Calanolides as tools to find new drug targets for Mycobacterium tuberculosis

描述(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。据报告，2012年可归因性死亡率为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完整的了解。由于耐药性的出现，以及结核分枝杆菌在被称为非复制(NR)的状态下保持较长时间潜伏的能力，治疗是具有挑战性的。大多数现有的治疗方法在根除NR Mtb方面无效，因为它有可能重新激活，导致疾病。需要针对复制(R)和NR Mtb的新疗法。我们已经确定了一类新的化合物，硝基呋喃甘露内酯(NCS)，可以实现这一目标。NCs对R和NR Mtb具有很强的抗药性，具有选择性，并能被原代人类细胞很好地耐受。此外，它们杀死原代人类巨噬细胞中的结核分枝杆菌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可用药靶点。我们将通过寻找在基本靶点(S)发生突变的耐NC细菌来实现这一点，此外，我们还将使用代谢组学来确定由NC改变的基本途径。我们的初步数据表明，Mtb中一个功能未知的基因rv2466c是一种激活NCS的酶，导致了体外几种转化产物的产生，表明该酶具有硝基还原酶功能。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。如果被激活，这些转化产物也将被用来识别在杀死R和NR Mtb方面重要的潜在靶点。此外，我们预测rv2466c除了在NC激活中发挥作用外，还在Mtb的发病和休眠中发挥作用。来自转录组研究的证据预测，在宿主遇到的压力条件下，它是重要的，这得到了我们的初步发现的支持，表明它在氧化应激抵抗中的重要性。我们将通过产生一株rv2466c-基因敲除菌株，并评估其在生理应激反应和体内感染过程中的表型，进一步评估Rv2466c的意义。此外，我们将使用免疫共沉淀策略评估其结合伙伴，同时使用已有的晶体结构信息来指导我们更好地描绘对酶和结合活性重要的位点。

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.