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

减去 克氏锥虫是恰加斯病的病原体，这种疾病影响了600万到700万人。 世界各地的人，主要是拉丁美洲的人。慢性恰加斯病没有有效的治疗方法， 已经出现了对当前一线治疗具有耐药性的菌株。越来越多的人需要新的 治疗这种疾病的药物替代品。 这项建议利用了一种基于目标的方法来寻找针对查加斯的先导化合物。 疾病。克鲁氏锥虫二氢罗酸脱氢酶(TcDHODH)是黄素单核苷酸 第四步，也是唯一一步，含有催化L-二氢甲酸甲酯氧化为甲酸乙酯的酶 嘧啶核苷酸从头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琥珀酸/富马酸代谢与从头合成嘧啶中的作用 生物合成。DHODH已被广泛用作治疗增殖和寄生虫的药物靶点 疾病。遗传学研究表明，DHODH对克氏锥虫的生存是必不可少的，这提供了证据 这种酶是开发抗脉络痛药物的一个有吸引力的靶点。 该项目的目标是从化学上验证TcDHODH作为治疗恰加斯病的新药靶点 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正在发展 TcDHODH的选择性和共价抑制剂。首先，我们会寻求更深入地了解 用溶剂映射法表征具有柔性活性中心的DHODH热点 环路打开和关闭。这些信息将有助于加强计算建模，以及后续 复合式设计。 基于鉴定一组靶向共价半胱氨酸的酶的共价抑制 包括丙烯酰胺、乙烯基砜和腈在内的弹头已被证明是一种成功的方法。 药物发现管道。在我们的提案中，最初的一组推定的抑制剂中包含了丙烯酰胺 弹头已经被合理地设计成瞄准TcDHODH活性部位的半胱氨酸。预测 将评估抑制剂对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射线结晶学、碎片筛选和药物化学 将结合起来为合成新一代有效的、选择性的和 类药物抑制剂。符合铅标准(包括PK和耐受性)的前3名抑制剂将有他们的 DNDI通过对该项目的实物捐助来评估疗效。 这一提议是一项史无前例的倡议，旨在显著促进 巴西的药物发现领域，这一研究领域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.