Identification of preclinical drug candidates for the treatment of schistosomiasis

项目摘要/摘要 血吸虫寄生虫感染2亿人，导致显著发病率和20多万人 每年的死亡人数。血吸虫病的控制策略几乎完全依赖于化疗和数十种 数百万人正在接受唯一可用的药物--吡喹酮(PZQ)的治疗。没有新的药物在 临床流水线。在大规模药物管理活动中获得的PZQ治愈率通常不到50%。 此外，随着PZQ使用量的预测，耐PZQ的寄生虫将不可避免地进化。 因此，寻找治疗血吸虫病的新药靶点和药物势在必行。我们确定了一个 前景看好的药物靶点：蠕虫含硒半胱氨酸酶硫氧还蛋白谷胱甘肽还原酶 (TGR)。我们确定TGR是蠕虫体内抗氧化防御的中心和必要的介体。这个 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，而血吸虫只依赖TGR。TGR是一个瓶颈， 它的抑制会导致虫子在所有发育阶段迅速死亡。相比之下，PZQ对 幼虫，通常导致部分治愈。我们已经证明TGR是可下药的，可以选择性地 靶向于人的同源酶及其在蠕虫中的抑制作用 血吸虫病会导致蠕虫死亡。PZQ模拟处于非活动状态，限制了模拟开发以避免或 对抗抗药性。与PZQ不同的是，TGR是一个定义的 分子靶标，以重组蛋白的形式活跃，已建立的生化分析适合于快速 化合物分析、合成孔径雷达和优化。我们最近完成了一个大型建筑群的多层高温超导 库(&gt；350,000化合物)，它鉴定了对脱靶无效的&gt；100 TGR抑制剂， 与人类同源的酶，对哺乳动物细胞无毒。这些命中的确认证明了 TGR的特异性抑制剂可以在没有靶外相互作用和细胞毒性的情况下获得。我们有 得到了TGR的配体和非配体的晶体结构，允许基于结构的方法来命中 优化。我们假设迭代的药物化学优化将产生有效的和选择性的 具有体内杀虫活性的小分子TGR抑制剂。在R21阶段，我们的目标是 确定具有强大(&lt；5微米)蠕虫杀灭活性的多层HTS的命中率，并对TGR进行表征 通过与TGR的共结晶和晶体结构测定，确定了这些缓蚀剂的结合部位。在R33公路上 阶段我们建议使用尖端技术、结构和配体来优化这些新的、有效的TGR抑制剂。 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。 这将得到X射线结晶学和化学蛋白质组学的补充，使用光反应探针来 表征分子TGR与化合物的相互作用。药用化学将由酶制剂提供信息 TGR和人类同源酶的分析、代谢稳定性、体外细胞毒性和抗肿瘤活性 体外蠕虫。最后，将评估选定的化合物的PK/PD性能和对抗 小鼠的血吸虫感染。为了实现这些变革性目标，一个创新的国际 具有血吸虫生物化学和药物发现专业知识的全球专家的协作，结构 生物学、计算机辅助分子设计和化学蛋白质组学已经组装完毕。多姿多彩的 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类药物化合物，适用于血吸虫病候选药物的选择 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.