Validation of Trypanosoma cruzi dihydroorotate dehydrogenase as a drug target for Chagas´disease.

提取 克氏锥虫是南美锥虫病的病原体， 世界各地的人，主要是在拉丁美洲。慢性恰加斯病没有有效的治疗方法， 对目前的前线治疗产生抗药性的菌株已经出现。越来越需要新的 治疗这种疾病的药物替代品。 该提案利用基于目标的方法来寻找针对恰加斯病的先导化合物 疾病克氏锥虫二氢乳清酸脱氢酶（TcDHODH）是一种黄素单核苷酸 含有催化L-二氢乳清酸氧化成乳清酸的酶，第四步， 嘧啶核苷酸从头生物合成中的氧化还原反应。TcDHODH也被描述为 可溶性富马酸还原酶在连接琥珀酸/富马酸代谢与从头嘧啶中起作用 生物合成DHODH已经被广泛开发作为增殖和寄生虫感染的药物靶标。 疾病遗传学研究表明，DHODH是T. cruzi生存提供了证据， 这种酶是开发抗痛风药物的有吸引力的靶点。 该项目的目标是化学验证TcDHODH作为南美锥虫病的新药靶点 并为药物开发提供线索。我们的方法结合了多种技术，包括 计算化学、酶学、结构生物学、寄生虫学和药物化学， TcDHODH的选择性和共价抑制剂。首先，我们将寻求更好地了解 通过使用溶剂映射来表征具有柔性活性位点的结合口袋的DHODH热点 循环打开和关闭。这些信息将有助于加强计算建模， 复合设计 基于半胱氨酸靶向共价键组的酶的共价抑制 包括丙烯酰胺、乙烯砜和腈在内的弹头，已被证明是一种成功的方法， 药物研发管道在我们的建议中，最初的一套假定的抑制剂， 弹头已经被合理地设计为靶向TcDHODH活性位点半胱氨酸。预测 将评价抑制剂对TcDHODH的抑制效力和抑制机制、细胞毒性 以及其在细胞培养中的抗寄生虫作用。X射线晶体学、碎片筛选和药物化学 将结合起来，为合成新一代有效的，选择性的， 类药物抑制剂。符合先导标准（包括PK和耐受性）的前3种抑制剂将有其 DNDi通过对该项目的实物捐助评估其功效。 这一提议是一项前所未有的举措，将大大促进联合国的能力建设， 在巴西，药物发现领域的研究在发展中国家仍处于起步阶段。

ABTRACT Trypanosoma cruzi is the etiological agent of Chagas disease, a pathogenesis that affects 6 to 7 million people worldwide, mainly in Latin America. There is no effective treatment for chronic Chagas disease and resistant strains to the current frontline treatment have already emerged. There is a growing need for new pharmacological alternatives to treat this disease. This proposal exploits a target-based approach to search for lead compounds against Chagas disease. Trypanosoma cruzi dihydroorotate dehydrogenase (TcDHODH) is a flavin mononucleotide containing enzyme, which catalyzes the oxidation of L-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. TcDHODH was also described as a soluble fumarate reductase playing a role in connecting succinate/fumarate metabolism to de novo pyrimidine biosynthesis. DHODH has already been extensively exploited as a drug target for proliferative and parasitic diseases. Genetic studies have shown that DHODH is essential for T. cruzi survival providing evidence that this enzyme is an attractive target for the development of antichagasic drugs. The goal of this project is to chemically validate TcDHODH as a new drug target for Chagas disease and to provide leads for drug development. Our approach combines multiple techniques including computational chemistry, enzymology, structural biology, parasitology, and medicinal chemistry to develop selective and covalent inhibitors of TcDHODH. Initially, we will seek to gain a greater understanding of the DHODH hot spots by using solvent mapping to characterize the binding pockets with the flexible active-site loop open and closed. This information will help strengthen the computational modeling, and subsequent compound design. Covalent inhibition of enzymes based on the identification of a set of cysteine targeting covalent warheads including acrylamides, vinyl sulfones and nitriles, has been shown to be a successful approach in drug discovery pipelines. In our proposal, an initial set of putative inhibitors that incorporate an acrylamide warhead have already been rationally designed to target the TcDHODH active-site cysteine. Predicted inhibitors will be evaluated for inhibitory potency and mechanism of inhibition against TcDHODH, cytotoxicity and its anti-parasitic effects in cell culture. X-ray crystallography, fragment screening and medicinal chemistry will be combined to provide the chemical basis for the synthesis of a new generation of potent, selective, and drug-like inhibitors. The top 3 inhibitors that meet lead criteria (including PK and tolerability) will have their efficacy evaluated by DNDi via an in-kind contribution to the project. This proposal represents an unprecedent initiative to significantly contribute to building capacity in the field of drug discovery in Brazil, a field of research still very incipient in developing countries.