Identification of the target of meclonazepam in schistosome worms

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。 许多具有抗血吸虫活性的极具前景的化合物已被鉴定。然而，进一步 这些化合物的药物进展受到阻碍，因为它们的目标未知。在这方面， 苯二氮卓类衍生物甲氯硝西潘(Ro11-3128)已被确定为首选候选药物。 甲硝西仑对曼氏血吸虫成熟期和未成熟期均显示出较强的治疗活性 和人类体内的嗜血杆菌。不幸的是，这种药物后来由于缺乏选择性而停用。 在治疗剂量下会产生不可接受的副作用。甲硝西潘是一种抗焦虑的苯二氮卓类药物 是哺乳动物γ-氨基丁酸受体的变构调节剂。血吸虫中没有同源物 在基因组数据库中已经发现了蠕虫。最接近的匹配(谷氨酸门控转运体) 已被研究，且未被甲氯硝西潘抑制。这表明甲硝西仑在体内的靶点 血吸虫不是已知的苯二氮卓类受体，至今仍未被发现。 这项修订后的R21提案旨在测试甲硝西仑通过以下途径杀死蠕虫的中心假设 与蠕虫特定的未知目标交互。我们建议确定甲氯硝西仑的靶点 用正交法和互补法研究血吸虫寄生虫(1)设计和合成新的 类药物甲氯西仑光反应探针，以及(2)结合蛋白的生物物理变化 抑制剂。我们预计这些分析将鉴定出多种蛋白质。蛋白质将在以下情况下进行进一步研究 (1)它们只在对甲氯西仑敏感的物种中被鉴定，而在那些不敏感的物种中不被鉴定；(2)它们是 在用甲氯硝西仑或活性类似物处理的蠕虫中发现，在用非活性处理的蠕虫中未发现 类似物，以及(3)甲氯硝西仑与结合蛋白的探针竞争。满足这些严格要求的蛋白质 将选择标准进行进一步调查。势函数分析、重组表达和 生化分析和基因沉默将用于确认靶标。分子鉴定 蠕虫中的靶点是开发新的治疗靶点和临床生产所需的必要的第一步 血吸虫病的相关治疗。

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. A number of highly promising compounds with antischistosomal activity have been identified. However, further advancement of these compounds to drugs is impeded because their targets are unknown. In this regard, the benzodiazepine derivative meclonazepam (Ro11-3128) had been identified as a lead candidate. Meclonazepam exhibited potent therapeutic activity against both mature and immature stages of S. mansoni and S. haematobium in humans. Unfortunately, the drug was later discontinued due to its lack of selectivity resulting in unacceptable side effects at therapeutic doses. Meclonazepam is an anxiolytic benzodiazepine that is an allosteric modulator of mammalian gamma-amino butyric acid receptors. No homologues in Schistosoma worms have been identified in genome databases. The closest matches (glutamate-gated transporters) have been investigated and were not inhibited by meclonazepam. This indicates that the target of meclonazepam in schistosomes is not a known benzodiazepine receptor and remains undiscovered. This revised R21 proposal is designed to test the central hypothesis that meclonazepam kills worms through interaction with a worm-specific and unknown target. We propose to identify the target of meclonazepam in Schistosoma parasites using orthogonal and complementary approaches (1) design and synthesis of novel, drug-like meclonazepam-based photoreactive probes, and (2) biophysical changes in proteins bound to inhibitors. We anticipate multiple proteins being identified by these analyses. Proteins will be studied further if (1) they were identified only in species sensitive to meclonazepam and not in those insensitive, (2) they were identified in worms treated with meclonazepam or active analogs and not in worms treated with inactive analogs, and (3) meclonazepam competes with probe binding to the protein. Proteins meeting these strict criteria will be selected for further investigation. Analysis of potential function, recombinant expression and biochemical analysis, and gene silencing will be used to confirm the targets. Identification of the molecular target in the worm is the essential first step needed to develop novel therapeutic targets and produce clinically relevant treatments for schistosomiasis.