A high throughput screen for inhibitors of nematode detoxification genes

描述(申请人提供)：线虫寄生在约25%的人类种群中。几十年来，针对蠕虫的药物或驱虫剂一直被用于控制寄生线虫，许多物种正在形成多重耐药性。在不同的生物体中，多药耐药性是由解毒外源物质的酶的表达和活性增加所介导的。针对异源解毒途径的药物化合物将为研究多药耐药性提供亟需的工具，并可能极大地延长当前和未来驱虫剂的使用寿命。很少有药理化合物可用于研究和靶向多重耐药，现有的那些不是针对线虫的，只针对一种酶或一类酶。转录因子SKN-1激活线虫体内异源解毒基因的表达。SKN-1的遗传抑制使线虫对不同的外源物质敏感，线虫可以通过增加受SKN-1调控的基因的表达而获得对驱虫剂的抗药性。SKN-1对胚胎的发育也是必不可少的。我们最近的研究发现了一个调控SKN-1的主要途径，该途径与调控哺乳动物异源解毒的途径非常不同。因此，SKN-1是一个很有希望的靶点，可以用来开发在不影响人类类似途径的情况下干扰胚胎发育、降低应激抗性、抑制线虫的异物解毒作用的药物。线虫的体积小、培养简单和遗传易操纵性使其成为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一种基于荧光的线虫SKN-1活性分析方法。该项目的第二个目标是开发四个二级筛查和计数器筛查，以快速确定命中化合物的优先顺序，并在2000种化合物的试点筛查中测试该分析。完成这些目标后，将提交一份优化后的分析的详细说明，以便进入分子图书馆探针生产中心网络(MLPCN)。 公共卫生相关性：线虫寄生在约25%的人类中，许多菌株对目前使用的所有抗寄生药都具有抗药性。在许多生物中，抗药性是由过度活跃的药物解毒途径引起的。本申请中提出的研究将开发、优化和验证线虫药物解毒药物抑制剂的筛选。

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.