Mycobacterium tuberculosis dUTPase as a tool in disease control

项目概述/摘要dUTP酶是一种独特的酶，可以催化dUTP的热磷酸化，从而调节尿嘧啶并入DNA的程度。大量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。目前的建议侧重于结核分枝杆菌（MTB） dutp酶，它在结核分枝杆菌dTTP生物合成中起核心作用，因此它可能对结核分枝杆菌的生存能力至关重要。结核分枝杆菌是引起结核病的病原体，随着新型多药（MDR）和广泛耐药（XDR）菌株的高发生率出现，结核病在全球造成越来越大的威胁。一些研究议程（包括美国国立卫生研究院的一项议程）阐明了成功的结核病管理所需的新措施，这涉及对新药靶点的深入研究和新药的开发。本建议有三个具体目标，所有这些目标都是为了评价MTB dutp酶作为防治结核病的有效工具：dUTPase功能消融术对分枝杆菌生存能力影响的研究[j]。阐明结核分枝杆菌dUTPase的酶促机制，探讨人（宿主）与结核分枝杆菌（病原体）dUTPase的机制差异；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，将采用几种瞬态动力学和平衡酶学以及光谱方法对野生型和突变型MTB dutp酶进行分析。将在dUTPase基因置换的非致病性分枝杆菌模型中研究dUTPase功能性消融在分枝杆菌中的生理效应。本项目的预期结果将对设计有效的MTB dUTPase物种选择性抑制剂以及预测此类抑制剂的体内机制具有重要意义。该项目方法的结合（参见目标1-3）可能为进一步研究潜在的dUTPase靶点（如恶性疟原虫dUTPase）提供有用的概念；锥虫属;牛痘、疱疹和爱泼斯坦-巴尔病毒目前的建议侧重于一种重要的DNA修复酶，称为dUTPase的细菌引起结核病。结核病对全球健康构成越来越大的威胁。因此，一些研究议程（包括一个强有力的NIH计划）阐明了成功管理结核病所需的新措施，这涉及对新药生理靶点的深入研究。本提案的目的是针对dUTPase作为结核病控制药物靶点的评价。

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.