The Dimeric Dutpase of Trypanosoma brucei as a therapeutic target

项目总结 布鲁氏锥虫是一种原生动物寄生虫，也是人类非洲肺炎的病原体。 锥虫病(HAT)，或“昏睡病”。HAT与其他疾病密切相关 致病寄生虫是一个重大的全球健康问题，特别是因为有一个 有效的抗寄生虫药物选择有限。新近发现dUTP焦磷酸酶 布氏毛滴虫的dUTPase酶与人类或其他哺乳动物的dUTPase酶有显著差异 系统公司已经提高了人们对这种酶作为可能的抗寄生虫药物靶点的兴趣。DUTP酶 是大多数已知生命形式的必需蛋白质；它是合成脱氧胸苷所必需的。 三磷酸(DTTP)，用于支持DNA复制和限制尿嘧啶在 基因组DNA，以减少尿嘧啶糖基酶引发的有害DNA断裂。从长远来看， 目的是开发一种毒性小、治疗指数高、有效的抗锥虫药物。 这项提议的具体目标是识别和表征具有细胞毒性的小分子 通过抑制dUTPase活性达到以下两个特定目的。在Aim1中，我们将采取 两种互补的方法来识别靶向布氏毛滴虫dUPTase的候选小分子。AS 年开展的尿嘧啶DNA掺入机制的综合工作的延伸 我们的实验室，我们设计了一种基于细胞的检测方法，其中布氏毛滴虫或人类dUTPase的功能 取代了酵母蛋白。使用这个系统，我们将筛选差异抑制分子 表达布氏支原体dUTPase的酵母细胞的生长，而不是表达人酶的酵母细胞的生长。 此外，通过使用虚拟对接程序，我们将识别与高结合的分子 对布氏毛滴虫有亲和力，但不对人dUTPase有亲和力。在AIM2中，候选分子的细胞毒作用 将在酵母菌和布氏锥虫培养中进一步鉴定。我们还将确认，正如预期的那样， DUTP-&dTTP代谢途径受阻，细胞毒性与升高水平相关 DNA中尿嘧啶的含量、DNA断裂和细胞周期缺陷。拟议研究的意义在于 强调了它的巨大潜力，提供了对抗寄生虫机制的进一步洞察 靶向细胞dUTP/dTTP代谢的药物，最后是改善 联合化疗方案治疗HAT等相关疾病的疗效观察

PROJECT SUMMARY Trypanosoma Brucei is a protozoan parasite and a pathogen responsible for the Human African Trypanosomiasis (HAT), or “sleeping sickness”. HAT and other diseases caused by closely related pathogenic parasites represent a substantial global health concern, especially because there is a limited choice of effective antiparasitic drugs. Recent discovery that the dUTP pyrophosphatase (dUTPase) enzyme in T. brucei is significantly divergent from that of human or other mammalian systems has heightened the interest in this enzyme as a possible anti-parasitic drug target. dUTPase is an essential protein in most known living forms; it is required for the synthesis of deoxythymidine triphosphate (dTTP) in support of DNA replication and for limiting the incorporation of uracil into genomic DNA to reduce the detrimental DNA breaks initiated by uracil glycosylase. In the long term, the goal is to develop an effective anti-trypanosome drug with limited toxicity and high therapeutic index. The specific objective of this proposal is to identify and characterize small molecules that are cytotoxic to T. brucei by inhibition of the dUTPase activity in the following two specific aims. In Aim1, we will take two complementary approaches to identify candidate small molecules targeting T. brucei dUPTase. As an extension of the comprehensive work into the mechanism of uracil DNA incorporation carried out in our laboratory, we designed a cell-based assay where T. brucei or human dUTPase functionally replaces the yeast protein. Using this system, we will screen for molecules differentially inhibiting the growth of yeast cells expressing T. brucei dUTPase and not the cells expressing the human enzyme. Additionally, through the use of virtual docking program, we will identify molecules binding with high affinity to T. brucei but not human dUTPase. In Aim2, the cytotoxic effect of the candidate molecules will be further characterized in yeast and T. brucei cultures. We will also confirm that, as expected from the disruption of dUTP->dTTP metabolic pathway, the cytotoxicity is correlated with the elevated level of uracil in DNA, DNA breaks, and cell-cycle defect. The significance of the proposed research is underscored by its significant potential to provide further insight into the mechanism of anti-parasitic drugs that target the cellular dUTP/dTTP metabolism and finally a mechanistic basis for the improved efficacy of the combination chemotherapeutic regimen against HAT and other related diseases.