A quantitative high throughput assay for identifying gametocytocidal compounds.

Methods Development

Abstract

Current antimalarial drug treatment does not effectively kill mature Plasmodium falciparum gametocytes, the parasite stage responsible for malaria transmission from human to human via a mosquito. Consequently, following standard therapy malaria can still be transmitted for over a week after the clearance of asexual parasites. A new generation of malaria drugs with gametocytocidal properties, or a gametocytocidal drug that could be used in combinational therapy with currently available antimalarials, is needed to control the spread of the disease and facilitate eradication efforts. We have developed a 1536-well gametocyte viability assay for the high throughput screening of large compound collections to identify novel compounds with gametocytocidal activity. The signal-to-basal ratio and Z'-factor for this assay were 3.2-fold and 0.68, respectively. The IC(50) value of epoxomicin, the positive control compound, was 1.42±0.09 nM that is comparable to previously reported values. This miniaturized assay significantly reduces the number of gametocytes required for the AlamarBlue viability assay, and enables high throughput screening for lead discovery efforts. Additionally, the screen does not require a specialized parasite line, gametocytes from any strain, including field isolates, can be tested. A pilot screen utilizing the commercially available LOPAC library, consisting of 1280 known compounds, revealed two selective gametocytocidal compounds having 54- and 7.8-fold gametocytocidal selectivity in comparison to their cell cytotoxicity effect against the mammalian SH-SY5Y cell line.

Authors

Tanaka, Takeshi Q; Dehdashti, Seameen J; Nguyen, Trung; McKew, John C; Zheng, Wei; Williamson, Kim C;

Keywords

  • Antimalarials/ isolation & purification
  • Antimalarials/ pharmacology
  • Cell Survival/ drug effects
  • High-Throughput Screening Assays
  • Humans
  • Inhibitory Concentration 50
  • Oxazines/ metabolism
  • Parasitic Sensitivity Tests/ methods
  • Plasmodium falciparum/ drug effects
  • Staining and Labeling/ methods
  • Xanthenes/ metabolism

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