A high-throughput screen for the identification of compounds that inhibit nematode gene expression by targeting spliced leader trans-splicing.
A high-throughput screen for the identification of compounds that inhibit nematode gene expression by targeting spliced leader trans-splicing.
复制标题
一种高通量筛选，用于鉴定通过靶向剪接前导反式剪接抑制线虫基因表达的化合物。
DOI：
10.1016/j.ijpddr.2019.04.001
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发表时间：
2019
期刊：
影响因子：
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通讯作者：
Pandarakalam GC
中科院分区：
文献类型：
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作者：
Pandarakalam GC
寄生线虫感染是最重要的被忽视的热带疾病之一，影响着主要生活在经济活动较低的热带地区的约10亿人，目前控制线虫感染最有效的策略是采用驱虫药进行大规模治疗。这种策略面临着抗药性寄生虫出现的风险。寄生线虫也会影响牲畜，这些牲畜也用同样有限的驱虫药物治疗。家畜寄生虫对单一药物耐药，甚至对多种药物耐药的寄生虫正在许多地区出现。因此，迫切需要新的驱虫药物。在这里，我们使用线虫秀丽隐杆线虫作为寄生线虫的模型，并证明sinefungin，甲基转移酶的竞争性抑制剂，导致发育延迟和生殖力降低，并抑制剪接的leadertrans-splicing。剪接的leadertrans-splicing是基因表达的一个重要步骤，不发生在宿主的寄生线虫，因此是一个潜在的目标，为新的驱虫drugs.We利用sinefungin抑制剪接leadertrans-splicing适应绿色荧光蛋白为基础的报告基因检测，监测剪接leadertrans-splicing高通量筛选新的驱虫化合物的能力。我们已经建立了一个强大的高通量筛选协议，结合livingC的机械分配。使用声学液体分配器添加化合物，将elegansin加入384或1536孔板中，并使用酶标仪检测SL反式剪接的抑制。我们已经测试了这个协议在第一个试点屏幕，并设想，这种测定将是一个有价值的工具，在寻找新的驱虫药。
Infections with parasitic nematodes are among the most significant of the neglected tropical diseases affecting about a billion people living mainly in tropical regions with low economic activity.The most effective current strategy to control nematode infections involves large scale treatment programs with anthelmintic drugs. This strategy is at risk from the emergence of drug resistant parasites. Parasitic nematodes also affect livestock, which are treated with the same limited group of anthelmintic drugs. Livestock parasites resistant to single drugs, and even multi-drug resistant parasites, are appearing in many areas. There is therefore a pressing need for new anthelmintic drugs.Here we use the nematodeCaenorhabditis elegansas a model for parasitic nematodes and demonstrate that sinefungin, a competitive inhibitor of methyltransferases, causes a delay in development and reduced fecundity, and inhibits spliced leadertrans-splicing. Spliced leadertrans-splicing is an essential step in gene expression that does not occur in the hosts of parasitic nematodes, and is therefore a potential target for new anthelmintic drugs.We have exploited the ability of sinefungin to inhibit spliced leadertrans-splicing to adapt a green fluorescent protein based reporter gene assay that monitors spliced leadertrans-splicing for high-throughput screening for new anthelmintic compounds. We have established a protocol for robust high-throughput screening, combining mechanical dispensing of livingC. elegansinto 384- or 1536- well plates with addition of compounds using an acoustic liquid dispenser, and the detection of the inhibition of SLtrans-splicing using a microplate reader. We have tested this protocol in a first pilot screen and envisage that this assay will be a valuable tool in the search for new anthelmintic drugs.