Validating novel drug targets in Trypanosoma cruzi polyamine biosynthesis

描述(申请人提供)：克氏锥虫是一种原生动物锥虫寄生虫，导致恰加斯病，影响22个拉丁美洲国家的1,000-1,200万贫困人口，美国估计有300,000人。即使在非流行国家，它也可以通过献血或器官捐赠传播，母婴感染导致20名受影响儿童中有1人死亡。慢性感染往往在未被发现的情况下，通过潜伏地破坏心脏的心肌和离子传导系统而导致致命的心肌病，还会导致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，需要长时间的治疗，而且据信只对疾病的急性阶段有效。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标的了解，并为新的药物线索获得合理的基础。在与克氏锥虫相关的锥虫寄生虫中，多胺的生物合成是必要的和可用药的。因此，我假设(1)克氏锥虫多胺生物合成途径的中断对毒力是致命的或严重有害的，(2)该途径含有与人类对应的酶不同的特征，足以允许它们的选择性抑制。为了验证这些假设，我建议评估和表征在克氏锥虫多胺生物合成中有希望的药物靶点：ADOMETDC、SPDSYN、SPMSYN和TRYSYN。为了测试克鲁兹锥虫合成多胺的重要性，我将：1.1)在克鲁兹锥虫中通过同源重组产生ADOMETDC、SPDSYN和SPMSYN的缺失突变，并通过补充亚精胺和精胺来分离含有这些潜在的有条件致死缺失的克隆，而精胺和精胺可被这种寄生虫清除；1.2)通过测量多胺和色胺(多胺途径下游的代谢物)的水平以及在各种多胺补充方案下培养的寄生虫的生长速度，评估这些缺失的影响；1.3)确定每个缺失菌株在哺乳动物细胞中的感染力。为了确定多胺生物合成酶的显著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，并开发和完善获得高纯度ADOMETDC、SPDSYN、SPMSYN和TRYSYN的方案；最后，2.2)解决和分析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。除了验证治疗弓形虫感染的新方法外，这项研究产生的对多胺代谢的新的基本见解可能适用于其他原虫寄生虫感染和非传染性疾病的生物学和治疗，如癌症。

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.