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

Pavithra Viswanath; Sapna Morayya; Nikhil Rautela; Achyut Sinha

doi:10.1515/ap-2016-0115

Article

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

Pavithra Viswanath , Sapna Morayya , Nikhil Rautela and Achyut Sinha

Published/Copyright: October 24, 2016

Published by

Become an author with De Gruyter Brill

Author Information

From the journal Acta Parasitologica Volume 61 Issue 4

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 IIntroduction

Acknowledgements

The authors were employees of AstraZeneca India Pvt Ltd and all the experimental work was carried out at the R & D site at Bangalore.

Conflict of Interests

Authors declares no conflict of interests.

References

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, 771–77610.4269/ajtmh.1994.50.771Search in Google Scholar PubMed

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. 10.1186/1475-2875-11-316Search in Google Scholar PubMed PubMed Central

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. 10.1016/j.molbiopara.2011.11.003Search in Google Scholar PubMed

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, 260–27110.1006/expr.1995.1032Search in Google Scholar PubMed

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, 193–19910.4269/ajtmh.1994.50.193Search in Google Scholar PubMed

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, 388–39110.4269/ajtmh.1995.53.388Search in Google Scholar PubMed

Burrows J.N., Chibale K., Wells T.N. 2011a. The state of the art in anti-malarial drug discovery and development. Current Topics in Medicinal Chemistry, 11, 1226–125410.2174/156802611795429194Search in Google Scholar PubMed

Burrows J.N., Leroy D., Lotharius J., Waterson D. 2011b. Challenges in antimalarial drug discovery. Future Medicinal Chemistry, 3, 1401–1412. 10.4155/fmc.11.91Search in Google Scholar PubMed

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. 10.1186/1475-2875-12-187Search in Google Scholar PubMed PubMed Central

Dahl E.L., Rosenthal P.J. 2007. Multiple Antibiotics Exert Delayed Effects against the Plasmodium falciparum Apicoplast. Antimicrobial Agents and Chemotherapy, 51, 3485–349010.1128/AAC.00527-07Search in Google Scholar PubMed PubMed Central

Diagana T.T. 2015. Supporting malaria elimination with 21st century antimalarial agent drug discovery. Drug Discovery Today, 20, 1265–1270. 10.1016/j.drudis.2015.06.009Search in Google Scholar

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. 10.1371/journal.pmed.0020204Search in Google Scholar

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. 10.1016/j.chembiol.2012.01.004Search in Google Scholar

Lambros C., Vanderberg J.P. 1979. Synchronization of Plasmodium falciparum erythrocytic stages in culture. Journal of Para-sitology, 65, 418–42010.2307/3280287Search in Google Scholar

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 stagespecificity of anti-malarials in Plasmodium falciparum. Malaria Journal, 12, 424. 10.1186/1475-2875-12-424Search in Google Scholar

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. 10.1016/ s0140-6736(14)60844-8Search in Google Scholar

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 sys tematic analysis. Lancet, 379, 413–431. 10.1016/s01406736(12)60034-8Search in Google Scholar

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. 10.1016/s0140-6736(16)00230-0Search in Google Scholar

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-3diarylethers: a new class of antimalarial drug. Science Translational Medicine, 5, 177ra137. 10.1126/scitranslmed. 3005029Search in Google Scholar

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. 10.1128/aac.00937-10Search in Google Scholar PubMed PubMed Central

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 C.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. 10.1126/scitranslmed.aaa6645Search in Google Scholar PubMed PubMed Central

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 D.A., Winzeler E.A., Diagana T.T. 2010. Spiroindolones, a potent compound class for the treatment of malaria. Science, 329, 1175–1180. 10.1126/science.1193225Search in Google Scholar PubMed PubMed Central

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. 10.1371/journal.pone.0030949Search in Google Scholar PubMed PubMed Central

Trager W., Jensen J.B. 1976. Human malaria parasites in continuous culture. Science, 193, 673–67510.1126/science.781840Search in Google Scholar PubMed

Visser B.J., van Vugt M., Grobusch M.P. 2014. Malaria: an update on current chemotherapy. Expert Opinion on Pharmacotherapy, 15, 2219–2254. 10.1517/14656566.2014.944499Search in Google Scholar PubMed

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. 10.1038/nrd2972Search in Google Scholar PubMed

White N.J. 1997. Assessment of the pharmacodynamic properties of antimalarial drugs in vivo. Antimicrobial Agents and Chemotherapy, 41, 1413–142210.1128/AAC.41.7.1413Search in Google Scholar PubMed PubMed Central

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. 10.1056/NEJ- Moal315860Search in Google Scholar

WHO 2015. World Malaria Report 2015. http://www.who.int/malaria/publications/world-malaria-report-2015/en/Search in Google Scholar

Received: 2016-6-1

Revised: 2016-7-18

Accepted: 2016-8-4

Published Online: 2016-10-24

Published in Print: 2016-12-1

You are currently not able to access this content.

Articles in the same Issue

https://doi.org/10.1515/ap-2016-0115

Keywords for this article

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