title: Comparison of a high-throughput high-content intracellular Leishmania donovani assay with an axenic amastigote assay.
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.
authors.affiliation: Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, United Kingdom. m.derycker@dundee.ac.uk
authors.forename: Manu
authors.initials: M
authors.lastname: De Rycker
authors.forename: Irene
authors.initials: I
authors.lastname: Hallyburton
authors.forename: John
authors.initials: J
authors.lastname: Thomas
authors.forename: Lorna
authors.initials: L
authors.lastname: Campbell
authors.forename: Susan
authors.initials: S
authors.lastname: Wyllie
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Joshi
authors.forename: Scott
authors.initials: S
authors.lastname: Cameron
authors.forename: Ian H
authors.initials: IH
authors.lastname: Gilbert
authors.forename: Paul G
authors.initials: PG
authors.lastname: Wyatt
authors.forename: Julie A
authors.initials: JA
authors.lastname: Frearson
authors.forename: Alan H
authors.initials: AH
authors.lastname: Fairlamb
authors.forename: David W
authors.initials: DW
authors.lastname: Gray
mesh_terms: D000981:Antiprotozoal Agents / Q000494:pharmacology*; D061245:Axenic Culture; D002460:Cell Line; D055808:Drug Discovery; D057166:High-Throughput Screening Assays; D006801:Humans; D007893:Leishmania donovani / Q000187:drug effects*; D007898:Leishmaniasis, Visceral / Q000188:drug therapy*; D008264:Macrophages / Q000469:parasitology; D021261:Parasitic Sensitivity Tests
chemical_list: D000981:Antiprotozoal Agents