
  11. Ferone R., Roland S. Dihydrofolate reductase: thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5802–5806. doi: 10.1073/pnas.77.10.5802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. 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]
  15. Hall S. J., Sims P. F., Hyde J. E. Functional expression of the dihydrofolate reductase and thymidylate synthetase activities of the human malaria parasite Plasmodium falciparum in Escherichia coli. Mol Biochem Parasitol. 1991 Apr;45(2):317–330. doi: 10.1016/0166-6851(91)90100-k. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. Ingraham H. A., Dickey L., Goulian M. DNA fragmentation and cytotoxicity from increased cellular deoxyuridylate. Biochemistry. 1986 Jun 3;25(11):3225–3230. doi: 10.1021/bi00359a022. [DOI] [PubMed] [Google Scholar]
  20. Jackman A. L., Taylor G. A., Gibson W., Kimbell R., Brown M., Calvert A. H., Judson I. R., Hughes L. R. ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent inhibitor of L1210 tumor cell growth in vitro and in vivo: a new agent for clinical study. Cancer Res. 1991 Oct 15;51(20):5579–5586. [PubMed] [Google Scholar]
  21. Kunz B. A., Kohalmi S. E., Kunkel T. A., Mathews C. K., McIntosh E. M., Reidy J. A. International Commission for Protection Against Environmental Mutagens and Carcinogens. Deoxyribonucleoside triphosphate levels: a critical factor in the maintenance of genetic stability. Mutat Res. 1994 Aug;318(1):1–64. doi: 10.1016/0165-1110(94)90006-x. [DOI] [PubMed] [Google Scholar]
  22. Lockshin A., Moran R. G., Danenberg P. V. Thymidylate synthetase purified to homogeneity from human leukemic cells. Proc Natl Acad Sci U S A. 1979 Feb;76(2):750–754. doi: 10.1073/pnas.76.2.750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Pattanakitsakul S. N., Ratanaphan A., Ruenwongsa P. Comparative studies on thymidylate synthetase from P. berghei and mouse reticulocytes. Comp Biochem Physiol B. 1985;80(3):583–587. doi: 10.1016/0305-0491(85)90295-0. [DOI] [PubMed] [Google Scholar]
  25. Radparvar S., Houghton P. J., Houghton J. A. Characteristics of thymidylate synthase purified from a human colon adenocarcinoma. Arch Biochem Biophys. 1988 Jan;260(1):342–350. doi: 10.1016/0003-9861(88)90458-4. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Rathod P. K., Leffers N. P., Young R. D. Molecular targets of 5-fluoroorotate in the human malaria parasite, Plasmodium falciparum. Antimicrob Agents Chemother. 1992 Apr;36(4):704–711. doi: 10.1128/aac.36.4.704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rathod P. K., Reshmi S. Susceptibility of Plasmodium falciparum to a combination of thymidine and ICI D1694, a quinazoline antifolate directed at thymidylate synthase. Antimicrob Agents Chemother. 1994 Mar;38(3):476–480. doi: 10.1128/aac.38.3.476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Reyes P., Heidelberger C. Fluorinated pyrimidines. XXVI. Mammalian thymidylate synthetase: its mechanism of action and inhibition by fluorinated nucleotides. Mol Pharmacol. 1965 Jul;1(1):14–30. [PubMed] [Google Scholar]
  31. 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]
  32. Roberts D. An isotopic assay for thymidylate synthetase. Biochemistry. 1966 Nov;5(11):3546–3548. doi: 10.1021/bi00875a022. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. Ward W. H., Kimbell R., Jackman A. L. Kinetic characteristics of ICI D1694: a quinazoline antifolate which inhibits thymidylate synthase. Biochem Pharmacol. 1992 May 8;43(9):2029–2031. doi: 10.1016/0006-2952(92)90646-z. [DOI] [PubMed] [Google Scholar]
  38. Yoshioka A., Tanaka S., Hiraoka O., Koyama Y., Hirota Y., Ayusawa D., Seno T., Garrett C., Wataya Y. Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death. J Biol Chem. 1987 Jun 15;262(17):8235–8241. [PubMed] [Google Scholar]
  39. Young R. D., Rathod P. K. Clonal viability measurements on Plasmodium falciparum to assess in vitro schizonticidal activity of leupeptin, chloroquine, and 5-fluoroorotate. Antimicrob Agents Chemother. 1993 May;37(5):1102–1107. doi: 10.1128/aac.37.5.1102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zolg J. W., Plitt J. R., Chen G. X., Palmer S. Point mutations in the dihydrofolate reductase-thymidylate synthase gene as the molecular basis for pyrimethamine resistance in Plasmodium falciparum. Mol Biochem Parasitol. 1989 Oct;36(3):253–262. doi: 10.1016/0166-6851(89)90173-4. [DOI] [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES