Mycobacterium tuberculosis dUTPase as a tool in disease control

描述(由申请人提供)：项目摘要/摘要dUTP酶是催化dUTP焦磷酸分解的唯一酶，从而调节尿嘧啶在DNA中的结合程度。大量的尿嘧啶掺入可能导致细胞死亡。因此，dUTPase已被认为是癌症、病毒和细菌疾病控制的高潜力药物靶点。目前的建议集中在结核分枝杆菌(MTB)dUTPase，它在分枝杆菌dTTP的生物合成中发挥核心作用，因此它可能是MTB生存所必需的。结核分枝杆菌是引起结核病的病原体，随着新的多药耐药(MDR)和广泛耐药(XDR)菌株的出现，结核病对全球构成越来越大的威胁。几个研究议程(包括美国国立卫生研究院的一个)阐明了成功的结核病管理所需的新措施，其中包括对新药靶点的深入研究和新药的开发。目前的建议有三个特定的目的都是为了评价MTB dUTPase作为一种有效的抗击结核病的工具：1.研究dUTPase功能消融对分枝杆菌生存的影响，2.阐明MTB dUTPase与人(宿主)和MTB(病原体)dUTPase之间的机制差异的酶机制，3)确定dUTPase中可能是物种特异性靶向的两个结构元件的催化作用。为了解决上述问题，将使用野生型和突变型MTB dUTPase酶的几种瞬时动力学和平衡酶以及光谱学方法。在一个非致病的分枝杆菌模型中，将研究dUTPase基因替换对分枝杆菌中dUTPase功能消融的生理影响。该项目的预期结果将对MTB dUTPase的有效物种选择性抑制剂的设计和对此类抑制剂的体内机制的预测非常有用。本项目各种方法的结合(参看。AIMS 1-3)可作为进一步研究潜在dUTP酶靶标的有用概念，如恶性疟原虫、锥虫、牛痘、疱疹和EB病毒的dUTP酶。公共卫生相关性本提案侧重于引起结核病的细菌的一种重要的DNA修复酶，称为dUTPase。结核病对全球健康构成越来越大的威胁。因此，几个研究议程(包括一个强有力的NIH项目)阐明了成功的结核病管理所需的新措施，这涉及到对新药的生理靶点的深入研究。这项提案的目的是评估dUTPase作为结核病控制的药物靶标的作用。

DESCRIPTION (provided by applicant): Project Summary / Abstract dUTPase is the unique enzyme that catalyses the pyrophosphorolysis of dUTP, thus regulating the extent of uracil incorporation into DNA. Massive uracil incorporation may lead to cell death. dUTPase has therefore been recognized as a high-potential drug target in cancer, viral and bacterial disease control. The present proposal focuses on the Mycobacterium tuberculosis (MTB) dUTPase that plays a central role in the mycobacterial dTTP biosynthesis and thus it is likely to be essential for the viability of MTB. MTB is the pathogen that causes tuberculosis, which imposes an increasing global threat with the high-rate emergence of novel multidrug (MDR) and extensively drug-resistant (XDR) strains. Several research agendas (including one at NIH) articulated new measures needed for successful tuberculosis management, which involves intensive research on new drug targets and the development of novel drugs. The present proposal has three Specific Aims all directed towards the evaluation of MTB dUTPase as a valid tool in fighting tuberculosis: 1. Study of the effect of dUTPase functional ablation on the viability of Mycobacterium, 2. Elucidation of the enzymatic mechanism of MTB dUTPase with regards to the mechanistic differences between the human (host) and MTB (pathogen) dUTPases, 3) Determination of the catalytic role of the two structural elements that may be species-specifically targeted in dUTPase. To address the above issues, several transient kinetic and equilibrium enzymological as well as spectroscopical methods will be employed using wild- type and mutant MTB dUTPase enzymes. The physiological effect of functional ablation of dUTPase in Mycobycterium will be investigated in a non-pathogenic Mycobacterium model subjected to dUTPase gene replacement. The expected results of the proposed project will be highly useful in effective species-selective inhibitor design for MTB dUTPase and in the prediction of the in vivo mechanism of such inhibitors. The combination of approaches of this project (cf. Aims 1-3) may serve as a useful concept for the investigation of further potential dUTPase targets such as dUTPases from Plasmodium falciparum; Trypanosoma; vaccinia, herpes and Epstein-Barr viruses. PUBLIC HEALTH RELEVANCE The present proposal focuses on an important DNA repair enzyme called dUTPase of the bacterium causing tuberculosis. Tuberculosis imposes an increasing threat on global health. Therefore several research agendas (including a robust NIH program) articulated new measures needed for successful tuberculosis management, which involves the intensive research of physiological targets for new drugs. The aims of this proposal are directed towards the evaluation of dUTPase as a drug target in tuberculosis control.