title: Validating a firefly luciferase-based high-throughput screening assay for antimalarial drug discovery.
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.
authors: 1 Division of Infectious Diseases, Department of Medicine, University of Alabama, Birmingham, Alabama 35294, USA.
authors: Pulin
authors: P
authors: Che
authors: Long
authors: L
authors: Cui
authors: Olaf
authors: O
authors: Kutsch
authors: Liwang
authors: L
authors: Cui
authors: Qianjun
authors: Q
authors: Li
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000032:analysis / Q000494:pharmacology*; D055808:Drug Discovery / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D049409:Luciferases, Firefly / Q000096:biosynthesis; D030781:Organisms, Genetically Modified; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics*
chemical_list: D000962:Antimalarials; D049409:Luciferases, Firefly