Validating novel drug targets in Trypanosoma cruzi polyamine biosynthesis

描述（由申请人提供）：克氏锥虫是一种引起恰加斯病的原生动物锥虫寄生虫，该疾病影响22个拉丁美洲国家的1000 - 1200万绝大多数贫困人口，估计在美国有30万人。即使在非流行国家，艾滋病也可通过血液或器官捐赠传播，每20名受感染儿童中就有1名死于母婴感染。慢性感染常常未被发现，通过对心肌和心脏离子传导系统的潜在破坏，经常引起致命的心肌病，还引起严重的内脏器官疾病。现有的治疗方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。为恰加斯病开发安全有效的新疗法需要提高对T。为新药先导物的开发提供合理依据。多胺生物合成是锥虫相关寄生虫的必需和药物。克鲁兹因此，我推测（1）T. Cruzi是致命的或对毒力严重有害的，以及（2）该途径包含具有与它们的人类对应物不同的特征的酶，这些特征足以允许它们的选择性抑制。为了验证这些假设，我建议在T. cruzi多胺生物合成：ADOMETDC、SPDSYN、SPMSYN和TRYSYN。为了验证多胺生物合成在T. cruzi等人的研究工作：1.在T. Cruzi的同源重组，并通过补充精脒和精胺分离携带这些潜在条件致死缺失的克隆，精脒和精胺可被寄生虫清除; 1.2）通过测量多胺和锥虫硫酮的水平评估这些缺失的影响（多胺途径下游的代谢物）以及在具有各种多胺补充方案的培养物中的寄生虫生长速率; 1.3）确定每种缺失菌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性，作为合理药物设计的基础，我将：2.1）开发有效的重组ADOMETDC、SPDSYN、SPMSYN和TRYSYN表达系统，并开发和改进获得高纯度ADOMETDC、SPDSYN、SPMSYN和TRYSYN的方案;最后，2.2）解析和分析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T. Cruzi感染，从这项研究中产生的多胺代谢的新的基本见解可能适用于其他原生动物寄生虫感染和非感染性疾病如癌症的生物学和治疗。

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.