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.

项目摘要布鲁氏锥虫是一种原生动物寄生虫，是负责人类非洲的病原体锥虫病（帽子）或“睡眠疾病”。帽子和其他由密切相关引起的疾病致病寄生虫代表了全球健康问题，尤其是因为有一个有效的抗寄生虫药物有限选择。最近发现DUTP焦磷酸酶（dutpase）T。brucei中的酶与人或其他哺乳动物的酶明显不同作为可能的抗寄生虫药物靶标，系统已提高了对该酶的兴趣。 dutpase 在最著名的生活形式中是必不可少的蛋白质。这是脱氧胸苷的合成所必需的三磷酸（DTTP）支持DNA复制并限制将尿嘧啶掺入基因组DNA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有效的抗肌体药物，具有有限的毒性和高治疗指数。该建议的具体目标是识别和表征细胞毒性的小分子通过在以下两个特定目标中抑制dutpase活性来抑制t. brucei。在AIM1中，我们将采取两种鉴定靶向t. brucei duptase的候选小分子的完成方法。作为将综合工作扩展到尿素DNA的机制中我们的实验室，我们设计了基于细胞的测定法取代酵母蛋白。使用此系统，我们将筛选分子以不同的抑制分子表达T. brucei dutpase而不是表达人类酶的细胞的生长。此外，通过使用虚拟对接程序，我们将确定分子的结合高对布鲁氏菌的亲和力，但不是人类杜特帕酶。在AIM2中，候选分子的细胞毒性作用将在酵母和布鲁氏菌文化中进一步描述。我们还将确认，如 DUTP-> DTTP代谢途径的破坏，细胞毒性与升高水平相关 DNA，DNA断裂和细胞周期缺陷中的尿嘧啶。拟议的研究的重要性是强调了它的巨大潜力，以进一步了解抗寄生虫的机制针对细胞DUTP/DTTP代谢的药物，最后是改进的机械基础组合化学治疗方案对HAT和其他相关疾病的功效。