High content, high throughput, diversity screen for novel anti-tubercular agents targeting intracellular bacteria

摘要 结核病仍然是全球主要的健康负担，2019年新增病例1000万例，潜在感染者 数十亿人口。结核病死亡人数现在超过了艾滋病毒死亡人数(2019年为120万人)。分枝杆菌 结核病是一种复杂的病原体，可在人体内持续数十年。 而且它需要长期的治疗才能用多种抗生素治愈。 结核分枝杆菌的特征之一是其在人类细胞内生存和复制的能力，包括 巨噬细胞是抵抗感染的正常宿主防御机制之一。胞内细菌是一种特殊的 可能很难杀死的种群，部分原因是分子必须进入真核细胞，而在 部分原因是细菌所处的不同生理状态。越来越多的证据表明 细胞内细菌对抗生素的耐受性水平，以及增加的异质性。 为了寻找新的药物，我们开发了一种表型筛选方法，利用高含量筛选来 同时监测细菌和真核细胞的存活情况。我们进行了初步筛选，以确定新的化学物质 分枝杆菌细胞内复制的抑制剂。我们找到了几个感兴趣的系列，并选择了三个系列 具有诱人的物理化学性质。在我们初步成功的基础上，我们建议扩大我们的努力，以运行 一个更大的多样化的放映集。 该方案的总体目标是筛选抗细胞内结核分枝杆菌的多样性分子集合，并评估 并确定未来工作的优先顺序。我们将把生物活性、轮廓、作用方式和物理化学结合起来 选择最有希望的系列的属性。这一探索性建议的主要结果将是新颖的。 化学物质准备进入发现号管道。

Summary Tuberculosis remains a major global health burden with 10 million new cases in 2019 and a latently-infected population of billions. Deaths from tuberculosis now exceed those from HIV (1.2 million in 2019). Mycobacterium tuberculosis, the causative agent, is a sophisticated pathogen which can persist for decades in the human host and which requires lengthy treatment for cure with multiple antibiotics. One of the features of M. tuberculosis is its ability to survive and replicate inside human cells, including macrophages, one of the normal host defense mechanisms against infection. Intracellular bacteria are a specific population which can be hard to kill, in part due to the requirement that molecules enter eukaryotic cells, and in part due to the different physiological state in which the bacteria persist. Increasing evidence points to a higher level of antibiotic tolerance in intracellular bacteria, as well as increased heterogeneity. In order to find new agents, we developed a phenotypic screening method utilizing high content screening to monitor bacterial and eukaryotic cell survival simultaneously. We ran a pilot screen to identify novel chemical inhibitors of mycobacterial intracellular replication. We found several series of interest and selected three series with attractive physicochemical properties. Based on our initial success we propose to expand our effort to run a larger diverse screening set. The overall aim of this proposal is to screen a diversity set of molecules against intracellular Mtb and to evaluate and prioritize hits for future work. We will combine biological activity, profile, mode of action and physicochemical properties to select series with the most promise. The major outcome of this exploratory proposal will be novel chemical matter ready to enter the discovery pipeline.