Calanolides as tools to find new drug targets for Mycobacterium tuberculosis

描述（由申请人提供）：结核分枝杆菌（Mtb）是一个重大的全球健康问题，感染了世界三分之一的人口，是一种可治愈的传染病导致死亡的主要原因。2012年报告的可归因死亡率为130万。尽管自古以来就有人类感染，但我们对结核分枝杆菌的发病机制缺乏全面的了解。由于耐药的出现，以及结核分枝杆菌在非复制（NR）状态下长时间潜伏的能力，治疗具有挑战性。大多数目前的治疗方法在根除NR结核分枝杆菌方面无效，这种结核分枝杆菌有可能重新激活，导致疾病。需要针对复制型(R)和NR结核分枝杆菌的新疗法。我们已经确定了一类新的化合物，硝基呋喃烷酸酯（NCs），实现了这一目标。NCs对R和NR结核分枝杆菌非常有效，具有选择性，并且被原代人细胞耐受良好。此外，它们在原代人巨噬细胞中杀死Mtb的能力使它们成为一个重要的研究类别。我们的项目重点是使用NCs作为工具来发现休眠NR状态的生物学和新的药物靶点。除了使用代谢组学鉴定被nc改变的基本途径外，我们还将通过发现在基本靶点上有突变的nc耐药细菌来实现这一目标。我们的初步数据显示，Mtb中功能未知的基因rv2466c是NCs的激活酶，在体外导致几种转化产物的产生，表明该酶具有硝化还原酶功能。我们将鉴定和合成这些转化产物，并测试它们对结核分枝杆菌的活性。如果激活，这些转化产物也将用于识别在杀死R和NR Mtb中重要的潜在目标。此外，我们预测rv2466c除了在NC激活中发挥作用外，还在Mtb的发病机制和休眠中发挥作用。来自转录组研究的证据预测它在宿主遇到的应激条件下是重要的，我们的初步发现表明它在氧化应激抵抗中的重要性支持了这一点。我们将通过产生Rv2466c敲除菌株，并评估其在生理应激反应和小鼠体内感染期间的表型，进一步评估Rv2466c的意义。此外，我们将使用共免疫沉淀策略评估其结合伙伴，同时使用来自已有晶体结构的信息来指导我们更好地描述酶和结合活性的重要位点。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb) is a significant global health problem, infecting one third of the world's population, and is the leading cause of death from a curable infectious disease. Attributable mortality rates were reported as 1,300,000 in 2012. Despite infection in humans since ancient times, we lack complete understanding of Mtb pathogenesis. Therapy is challenging due to the emergence of drug resistance, and the ability of Mtb to remain latent for extended periods in a state referred to as non-replicating (NR) Most current therapies are ineffective at eradicating NR Mtb, which has the potential to reactivate, causing disease. New therapeutics are needed that target both replicating (R) and NR Mtb. We have identified a novel class of compounds, nitrofuranyl calanolides (NCs), which achieve this goal. NCs are very potent against R and NR Mtb, are selective, and tolerated well by primary human cells. In addition, their ability to kill Mtb in primary human macrophages makes them an important class to study. Our project focuses on using NCs as tools to discover the biology of the dormant NR state and new druggable targets. We will accomplish this by finding NC-resistant bacteria that have mutations in the essential target(s), in addition to using metabolomics to identify essential pathways that are altered by NCs. Our preliminary data show that rv2466c, a gene of unknown function in Mtb, is an activating enzyme for NCs, leading to production of several conversion products in vitro, proposing a nitroreductase function for this enzyme. We will identify and synthesize these conversion products, and test their activity against Mtb. If active, these conversion products will be also used to identify potential targets important in killing R and NR Mtb. Moreover, we predict that in addition to its role in NC activation, rv2466c also plays a role in Mtb pathogenesis and dormancy. Evidence from transcriptome studies predict it to be important during stress conditions encountered in the host, which is supported by our preliminary findings showing its importance in oxidative stress resistance. We will further evaluate significance of Rv2466c by generating an rv2466c-knockout strain, and evaluating its phenotype in response to physiologic stresses and during in vivo mouse infections. Furthermore, we will evaluate its binding partners using co- immunoprecipitation strategies, while using information from the already available crystal structure to guide us better delineate sites important for enzymatic and binding activity.