Advertisement

Acta Parasitologica

, Volume 61, Issue 4, pp 828–835 | Cite as

Development of a rapid and reliable assay for in vitro determination of compound cidality against the asexual stages of Plasmodium falciparum

  • Pavithra ViswanathEmail author
  • Sapna Morayya
  • Nikhil Rautela
  • Achyut Sinha
Article

Abstract

The pace of anti-malarial drug discovery is often impeded due to the lack of tools to determine the cidality of compounds in vitro. An anti-malarial compound must have a cidal mode of action, i.e. kill parasites, in order to quickly reduce parasite load. A static compound that merely inhibits growth must be identified early on in the discovery cascade. In this paper, we describe a high-throughput fluorescent assay for determination of the cidality of an anti-malarial compound. The assay works on the principle that cultures treated with a static compound will exhibit re-growth while treatment with a cidal compound leads to a marked reduction in parasite number. Parasite cultures are treated with the drug for 48 or 72 h following which the drug is washed off. Cultures are allowed to recover in drug-free media for 72 h and DNA content estimated using the fluorescent dye SyBR Green I. Following estimation of IC50 and IC99 values, we find that the IC99/IC50 ratio is a reliable indicator of the cidality of a compound. Cidal compounds like artemisinin and chloroquine display an IC99/IC50 ratio <5 while the ratio for a static compound like atovaquone is >5. This correlation holds true for various anti-malarial drugs with known modes of action. Importantly, the IC99/IC50 ratio drops to <5 when a compound becomes cidal in action with longer duration of treatment. The assay is robust, reliable and provides a fast and effective means for prioritizing cidal compounds for progression along the drug discovery cascade.

Keywords

Anti-malarial cidality mode of action malaria drug discovery SyBR Green I 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Alin M.H., Bjorkman A. 1994. Concentration and time dependency of artemisinin efficacy against Plasmodium falciparum in vitro. The American Journal of Tropical Medicine and Hygiene, 50, 771–776CrossRefGoogle Scholar
  2. Anthony M.P., Burrows J.N., Duparc S., Moehrle J.J., Wells T.N. 2012. The global pipeline of new medicines for the control and elimination of malaria. Malaria Journal, 11, 316. DOI: 10.1186/1475-2875-11-316CrossRefGoogle Scholar
  3. Bahamontes-Rosa N., Rodriguez-Alejandre A., Gonzalez-del-Rio R., Garcia-Bustos J.F., Mendoza-Losana A. 2012. A new molecular approach for cidal vs static antimalarial determination by quantifying mRNA levels. Molecular and Biochemical Parasitology, 181, 171–177. DOI: 10.1016/j.molbiopara.2011.11.003CrossRefGoogle Scholar
  4. Basco L.K., Marquet F., Makler M.M., Le Bras J. 1995a. Plasmodium falciparum and Plasmodium vivax: lactate dehydrogenase activity and its application for in vitro drug susceptibility assay. Experimental Parasitology, 80, 260–271CrossRefGoogle Scholar
  5. Basco L.K., Ramiliarisoa O., Le Bras J. 1994. In vitro activity of pyrimethamine, cycloguanil, and other antimalarial drugs against African isolates and clones of Plasmodium falciparum. The American Journal of Tropical Medicine and Hygiene, 50, 193–199CrossRefGoogle Scholar
  6. Basco L.K., Ramiliarisoa O., Le Bras J. 1995b. In vitro activity of atovaquone against the African isolates and clones of Plasmodium falciparum. The American Journal of Tropical Medicine and Hygiene, 53, 388–391CrossRefGoogle Scholar
  7. Burrows J.N., Chíbale K., Wells T.N. 2011a. The state of the art in anti-malarial drug discovery and development. Current Topics in Medicinal Chemistry, 11, 1226–1254CrossRefGoogle Scholar
  8. Burrows J.N., Leroy D., Lotharius J., Waterson D. 2011b. Challenges in antimalarial drug discovery. Future Medicinal Chemistry, 3, 1401–1412. DOI: 10.4155/fmc.11.91CrossRefGoogle Scholar
  9. Burrows J.N., van Huijsduijnen R.H., Mohrle J.J., Oeuvray C., Wells T.N. 2013. Designing the next generation of medicines for malaria control and eradication. Malaria Journal, 12, 187. DOI: 10.1186/1475-2875-12-187CrossRefGoogle Scholar
  10. Dahl E.L., Rosenthal PJ. 2007. Multiple Antibiotics Exert Delayed Effects against the Plasmodium falciparum Apicoplast. Antimicrobial Agents and Chemotherapy, 51, 3485–3490CrossRefGoogle Scholar
  11. Diagana T.T. 2015. Supporting malaria elimination with 21st century antimalarial agent drug discovery. Drug Discovery Today, 20, 1265–1270. DOI: 10.1016/j.drudis.2015.06.009CrossRefGoogle Scholar
  12. Dondorp A.M., Desakorn V., Pongtavornpinyo W., Sahassananda D., Silamut K., Chotivanich K., Newton P.N., Pitisuttithum P., Smithyman A.M., White N.J., Day N.P. 2005. Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2. PLoSMedicine, 2, e204. DOI: 10.1371/journal.pmed.0020204Google Scholar
  13. Guiguemde W.A., Shelat A.A., Garcia-Bustos J.F., Diagana T.T., Gamo F.J., Guy R.K. 2012. Global phenotypic screening for antimalarials. Chemistry & Biology, 19, 116–129. DOI: 10.1016/j.chembiol.2012.01.004CrossRefGoogle Scholar
  14. Lambros C., Vanderberg J.P. 1979. Synchronization of Plasmodium falciparum erythrocytic stages in culture. Journal of Parasitology, 65, 418–420CrossRefGoogle Scholar
  15. Le Manach C., Scheurer C., Sax S., Schleiferbock S., Cabrera D.G., Younis Y., Paquet T., Street L., Smith P., Ding X.C., Waterson D., Witty M.J., Leroy D., Chibale K., Wittlin S. 2013. Fast in vitro methods to determine the speed of action and the stage- specificity of anti-malarials in Plasmodium falciparum. Malaria Journal, 12, 424. DOI: 10.1186/1475-2875-12-424CrossRefGoogle Scholar
  16. Murray C.J., Ortblad K.F., Guinovart C., Lim S.S., Wolock T.M., Roberts D.A., et al. 2014. Global, regional, and national incidence and mortality for HIV, tuberculosis, and malaria during 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet, 384, 1005–1070. DOI: 10.1016/s0140-6736(14)60844-8CrossRefGoogle Scholar
  17. Murray C.J., Rosenfeld L.C., Lim S.S., Andrews K.G., Foreman K.J., Haring D., Fullman N., Naghavi M., Lozano R., Lopez A.D. 2012. Global malaria mortality between 1980 and 2010: a systematic analysis. Lancet, 379, 413–431. DOI: 10.1016/s0140-6736(12)60034-8CrossRefGoogle Scholar
  18. Newby G., Bennett A., Larson E., Cotter C., Shretta R., Phillips A.A., Feachem R.G. 2016. The path to eradication: a progress report on the malaria-eliminating countries. Lancet, 387, 1775–1784. DOI: 10.1016/s0140-6736(16)00230-0CrossRefGoogle Scholar
  19. Nilsen A., LaCrue A.N., White K.L., Forquer I.P., Cross R.M., Marfurt J., Mather M.W., Delves M.J., Shackleford D.M., Saenz F.E., Morrisey J.M., Steuten J., Mutka T., Li Y., Wirjanata G., Ryan E., Duffy S., Kelly J.X., Sebayang B.F., Zeeman A.M., Noviyanti R., Sinden R.E., Kocken C.H., Price R.N., Avery V.M., Angulo-Barturen I., Jimenez-Diaz M.B., Ferrer S., Herreros E., Sanz L.M., Gamo F.J., Bathurst I., Burrows J.N., Siegl P., Guy R.K., Winter R.W., Vaidya A.B., Charman S.A., Kyle D.E., Manetsch R., Riscoe M.K. 2013. Quinolone-3-diarylethers: a new class of antimalarial drug. Science Translational Medicine, 5, 177ra137. DOI: 10.1126/scitranslmed. 3005029Google Scholar
  20. Painter H.J., Morrisey J.M., Vaidya A.B. 2010. Mitochondrial electron transport inhibition and viability of intraerythrocytic Plasmodium falciparum. Antimicrobial Agents and Chemotherapy, 54, 5281–5287. DOI: 10.1128/aac.00937-10CrossRefGoogle Scholar
  21. Phillips M.A., Lotharius J., Marsh K., White J., Dayan A., White K.L., Njoroge J.W., El Mazouni F., Lao Y., Kokkonda S., Tomchick D.R., Deng X., Laird T., Bhatia S.N., March S., Ng L., Fidock D.A., Wittlin S., Lafuente-Monasterio M., Benito F.J., Alonso L.M., Martinez M.S., Jimenez-Diaz M.B., Bazaga S.F., Angulo-Barturen I., Haselden J.N., Louttit J., Cui Y., Sridhar A., Zeeman A.M., Kocken C., Sauerwein R., Dechering K., Avery V.M., Duffy S., Delves M., Sinden R., Ruecker A., Wickham K.S., Rochford R., Gahagen J., Iyer L., Riccio E., Mirsalis J., Bathhurst I., Rueckle T., Ding X., Campo B., Leroy D., Rogers M.J., Rathod P.K., Burrows J.N., Charman S.A. 2015. A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria. Science Translational Medicine, 7, 296ra111. DOI: 10.1126/scitranslmed.aaa6645CrossRefGoogle Scholar
  22. Rottmann M., McNamara C., Yeung B.K., Lee M.C., Zou B., Russell B., Seitz P., Plouffe D.M., Dharia N.V., Tan J., Cohen S.B., Spencer K.R., Gonzalez-Paez G.E., Lakshminarayana S.B., Goh A., Suwanarusk R., Jegla T., Schmitt E.K., Beck H.P., Brun R., Nosten F., Renia L., Dartois V., Keller T.H., Fidock A., Winzeler E.A., Diagana T.T. 2010. Spiroindolones, a potent compound class for the treatment of malaria. Science, 329, 1175–1180. DOI: 10.1126/science.1193225CrossRefGoogle Scholar
  23. Sanz L.M., Crespo B., De-Cozar C., Ding X.C., Llergo J.L., Burrows J.N., Garcia-Bustos J.F., Gamo F.J. 2012. P. falciparum in vitro killing rates allow to discriminate between different antimalarial mode-of-action. PLoS One, 7, e30949. DOI: 10.1371/journal.pone.0030949CrossRefGoogle Scholar
  24. Trager W., Jensen J.B. 1976. Human malaria parasites in continuous culture. Science, 193, 673–675CrossRefGoogle Scholar
  25. Visser B.J., van Vugt M., Grobusch M.P. 2014. Malaria: an update on current chemotherapy. Expert Opinion on Pharmacotherapy, 15, 2219–2254. DOI: 10.1517/14656566.2014.944499CrossRefGoogle Scholar
  26. Wells T.N., Alonso P.L., Gutteridge W.E. 2009. New medicines to improve control and contribute to the eradication of malaria. Nature Reviews Drug Discovery, 8, 879–891. DOI: 10.1038/nrd2972CrossRefGoogle Scholar
  27. White N.J. 1997. Assessment of the pharmacodynamic properties of antimalarial drugs in vivo. Antimicrobial Agents and Chemotherapy, 41, 1413–1422CrossRefGoogle Scholar
  28. White N.J., Pukrittayakamee S., Phyo A.P., Rueangweerayut R., Nosten F., Jittamala P., Jeeyapant A., Jain J.P., Lefèvre G., Li R., Magnusson B., Diagana T.T., Leong F.J. 2014. Spiroindolone KAE609 for Falciparum and Vivax Malaria. New England Journal of Medicine, 371, 403–410. DOI: 10.1056/NEJMoa1315860CrossRefGoogle Scholar
  29. WHO 2015. World Malaria Report 2015. http://www.who.int/malaria/publications/world-malaria-report-2015/en/Google Scholar

Copyright information

© Witold Stefański Institute of Parasitology, Polish Academy of Sciences 2016

Authors and Affiliations

  • Pavithra Viswanath
    • 1
    Email author
  • Sapna Morayya
    • 1
  • Nikhil Rautela
    • 1
  • Achyut Sinha
    • 1
  1. 1.Department of Radiology and Biomedical ImagingUniversity of California San FranciscoSan FranciscoUSA

Personalised recommendations