title: Simple colorimetric trypanothione reductase-based assay for high-throughput screening of drugs against Leishmania intracellular amastigotes.
abstract: Critical to the search for new anti-leishmanial drugs is the availability of high-throughput screening (HTS) methods to test chemical compounds against the relevant stage for pathogenesis, the intracellular amastigotes. Recent progress in automated microscopy and genetic recombination has produced powerful tools for drug discovery. Nevertheless, a simple and efficient test for measuring drug activity against Leishmania clinical isolates is lacking. Here we describe a quantitative colorimetric assay in which the activity of a Leishmania native enzyme is used to assess parasite viability. Enzymatic reduction of disulfide trypanothione, monitored by a microtiter plate reader, was used to quantify the growth of Leishmania parasites. An excellent correlation was found between the optical density at 412 nm and the number of parasites inoculated. Pharmacological validation of the assay was performed against the conventional alamarBlue method for promastigotes and standard microscopy for intracellular amastigotes. The activity of a selected-compound panel, including several anti-leishmanial reference drugs, demonstrated high consistency between the newly developed assay and the reference method and corroborated previously published data. Quality assessment with standard measures confirmed the robustness and reproducibility of the assay, which performed in compliance with HTS requirements. This simple and rapid assay provides a reliable, accurate method for screening anti-leishmanial agents, with high throughput. The basic equipment and manipulation required to perform the assay make it easy to implement, simplifying the method for scoring inhibitor assays.
authors.affiliation: Department of Biomedical Research, Royal Tropical Institute, Amsterdam, the Netherlands.
authors.forename: Erika
authors.initials: E
authors.lastname: van den Bogaart
authors.forename: Gerard J
authors.initials: GJ
authors.lastname: Schoone
authors.forename: Paul
authors.initials: P
authors.lastname: England
authors.forename: Dorien
authors.initials: D
authors.lastname: Faber
authors.forename: Kristina M
authors.initials: KM
authors.lastname: Orrling
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Dujardin
authors.forename: Shyam
authors.initials: S
authors.lastname: Sundar
authors.forename: Henk D F H
authors.initials: HD
authors.lastname: Schallig
authors.forename: Emily R
authors.initials: ER
authors.lastname: Adams
mesh_terms: D002460:Cell Line; D003124:Colorimetry / Q000379:methods*; D004353:Drug Evaluation, Preclinical; D057166:High-Throughput Screening Assays; D006801:Humans; D007891:Leishmania / Q000187:drug effects* / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D015203:Reproducibility of Results; D014344:Trypanocidal Agents / Q000494:pharmacology*
chemical_list: D014344:Trypanocidal Agents; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase