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The Dimeric Dutpase of Trypanosoma brucei as a therapeutic target

布氏锥虫二聚体脱氨酶作为治疗靶点

基本信息

批准号：
10362613
负责人：
Nayun Kim
金额：
$ 23.07万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-03-02 至 2023-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10362613
关键词：
2&apos -Deoxythymidine Affect Affinity African Trypanosomiasis Antiparasitic Agents Binding Biochemical Biological Assay Biology Caring Cell Culture Techniques Cell Cycle Cells Clinical Communicable Diseases Crystallization Cytostatics DNA DNA Damage DNA Repair DNA biosynthesis DNA lesion Defect Development Disease Docking Drug Targeting Engineering Enzymes Etiology Event Excision Fluorouracil Genome Genomic DNA Goals Growth Health Human Kinetics Knowledge Laboratories Lead Lesion Ligands Mediating Metabolic Pathway Metabolism Methods Methotrexate Mission Organism Parasites Pathogenicity Pathway interactions Pentamidine Pharmaceutical Preparations Pharmacology Production Proteins Protozoan Infections Public Health Recombinants Regimen Research Resolution Site Structural Biochemistry Structure System Therapeutic Index Thymidine Toxic effect Trypanocidal Agents Trypanosoma Trypanosoma brucei brucei United States National Institutes of Health Uracil Work Yeasts base cancer therapy cellular targeting combat cytotoxic cytotoxicity dUTP pyrophosphatase design dimer efficacy testing genome integrity genotoxicity global health high risk improved insight interest mortality novel novel strategies nucleotide metabolism pathogen prevent programs screening small molecule small molecule inhibitor targeted agent therapeutic target tripolyphosphate virtual virtual screening yeast genetics yeast protein

项目摘要

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.

项目摘要布氏锥虫是一种原生动物寄生虫，也是导致非洲人类感染的病原体。锥虫病（HAT），或“昏睡病”。HAT与其他引起的疾病密切相关致病性寄生虫代表了一个重要的全球健康问题，特别是因为有效抗寄生虫药物的选择有限。最近发现dUTP焦磷酸酶（dUTR）酶。布氏杆菌与人或其它哺乳动物的布氏杆菌显著不同系统已经提高了这种酶作为可能的抗寄生虫药物靶点的兴趣。dutpase 是大多数已知生命形式中的必需蛋白质;它是合成脱氧胸苷所必需的三磷酸（dTTP），以支持DNA复制和限制尿嘧啶掺入基因组DNA，以减少尿嘧啶糖基化酶引发的有害DNA断裂。从长远来看，目的是开发一种具有有限毒性和高治疗指数的有效抗锥虫药物。本提案的具体目标是识别和表征具有细胞毒性的小分子随时为我们提供帮助布氏杆菌通过抑制dUTR的活性实现以下两个具体目的。在Aim 1中，我们将两种互补的方法来鉴定靶向T的候选小分子。布氏杆菌duptase。作为一个扩展的全面工作到尿嘧啶DNA掺入的机制进行，我们的实验室，我们设计了一种基于细胞的检测方法，其中T。布鲁氏菌或人脱氧尿苷酶功能上代替了酵母蛋白。利用这个系统，我们将筛选出差异抑制表达T.而不是表达人类酶的细胞。此外，通过使用虚拟对接程序，我们将识别与高分子结合的分子亲和T.而不是人结核菌素。在Aim2中，候选分子的细胞毒性作用将在酵母和T.布鲁氏菌培养物。我们还将确认，正如预期的那样， dUTP-> dTTP代谢途径的破坏，细胞毒性与升高的水平相关尿嘧啶的DNA，DNA断裂和细胞周期缺陷。本研究的意义在于强调其重要的潜力，提供进一步深入了解抗寄生虫的机制，靶向细胞dUTP/dTTP代谢的药物，并最终为改善联合化疗方案对HAT和其他相关疾病的疗效。