A high-throughput drug screen for Entamoeba histolytica identifies a new lead and target


Entamoeba histolytica, a protozoan intestinal parasite, is the causative agent of human amebiasis. Amebiasis is the fourth leading cause of death and the third leading cause of morbidity due to protozoan infections worldwide1, resulting in 70,000 deaths annually. E. histolytica has been listed by the National Institutes of Health as a category B priority biodefense pathogen in the United States. Treatment relies on metronidazole2, which has adverse effects3, and potential resistance of E. histolytica to the drug is an increasing concern4,5. To facilitate drug screening for this anaerobic protozoan, we developed and validated an automated, high-throughput screen (HTS). This screen identified auranofin, a US Food and Drug Administration (FDA)-approved drug used therapeutically for rheumatoid arthritis, as active against E. histolytica in culture. Auranofin was ten times more potent against E. histolytica than metronidazole. Transcriptional profiling and thioredoxin reductase assays suggested that auranofin targets the E. histolytica thioredoxin reductase, preventing the reduction of thioredoxin and enhancing sensitivity of trophozoites to reactive oxygen-mediated killing. In a mouse model of amebic colitis and a hamster model of amebic liver abscess, oral auranofin markedly decreased the number of parasites, the detrimental host inflammatory response and hepatic damage. This new use of auranofin represents a promising therapy for amebiasis, and the drug has been granted orphan-drug status from the FDA.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Assay development for HTS and scatter plot of percentage inhibition of each well from plates of compound library.
Figure 2: Inhibition of EhTrxR by auranofin and its analogs and in vitro and in vivo effects of auranofin on trophozoites.
Figure 3: Effect of auranofin or metronidazole on animal models of amebic colitis and liver abscesses.

Accession codes

Primary accessions



  1. 1

    World Health Organization. The World Health Report 1998: life in the 21st century: a vision for all. <http://www.who.int/whr/1998/en/whr98_en.pdf> (1998).

  2. 2

    Freeman, C.D., Klutman, N.E. & Lamp, K.C. Metronidazole. A therapeutic review and update. Drugs 54, 679–708 (1997).

    CAS  Article  Google Scholar 

  3. 3

    Krogstad, D.J. & Cedeno, J.R. Problems with current therapeutic regimens. in Amebiasis: Human Infection by Entamoeba Histolytica (ed. Ravdin, J.I.) 741–748 (John Wiley & Sons, New York, 1988).

  4. 4

    Samarawickrema, N.A., Brown, D.M., Upcroft, J.A., Thammapalerd, N. & Upcroft, P. Involvement of superoxide dismutase and pyruvate:ferredoxin oxidoreductase in mechanisms of metronidazole resistance in Entamoeba histolytica. J. Antimicrob. Chemother. 40, 833–840 (1997).

    CAS  Article  Google Scholar 

  5. 5

    Wassmann, C., Hellberg, A., Tannich, E. & Bruchhaus, I. Metronidazole resistance in the protozoan parasite Entamoeba histolytica is associated with increased expression of iron-containing superoxide dismutase and peroxiredoxin and decreased expression of ferredoxin 1 and flavin reductase. J. Biol. Chem. 274, 26051–26056 (1999).

    CAS  Article  Google Scholar 

  6. 6

    Seifert, K. et al. Effects of miltefosine and other alkylphosphocholines on human intestinal parasite Entamoeba histolytica. Antimicrob. Agents Chemother. 45, 1505–1510 (2001).

    CAS  Article  Google Scholar 

  7. 7

    Ghosh, S. et al. Effects of bisphosphonates on the growth of Entamoeba histolytica and Plasmodium species in vitro and in vivo. J. Med. Chem. 47, 175–187 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Singh, S., Athar, F. & Azam, A. Synthesis, spectral studies and in vitro assessment for antiamoebic activity of new cyclooctadiene ruthenium(II) complexes with 5-nitrothiophene-2-carboxaldehyde thiosemicarbazones. Bioorg. Med. Chem. Lett. 15, 5424–5428 (2005).

    CAS  Article  Google Scholar 

  9. 9

    Ashburn, T.T. & Thor, K.B. Drug repositioning: identifying and developing new uses for existing drugs. Nat. Rev. Drug Discov. 3, 673–683 (2004).

    CAS  Article  Google Scholar 

  10. 10

    Makioka, A., Kumagai, M., Ohtomo, H., Kobayashi, S. & Takeuchi, T. Effect of calcium antagonists, calcium channel blockers and calmodulin inhibitors on the growth and encystation of Entamoeba histolytica and E. invadens. Parasitol. Res. 87, 833–837 (2001).

    CAS  Article  Google Scholar 

  11. 11

    Gottlieb, N.L. Pharmacology of auranofin: overview and update. Scand. J. Rheumatol. Suppl. 63, 19–28 (1986).

    CAS  PubMed  Google Scholar 

  12. 12

    Kuntz, A.N. et al. Thioredoxin glutathione reductase from Schistosoma mansoni: an essential parasite enzyme and a key drug target. PLoS Med. 4, e206 (2007).

    Article  Google Scholar 

  13. 13

    Lobanov, A.V., Gromer, S., Salinas, G. & Gladyshev, V.N. Selenium metabolism in Trypanosoma: characterization of selenoproteomes and identification of a kinetoplastida-specific selenoprotein. Nucleic Acids Res. 34, 4012–4024 (2006).

    CAS  Article  Google Scholar 

  14. 14

    Bonilla, M. et al. Platyhelminth mitochondrial and cytosolic redox homeostasis is controlled by a single thioredoxin glutathione reductase and dependent on selenium and glutathione. J. Biol. Chem. 283, 17898–17907 (2008).

    CAS  Article  Google Scholar 

  15. 15

    Sannella, A.R. et al. New uses for old drugs. Auranofin, a clinically established antiarthritic metallodrug, exhibits potent antimalarial effects in vitro: Mechanistic and pharmacological implications. FEBS Lett. 582, 844–847 (2008).

    CAS  Article  Google Scholar 

  16. 16

    Ilari, A. et al. A gold-containing drug against parasitic polyamine metabolism: the X-ray structure of trypanothione reductase from Leishmania infantum in complex with auranofin reveals a dual mechanism of enzyme inhibition. Amino Acids 42, 803–811 (2012).

    CAS  Article  Google Scholar 

  17. 17

    Davis, P.H., Schulze, J. & Stanley, S.L. Jr. Transcriptomic comparison of two Entamoeba histolytica strains with defined virulence phenotypes identifies new virulence factor candidates and key differences in the expression patterns of cysteine proteases, lectin light chains and calmodulin. Mol. Biochem. Parasitol. 151, 118–128 (2007).

    CAS  Article  Google Scholar 

  18. 18

    Blower, M.D., Nachury, M., Heald, R. & Weis, K.A. Rae1-containing ribonucleoprotein complex is required for mitotic spindle assembly. Cell 121, 223–234 (2005).

    CAS  Article  Google Scholar 

  19. 19

    Parks, R.E. Jr. et al. Purine metabolism in primitive erythrocytes. Comp. Biochem. Physiol. B 45, 355–364 (1973).

    CAS  Article  Google Scholar 

  20. 20

    Créchet, J.B., Cool, R.H., Jacquet, E. & Lallemand, J.Y. Characterization of Saccharomyces cerevisiae Ras1p and chimaeric constructs of Ras proteins reveals the hypervariable region and farnesylation as critical elements in the adenylyl cyclase signaling pathway. Biochemistry 42, 14903–14912 (2003).

    Article  Google Scholar 

  21. 21

    Arnaud-Dabernat, S. et al. Nm23-M2/NDP kinase B induces endogenous c-myc and nm23-M1/NDP kinase A overexpression in BAF3 cells. Both NDP kinases protect the cells from oxidative stress-induced death. Exp. Cell Res. 301, 293–304 (2004).

    CAS  Article  Google Scholar 

  22. 22

    Sok, J. et al. Arsenite-inducible RNA-associated protein (AIRAP) protects cells from arsenite toxicity. Cell Stress Chaperones 6, 6–15 (2001).

    CAS  Article  Google Scholar 

  23. 23

    Lu, J., Chew, E.H. & Holmgren, A. Targeting thioredoxin reductase is a basis for cancer therapy by arsenic trioxide. Proc. Natl. Acad. Sci. USA 104, 12288–12293 (2007).

    CAS  Article  Google Scholar 

  24. 24

    Angelucci, F. et al. Inhibition of Schistosoma mansoni thioredoxin-glutathione reductase by auranofin: structural and kinetic aspects. J. Biol. Chem. 284, 28977–28985 (2009).

    CAS  Article  Google Scholar 

  25. 25

    Talbot, S., Nelson, R. & Self, W.T. Arsenic trioxide and auranofin inhibit selenoprotein synthesis: implications for chemotherapy for acute promyelocytic leukaemia. Br. J. Pharmacol. 154, 940–948 (2008).

    CAS  Article  Google Scholar 

  26. 26

    Townsend, D.M., Tew, K.D. & Tapiero, H. The importance of glutathione in human disease. Biomed. Pharmacother. 57, 145–155 (2003).

    CAS  Article  Google Scholar 

  27. 27

    Lillig, C.H. & Holmgren, A. Thioredoxin and related molecules—from biology to health and disease. Antioxid. Redox Signal. 9, 25–47 (2007).

    CAS  Article  Google Scholar 

  28. 28

    Sayed, A.A. et al. Identification of oxadiazoles as new drug leads for the control of schistosomiasis. Nat. Med. 14, 407–412 (2008).

    CAS  Article  Google Scholar 

  29. 29

    Fahey, R.C., Newton, G.L., Arrick, B., Overdank-Bogart, T. & Aley, S.B. Entamoeba histolytica: a eukaryote without glutathione metabolism. Science 224, 70–72 (1984).

    CAS  Article  Google Scholar 

  30. 30

    Arias, D.G., Gutierrez, C.E., Iglesias, A.A. & Guerrero, S.A. Thioredoxin-linked metabolism in Entamoeba histolytica. Free Radic. Biol. Med. 42, 1496–1505 (2007).

    CAS  Article  Google Scholar 

  31. 31

    Hirt, R.P., Muller, S., Embley, T.M. & Coombs, G.H. The diversity and evolution of thioredoxin reductase: new perspectives. Trends Parasitol. 18, 302–308 (2002).

    CAS  Article  Google Scholar 

  32. 32

    Jeelani, G. et al. Two atypical L-cysteine-regulated NADPH-dependent oxidoreductases involved in redox maintenance, L-cystine and iron reduction and metronidazole activation in the enteric protozoan Entamoeba histolytica. J. Biol. Chem. 285, 26889–26899 (2010).

    CAS  Article  Google Scholar 

  33. 33

    Sen, A., Chatterjee, N.S., Akbar, M.A., Nandi, N. & Das, P. The 29-kilodalton thiol-dependent peroxidase of Entamoeba histolytica is a factor involved in pathogenesis and survival of the parasite during oxidative stress. Eukaryot. Cell 6, 664–673 (2007).

    CAS  Article  Google Scholar 

  34. 34

    Williams, C.H. et al. Thioredoxin reductase: two modes of catalysis have evolved. Eur. J. Biochem. 267, 6110–6117 (2000).

    CAS  Article  Google Scholar 

  35. 35

    Houpt, E.R. et al. The mouse model of amebic colitis reveals mouse strain susceptibility to infection and exacerbation of disease by CD4+ T cells. J. Immunol. 169, 4496–4503 (2002).

    CAS  Article  Google Scholar 

  36. 36

    He, C. et al. A novel Entamoeba histolytica cysteine proteinase, EhCP4, is key for invasive amebiasis and a therapeutic target. J. Biol. Chem. 285, 18516–18527 (2010).

    CAS  Article  Google Scholar 

  37. 37

    Markiewicz, V.R., Saunders, L.A., Geus, R.J., Payne, B.J. & Hook, J.B. Carcinogenicity study of auranofin, an orally administered gold compound in mice. Fundam. Appl. Toxicol. 11, 277–284 (1988).

    CAS  Article  Google Scholar 

  38. 38

    Bersani, N.A., Merwin, J.R., Lopez, M.I., Pearson, G.D. & Merrill, G.F. Protein electrophoretic mobility shift assay to monitor redox state of thioredoxin in cells. Methods Enzymol. 347, 317–326 (2002).

    CAS  Article  Google Scholar 

  39. 39

    Diamond, L.S., Harlow, D.R. & Cunnick, C.C. A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans. R. Soc. Trop. Med. Hyg. 72, 431–432 (1978).

    CAS  Article  Google Scholar 

  40. 40

    Mulrooney, S.B. Application of a single-plasmid vector for mutagenesis and high-level expression of thioredoxin reductase and its use to examine flavin cofactor incorporation. Protein Expr. Purif. 9, 372–378 (1997).

    CAS  Article  Google Scholar 

  41. 41

    Meléndez-López, S.G. et al. Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model. Eukaryot. Cell 6, 1130–1136 (2007).

    Article  Google Scholar 

Download references


This work was supported by the Sandler Foundation and US National Institute of Allergy and Infectious Diseases grant 5U01AI077822; we also acknowledge support from R01 GM050389. E. histolytica microarray slides were kindly provided by S.L. Stanley Jr. (Stony Brook University, New York), and the E. histolytica thioredoxin–specific antibody was a kind gift from S. Adrian-Guerrero (Universidad Nacional del Litoral). We thank G. Hwang and C. Le for their help with the mouse surgery and K. Ang and J. Gut for sharing their expertise.

Author information




A.D. and J.H.M. designed the HTS screening studies and arrays. A.D. performed HTS and array experiments. D.P. and L.B.P. performed the enzymatic assays. R.M.A. performed the oxidant studies. C.H., E.R.C., K.H., G.G.-R., E.O. and M.B.M. did the in vivo studies. K.H. purified recombinant EhTrxR. S.C. and M.R.A. provided compound libraries and edited the manuscript. S.S.G. and A.M.B. synthesized auranofin analogs. S.L.R. designed the EhTrxR and oxidant studies. A.D., L.B.P., J.H.M. and S.L.R. wrote the manuscript.

Corresponding authors

Correspondence to Anjan Debnath or Sharon L Reed.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 1–3 (PDF 681 kb)

Supplementary Data 1

HTS raw data (XLS 299 kb)

Supplementary Data 2

HTS Analyst HT plate heat maps (DOC 1620 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Debnath, A., Parsonage, D., Andrade, R. et al. A high-throughput drug screen for Entamoeba histolytica identifies a new lead and target. Nat Med 18, 956–960 (2012). https://doi.org/10.1038/nm.2758

Download citation

Further reading


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing