TY - JOUR
T1 - Validating a firefly luciferase-based high-throughput screening assay for antimalarial drug discovery
AU - Che, Pulin
AU - Cui, Long
AU - Kutsch, Olaf
AU - Cui, Liwang
AU - Li, Qianjun
N1 - Copyright:
Copyright 2012 Elsevier B.V., All rights reserved.
PY - 2012/2/1
Y1 - 2012/2/1
N2 - 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.
AB - 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.
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U2 - 10.1089/adt.2011.0378
DO - 10.1089/adt.2011.0378
M3 - Article
C2 - 22050430
AN - SCOPUS:84863233246
VL - 10
SP - 61
EP - 68
JO - Assay and Drug Development Technologies
JF - Assay and Drug Development Technologies
SN - 1540-658X
IS - 1
ER -