Identification of the target of meclonazepam in schistosome worms

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。 已经鉴定了许多具有抗抗肿瘤活性的非常有前途的化合物。但进一步 这些化合物向药物的发展受到阻碍，因为它们的靶点未知。在这方面 苯二氮卓衍生物甲氯硝西泮（Ro 11 -3128）已被确定为主要候选药物。 甲氯硝西泮对成熟期和未成熟期的S.曼氏 和S.人体内的嗜血杆菌不幸的是，由于缺乏选择性，该药物后来被停用 导致治疗剂量下不可接受的副作用。甲氯安定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。血吸虫没有同源物 蠕虫已经在基因组数据库中被识别。最接近的匹配（谷氨酸门控转运蛋白） 已进行研究，且不受甲氯硝西泮抑制。这表明甲氯硝西泮的靶点 β受体不是已知的苯二氮卓类受体，并且仍然未被发现。 这项修订后的R21提案旨在检验中心假设，即甲氯硝西泮通过以下途径杀死蠕虫： 与蠕虫特定的未知目标相互作用。我们建议确定甲氯硝西泮的靶点， 使用正交和互补方法的血吸虫寄生虫（1）新的， 药物样甲氯硝西泮为基础的光反应性探针，和（2）结合蛋白质的生物物理变化， 抑制剂的我们预计通过这些分析鉴定出多种蛋白质。蛋白质将进一步研究，如果 (1)它们仅在对甲氯硝西泮敏感的物种中被鉴定，而在不敏感的物种中未被鉴定;（2）它们是 在用甲氯硝西泮或活性类似物处理的蠕虫中发现，而在用非活性 类似物，和（3）甲氯硝西泮与探针竞争结合蛋白质。满足这些严格 我们会选定准则作进一步研究。潜在功能分析、重组表达和 生物化学分析和基因沉默将用于确认目标。分子鉴别 在蠕虫中的靶点是开发新的治疗靶点和临床生产所需的基本第一步 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.