Validating novel drug targets in Trypanosoma cruzi polyamine biosynthesis

描述（由申请人提供）：克氏锥虫是一种导致恰加斯病的原生锥虫寄生虫，影响着22个拉丁美洲国家的1000万至1200万极度贫困人口，估计在美国有30万人。即使在非流行国家，它也可以通过血液或器官捐献传播，每20名受感染儿童中就有1人因母婴感染而死亡。慢性感染常常不被发现，通过对心脏的心肌和离子传导系统的潜在破坏，经常引起致命的心肌病，也引起严重的内脏器官疾病。现有的治疗方法有严重的毒性问题，需要长时间的治疗，并且被认为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点的认识，并获得新药先导的合理基础。多胺生物合成是与克氏锥虫有关的锥虫虫必不可少的药物。因此，我假设：(1)克氏锥虫多胺生物合成途径的破坏是致命的或对毒性严重有害的；(2)该途径含有与人类相应的酶具有不同特征的酶，足以允许其选择性抑制。为了验证这些假设，我建议评估和表征克氏多胺生物合成中有希望的药物靶点：ADOMETDC， SPDSYN， SPMSYN和TRYSYN。为了检验克氏体多胺生物合成的必要性，我将：1.1)通过同源重组在克氏体菌株中产生ADOMETDC、SPDSYN和SPMSYN缺失突变体，并通过补充可被寄生虫清除的亚精胺和精胺分离出具有潜在条件致死缺失的克隆；1.2)通过测量多胺和锥虫硫酮（多胺途径下游的代谢物）的水平以及不同多胺补充方案培养中寄生虫的生长速度来评估这些缺失的影响；1.3)测定各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征，为合理的药物设计提供依据，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纯度的ADOMETDC、SPDSYN、SPMSYN和TRYSYN；最后，2.2)求解和分析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。除了验证克氏锥虫感染的新治疗方法外，本研究产生的关于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（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.