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

寄生虫会导致毁灭性的疾病，威胁人和牲畜的健康。锥虫(Trypanosomatids)，包括引起非洲睡眠病(Trypanosoma brucei spp.)、恰加斯病(T.ruzi)和内脏及皮肤利什曼病(Leishmania spp.)的病原体。是单细胞真核原生动物，其特征是以台盼硫酮为中心的独特的硫醇氧化还原代谢，作为DNA合成、蛋白质修复和氢过氧化氢解毒的全球电子供体。这些反应是由PxIII等过氧化物酶进行的，这些过氧化物酶通过色氨酸还蛋白(TPX)还原，色氨酸是一种基本的寄生性氧化还原酶。我们最近确定了TPX的第一个结构，它与一种小的共价抑制剂在体外和体内显示出抗锥虫的潜力。有趣的是，这种抑制物(CFT)通过复杂的蛋白质-抑制物、蛋白质-蛋白质和抑制物-抑制物相互作用诱导TPX二聚化，从而构成了化学诱导二聚化领域的一个新概念。我们进一步描述了影响氧化还原酶功能和/或其与CFT、色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，但由于缺乏可靠的结构数据，目前对TPX催化的氧化还原反应的详细机理理解受到阻碍。最后，改进的TPX抑制剂的开发需要对决定蛋白质-抑制剂相互作用和观察到的CFT诱导的蛋白质二聚的相关参数进行详细的分子描述。因此，我们的目标是使用一种综合的结构生物学方法来阐明TPX与抑制剂CFT、色硫酮和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，因此不能通过突变进行研究。在这些情况下，我们将对抑制剂进行化学修饰，以探索分子间CFT-TPX相互作用的特定决定因素。TPX在锥虫中高度保守。这包括分布最广、与医学关系最密切的人类致病物种克氏锥虫、婴儿锥虫、多诺瓦尼锥虫和梅杰锥虫。因此，对小分子抑制剂TPX相互作用的详细分子表征对于开发治疗这些被忽视的热带病的新药具有前所未有的潜力。因此，我们将在结构和功能水平上研究我们的抑制剂与这些生物体中的Tryparedosin氧化还原酶之间的相互作用。

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.