High Content Screening of Mycobacterium Tuberculosis

总结 结核病（TB）仍然是一个主要的全球健康负担，2014年有960万新病例， 结核病感染人数和死亡人数现已超过艾滋病毒感染人数。结核病是一种复杂的病原体 其可在人类宿主中持续数十年并且需要长期治疗以治愈。之一 结核分枝杆菌的特征是它能够在人体细胞内生存和复制，包括巨噬细胞，巨噬细胞是结核分枝杆菌的主要致病因子之一。 正常的宿主防御机制。 迫切需要结核病的新药和结核分枝杆菌的新药靶标。加大禁毒力度 这一发现导致了Mtb筛查技术的发展和应用;特别是筛查 对抗无菌培养的细菌然而，实验室培养基和培养条件并不准确 再现体内设置。高含量分析（HCA）是一种强大的筛选方法， 生物学相关的基于细胞的测定以高通量方式鉴定活性化合物。为 对于细胞内病原体，高含量筛选具有能够监测细菌和 巨噬细胞数量，同时和在相同的威尔斯，从而导致更可靠的数据和 快速评估复合吸引力。 本提案针对NIH公告PAR-13-364 -高通量测定的开发 用于探针和治疗前发现的筛选（R 01）。我们建议开发和运行强大的 和用于筛选抗细胞内Mtb的可再现的高含量测定。我们将运行一个试点屏幕， 鉴定对细胞内细菌差异有效但缺乏细胞毒性的抑制剂，和 因此可能靶向与感染相关的新途径。我们将进行正交试验和二级试验， 优先考虑药物发现和开发双重方面的化合物，并作为化学探针， 启动目标识别研究。 这项建议将利用我们研究所最近建立的一个新设施， 使用最先进的成像设备，在BSL 3下进行高通量、高内容筛选， 机器人

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.