Mycobacterium tuberculosis dUTPase as a tool in disease control

描述（由申请人提供）：项目摘要/摘要dUTP是催化dUTP焦磷酸解的独特酶，从而调节尿嘧啶掺入DNA的程度。大量尿嘧啶掺入可导致细胞死亡。因此，dUTR已被认为是癌症、病毒和细菌疾病控制中的高潜力药物靶标。目前的建议集中于结核分枝杆菌（MTB）dUTR，它在分枝杆菌dTTP生物合成中起着核心作用，因此它可能对MTB的生存力至关重要。结核分枝杆菌是引起结核病的病原体，随着新型多药耐药（MDR）和广泛耐药（XDR）菌株的高发生率，结核病对全球构成越来越大的威胁。几项研究议程（包括美国国立卫生研究院的一项研究议程）阐明了成功管理结核病所需的新措施，其中涉及对新药靶点的深入研究和新药的开发。本提案有三个具体目标，都是为了评价结核分枝杆菌dUTR作为抗击结核病的有效工具：1. dUTR功能性去除对分枝杆菌活力的影响的研究，2。关于人（宿主）和MTB（病原体）dUTPases之间的机制差异阐明MTB dUTPases的酶机制，3）确定可能在dUTPases中物种特异性靶向的两种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脱氧尿苷酶采用几种瞬时动力学和平衡酶学以及光谱学方法.将在进行dUTR基因置换的非致病性分枝杆菌模型中研究分枝杆菌中dUTR功能性消融的生理效应。该项目的预期结果将在MTB dUTR的有效物种选择性抑制剂设计和预测这种抑制剂的体内机制方面非常有用。本项目的方法组合（参见目的1-3）可以作为研究进一步的潜在dUTPs靶点（例如来自恶性疟原虫、锥虫、牛痘、疱疹和EB病毒的dUTPs）的有用概念。公共卫生相关性本提案集中于引起结核病的细菌的一种称为dUTR的重要DNA修复酶。结核病对全球健康构成越来越大的威胁。因此，几项研究议程（包括一个强大的NIH计划）阐明了成功的结核病管理所需的新措施，其中包括对新药生理靶点的深入研究。本提案的目的是评价dUTR作为结核病控制药物靶标的作用。

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.