A high throughput screen for inhibitors of nematode detoxification genes

描述（由申请人提供）：线虫寄生在约25%的人口中。针对寄生虫的药物或驱虫药已用于控制寄生线虫数十年，许多物种正在进化出多药耐药性。在多种生物中，多药耐药是由解毒外源酶的表达和活性增加介导的。针对外源解毒途径的药理学化合物将为研究多药耐药性提供急需的工具，并可以大大增加当前和未来驱虫剂的使用寿命。很少有药物化合物可用于研究和靶向多药耐药，并且那些可用的化合物不是针对线虫的特异性的，并且仅针对单一酶或一类酶。转录因子SKN-1激活秀丽隐杆线虫中外源解毒基因的表达。遗传抑制SKN-1使秀丽隐杆线虫对多种外源药物敏感，秀丽隐杆线虫可以通过增加SKN-1调控基因的表达获得对驱虫药的抗性。SKN-1对于胚胎的发育也是必不可少的。我们最近的研究已经确定了调节SKN-1的主要途径，该途径与哺乳动物调节外源解毒的途径高度不同。因此，SKN-1是一个很有希望的靶标，可以用于开发药物，破坏胚胎发育，降低应激抗性，抑制线虫的外源解毒，而不影响人类的类似途径。秀丽隐杆线虫具有体积小、培养简单、遗传易感等特点，是筛选外源解毒基因抑制剂的理想系统。该项目的第一个目标是开发和优化秀丽隐杆线虫中SKN-1活性的荧光检测方法。该项目的第二个目标是开发四种二级和反筛，以快速确定命中化合物的优先顺序，并在2000种化合物的试点筛选中测试该分析方法。完成这些目标后，将提交优化分析的详细描述，以进入分子文库探针生产中心网络（MLPCN）。

DESCRIPTION (provided by applicant): Nematodes parasitize ~25% of the human population. Helminth targeting drugs, or anthelmintics, have been used to control parasitic nematodes for decades and many species are evolving multidrug resistance. In diverse organisms, multidrug resistance is mediated by increased expression and activity of enzymes that detoxify xenobiotics. Pharmacological compounds that target xenobiotic detoxification pathways would provide much needed tools for studying multidrug resistance and could greatly increase the useful life of current and future anthelmintics. Few pharmacological compounds are available for studying and targeting multidrug resistance and those that are available are not specific for nematodes and only target a single enzyme or class of enzymes. The transcription factor SKN-1 activates the expression of xenobiotic detoxification genes in the nematode Caenorhabditis elegans. Genetic inhibition of SKN-1 sensitizes C. elegans to diverse xenobiotics, and C. elegans can acquire resistance to anthelmintics by increasing the expression of genes that are regulated by SKN-1. SKN-1 is also essential for the development of embryos. Our recent studies have identified a principal pathway regulating SKN-1 that is highly divergent from pathways that regulate xenobiotic detoxification in mammals. Therefore, SKN-1 is a promising target for the development of drugs that disrupt embryonic development, decrease stress resistance, and inhibit xenobiotic detoxification in nematodes without affecting analogous pathways in humans. The small size, simple culturing characteristics, and genetic tractability of C. elegans make it an ideal system to screen for inhibitors of xenobiotic detoxification genes. The first goal of this project is to develop and optimize a fluorescence-based assay of SKN-1 activity in C. elegans. The second goal of this project is to develop four secondary and counter screens to rapidly prioritize hit compounds and to test the assay in a pilot screen of 2000 compounds. After completion of these goals, a detailed description of the optimized assay will be submitted for entry into the Molecular Libraries Probe Production Centers Network (MLPCN). PUBLIC HEALTH RELEVANCE: Nematodes parasitize ~25% of humans and many strains are resistant to all currently used antiparasitic drugs. In many organisms, drug resistance is caused by over active drug detoxification pathways. The studies proposed in this application will develop, optimize, and validate a screen for pharmacological inhibitors of drug detoxification in nematodes.