Calanolides as tools to find new drug targets for Mycobacterium tuberculosis

Calanolides作为寻找结核分枝杆菌新药物靶点的工具

• 批准号: 8767616
• 负责人: Selin S Somersan-Karakaya
• 金额: $ 17.35万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8767616
• 关键词: Acute; Affect; Alkynes; Anti-HIV Agents; Antibiotics; Bacteria; Binding; Binding Proteins; Biological Factors; Biology; Biotin; Cause of Death; Cells; Chemistry; Chronic; Clinical; Co-Immunoprecipitations; Communicable Diseases; Complement; Data; Development; Disease; Drug Targeting; Drug resistance; Enzymes; Face; Gene Dosage; Gene Expression Profile; Genes; Genome; Genus Mycobacterium; Goals; Growth; Homologous Gene; Hour; Human; In Vitro; Infection; Knock-out; Mentors; Metabolic Pathway; Mus; Mutation; Mycobacterium tuberculosis; Nature; Nitrofurans; Nitroreductases; Oxidative Stress; Oxidoreductase; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Phenotype; Physiological; Play; Population; Predisposition; Prodrugs; Production; Proteins; Reporting; Resistance; Risk; Role; Site; Stress; Structure; Testing; Time; Tuberculosis; attributable mortality; bacterial resistance; bactericide; career development; cofactor; genome sequencing; global health; improved; in vitro Assay; in vivo; indexing; killings; loss of function; macrophage; metabolomics; microbial; mouse model; mutant; mycobacterial; nitrosative stress; non-tuberculosis mycobacteria; novel; novel therapeutics; overexpression; prevent; public health relevance; response; tool

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.

描述（由申请人提供）：结核分枝杆菌（Mtb）是一个重要的全球健康问题，感染了世界三分之一的人口，并且是可治愈的传染病的主要死因。2012年报告的归因死亡率为1，300，000人。尽管自古以来人类就感染结核病，但我们对结核病的发病机制缺乏完整的了解。由于耐药性的出现，以及Mtb在称为非复制（NR）的状态下保持潜伏延长时间的能力，治疗是具有挑战性的。大多数目前的疗法在根除NR Mtb方面是无效的，NR Mtb具有重新激活的潜力，从而引起疾病。需要靶向复制（R）和NR Mtb两者的新疗法。我们已经确定了一类新的化合物，硝基呋喃基calanolides（NC），实现这一目标。NC对R和NR Mtb非常有效，具有选择性，并且被原代人细胞良好耐受。此外，它们在原代人类巨噬细胞中杀死Mtb的能力使它们成为研究的重要类别。我们的项目侧重于使用NC作为工具来发现休眠NR状态的生物学和新的可药用靶标。我们将通过寻找在基本靶标中具有突变的NC耐药细菌来实现这一目标，此外还将使用代谢组学来鉴定被NC改变的基本途径。我们的初步数据显示，rv 2466 c（Mtb中功能未知的基因）是NC的激活酶，导致体外产生多种转化产物，从而提出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。如果有活性，这些转化产物也将用于鉴定在杀死R和NR Mtb中重要的潜在靶标。此外，我们预测rv2466c除了在NC激活中的作用外，还在Mtb发病和休眠中发挥作用。来自转录组研究的证据预测它在宿主中遇到的应激条件下是重要的，这得到了我们的初步研究结果的支持，表明它在抗氧化应激中的重要性。我们将通过产生rv2466c敲除菌株，并评估其对生理应激的反应和体内小鼠感染期间的表型，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伴侣，同时使用来自已经可用的晶体结构的信息来指导我们更好地描绘对酶和结合活性重要的位点。