The oxidoreductase Tpx: dissection of an unusual inhibitor binding mode and structural basis for its function in the hydroperoxide clearance cascade in trypanosomatids

氧化还原酶 Tpx：解析一种不寻常的抑制剂结合模式及其在锥虫类氢过氧化物清除级联中的功能的结构基础

• 批准号: 438511573
• 负责人: Professorin Dr. Ute Hellmich
• 金额: --
• 依托单位: Institut für Organische Chemie und Makromolekulare Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/438511573?language=en
• 关键词: oxidoreductase; Tpx; dissection; unusual; inhibitor

Parasites cause devastating diseases and threathen the health of both people and their livestock. Trypanosomatids, including the causative agents of African Sleeping Sickness (Trypanosoma brucei spp.), Chagas disease (T. cruzi) and visceral and cutaneous Leishmaniosis (Leishmania spp.) are unicellular eukaryotic protozoans that are characterized by a unique thiol redox metabolism centered on trypanothione as the global electron donor for DNA synthesis, protein repair and hydroperoxide detoxification. These reactions are carried out by peroxidases such as PxIII that are reduced via tryparedoxin (Tpx), an essential parasitic oxidoreductase. We recently determined the first structure of Tpx in complex with a small, covalent inhibitor that shows anti-trypanosomal potential in vitro and in vivo. Intriguingly, this inhibitor (CFT) induces Tpx dimerization through intricate protein-inhibitor, protein-protein and inhibitor-inhibitor interactions thus constituting a novel concept in the area of chemically induced dimerization. We further described multiple Tpx point mutations that affect oxidoreductase function and/or its interaction with CFT, trypanothione and peroxidases. Despite these advances, a detailed mechanistic understanding of Tpx-catalyzed redox reactions is currently hampered by the lack of reliable structural data for Tpx in complex with its upstream and downstream partners. Finally, the development of improved Tpx inhibitors requires a detailed molecular description of the relevant parameters dictating protein-inhibitor interactions and the observed CFT-induced protein dimerization. We thus aim to employ an integrated structural biology approach to elucidate the structural and dynamical basis of Tpx interaction with the inhibitor CFT, trypanothione and PxIII. Some of the Tpx-CFT interactions involve the protein backbone and can therefore not be investigated by mutagenesis. In these cases, we will chemically modify the inhibitor to probe specific determinants of the intermolecular CFT-Tpx interaction. Tpx is highly conserved across trypanosomatids. This includes the most widely distributed and the most medically relevant human pathogenic species T. cruzi, L. infantum, L. donovani and L. major. Thus, a detailed molecular characterization of small molecule inhibitor Tpx interactions has unprecedented potential for the development of novel drugs against these neglected tropical diseases. Consequently, we will investigate the interaction of our inhibitors with the tryparedoxin oxidoreductases from these organisms on a structural and functional level.

寄生虫引起毁灭性的疾病，并损害人和牲畜的健康。锥虫，包括非洲昏睡病的病原体（布氏锥虫属），恰加斯病（Chagas disease，T. cruzi）和内脏和皮肤利什曼病（利什曼原虫属（Leishmania spp.））是单细胞真核原生动物，其特征在于以锥虫硫酮为中心的独特硫醇氧化还原代谢，锥虫硫酮作为DNA合成、蛋白质修复和氢过氧化物解毒的全局电子供体。这些反应是通过过氧化物酶如PxIII进行的，PxIII通过Tryparedoxin（Tpx）（一种必需的寄生虫氧化还原酶）还原。我们最近确定了第一个结构的Tpx在复杂的一个小的，共价的抑制剂，显示抗锥虫的潜力，在体外和体内。有趣的是，这种抑制剂（CFT）通过复杂的蛋白质-抑制剂，蛋白质-蛋白质和底物-抑制剂相互作用诱导Tpx二聚化，从而构成了化学诱导二聚化领域的新概念。我们进一步描述了影响氧化还原酶功能和/或其与CFT、锥虫硫酮和过氧化物酶相互作用的多个Tpx点突变。尽管取得了这些进展，详细的机理TPX催化的氧化还原反应的理解是目前阻碍了TPX与其上游和下游合作伙伴的复杂的缺乏可靠的结构数据。最后，改进的Tpx抑制剂的开发需要详细的分子描述的相关参数决定蛋白质-抑制剂的相互作用和观察到的CFL诱导的蛋白质二聚化。因此，我们的目标是采用一个综合的结构生物学方法来阐明Tpx与抑制剂CFT，锥虫硫酮和PxIII相互作用的结构和动力学基础。一些Tpx-CFT相互作用涉及蛋白质骨架，因此不能通过诱变进行研究。在这些情况下，我们将化学修饰抑制剂，以探测分子间CFT-Tpx相互作用的特定决定因素。Tpx在锥虫中是高度保守的。这包括分布最广泛和医学上最相关的人类致病种T。克鲁济湖婴儿湖donovani和L.少校因此，详细的小分子抑制剂Tpx相互作用的分子表征，对这些被忽视的热带疾病的新药开发具有前所未有的潜力。因此，我们将研究我们的抑制剂与这些生物体的tryparedoxin氧化还原酶在结构和功能水平上的相互作用。