Comparison of a high-throughput high-content intracellular Leishmania donovani assay with an axenic amastigote assay

Manu De Rycker*, Irene Hallyburton, John Thomas, Lorna Campbell, Susan Wyllie, Dhananjay Joshi, Scott Cameron, Ian H. Gilbert, Paul G. Wyatt, Julie A. Frearson, Alan H. Fairlamb, David W. Gray

*Corresponding author for this work

    Research output: Contribution to journalArticle

    76 Citations (Scopus)

    Abstract

    Visceral leishmaniasis is a neglected tropical disease with significant health impact. The current treatments are poor, and there is an urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-living forms of the Leishmania parasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiological situation, as the disease-causing stage of the parasite resides inside human host cells. Assessing the drug sensitivity of intracellular parasites on scale has recently become feasible with the advent of high-content screening methods. We describe here a 384-well microscopy-based intramacrophage Leishmania donovani assay and compare it to an axenic amastigote system. A panel of eight reference compounds was tested in both systems, as well as a human counterscreen cell line, and our findings show that for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 diverse compounds was also screened using both systems. This resulted in the identification of seven new antileishmanial compounds and revealed a high false-positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited as a primary hit-discovery platform than the current form of axenic assay, and we discuss how modifications to the axenic assay may render it more suitable for hit-discovery.

    Original languageEnglish
    Pages (from-to)2913-2922
    Number of pages10
    JournalAntimicrobial Agents and Chemotherapy
    Volume57
    Issue number7
    Early online date9 Apr 2013
    DOIs
    Publication statusPublished - Jul 2013

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    Leishmania donovani
    Parasites
    Neglected Diseases
    Visceral Leishmaniasis
    Leishmania
    Drug Discovery
    Pharmaceutical Preparations
    Microscopy
    Cell Line
    Health

    Cite this

    De Rycker, M., Hallyburton, I., Thomas, J., Campbell, L., Wyllie, S., Joshi, D., ... Gray, D. W. (2013). Comparison of a high-throughput high-content intracellular Leishmania donovani assay with an axenic amastigote assay. Antimicrobial Agents and Chemotherapy, 57(7), 2913-2922. https://doi.org/10.1128/AAC.02398-12
    De Rycker, Manu ; Hallyburton, Irene ; Thomas, John ; Campbell, Lorna ; Wyllie, Susan ; Joshi, Dhananjay ; Cameron, Scott ; Gilbert, Ian H. ; Wyatt, Paul G. ; Frearson, Julie A. ; Fairlamb, Alan H. ; Gray, David W. / Comparison of a high-throughput high-content intracellular Leishmania donovani assay with an axenic amastigote assay. In: Antimicrobial Agents and Chemotherapy. 2013 ; Vol. 57, No. 7. pp. 2913-2922.
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    abstract = "Visceral leishmaniasis is a neglected tropical disease with significant health impact. The current treatments are poor, and there is an urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-living forms of the Leishmania parasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiological situation, as the disease-causing stage of the parasite resides inside human host cells. Assessing the drug sensitivity of intracellular parasites on scale has recently become feasible with the advent of high-content screening methods. We describe here a 384-well microscopy-based intramacrophage Leishmania donovani assay and compare it to an axenic amastigote system. A panel of eight reference compounds was tested in both systems, as well as a human counterscreen cell line, and our findings show that for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 diverse compounds was also screened using both systems. This resulted in the identification of seven new antileishmanial compounds and revealed a high false-positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited as a primary hit-discovery platform than the current form of axenic assay, and we discuss how modifications to the axenic assay may render it more suitable for hit-discovery.",
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    De Rycker, M, Hallyburton, I, Thomas, J, Campbell, L, Wyllie, S, Joshi, D, Cameron, S, Gilbert, IH, Wyatt, PG, Frearson, JA, Fairlamb, AH & Gray, DW 2013, 'Comparison of a high-throughput high-content intracellular Leishmania donovani assay with an axenic amastigote assay', Antimicrobial Agents and Chemotherapy, vol. 57, no. 7, pp. 2913-2922. https://doi.org/10.1128/AAC.02398-12

    Comparison of a high-throughput high-content intracellular Leishmania donovani assay with an axenic amastigote assay. / De Rycker, Manu; Hallyburton, Irene; Thomas, John; Campbell, Lorna; Wyllie, Susan; Joshi, Dhananjay; Cameron, Scott; Gilbert, Ian H.; Wyatt, Paul G.; Frearson, Julie A.; Fairlamb, Alan H.; Gray, David W.

    In: Antimicrobial Agents and Chemotherapy, Vol. 57, No. 7, 07.2013, p. 2913-2922.

    Research output: Contribution to journalArticle

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    T1 - Comparison of a high-throughput high-content intracellular Leishmania donovani assay with an axenic amastigote assay

    AU - De Rycker, Manu

    AU - Hallyburton, Irene

    AU - Thomas, John

    AU - Campbell, Lorna

    AU - Wyllie, Susan

    AU - Joshi, Dhananjay

    AU - Cameron, Scott

    AU - Gilbert, Ian H.

    AU - Wyatt, Paul G.

    AU - Frearson, Julie A.

    AU - Fairlamb, Alan H.

    AU - Gray, David W.

    PY - 2013/7

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    N2 - Visceral leishmaniasis is a neglected tropical disease with significant health impact. The current treatments are poor, and there is an urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-living forms of the Leishmania parasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiological situation, as the disease-causing stage of the parasite resides inside human host cells. Assessing the drug sensitivity of intracellular parasites on scale has recently become feasible with the advent of high-content screening methods. We describe here a 384-well microscopy-based intramacrophage Leishmania donovani assay and compare it to an axenic amastigote system. A panel of eight reference compounds was tested in both systems, as well as a human counterscreen cell line, and our findings show that for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 diverse compounds was also screened using both systems. This resulted in the identification of seven new antileishmanial compounds and revealed a high false-positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited as a primary hit-discovery platform than the current form of axenic assay, and we discuss how modifications to the axenic assay may render it more suitable for hit-discovery.

    AB - Visceral leishmaniasis is a neglected tropical disease with significant health impact. The current treatments are poor, and there is an urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-living forms of the Leishmania parasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiological situation, as the disease-causing stage of the parasite resides inside human host cells. Assessing the drug sensitivity of intracellular parasites on scale has recently become feasible with the advent of high-content screening methods. We describe here a 384-well microscopy-based intramacrophage Leishmania donovani assay and compare it to an axenic amastigote system. A panel of eight reference compounds was tested in both systems, as well as a human counterscreen cell line, and our findings show that for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 diverse compounds was also screened using both systems. This resulted in the identification of seven new antileishmanial compounds and revealed a high false-positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited as a primary hit-discovery platform than the current form of axenic assay, and we discuss how modifications to the axenic assay may render it more suitable for hit-discovery.

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    DO - 10.1128/AAC.02398-12

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