Identification of preclinical drug candidates for the treatment of schistosomiasis

项目总结/摘要 血吸虫寄生虫感染2亿人，导致显著的发病率和20多万 每年死亡。血吸虫病控制策略几乎完全依赖于化疗和数十种 数以百万计的人用唯一可用的药物吡喹酮（PZQ）治疗。没有新的药物 临床管道在大规模药物管理活动中获得的PZQ治愈率通常低于50%。 此外，随着PZQ使用的预计水平，PZQ抗性寄生虫将不可避免地进化。 因此，寻找新的治疗血吸虫病的药物靶标和药物势在必行。我们确定了一个 极具前景的药物靶点：蠕虫含硒半胱氨酸的酶硫氧还蛋白谷胱甘肽还原酶 （TGR）。我们确定TGR是蠕虫抗氧化防御的中心和必需介质。的 脊椎动物的抗氧化防御多样化为三种独立的酶，谷胱甘肽还原酶， 硫氧还蛋白还原酶和谷氧还蛋白，而溶酶体仅依赖于TGR。TGR是一个瓶颈， 其抑制导致蠕虫在所有发育阶段快速死亡。相比之下，PZQ对 蠕虫，通常导致部分治愈。我们已经证明TGR是可药用的，可以选择性地使用 靶向于人类正磷酸酶，并在动物模型中的蠕虫中抑制 血吸虫病导致蠕虫死亡。PZQ类似物是无活性的，限制了类似物的开发，以避免或 抵抗抗药性与作用机制未知的PZQ不同，TGR是一种定义明确的 分子靶点，作为重组蛋白具有活性，具有适合快速检测的已建立的生化测定法， 化合物分析、SAR和优化。我们最近完成了一个大型化合物的多层HTS 文库（> 350，000种化合物），其鉴定了>100种对脱靶无活性的TGR抑制剂， 对人体酶无毒性，对哺乳动物细胞无毒。这些命中的识别表明 TGR的特异性抑制剂可以在没有脱靶相互作用和细胞毒性的情况下获得。我们有 获得了TGR的配体和无配体晶体结构，允许基于结构的方法击中 优化.我们假设迭代药物化学优化将产生有效且有选择性的结果 小分子TGR抑制剂将具有体内杀虫活性。在R21阶段，我们的目标是 识别具有强效（< 5 µM）蠕虫杀灭活性的多层HTS命中，并表征TGR 通过与TGR共结晶和晶体结构测定，确定了这些抑制剂的结合位点。在R33 阶段，我们建议优化这些新的，有效的TGR抑制剂使用尖端，结构和配体- 基于计算机辅助设计和药物化学，以提高效力，稳定性和口服生物利用度。 这将通过X射线晶体学和化学蛋白质组学进行补充，使用光反应探针， 表征分子TGR-化合物相互作用。药物化学将通过酶 分析TGR和orthophosphate人酶、代谢稳定性、体外细胞毒性和抗肿瘤活性 离体蠕虫。最后，将评估所选化合物的PK/PD特性和针对以下的功效： 在小鼠中的一些感染。为了实现这些变革目标，一个创新的国际 在生物化学和药物发现、结构和生物技术领域拥有专业知识的全球专家 生物学、计算机辅助分子设计和化学蛋白质组学已经组装。各种各样的， 该小组的协同专业知识将有助于克服药物开发的关键障碍。完成 该项目将确定临床前药物样化合物，适合于血吸虫病候选药物选择 治疗

PROJECT SUMMARY/ABSTRACT Schistosome parasites infect 200 million people, resulting in significant morbidity and more than 200,000 deaths annually. Schistosomiasis control strategies rely almost exclusively on chemotherapy and tens of millions of people are treated with the only available drug, praziquantel (PZQ). There are no new drugs in the clinical pipeline. PZQ cure rates obtained in mass drug administration campaigns are typically less than 50%. Furthermore, with projected levels of PZQ use it is inevitable that PZQ-resistant parasites will evolve. Therefore, it is imperative to identify new drug targets and drugs for schistosomiasis treatment. We identified a highly promising drug target: the worm selenocysteine-containing enzyme thioredoxin glutathione reductase (TGR). We established that TGR is a central and essential mediator of antioxidant defenses in the worm. The antioxidant defenses of vertebrates are diversified to three independent enzymes, glutathione reductase, thioredoxin reductase, and glutaredoxin, whereas schistosomes rely solely on TGR. TGR is a chokepoint and its inhibition leads to rapid worm death in all developmental stages. In contrast, PZQ has poor activity against juvenile worms, often resulting in partial cures. We have shown that TGR is druggable, can be selectively targeted over human orthologous enzymes and that its inhibition in worms in an animal model of schistosomiasis leads to worm death. PZQ analogs are inactive, restricting analog development to avoid or counteract drug resistance. Unlike PZQ for which the mechanism of action is not known, TGR is a defined molecular target, active as a recombinant protein, with established biochemical assays amenable to rapid compound analysis, SAR, and optimization. We recently completed a multi-tiered HTS of a large compound library (>350,000 compounds), which identified >100 TGR inhibitors that were inactive against off-target, orthologous human enzymes and nontoxic to mammalian cells. The identification of these hits demonstrates that specific inhibitors of TGR can be obtained without off-target interactions and cytotoxicity. We have obtained both liganded and ligand-free crystal structures of TGR, allowing a structure based approach to hit optimization. We hypothesize that iterative medicinal chemistry optimization will yield potent and selective small molecule TGR inhibitors that will have in vivo worm killing activity. In the R21 phase our aims are to identify hits from the multi-tiered HTS with potent (< 5 µM) worm killing activity and to characterize the TGR binding site of these inhibitors by co-crystallization with TGR and crystal structure determination. In the R33 phase we propose to optimize these novel, potent TGR inhibitors using cutting-edge, structure and ligand- based computer-aided design and medicinal chemistry to improve potency, stability, and oral bioavailability. This will be complemented by X-ray crystallography and chemoproteomics using photoreactive probes to characterize molecular TGR-compound interactions. Medicinal chemistry will be informed by enzymatic analysis of TGR and orthologous human enzymes, metabolic stability, in vitro cell toxicity, and activity against ex vivo worms. Finally, select compounds will be assessed for PK/PD properties and efficacy against schistosome infections in mice. To accomplish these transformative aims, an innovative international collaboration of global experts with expertise in schistosome biochemistry and drug discovery, structural biology, computer-aided molecular design, and chemoproteomics has been assembled. The varied and synergistic expertise of the team will facilitate overcoming critical barriers to drug development. Completion of the project will identify preclinical drug-like compounds, suitable for candidate selection for schistosomiasis treatment.