High Content Screening of Mycobacterium Tuberculosis

摘要 结核病(TB)仍然是全球主要的健康负担，2014年新增病例960万例，潜在的- 数十亿受感染人口和死于结核病的人数现在超过了艾滋病毒感染人数。结核分枝杆菌是一种复杂的病原体 它可以在人类宿主中持续数十年，需要长期治疗才能治愈。其中一个 结核分枝杆菌的特征是它能够在包括巨噬细胞在内的人类细胞内生存和复制，巨噬细胞是 正常的宿主抵抗感染的防御机制。 目前迫切需要治疗结核病的新药和结核分枝杆菌的新药靶标。加大对毒品的打击力度 发现导致了结核分枝杆菌筛查技术的发展和应用；特别是筛查 抗轴突培养的细菌。然而，实验室的培养基和培养条件并不准确 重现活体环境。高含量分析(HCA)是一种强大的筛选方法，它使用 生物相关的基于细胞的分析，以高通量的方式鉴定活性化合物。为 细胞内病原体，高含量筛选的优势是能够同时监控细菌和 巨噬细胞数量的同时和在相同的井，从而导致更可靠的数据和 更快地评估复合吸引力。 该提案涉及NIH公告PAR-13-364-高通量分析的开发 用于探针和治疗前发现的筛查(R01)。我们建议开发并运行健壮的 和可重复的高含量试验(S)，用于筛选抗胞内结核分枝杆菌。我们将运行引导屏幕以 确定对细胞内细菌有不同效果但没有细胞毒性的抑制剂，以及 因此可能针对与感染相关的新途径。我们将进行正交和二次检测，以 优先考虑药物发现和开发的两个方面的化合物，以及作为化学探针和 启动目标识别研究。 这项提议将利用我们研究所最近建立的一个新设施，该设施有能力 使用最先进的成像设备在BSL3下进行高通量、高内容筛选，并 机器人学。

SUMMARY Tuberculosis (TB) remains a major global health burden with 9.6 million new cases in 2014 and a latently- infected population of billions and deaths from TB now exceed those from HIV. Mtb is a sophisticated pathogen which can persist for decades in the human host and which requires lengthy treatment for cure. One of the features of Mtb is its ability to survive and replicate inside human cells, including macrophages, one of the normal host defense mechanisms against infection. There is an urgent need for new drugs for TB and new drug targets for Mtb. An increased effort in drug discovery has led to the development and application of screening technologies to Mtb; in particular screening against axenically-cultured bacteria. However, laboratory medium and culture conditions do not accurately reproduce the in vivo setting. High-content analysis (HCA) is a powerful screening methodology which uses biologically relevant cell-based assays to identify active compounds in a high throughput manner. For intracellular pathogens, high content screening has the advantage of being able to monitor both bacterial and macrophage cell numbers simultaneously and in the same wells, thus leading to more reliable data and a quicker assessment of compound attractiveness. This proposal addresses NIH announcement PAR-13-364 - Development of Assays for High-Throughput Screening for Use in Probe and Pre-therapeutic Discovery (R01). We propose to develop and run robust and reproducible high content assay(s) for screening against intracellular Mtb. We will run a pilot screen to identify inhibitors which are differentially effective against intracellular bacteria, but which lack cytotoxicity, and so may target novel pathways relevant to infection. We will conduct orthogonal and secondary assays to prioritize compounds for the dual aspects of drug discovery and development, and as chemical probes and initiate target identification studies. This proposal will take advantage of a new facility recently established at our institute with the capability to conduct high throughput, high content screening under BSL3 using state of the art imaging equipment and robotics.