Validating novel drug targets in Trypanosoma cruzi polyamine biosynthesis

验证克氏锥虫多胺生物合成中的新药物靶点

基本信息

批准号：
8525512
负责人：
Michael C. Gretes
金额：
$ 5.39万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8525512
关键词：
Ablation Acute Adenosylmethionine Decarboxylase Adverse effects Affect African Trypanosomiasis American Amino Acids Anabolism Back Biology Blood Donations Cadaverine Cardiac conduction system Cardiomyopathies Cell Line Cells Chagas Disease Child Chronic Complement Complex Country Discipline Disease Dissection Drug Design Drug Targeting Educational workshop Environment Enzymes Episome Eukaryota Genes Goals Grant Growth High Pressure Liquid Chromatography Human Infection Interdisciplinary Study International Ions Knowledge Laboratories Latin America Left Life Cycle Stages Ligands Ligase Malignant Neoplasms Mammalian Cell Measures Medicine Mentors Metabolic Pathway Metabolism Methods Molecular Mothers Myocardium Nature Nutritional Nutritional Requirements Organ Organ Donations Outcomes Research Oxidation-Reduction Parasites Parasitic Diseases Parasitology Pathway interactions Patients Pharmaceutical Preparations Polyamines Proteins Protocols documentation Putrescine Recombinants Regimen Research Resolution Spermidine Spermidine Synthase Spermine Spermine Synthase Staging Structure Supplementation System Techniques Technology Testing Therapeutic Toxic effect Training Transfection Treatment Protocols Trypanosoma brucei brucei Trypanosoma cruzi United States Ursidae Family Virulence Visceral Work Writing analog base cell growth design dithiol drug discovery experience genetic manipulation homologous recombination improved inhibitor/antagonist insight killings mutant neglect novel novel therapeutic intervention pathogen protein structure public health relevance skills symposium trypanothione

项目摘要

DESCRIPTION (provided by applicant): Trypanosoma cruzi is the protozoan trypanosomatid parasite that causes Chagas disease, which affects 10-12 million overwhelmingly poor people in 22 Latin American countries and an estimated 300,000 in the United States. It is transmissible even in non-endemic countries by blood or organ donation, and mother-to-child infection kills 1 in 20 affected children. Often undetected, chronic infection frequently causes fatal cardiomyopathy via insidious destruction of the myocardium and ion conduction systems of the heart, and also causes severe visceral organ disease. Existing therapies have major toxicity problems, require lengthy treatment, and are believed to be effective only against the acute stage of the disease. Developing safe and effective new therapies for Chagas disease requires improving knowledge of valid drug targets in T. cruzi and obtaining rational bases for new drug leads. Polyamine biosynthesis is essential and druggable in trypanosomatid parasites related to T. cruzi. Therefore, I hypothesize (1) that disruption of the polyamine biosynthetic pathway in T. cruzi is lethal or severely detrimental to virulence and (2) that this pathway contains enzymes with distinct features from their human counterparts sufficient to permit their selective inhibitio. To test these hypotheses, I propose to evaluate and characterize promising drug targets in T. cruzi polyamine biosynthesis: ADOMETDC, SPDSYN, SPMSYN, and TRYSYN. To test the essentiality of polyamine biosynthesis in T. cruzi, I will: 1.1) generate deletion mutants of ADOMETDC, SPDSYN and SPMSYN in strains of T. cruzi by homologous recombination and isolate clones bearing these potentially conditionally lethal deletions by supplementation with spermidine and spermine, which can be scavenged by the parasite; 1.2) evaluate the effects of these deletions by measuring levels of polyamines and trypanothione (a metabolite immediately downstream of polyamine pathway) as well as parasite growth rates in culture with various polyamine supplementation regimens; 1.3) determine infectivity of each deletion strain in mammalian cells. To identify distinct features of polyamine biosynthetic enzymes as a basis for rational drug design, I will: 2.1) develop efficient recombinant ADOMETDC, SPDSYN, SPMSYN and TRYSYN expression systems, and develop and refine protocols to obtain highly purified ADOMETDC, SPDSYN, SPMSYN and TRYSYN; and finally, 2.2) solve and analyze crystal structures of ADOMETDC, SPMSYN, SPDSYN, and TRYSYN alone and in complex with substrates and substrate analogs. In addition to validating new therapeutic approaches to T. cruzi infection, new fundamental insights into polyamine metabolism arising from this study may be applicable to the biology and treatment of other protozoan parasite infections and non-infectious diseases such as cancer.

描述（由申请人提供）：克氏锥虫是一种原生动物锥虫寄生虫，可引起恰加斯病，影响 22 个拉丁美洲国家的 10-1200 万贫困人口，估计在美国有 30 万贫困人口。即使在非流行国家，这种病毒也可以通过血液或器官捐赠传播，母婴感染会导致二十分之一的受影响儿童死亡。通常未被发现的慢性感染经常通过对心肌和心脏离子传导系统的潜在破坏而导致致命的心肌病，并且还会导致严重的内脏器官疾病。现有疗法存在严重的毒性问题，需要长期治疗，并且被认为仅对疾病的急性期有效。开发安全有效的恰加斯病新疗法需要提高对克氏锥虫有效药物靶点的了解，并为新药物先导物获得合理的基础。多胺生物合成对于与克氏锥虫相关的锥虫寄生虫来说是必需的且可药物化。因此，我假设（1）克氏锥虫中多胺生物合成途径的破坏是致命的或严重损害毒力；（2）该途径含有与人类对应物具有明显特征的酶，足以允许其选择性抑制。为了检验这些假设，我建议评估和表征克氏锥虫多胺生物合成中有前景的药物靶点：ADOMETDC、SPDSYN、SPMSYN 和 TRYSYN。为了测试克氏锥虫中多胺生物合成的必要性，我将： 1.1) 通过同源重组在克氏锥虫菌株中产生 ADOMETDC、SPDSYN 和 SPMSYN 的缺失突变体，并通过补充亚精胺和精胺分离带有这些潜在条件致死缺失的克隆。，可以被寄生虫清除； 1.2) 通过测量多胺和锥硫酮（多胺途径下游的代谢物）的水平以及使用各种多胺补充方案培养的寄生虫生长率来评估这些缺失的影响； 1.3)确定每个缺失株在哺乳动物细胞中的感染性。为了确定多胺生物合成酶的独特特征作为合理药物设计的基础，我将： 2.1) 开发高效的重组 ADOMETDC、SPDSYN、SPMSYN 和 TRYSYN 表达系统，并开发和完善方案以获得高度纯化的 ADOMETDC、SPDSYN、SPMSYN 和 TRYSYN ;最后，2.2) 求解并分析 ADOMETDC、SPMSYN、SPDSYN 和 TRYSYN 单独以及与底物和底物类似物复合的晶体结构。除了验证克氏锥虫感染的新治疗方法外，这项研究对多胺代谢的新基本见解可能适用于其他原生动物寄生虫感染和非传染性疾病（例如癌症）的生物学和治疗。