Screening of the Pathogen Box for inhibitors with dual efficacy against Giardia lamblia and Cryptosporidium parvum
Screening of the Pathogen Box for inhibitors with dual efficacy against Giardia lamblia and Cryptosporidium parvum
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DOI：
10.1371/journal.pntd.0006673
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发表时间：
2018-08-01
影响因子：
3.8
通讯作者：
Ojo, Kayode K.
中科院分区：
文献类型：
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作者：
Hennessey, Kelly M.;Rogiers, Ilse C.;Ojo, Kayode K.
需要一种更有效的基于细胞的检测方法来进行针对贾第虫的高通量药物筛选。在这里，我们报告的筛选方法，利用G。工程化以表达红移萤火虫荧光素酶的Lamblia。在生物发光读出平台中，使用D-曲马多蛋白作为底物定量寄生虫生长和复制。针对疟疾风险药物（MMV）病原体盒化合物，验证了该试验的重现性和可靠性。对于嗜热脂肪地芽孢杆菌Lamblia，43种化合物在初始筛选（16 μ M）中显示>= 75%的寄生虫生长抑制，其中15种显示>= 95%的抑制。还针对表达纳米荧光素酶的（Nluc）C筛选病原体盒。parvum，得到85种化合物，在10 μ M时具有>= 75%的寄生虫生长抑制，其中6种显示>= 95%的抑制。进一步分析了对两种寄生虫具有活性的七种化合物的代表性组，以确定引起对哺乳动物HepG2细胞的50%生长抑制（EC50）和细胞毒性的有效浓度。这七种化合物中有四种以前已知对治疗贾第虫或隐孢子虫有效。其余三个没有明显的化学相似性与任何以前的特点，抗寄生虫治疗药物，并提供新的药物化学治疗的发展机会。这些结果表明，生物发光法适用于大规模筛选针对两种C. parvum和G.兰布利亚
There is need for a more efficient cell-based assay amenable to high-throughput drug screening against Giardia lamblia. Here, we report the development of a screening method utilizing G. lamblia engineered to express red-shifted firefly luciferase. Parasite growth and replication were quantified using D-luciferin as a substrate in a bioluminescent read-out plateform. This assay was validated for reproducibility and reliability against the Medicines for Malaria Venture (MMV) Pathogen Box compounds. For G. lamblia, forty-three compounds showed >= 75% inhibition of parasite growth in the initial screen (16 mu M), with fifteen showing >= 95% inhibition. The Pathogen Box was also screened against Nanoluciferase expressing (Nluc) C. parvum, yielding 85 compounds with >= 75% parasite growth inhibition at 10 mu M, with six showing >= 95% inhibition. A representative set of seven compounds with activity against both parasites were further analyzed to determine the effective concentration that causes 50% growth inhibition (EC50) and cytotoxicity against mammalian HepG2 cells. Four of the seven compounds were previously known to be effective in treating either Giardia or Cryptosporidium. The remaining three shared no obvious chemical similarity with any previously characterized anti-parasite diarrheal drugs and offer new medicinal chemistry opportunities for therapeutic development. These results suggest that the bioluminescent assays are suitable for large-scale screening of chemical libraries against both C. parvum and G. lamblia.