Validating a firefly luciferase-based high-throughput screening assay for antimalarial drug discovery

Assay Drug Dev Technol. 2012 Feb;10(1):61-8. doi: 10.1089/adt.2011.0378. Epub 2011 Nov 3.

Abstract

The emergence and spread of multidrug-resistant Plasmodium falciparum and recent detection of potential artemisinin-resistant strains in Southeast Asia highlight the importance of developing novel antimalarial therapies. Using a previously generated stable transgenic P. falciparum line with high-level firefly luciferase expression, we report the adaptation, miniaturization, optimization, and validation of a high-throughput screening assay in 384-well plates. Assay conditions, including the percentage of parasitemia and hematocrit, were optimized. Parameters of assay robustness, including Z'-value, coefficient variation (CV), and signal-to-background (S/B) ratio, were determined. The LOPAC(1280) small-compound library was used to validate this assay. Our results demonstrated that this assay is robust and reliable, with an average Z'-value of >0.7 and CV of <10%. Moreover, this assay showed a very low background, with the S/B ratio up to 71. Further, identified hits were selected and confirmed using a SYBR Green I-based confirmatory assay. It is evident that this assay is suitable for large-scale screening of chemical libraries for antimalarial drug discovery.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Animals
  • Antimalarials / analysis
  • Antimalarials / pharmacology*
  • Drug Discovery / methods*
  • High-Throughput Screening Assays / methods*
  • Luciferases, Firefly* / biosynthesis
  • Organisms, Genetically Modified
  • Plasmodium falciparum / drug effects*
  • Plasmodium falciparum / genetics*

Substances

  • Antimalarials
  • Luciferases, Firefly