Skip to main content
NCBI home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1993 May;37(5):1102–1107. doi: 10.1128/aac.37.5.1102

Clonal viability measurements on Plasmodium falciparum to assess in vitro schizonticidal activity of leupeptin, chloroquine, and 5-fluoroorotate.

R D Young 1, P K Rathod 1
PMCID: PMC187909  PMID: 8517698

Abstract

Until now, the in vitro activity of potential antimalarial agents has been evaluated primarily by monitoring decreases in parasite proliferation. These traditional assays do not distinguish between compounds that arrest proliferation of parasites and compounds that kill them. In this report, a more complex in vitro cytocidal assay for Plasmodium falciparum is described. This assay measures the clonal viability of P. falciparum after the parasites have been treated with an antimalarial agent. The new assay was used to assess cytocidal activities of three antimalarial agents that work through unrelated mechanisms. Leupeptin, a protease inhibitor, arrested the proliferation of W2 clones of P. falciparum at a MIC of 50 microM, but at least 80% of leupeptin-treated cells were viable as judged by the cytocidal assay. On the other hand, chloroquine at 1 microM, its MIC for W2 cells, not only arrested parasite proliferation but also killed more than 99% of the cells. Earlier studies had shown that treatment of P. falciparum with 100 nM 5-fluoroorotate for 48 h was sufficient to inhibit parasite proliferation and parasite thymidylate synthase but not enough to cause significant incorporation of 5-fluoropyrimidines in parasite nucleic acids. By using the new schizonticidal assay, these conditions were found to be necessary and sufficient to kill all parasites in culture. Results of these studies are consistent with the hypothesis that 5-fluoroorotate-based inactivation of P. falciparum thymidylate synthase triggers a lethal mechanism against malarial parasites.

Full text

PDF
1102

Images in this article

1104Image
on p.1104

Selected References

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

  1. Barker R. H., Jr, Brandling-Bennett A. D., Koech D. K., Mugambi M., Khan B., David R., David J. R., Wirth D. F. Plasmodium falciparum: DNA probe diagnosis of malaria in Kenya. Exp Parasitol. 1989 Oct;69(3):226–233. doi: 10.1016/0014-4894(89)90069-6. [DOI] [PubMed] [Google Scholar]
  2. Coller H. A., Coller B. S. Poisson statistical analysis of repetitive subcloning by the limiting dilution technique as a way of assessing hybridoma monoclonality. Methods Enzymol. 1986;121:412–417. doi: 10.1016/0076-6879(86)21039-3. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Dluzewski A. R., Rangachari K., Wilson R. J., Gratzer W. B. Plasmodium falciparum: protease inhibitors and inhibition of erythrocyte invasion. Exp Parasitol. 1986 Dec;62(3):416–422. doi: 10.1016/0014-4894(86)90050-0. [DOI] [PubMed] [Google Scholar]
  5. Ginsburg H., Krugliak M. Quinoline-containing antimalarials--mode of action, drug resistance and its reversal. An update with unresolved puzzles. Biochem Pharmacol. 1992 Jan 9;43(1):63–70. doi: 10.1016/0006-2952(92)90662-3. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Lal A. A., Changkasiri S., Hollingdale M. R., McCutchan T. F. Ribosomal RNA-based diagnosis of Plasmodium falciparum malaria. Mol Biochem Parasitol. 1989 Aug;36(1):67–71. doi: 10.1016/0166-6851(89)90201-6. [DOI] [PubMed] [Google Scholar]
  9. McLaughlin G. L., Decrind C., Dayal-Drager R., Hassan-King M., Subramanian S., Greenwood B. M. Optimization of a rapid nonisotopic DNA probe assay for Plasmodium falciparum in the Gambia. J Clin Microbiol. 1991 Jul;29(7):1517–1519. doi: 10.1128/jcm.29.7.1517-1519.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. Richards W. H., Maples B. K. Studies on Plasmodium falciparum in continuous cultivation. I. The effect of chloroquine and pyrimethamine on parasite growth and viability. Ann Trop Med Parasitol. 1979 Apr;73(2):99–108. [PubMed] [Google Scholar]
  16. Rosario V. Cloning of naturally occurring mixed infections of malaria parasites. Science. 1981 May 29;212(4498):1037–1038. doi: 10.1126/science.7015505. [DOI] [PubMed] [Google Scholar]
  17. Rosenthal P. J., McKerrow J. H., Aikawa M., Nagasawa H., Leech J. H. A malarial cysteine proteinase is necessary for hemoglobin degradation by Plasmodium falciparum. J Clin Invest. 1988 Nov;82(5):1560–1566. doi: 10.1172/JCI113766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]

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

RESOURCES