Development of small molecule inhibitors of metabolic enzymes as broad spectrum anthelmintic drugs

小分子代谢酶抑制剂作为广谱驱虫药的研究进展 摘要 肠道寄生线虫包括钩虫，蛔虫和鞭虫，感染超过20亿人 在世界范围内，造成严重的发病率、贫困的延续和生命的丧失。线虫的特征 基因组提供了加速基础和转化研究所必需的基本分子信息， 这是一个公共卫生优先事项，因为目前可用的有效药物数量有限， 耐药性在这项提案中，我们将继续进行后基因组药物发现研究，以开发小分子 作为治疗这些毁灭性寄生虫引起的感染的新疗法的药物。 我们已经建立了一个广泛的人类线虫寄生虫的组学/生物信息学数据库 跨越线虫类的主要分类分支。利用系统生物学和进化原理，我们 重建代谢网络的56种不同的线虫寄生虫和确定的瓶颈酶，即。 唯一消耗特定底物或产生独特产物的代谢酶。这导致了我们的 一个中心假设是，抑制保守的阻塞点酶的化合物具有广泛应用的强大潜力， 控制各种线虫。为了验证这一点，我们确定了保守的目标和初始抑制剂， 广谱活性，因此，在寄生虫发生的极端情况下对寄生虫进行表型筛选， 证实了我们的预测。此外，我们还建立了线虫特异性分子生物学数据库， 特性之间的阻塞点酶的目标，并通过实验确定，活性位点的差异， 线虫酶相对于它们的人类直向同源物可以合理地指导选择性抑制剂的设计。 在我们的表型筛选中具有最佳活性的化合物是预测靶向三个靶点的抑制剂。 已知的酶类别（CPT、mTOR/PI 3 K和PDE）。为了确认假定的线虫靶标，我们将 表达线虫蛋白并实施生化酶抑制测定，采用基于亲和性的标记， 技术，并测试针对靶标敲减蠕虫的活性（目标1）。通过利用寄生虫特异性活性- 确定的蛋白质靶点的位点特征，我们将使用基于X射线结构的药物设计（SBDD）来优化 三个已确定目标类别的主要抑制剂（目标2）。优化的铅化合物最有效地对抗 将在体外对人钩虫锡兰钩虫和鞭虫进行体内试验 它们在线虫感染的仓鼠和小鼠动物模型中的全肠道功效（目的3）。 我们的初步结果，结合这项拟议的研究，是非常重要的，因为他们提供了 更好地了解线虫生存所必需的代谢功能，这可以成为药物治疗的目标。 的发现代谢阻塞点酶作为驱虫剂的合理靶向是创新的， 利用特定的泛门保守靶点来开发具有广泛生物活性的抗蠕虫药物的概念， 对线虫的广谱功效。总的来说，这项工作具有很高的潜力，可以提供一个或多个新的小型 抗寄生线虫感染的广谱活性的分子治疗剂。

Development of small molecule inhibitors of metabolic enzymes as broad spectrum anthelmintic drugs Abstract Parasitic intestinal nematodes including hookworms, roundworm and whipworms, infect over two billion people worldwide, causing significant morbidity, perpetuation of poverty, and loss of life. Characterization of nematode genomes provides fundamental molecular information essential for accelerating basic and translational research, which is a public health priority due to the limited number of currently available effective drugs and increasing drug resistance. In this proposal, we will pursue post-genomic drug discovery studies to develop small molecule drugs as novel therapeutics to treat infections caused by these devastating parasites. We have established an extensive omics/bioinformatics database for human nematode parasites spanning the major taxonomic clades of Nematoda. Using systems biology and evolutionary principles, we reconstructed metabolic networks for 56 diverse nematode parasites and identified chokepoint enzymes, i.e. metabolic enzymes that uniquely consume a specific substrate or generate a unique product. This led to our central hypothesis that compounds that inhibit conserved chokepoint enzymes have a strong potential for broad control of diverse nematodes. To test this, we identified conserved targets and initial inhibitors with potential for broad-spectrum activity, for which phenotypic screening of parasites at the extremes of the phylogeny have validated our predictions. Furthermore, we established a unique database of nematode-specific molecular features among the chokepoint enzyme targets and experimentally established that active-site differences in the nematode enzymes relative to their human orthologs can rationally guide the design of selective inhibitors. The compounds with the best activity in our phenotypic screens are inhibitors predicted to target three known enzyme classes (CPT, mTOR/PI3K, and PDE). To confirm the putative nematode target(s), we will express nematode proteins and implement biochemical enzyme inhibition assays, employ affinity-based labeling techniques, and test for activity against target knockdown worms (Aim 1). By leveraging parasite-specific active- site features of the confirmed protein targets, we will use a X-ray structure-based drug design (SBDD) to optimize lead inhibitors of the three identified target classes (Aim 2). Optimized lead compounds most effective against the human hookworm Ancylostoma ceylanicum and the whipworm Trichuris muris in vitro will be tested in vivo for their pan-intestinal efficacy in hamster and mouse animal models of nematode infection (Aim 3). Our preliminary results, combined with this proposed research, are highly significant since they provide a better understanding of metabolic functions essential for nematode survival, which can be targeted for drug discovery. The rational targeting of metabolic chokepoint enzymes as anthelminthic agents is innovative, as is the concept of utilizing specific pan-phylum conserved targets to develop anthelmintic drug or drugs with broad spectrum efficacy against nematodes. Collectively, this work has high potential to provide one or more new small molecule therapeutics with broad spectrum activity against parasitic nematode infections.