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

Abstract

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.

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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.

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Acknowledgements

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.

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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.

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Correspondence to Anjan Debnath or Sharon L Reed.

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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)

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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

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