Che Pulin, Cui Long, Kutsch Olaf, Cui Liwang, Li Qianjun
1 Division of Infectious Diseases, Department of Medicine, University of Alabama, Birmingham, Alabama 35294, USA.
Assay Drug Dev Technol. 2012 Feb;10(1):61-8. doi: 10.1089/adt.2011.0378. Epub 2011 Nov 3.
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.
多重耐药恶性疟原虫的出现与传播，以及近期在东南亚检测到潜在的青蒿素耐药菌株，凸显了开发新型抗疟疗法的重要性。利用先前构建的具有高水平萤火虫荧光素酶表达的稳定转基因恶性疟原虫系，我们报告了在384孔板中高通量筛选试验的适配、小型化、优化及验证情况。对包括疟原虫血症百分比和血细胞比容在内的试验条件进行了优化。确定了试验稳健性参数，包括Z'值、变异系数（CV）和信号背景比（S/B）。使用LOPAC(1280)小分子化合物库对该试验进行验证。我们的结果表明，该试验稳健可靠，平均Z'值>0.7，CV<10%。此外，该试验背景极低，S/B比高达71。此外，使用基于SYBR Green I的验证试验对筛选出的命中化合物进行选择和确认。显然，该试验适用于大规模筛选化学文库以发现抗疟药物。
