Determination of the Cytostatic and Cytocidal Activities of Antimalarial Compounds and Their Combination Interactions

Katy S. Sherlach1, Paul D. Roepe1

1 Department of Chemistry and Department of Biochemistry and Cellular and Molecular Biology, Georgetown University, Washington, D.C.
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch140125
Online Posting Date:  December, 2014
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Abstract

Determining the antiplasmodial activity of candidate antimalarial drugs in vitro identifies new therapies for drug‐resistant malaria. Importantly though, activity can be either growth‐inhibitory (cytostatic) or parasite‐kill (cytocidal), or both. The simple methods described here can allow for distinction between these activities, as well as definition of drug interactions between two or more compounds. The latter is important in the definition of novel drug combination therapy for malaria. These methods involve live malarial parasite red blood cell culture, routine pharmacology, high‐throughput detection of parasite DNA with fluorescent reporters, and routine mathematical analysis of dose‐response curves. The techniques and approaches are accessible to most laboratories and require minimal special equipment beyond a fluorescent plate reader and tissue culture facilities. © 2014 by John Wiley & Sons, Inc.

Keywords: Plasmodium falciparum; antiplasmodial activity; cytostatic; cytocidal; drug combination analysis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cytostatic (IC50) or Growth‐Inhibition Assay (Adapted from Bennett et al., )
  • Basic Protocol 2: Cytocidal (LD50) or Parasite‐Kill Assay (Paguio et al., )
  • Basic Protocol 3: Limiting Serial‐Dilution Assay to Determine Efficacy of Parasite Kill (Adapted From Sanz et al., )
  • Basic Protocol 4: Modifying the Cytostatic and Cytocidal Assays for Assessing Synergy of Drug Combinations—Adapted From Chou and Talalay () and Suberu et al. ()
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Cytostatic (IC50) or Growth‐Inhibition Assay (Adapted from Bennett et al., )

  Materials
  • 10% Giemsa dye (Sigma)
  • Plasmodium falciparum culture at 2% hematocrit (volume packed RBC/volume medium); if needed, stock parasite culture can be obtained from MR4 (http://www.mr4.org/) or other suppliers
  • Complete medium (see recipe)
  • Fresh O+ human whole blood, e.g., Biochemed (http://www.biochemed.com/), washed to isolate the erythrocytes, and stored in incomplete medium (see recipe)
  • Drug stock solutions (in DMSO, deionized water, or 50% ethanol)
  • 5% CO 2, 5% O 2, and 90% N 2 gas mix, e.g., Roberts Oxygen (http://www.robertsoxygen.com/)
  • 10,000× SYBR Green I stock from the vendor (Invitrogen), or synthesized (see Bennett et al., )
  • 96‐well plate, clear well bottom with opaque well sides to prevent fluorescence interference
  • Air‐tight chamber for storing properly gassed 96‐well plates: the chamber should be large enough to accommodate several plates and have convenient ports for gassing and de-gassing (e.g., a Billups-Rothenberg modular incubator chamber; see http://www.brincubator.com/incubatorchamber.htm)
  • 37°C Incubator
  • Light microscope
  • Spectra Max Gemini EM Microplate Reader, or similar bottom‐read fluorescence plate‐reader
  • Microsoft Excel, Sigma Plot, or other data analysis program that allows nonlinear curve fitting

Basic Protocol 2: Cytocidal (LD50) or Parasite‐Kill Assay (Paguio et al., )

  Additional Materials
  • Eppendorf 5804 centrifuge with A‐2‐DWP rotor
  • Multichannel pipets
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Figures

Videos

Literature Cited

Literature Cited
  Bennett, T.N., Paguio, M., Gligorijevic, B., Seudieu, C., Kosar, A.D., Davidson, E., and Roepe, P.D. 2004. Novel, rapid, and inexpensive cell‐based quantification of antimalarial drug efficacy. Antimicrob. Agents Chemother. 48:1807‐1810.
  Berenbaum, M.C. 1978. A method for testing for synergy with any number of agents. J. Infect. Dis. 137:122‐130.
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  Burrows, J.N., van Huijsduijnen, R.H., Möhrle, J.J., Oeuvray, C., and Wells, T.N.C. 2013. Designing the next generation of medicines for malaria control and eradication. Malaria J. 12:187‐207.
  Cabrera, M., Paguio, M.F., Xie, C., and Roepe, P.D. 2009. Reduced digestive vacuolar accumulation of chloroquine is not linked to resistance to chloroquine toxicity. Biochemistry 48:11152‐11154.
  Chou, T.C. and Talalay, P. 1984. Quantitative analysis of dose‐effect relationships: The combined effects of multiple drugs of enzyme inhibitors. Adv. Enzyme Regul. 22:27‐55.
  Desjardins, R.E., Canfield, C.J., Haynes, J.D., and Chulay, J.D. 1979. Antimicrob. Agents Chemother. 16:710‐718.
  Druilhe, P., Moreno, A., Blanc, C., Brasseur, P.H., and Jaquier, P. 2001. A colorimetric in vitro drug sensitivity assay for Plasmodium falciparum based on a highly sensitive double‐site lactate dehydrogenase antigen‐capture enzyme‐linked immunosorbent assay. Am. J. Trop. Med. Hyg. 64:233‐241.
  Gaviria, D., Paguio, M.F., Turnbull, L.B., Tan, A., Siriwardana, A., Ghosh, D., Ferdig, M.T., Sinai, A.P., and Roepe, P.D. 2013. A process similar to autophagy is associated with cytocidal chloroquine resistance in Plasmodium falciparum. PLoS One 8:e79059.
  Gligorijevic, B., Purdy, K., Elliot, D.A., Cooper, R.A., and Roepe, P.D. 2008. Stage independent chloroquine resistance and chloroquine toxicity revealed via spinning disk confocal microscopy. Mol. Biochem. Parasitol. 159:7‐23.
  Gorka, A.P., Alumasa, J.N., Sherlach, K.S., Jacobs, L.M., Nickley, K.B., Brower, J.P., de Dios, A.C., and Roepe, P.D. 2013a. Cytostatic versus cytocidal activities of chloroquine analogues and inhibition of hemozoin crystal growth. Antimicrob. Agents Chemother. 57:356‐364.
  Gorka, A.P., Sherlach, K.S., de Dios, A.C., and Roepe, P.D. 2013b. Relative to quinine and quinidine, their 9‐epimers exhibit decreased cytostatic activity and altered heme binding but similar cytocidal activity versus Plasmodium falciparum. Antimicrob. Agents. Chemother. 57:365‐374.
  Hasenkamp, S., Sidaway, A., Devine, O., Roye, R., and Horrocks, P. 2013. Evaluation of bioluminescence‐based assays of anti‐malarial drug activity. Malaria J. 12:58‐67.
  Lukens, A.K., Ross, L.S., Heidebrecht, R., Gamo, F.J., Lafuente‐Monasterio, M.J., Booker, M.L., Hartl, D.L., Wiegand, R.C., and Wirth, D.F. 2014. Harnessing evolutionary fitness in Plasmodium falciparum for drug discovery and suppressing resistance. Proc. Natl. Acad. Sci. U.S.A. 111:799‐804.
  Matthews, H., Usman‐Idris, M., Khan, F., Read, M., and Nirmalan, N. 2013. Drug repositioning as a route to anti‐malarial drug discovery: Preliminary investigation of the in vitro anti‐malarial efficacy of emetine dihydrochloride hydrate. Malaria J. 12:359‐370.
  Paguio, M.F., Bogle, K.L., and Roepe, P.D. 2011. Plasmodium falciparum resistance to cytocidal versus cytostatic effects of chloroquine. Mol. Biochem. Parasitol. 178:1‐6.
  Quashie, N.B., de Koning, H.P., and Ranford‐Cartwright, L.C. 2006. An improved and highly sensitive microfluorimetric method for assessing susceptibility of Plasmodium falciparum to antimalarial drugs in vitro. Malaria J. 5:95‐101.
  Sanz, L.M., Crespo, B., De‐Cósar, C., Ding, X.C., Llergo, J.L., Burrows, J.N.; García‐Bustos, J.F., and Gamo, F.J. 2012. P. falciparum in vitro killing rates allow to discriminate between different antimalarial mode‐of action. PLoS One 7:e30949.
  Suberu, J.O., Gorka, A.P., Jacobs, L., and Roepe, P.D., Sullivan, N., Barker, G.C., and Lapkin, A.A. 2013. Anti‐plasmodial polyvalent interactions in Artemisia annua L. aqueous extract—possible synergistic and resistance mechanisms. PLoS One 8:e80790.
Key References
  Bennett et al., 2004. See above.
  This article describes the design and optimization of the SYBR Green I‐based cytostatic assay, and the advantages this measurement technique has compared to the other published assays. A similar assay was published simultaneously in the same issue of the same journal by Riscoe and colleagues: Smilkstein, M., Sriwilaijaroen, N., Kelly, J.X., Wilairat, P., and Riscoe, M. 2004. Simple and inexpensive fluorescence‐based technique for high‐throughput antimalarial drug screening. Antimicrob Agents Chemother. 48:1803‐1806.
  Gaviria et al., 2013. See above.
  This article shows that the genetic determinants for cytostatic vs. cytocidal potency in CQR P. falciarpum are distinct. This underlines the conclusions from several other papers from the Roepe laboratory showing that mechanisms of cytostatic vs. cytocidal drug action are likely distinct for quinoline antimalarial drugs.
  Paguio et al., 2011. See above.
  This article describes the design and optimization of the cytocidal SYBR Green I cytocidal assay, as well as initial observations related to differences between resistance to the cytostatic versus cytocidal effects of chloroquine.
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