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

验证克氏锥虫二氢乳清酸脱氢酶作为恰加斯病的药物靶点。

• 批准号: 10454280
• 负责人: Flavio da Silva Emery
• 金额: $ 13.25万
• 依托单位: UNIVERSITY OF SAO PAULO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-21 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10454280
• 关键词: Acrylamides; Active Sites; Acute; Address; Affect; Anabolism; Animal Disease Models; Antiparasitic Agents; Binding; Biological Assay; Blood; Brazil; Cell Culture Techniques; Cells; Chagas Disease; Chemical Structure; Chemicals; Chronic; Chronic Phase; Collaborations; Communities; Complex; Computer Assisted; Computer Models; Country; Crystallization; Cysteine; DHODH gene; Data; Developing Countries; Development; Dihydroorotate dehydrogenase; Disease; Dose; Drug Kinetics; Drug Targeting; Enzymatic Biochemistry; Enzyme Inhibition; Enzymes; Equilibrium; Etiology; Flavin Mononucleotide; Fumarates; Funding; Generations; Genetic study; Goals; Hot Spot; Infection; Institution; Kinetics; Knowledge; Latin America; Lead; Libraries; Ligands; Mammalian Cell; Maps; Measures; Metabolic Activation; Metabolism; Methods; Nitriles; Outcome; Oxidation-Reduction; Parasitic Diseases; Parasitology; Pathogenesis; Persons; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Play; Poverty; Predisposition; Property; Public Health; Pyrimidine; Pyrimidine Nucleotides; Reaction; Regimen; Research; Research Personnel; Role; Route; Running; Societies; Solvents; Structure; Succinate Dehydrogenase; Succinates; Techniques; Testing; Time; Toxic effect; Treatment Protocols; Trypanosoma cruzi; United States; Validation; Vulnerable Populations; X-Ray Crystallography; base; computational chemistry; cytotoxicity; data portal; design; dihydroorotate; divinyl sulfone; drug development; drug discovery; drug metabolism; effective therapy; efficacy evaluation; fighting; flexibility; in silico; in vitro activity; in vivo; inhibitor; lead optimization; mouse model; multidisciplinary; neglect; new therapeutic target; novel; orotate; oxidation; public-private partnership; rational design; resistant strain; screening; side effect; structural biology; tool

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.

摘要 克氏锥虫是恰加斯病的病原体，这种疾病影响了 6 至 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， 对当前一线治疗的耐药菌株已经出现。对新产品的需求日益增长 治疗这种疾病的药物替代方案。 该提案利用基于目标的方法来寻找针对恰加斯病毒的先导化合物 疾病。克氏锥虫二氢乳清酸脱氢酶 (TcDHODH) 是一种黄素单核苷酸 含有酶，催化L-二氢乳清酸氧化为乳清酸，第四步也是唯一的 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将琥珀酸/富马酸代谢与从头嘧啶连接起来方面发挥作用 生物合成。 DHODH 已被广泛用作增殖和寄生药物靶标 疾病。遗传学研究表明，DHODH 对于克氏锥虫的生存至关重要，这提供了以下证据： 这种酶是抗恰加斯药物开发的一个有吸引力的靶点。 该项目的目标是通过化学方法验证 TcDHODH 作为恰加斯病的新药物靶点 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的发展 TcDHODH 的选择性和共价抑制剂。首先，我们将寻求对 DHODH 热点，通过使用溶剂图谱来表征具有灵活活性位点的结合口袋 循环打开和关闭。这些信息将有助于加强计算建模，以及后续 复合设计。 基于一组半胱氨酸靶向共价键的酶共价抑制 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，最初的一组假定抑制剂包含丙烯酰胺 弹头已经经过合理设计，可以瞄准TcDHODH活性位点半胱氨酸。预测 将评估抑制剂的抑制效力和 TcDHODH 抑制机制、细胞毒性 及其在细胞培养中的抗寄生虫作用。 X 射线晶体学、片段筛选和药物化学 将结合起来，为合成新一代有效的、选择性的和 药物样抑制剂。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来评估功效。 该提案是一项史无前例的举措，将为该领域的能力建设做出重大贡献。 巴西的药物发现领域是一个在发展中国家仍处于起步阶段的研究领域。