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

广谱驱虫药代谢酶小分子抑制剂的研究进展 摘要 包括钩虫、蛔虫和鞭虫在内的寄生肠道线虫感染着超过20亿人 在全世界范围内，造成严重的发病率、长期贫困和生命损失。线虫的特性研究 基因组提供了加速基础和翻译研究所必需的基本分子信息， 由于目前可用的有效药物数量有限，这是公共卫生的优先事项，而且还在不断增加 抗药性。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 药物作为治疗由这些毁灭性寄生虫引起的感染的新疗法。 我们已经为人类线虫寄生虫建立了一个广泛的组学/生物信息学数据库 横跨线虫的主要分类支系。利用系统生物学和进化原理，我们 重建了56种不同线虫的代谢网络，并鉴定了瓶颈酶，即 唯一消耗特定底物或产生独特产品的代谢酶。这导致了我们的 中心假设，抑制保守的咽喉节点酶的化合物有很强的广泛应用潜力 控制各种线虫。为了测试这一点，我们确定了保守的靶点和最初的抑制物，具有潜在的 广谱活性，在系统发育的极端对寄生虫的表型筛选具有 证实了我们的预测。此外，我们还建立了一个独特的线虫特异分子数据库 在瓶颈酶靶标之间的特征和实验确定的活性部位的差异 线虫酶相对于其人类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，活性最好的化合物是预测靶向三个目标的抑制剂 已知的酶类(CPT、mTOR/PI3K和PDE)。为了确认假定的线虫目标(S)，我们将 表达线虫蛋白并进行生化酶抑制试验，采用亲和力标记 技术，以及针对目标击倒蠕虫的活性测试(目标1)。通过利用寄生虫特定的主动- 针对确认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)进行优化 已确定的三个目标类别的铅抑制剂(目标2)。优化的先导化合物最有效地对抗 人体钩虫、线虫和鞭虫的体外试验将在体内进行 在线虫感染的仓鼠和小鼠动物模型中的泛肠道疗效(目标3)。 我们的初步结果，结合这项拟议的研究，具有非常重要的意义，因为它们提供了 更好地了解线虫生存所必需的代谢功能，这可以作为药物的靶点 发现号。合理地将代谢瓶颈酶作为驱虫剂的目标是创新的，就像是一样 利用特定的泛门保守靶点开发驱虫药或广谱药物的概念 对线虫的光谱药效。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 具有广谱抗寄生性线虫感染活性的分子疗法。

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.